# Patent

## Title
Compositions and methods for the suppression of target polynucleotides from lepidoptera

## Abstract

Methods and compositions are provided which employ a silencing element that, when ingested by a pest, such as a pest from the Lepidoptera order, they are capable of decreasing the expression of a target sequence in the pest. Further provided are silencing elements which when ingested by the pest decrease the level of the target polypeptide and thereby control the pest. In specific embodiment, the pest is Spodoptera frugiperda. Plants, plant part, bacteria and other host cells comprising the silencing elements or an active variant or fragment thereof of the invention are also provided.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to, U.S. Provisional Application No. 61/021,699, filed Jan. 17, 2008, and U.S. Provisional Application No. 61/021,676, filed Jan. 17, 2008; and is a continuation of U.S. Non Provisional application Ser. No. 12/351,267 filed Jan. 9, 2009, now granted as U.S. Pat. No. 8,847,013, which are herein incorporated by reference in their entirety.


FIELD OF THE INVENTION
The present invention relates generally to methods of molecular biology and gene silencing to control pests.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB
The official copy of the sequence listing is submitted concurrently with the specification as a text file via EFS-Web, in compliance with the American Standard Code for Information Interchange (ASCII), with a file name of 366590seqlist.txt, a creation date of Jan. 9, 2009, and a size of 102 Kb. The sequence listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein.
BACKGROUND OF THE INVENTION
Insect pests are a serious problem in agriculture. They destroy millions of acres of staple crops such as corn, soybeans, peas, and cotton. Yearly, these pests cause over $100 billion dollars in crop damage in the U.S. alone. In an ongoing seasonal battle, farmers must apply billions of gallons of synthetic pesticides to combat these pests. Other methods employed in the past delivered insecticidal activity by microorganisms or genes derived from microorganisms expressed in transgenic plants. For example, certain species of microorganisms of the genus Bacillus are known to possess pesticidal activity against a broad range of insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera, and others. In fact, microbial pesticides, particularly those obtained from Bacillus strains, have played an important role in agriculture as alternatives to chemical pest control. Agricultural scientists have developed crop plants with enhanced insect resistance by genetically engineering crop plants to produce insecticidal proteins from Bacillus. For example, corn and cotton plants genetically engineered to produce Cry toxins (see, e.g., Aronson (2002) Cell Mol. Life Sci. 59(3):417-425; Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62(3):775-806) are now widely used in American agriculture and have provided the farmer with an alternative to traditional insect-control methods. However, these Bt insecticidal proteins only protect plants from a relatively narrow range of pests. Moreover, these modes of insecticidal activity provided varying levels of specificity and, in some cases, caused significant environmental consequences. Thus, there is an immediate need for alternative methods to control pests.
BRIEF SUMMARY OF THE INVENTION
Methods and compositions are provided which employ a silencing element that, when ingested by a pest, such as a pest from the Lepidoptera order, is capable of decreasing the expression of a target sequence in the pest. In specific embodiments, the decrease in expression of the target sequence controls the pest and thereby the methods and compositions are capable of limiting damage to a plant. The present invention provides various target polynucleotides encoding polypeptides from specific families as disclosed elsewhere herein and various target polynucleotides set forth in SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or active variants or fragments thereof, wherein a decrease in expression of one or more the sequences in the target pest controls the pest (i.e., has insecticidal activity). Further provided are silencing elements, which when ingested by the pest, decrease the level of expression of one or more of the target polynucleotides. In specific embodiments, the silencing element comprises at least 15, 20, or 22 consecutive nucleotides of any one of SEQ ID NO:1-50. In specific embodiments, the pest that is controlled is Spodoptera frugiperda. Plants, plant parts, plant cells, bacteria and other host cells comprising the silencing elements or an active variant or fragment thereof are also provided.
In another embodiment, a method for controlling a pest, such as a pest from the Lepidoptera order, is provided. The method comprises feeding to a pest a composition comprising a silencing element, wherein the silencing element, when ingested by the pest, reduces the level of a target sequence in the pest and thereby controls the pest. Further provided are methods to protect a plant from a pest. Such methods comprise introducing into the plant or plant part a silencing element of the invention. When the plant expressing the silencing element is ingested by the pest, the level of the target sequence is decreased and the pest is controlled.


DETAILED DESCRIPTION OF THE INVENTION
The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
I. Overview
Methods and compositions are provided which employ a silencing element that, when ingested by a pest, such as a pest from the Lepidoptera order, is capable of decreasing the expression of a target sequence in the pest. In specific embodiments, the decrease in expression of the target sequence controls the pest and thereby the methods and compositions are capable of limiting damage to a plant or plant part. The present invention provides target polynucleotides which encode polypeptides from a variety of protein classes including, for example, a juvenile hormone polypeptide, a vacuolar polypeptide, a cadherin polypeptide, a cuticle polypeptide, a translation initiation factor, a SARI polypeptide, an elongation factor, a phosphooligosaccharide, a myosin polypeptide, a potassium channel amino acid transporter, a potassium inwardly rectifier polypeptide, an amino acid transporter, a tubulin polypeptide, a ubiquitin polypeptide, and small nuclear ribonucleoprotein. In other embodiments the target polynucleotides are set forth in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or active variants and fragments thereof. Silencing elements designed in view of these target polynucleotides are provided which, when ingested by the pest, decrease the expression of one or more of the target sequences and thereby controls the pest (i.e., has insecticidal activity). See, for example, SEQ ID NOS:51-465.
As used herein, by “controlling a pest” or “controls a pest” is intended any affect on a pest that results in limiting the damage that the pest causes. Controlling a pest includes, but is not limited to, killing the pest, inhibiting development of the pest, altering fertility or growth of the pest in such a manner that the pest provides less damage to the plant, decreasing the number of offspring produced, producing less fit pests, producing pests more susceptible to predator attack, or deterring the pests from eating the plant.
By “disease resistance” is intended that the plants avoid the disease symptoms that are the outcome of plant-pathogen interactions. That is, pathogens are prevented from causing plant diseases and the associated disease symptoms, or alternatively, the disease symptoms caused by the pathogen is minimized or lessened.
Reducing the level of expression of the target polynucleotide or the polypeptide encoded thereby, in the pest results in the suppression, control, and/or killing the invading pathogenic organism. Reducing the level of expression of the target sequence of the pest will reduce the disease symptoms resulting from pathogen challenge by at least about 2% to at least about 6%, at least about 5% to about 50%, at least about 10% to about 60%, at least about 30% to about 70%, at least about 40% to about 80%, or at least about 50% to about 90% or greater. Hence, the methods of the invention can be utilized to protect plants from disease, particularly those diseases that are caused by pests from the Lepidoptera order.
Assays that measure the control of a pest are commonly known in the art, as are methods to quantitate disease resistance in plants following pathogen infection. See, for example, U.S. Pat. No. 5,614,395, herein incorporated by reference. Such techniques include, measuring over time, the average lesion diameter, the pathogen biomass, and the overall percentage of decayed plant tissues. See, for example, Thomma et al. (1998) Plant Biology 95:15107-15111, herein incorporated by reference. See, also the examples below.
The invention is drawn to compositions and methods for protecting plants from a plant pest, such as pests from the Lepidoptera order or inducing resistance in a plant to a plant pest, such as pests from the Lepidoptera order. Caterpillars and related forms of lepidopteran insects comprise an important group of plant-feeding agricultural pests, especially during the larvae stage of growth. Feeding methods of Lepidoptera larvae typically include chewing plants or plant parts. As used herein, the term “Lepidoptera” is used to refer to any member of the Lepidoptera order. In particular embodiments, compositions and methods of the invention control Lepidoptera larvae (i.e. caterpillars). Accordingly, the compositions and methods are also useful in protecting plants against any Lepidoptera including, for example, Pieris rapae, Pectinophora gossypiella, Synanthedon exitiosa, Melittia cucurbitae, Cydia pomonella, Grapholita molesta, Ostrinia nubilalis, Plodia interpunctella, Galleria mellonella, Manduca sexta, Manduca quinquemaculata, Lymantria dispar, Euproctis chrysorrhoea, Trichoplusia ni, Mamestra brassicae, Agrotis ipsilon, Plutella xylostella, Anticarsia gemmatalis, Psuedoplusia includens, Epinotia aporema, Helicoverpa zea, Heliothis virescens, Heliothis armigera, Spodoptera exigua, Scirpophaga incertulus, Sesamia spp., Buseola fusca, Cnaphalocrocis medinalis, Chilo suppressalis, or Spodoptera littoralis. In particular embodiments, methods control Spodoptera frugiperda. 
II. Target Sequences
As used herein, a “target sequence” or “target polynucleotide” comprises any sequence in the pest that one desires to reduce the level of expression. In specific embodiments, decreasing the level of the target sequence in the pest controls the pest. For instance, the target sequence can be essential for growth and development. While the target sequence can be expressed in any tissue of the pest, in specific embodiments of the invention, the sequences targeted for suppression in the pest are expressed in cells of the gut tissue of the pest, cells in the midgut of the pest, and cells lining the gut lumen or the midgut. Such target sequences can be involved in gut cell metabolism, growth or differentiation.
In one embodiment of the invention the target sequence comprises a polypeptide belonging to one or more classes of enzymes such as a juvenile hormone polypeptide, a vacuolar polypeptide, a cadherin polypeptide, a cuticle polypeptide, a translation initiation factor, a SARI polypeptide, an elongation factor, a phosphooligosaccharide, a myosin polypeptide, a potassium channel amino acid transporter, a potassium inwardly rectifier, an amino acid transporter, a tubulin polypeptide, a ubiquitin polypeptide, and a small nuclear ribonucleoprotein. Non-limiting examples of target sequences of the invention include a polynucleotide set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. As exemplified elsewhere herein, decreasing the level of expression of these target sequence or members of the recited enzyme classes in Lepidoptera controls the pest.
III. Silencing Elements
By “silencing element” is intended a polynucleotide which when ingested by a pest, is capable of reducing or eliminating the level or expression of a target polynucleotide or the polypeptide encoded thereby. The silencing element employed can reduce or eliminate the expression level of the target sequence by influencing the level of the target RNA transcript or, alternatively, by influencing translation and thereby affecting the level of the encoded polypeptide. Methods to assay for functional silencing elements that are capable of reducing or eliminating the level of a sequence of interest are disclosed elsewhere herein. A single polynucleotide employed in the methods of the invention can comprises one or more silencing elements to the same or different target polynucleotides.
In specific embodiments, the target sequence is not a plant endogenous gene. In other embodiments, while the silencing element controls pests, preferably the silencing element has no effect on the normal plant or plant part.
As discussed in further detail below, silencing elements can include, but are not limited to, a sense suppression element, an antisense suppression element, a double stranded RNA, a miRNA, or a hairpin suppression element. Non-limiting examples of silencing elements that can be employed to decrease expression of these target Lepidoptera sequences comprise fragments and variants of the sense or antisense sequence or consists of the sense or antisense sequence of the sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363, 366, 369, 372, 375, 378, 381, 384, 387, 390, 393, 396, 399, 402, 405, 408, 411, 415, 418, 421, 424, 427, 430, 433, 436, 439, 442, 457, 460, and/or 463 or a biologically active variant or fragment thereof. In specific embodiments, the silencing element comprises or consists of at least one of the sequences set forth in any one of SEQ ID NOS: 51-465. In further embodiments, the silencing elements can comprise at least one thymine residue at the 3′ end. This can aid in stabilization. Thus, the silencing elements can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more thymine residues at the 3′ end.
In further embodiments, the silencing element comprises SEQ ID NO: 52 and 53; 55 and 56; 58 and 59; 61 and 62; 64 and 65; 67 and 68; 70 and 71; 73 and 74; 76 and 77; 79 and 80; 82 and 83; 85 and 86; 88 and 89; 91 and 92; 94 and 95; 97 and 98; 100 and 101; 103 and 104; 106 and 107; 109 and 110; 112 and 113; 115 and 116; 118 and 119; 121 and 122; 124 and 125; 127 and 128; 130 and 131; 133 and 134; 136 and 137; 139 and 140; 142 and 143; 145 and 146; 148 and 149; 151 and 152; 154 and 155; 157 and 158; 160 and 161; 163 and 164; 166 and 167; 169 and 170; 172 and 173; 175 and 176; 178 and 179; 181 and 182; 184 and 185; 187 and 188; 190 and 191; 193 and 194; 196 and 197; 199 and 200; 202 and 203; 205 and 206; 208 and 209; 211 and 212; 214 and 215; 217 and 218; 220 and 221; 223 and 224; 226 and 227; 229 and 230; 232 and 233; 235 and 236; 238 and 239; 241 and 242; 244 and 245; 247 and 248; 250 and 251; 253 and 254; 256 and 257; 259 and 260; 262 and 263; 265 and 266; 268 and 269; 271 and 272; 274 and 275; 277 and 278; 280 and 281; 283 and 284; 286 and 287; 289 and 290; 292 and 293; 295 and 296; 298 and 299; 301 and 302; 304 and 305; 307 and 308; 310 and 311; 313 and 314; 316 and 317; 139 and 320; 322 and 323; 325 and 326; 328 and 329; 331 and 332; 334 and 335; 337 and 338; 340 and 341; 343 and 344; 346 and 347; 349 and 350; 352 and 353; 355 and 356; 358 and 359; 361 and 362; 364 and 365; 367 and 368; 370 and 371; 373 and 374; 376 and 377; 379 and 380; 382 and 383; 385 and 386; 388 and 389; 391 and 392; 394 and 395; 397 and 398; 400 and 401; 403 and 404; 406 and 407; 409 and 410; 412 and 413; 416 and 417; 419 and 420; 422 and 423; 425 and 426; 428 and 429; 431 and 432; 434 and 435; 437 and 438; 440 and 441; 443 and 444; 458 and 459; 461 and 462; and/or 464 and 465.
By “reduces” or “reducing” the expression level of a polynucleotide or a polypeptide encoded thereby is intended to mean, the polynucleotide or polypeptide level of the target sequence is statistically lower than the polynucleotide level or polypeptide level of the same target sequence in an appropriate control pest which is not exposed to (i.e., has not ingested) the silencing element. In particular embodiments of the invention, reducing the polynucleotide level and/or the polypeptide level of the target sequence in a pest according to the invention results in less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the polynucleotide level, or the level of the polypeptide encoded thereby, of the same target sequence in an appropriate control pest. Methods to assay for the level of the RNA transcript, the level of the encoded polypeptide, or the activity of the polynucleotide or polypeptide are discussed elsewhere herein.
i. Sense Suppression Elements
As used herein, a “sense suppression element” comprises a polynucleotide designed to express an RNA molecule corresponding to at least a part of a target messenger RNA in the “sense” orientation. Expression of the RNA molecule comprising the sense suppression element reduces or eliminates the level of the target polynucleotide or the polypeptide encoded thereby. The polynucleotide comprising the sense suppression element may correspond to all or part of the sequence of the target polynucleotide, all or part of the 5′ and/or 3′ untranslated region of the target polynucleotide, all or part of the coding sequence of the target polynucleotide, or all or part of both the coding sequence and the untranslated regions of the target polynucleotide.
Typically, a sense suppression element has substantial sequence identity to the target polynucleotide, typically greater than about 65% sequence identity, greater than about 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. See, U.S. Pat. Nos. 5,283,184 and 5,034,323; herein incorporated by reference. The sense suppression element can be any length so long as it allows for the suppression of the targeted sequence. The sense suppression element can be, for example, 15, 20, 22, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 900 or longer.
ii. Antisense Suppression Elements
As used herein, an “antisense suppression element” comprises a polynucleotide which is designed to express an RNA molecule complementary to all or part of a target messenger RNA. Expression of the antisense RNA suppression element reduces or eliminates the level of the target polynucleotide. The polynucleotide for use in antisense suppression may correspond to all or part of the complement of the sequence encoding the target polynucleotide, all or part of the complement of the 5′ and/or 3′ untranslated region of the target polynucleotide, all or part of the complement of the coding sequence of the target polynucleotide, or all or part of the complement of both the coding sequence and the untranslated regions of the target polynucleotide. In addition, the antisense suppression element may be fully complementary (i.e., 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target polynucleotide. In specific embodiments, the antisense suppression element comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence complementarity to the target polynucleotide. Antisense suppression may be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Pat. No. 5,942,657. Furthermore, the antisense suppression element can be complementary to a portion of the target polynucleotide. Generally, sequences of at least 15, 20, 22, 25, 50, 100, 200, 300, 400, 450 nucleotides or greater may be used. Methods for using antisense suppression to inhibit the expression of endogenous genes in plants are described, for example, in Liu et al (2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829 and 5,942,657, each of which is herein incorporated by reference.
iii. Double Stranded RNA Suppression Element
A “double stranded RNA silencing element” or “dsRNA” comprises at least one transcript that is capable of forming a dsRNA either before or after ingestion by a pest. Thus, a “dsRNA silencing element” includes a dsRNA, a transcript or polyribonucleotide capable of forming a dsRNA or more than one transcript or polyribonucleotide capable of forming a dsRNA. “Double stranded RNA” or “dsRNA” refers to a polyribonucleotide structure formed either by a single self-complementary RNA molecule or a polyribonucleotide structure formed by the expression of least two distinct RNA strands. The dsRNA molecule(s) employed in the methods and compositions of the invention mediate the reduction of expression of a target sequence, for example, by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner. In the context of the present invention, the dsRNA is capable of reducing or eliminating the level or expression of a target polynucleotide or the polypeptide encoded thereby in a pest.
The dsRNA can reduce or eliminate the expression level of the target sequence by influencing the level of the target RNA transcript, by influencing translation and thereby affecting the level of the encoded polypeptide, or by influencing expression at the pre-transcriptional level (i.e., via the modulation of chromatin structure, methylation pattern, etc., to alter gene expression). See, for example, Verdel et al. (2004) Science 303:672-676; Pal-Bhadra et al. (2004) Science 303:669-672; Allshire (2002) Science 297:1818-1819; Volpe et al. (2002) Science 297:1833-1837; Jenuwein (2002) Science 297:2215-2218; and Hall et al. (2002) Science 297:2232-2237. Methods to assay for functional iRNA that are capable of reducing or eliminating the level of a sequence of interest are disclosed elsewhere herein. Accordingly, as used herein, the term “dsRNA” is meant to encompass other terms used to describe nucleic acid molecules that are capable of mediating RNA interference or gene silencing, including, for example, short-interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), hairpin RNA, short hairpin RNA (shRNA), post-transcriptional gene silencing RNA (ptgsRNA), and others.
In specific embodiments, at least one strand of the duplex or double-stranded region of the dsRNA shares sufficient sequence identity or sequence complementarity to the target polynucleotide to allow for the dsRNA to reduce the level of expression of the target sequence. As used herein, the strand that is complementary to the target polynucleotide is the “antisense strand” and the strand homologous to the target polynucleotide is the “sense strand.”
In one embodiment, the dsRNA comprises a hairpin RNA. A hairpin RNA comprises an RNA molecule that is capable of folding back onto itself to form a double stranded structure. Multiple structures can be employed as hairpin elements. In specific embodiments, the dsRNA suppression element comprises a hairpin element which comprises in the following order, a first segment, a second segment, and a third segment, where the first and the third segment share sufficient complementarity to allow the transcribed RNA to form a double-stranded stem-loop structure.
The “second segment” of the hairpin comprises a “loop” or a “loop region.” These terms are used synonymously herein and are to be construed broadly to comprise any nucleotide sequence that confers enough flexibility to allow self-pairing to occur between complementary regions of a polynucleotide (i.e., segments 1 and 3 which form the stem of the hairpin). For example, in some embodiments, the loop region may be substantially single stranded and act as a spacer between the self-complementary regions of the hairpin stem-loop. In some embodiments, the loop region can comprise a random or nonsense nucleotide sequence and thus not share sequence identity to a target polynucleotide. In other embodiments, the loop region comprises a sense or an antisense RNA sequence or fragment thereof that shares identity to a target polynucleotide. See, for example, International Patent Publication No. WO 02/00904, herein incorporated by reference. In specific embodiments, the loop region can be optimized to be as short as possible while still providing enough intramolecular flexibility to allow the formation of the base-paired stem region. Accordingly, the loop sequence is generally less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 25, 20, 15, 10 nucleotides or less.
The “first” and the “third” segment of the hairpin RNA molecule comprise the base-paired stem of the hairpin structure. The first and the third segments are inverted repeats of one another and share sufficient complementarity to allow the formation of the base-paired stem region. In specific embodiments, the first and the third segments are fully complementary to one another. Alternatively, the first and the third segment may be partially complementary to each other so long as they are capable of hybridizing to one another to form a base-paired stem region. The amount of complementarity between the first and the third segment can be calculated as a percentage of the entire segment. Thus, the first and the third segment of the hairpin RNA generally share at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to and including 100% complementarity.
The first and the third segment are at least about 1000, 500, 400, 300, 200, 100, 50, 40, 30, 25, 22, 20, 15 or 10 nucleotides in length. In specific embodiments, the length of the first and/or the third segment is about 10-100 nucleotides, about 10 to about 75 nucleotides, about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 35 nucleotides, about 10 to about 30 nucleotides, about 10 to about 25 nucleotides, about 10 to about 20 nucleotides. In other embodiments, the length of the first and/or the third segment comprises at least 10-20 nucleotides, 20-35 nucleotides, 30-45 nucleotides, 40-50 nucleotides, 50-100 nucleotides, or 100-300 nucleotides. See, for example, International Publication No. WO 0200904. In specific embodiments, the first and the third segment comprise at least 20 nucleotides having at least 85% complementary to the first segment. In still other embodiments, the first and the third segments which form the stem-loop structure of the hairpin comprises 3′ or 5′ overhang regions having unpaired nucleotide residues.
In specific embodiments, the sequences used in the first, the second, and/or the third segments comprise domains that are designed to have sufficient sequence identity to a target polynucleotide of interest and thereby have the ability to decrease the level of expression of the target polynucleotide. The specificity of the inhibitory RNA transcripts is therefore generally conferred by these domains of the silencing element. Thus, in some embodiments of the invention, the first, second and/or third segment of the silencing element comprise a domain having at least 10, at least 15, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 500, at least 1000, or more than 1000 nucleotides that share sufficient sequence identity to the target polynucleotide to allow for a decrease in expression levels of the target polynucleotide when expressed in an appropriate cell. In other embodiments, the domain is between about 15 to 50 nucleotides, about 20-35 nucleotides, about 25-50 nucleotides, about 20 to 75 nucleotides, about 40-90 nucleotides about 15-100 nucleotides.
In specific embodiments, the domain of the first, the second, and/or the third segment has 100% sequence identity to the target polynucleotide. In other embodiments, the domain of the first, the second and/or the third segment having homology to the target polypeptide have at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to a region of the target polynucleotide. The sequence identity of the domains of the first, the second and/or the third segments to the target polynucleotide need only be sufficient to decrease expression of the target polynucleotide of interest. See, for example, Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijk et al. (2002) Plant Physiol. 129:1723-1731; Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38; Pandolfini et al. BMC Biotechnology 3:7, and U.S. Patent Publication No. 20030175965; each of which is herein incorporated by reference. A transient assay for the efficiency of hpRNA constructs to silence gene expression in vivo has been described by Panstruga et al. (2003) Mol. Biol. Rep. 30:135-140, herein incorporated by reference.
The amount of complementarity shared between the first, second, and/or third segment and the target polynucleotide or the amount of complementarity shared between the first segment and the third segment (i.e., the stem of the hairpin structure) may vary depending on the organism in which gene expression is to be controlled. Some organisms or cell types may require exact pairing or 100% identity, while other organisms or cell types may tolerate some mismatching. In some cells, for example, a single nucleotide mismatch in the targeting sequence abrogates the ability to suppress gene expression. In these cells, the suppression cassettes of the invention can be used to target the suppression of mutant genes, for example, oncogenes whose transcripts comprise point mutations and therefore they can be specifically targeted using the methods and compositions of the invention without altering the expression of the remaining wild-type allele.
Any region of the target polynucleotide can be used to design the domain of the silencing element that shares sufficient sequence identity to allow expression of the hairpin transcript to decrease the level of the target polynucleotide. For instance, the domain can be designed to share sequence identity to the 5′ untranslated region of the target polynucleotide(s), the 3′ untranslated region of the target polynucleotide(s), exonic regions of the target polynucleotide(s), intronic regions of the target polynucleotide(s), and any combination thereof. In specific embodiments a domain of the silencing element shares sufficient homology to at least about 15, 20, 22, 25 or 30 consecutive nucleotides from about nucleotides 1-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 550-600, 600-650, 650-700, 750-800, 850-900, 950-1000, 1000-1050, 1050-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000 of the target sequence. In some instances to optimize the siRNA sequences employed in the hairpin, the synthetic oligodeoxyribonucleotide/RNAse H method can be used to determine sites on the target mRNA that are in a conformation that is susceptible to RNA silencing. See, for example, Vickers et al. (2003) J. Biol. Chem 278:7108-7118 and Yang et al. (2002) Proc. Natl. Acad. Sci. USA 99:9442-9447, herein incorporated by reference. These studies indicate that there is a significant correlation between the RNase-H-sensitive sites and sites that promote efficient siRNA-directed mRNA degradation.
The hairpin silencing element may also be designed such that the sense sequence or the antisense sequence do not correspond to a target polynucleotide. In this embodiment, the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the target polynucleotide. Thus, it is the loop region that determines the specificity of the RNA interference. See, for example, WO 02/00904, herein incorporated by reference.
In specific embodiments, the silencing element comprising the hairpin comprises sequences selected from the group consisting of SEQ ID NO: 52 and 53; 55 and 56; 58 and 59; 61 and 62; 64 and 65; 67 and 68; 70 and 71; 73 and 74; 76 and 77; 79 and 80; 82 and 83; 85 and 86; 88 and 89; 91 and 92; 94 and 95; 97 and 98; 100 and 101; 103 and 104; 106 and 107; 109 and 110; 112 and 113; 115 and 116; 118 and 119; 121 and 122; 124 and 125; 127 and 128; 130 and 131; 133 and 134; 136 and 137; 139 and 140; 142 and 143; 145 and 146; 148 and 149; 151 and 152; 154 and 155; 157 and 158; 160 and 161; 163 and 164; 166 and 167; 169 and 170; 172 and 173; 175 and 176; 178 and 179; 181 and 182; 184 and 185; 187 and 188; 190 and 191; 193 and 194; 196 and 197; 199 and 200; 202 and 203; 205 and 206; 208 and 209; 211 and 212; 214 and 215; 217 and 218; 220 and 221; 223 and 224; 226 and 227; 229 and 230; 232 and 233; 235 and 236; 238 and 239; 241 and 242; 244 and 245; 247 and 248; 250 and 251; 253 and 254; 256 and 257; 259 and 260; 262 and 263; 265 and 266; 268 and 269; 271 and 272; 274 and 275; 277 and 278; 280 and 281; 283 and 284; 286 and 287; 289 and 290; 292 and 293; 295 and 296; 298 and 299; 301 and 302; 304 and 305; 307 and 308; 310 and 311; 313 and 314; 316 and 317; 139 and 320; 322 and 323; 325 and 326; 328 and 329; 331 and 332; 334 and 335; 337 and 338; 340 and 341; 343 and 344; 346 and 347; 349 and 350; 352 and 353; 355 and 356; 358 and 359; 361 and 362; 364 and 365; 367 and 368; 370 and 371; 373 and 374; 376 and 377; 379 and 380; 382 and 383; 385 and 386; 388 and 389; 391 and 392; 394 and 395; 397 and 398; 400 and 401; 403 and 404; 406 and 407; 409 and 410; 412 and 413; 416 and 417; 419 and 420; 422 and 423; 425 and 426; 428 and 429; 431 and 432; 434 and 435; 437 and 438; 440 and 441; 443 and 444; 458 and 459; 461 and 462; and/or 464 and 465.
In addition, transcriptional gene silencing (TGS) may be accomplished through use of a hairpin suppression element where the inverted repeat of the hairpin shares sequence identity with the promoter region of a target polynucleotide to be silenced. See, for example, Aufsatz et al. (2002) PNAS 99 (Suppl. 4):16499-16506 and Mette et al. (2000) EMBO J 19(19):5194-5201.
In other embodiments, the dsRNA can comprise a small RNA (sRNA). sRNAs can comprise both micro RNA (miRNA) and short-interfering RNA (siRNA) (Meister and Tuschl (2004) Nature 431:343-349 and Bonetta et al. (2004) Nature Methods 1:79-86). miRNAs are regulatory agents comprising about 19 ribonucleotides which are highly efficient at inhibiting the expression of target polynucleotides. See, for example Javier et al. (2003) Nature 425: 257-263, herein incorporated by reference. For miRNA interference, the silencing element can be designed to express a dsRNA molecule that forms a hairpin structure containing a 19-nucleotide sequence that is complementary to the target polynucleotide of interest. The miRNA can be synthetically made, or transcribed as a longer RNA which is subsequently cleaved to produce the active miRNA. Specifically, the miRNA can comprise 19 nucleotides of the sequence having homology to a target polynucleotide in sense orientation and 19 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence.
When expressing an miRNA, it is recognized that various forms of an miRNA can be transcribed including, for example, the primary transcript (termed the “pri-miRNA”) which is processed through various nucleolytic steps to a shorter precursor miRNA (termed the “pre-miRNA”); the pre-miRNA; or the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA (the strand that will eventually basepair with the target) and miRNA*. The pre-miRNA is a substrate for a form of dicer that removes the miRNA/miRNA* duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex. It has been demonstrated that miRNAs can be transgenically expressed and be effective through expression of a precursor form, rather than the entire primary form (Parizotto et al. (2004) Genes & Development 18:2237-2242 and Guo et al. (2005) Plant Cell 17:1376-1386).
The methods and compositions of the invention employ silencing elements that when transcribed “form” a dsRNA molecule. Accordingly, the heterologous polynucleotide being expressed need not form the dsRNA by itself, but can interact with other sequences in the plant cell or in the pest gut after ingestion to allow the formation of the dsRNA. For example, a chimeric polynucleotide that can selectively silence the target polynucleotide can be generated by expressing a chimeric construct comprising the target sequence for a miRNA or siRNA to a sequence corresponding to all or part of the gene or genes to be silenced. In this embodiment, the dsRNA is “formed” when the target for the miRNA or siRNA interacts with the miRNA present in the cell. The resulting dsRNA can then reduce the level of expression of the gene or genes to be silenced. See, for example, U.S. Provisional Application No. 60/691,613, filed Jun. 17, 2005, entitled “Methods and Compositions for Gene Silencing, herein incorporated by reference. The construct can be designed to have a target for an endogenous miRNA or alternatively, a target for a heterologous and/or synthetic miRNA can be employed in the construct. If a heterologous and/or synthetic miRNA is employed, it can be introduced into the cell on the same nucleotide construct as the chimeric polynucleotide or on a separate construct. As discussed elsewhere herein, any method can be used to introduce the construct comprising the heterologous miRNA.
IV. Variants and Fragments
By “fragment” is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a polynucleotide may encode protein fragments that retain the biological activity of the native protein. Alternatively, fragments of a polynucleotide that are useful as a silencing element do not need to encode fragment proteins that retain biological activity. Thus, fragments of a nucleotide sequence may range from at least about 10, about 15, about 20 nucleotides, about 22 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides and up to the full-length polynucleotide employed in the invention. Methods to assay for the activity of a desired silencing element or a suppressor enhancer element are described elsewhere herein.
“Variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides employed in the invention. Variant polynucleotides also include synthetically derived polynucleotide, such as those generated, for example, by using site-directed mutagenesis, but continue to retain the desired activity. Generally, variants of a particular polynucleotide of the invention (i.e., a silencing element) will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.
Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides employed in the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
“Variant” protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more internal sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, as discussed elsewhere herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native protein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
The following terms are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, and, (d) “percentage of sequence identity.”
(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
(b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two polynucleotides. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
(c) As used herein, “sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
(d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
V. DNA Constructs
The use of the term “polynucleotide” is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
The polynucleotide encoding the silencing element or in specific embodiments employed in the methods and compositions of the invention can be provided in expression cassettes for expression in a plant or organism of interest. It is recognized that multiple silencing elements including multiple identical silencing elements, multiple silencing elements targeting different regions of the target sequence, or multiple silencing elements from different target sequences can be used. In this embodiment, it is recognized that each silencing element can be contained in a single or separate cassette, DNA construct, or vector. As discussed, any means of providing the silencing element is contemplated. A plant or plant cell can be transformed with a single cassette comprising DNA encoding one or more silencing elements or separate cassettes comprising each silencing element can be used to transform a plant or plant cell or host cell. Likewise, a plant transformed with one component can be subsequently transformed with the second component. One or more silencing elements can also be brought together by sexual crossing. That is, a first plant comprising one component is crossed with a second plant comprising the second component. Progeny plants from the cross will comprise both components.
The expression cassette can include 5′ and 3′ regulatory sequences operably linked to the polynucleotide of the invention. “Operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of the invention and a regulatory sequence (i.e., a promoter) is a functional link that allows for expression of the polynucleotide of the invention. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional polynucleotide to be cotransformed into the organism. Alternatively, the additional polypeptide(s) can be provided on multiple expression cassettes. Expression cassettes can be provided with a plurality of restriction sites and/or recombination sites for insertion of the polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.
The expression cassette will include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a polynucleotide comprising the silencing element employed in the methods and compositions of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in plants. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the polynucleotides employed in the invention may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the polynucleotide employed in the invention may be heterologous to the host cell or to each other. As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
The termination region may be native with the transcriptional initiation region, may be native with the operably linked polynucleotide encoding the silencing element, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the polynucleotide comprising silencing element, the plant host, or any combination thereof. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.
Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.
A number of promoters can be used in the practice of the invention. The polynucleotide encoding the silencing element can be combined with constitutive, tissue-preferred, or other promoters for expression in plants.
Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.
An inducible promoter, for instance, a pathogen-inducible promoter could also be employed. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116. See also WO 99/43819, herein incorporated by reference.
Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386 (nematode-inducible); and the references cited therein. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).
Additionally, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter may be used in the constructions of the invention. Such wound-inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology 14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76); MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like, herein incorporated by reference.
Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.
Tissue-preferred promoters can be utilized to target enhanced expression within a particular plant tissue. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.
Leaf-preferred promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633-641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-specific promoter activity was preserved. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri et al. (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2′ gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused to nptII (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.
In one embodiment of this invention the plant-expressed promoter is a vascular-specific promoter such as a phloem-specific promoter. A “vascular-specific” promoter, as used herein, is a promoter which is at least expressed in vascular cells, or a promoter which is preferentially expressed in vascular cells. Expression of a vascular-specific promoter need not be exclusively in vascular cells, expression in other cell types or tissues is possible. A “phloem-specific promoter” as used herein, is a plant-expressible promoter which is at least expressed in phloem cells, or a promoter which is preferentially expressed in phloem cells.
Expression of a phloem-specific promoter need not be exclusively in phloem cells, expression in other cell types or tissues, e.g., xylem tissue, is possible. In one embodiment of this invention, a phloem-specific promoter is a plant-expressible promoter at least expressed in phloem cells, wherein the expression in non-phloem cells is more limited (or absent) compared to the expression in phloem cells. Examples of suitable vascular-specific or phloem-specific promoters in accordance with this invention include but are not limited to the promoters selected from the group consisting of: the SCSV3, SCSV4, SCSV5, and SCSV7 promoters (Schunmann et al. (2003) Plant Functional Biology 30:453-60; the rolC gene promoter of Agrobacterium rhizogenes(Kiyokawa et al. (1994) Plant Physiology 104:801-02; Pandolfini et al. (2003) BioMedCentral (BMC) Biotechnology 3:7; Graham et al. (1997) Plant Mol. Biol. 33:729-35; Guivarc'h et al. (1996); Almon et al. (1997) Plant Physiol. 115:1599-607; the rolA gene promoter of Agrobacterium rhizogenes (Dehio et al. (1993) Plant Mol. Biol. 23:1199-210); the promoter of the Agrobacterium tumefaciens T-DNA gene 5 (Korber et al. (1991) EMBO J. 10:3983-91); the rice sucrose synthase RSs1 gene promoter (Shi et al. (1994) J. Exp. Bot. 45:623-31); the CoYMV or Commelina yellow mottle badnavirus promoter (Medberry et al. (1992) Plant Cell 4:185-92; Zhou et al. (1998) Chin. J. Biotechnol. 14:9-16); the CFDV or coconut foliar decay virus promoter (Rohde et al. (1994) Plant Mol. Biol. 27:623-28; Hehn and Rhode (1998) J. Gen. Virol. 79:1495-99); the RTBV or rice tungro bacilliform virus promoter (Yin and Beachy (1995) Plant J. 7:969-80; Yin et al. (1997) Plant J. 12:1179-80); the pea glutamin synthase GS3A gene (Edwards et al. (1990) Proc. Natl. Acad. Sci. USA 87:3459-63; Brears et al. (1991) Plant J. 1:235-44); the inv CD111 and inv CD141 promoters of the potato invertase genes (Hedley et al. (2000) J. Exp. Botany 51:817-21); the promoter isolated from Arabidopsis shown to have phloem-specific expression in tobacco by Kertbundit et al. (1991) Proc. Natl. Acad. Sci. USA 88:5212-16); the VAHOX1 promoter region (Tornero et al. (1996) Plant J. 9:639-48); the pea cell wall invertase gene promoter (Zhang et al. (1996) Plant Physiol. 112:1111-17); the promoter of the endogenous cotton protein related to chitinase of US published patent application 20030106097, an acid invertase gene promoter from carrot (Ramloch-Lorenz et al. (1993) The Plant J. 4:545-54); the promoter of the sulfate transporter geneSultr1; 3 (Yoshimoto et al. (2003) Plant Physiol. 131:1511-17); a promoter of a sucrose synthase gene (Nolte and Koch (1993) Plant Physiol. 101:899-905); and the promoter of a tobacco sucrose transporter gene (Kuhn et al. (1997) Science 275-1298-1300).
Possible promoters also include the Black Cherry promoter for Prunasin Hydrolase (PH DL1.4 PRO) (U.S. Pat. No. 6,797,859), Thioredoxin H promoter from cucumber and rice (Fukuda A et al. (2005). Plant Cell Physiol. 46(11):1779-86), Rice (RSs1) (Shi, T. Wang et al. (1994). J. Exp. Bot. 45(274): 623-631) and maize sucrose synthese-1 promoters (Yang., N-S. et al. (1990) PNAS 87:4144-4148), PP2 promoter from pumpkin Guo, H. et al. (2004) Transgenic Research 13:559-566), At SUC2 promoter (Truernit, E. et al. (1995) Planta 196(3):564-70., At SAM-1 (S-adenosylmethionine synthetase) (Mijnsbrugge K V. et al. (1996) Planr. Cell. Physiol. 37(8): 1108-1115), and the Rice tungro bacilliform virus (RTBV) promoter (Bhattacharyya-Pakrasi et al. (1993) Plant J. 4(1):71-79).
The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and yellow florescent protein (PhiYFPTM from Evrogen, see, Bolte et al. (2004) J. Cell Science 117:943-54). For additional selectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference. The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention.
VI. Compositions Comprising Silencing Elements
One or more of the polynucleotides comprising the silencing element can be provided as an external composition such as a spray or powder to the plant, plant part, seed, a pest, or an area of cultivation. In another example, a plant is transformed with a DNA construct or expression cassette for expression of at least one silencing element. In either compositions, the silencing element, when ingested by an insect, can reduce the level of a target pest sequence and thereby control the pest (i.e., any pest from the Lepidoptera order, such as, Spodoptera frugiperda). It is recognized that the composition can comprise a cell (such as plant cell or a bacterial cell), in which a polynucleotide encoding the silencing element is stably incorporated into the genome and operably linked to promoters active in the cell. Compositions comprising a mixture of cells, some cells expressing at least one silencing element are also encompassed. In other embodiments, compositions comprising the silencing elements are not contained in a cell. In such embodiments, the composition can be applied to an area inhabited by a pest. In one embodiment, the composition is applied externally to a plant (i.e., by spraying a field or area of cultivation) to protect the plant from the pest.
In one embodiment, the composition comprising the silencing element that controls a pest from the Lepidoptera order does not comprise a heterologous cationic oligopeptide to facilitate uptake of the RNAi into the insect cells. Accordingly, in such embodiments, insecticidal activity occurs in the compositions of the invention (i.e., the plant, plant part, plant cell, or microbe) in the absence of a cationic oligopeptide that is heterologous to the plant, plant part or microbe. The cationic oligopeptide is target non-specific and interacts non-specifically with RNA via electrostatic interactions and neutralization of charge to penetrate membranes and lacks a specific activity that promotes a specific interaction with a cell membrane.
The composition of the invention can further be formulated as bait. In this embodiment, the compositions comprise a food substance or an attractant which enhances the attractiveness of the composition to the pest.
The composition comprising the silencing element can be formulated in an agriculturally suitable and/or environmentally acceptable carrier. Such carriers can be any material that the animal, plant or environment to be treated can tolerate. Furthermore, the carrier must be such that the composition remains effective at controlling a pest. Examples of such carriers include water, saline, Ringer's solution, dextrose or other sugar solutions, Hank's solution, and other aqueous physiologically balanced salt solutions, phosphate buffer, bicarbonate buffer and Tris buffer. In addition, the composition may include compounds that increase the half-life of a composition.
It is recognized that the polynucleotides comprising sequences encoding the silencing element can be used to transform organisms to provide for host organism production of these components, and subsequent application of the host organism to the environment of the target pest(s). Such host organisms include baculoviruses, bacteria, and the like. In this manner, the combination of polynucleotides encoding the silencing element may be introduced via a suitable vector into a microbial host, and said host applied to the environment, or to plants or animals.
The term “introduced” in the context of inserting a nucleic acid into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be stably incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
Microbial hosts that are known to occupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops of interest may be selected. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild-type microorganisms, provide for stable maintenance and expression of the sequences encoding the silencing element, and desirably, provide for improved protection of the components from environmental degradation and inactivation.
Such microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms such as bacteria, e.g., Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi, particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli and Azotobacter vinlandir, and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces rosues, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.
A number of ways are available for introducing the polynucleotide comprising the silencing element into the microbial host under conditions that allow for stable maintenance and expression of such nucleotide encoding sequences. For example, expression cassettes can be constructed which include the nucleotide constructs of interest operably linked with the transcriptional and translational regulatory signals for expression of the nucleotide constructs, and a nucleotide sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system that is functional in the host, whereby integration or stable maintenance will occur.
Transcriptional and translational regulatory signals include, but are not limited to, promoters, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (2000); Molecular Cloning: A Laboratory Manual (3rd ed.; Cold Spring Harbor Laboratory Press, Plainview, N.Y.); Davis et al. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); and the references cited therein.
Suitable host cells include the prokaryotes and the lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and Gram-positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.
Characteristics of particular interest in selecting a host cell for purposes of the invention include ease of introducing the coding sequence into the host, availability of expression systems, efficiency of expression, stability in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.
Host organisms of particular interest include yeast, such as Rhodotorula spp., Aureobasidium spp., Saccharomyces spp., and Sporobolomyces spp., phylloplane organisms such as Pseudomonas spp., Erwinia spp., and Flavobacterium spp., and other such organisms, including Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, and the like.
The sequences encoding the silencing elements encompassed by the invention can be introduced into microorganisms that multiply on plants (epiphytes) to deliver these components to potential target pests. Epiphytes, for example, can be gram-positive or gram-negative bacteria.
The silencing element can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray in the same manner that Bacillus thuringiensis strains have been used as insecticidal sprays. Any suitable microorganism can be used for this purpose. Pseudomonas has been used to express Bacillus thuringiensis endotoxins as encapsulated proteins and the resulting cells processed and sprayed as an insecticide Gaertner et al. (1993), in Advanced Engineered Pesticides, ed. L. Kim (Marcel Decker, Inc.).
Alternatively, the components of the invention are produced by introducing heterologous genes into a cellular host. Expression of the heterologous sequences results, directly or indirectly, in the intracellular production of the silencing element. These compositions may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. See, for example, EPA 0192319, and the references cited therein.
In the present invention, a transformed microorganism can be formulated with an acceptable carrier into separate or combined compositions that are, for example, a suspension, a solution, an emulsion, a dusting powder, a dispersible granule, a wettable powder, and an emulsifiable concentrate, an aerosol, an impregnated granule, an adjuvant, a coatable paste, and also encapsulations in, for example, polymer substances.
Such compositions disclosed above may be obtained by the addition of a surface-active agent, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protectant, a buffer, a flow agent or fertilizers, micronutrient donors, or other preparations that influence plant growth. One or more agrochemicals including, but not limited to, herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaracides, plant growth regulators, harvest aids, and fertilizers, can be combined with carriers, surfactants or adjuvants customarily employed in the art of formulation or other components to facilitate product handling and application for particular target pests. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g., natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders, or fertilizers. The active ingredients of the present invention (i.e., at least one silencing element) are normally applied in the form of compositions and can be applied to the crop area, plant, or seed to be treated. For example, the compositions may be applied to grain in preparation for or during storage in a grain bin or silo, etc. The compositions may be applied simultaneously or in succession with other compounds. Methods of applying an active ingredient or a composition that contains at least one silencing element include, but are not limited to, foliar application, seed coating, and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest.
Suitable surface-active agents include, but are not limited to, anionic compounds such as a carboxylate of, for example, a metal; carboxylate of a long chain fatty acid; an N-acylsarcosinate; mono- or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate, or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkylphenol sulfates; lignin sulfonates; petroleum sulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates or lower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate; salts of sulfonated naphthalene-formaldehyde condensates; salts of sulfonated phenol-formaldehyde condensates; more complex sulfonates such as the amide sulfonates, e.g., the sulfonated condensation product of oleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g., the sodium sulfonate or dioctyl succinate. Non-ionic agents include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- or alkenyl-substituted phenols with ethylene oxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fatty acid esters, condensation products of such esters with ethylene oxide, e.g., polyoxyethylene sorbitan fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols such as 2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols. Examples of a cationic surface-active agent include, for instance, an aliphatic mono-, di-, or polyamine such as an acetate, naphthenate or oleate; or oxygen-containing amine such as an amine oxide of polyoxyethylene alkylamine; an amide-linked amine prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt.
Examples of inert materials include, but are not limited to, inorganic minerals such as kaolin, phyllosilicates, carbonates, sulfates, phosphates, or botanical materials such as cork, powdered corncobs, peanut hulls, rice hulls, and walnut shells.
The compositions comprising the silencing element can be in a suitable form for direct application or as a concentrate of primary composition that requires dilution with a suitable quantity of water or other dilutant before application.
The compositions (including the transformed microorganisms) can be applied to the environment of an insect pest (such as a pest from the Lepidoptera order) by, for example, spraying, atomizing, dusting, scattering, coating or pouring, introducing into or on the soil, introducing into irrigation water, by seed treatment or general application or dusting at the time when the pest has begun to appear or before the appearance of pests as a protective measure. For example, the composition(s) and/or transformed microorganism(s) may be mixed with grain to protect the grain during storage. It is generally important to obtain good control of pests in the early stages of plant growth, as this is the time when the plant can be most severely damaged. The compositions can conveniently contain another insecticide if this is thought necessary. In an embodiment of the invention, the composition(s) is applied directly to the soil, at a time of planting, in granular form of a composition of a carrier and dead cells of a Bacillus strain or transformed microorganism of the invention. Another embodiment is a granular form of a composition comprising an agrochemical such as, for example, a herbicide, an insecticide, a fertilizer, in an inert carrier, and dead cells of a Bacillus strain or transformed microorganism of the invention.
VII. Plants, Plant Parts, and Methods of Introducing Sequences into Plants
In one embodiment, the methods of the invention involve introducing a polypeptide or polynucleotide into a plant. “Introducing” is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a sequence into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
“Stable transformation” is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.
Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, U.S. Pat. No. 4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. Nos. 5,886,244; and, 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.
In specific embodiments, the silencing element sequences of the invention can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the protein or variants and fragments thereof directly into the plant or the introduction of the transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107:775-784, all of which are herein incorporated by reference. Alternatively, polynucleotides can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, the transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with polyethylimine (PEI; Sigma #P3143).
In other embodiments, the polynucleotide of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the invention within a viral DNA or RNA molecule. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology 5:209-221; herein incorporated by reference.
Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are herein incorporated by reference. Briefly, the polynucleotide of the invention can be contained in transfer cassette flanked by two non-recombinogenic recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its genome a target site which is flanked by two non-recombinogenic recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.
The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as “transgenic seed”) having a polynucleotide of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.
As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides.
The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.
Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.
Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). In specific embodiments, plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments, corn and soybean plants are optimal, and in yet other embodiments corn plants are optimal.
Other plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
VIII. Methods of Use
The methods of the invention comprise methods for controlling a pest (i.e., pest from the Lepidoptera order, such as, Spodoptera frugiperda). The method comprises feeding to a pest a composition comprising a silencing element of the invention, wherein said silencing element, when ingested by a pest (i.e., pests from the Lepidoptera order, such as, Spodoptera frugiperda), reduces the level of a target polynucleotide of the pest and thereby controls the pest. The pest can be fed the silencing element in a variety of ways. For example, in one embodiment, the polynucleotide comprising the silencing element is introduced into a plant. As the Lepidoptera feeds on the plant or part thereof expressing these sequences, the silencing element is delivered to the pest. When the silencing element is delivered to the plant in this manner, it is recognized that the silencing element can be expressed constitutively or alternatively, it may be produced in a stage-specific manner by employing the various inducible or tissue-preferred or developmentally regulated promoters that are discussed elsewhere herein. In specific embodiments, the silencing element expressed in the roots, stalk or stem, leaf including pedicel, xylem and phloem, fruit or reproductive tissue, silk, flowers and all parts therein or any combination thereof.
In another method, a composition comprising at least one silencing element of the invention is applied to a plant. In such embodiments, the silencing element can be formulated in an agronomically suitable and/or environmentally acceptable carrier, which is preferably, suitable for dispersal in fields. In addition, the carrier can also include compounds that increase the half life of the composition. In specific embodiments, the composition comprising the silencing element is formulated in such a manner such that it persists in the environment for a length of time sufficient to allow it to be delivered to a pest. In such embodiments, the composition can be applied to an area inhabited by a pest. In one embodiment, the composition is applied externally to a plant (i.e., by spraying a field) to protect the plant from pests.
In certain embodiments, the constructs of the present invention can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired trait. A trait, as used herein, refers to the phenotype derived from a particular sequence or groups of sequences. For example, the polynucleotides of the present invention may be stacked with any other polynucleotides encoding polypeptides having pesticidal and/or insecticidal activity, such as other Bacillus thuringiensis toxic proteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109), lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825, pentin (described in U.S. Pat. No. 5,981,722), and the like. The combinations generated can also include multiple copies of any one of the polynucleotides of interest. The polynucleotides of the present invention can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including, but not limited to, traits desirable for animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409); barley high lysine (Williamson et al. (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122) and high methionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; and Musumura et al. (1989) Plant Mol. Biol. 12:123)); increased digestibility (e.g., modified storage proteins (U.S. Pat. No. 6,858,778); and thioredoxins (U.S. Pat. No. 7,009,087)); the disclosures of which are herein incorporated by reference.
The polynucleotides of the present invention can also be stacked with traits desirable for disease or herbicide resistance (e.g., fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence and disease resistance genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089); acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations; inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene); and glyphosate resistance (EPSPS gene)); and traits desirable for processing or process products such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)); the disclosures of which are herein incorporated by reference. One could also combine the polynucleotides of the present invention with polynucleotides providing agronomic traits such as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g., WO 99/61619, WO 00/17364, and WO 99/25821); the disclosures of which are herein incorporated by reference.
These stacked combinations can be created by any method including, but not limited to, cross-breeding plants by any conventional or TopCross methodology, or genetic transformation. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are herein incorporated by reference.
Methods and compositions are further provided which allow for an increase in RNAi produced from the silencing element. In such embodiments, the methods and compositions employ a first polynucleotide comprising a silencing element for a target pest sequence operably linked to a promoter active in the plant cell; and, a second polynucleotide comprising a suppressor enhancer element comprising the target pest sequence or an active variant or fragment thereof operably linked to a promoter active in the plant cell. The combined expression of the silencing element with suppressor enhancer element leads to an increased amplification of the inhibitory RNA produced from the silencing element over that achievable with only the expression of the silencing element alone. In addition to the increased amplification of the specific RNAi species itself, the methods and compositions further allow for the production of a diverse population of RNAi species that can enhance the effectiveness of disrupting target gene expression. As such, when the suppressor enhancer element is expressed in a plant cell in combination with the silencing element, the methods and composition can allow for the systemic production of RNAi throughout the plant; the production of greater amounts of RNAi than would be observed with just the silencing element construct alone; and, the improved loading of RNAi into the phloem of the plant, thus providing better control of phloem feeding insects by an RNAi approach. Thus, the various methods and compositions provide improved methods for the delivery of inhibitory RNA to the target organism. See, for example, U.S. Provisional Application No. 61/021,676, entitled “Compositions and Methods for the Suppression of Target Polynucleotides”, filed Jan. 17, 2008 and herein incorporated by reference in its entirety.
As used herein, a “suppressor enhancer element” comprises a polynucleotide comprising the target sequence to be suppressed or an active fragment or variant thereof. It is recognize that the suppressor enhancer element need not be identical to the target sequence, but rather, the suppressor enhancer element can comprise a variant of the target sequence, so long as the suppressor enhancer element has sufficient sequence identity to the target sequence to allow for an increased level of the RNAi produced by the silencing element over that achievable with only the expression of the silencing element. Similarly, the suppressor enhancer element can comprise a fragment of the target sequence, wherein the fragment is of sufficient length to allow for an increased level of the RNAi produced by the silencing element over that achievable with only the expression of the silencing element. Thus, in specific embodiments, the suppressor enhancer element comprises a fragment or a variant of a polynucleotide encoding a juvenile hormone polypeptide, a vacuolar polypeptide, a cadherin polypeptide, a cuticle polypeptide, a translation initiation factor, a SARI polypeptide, an elongation factor, a phosphooligosaccharide, a myosin polypeptide, a potassium channel amino acid transporter, a potassium inwardly rectifier polypeptide, an amino acid transporter, a tubulin polypeptide, a ubiquitin polypeptide, small nuclear ribonucleoprotein, or any other polynucleotide of interest disclosed herein. In still other embodiments, the suppressor enhancer element comprises a polynucleotide set forth in SEQ ID NO: 1-50 or an active variant or fragment thereof.
It is recognized that multiple suppressor enhancer elements from the same target sequence or from different target sequences, or from different regions of the same target sequence can be employed. For example, the suppressor enhancer elements employed can comprise fragments of the target sequence derived from different region of the target sequence (i.e., from the 3′UTR, coding sequence, intron, and/or 5′UTR). Further, the suppressor enhancer element can be contained in an expression cassette, as described elsewhere herein, and in specific embodiments, the suppressor enhancer element is on the same or on a different DNA vector or construct as the silencing element. The suppressor enhancer element can be operably linked to a promoter as disclosed herein. It is recognized that the suppressor enhancer element can be expressed constitutively or alternatively, it may be produced in a stage-specific manner employing the various inducible or tissue-preferred or developmentally regulated promoters that are discussed elsewhere herein.
In specific embodiments, employing both a silencing element and the suppressor enhancer element the systemic production of RNAi occurs throughout the entire plant. In further embodiments, the plant or plant parts of the invention have an improved loading of RNAi into the phloem of the plant than would be observed with the expression of the silencing element construct alone and, thus provide better control of phloem feeding insects by an RNAi approach.
In specific embodiments, the plants, plant parts, and plant cells of the invention can further be characterized as allowing for the production of a diversity of RNAi species that can enhance the effectiveness of disrupting target gene expression.
In specific embodiments, the combined expression of the silencing element and the suppressor enhancer element increases the concentration of the inhibitory RNA in the plant cell, plant, plant part, plant tissue or phloem over the level that is achieved when the silencing element is expressed alone.
As used herein, an “increased level of inhibitory RNA” comprises any statistically significant increase in the level of RNAi produced in a plant having the combined expression when compared to an appropriate control plant. For example, an increase in the level of RNAi in the plant, plant part or the plant cell can comprise at least about a 1%, about a 1%-5%, about a 5%-10%, about a 10%-20%, about a 20%-30%, about a 30%-40%, about a 40%-50%, about a 50%-60%, about 60-70%, about 70%-80%, about a 80%-90%, about a 90%-100% or greater increase in the level of RNAi in the plant, plant part, plant cell, or phloem when compared to an appropriate control. In other embodiments, the increase in the level of RNAi in the plant, plant part, plant cell, or phloem can comprise at least about a 1 fold, about a 1 fold-5 fold, about a 5 fold-10 fold, about a 10 fold-20 fold, about a 20 fold-30 fold, about a 30 fold-40 fold, about a 40 fold-50 fold, about a 50 fold-60 fold, about 60 fold-70 fold, about 70 fold-80 fold, about a 80 fold-90 fold, about a 90 fold-100 fold or greater increase in the level of RNAi in the plant, plant part, plant cell or phloem when compared to an appropriate control. Methods to assay for an increase in the level of RNAi are discussed elsewhere herein.
The following examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL
Example 1
Specific Target Genes and Silencing Elements that Cause Insecticidal Activity Against Spodoptera Frugiperda 
Disruption of insect gene function via RNAi can produce specific activity against target insects. This specificity is enhanced by delivery of the dsRNAs via transgenic plants. Identification of gene function in insects via RNAi has been largely limited to injection of dsRNAs. In fact, past experiments have indicated that insects are not capable of systemic RNAi response based on exposure to dsRNAs.
As described below, we have demonstrated acute activity of numerous dsRNA pairs through injection experiments and additionally have demonstrated insect antagonism through ingestion of dsRNAs. This evidence identifies several gene/primer pair combinations with clear insecticidal properties. The use of dsRNAs in transgenic plants also addresses the potential complication of heterologous protein expression and the possible risks of allergic reaction, non-target activity, and environmental- or bioaccumulation. The data presented below represents the first test of disruption of these particular genes resulting in insecticidal activity in whole organisms and the first report of insecticidal activity of dsRNAs against Spodoptera frugiperda. 
The invention describes specific target genes and the dsRNA sequences causing insecticidal activity against the Lepidopteran Spodoptera frugiperda through RNA interference of the target gene's expression. Disruption of the genes targeted by the dsRNA sequences may be broadly insecticidal in numerous species. The specific dsRNA sequences display insecticidal activity upon ingestion and can be utilized with a transgenic plant mode of delivery. Table 1 provides the polynucleotide of non-limiting examples of target sequence from Spodoptera frugiperda, a brief description of the function of the protein encoded by the target sequence, and a SEQ ID NO. Table 2 provides a summary of primers used to suppress the target polynucleotides.







TABLE 1





Target Polynucleotides from Spodoptera frugiperda.















>ise1c.pk002.m13 Juvenile hormone query


SEQ ID NO: 1


CAAGCATCCAACATGGTATCCGACTTCAGGAAGAAGAAGCTCCTCCACGTGTTCAAGTCCTTCTTCGACACGGAC


GGCAGCGGCAACATCGAGAAGGATGACTTCCTGATGGCCATCGAAAGGATAACCAAGACCAGAGGCTGGAAAGCT


GGAGACGACAAATACAAATTTGTCGAGGAGACCCTATTGAAGATCTGGGACGGCATCCAGAAGGTCGCTGACGAG


AACAAGGACGGACAGGTCAGCCAGGACGAGTGGATCGCTATGTGGGACAAGTACTCCAAGAACCCGTCCGAGGCG


TTCGAGTGGCAGACCCTGTACTGCAAGTTCGCGTTCACTCTTGAAGACGCCAGCGACGATGGATCCATCGACAGC


GAGGAGTTCTCCTCTGTGTACGCCTCCTTCGGCCTGGACAANGACGANGCTGTGGCTGCCTTCAAGAAAGATGGC


TAACGGTAAGTCCGAAGTGTCCTGGGCTTGAGTTCCACGACCTGTGGAANGAGTACTTCTCATCCGGAAGACTNG


AACGCTGCCGGCAAN


 


>ise1c.pk003.f7 Juvenile hormone query


SEQ ID NO: 2


CCAACATGGTATCCGACTTCAGGAAGAAGAAGCTCCTCCACGTGTTCAAGTCCTTCTTCGACACGGACGGCAGCG


GCAACATCGAGAAGGATGATTTCCTGATGGCCATCGAAAGGATAACCAAGACCAGAGGCTGGAAAGCTGGAGACA


ACAAATACAAATTTGTCGAGGAAACCCTATTGAAGATCTGGGACGGCATCCAGAAGGTCGCTGACGAGAACAAGG


ACGGACAGGTCAGCCAGGACGAGTGGATCGCTATGTGGGACAAGTACTCCAAGAACCCATCCGAGGCGTTCGAGT


GGCAGACCCTGTACTGCAAGTTCGCGTTCACTCTTGAAGACGCCAGCGACGACGGATNCATCGACAGCGAAGAGT


TCTCCTCTGTGTACGCCTCCTTCGGGCTGGACAANGGACGAGGCGGTGGCTGCCTTCAAGAAGATGNTAACGGTA


AGTCCGAATGTCCTGGGGCTGAGTTTCAAGANCTGTTGGAAGGATACTTCTCAAC


 


>ise1c.pk005.a15 Juvenile hormone query


SEQ ID NO: 3


CAACATGGTATCCGACTTCAGGAAGAATAAGCTCCTCCACGTGTTCAAGTCCTTCTTCGACACGGACGGCAGCGG


CAACATCGAGAAGGATGACTTCCTGATGGCCATCGAAAGGATAACCAAGACCAGAGGCTGGAAAGCTGGAGACGA


CAAATACAAATTTGTCGAGGAGACCCTATTGAAGATCTGGGACGGCATCCAGAAGGTCGCTGACGAGAACAAGGA


CGGACAGGTCAGCCAGGACGAGTGGATCGCTATGTGGGACAAGTANTCCAAGAACCCGTCCGAGGCGTTCGAGTG


GCAGACCCTGTACTGCAAGTTCGCGTTCACTCTTGAAGACGCCAGNGACGATGGATCCATCGACAGCGAGGAGTT


CTCCTCTGTGTACGCCTCCTTCGGCCTGGACAAGG


 


>ise1c.pk006.d24 Juvenile hormone query


SEQ ID NO: 4


GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTNGGAAANTAC


TATTTTATTGTACCAACTGCCCCTTAACCTCATCTATGAGTCACCCATAAATGTTATTTTGGTAAAATGTTTGAC


ACACTTCACACTAATATTTATAAATGTGAAAGTTTGTTTGTTTGAATGTTTGTATATTTGTCTGTCAATCACGCT


GAAACCACTGTATAGAATTTGACCTAATTTGGTATACANACAGGGTATGAGCTGACTTGGGTGATAGGATACTTT


TTATCCCACAGGAACGCGGGTAAAGTCCNTGGGCAGAAGCTAGTATGTAATAATTATNTCCCTCTACCTACCCTA


TATGGGGGTGGACCGTCATGTTCTTTACNCNACAACCNGTTTGTCCACCTCNCCTTTAAAGTTTTGTNAG


 


>ise2c.pk009.i4 Juvenile hormone query


SEQ ID NO: 5


GCACGAGGGCCGTGTCGACTTCGCACCAGTCCCCTATTTATTTACCTTGACAAAAATATGGCGCGCCTATTGTTT


ATTGCGCCTATCCTGGCGTTGGCTATAATGCCAGTATACTTCTTATTCCTAAAGGGACCACCCCCACTACCCGAA


CTAGATATGAACGAGTGGTGGGGCCCAGAGAAGCTAAAAGCAAAACCTGACACTAGTATAAAACCCTTTAAAATT


GCTTTTGGAGACACTGTTGTAAAAGACTTAAAAGACCGTCTCAAACGTTCTCGGTCTTTCACTGCTCCGCTGGAG


GGTGTGGCATTCCAGTACGGCTTCAACACTGCTCAGCTGGATGGTTGGCTGAAGTACTGGGCTAATGAGTATAAG


TTCAAGGAGAGAGAGACCTTCCTCAACCAGTACCCTCAGTACAAAACCAATATCCAGGGTCTTGACATCCACTTC


ATCAGGGTTACACCGAAGGTACCGGCAGGAGTGGAGGTGGTACCCATGCTACTCCTCCACGGCTGGCCAGGCTCT


GTCAGGGAGTTCTACGAGGCTATTCCTCTCATCACAGCAGTCAGCAAGGACCGTGACTTCGCTGTGGAAGTCATC


GTTCCAAGTCTACCTGGCTATGGATTCTCTGATGCCGCAGTTCGTCCCGGCnnnnnnnCCCCACAAATGnnn


 


>ise2c.pk001.d19 vacuolar query


SEQ ID NO: 6


GCACGAGGCTTGGACGTGATGTTACCTGGGAATTCAACCCCTTGAATGTTAAGGTCGGCTCCCACATCACCGGAG


GAGACTTGTACGGTATCGTACACGAGAACACATTGGTTAAGCACAAGATGTTGATCCCACCCAAGGCCAAGGGTA


CCGTCACCTACGTCGCGCCCTCCGGCAACTACAAAGTCACTGACGTAGTGTTGGAGACGGAGTTCGACGGCGAGA


AGGAGAAGTACACCATGTTGCAAGTATGGCCGGTGCGCCAGCCGCGCCCCGTCACTGAGAAGCTGTCCGCCAACC


ACCCCCTGCTCACCGGACAGAGAGTGCTCGACTCTCTCTTCCCTTGTGTCCAGGGTGGTACCACGGCCATCCCCG


GCGCCTTCGGTTGTGGCAAGACTGTCGTCTCACAGGCTCTGTCCAAGTACTCCAACTCTGACGTCATCATCTACG


TCGGATGCGGTGAACGTGGTAACGAGATGTCTGAGGTACTGCGTGACTTCCCCGAGCTGACGGTGGAGATCGAGG


GCATGACCGAGTCCATCATGAAGCGTACCGCGCTCGTCGCCAACACCTCCAACATGCCTGTAGCCGCCCGAGAGG


CTTCCATCTACACCGGTATCACCCTCTCCGAGTACTTCCGTGACATGGGTTACAACGTGTCCATGATGGCTGACT


CCACCTCTCGTTGGGCCG


 


>ise2c.pk001.e14 vacuolar query


SEQ ID NO: 7


GCACGAGGCAGATAGTCATCACTGTTTTTGGGACCTGTnnnTACTCCCTCAATAAACCTACAAAATGGCCGAAAA


CCCAATCTACGGACCCTTCTTTGGAGTTATGGGGGCGGCGTCTGCTATCATCTTTAGCGCGCTGGGAGCTGCCTA


TGGAACTGCTAnGnCnnnnACCGGTATCGCCGCCATGTCGGTGATGCGGCCCGAGCTCATCATGAAGTCCAACAA


CTACACCCTTTACAAGnGGTTCATCCACCTTGGCGCTGGTCTnnnCGTAAGTTTCTCCGGTCTAGCGnnnGGCnn


 


>ise2c.pk001.f20 vacuolar query


SEQ ID NO: 8


GCACGAGGCTCACAGGCTCTGTCCAAGTACTCCAACTCTGACGTCATCATCTACGTCGGATGCGGTGAACGTGGT


AACGAGATGTCTGAGGTACTGCGTGACTTCCCCGAGCTGACGGTGGAGATCGAGGGCATGACCGAGTCCATCATG


AAGCGTACCGCGCTCGTCGCCAACACCTCCAACATGCCTGTAGCCGCCCGAGAGGCTTCCATCTACACCGGTATC


ACCCTCTCCGAGTACTTCCGTGACATGGGTTACAACGTGTCCATGATGGCTGACTCCACCTCTCGTTGGGCCGAG


GCTCTTCGTGAGATCTCnnnnCGTCTGGCTGAGATGCCTGCCGACTCGGGTTACCCCGCCTACCTGGGAGCCCGT


CTGGCCTCGTTCTACGAGCGTGCCGGACGTGTGAAGTGCTTGGGTAACCCCGACAGGGAGGGCTCCGTGTCCATC


GTGGGCGCCGTGTCGCCGCCCGGAGGTGACTTCTCCGACCCCGTGACGGCCGCCACGCTGGGTATCGTGCAGGTG


TTCTGGGGGTTGGACAAGAAGCTCGCGCAGCGCAAGCACTTCCCCGCCATCAACTGGCTCATCTCCTACAGCAAG


TACATGCGAGCGCTGGACGACTTCTATGAGAAGAACTACCCCGAGTTCGTGCCCCTCnnnnnCAAGGGTCAAGGA


GATCCTGCAGnnn


 


>ise2c.pk010.h3 cadherin query


SEQ ID NO: 9


GCACGAGGTATCTAAAACAGTGCGTCGTAATATATTCAAGATGTCTCGTCTTAGGTTTTGTTTTTTATTAGCAGT


ACTATGCAGTTGTTTGCAGAATGGTTACGGTTTTACAACAGAAAAGCCAGTTACCCAGCATGTAGATCCTAAACC


AGAAGTTCCTGAAACGTTGCCTGAAACAACACGAGTGCCTGCGCCGAGCTCGTCGACGGCAGCGCCGACCACACC


AGCTCCGACACCGGCACCAACGCCAGCACCCACACCAGCTCCTACACCAGCTCCTACTCCAGCTCCTACCCCTGC


GCCTACTCCTGCGCCTACTCCTGCGCCTACTCCTGCGCCTACCCCCGCACCTACACCAGCGCCCACTCCTGCTCC


CACCCCAGCTCCCCTCCCCGCCCCCGACCAAGGCACATGGTCCTTCACTGATGAAAAGGCCAATCAGACATGCAT


TGTGGCCCAATTCGCAGCCCAACTGAATGTCACATACACCAAGTTAGTGGAGAATGCAACGTCTCTATCGTACGT


GAGGCTCAACGTGCCCGCGAACGCGTCGGTCCTCAACGGCAGCTGTTCGGACCCCGACCAATGGATCCAGATCAC


CTGGAAGACCAACGACGACAGCGAGACGAACAACACCATGACCCTCGTGTACAACAAGAATGCCACCACCAAGnn


CTACGGCCTG


 


>ise2c.pk011.a10 cuticle protein


SEQ ID NO: 10


GCACGAGGGCGGTTTGAAGTGATCTAGTTCGTCAGAAAAAACACAGACCACGTTCACAATGAAATCGATGGTGGT


GTTATTCGCTGTGTGCGCCGTGGCGTGCGGCTCCCTGGTGCCGCTGGCGCAGCCTCCTCATCACCCCGCCGTCGT


GCTGGACCCGCACGGCCGCCCGCTCGACACCGCCGAGGTGATCAACGCCCGCGCCCTCCACCTGCAGGCTAAGGC


CCTGGATGGACACTACGCTCCCCTCGCGCACGCTGCCGTCGTGCCTGTTGCCCACTCCGTGGTAGCCGCCCCCGC


TGTGGTCGCCGCTCCCGCCGCCGTGTCCCACCAGTCCCGTGTGGATGTGCGCACCAGCCCCGCCATCGTGAGCCA


CGCCGTCGCTGCTCCCGTAGTAGCCCACGGTGTCTACTCCGCTCCCCTGCTGGCCCACTCCGCTCTCGGCTACGC


CGGTCACGGACACTACCTGAAGAAGCGCTCCCTGGGACACCTCGCCTACGCCGCTCCCGTCGTCGCCCACGTAGC


TCCCTCCGCGGTGTCGCACCAGTCCCGCGTGGnCnTCGTCTCCAGCCnnnCTGTCGTGTCTCAnnnnnTnnnTnC


CGTnnTGTCCCn


 


>ise2c.pk011.h12 cuticle protein


SEQ ID NO: 11


GCACGAGGGGACGTTGAACGAAAGAAAATGCTACGCGTTACGATTTTAGCCGCAGTGGTGGTGTTCGCCTCAGGC


GCGCCCCAGAACAACTTCATCTTCAAGAATGACATCACTCCTGAGGAAGCCCAGCAGTACCTCAAACAACTGCCG


TTCACCTCACCCCAGCTCTCTGGACGCACCGCTGTACTGCCTCTGGTTCGCTACGACGACCCCAGGTTTCGTTCA


GCTGAAGCTGGCCCAACCCTTGGACACTACTGGAAGAATGGACAGGAGATCCAGAACACAGAGGACTACTTAGAA


GAGGTCTACAACGCGGCTCAATACCACGGCCAGGACGGTCTTGGCAACTACGCCTACGGTTATGAGACCCCTGAA


TCTTCCAAGGTTGAGAACCGTGAAGGTTCCGGAGTCGTCCAAGGATCCTATGTGTACCAGGTTCCCGGAATGAAG


GATCTCGTCnnGGTCCGTTACTGGGCTGACAGCCnnnnnTTCCACCAGnAnGACAATCTTCCCAAGGTTGAACTG


AnnnCCGCTnnnnnnnnCCCGCTCT


 


>ise2c.pk001.d22 translation initiation factor


SEQ ID NO: 12


GCACGAGGTATCACTCCTGACCGTATCTAAAACTCGGCACACAACACAATGGCTGACATCGAAGATACACATTTC


GAGACCGGGGACTCCGGTGCCTCCGCCACCTTCCCTATGCAATGCTCGGCCCTGCGCAAGAACGGTTTCGTCATG


CTTAAGGGTCGCCCCTGCAAAATCGTCGAGATGTCCACTTCCAAAACCGGAAAGCACGGCCACGCTAAAGTTCAC


TTGGTTGGAATCGATATTTTTAACGGCAAGAAATACGAAGATATCTGCCCTTCCACCCACnnnCATGGACGTGCC


CCACGTGAAGCGTGAGGACTACCAGCTCACCGATATCTCTGACGACGGCTACCTTACCCTCATGGCTGACAACGG


CGATCTCCGCGAGGACCTCAAGATCCCAGACGGTGACCTCGGCACCCAGTTGCGTTCTGACTTCGATAGCGGCAA


AGAGCTGTTGTGCACTGTGCTGAAGTCTTGCGGTGAGGAGTGTGTAATCGCAGTCAAGGCAAACACAGCTCTCGA


CAAATAAACCAACTCAGCATTTATAGGGATATACATACATATAATTTTTTTACAATCAACAGCTCTTACATAAAT


GTAAAACATAATACTATGTATAATTTAACATnnnnnATTATGGTGTGACGCGGTGCTGGCTTGTCGCCGTCCACT


CCACCCCCGAAG


 


>ise2c.pk001.d9 translation initiation factor


SEQ ID NO: 13


GCACGAGGCGCGATTGTAACATGTCGTATTCACCAGAAAGAAGATCAGAAGATTGGCCGGAAGATTCCAAAAATG


GCCCGTCTAAGGATCAAGGCAACTATGATGGGCCTCCAGGAATGGAACCCCAAGGGGCACTTGATACAAACTGGC


ACCAGGTCGTGGAAAGCTTTGACGACATGAATCTGAAGGAAGAATTGTTGAGAGGAATTTATGCTTACGGTTTTG


AAAAGCCGTCTGCTATCCAACAACGCGCTATTATGCCTTGCATTCAAGGCCGTGATGTCATAGCTCAAGCCCAGT


CTGGTACTGGGAAGACTGCTACCTTCTCTATTTCAATTCTTCAGCAAATCGATACCAGTATTCGTGAATGCCAAG


CACTGATTTTGGCCCCTACTAGAGAGCTGGCTCAGCAGATCCAAAAGGTGGTGATTGCTCTTGGGGATCACTTGA


ATGCTAAATGCCATGCTTGCATCGGCGGCACTAATnnnGCGCGAAGATGTTCGTCAGCTnnnnn


 


>ise2c.pk001.i23 translation initiation factor


SEQ ID NO: 14


GCACGAGGGTCGTATTCACCAGAAAGAAGATCAGAAGATTGGCCGGAAGATTCCAAAAATGGCCCGTCTAAGGAT


CAAGGCAACTATGATGGGCCTCCAGGAATGGAACCCCAAGGGGCACTTGATACAAACTGGCACCAGGTCGTGGAA


AGCTTCGACGACATGAATCTGAAGGAAGAATTGTTGAGAGGAATTTATGCTTACGGTTTTGAAAAGCCGTCTGCT


ATCCAACAACGCGCTATTATGCCTTGCATTCAAGGCCGTGATGTCATAGCTCAAGCCCAGTCTGGTACTGGGAAG


ACTGCTACCTTCTCTATTTCAATTCTTCAGCAAATCGATACCAGTATTCGTGAATGCCAAGCACTGATTTTGGCC


CCTACTAGAGAGCTGGCTCAGCAGATCCAAAAGGTGGTGATTGCTCTTGGGGATCACTTGAATGCTAAATGCCAT


GCTTGCATCGGCGGCACTAATGTGCGCGAAGATGTTCGTCAGCTGGAGAGTGGTGTGCATGTGGTGGTGGGTACA


CCTGGTCGCGTGTACGACATGATAACTCGTCGTGCTCTCCGTGCTAACACTATCAAGCTGTTTGTACTTGATGAA


GCTGATGAAATGCTGTCAAGAGGATTTAAAGATCnn


 


>ise2c.pk001.l24 translation initiation factor


SEQ ID NO: 15


GCACGAGGGCCATCCTGTCACACATCTACCACCACGCCCTGCACGATAACTGGTTCCAAGCTCGAGACTTGCTCT


TGATGTCACACTTGCAAGAGACTGTTCAACATTCAGACCCGAGCACTCAGATTTTGTACAATCGTACTATGGCCA


ATCTAGGTTTGTGCGCTTTTCGAAGGGGCAATGTTAAAGAAGCCCATGGCTGCCTAGCTGAACTGATGATGACTG


GCAAACCCAAGGAACTGTTAGCTCAAGGTCTGCTACCTCAGCGTCAACACGAGCGTTCAAAGGAACAGGAAAAGA


TAGAGAAGCAACGCCAAATGCCGTTCCACATGCACATCAACTTGGAACTGCTTGAATGTGTGTATTTAGTGTCTG


CCATGCTGATTGAAATTCCATACATGGCCGCCCACGAATTCGATGCTCGCCGGCGCATGATTAGTAAGACTTTCT


ATCAGAATTTGCGCGCAAGTGAGCGTCAGGCTTTGGTAGGCCCGCCCGAATCCATGCGTGAGCATGCTGTGGCTG


CCGCCAGGGCGATGCGCCGCGGAGACTGGCGTGCTTGCCTCAATTTTATTGTnnnTGnnnAATGAAT


 


>ise2c.pk005.b9 translation initiation factor


SEQ ID NO: 16


GCACGAGGCTGATAGCCACCTGCCAAATTATCTTGAAATATAACCATTCACTAAAATATTTAACGTAATTTAGTG


GTTAATTCTAAACTTAATCATGGACGACGACATGGTATTTGATCCATCTTTAAAGAAAAAGAAGAAGAAGAAGAC


CGGTTTCGACTTAGATGCCGCTCTCGCAGGCGAACAAGGTGAGAGCACGAGCGTGGAGGCGCCCGCTGGGTCGGG


TGACGTCGACTTGCCTGAGGATGATAACCTCGATTTGGATAATTTTGGAAAGAAAAAGAAGAAGAAGAAGAAGGG


AGTCTTCAACATGGAAGAACTTGAAAGTACGTTACCGGAAACACCTCCGGCCGAAGAGCCGGAACAGCAGGAGGA


CGAAGTTATTGACGATTTAGATCTAGATATTGACTTCTCTAAAACGAAAAAGAAGAAGAAGAAGAAAAACATnnn


AnGAGCTCGTCCTTGAAGATGACACCAAGGGAGAAGATCAAGAGAATGTCGAGGATGTTAGTGGTGATTTATGGA


GCGGCACAGACCGTGACTACACGTACGACGAGCTACTAGAGCGAGTGTTCGACATCATGCGAGAAAAGAnnnnnA


GCATGGTTT


 


>ise2c.pk002.m10 SAR1


SEQ ID NO: 17


GCACGAGGCAGATTCATATTTCCATCGCTTATTCGTTGCTGAGAAAAATCGTCGGTTTTAGCGACGTAACATATT


GCTAATAAGTGTGAAATATTGTGATAAACTTCCTTTTAGCATTAGTTAATCTAGTTCAATTTTAAATAATTCAAA


ATGTTTATCTTGGATTGGTTCACTGGTGTTCTCGGATTCCTTGGTCTGTGGAAGAAATCAGGCAAGCTACTGTTC


CTGGGACTGGACAATGCTGGCAAGACCACACTCCTGCACATGCTGAAGGATGACAGATTGGCGCAGCATGTACCC


ACATTGCATCCCACGTCGGAGGAACTGTCAATAGGCAGTATGCGTTTCACGACGTTCGACTTGGGCGGGCATCAG


CAGGCGCGGCGCGTGTGGCGCGACTACTTCCCGGCGGTGGACGCCATCGTGTTCCTGGTGGACGCGTGCGACCGC


CCGCGCCTGCCCGAGTCCAAGGCCGAGCTGGACTCGCTGCTCACTGACGAGACGCTCAGCnnACTGCCCCGTGCT


CATCCTCGGCAACAAGATCGACAAGCCCGGCGCAGCTAGTGAGGACGAGCTCCGTCAGTTCTTCAACCTGTACCA


ACAGACCACTGGAnAnGnCAAAGTATCnAGnTCAnnnnT


 


>ise2c.pk001.c14 Elongation factor


SEQ ID NO: 18


GCACGAGGGTCTATCTCGGATATTACACGTGGATTGTAATCCGTGACTAACCAAAAATGGGCAAGGAAAAGnnnC


ACATTAACATTGTCGTCATTGGACACGTCGACTCCGGCAAGTCCACCACCACCGGTCACTTGATCTACAAATGCG


GTGGTATCGACAAACGTACCATCGAGAAGTTCGAGAAGGnnnCCCAGGAAATGGGGTAAGGGTTCCTTCAAATAC


GCCTGGGTATTGGACAAACTGAAGGCTGAGCGTGAACGTGGTATCACCATCGATATTGCTCTGTGGAAGTTCGAA


ACCGCTAAATACTATGTCACCATCATTGACGCTCCCGGACACAGAGATTTCATCAAGAACATGATCACTGGAACT


TCCCAGGCTGATTGCGCCGTACTCATTGTCGCCGCTGGTACCGGTGAGTTCGAGGCTGGTATCTCGAAGAACGGA


CAGACCCGTGAGCACGCTCTGCTCGCTTTCACACTCGGTGTCAAGCAGCTGATTGTGGGCGTCAACAAAATGGAC


TCCACTGAGCCCCCATACAGCGAATCCCGTTTCGAGGAAATCnnnnnnn


 


>ise2c.pk001.d16 Elongation factor


SEQ ID NO: 19


GCACGAGGCGGATATTACACGTGGATTGTAATCCGTGACTAACCAAAAATGGGCAAGGAAAAGnTTCACATTAAC


ATTGTCGTCATTGGACACGTCGACTCCGGCAAGTCCACCACCACCGGTCACTTGATCTACAAATGCGGTGGTATC


GAnnnACGTACCATnnnnn


 


>ise2c.pk001.j9 myosin


SEQ ID NO: 20


GCACGAGGCTCTAGTCCCGTCACCGTCGCCAGTAGGGGGCGCCACAAGAACAGAAAGAGAATTATTTCAAACTCC


AATTATAACCTACTAGATAACTCCAAAAGTTCTGTCAGTTCTAACTTTAATTTAACGGGGACGTCAGAGTTTATG


GATAGGACCGATAAGATAATATCGGACGCGACTGAGCTACAAGCAATGCAGAACTTTATCATGGAGAAGATTTAC


GAAATGGAACCTAATGAGAAGAAGAAGCAATCTGAGGTCGACAGGGTATTCAAACACGCATTATTAGAATTCAAA


GACAATTTAGTAGCGACGTACAGCATAGTGGAGACGCGGGGCTCTGCGCTGAAGTACAAGGATCTGATCGGCAAC


TTCCTGCACGTCATGGAGACGGTTTGTGCCAGGGAGGGGTCCACGCTCTCCATCACCATGGGGGTCAACGCCTTT


AGGGGTTTCATGGACGAGTTTATGAGCCAACATGACACTGATAAAGCTAGGACGnnnnGnnnAAGGATAAAAAGA


nnnnnnTGGACGATCCAATACAATACAAAGGCCATACGTTCATACTGTCCATGATCAACATACCAAnnnnAGTGT


GAGATCTGCAAGACTTTCTTCATGTGGCCCATAGAGCGGTCACTCATATGCCAGACGTGTAAACTTGCCTCGCAT


AAnnnTnnnACACTA


 


>ise2c.pk001.b14 potassium channel amino acid transporter


SEQ ID NO: 21


TGACTCCACAGTGGGACAAACTCATAGAGCTTGATGTGTGGTACGCTGCTGTGACCCAAGTGTTCTTCTCTCTGT


CTGTGTGCACCGGTGCCATCATTATGTTCTCGTCCTACAATGGATTCAGACAAAATGTTTACAGAGACGCGATGA


TTGTCACTACTTTGGACACCTTCACCAGTTTGTTATCCGGTTTCACGATCTTCGGTATCCTGGGTAACTTGGCGT


ACGAGTTGGACAAAGATGTGGATGACGTCACTGGTTCTGCAGGAACTGGACTTGCCTTCATTTCATACCCTGACG


CGATCTCCAAAACTTTCCAGCCACAGTTGTTCGCAGTGCTGTTCTTCTTGATGATGACGGTACTAGGTATCGGAT


CAGCAGTTGCTTTACTTTCCACCATCAACACCGTGATGATGGACGCGTTCCCTCGCATCAAGACCATCTACATGT


CCGCCTTCTGCTGCACTATTGGATTTGCCATCGGTCTCATTTACGTCACACCTGGTGGCCAATATATTCTCGAGC


TGGTGGATTACTTCGGTGGAACCTTCCTGATTCTCTTCTGTGCTATCGCTGAAATTATTGGTGTATTCTGGATTT


ACGGCTTGGAGnnnTATGCCTGGATATTGAGTACATGTTGGGAGTTAAACTTCTTCTACTGGnnnTnnTGTTGGG


GCGTTATTATGCCTGCCATGATGATnACCGnnnnnn


 


>ise2c.pk003.f2 potassium inwardly rectifier . . .


SEQ ID NO: 22


GCACGAGGGTAAACAGATTTTAACACTACATTAATTTGTTCTAGAGTTAAATGTATTAATTCCGACTTAAAAACA


GTGCTTGTGATAAGTGAACACAAATTATTGAGCAATGACTGACTTTATAAAACAATATTTCAAGGAACAATATGA


AATAAATGAAAAAATGCTTTCGAAAATTGACGCGGATCTGCGAACCTGCGGAGCACACTTAGTAGCAGTGAAGTT


AATGGTGACTGCCCTCGAGTTGAAAATGACTTCGATGAAGACAATGTATCAGGATCTAATGGAACTCAGAGAAAT


AATCGTTCTTTTAAATCCACACTTGAAGAAACCGAGATAATAATACAATACAGTAAGGTTAACGAATACTATCTT


TTAATTTCCTTAAATTATGTTCATAAAAATGATTAAGTTGTTTAGCTGAACACAGTGGTGTACTGACAGGATAGG


TTTCATTAAACTTTGCATAATCGATCAGAAAACCGTGCTTTTCTTTTTTGTACTCGACCATTTCAATAAAGCGAT


GACCCCATAGGATTnnnnTGGGTGGTGTAGCTCGACTTCGCTTGGACAGGCTGACCAGTTGATTCTATAGTGCCT


TCAAACACTACGAnnATTTCCATAT


 


>ise2c.pk005.l20 amino acid transporter


SEQ ID NO: 23


GCACGAGGATTTTCTTAAAACGGTACTGCAGCAAAAAGACGGCATTGAAGGTGGACTCGGTCTGCCTATCTGGTA


CCTGGTGGTTTGTCTGTTCGGGTCATGGTTTATCATCTTCGTGATTGTGTCCCGAGGTGTAAAGAGTTCCGGTAA


AGCTGCATACTTCTTGGCTCTCTTCCCCTACGTTGTGATGCTCATTTTGCTTATAACGACCTCTATTCTGCCCGG


AGCCGGCACCGGCATTCTTTTCTTCCTGACTCCACAGTGGGACAAACTCATAGAGCTTGATGTGTGGTACGCTGC


CGTGACCCAAGTGTTCTTCTCTCTGTCTGTGTGCACCGGTGCCATCATTATGTTCTCGTCCTACAATGGATTCAG


ACAAAATGTTTACAGAGACGCGATGATTGTCACTACTTTGGACACCTTCACCAGTTTGTTATCCGGTTTCACGAT


CTTnnnTATCCTnnnTAACTTG


 


>ise2c.pk001.d1 tubulin


SEQ ID NO: 24


GCACGAGGCCGGTCTTCAGGGCTTCCTTATCTTCCACTCCTTCGGTGGAGGTACTGGATCTGGTTTCACTTCCCT


CCTGATGGAGCGACTCTCCGTGGACTACGGCAAGAAGTCCAAGCTGGAGTTCGCCATCTACCCGGCGCCTCAGGT


GTCCACCGCTGTCGTGGAGCCCTACAACTCCATCCTCACCACCCACACCACCCTTGAGCACTCCGACTGCGCCTT


CATGGTCGACAACGAGGCCATCTACGACATCTGCCGCCGCAACCTCGACATCGAGCGCCCCACGTACACCAACCT


GAACCGTCTCATCGGGCAGATCGTGTCCTCCATCACGGCCTCCCTGCGCTTCGACGGCGCCCTCAACGTCGATCT


TACCGAGTTCCAGACCAACTTGGTGCCCTACCCCCGTATCCACTTCCCTCTGGTCACATACGCCCCGGTCATCTC


TGCCGAGAAGGCGTACCACGAGCAGCTGTCGGTGGCTGAAATCACCAACGCATGCTTCGAGCCCGCCAACCAGAT


GGTCAAGTGCGACCCTCGTCACGGCAAGTACATGGCTnnnnTGCATGTTGTACCGTGGTGACGTCGTCCCCAAGG


ACGTGAACGCCGCCATCGCCACCATCAAGACCAAGCGTACCATCCAGnnnCGTCnnTTGGTGTCCnnCnnnGTnn


n


 


>ise2c.pk001.k6 tubulin


SEQ ID NO: 25


GCACGAGGATTCGTTTGGCAAGCCTCTTAACCGGTCGCGCTGAACGACGACTGATATTTAATTAATTTATATTCT


ACGTTAAGTTCAACAAAACTCAATTCAAAATGCGTGAGTGCATCTCAGTACACGTTGGACAAGCCGGAGTCCAGA


TCGGTAATGCCTGCTGGGAATTATATTGCCTTGAGCATGGAATCCAGCCTGACGGCCAGATGCCCACAGACAAGA


CCGTGGGCGGTGGTGATGACTCCTTCAACACCTTCTTCAGCGAGACCGGTGCCGGCAAGCACGTCCCCAGGGCTG


TGTTTGTTGACTTGGAACCCACAGTAGTTGATGAGGTCCGCACTGGCACATACAGACAGTTGTTTCATCCAGAAC


AACTTATCACTGGTAAGGAAGATGCGGCCAACAACTACGCCCGTGGTCACTACACCATCGGCAAGGAAATCGTAG


ACCTAGTCCTCGACCGCATCCGTAAGCTCGCCGACCAGTGCACCGGTCTCCAGGGCTTCCTTATCTTCCACnnnn


nTCGGTGnnnnnACTGGGATCTGGTTTCACTTCCCTCCTGATGGAGCGACTCTCCGTGGACTACGGCAAGAAGTn


nAAGCTGGAGTTCGCCATCTnnnCnGCnnCTCnnnnnTCnnnnnnCTGTC


 


>ise2c.pk001.l2 tubulin


SEQ ID NO: 26


TTCGGCACGAGGGGCAAGCCTCTTAACCGGTCGCGCTGAACGACGACTGATATTTAATTAATTTATATTCTACGT


TAAGTTCAACAAAACTCAATTCAAAATGCGTGAGTGCATCTCAGTACACGTTGGACAAGCCGGAGTCCAGATCGG


TAATGCCTGCTGGGAATTATATTGCCTTGAGCATGGAATCCAGCCTGATGGCCAGATGCCCACAGACAAGACCGT


GGGCGGTGGTGATGACTCCTTCAACACCTTCTTCAGCGAGACCGGTGCCGGCAAGCACGTCCCCAGGGCTGTGTT


TGTTGACTTGGAACCCACAGTAGTTGATGAGGTCCGCACTGGCACATACAGACAGTTGTTTCATCCAGAACAACT


TATCACTGGTAAGGAAGATGCGGCCAACAACTACGCCCGTGGTCACTACACCATCGGCAAGGAAATCGTAGACCT


AGTCCTCGACCGCATCCGTAAGCTCGCCGACCAGTGCACCGGTCTCCAGGGCTTCCTTATCTTCCACTCCnnnCn


GTGGAGnTnnnTGGATCTGGTTTCACTTCCCTCCTGATGGAGCGACTCTCCGTGGACTACGGCAAGAAGTnnAAG


CTGGAGTTCGCCATCTAnnnn


 


>ise2c.pk002.b4 ubiquitin


SEQ ID NO: 27


GCACGAGGATCAAAGAGTTACGAACCGTCACCATACTGAAGGAGATACCATTCGTCGTGCCATTCTCAACACGCG


TCCTTATATTCCAAGGACTTTTAGCGAGAGAGAAGCACGACCACTGGTACGAAATGACGAACTTCAACGAGGGGC


CCTCGATCAACATCAGTGTTCGAAGGACGCATTTATATGAAGATGCATTTGATAAACTTAGTCCGGATAATGAAC


CTGATTTGAAGTTGAAACTTCGCGTGCAACTGATCAACCAGGCCGGTGCGGAGGAAGCTGGTGTCGACGGCGGTG


GACTATTCCGAGAGTTTCTTTCTGAGCTCTTAAAATCTGCATTTGATCCGAACAGGGGTCTGTTCCGGCTGACAA


TAGACAACATGTTGTATCCGAACCCCGCCGTACATCTACTGTACGATGACTTCCCCATGCACTACTACTTCGTCG


GCAGGATGCTGGGAAAGGCGATGTACGAGAACCTGTTGGTGGAGCTGCCGCTGGCGGAGTTCTTCCTGGGCAAGC


TGTGCGGCTGCGGGGAGGCCGACGTGCACGCGCTGGCCTCGCTCGACCCCGCGCTGCACCGCGGGTTGTTACTAC


TC


 


>ise2c.pk001.j16 small nuclear ribonucleoprotein


SEQ ID NO: 28


GCACGAGGGCCGGCCGCCGTGTTCGTGCCGTCCCGCCGGGCCGCGCGCCTACTGGCCGCCGACCTGCTGGCGCTG


GCCGCGGCGCACGCGCAGCCCGCCGCCTTCCTGCGCGCGCGCCCCGACGTGCTGCAGCCCTTCCTCAAGAGGATC


AACGACAAGATGCTGAAGGAGACGGTGGCTGCGGGCGTGGCGTACCTGCACGAGGGCGTGGACCCGGCGGAnnGG


CGCCTGGTGCAACAACTGCTGGAGTCGGGCGCGCTGGCGCTCTGCGTCGTGGCCGCCGAGCTGGCCTGGGGACT


 


>ise2c.pk006.h23 small nuclear ribonucleoprotein


SEQ ID NO: 29


GCACGAGGCGAAGATAAAGGTCGCGTGTGGACCTTAGGTTTAAGTTTATTATTAAATAATTTAGCCTAAACATAA


GTCATGGCCAATAACGACAACTTTGCACAAGATGTTACTGATAATCAACTAAATGGAAATGCCGAAAATGGTGGT


GGCGATACGCAAGAACATAATAGTGCCGAAGCCCCTGGGCGTGATGATGACAGAAAACTTTTTGTCGGAGGCCTG


AGCTGGGAAACCACAGACAAGGAGTTACGTGACCACTTCAGTGCATATGGTGAGATTGAGAGCATCAATGTCAAG


ACTGATCCAAACACTGGCAGATCAAGAGGATTTGCCTTTATTGTGTTCAAGGCACCAGATTCAATAGACAAAGTG


ATGGCTGCTGGAGAGCACACTATTAACAACAAAAAAGTTGATCCGAAAAAAGCAAAGGCTAGACATGGAAAGATC


TTTGTTGGTGGTCTTAGCAGTGAAATATCAGATGATGAGATCAAAAACTTCTTCAGTAATTTTGGAACAATAATT


GAAGTCGAGATGCCCTTTGACAAAACCAAGAATCAGnnnAAGGGATTCTGCTTTATAACATTCGAGTCTGAACAG


GTGGTCAATGAGCTGCTGAnnnCn


 


>ise2c.pk006.m8


SEQ ID NO: 30


GCACGAGGCGCGTGTGGACCTTAGGTTTAAGTTTATTATTAAATAATTTAGCCTAAACATAAGTCATGGCCAATA


ACGACAACTTTGCACAAGATGTTACTGATAATCAACTAAATGGAAATGCCGAAAATGGTGGTGGCGATACGCAAG


AACATAATAGTGCCGAAGCCCCTGGGCGTGATGATGACAGAAAACTTTTTGTCGGAGGCCTGAGCTGGGAAACCA


CAGACAAGGAGTTACGTGACCACTTCAGTGCATATGGTGAGATTGAGAGCATCAATGTCAAGACTGATCCAAACA


CTGGCAGATCAAGAGGATTTGCCTTTATTGTGTTCAAGGCACCAGATTCAATAGACAAAGTGATGGCTGCTGGAG


AGCACACTATTAACAACAAAAAAGTTGATCCGAAAAAAGCAAAGGCTAGACATGGAAAGATCTTTGTTGGTGGTC


TTAGCAGTGAAATATCAGATGATGAGATCAAAAACTTCTTCAGTAATTTTGGAACAATAATTGAAGTCGAGATGC


CCTTTGACAAAACTAAGAATCAGAGGAAGGGATTCTGCTTTATAACATTCGAGTCTGAACAGGTGGTCAATGAGC


TGCTGAnGACTCCTAAGCAGnnnATTGGTGGCAnnnnnnnCGAC


 


>ise2c.pk001.a23


SEQ ID NO: 31


GCACGAGGATGAAGTTGGCTCTGACACTCTTGGCTCTGGCGGCGGTGGCCACCGCTAAAAACATCAACGTCGAGG


ATGCCATCGACCTAGAGGACATCACCGCCTACGGATACTTGGCTAAGATCGGTAAACCTCTTGCCGACGAAATCC


GCAAAGCTGAGGAGGCAGAGAGCGCATCCAGAATTGTTGGTGGTCAGGCCTCCAGCCTCGGACAGTTCCCCTACC


AGGCTGGTCTTCTCGCTGACTTCTCCGCTGGCCAAGGTGTGTGTGGTGGTTCCTTGGTGCGTGCCAACCGTGTTC


TTACTGCTGCTCACTGCTGGTTCGATGGCCAGAACCAGGCCTGGAGATTCACCGTTGTTCTTGGCTCCATCCGTT


TGTTCTCCGGTGGTACCAGAGTTCAAACCTCCAACGTTGTTATGCATGGAAGCTGGAACCCCAGTAACATCCGTA


ATGACGTCGCCATGATCAGGCTGAACTCCAACGTTGGTCTTTCAAACACCATTGCACTCATCGCTCTGCCCAGCG


GTAGCCAGCTCAACGAAAACTTCGCCGGTGAAAACGCCGTCGCnnnCTGGATTCG


 


>ise2c.pk001.a7


SEQ ID NO: 32


GCACGAGGATCAAAATGAAACTGTTCCTCGCAGTCGTGTGCTTGGCCGTTGCCGCATCCGCGGTGGAGATTGGAG


TTCCGTCTCAGGAAAACCCAGTCTTTGGCTACCATCAAAACTTCGGTATTGCCGAAGCTGCCAGGATCAAGAAGG


CTGAGGAAGAAACCAGCCCTAGCGCCCAGAGGATCGTCGGAGGATCTGTCACTGACATTTCCAACGTCCCTTACC


AGGCTGGTCTCGTGATCCAAGTTTTGGTCATCTTCCAATCCGTGTGCGGTGGTTCCATCATCTCCCACAACCGCA


TCGTGACCGCTGCTCACTGCAACTGGGACGGTTCTATCACCGCTAACTCTTTCACCGTCGTACTTGGCTCCAACT


TCCTCTTCTCCGGCGGTAACCGCATCACCACCAGAGATGTTGTCATGCACCCCAACTGGACCCCAACCACCGCTG


CCAACGACATTGCTGTCCTCCGCATTAGCTCCGTTACTTTCACCAACGTGATCCAGCCCATCGCTCTGCCCAGCG


GCAACGAGCTCAACAACGACTTCGTCAACTGGAACGCTATCGCTTCCGGATACGGTCTTACCGCTGATGGTGCTA


ACATCGGTACTACCCAACGTGTCAGCTCCGTGGTACTCCCCGTGATCnnnnnnCGCCAGnnCGCTACCGTnnnnn


n


 


>ise2c.pk004.c4


SEQ ID NO: 33


GCACGAGGAATCTTAGTTACATTGGAGTGACTTTTATTTATCAATAACATTTTTATTTGAAGACTCAGTACGTAT


TATCGCGTAGTTCAACAGAGTTGCTAGTGTAGTTTTCTGAAAGTTGCCATCTTGCTTTTGCAACTTTTAAATATA


AAAGTCTTATTAGATCGTTTTTACTACCGATAAATTTACTAAAAATATAAAAGTGCAATTTACAATTACTCTGTT


AGTGTCAGTTTGTGTGAATTTGTCGTAGTTATAAAAGGACACTGTATTGATTTTGTCAATCAGTTTGACGCATGC


GCTCATTGGGTGCCGTAAAAAAGGGTTGGCCAACATTCCGAACAGTGTCGTTCCGGTCGCCGTTGTCGTGGTGTC


GGTGAAGTTAGTGGTGGAATTTTTACGTGTATAACATCAAAAAATGGCGTCTGGTGTGACAGTTTCGGACGCGTG


CAAAACGACGTACGAGGAGATTAAGAAAGACAAGAAGCACCGCTACGTGGTGTTCTACATCAGGGATGAGAAACA


AATTGACGTAGAGACCGTCGGCGAACGTAACGCGGAATACGATCAGTTCCTTGAGGATCTGCAGnnnGGTGGCAC


CGGnnAGTGCn


 


>ise2c.pk004.l4


SEQ ID NO: 34


GCACGAGGCTGATATCTAATCTTAGTTACATTGGATTGACTTTTATTTATCAATAACATTTTTATTTGAAGACTC


AGTACGTATTATCGCGTAGTTCAACGGAGTTGCTAGTGTAGTTTTCTGAAAGTTGCCATCTTGCTTTTGCAACTT


TTAAATATAAAAGTCTTATTAGATCGTTTTTACTACCGATAAATTTATCAAAAATATAAAAGTGCAATTTACAAT


TACTCTGTTAGTGTCAGTTTGTGTGAATTTGTCTTAGTTATAAAAGGACACTGTATTGATTTTGTCAATCAGTTT


GACGCATGCGCTCATTGGGTGCCGTAAAAAAGGGTTGGCCAACATTCCGAACAGTGTCGTTCCGGTCGCCGTTGT


CGTGGTGTCGGTGAAGTTAGTGGTGGAATTTTTACGTGTATAACATCAAAAAATGGCGTCTGGTGTGACAGTTTC


GGACGCGTGCAAAACGACGTACGAGGAGATTAAGAAAGACAAGAAGnnnCCGCTACGTGGTGTTCTACATCAGGG


ATGAGAAACAAATTGACGTAGAGACCGTCGGCGAACGTAACGCGGAATACGATCAGTTCCTTGAGGATCTGCAGA


AGGGTGGCACCGGAGAGTGCAGATATGGCCTCTTCGACTTCGAGTACACGCACCAGTGCCAAGGCACGTCGnnn


 


>ise2c.pk004.n19


SEQ ID NO: 35


GCACGAGGCCTCGTGCCGCGCGAATAGACAGTTTTGTGTGCACAATGTTGATCCTTTGGCTAAATATCATCGCAA


TAATTTGTGTCATACCCTACGCAAATGGAGAAGGAAGGGTTGCAATAGCGCATTTACAATCGCTAAAGTCAGTGA


CTGGTCAAATTCAATTTACGGAGACGGCAAAAGGGCTTCATGTCGAAGGAGTTATATTTGGTTTACCACCCGGTG


CCTACGGGTTTCACGTTCACGAATTAGGAGATGTTGCACCTGGTTGCGACCAGGCGGGCCGGCACTTCAACCCTG


AGGGATCCACCCACGGTGGCAGGAACTCCACCGTACGCCATGTCGGTGACCTCGGAAATGTAGTGTTCGTTAGCG


AGCGAGCCGCTTATGCTACAGTAGACTTTGTAGATAGTCTATTGGCACTTCAAGGACGTAATAGTATATTGGGGC


GCTCTTTGGTCTTGCATGAACAAACGGATGACCTAGGTTTGGGAGGAAACGCGACGTCTTTGACTACAGGTAACT


CGGGGCCCCGGATAGCATGTGGTGCTATTGGAATCAAATCACCTTATGACCCTTGGAATGCTGCTAGCTCTATGT


CTCCGTCGATGCTACTATTTATCACATCTTTAACTTTATTTACTTTAnnnTnnnAAnTnnnnGTATnAGTATTTA


ATTTnnnnn


 


>ise2c.pk005.f21


SEQ ID NO: 36


GCACGAGGCTTCCACATACGCGAATAGACAGTTTTGTGTGCACAATGTTGGTCCTTTGGCTAAATATCATCGCAA


TAATTTGTGTCATACCCTACGCAAATGGAGAAGGAAGGGTTGCAATAGCGCATTTACAATCGCTAAAGTCAGTGA


CTGGTCAAATTCAATTTACGGAGACGGCAAAAGGGCTTCATGTCGAAGGAGTTATATTTGGTTTACCACCCGGTG


CCTACGGGTTTCATGTTCACGAATTAGGAGATGTTGCACCTGGTTGCGACCAGGCGGGCCGGCACTTCAACCCTG


AGGGATCCAACCACGGTGGCnnnnnCTCCACCGTGCGCCATGTCGGTGACCTCnnAAATGTAGTGnTTGTTAGCG


AGCGAGCCGCTTATGCTACAGTnnnCn


 


>ise2c.pk010.h5


SEQ ID NO: 37


GCACGAGGGTCGAGAGATACGGTGCGCACATAGCAACAATATCAAAGTACAAAGGTCAGTAACTATGAGTGGTAA


ATTGTTAAAAACTCTAATCCTTGGGGCACCTGCTTCAGGCAAGGGGACTATATCGTCTCGGATAGTGAAGAAATA


TGCTGTGGCACACGTGTCCAGTGGGGACAAGCTGAGGGACCACATTGAGAAACAAACTGACCTAGGTAAAGAAGT


CAAAAAGTACTTGAATGAAGGGAAACTTGTACCTGATGATGTCATGATAAAGTTTATGATCACAGAATTAAAAAA


AGTTGAAGATAAACCATGGCTACTGGATGGATTCCCGAGGACTGTGGGACAGGCTGATGCTTTGTGGAAGGTACA


ACCTGTTGATGTAGTAGnnnnnTTAGTAGTGCCTTTTGAGGTAATCATAGACAGAGTGAnnnAnCGCTGGGTGCA


CTTGCCTTCGGGCCGAGTGTATAACATTGGCTTCAACACTCCTAAAGTGGAAGGTAAGGATGATGAGACAGGTGA


GGACTTGGTTCAGAGACCTGACGACAAGCCAGAGGCTGTGCGCAAGCGGCTGGAGATCTATGAGAGTGTGACGAG


GCCAGTCATAGAGTTCTAnnnnGCTAA


 


>ise2c.pk001.c18


SEQ ID NO: 38


TGCTGCTGCTGGAAGCTGGGCCCAACCCTCCCGAGGAGAGCATTATACCAGGCTTAAGACAAACCTTGAAAGAAA


CGCCCTACGACTGGAACTTCACCACCATTGACGACGGGGTCACGAGCCAGGCGCTGGCGGGCCACGTGCAGAGAC


AGCCGCGGGGCAAGATGCTGGGCGGCAGCGGCTCGCTCAACGACATGGTGTACGCGCGGGGCCACCCCGAGGACT


ACTACGAGTGGGCCGACATCGCCGGCGACGTCTGGAACTGGACCAACGTGCTGGACTACTTCAAGCGGACGGAGC


ACATGACGGACGCCAATATCGTTCACAACnnnnAGCTCATGCAGTACCACGGCACGGnnnnnnCCATnnnnnnnT


nnnGnnnCCAnTnnnnnnnn


 


>ise2c.pk004.p1


SEQ ID NO: 39


GCACGAGGGGAAAACATGGGAAGGAGGTCGCATCAAGATGTTAGTGCTCGACTTGAACTGCCCGGTCGTTGGAGA


CGACTGCAAAGACAGCCGCAAGAAGTTGCTTGTGGACTACTTCCATACAAACCTGCATACCCAGAACTTCTACGC


GTTCCGCTTCTTTATCTGCGAAGTGTTGAACTTCATCAACGTCGTGGGCCAGATCTTCTTCATGGACTTTTTCCT


GGACGGCGAGTTCTCCACGTACGGCAGTGACGTGGTCAGTTTCACCGAGATGGAGCCCGAGGAGCGTGTGGACCC


GATGGCTAGAGTGTTCCCGAAAGTGACCAAGTGCACCTTCCACAAATACGGTCCTTCAGGAACCGTGCAGAAGTT


CGACGGTCTGTGCGTGCTGCCATTGAACATCGTCAATGAAAAGATCTACGTGTTCCTGTGGTTCTGGTTTATGAT


CCTGTCGATCCTGAGTGGAATTTCGCTGATTTACCGCATGGCCGTGGTGGCTGGACCGCGCGTGCGCCTGTACCT


GCTGCGTGCGCGCAGCCGCCTGGCCCCGCnnnCGCnnnnnGnnnn


 


>ise2c.pk005.p13


SEQ ID NO: 40


GCACGAGGATTTTAATAGCTATTATGACTTTACAGACTAGACGGATCAAGGCCATGCCTCTCGCTTGCATACTCA


CCATCCGCACATACCGTATTGCGGTATGTCAATAAGTTGCAAATAATGTCTGTTCAGTTTTACAAGGATAAGATC


AGCAGTATTTGCGAACTGTACCTACTACTAAGCTGATAATGTAATAATTAAACTTTATTATTGAAATAGATATGT


ATAATTGACATCTTTCTCAAATGGGTGTCAATACTGCCAACTCTATTACCACAATTTCTTTTCGTATTTGCTTTT


ATACTGAGCCTGATGACGTACTGTACTTTTTATTAGAATTTAATTTTTCTTATTTTTCTTACTACGTAGTCATTA


AATCTGAGAAATTAAAAATTACTAATTTAGAACTCCCAAATTCTGAATGAGGTTCTAAAAAGTTGTTAGGAATAC


TAAATACCATTTTACCAACATAAATCTAATTTCGTTACTTAAAATATTAAATGTATAATGAAATGTCTATGATAA


GTGTTTACTATCTTTATATCGACAAAATTTATTTTCCATGTTTTAAAATTTATTTTTCAGATGTTTTGACGTGAT


AAGTTTGTATTTTATCAATATCTGATAGTCGAGAGTTAnnnAnTATTG


 


>ise2c.pk001.f12


SEQ ID NO: 41


GCACGAGGGAGGAGAGGTGGTGGCTGGCTTCCTTGCAAACGAAGCGTCGTAAATTACATCTTATTTGTAAATTTT


AATAAAAATTTGATCGTTAAACGATCGAATCAGTAGTGATTTAAGTGCTCAAGCAGTTTCACATCCAATCGACAA


TGAGTTCGAGTGTATGCTACAAGTGTAACCGGACAGGGCACTTCGCCCGCGAGTGCACCCAGGGTGGTGTTGCCG


CTCGTGACTCTGGTTTCAACCGTCAGCGCGAAAAGTGCTTCAAGTGCAACCGCGCTGGGCACTTCGCTCGGGATT


GCAAGGAGGAGGCCGACCGTTGCTACAGATGTAACGGCACGGGACACATAGCGCGTGAGTGCGCGCAAAGTCCGG


ACGAGCCGTCGTGTTACACTTGCAACAAGACCGGGCACATCGCACGGAACTGCCCAGAGGGCGGGCGCGACAGCT


CCAACCAGACCTGCTACAACTGCAACAAGTCCGGCCACATCTCACGCAACTGCCCCGACGGCACCAAGACTTGTT


ACGTGTGCGGAAAGCCCGGACACATCTCCCGCGATTGCGATGAGGAGCGGAACTAACACACGCCTCTTCGCGACT


GCCTATATATAnnnTAAACTATGTATATTATGATGCCACGCACGGACGATAAGCAAAGGACGCGATACGCGACAC


TAGATCGTAAGACCACACGACTGTATGnnnnTAATGCAACG


 


>ise2c.pk001.n21


SEQ ID NO: 42


GCACGAGGATAATAAACGTTAATATTTAACAAGTTGAAAAGTTTGTCTTTCAATTTGTGATTTTGTAAAGATCAT


TCTATGGAATGGACAGTTTGCTATCTGTGAAACATCCATTAGCTTTGTGTTGAGAGCAGAGGTCGCGGCGGCGGG


GTGATGCGGCCATGGCTTCGCGGCGCGTGACGCGCAAGTGGGAGGTGTTCGCGGGACGGAACCGATTCTGGTGCG


ACGGCCGCCTCATGACGGCGCCGCACCCCGGCGTGTTCCTGCTCACGCTCGCGCTCATCTGCGGCACGTGCGCCC


TGCACTTCGCCTTCGACTGCCCCTTCCTGGCCGTGCGCGTGTCGCCCGCCGTGCCCGCGGCCGGCGCCGCGCTGT


GCGCGCTGACGCTGGCGGCGCTGCTGCGCACGGCGCTGTCCGACCCCGGCATCATCCCGCGCGCCGCCGCGGCCG


AGGCGGCGGCGCTGGAGGCGGnG


 


>ise2c.pk004.e20


SEQ ID NO: 43


CGCGCACGTCGCTCnnCAAGCCCGCTGCAGCGCCGGCCAAGCCCGCCCCCGCGGCGGCGCGCGCCACCAGTGCGA


CCAGCCGCGCGGCCCCCGCGGCCCGGCCGGCCCCCAAGTCCGCAGTAGGCGCAGCGCGGCCCGCAGCACAAAAGA


CAGATGCGGCCGCCAAACCCGCGGCGACCCGGGTTGCGGCTCCGCGTCCCGCGCTGTCGGCGCCCAGGCCCCAGC


CTAAGCCGGCAGACAAGAAGCCAGTACCGAATGGTGACGTGAAAGACTCCAAGCCAGCCGCGCGGCCCGCGCCCC


GGCCGGCCGCGGCCGCGCGCCCCGCGCCGCGCCCCACTCCCCGCGCCCCCGCCGCACnGGTCGCACCCACTACTn


nnnnGAGTGCCCCCAAGCCGGCGCCGCGTGCTCCCCTGGACAAGCAGAGCnnnGACCTCGCTAACAAACGCATCn


nnGnCAnGGCAGCACCGCCTAGGACTGCTCCCCCTAAGACGACAACGACGACAACAGGnnnnnnnnnnnnnGTnn


CGAAGnnnnn


 


>ise2c.pk005.n11


SEQ ID NO: 44


GCACGAGGCTATAACAAGCAGCATATAAAAATGAAATTCTTGCTGTCTTTCGCTGCCGTCATCGCCGTGGCCGCC


GCTGGCCTGGTGCCCGTTGGACCCGCCGGCCCTGCGCCCGCTCCTGAGGCCCCTGAGGTCTTCGAGCCCGTCGCT


ATTGGACCCGCTGTCATTGACTCCTTCGAGCCCATCGCCATCGGACCCGCTATCATCGACTCCTTCGAGCCCATC


GCCATCGGACCCGCTATTGTTCCATCTCCCGAGCCCGTCGCCATCGGACCCGCCATCATTGAGAGCCCAGAGCCC


GTTGCTGTCGGACCTGCATGGATTGACTTCCCCCTGCCCGACGGTGGTGCTGCCGTTGCCCCCGTTGAGCCCTCT


CCCGTGGCTGTTATCCCCGGTCCCGTGTCCACTGAGGTTGCTTCAGGCACTCCCCTCGTTCAGATCATCCTGAAC


ATCAACnnnnnnTCTGCTGACGTTAGCCCCGTTGCTGTnGGCCCCGCTGTCGAGnnnACACCCGTGCACGTTGTG


GACTCTGCCCCTGAACCCGTCCACGTTGTGnnnnnnGCCCCnnnnnCnATCnnnnnGTCGn


 


>ise2c.pk003.l14


SEQ ID NO: 45


GCACGAGGCTTAGAGTAAGCATAGGTGTATTTATGTATTGAGTCGGAAGAAGCAATGGACGATCCAAATAGGATG


ATGGCGCATAGCGGCGGGCTTATGGGGCCGCAGGGCTACGGCCTGCCTGGCGGCGAGGGAACTCCAACCGCAGGC


GAAGGTGAAGCCCGCAAGCAAGATATTGGTGAAATATTGCAACAGATCATGAATATTACAGATCAAAGTCTTGAT


GAAGCGCAAGCGAGAAAACATACTCTCAACTGTCACAGAATGAAGCCTGCCCTATTTTCAGTGTTGTGTGAAATC


AAAGAGAAAACAGTGCTGTCCCTCCGCAACACGCAAGAGGAGGAGCCCCCAGATCCCCAGCTGATGCGCTTGGAC


AACATGCTCATAGCCGAGGGGGTCGCTGGCCCTGAAAAGGGTGGTGGTGCGGGCGCTGCAGCTTCGGCATCAGCT


GCTGCTGGTGAATGGGACAATGCCATCGAGCACTCTGACTACCGTGCGAAGTTGGCGCAGATCCGCCAGATCTAC


CACCAGGAGCTGGACAAGTATGAGAATGCTTGTAATGnnnnnnCCACCCACGTGATGAACTTACTCCGCGAGCAG


AGCCGCACCAGGCCTATCACAnn


 


>ise2c.pk003.e24


SEQ ID NO: 46


GCACGAGGCCAGGTTTGAGAAAAACGCTTAAACTGCCACAAAATCCCGTTCTCGAAGAAGCACTTTTCACTTATT


AATAAGTAACTTGTGTAAAATGTGGTTTAAATGTGTATTTTACTAAACCTCAATAAATATATTTATATCAAAATA


TTTTTTTTCTATACTGTATTATTTATTCCTATAGTACATATTATAATCCGAACGCTCCGTGAGTCCGAACAGGGG


TAATTTTTTGGTAGTTCGGATTATCGAGGCTCTACTGTATACCTACTTTTTGTTAAAATATTTTAGTCTTATATA


CGACTTCCTAACTAATCCATATCTCTTAGAGCTTTCGAATATCCATTTGCCTTTTTCTTAAAAAGATTAATAACT


ATTTATATATATCCCAAATATATAAAAACAACCACTCCAATTATTATTATTCAAATATGACAAACTAGATAGAAT


GTCCCAAGAAATTTGCAAAAAAGTAATGTTCAAATTATTAACCGAAGAACGAATTnnnGAGTGTATAATATTATA


CAGACATTTAGAAATTTTTAATAGGCTCCAATCGCATGAGAGGTCGCTTTAAAATTCGGCATTGGTGTGTGCGTT


GCAATTTAATCTTTAACACCCnnn


 


>ise2c.pk005.l5


SEQ ID NO: 47


GCACGAGGGGACGTGTTTACAATTTACTTTCGTGCTCGTGTGATTTTAATTAAAACAGTGCTAAGTGCTCTAGGA


CGCTGAATAACTGATATTTGTTTTAAAAGTTGATATAAATTAATCACAATGAATAGAGATAAACGAGAACCAGAG


TATCCAACGGAGTTGGAGTCTCAATTCGTAATGCGTTTACCTGAGGAGCCTGCAAAAGTTTTGAGAGAAGTGTTG


AAATCCGGAGAGAACCTGAAAAACAGACTGACGATACAAATAGAAAACGACATGCGCACGGGCGAGGTAAGGTTT


GATCACTGGTTGATGCACGCCAAGATCGTGGATCTACCAACCATCATAGAATCTCTAAAAACGATCGACAACAAG


AGTTTCTACAAAACAGCAGATATATGCCAAATGATGATTTGTAAAGAAGAACCTGACCAACCATCCACAGAGGAA


GAGTCACCAGCTAAAAATAAGAAAAAAGATCCATACAAAGTTGACAAAAAGTTCCTATGGCCACACGGCATCACA


CCGCCTACGAAGAACGTACGGAAGCGTCGATTTAGAAAAACCCTTAAAAAGAAATATGTAGAAGCACCAGAAATT


GAAAAGGAAGTGAAGAGGCTGCTGAGnGCAnnCnATGAGGCTGTTAGTGTTAACTGGGAGGTCATCAAnnnnnnn


GAT


 


>ise2c.pk006.k12


SEQ ID NO: 48


GCACGAGGGTCGAATGGAACATGGCGGTGCTAGGCAGGATGTGCATAAGTTTTTGATTTTTGCATTTTTAACGAG


TTGCTTATATCAGTTAGCTTTCTAAATAATTTCTGACTTATTTCGTGTGTTATAATATTTGTTATAGTGTAAAAG


CTTATCCACCCCAGGAATTTCCTATCTGGACTTACTTAGTTCTGCAATGAAAATTATTATTCGTTGGTAGTGTAA


AAATAATTGTGACAAATATATCACTTTGCTTCAGTGTGCCGTGTTGGTCATGGCTACGCTCCTCCAAGAGAATGG


TATAAAGGAGTTAAGCAAAGTTGTGCCTAACCGTGGTATATCCTCACATAGTGTAACAAATCATATGGTGCCTGA


TCATGAATATTGCGAAGCTGGGTCAACTAGCACGTCACAGATGAAGTGTACCGATACAAGTGAGGCGATGGCGCC


ACCCGCCGCCATTGAAGAAGAGGAGGATACACCAGAAATAGATATAATGATAAACAATGTTGTGTGCAGTTTTAG


TGTTAAGTGCCACCTGAACCTTAGACAGATAGCATTnnnTGGTGTGAACGTTGAATTTCGCCGCGAGAACGGCAT


GGTAACTATGAAGTTACGGCGTCCATACACTACTGCGTCCATCTGGTCGTCCGGCCGCGTGACGTGCACTGGTGC


AACCAGCG


 


>ise2c.pk010.i8


SEQ ID NO: 49


GCACGAGGGAATATTGCGAAGCTGGGTCAACTAGCACGTCACAGATGAAGTGTACCGATACAAGTGAGGCGATGG


CGCCACCCGCCGCCATTGAAGAAGAGGAGGATACACCAGAAATAGATATAATGATAAACAATGTTGTGTGCAGTT


TTAGTGTTAAGTGCCACCTGAACCTTAGACAGATAGCATTAAATGGTGTGAACGTTGAATTTCGCCGCGAGAACG


GCATGGTAACTATGAAGTTACGGCGTCCATACACTACTGCGTCCATCTGGTCGTCCGGCCGCGTGACGTGCACTG


GTGCAACCAGCGAGGACCAGGCGAAGGTTGCCGCACGACGGTATGCGCGCGCCCTTCAGAAGCTCGGCTTCCAAG


TGCGTTTCCGCAATTTCCGTGTAGTCAATGTATTAGGCACCTGTCGGATGCCGTTTGGTATAAGGATCATATCTT


TTTCGAAAAAATACAAGGAAGCAGACTATGAACCTGAGCTCCATCCTGGAGTCACATATAAGTTATACAATCCTA


AAGCCACACTCAAGATATTCTCCACTGGTGGTGTGACTATCACAGCTCGGAGTGTGAGTGACGTTCAGTCAGCCG


TGGAACGCATCTTCCCTTTGnTGTACGAGTTCCGCAAGCCTCnnnnACCGGCAnnnnA


 


>ise2c.pk010.b12


SEQ ID NO: 50


GCACGAGGGTACCAAAAGCTCTTTTCATTGCAGCTGAAGGGTCACTGCAACTTGGCCAATCAGAATTAGCATTGA


AACTATTCAAAGAACTAAAACAAGAAGGAATGGAAATCAGGCAACATTTCTATTGGCCTTTGTTAGTTCAGAAGG


CAAAGGAAAATGATGAGGAAGGCCTCTTGCAAATTTTAAAAGAAATGAGCAGCAATGACTTTACTGTTACTGGAG


AAGCGTTAAGAGACTATGTTATCCCTTACTTGATAAAAAAAGATTCTCCACAGAATGTCTTACTTAAACTTCAAA


TTGCAAATGTACCAACAATCCATGCTGCAAGAAATCTAATGGTTGATCTTTTGGATTCTGGAGACATAAAAGGCG


CAGCGGAAATAGCTCTGCAATATAGACCTTGGGGCAACTACTCTCTTGTTGCCAGGTCCCTCATCAATGCAGTGA


ATAAGACAAAAGATGTAGAATCGTTTGCTAAAATTCTTCATGCTATAAGCAGTAAACCTTTGTCACAGGGTGAAG


AAGATGTTGCTGCCAACAATGAGGAAGGTCAAAGTGATGAAAATAATGATATTCATGAAGTCGGCCGTATTGTGA


GGTCGTCTGCCAAGAGTTTGGCTAAACCAGACTTAATAGnAAnnnnTTTAGA
















TABLE 2







List of dsRNA primers. 



















SEQ ID NO


Primer



Sense
Antisense
Target/sense/


#
Target Gene ID
Seq ID
Target
strand
strand
antisense





0075
juvenile
ise1c.pk002.m13
AACATGGTATCC
CAUGGUAUCC
CCUGAAGUCG
51/52/53



hormone diol

GACTTCAGGAA
GACUUCAGG
GAUACCAUG




kinase







 


0076
juvenile
ise1c.pk002.m13
AAGGTCGCTGAC
GGUCGCUGAC
CUUGUUCUCG
54/55/56



hormone diol

GAGAACAAGGA
GAGAACAAG
UCAGCGACC




kinase







 


0077
juvenile
ise1c.pk002.m13
AAGTGTCCTGGG
GUGUCCUGGG
GAACUCAAGC
57/58/59



hormone diol

CTTGAGTTCCA
CUUGAGUUC
CCAGGACAC




kinase







 


0078
juvenile
ise1c.pk003.f7 
AAGAAGAAGCTC
GAAGAAGCUC
CACGUGGAGG
60/61/62



hormone diol

CTCCACGTGTT
CUCCACGUG
AGCUUCUUC




kinase







 


0079
juvenile
ise1c.pk003.f7 
AAGGTCGCTGAC
GGUCGCUGAC
CUUGUUCUCG
63/64/65



hormone diol

GAGAACAAGGA
GAGAACAAG
UCAGCGACC




kinase







 


0080
juvenile
ise1c.pk003.f7 
AATGTCCTGGGG
UGUCCUGGGG
GAAACUCAGC
66/67/68



hormone diol

CTGAGTTTCAA
CUGAGUUUC
CCCAGGACA




kinase







 


0081
juvenile
ise1c.pk005.a15
AAGAATAAGCTC
GAAUAAGCUC
CACGUGGAGG
69/70/71



hormone diol

CTCCACGTGTT
CUCCACGUG
AGCUUAUUC




kinase







 


0082
juvenile
ise1c.pk005.a15
AATTTGTCGAGG
UUUGUCGAGG
AUAGGGUCUC
72/73/74



hormone diol

AGACCCTATTG
AGACCCUAU
CUCGACAAA




kinase







 


0083
juvenile
ise1c.pk005.a15
AAGTTCGCGTTC
GUUCGCGUUC
UUCAAGAGUG
75/76/77



hormone diol

ACTCTTGAAGA
ACUCUUGAA
AACGCGAAC




kinase







 


0084
ribosomal
ise1c.pk006.d24
AACTGCCCCTTA
CUGCCCCUUA
AGAUGAGGUU
78/79/80



protein L18a

ACCTCATCTAT
ACCUCAUCU
AAGGGGCAG



 


0085
ribosomal
ise1c.pk006.d24
AATCACGCTGAA
UCACGCUGAA
UACAGUGGUU
81/82/83



protein L18a

ACCACTGTATA
ACCACUGUA
UCAGCGUGA



 


0086
epoxide
ise2c.pk009.i4 
AAAATATGGCGC
AAUAUGGCGC
ACAAUAGGCG
84/85/86



hydrolase

GCCTATTGTTT
GCCUAUUGU
CGCCAUAUU



 


0087
epoxide
ise2c.pk009.i4 
AACGTTCTCGGT
CGUUCUCGGU
CAGUGAAAGA
87/88/89



hydrolase

CTTTCACTGCT
CUUUCACUG
CCGAGAACG



 


0088
epoxide
ise2c.pk009.i4 
AAGTCATCGTTC
GUCAUCGUUC
GUAGACUUGG
90/91/92



hydrolase

CAAGTCTACCT
CAAGUCUAC
AACGAUGAC



 


0089
V-ATPase A
ise2c.pk001.d19
AACCCCTTGAAT
CCCCUUGAAU
GACCUUAACA
93/94/95



subunit

GTTAAGGTCGG
GUUAAGGUC
UUCAAGGGG



 


0090
V-ATPase A
ise2c.pk001.d19
AAGTACACCATG
GUACACCAUG
UACUUGCAAC
96/97/98



subunit

TTGCAAGTATG
UUGCAAGUA
AUGGUGUAC



 


0091
V-ATPase A
ise2c.pk001.d19
AACGTGTCCATG
CGUGUCCAUG
GUCAGCCAUC
 99/100/101



subunit

ATGGCTGACTC
AUGGCUGAC
AUGGACACG



 


0092
H+-ATPase V-
ise2c.pk001.e14
AAACCTACAAAA
ACCUACAAAA
UUUCGGCCAU
102/103/104



type subunit

TGGCCGAAAAC
UGGCCGAAA
UUUGUAGGU



 


0093
H+-ATPase V-
ise2c.pk001.e14
AATCTACGGACC
UCUACGGACC
CCAAAGAAGG
105/106/107



type subunit

CTTCTTTGGAG
CUUCUUUGG
GUCCGUAGA



 


0094
V-ATPase A
ise2c.pk001.f20
AACTCTGACGTC
CUCUGACGUC
GUAGAUGAUG
108/109/110



subunit

ATCATCTACGT
AUCAUCUAC
ACGUCAGAG



 


0095
V-ATPase A
ise2c.pk001.f20
AAGTGCTTGGGT
GUGCUUGGGU
GUCGGGGUUA
111/112/113



subunit

AACCCCGACAG
AACCCCGAC
CCCAAGCAC



 


0096
V-ATPase A
ise2c.pk001.f20
AACTGGCTCATC
CUGGCUCAUC
GCUGUAGGAG
114/115/116



subunit

TCCTACAGCAA
UCCUACAGC
AUGAGCCAG



 


0097
novel sequence
ise2c.pk010.h3
AAACAGTGCGTC
ACAGUGCGUC
AUAUAUUACG
117/118/119





GTAATATATTC
GUAAUAUAU
ACGCACUGU



 


0098
novel sequence
ise2c.pk010.h3
AAGGCACATGGT
GGCACAUGGU
CAGUGAAGGA
120/121/122





CCTTCACTGAT
CCUUCACUG
CCAUGUGCC



 


0099
novel sequence
ise2c.pk010.h3
AACACCATGACC
CACCAUGACC
GUACACGAGG
123/124/125





CTCGTGTACAA
CUCGUGUAC
GUCAUGGUG



 


0100
Larval cuticle
ise2c.pk007.k24
AACGAGGCCGGA
CGAGGCCGGA
CUUAAGAGAU
457/458/459



protein LCP-17

TCTCTTAAGCA
UCUCUUAAG
CCGGCCUCG



 


0101
Larval cuticle
ise2c.pk007.k24
AACTTCACACAT
CUUCACACAU
UGUCUAGUUA
460/461/462



protein LCP-17

AACTAGACAAA
AACUAGACA
UGUGUGAAG



 


0102
Larval cuticle
ise2c.pk007.k24
AATGCGTGGCGA
UUAGAAAUUA
CUGGGCUUAU
463/464/465



protein LCP-17

TTTCAAACTTA
UAAGCCCAG
AAUUUCUAA



 


0103
transcriptional
ise2c.pk011.a10
AAAAAACACAGA
AAAACACAGA
UGAACGUGGU
126/127/128



repressor

CCACGTTCACA
CCACGUUCA
CUGUGUUUU



 


0104
transcriptional
ise2c.pk011.a10
AATCGATGGTGG
UCGAUGGUGG
CGAAUAACAC
129/130/131



repressor

TGTTATTCGCT
UGUUAUUCG
CACCAUCGA



 


0105
novel sequence
ise2c.pk011.h12
AAAGAAAATGCT
AGAAAAUGCU
GUAACGCGUA
132/133/134





ACGCGTTACGA
ACGCGUUAC
GCAUUUUCU



 


0106
novel sequence
ise2c.pk011.h12
AACCCTTGGACA
CCCUUGGACA
UUCCAGUAGU
135/136/137





CTACTGGAAGA
CUACUGGAA
GUCCAAGGG



 


0107
novel sequence
ise2c.pk011.h12
AAGGATCCTATG
GGAUCCUAUG
CCUGGUACAC
138/139/140





TGTACCAGGTT
UGUACCAGG
AUAGGAUCC



 


0108
translation
ise2c.pk001.d22
AAACTCGGCACA
ACUCGGCACA
AUUGUGUUGU
141/142/143



initiation 

CAACACAATGG
CAACACAAU
GUGCCGAGU




factor 5A







 


0109
translation
ise2c.pk001.d22
AATACGAAGATA
UACGAAGAUA
AAGGGCAGAU
144/145/146



initiation 

TCTGCCCTTCC
UCUGCCCUU
AUCUUCGUA




factor 5A







 


0110
translation
ise2c.pk001.d22
AATCAACAGCTC
UCAACAGCUC
UUUAUGUAAG
147/148/149



initiation 

TTACATAAATG
UUACAUAAA
AGCUGUUGA




factor 5A







 


0111
eukaryotic
ise2c.pk001.d9
AAAGAAGATCAG
AGAAGAUCAG
GCCAAUCUUC
150/151/152



initiation 

AAGATTGGCCG
AAGAUUGGC
UGAUCUUCU




factor eIF-4A







 


0112
eukaryotic
ise2c.pk001.d9
AAAAGCCGTCTG
AAGCCGUCUG
GUUGGAUAGC
153/154/155



initiation 

CTATCCAACAA
CUAUCCAAC
AGACGGCUU




factor eIF-4A







 


0113
eukaryotic
ise2c.pk001.d9
AATGCTAAATGC
UGCUAAAUGC
GCAAGCAUGG
156/157/158



initiation 

CATGCTTGCAT
CAUGCUUGC
CAUUUAGCA




factor eIF-4A







 


0114
Eukaryotic
ise2c.pk001.i23
AAGATCAGAAGA
GAUCAGAAGA
UCCGGCCAAU
159/160/161



initiation 

TTGGCCGGAAG
UUGGCCGGA
CUUCUGAUC




factor 4A







 


0115
Eukaryotic
ise2c.pk001.i23
AATTCTTCAGCA
UUCUUCAGCA
GUAUCGAUUU
162/163/164



initiation 

AATCGATACCA
AAUCGAUAC
GCUGAAGAA




factor 4A







 


0116
Eukaryotic
ise2c.pk001.i23
AAATGCTGTCAA
AUGCUGUCAA
UAAAUCCUCU
165/166/167



initiation 

GAGGATTTAAA
GAGGAUUUA
UGACAGCAU




factor 4A







 


0117
RNA
ise2c.pk001.l24
AAGCTCGAGACT
GCUCGAGACU
UCAAGAGCAA
168/169/170



polymerase

TGCTCTTGATG
UGCUCUUGA
GUCUCGAGC




sigma subunit








SigE







 


0118
RNA
ise2c.pk001.l24
AACTGTTAGCTC
CUGUUAGCUC
GCAGACCUUG
171/172/173



polymerase

AAGGTCTGCTA
AAGGUCUGC
AGCUAACAG




sigma subunit








SigE







 


0119
RNA
ise2c.pk001.l24
AAGACTTTCTAT
GACUUUCUAU
CAAAUUCUGA
174/175/176



polymerase

CAGAATTTGCG
CAGAAUUUG
UAGAAAGUC




sigma subunit








SigE







 


0120
translation
ise2c.pk005.b9
AAACTTAATCAT
ACUUAAUCAU
UCGUCGUCCA
177/178/179



initiation 

GGACGACGACA
GGACGACGA
UGAUUAAGU




factor 2, 








subunit 2 beta







 


0121
translation
ise2c.pk005.b9
AAAGAAGAAGAA
AGAAGAAGAA
CCCUUCUUCU
180/181/182



initiation 

GAAGAAGGGAG
GAAGAAGGG
UCUUCUUCU




factor 2, 








subunit 2 beta







 


0122
translation
ise2c.pk005.b9
AAGATCAAGAGA
GAUCAAGAGA
CCUCGACAUU
183/184/185



initiation 

ATGTCGAGGAT
AUGUCGAGG
CUCUUGAUC




factor 2, 








subunit 2 beta







 


0123
putative sar1
ise2c.pk002.m10
AAAATCGTCGGT
AAUCGUCGGU
GUCGCUAAAA
186/187/188



protein

TTTAGCGACGT
UUUAGCGAC
CCGACGAUU



 


0124
putative sar1
ise2c.pk002.m10
AACTGTCAATAG
CUGUCAAUAG
GCAUACUGCC
189/190/191



protein

GCAGTATGCGT
GCAGUAUGC
UAUUGACAG



 


0125
putative sar1
ise2c.pk002.m10
AACCTGTACCAA
CCUGUACCAA
AGUGGUCUGU
192/193/194



protein

CAGACCACTGG
CAGACCACU
UGGUACAGG



 


0126
elongation 
ise2c.pk001.c14
AACCAAAAATGG
CCAAAAAUGG
UUUCCUUGCC
195/196/197



factor 1-alpha

GCAAGGAAAAG
GCAAGGAAA
CAUUUUUGG



 


0127
elongation 
ise2c.pk001.c14
AACGTGGTATCA
CGUGGUAUCA
UAUCGAUGGU
198/199/200



factor 1-alpha

CCATCGATATT
CCAUCGAUA
GAUACCACG



 


0128
elongation 
ise2c.pk001.c14
AACAAAATGGAC
CAAAAUGGAC
CUCAGUGGAG
201/202/203



factor 1-alpha

TCCACTGAGCC
UCCACUGAG
UCCAUUUUG



 


0129
elongation
ise2c.pk001.d16
AATCCGTGACTA
UCCGUGACUA
AUUUUUGGUU
204/205/206



factor-1alpha F2 

ACCAAAAATGG
ACCAAAAAU
AGUCACGGA



 


0130
elongation
ise2c.pk001.d16
AACATTGTCGTC
CAUUGUCGUC
GUGUCCAAUG
207/208/209



factor-1alpha F3 

ATTGGACACGT
AUUGGACAC
ACGACAAUG



 


0131
Oligosaccharyl
ise2c.pk005.h3
AATTTGTGAGAC
UUUGUGAGAC
CGGCCACCAG
421/422/423



transferase 48

TGGTGGCCGAA
UGGUGGCCG
UCUCACAAA




kDa subunit







 


0132
Oligosaccharyl
ise2c.pk005.h3
AATCTGATTGTA
UCUGAUUGUA
GGGGGCGAAU
424/425/426



transferase 48

TTCGCCCCCTC
UUCGCCCCC
ACAAUCAGA




kDa subunit







 


0133
Oligosaccharyl
ise2c.pk005.h3
AACACTCTAGTT
CACUCUAGUU
AAUAGGCAGA
427/428/429



transferase 48

CTGCCTATTCT
CUGCCUAUU
ACUAGAGUG




kDa subunit







 


0134
Myosin
ise2c.pk001.d21
AACACACATCAC
CACACAUCAC
UCCGCCAUUG
430/431/432



regulatory light

AATGGCGGATA
AAUGGCGGA
UGAUGUGUG




chain







 


0135
Myosin
ise2c.pk001.d21
AAGGATGGCATC
GGAUGGCAUC
CUUGCCGAUG
433/434/435



regulatory light

ATCGGCAAGAA
AUCGGCAAG
AUGCCAUCC




chain







 


0136
Myosin
ise2c.pk001.d21
AAAGGCTTCATC
AGGCUUCAUC
CGCGGUGUCG
436/437/438



regulatory light

GACACCGCGAA
GACACCGCG
AUGAAGCCU




chain







 


0137
novel sequence
ise2c.pk001.j9
AAACTCCAATTA
ACUCCAAUUA
AGUAGGUUAU
210/211/212





TAACCTACTAG
UAACCUACU
AAUUGGAGU



 


0138
novel sequence
ise2c.pk001.j9
AAGTACAAGGAT
GUACAAGGAU
GCCGAUCAGA
213/214/215





CTGATCGGCAA
CUGAUCGGC
UCCUUGUAC



 


0139
novel sequence
ise2c.pk001.j9
AAGACTTTCTTC
GACUUUCUUC
GGGCCACAUG
216/217/218





ATGTGGCCCAT
AUGUGGCCC
AAGAAAGUC



 


0140
novel sequence
ise2c.pk002.f12 
AAACAAAGTATC
ACAAAGUAUC
GGUGUAGGCG
439/440/441





GCCTACACCGC
GCCUACACC
AUACUUUGU



 


0141
novel sequence
ise2c.pk002.f12 
AATAGCGTCGAT
UAGCGUCGAU
UCGUUGAAGA
442/443/444





CTTCAACGACT
CUUCAACGA
UCGACGCUA



 


0142
potassium
ise2c.pk001.b14 
AACTCATAGAGC
CUCAUAGAGC
ACACAUCAAG
219/220/221



coupled amino

TTGATGTGTGG
UUGAUGUGU
CUCUAUGAG




acid transporter







 


0143
potassium
ise2c.pk001.b14
AAGATGTGGATG
GAUGUGGAUG
CAGUGACGUC
222/223/224



coupled amino

ACGTCACTGGT
ACGUCACUG
AUCCACAUC




acid transporter 







 


0144
potassium
ise2c.pk001.b14 
AACCTTCCTGAT
CCUUCCUGAU
CAGAAGAGAA
225/226/227



coupled amino

TCTCTTCTGTG
UCUCUUCUG
UCAGGAAGG




acid transporter







 


0145
inwardly
ise2c.pk003.f2
AACAGTGCTTGT
CAGUGCUUGU
UCACUUAUCA
228/229/230



rectifying K+

GATAAGTGAAC
GAUAAGUGA
CAAGCACUG




channel protein 







 


0146
inwardly
ise2c.pk003.f2
AAGTTAATGGTG
GUUAAUGGUG
GAGGGCAGUC
231/232/233



rectifying K+

ACTGCCCTCGA
ACUGCCCUC
ACCAUUAAC




channel protein 







 


0147
inwardly
ise2c.pk003.f2
AATAAAGCGATG
UAAAGCGAUG
CUAUGGGGUC
234/235/236



rectifying K+

ACCCCATAGGA
ACCCCAUAG
AUCGCUUUA




channel protein 







 


0148
potassium
ise2c.pk005.l20 
AAACGGTACTGC
ACGGUACUGC
CUUUUUGCUG
237/238/239



coupled amino

AGCAAAAAGAC
AGCAAAAAG
CAGUACCGU




acid transporter







 


0149
potassium
ise2c.pk005.l20 
AAGCTGCATACT
GCUGCAUACU
GAGCCAAGAA
240/241/242



coupled amino

TCTTGGCTCTC
UCUUGGCUC
GUAUGCAGC




acid transporter







 


0150
potassium
ise2c.pk005.l20
AAATGTTTACAG
AUGUUUACAG
AUCGCGUCUC
243/244/245



coupled amino

AGACGCGATGA
AGACGCGAU
UGUAAACAU




acid transporter 







 


0151
alpha tubulin
ise2c.pk001.d1
AACGTCGATCTT
CGUCGAUCUU
GAACUCGGUA
246/247/248





ACCGAGTTCCA
ACCGAGUUC
AGAUCGACG



 


0152
tubulin alpha
ise2c.pk001.k6
AATTCAAAATGC
UUCAAAAUGC
UGCACUCACG
249/250/251



chain

GTGAGTGCATC
GUGAGUGCA
CAUUUUGAA



 


0153
tubulin alpha
ise2c.pk001.k6
AAATCGTAGACC
AUCGUAGACC
CGAGGACUAG
252/253/254



chain

TAGTCCTCGAC
UAGUCCUCG
GUCUACGAU



 


0154
tubulin alpha
ise2c.pk001.l2
AAACTCAATTCA
ACUCAAUUCA
CACGCAUUUU
255/256/257



chain

AAATGCGTGAG
AAAUGCGUG
GAAUUGAGU



 


0155
tubulin alpha
ise2c.pk001.l2
AACTTATCACTG
CUUAUCACUG
CUUCCUUACC
258/259/260



chain

GTAAGGAAGAT
GUAAGGAAG
AGUGAUAAG



 


0156
ubiquitin kinase
ise2c.pk002.b4
AAGAGTTACGAA
GAGUUACGAA
UGGUGACGGU
261/262/263





CCGTCACCATA
CCGUCACCA
UCGUAACUC



 


0157
ubiquitin kinase
ise2c.pk002.b4
AAACTTAGTCCG
ACUUAGUCCG
UUCAUUAUCC
264/265/266





GATAATGAACC
GAUAAUGAA
GGACUAAGU



 


0158
ubiquitin kinase
ise2c.pk002.b4
AAGGCGATGTAC
GGCGAUGUAC
CAGGUUCUCG
267/268/269





GAGAACCTGTT
GAGAACCUG
UACAUCGCC



 


0159
nuclear
ise2c.pk001.j16 
AACGACAAGATG
CGACAAGAUG
CUCCUUCAGC
270/271/272



ribonucleoprotein

CTGAAGGAGAC
CUGAAGGAG
AUCUUGUCG




200 kDa








helicase







 


0160
Sqd protein
ise2c.pk006.h23 
AAGATAAAGGTC
GAUAAAGGUC
UCCACACGCG
273/274/275



homologue

GCGTGTGGACC
GCGUGUGGA
ACCUUUAUC




(RNA binding)







 


0161
Sqd protein
ise2c.pk006.h23 
AATGTCAAGACT
UGUCAAGACU
GUUUGGAUCA
276/277/278



homologue

GATCCAAACAC
GAUCCAAAC
GUCUUGACA




(RNA binding)







 


0162
Sqd protein
ise2c.pk006.h23 
AACATTCGAGTC
CAUUCGAGUC
ACCUGUUCAG
279/280/281



homologue

TGAACAGGTGG
UGAACAGGU
ACUCGAAUG




(RNA binding)







 


0163
pre-mRNA-
ise2c.pk006.m8
AACATAAGTCAT
CAUAAGUCAU
UUAUUGGCCA
282/283/284



binding protein 

GGCCAATAACG
GGCCAAUAA
UGACUUAUG



 


0164
pre-mRNA-
ise2c.pk006.m8
AAGAACATAATA
GAACAUAAUA
CUUCGGCACU
285/286/287



binding protein 

GTGCCGAAGCC
GUGCCGAAG
AUUAUGUUC



 


0165
pre-mRNA-
ise2c.pk006.m8
AAACACTGGCAG
ACACUGGCAG
CCUCUUGAUC
288/289/290



binding protein 

ATCAAGAGGAT
AUCAAGAGG
UGCCAGUGU



 


0166
pre-mRNA-
ise2c.pk006.m8
AAGATCTTTGTT
GAUCUUUGUU
AAGACCACCA
291/292/293



binding protein 

GGTGGTCTTAG
GGUGGUCUU
ACAAAGAUC



 


0167
pre-mRNA-
ise2c.pk006.m8
AACAGGTGGTCA
CAGGUGGUCA
GCAGCUCAUU
294/295/296



binding protein 

ATGAGCTGCTG
AUGAGCUGC
GACCACCUG



 


0168
chymotrypsin-
ise2c.pk001.a23 
AAGTTGGCTCTG
GUUGGCUCUG
CAAGAGUGUC
297/298/299



like; protease

ACACTCTTGGC
ACACUCUUG
AGAGCCAAC



 


0169
chymotrypsin-
ise2c.pk001.a23 
AAATCCGCAAAG
AUCCGCAAAG
CCUCCUCAGC
300/301/302



like; protease

CTGAGGAGGCA
CUGAGGAGG
UUUGCGGAU



 


0170
chymotrypsin-
ise2c.pk001.a23
AACCGTGTTCTT
CCGUGUUCUU
AGCAGCAGUA
303/304/305



like; protease

ACTGCTGCTCA
ACUGCUGCU
AGAACACGG



 


0171
chymotrypsin-
ise2c.pk001.a23
AACGTTGTTATG
CGUUGUUAUG
GCUUCCAUGC
306/307/308



like; protease

CATGGAAGCTG
CAUGGAAGC
AUAACAACG



 


0172
chymotrypsin-
ise2c.pk001.a23
AAAACTTCGCCG
AACUUCGCCG
CGUUUUCACC
309/310/311



like; protease

GTGAAAACGCC
GUGAAAACG
GGCGAAGUU



 


0173
chymotrypsinogen;
ise2c.pk001.a7 
AAATGAAACTGT
AUGAAACUGU
CUGCGAGGAA
312/313/314



protease

TCCTCGCAGTC
UCCUCGCAG
CAGUUUCAU



 


0174
chymotrypsinogen;
ise2c.pk001.a7 
AAGAAGGCTGAG
GAAGGCUGAG
GGUUUCUUCC
315/316/317



protease

GAAGAAACCAG
GAAGAAACC
UCAGCCUUC



 


0175
chymotrypsinogen;
ise2c.pk001.a7 
AACTCTTTCACC
CUCUUUCACC
AAGUACGACG
318/319/320



protease

GTCGTACTTGG
GUCGUACUU
GUGAAAGAG



 


0176
chymotrypsinogen;
ise2c.pk001.a7 
AACGACATTGCT
CGACAUUGCU
GCGGAGGACA
321/322/323



protease

GTCCTCCGCAT
GUCCUCCGC
GCAAUGUCG



 


0177
chymotrypsinogen;
ise2c.pk001.a7 
AACATCGGTACT
CAUCGGUACU
ACGUUGGGUA
324/325/326



protease

ACCCAACGTGT
ACCCAACGU
GUACCGAUG



 


0178
actin-
ise2c.pk004.c4
AAGACTCAGTAC
GACUCAGUAC
CGAUAAUACG
327/328/329



depolymerizing 

GTATTATCGCG
GUAUUAUCG
UACUGAGUC



 


0179
actin-
ise2c.pk004.c4 
AAGTTGCCATCT
GUUGCCAUCU
GCAAAAGCAA
330/331/332



depolymerizing

TGCTTTTGCAA
UGCUUUUGC
GAUGGCAAC



 


0180
actin-
ise2c.pk004.c4 
AATCAGTTTGAC
UCAGUUUGAC
AGCGCAUGCG
333/334/335



depolymerizing

GCATGCGCTCA
GCAUGCGCU
UCAAACUGA



 


0181
actin-
ise2c.pk004.c4 
AATGGCGTCTGG
UGGCGUCUGG
ACUGUCACAC
336/337/338



depolymerizing

TGTGACAGTTT
UGUGACAGU
CAGACGCCA



 


0182
actin-
ise2c.pk004.c4 
AACGCGGAATAC
CGCGGAAUAC
GAACUGAUCG
339/340/341



depolymerizing

GATCAGTTCCT
GAUCAGUUC
UAUUCCGCG



 


0183
actin
ise2c.pk004.l4 
AAGACTCAGTAC
GACUCAGUAC
CGAUAAUACG
342/343/344



depolymerizing

GTATTATCGCG
GUAUUAUCG
UACUGAGUC




factor







 


0184
actin
ise2c.pk004.l4 
AAGTTGCCATCT
GUUGCCAUCU
GCAAAAGCAA
345/346/347



depolymerizing

TGCTTTTGCAA
UGCUUUUGC
GAUGGCAAC




factor







 


0185
actin
ise2c.pk004.l4 
AATCAGTTTGAC
UCAGUUUGAC
AGCGCAUGCG
348/349/350



depolymerizing

GCATGCGCTCA
GCAUGCGCU
UCAAACUGA




factor







 


0186
actin
ise2c.pk004.l4 
AAAAATGGCGTC
AAAUGGCGUC
GUCACACCAG
351/352/353



depolymerizing

TGGTGTGACAG
UGGUGUGAC
ACGCCAUUU




factor







 


0187
actin
ise2c.pk004.l4 
AATACGATCAGT
UACGAUCAGU
CCUCAAGGAA
354/355/356



depolymerizing

TCCTTGAGGAT
UCCUUGAGG
CUGAUCGUA




factor







0188
dismutase;
ise2c.pk004.n19
AATAATTTGTGT
UAAUUUGUGU
UAGGGUAUGA
357/358/359



superoxide

CATACCCTACG
CAUACCCUA
CACAAAUUA



 


0189
dismutase;
ise2c.pk004.n19
AAGTCAGTGACT
GUCAGUGACU
AAUUUGACCA
360/361/362



superoxide

GGTCAAATTCA
GGUCAAAUU
GUCACUGAC



 


0190
dismutase;
ise2c.pk004.n19
AATTAGGAGATG
UUAGGAGAUG
CAGGUGCAAC
363/364/365



superoxide

TTGCACCTGGT
UUGCACCUG
AUCUCCUAA



 


0191
dismutase;
ise2c.pk004.n19
AACAAACGGATG
CAAACGGAUG
AACCUAGGUC
366/367/368



superoxide

ACCTAGGTTTG
ACCUAGGUU
AUCCGUUUG



 


0192
dismutase;
ise2c.pk004.n19
AATGCTGCTAGC
UGCUGCUAGC
AGACAUAGAG
369/370/371



superoxide

TCTATGTCTCC
UCUAUGUCU
CUAGCAGCA



 


0193
superoxide
ise2c.pk005.f21
AATTTGTGTCAT
UUUGUGUCAU
GCGUAGGGUA
372/373/374



dismutase

ACCCTACGCAA
ACCCUACGC
UGACACAAA



 


0194
superoxide
ise2c.pk005.f21
AAAAGGGCTTCA
AAGGGCUUCA
CCUUCGACAU
375/376/377



dismutase

TGTCGAAGGAG
UGUCGAAGG
GAAGCCCUU



 


0195
superoxide
ise2c.pk005.f21
AATTAGGAGATG
UUAGGAGAUG
CAGGUGCAAC
378/379/380



dismutase

TTGCACCTGGT
UUGCACCUG
AUCUCCUAA



 


0196
adenylate kinase
ise2c.pk010.h5 
AAAGGTCAGTAA
AGGUCAGUAA
CACUCAUAGU
381/382/383



isozyme 3

CTATGAGTGGT
CUAUGAGUG
UACUGACCU



 


0197
adenylate kinase
ise2c.pk010.h5 
AAGAAATATGCT
GAAAUAUGCU
GUGUGCCACA
384/385/386



isozyme 3

GTGGCACACGT
GUGGCACAC
GCAUAUUUC



 


0198
adenylate kinase
ise2c.pk010.h5 
AACTTGTACCTG
CUUGUACCUG
UGACAUCAUC
387/388/389



isozyme 3

ATGATGTCATG
AUGAUGUCA
AGGUACAAG



 


0199
adenylate kinase
ise2c.pk010.h5 
AACATTGGCTTC
CAUUGGCUUC
AGGAGUGUUG
390/391/392



isozyme 3

AACACTCCTAA
AACACUCCU
AAGCCAAUG



 


0200
adenylate kinase
ise2c.pk010.h5 
AAGCGGCTGGAG
GCGGCUGGAG
CUCAUAGAUC
393/394/395



isozyme 3

ATCTATGAGAG
AUCUAUGAG
UCCAGCCGC



 


0201
ecdysone
ise2c.pk001.c18
AACCCTCCCGAG
CCCUCCCGAG
AAUGCUCUCC
396/397/398



oxidase

GAGAGCATTAT
GAGAGCAUU
UCGGGAGGG



 


0202
ecdysone
ise2c.pk001.c18
AACGCCCTACGA
CGCCCUACGA
AAGUUCCAGU
399/400/401



oxidase

CTGGAACTTCA
CUGGAACUU
CGUAGGGCG



 


0203
ecdysone
ise2c.pk001.c18
AACTGGACCAAC
CUGGACCAAC
GUCCAGCACG
402/403/404



oxidase

GTGCTGGACTA
GUGCUGGAC
UUGGUCCAG



 


0204
innexin-2
ise2c.pk004.p1 
AAAACATGGGAA
AACAUGGGAA
GCGACCUCCU
405/406/407





GGAGGTCGCAT
GGAGGUCGC
UCCCAUGUU



 


0205
innexin-2
ise2c.pk004.p1 
AAGTTGCTTGTG
GUUGCUUGUG
GAAGUAGUCC
408/409/410





GACTACTTCCA
GACUACUUC
ACAAGCAAC



 


0206
innexin-2
ise2c.pk004.p1 
AACGTCGTGGGC
CGUCGUGGGC
GAAGAUCUGG
411/412/413





CAGATCTTCTT
CAGAUCUUC
CCCACGACG



 


0207
innexin-2
ise2c.pk004.p1 
AATACGGTCCTT
UACGGUCCUU
CGGUUCCUGA
415/416/417





CAGGAACCGTG
CAGGAACCG
AGGACCGUA



 


0208
innexin-2
ise2c.pk004.p1
AATTTCGCTGAT
UUUCGCUGAU
AUGCGGUAAA
418/419/420





TTACCGCATGG
UUACCGCAU
UCAGCGAAA





(Note: the sense RNA primer sequence and the antisense RNA primer sequences shown in table 2 were generated with 2 thymine residues at the 3′ end.)






Droplet Feeding Assay for Evaluation of 21mer dsRNA Insecticidal Properties Against the Fall Armyworm Spodoptera frugiperda 

10 nanoMole quantities of 21mer desalted primers were purchased from Proligo (Sigma Aldrich, St. Louis, Miss.). The lyophilized sample is solubilized in nuclease free water at a 100 uMolar concentration. The stock solution was then diluted in 20% sucrose containing blue McCormick food coloring. O.5 ul droplets of this solution were dispensed in a circle in a parafilm-lined 65 mm petridish. Sucrose blanks were used as controls. Between 20 and 30 neonate fall armyworms were then added to the middle of the droplet circle and the petri dish sealed with parafilm. After two hours, the neonates with blue digestive tracts were removed and placed on standard multispecies lepidopteran insect diet. Insects were evaluated at 48, 72, and 96 hours post challenge for mortality and growth inhibition.
Serial dilution assays starting with a high dose of 20 uM and including 10, 5, 2.5, 1.25, 0.6, and 0 uMolar concentrations were also performed in this manner.











TABLE 3









Rep 1
Rep 2

















72 H

72 H



Primer

Insects
ave.
Insects
ave.
Combined


#
Target gene
treated
weight
treated
weight
ave.
















75
juvenile hormone diol kinase
9
10
11
11
11


83
juvenile hormone diol kinase
15
14
14
12
13


91
V-ATPase A subunit
14
14
11
15
14


99
conserved hypothetical
16
15
19
16
16



protein


107
novel sequence (cuticular
16
14
13
15
14



protein?)


115
Eukaryotic initiation factor 4A
16
9
18
14
12


123
putative sar1 protein
16
15
16
17
16


131
Oligosaccharyl transferase
17
13
14
15
14



48 kDa subunit


139
myosin protein
13
15
16
18
17


147
inwardly rectifying K+
15
11
15
16
14



channel protein


155
alpha tubulin chain
15
10
16
19
15



sucrose control
14
17
15
18
18









Sucrose Droplet Feeding Assay.

Neonate larvae were fed 25 uMolar dsRNAs. Treated insects were weighed en masse at 72 hours and compared to sucrose controls. 2 replicates of the experiment were averaged.
Injection Feeding Assay for Evaluation of 21mer dsRNA Insecticidal Properties Against the Fall Armyworm Spodoptera frugiperda 
Second instar fall armyworm were injected using a micromanipulator and microinjection needles pulled on a Sutter Instrument (Novato, Calif.) P-2000 horizontal needle puller. The needle was back loaded with dsRNA solution. Initial injection experiments employed a concentration of 2 ug/ul (see Table X). This rate produced high mortality across all primers tested. Subsequent assays were performed with lower concentrations. Blue McCormick food coloring was included in the dsRNA solution to better visualize the injection process. Prior to injection, the insects were affixed to a microscope slide using a glue stick (Office Depot, Delray Beach, Fla.). The injection needle was connected to a 20 ml hypodermic syringe via Teflon tubing. The injection needle was then mounted on a Leitz micromanipulator. The dsRNA solution was dispensed from the microinjection needle by pressing on the plunger of the 20 ml syringe. Injection volumes were variable but averaged approximately 250 nL (based on injection of approximately 20 insects injected from a 5 ul volume loaded into the needle). Following injection, insects were removed from the microscope slide with the aid of a moistened fine camelhair brush. The insects were then placed on multispecies diet and were evaluated for mortality at 24 and 48 hours. Water injections were used as controls. Silencer® Negative Control #1, 2, and 3 siRNA control primers from Ambion (Austin, Tex.) were also included as negative controls.











TABLE 4





Primer

No.




#
Target gene
injected
Alive
Dead



















75
juvenile hormone diol kinase
6
0
6


83
juvenile hormone diol kinase
6
0
6


91
V-ATPase A subunit
8
3
5


99
conserved hypothetical sequence
8
0
8


107
novel sequence (cuticular protein
8
0
8


115
Eukaryotic initiation factor 4A
8
0
8


123
putative sar1 protein
8
1
8


131
Oligosaccharyl transferase
8
2
8



48 kDa subunit


147
inwardly rectifying K+
8
1
7



channel protein



Water
8
7
1









Microinjection of dsRNAs [2 ug/ul].








TABLE 5







Microinjection of dsRNAs [0.7 ug/ul] into FAW neonate larvae










Rep 1
Rep 2














Primer #
Target gene
# Injected
24 H dead
48 H dead
# Injected
24 H dead
48 H dead

















75
juvenile hormone diol kinase 1
7
2
3
11
7
7


83
juvenile hormone diol kinase 2
11
10
10
12
6
5


91
V-ATPase A subunit
13
9
7
9
9
9


99
conserved hypothetical protein 1



9
2
2


107
novel sequence (cuticular protein?)



11
5
6


115
Eukaryotic initiation factor 4A



8
6
6


123
putative sar1 protein



13
9
9


131
Oligosaccharyl transferase 48 kDa subunit



8
6
4


139
myosin protein



10
4
4


147
inwardly rectifying K+ channel protein



14
8
9


155
alpha tubulin chain



11
9
9



Ambion control primer 1
6
0
0
11
0
0



Ambion control primer 2
11
0
0
8
0
0



Ambion control primer 3
12
1
1
9
1
1









Microinjection Assay using 0.7 ug/ul dose of dsRNA 21mers. Note; On some occasions, the mortality was lower at 48 hours than at 24 hours. This is due to moribund insects recovering at the later time point.

Topical Diet Assay for Evaluation of 21Mer dsRNA Insecticidal Properties Against the Fall Armyworm Spodoptera frugiperda 

The term “topical diet assay” refers to assays where artificial diets are pipetted into microtiter plates and the dsRNA solution is dispensed on the surface of the diet. In the dsRNA experiments, 100 ul of diet was dispensed per well. The surface of the well was then treated with 10 ul of a dsRNA solution of varying concentrations. The plates were then infested with 1 neonate fall armyworm per well and sealed with mylar. The mylar seal was punctured with a small insect pin to allow for air exchange. Plates were then stored in a growth chamber at 28 C and the assay was scored for stunting or mortality at 4 days. Table 6-12 represents several experiments using this method. Table 13 provides a summary of the data.
In topical assay #1, the primers that previously showed activity in injection assays were tested in a FAW topical diet assay. These results are shown in Table 6. A 50 uMolar solution (0.66 ug/ul) was used as the test concentration. 5 ul of this sample was loaded onto the top of 100 ul of diet producing a final concentration of 2.5 uMolar or 30 ppm. In addition to A1-A11 (A12 is a negative control), the other samples are those with no known human orthologs. The plate was infested with aprox. 5 neonates/well. The scoring period was 72 hours.
In topical assay #2, primers were tested in a FAW topical diet assay, and the results are shown in table 7. In this experiment, the 2.7 ug/ul stock was diluted to a starting concentration of 0.67 ug/ul. 2 fold serial dilution was carried out to produce stocks of 0.32 ug/ul and 0.16 ug/ul. 5 ul of these stocks were added to the 100 ul of diet producing final concentrations of 30, 15, and 8 ppm in diet. The scoring period was 72 hours.
In topical assay #3, primers were tested in a FAW topical diet assay, and the results are shown in table 8. In this experiment, the 2.7 ug/ul stock was diluted to a starting concentration of 0.67 ug/ul. 2 fold serial dilution was carried out to produce stocks of 0.32 ug/ul and 0.16 ug/ul. 5 ul of these stocks were added to the 100 ul of diet producing final concentrations of 30, 15, and 8 ppm in diet. This is a replicate of the previous experiment. The scoring period was 72 hours.
In topical assay #4, primers were tested in a FAW topical diet assay, and the results are shown in table 9. In this experiment, the 2.7 ug/ul stock was diluted to a starting concentration of 0.67 ug/ul. 2 fold serial dilution was carried out to produce stocks of 0.32 ug/ul and 0.16 ug/ul. 5 ul of these stocks were added to the 100 ul of diet producing final concentrations of 30, 15, and 8 ppm in diet. The scoring period was 72 hours.
A summary of the topical assay data shown in tables 6-9 appears in Table 10.
In topical assay #5, primers were tested in a FAW topical diet assay and the results are shown in Table 11. 50 ul of 0.16 ug/ul primers were mixed with 50 ul water and then serially diluted. 10 ul of the sample then added to the wells. Therefore the first concentration was 10 ul×0.08 ug/ul=0.8 ug total dsRNA/100 ul diet=8 ppm. This was 1⁄2 the rate of previous experiments (assays #1-4) where 5 ul of 0.32 showed activity. The scoring period was 72 hours. A score of “S” indicates clear stunting compared to untreated controls. A score of “ss” indicates live insects but exhibiting severe stunting defined as little or no growth beyond the neonate body size.
In topical assay #6, primers were tested in a FAW topical diet assay and the results are shown in Table 12. The first rate is 10 ul of the 0.16 ug/ul primer stock. From there, 50 ul of 0.16 ug/ul primer mixed with 50 ul water and then serially diluted. 10 ul of the sample was then added to the wells. Therefore first concentration was 10 ul×0.16 ug/ul=1.6 ug total dsRNA/100 ul diet=16 ppm. The scoring period was 72 hours.














TABLE 6












SEQ ID NO





Target


30
Target region/


Sample
seq id
gene id
sequence
forward
reverse
ppm
sense/antisense







0075
ise1c.pk002.m13 
Juvenile
AACATGGTATCC
CAUGGUAUCC
CCUGAAGUCG

51/52/53




hormone
GACTTCAGGAA
GACUUCAGG
GAUACCAUG






query







 


0083
ise1c.pk005.a15
Juvenile
AAGTTCGCGTTC
GUUCGCGUUC
UUCAAGAGUG
+
75/76/77




hormone
ACTCTTGAAGA
ACUCUUGAA
AACGCGAAC






query







 


0085
ise1c.pk006.d24 
Juvenile
AATCACGCTGAA
UCACGCUGAA
UACAGUGGUU
+
81/82/83




hormone
ACCACTGTATA
ACCACUGUA
UCAGCGUGA






query







 


0086
ise2c.pk009.i4
Juvenile
AAAATATGGCGC
AAUAUGGCGC
ACAAUAGGCG

84/85/86




hormone
GCCTATTGTTT
GCCUAUUGU
CGCCAUAUU






query







 


0088
ise2c.pk009.i5
Juvenile
AAGTCATCGTTC
GUCAUCGUUC
GUAGACUUGG
+
90/91/92




hormone
CAAGTCTACCT
CAAGUCUAC
AACGAUGAC






query







 


0089
ise2c.pk001.d19
vacuolar
AACCCCTTGAAT
CCCCUUGAAU
GACCUUAACA
+
93/94/95




query
GTTAAGGTCGG
GUUAAGGUC
UUCAAGGGG




 


0091
ise2c.pk001.d20
vacuolar
AACGTGTCCATG
CGUGUCCAUG
GUCAGCCAUC
+
 99/100/101




query
ATGGCTGACTC
AUGGCUGAC
AUGGACACG




 


0094
ise2c.pk001.f20
vacuolar
AACTCTGACGTC
CUCUGACGUC
GUAGAUGAUG
+
108/109/110




query
ATCATCTACGT
AUCAUCUAC
ACGUCAGAG




 


0095
ise2c.pk001.f21
vacuolar
AAGTGCTTGGGT
GUGCUUGGGU
GUCGGGGUUA
+
111/112/113




query
AACCCCGACAG
AACCCCGAC
CCCAAGCAC




 


0099
ise2c.pk010.h3
cadherin
AACACCATGACC
CACCAUGACC
GUACACGAGG

123/124/125




query
CTCGTGTACAA
CUCGUGUAC
GUCAUGGUG




 


0107
ise2c.pkOl1.h12
cuticle
AAGGATCCTATG
GGAUCCUAUG
CCUGGUACAC
+
138/139/140




protein
TGTACCAGGTT
UGUACCAGG
AUAGGAUCC




 


0115
ise2c.pk001.i23
Translation
AATTCTTCAGCA
UUCUUCAGCA
GUAUCGAUUU

162/163/164




initiation
AATCGATACCA
AAUCGAUAC
GCUGAAGAA






factor







 


0123
ise2c.pk002.m10
SAR1
AAAATCGTCGGT
AAUCGUCGGU
GUCGCUAAAA

186/187/188





TTTAGCGACGT
UUUAGCGAC
CCGACGAUU




 


0131
ise2c.pk005.h3 
phosphooligo-
AATTTGTGAGAC
UUUGUGAGAC
CGGCCACCAG

421/422/423




saccharide
TGGTGGCCGAA
UGGUGGCCG
UCUCACAAA






. . .







 


0139
ise2c.pk001.j9 
Myosin
AAGACTTTCTTC
GACUUUCUUC
GGGCCACAUG

216/217/218





ATGTGGCCCAT
AUGUGGCCC
AAGAAAGUC




 


0147
ise2c.pk003.f2 
Potassium
AATAAAGCGATG
UAAAGCGAUG
CUAUGGGGUC
+
234/235/236




inwardly
ACCCCATAGGA
ACCCCAUAG
AUCGCUUUA






rectifying









protein







 


0155
ise2c.pk001.l2 
Tubulin
AACTTATCACTG
CUUAUCACUG
CUUCCUUACC

258/259/260





GTAAGGAAGAT
GUAAGGAAG
AGUGAUAAG





(Note: the sense RNA primer sequence and the antisen RNA primer sequences shown in table 6 were generated with 2 thymine residues at the 3' end.)




















TABLE 7 












SEQ ID NO





Target


30
Target region/


Sample
seq id
gene id
sequence
forward
reverse
ppm
sense/antisense







0075
ise1c.pk002.m13
Juvenile
AACATGGTATCC
CAUGGUAUCC
CCUGAAGUCG

51/52/53




hormone
GACTTCAGGAA
GACUUCAGG
GAUACCAUG






query







 


0076


AAGGTCGCTGAC
GGUCGCUGAC
CUUGUUCUCG

54/55/56





GAGAACAAGGA
GAGAACAAG
UCAGCGACC




 


0077


AAGTGTCCTGGG
GUGUCCUGGG
GAACUCAAGC

57/58/59





CTTGAGTTCCA
CUUGAGUUC
CCAGGACAC




 


0078
ise1c.pk003.f7
Juvenile
AAGAAGAAGCTC
GAAGAAGCUC
CACGUGGAGG

60/61/62




hormone
CTCCACGTGTT
CUCCACGUG
AGCUUCUUC






query







 


0079


AAGGTCGCTGAC
GGUCGCUGAC
CUUGUUCUCG

63/64/65





GAGAACAAGGA
GAGAACAAG
UCAGCGACC




 


0080


AATGTCCTGGGG
UGUCCUGGGG
GAAACUCAGC

66/67/68





CTGAGTTTCAA
CUGAGUUUC
CCCAGGACA




 


0081
ise1c.pk005.a15
Juvenile
AAGAATAAGCTC
GAAUAAGCUC
CACGUGGAGG

69/70/71




hormone
CTCCACGTGTT
CUCCACGUG
AGCUUAUUC






query







 


0082


AATTTGTCGAGG
UUUGUCGAGG
AUAGGGUCUC

72/73/74





AGACCCTATTG
AGACCCUAU
CUCGACAAA




 


0083


AAGTTCGCGTTC
GUUCGCGUUC
UUCAAGAGUG
+
75/76/77





ACTCTTGAAGA
ACUCUUGAA
AACGCGAAC




 


0084
ise1c.pk006.d24 
Juvenile
AACTGCCCCTTA
CUGCCCCUUA
AGAUGAGGUU

78/79/80




hormone
ACCTCATCTAT
ACCUCAUCU
AAGGGGCAG






query







 


0085


AATCACGCTGAA
UCACGCUGAA
UACAGUGGUU

81/82/83





ACCACTGTATA
ACCACUGUA
UCAGCGUGA




 


0086
ise2c.pk009.i4 
Juvenile
AAAATATGGCGC
AAUAUGGCGC
ACAAUAGGCG

84/85/86




hormone
GCCTATTGTTT
GCCUAUUGU
CGCCAUAUU






query







 


0087


AACGTTCTCGGT
CGUUCUCGGU
CAGUGAAAGA

87/88/89





CTTTCACTGCT
CUUUCACUG
CCGAGAACG




 


0088


AAGTCATCGTTC
GUCAUCGUUC
GUAGACUUGG

90/91/92





CAAGTCTACCT
CAAGUCUAC
AACGAUGAC




 


0089
ise2c.pk001.d19 
vacuolar
AACCCCTTGAAT
CCCCUUGAAU
GACCUUAACA

93/94/95




query
GTTAAGGTCGG
GUUAAGGUC
UUCAAGGGG




 


0090


AAGTACACCATG
GUACACCAUG
UACUUGCAAC

96/97/98





TTGCAAGTATG
UUGCAAGUA
AUGGUGUAC




 


0091


AACGTGTCCATG
CGUGUCCAUG
GUCAGCCAUC

 99/100/101





ATGGCTGACTC
AUGGCUGAC
AUGGACACG




 


0092
ise2c.pk001.e14 
vacuolar
AAACCTACAAAA
ACCUACAAAA
UUUCGGCCAU

102/103/104




query
TGGCCGAAAAC
UGGCCGAAA
UUUGUAGGU




 


0093


AATCTACGGACC
UCUACGGACC
CCAAAGAAGG

105/106/107





CTTCTTTGGAG
CUUCUUUGG
GUCCGUAGA




 


0094
ise2c.pk001.f20 
vacuolar
AACTCTGACGTC
CUCUGACGUC
GUAGAUGAUG

108/109/110




query
ATCATCTACGT
AUCAUCUAC
ACGUCAGAG




 


0095


AAGTGCTTGGGT
GUGCUUGGGU
GUCGGGGUUA

111/112/113





AACCCCGACAG
AACCCCGAC
CCCAAGCAC




 


0096


AACTGGCTCATC
CUGGCUCAUC
GCUGUAGGAG

114/115/116





TCCTACAGCAA
UCCUACAGC
AUGAGCCAG




 


0097
ise2c.pk010.h3 
cadherin
AAACAGTGCGTC
ACAGUGCGUC
AUAUAUUACG

117/118/119




query
GTAATATATTC
GUAAUAUAU
ACGCACUGU




 


0098


AAGGCACATGGT
GGCACAUGGU
CAGUGAAGGA
+
120/121/122





CCTTCACTGAT
CCUUCACUG
CCAUGUGCC




 


0099


AACACCATGACC
CACCAUGACC
GUACACGAGG

123/124/125





CTCGTGTACAA
CUCGUGUAC
GUCAUGGUG




 


0100
ise2c.pk007.k24 
cuticle
AACGAGGCCGGA
CGAGGCCGGA
CUUAAGAGAU

457/458/459




protein
TCTCTTAAGCA
UCUCUUAAG
CCGGCCUCG




 


0101


AACTTCACACAT
CUUCACACAU
UGUCUAGUUA

460/461/462





AACTAGACAAA
AACUAGACA
UGUGUGAAG




 


0102


AATGCGTGGCGA
UUAGAAAUUA
CUGGGCUUAU

463/464/465





TTTCAAACTTA
UAAGCCCAG
AAUUUCUAA




 


0103
ise2c.pk011.a10 
cuticle
AAAAAACACAGA
AAAACACAGA
UGAACGUGGU
+
126/127/128




protein
CCACGTTCACA
CCACGUUCA
CUGUGUUUU




 


0104


AATCGATGGTGG
UCGAUGGUGG
CGAAUAACAC
+
129/130/131





TGTTATTCGCT
UGUUAUUCG
CACCAUCGA




 


0105
ise2c.pk011.h12 
cuticle
AAAGAAAATGCT
AGAAAAUGCU
GUAACGCGUA

132/133/134




protein
ACGCGTTACGA
ACGCGUUAC
GCAUUUUCU




 


0106


AACCCTTGGACA
CCCUUGGACA
UUCCAGUAGU
+
135/136/137





CTACTGGAAGA
CUACUGGAA
GUCCAAGGG




 


0107


AAGGATCCTATG
GGAUCCUAUG
CCUGGUACAC

138/139/140





TGTACCAGGTT
UGUACCAGG
AUAGGAUCC




 


0108
ise2c.pk001.d22 
translation 
AAACTCGGCACA
ACUCGGCACA
AUUGUGUUGU

141/142/143




initiation
CAACACAATGG
CAACACAAU
GUGCCGAGU






factor







 


0109


AATACGAAGATA
UACGAAGAUA
AAGGGCAGAU
+
144/145/146





TCTGCCCTTCC
UCUGCCCUU
AUCUUCGUA




 


0110


AATCAACAGCTC
UCAACAGCUC
UUUAUGUAAG

147/148/149





TTACATAAATG
UUACAUAAA
AGCUGUUGA




 


0111
ise2c.pk001.d9 
translation 
AAAGAAGATCAG
AGAAGAUCAG
GCCAAUCUUC

150/151/152




initiation
AAGATTGGCCG
AAGAUUGGC
UGAUCUUCU






factor







 


0112


AAAAGCCGTCTG
AAGCCGUCUG
GUUGGAUAGC
+
153/154/155





CTATCCAACAA
CUAUCCAAC
AGACGGCUU




 


0113


AATGCTAAATGC
UGCUAAAUGC
GCAAGCAUGG

156/157/158





CATGCTTGCAT
CAUGCUUGC
CAUUUAGCA




 


0114
ise2c.pk001.i23 
translation 
AAGATCAGAAGA
GAUCAGAAGA
UCCGGCCAAU
+
159/160/161




initiation
TTGGCCGGAAG
UUGGCCGGA
CUUCUGAUC






factor







 


0115


AATTCTTCAGCA
UUCUUCAGCA
GUAUCGAUUU

162/163/164





AATCGATACCA
AAUCGAUAC
GCUGAAGAA




 


0116


AAATGCTGTCAA
AUGCUGUCAA
UAAAUCCUCU

165/166/167





GAGGATTTAAA
GAGGAUUUA
UGACAGCAU




 


0117
ise2c.pk001.l24 
translation 
AAGCTCGAGACT
GCUCGAGACU
UCAAGAGCAA

168/169/170




initiation
TGCTCTTGATG
UGCUCUUGA
GUCUCGAGC






factor







 


0118


AACTGTTAGCTC
CUGUUAGCUC
GCAGACCUUG

171/172/173





AAGGTCTGCTA
AAGGUCUGC
AGCUAACAG




 


0119


AAGACTTTCTAT
GACUUUCUAU
CAAAUUCUGA
+
174/175/176





CAGAATTTGCG
CAGAAUUUG
UAGAAAGUC




 


0120
ise2c.pk005.b9 
translation 
AAACTTAATCAT
ACUUAAUCAU
UCGUCGUCCA

177/178/179




initiation
GGACGACGACA
GGACGACGA
UGAUUAAGU






factor







 


0121


AAAGAAGAAGAA
AGAAGAAGAA
CCCUUCUUCU
+
180/181/182





GAAGAAGGGAG
GAAGAAGGG
UCUUCUUCU




 


0122


AAGATCAAGAGA
GAUCAAGAGA
CCUCGACAUU
+
183/184/185





ATGTCGAGGAT
AUGUCGAGG
CUCUUGAUC




 


0123
ise2c.pk002.m10
SARI
AAAATCGTCGGT
AAUCGUCGGU
GUCGCUAAAA

186/187/188





TTTAGCGACGT
UUUAGCGAC
CCGACGAUU




 


0124


AACTGTCAATAG
CUGUCAAUAG
GCAUACUGCC

189/190/191





GCAGTATGCGT
GCAGUAUGC
UAUUGACAG




 


0125


AACCTGTACCAA
CCUGUACCAA
AGUGGUCUGU
+
192/193/194





CAGACCACTGG
CAGACCACU
UGGUACAGG




 


0126
ise2c.pk001.c14 
Elongation
AACCAAAAATGG
CCAAAAAUGG
UUUCCUUGCC
+
195/196/197




factor
GCAAGGAAAAG
GCAAGGAAA
CAUUUUUGG




 


0127


AACGTGGTATCA
CGUGGUAUCA
UAUCGAUGGU
+
198/199/200





CCATCGATATT
CCAUCGAUA
GAUACCACG




 


0128


AACAAAATGGAC
CAAAAUGGAC
CUCAGUGGAG

201/202/203





TCCACTGAGCC
UCCACUGAG
UCCAUUUUG




 


0129
ise2c.pk001.d16
Elongation
AATCCGTGACTA
UCCGUGACUA
AUUUUUGGUU
+
204/205/206




factor
ACCAAAAATGG
ACCAAAAAU
AGUCACGGA




 


0130


AACATTGTCGTC
CAUUGUCGUC
GUGUCCAAUG

207/208/209





ATTGGACACGT
AUUGGACAC
ACGACAAUG




 


0131
ise2c.pk005.h3 
phosphooligo-
AATTTGTGAGAC
UUUGUGAGAC
CGGCCACCAG

421/422/423




saccharide 
TGGTGGCCGAA
UGGUGGCCG
UCUCACAAA






. . .







 


0132


AATCTGATTGTA
UCUGAUUGUA
GGGGGCGAAU

424/245/426





TTCGCCCCCTC
UUCGCCCCC
ACAAUCAGA




 


0133


AACACTCTAGTT
CACUCUAGUU
AAUAGGCAGA

427/228/429





CTGCCTATTCT
CUGCCUAUU
ACUAGAGUG




 


0134
ise2c.pk001.d21
myosin
AACACACATCAC
CACACAUCAC
UCCGCCAUUG

430/431/432





AATGGCGGATA
AAUGGCGGA
UGAUGUGUG




 


0135


AAGGATGGCATC
GGAUGGCAUC
CUUGCCGAUG

433/434/435





ATCGGCAAGAA
AUCGGCAAG
AUGCCAUCC




 


0136


AAAGGCTTCATC
AGGCUUCAUC
CGCGGUGUCG

436/437/438





GACACCGCGAA
GACACCGCG
AUGAAGCCU




 


0137
ise2c.pk001.j9
myosin
AAACTCCAATTA
ACUCCAAUUA
AGUAGGUUAU

210/211/212





TAACCTACTAG
UAACCUACU
AAUUGGAGU




 


0138


AAGTACAAGGAT
GUACAAGGAU
GCCGAUCAGA
+
213/214/215





CTGATCGGCAA
CUGAUCGGC
UCCUUGUAC




 


0139


AAGACTTTCTTC
GACUUUCUUC
GGGCCACAUG

216/217/218





ATGTGGCCCAT
AUGUGGCCC
AAGAAAGUC




 


0140
ise2c.pk002.f12
myosin
AAACAAAGTATC
ACAAAGUAUC
GGUGUAGGCG

439/440/441





GCCTACACCGC
GCCUACACC
AUACUUUGU




 


0141


AATAGCGTCGAT
UAGCGUCGAU
UCGUUGAAGA

442/443/444





CTTCAACGACT
CUUCAACGA
UCGACGCUA




 


0142
ise2c.pk001.b14  
potassium
AACTCATAGAGC
CUCAUAGAGC
ACACAUCAAG

219/220/221




channel
TTGATGTGTGG
UUGAUGUGU
CUCUAUGAG






amino acid









transporter







 


0143


AAGATGTGGATG
GAUGUGGAUG
CAGUGACGUC

221/223/224





ACGTCACTGGT
ACGUCACUG
AUCCACAUC




 


0144


AACCTTCCTGAT
CCUUCCUGAU
CAGAAGAGAA

225/226/227





TCTCTTCTGTG
UCUCUUCUG
UCAGGAAGG




 


0145
ise2c.pk003.f2
potassium
AACAGTGCTTGT
CAGUGCUUGU
UCACUUAUCA
+
228/229/230




inwardly
GATAAGTGAAC
GAUAAGUGA
CAAGCACUG






rectifier









. . .







 


0146


AAGTTAATGGTG
GUUAAUGGUG
GAGGGCAGUC
+
231/232/233





ACTGCCCTCGA
ACUGCCCUC
ACCAUUAAC




 


0147


AATAAAGCGATG
UAAAGCGAUG
CUAUGGGGUC
+
234/235/236





ACCCCATAGGA
ACCCCAUAG
AUCGCUUUA




 


0148
ise2c.pk005.l20
amino acid
AAACGGTACTGC
ACGGUACUGC
CUUUUUGCUG
+
237/238/239




transporter
AGCAAAAAGAC
AGCAAAAAG
CAGUACCGU




 


0149


AAGCTGCATACT
GCUGCAUACU
GAGCCAAGAA
+
240/241/242





TCTTGGCTCTC
UCUUGGCUC
GUAUGCAGC




 


0150


AAATGTTTACAG
AUGUUUACAG
AUCGCGUCUC

243/244/245





AGACGCGATGA
AGACGCGAU
UGUAAACAU




 


0151
ise2c.pk001.d1
tubulin
AACGTCGATCTT
CGUCGAUCUU
GAACUCGGUA
+
246/247/248





ACCGAGTTCCA
ACCGAGUUC
AGAUCGACG




 


0152
ise2c.pk001.k6
tubulin
AATTCAAAATGC
UUCAAAAUGC
UGCACUCACG

249/250/251





GTGAGTGCATC
GUGAGUGCA
CAUUUUGAA




 


0153


AAATCGTAGACC
AUCGUAGACC
CGAGGACUAG
+
252/253/254





TAGTCCTCGAC
UAGUCCUCG
GUCUACGAU




 


0154
ise2c.pk001.l2
tubulin
AAACTCAATTCA
ACUCAAUUCA
CACGCAUUUU
+
255/256/257





AAATGCGTGAG
AAAUGCGUG
GAAUUGAGU




 


0155


AACTTATCACTG
CUUAUCACUG
CUUCCUUACC

258/259/260





GTAAGGAAGAT
GUAAGGAAG
AGUGAUAAG




 


0156
ise2c.pk002.b4
ubiquitin
AAGAGTTACGAA
GAGUUACGAA
UGGUGACGGU

261/262/263





CCGTCACCATA
CCGUCACCA
UCGUAACUC




 


0157


AAACTTAGTCCG
ACUUAGUCCG
UUCAUUAUCC
+
264/265/266





GATAATGAACC
GAUAAUGAA
GGACUAAGU




 


0158


AAGGCGATGTAC
GGCGAUGUAC
CAGGUUCUCG
+
267/268/269





GAGAACCTGTT
GAGAACCUG
UACAUCGCC




 


0159
ise2c.pk001.j16
small
AACGACAAGATG
CGACAAGAUG
CUCCUUCAGC
+
270/271/272




nuclear
CTGAAGGAGAC
CUGAAGGAG
AUCUUGUCG






ribonucleo-









protein







 


0160
ise2c.pk006.h23
small
AAGATAAAGGTC
GAUAAAGGUC
UCCACACGCG

273/274/275




nuclear
GCGTGTGGACC
GCGUGUGGA
ACCUUUAUC






ribonucleo-









protein







 


0161


AATGTCAAGACT 
UGUCAAGACU
GUUUGGAUCA

276/277/278





GATCCAAACAC
GAUCCAAAC
GUCUUGACA




 


0162


AACATTCGAGTC
CAUUCGAGUC
ACCUGUUCAG
+
279/280/281





TGAACAGGTGG
UGAACAGGU
ACUCGAAUG





(Note: the sense RNA primer sequence and the antisense RNA primer sequences shown in table 7 were generated with 2 thymine residues at the 3′ end.)




















TABLE 8












SEQ ID NO





Target


30
Target region/


Sample
seq id
gene id
sequence
forward
reverse
ppm
sense/antisense







0075
ise1c.pk002.m13
Juvenile
AACATGGTATCC
CAUGGUAUCC
CCUGAAGUCG

51/52/53




hormone
GACTTCAGGAA
GACUUCAGG
GAUACCAUG






query







 


0076


AAGGTCGCTGAC
GGUCGCUGAC
CUUGUUCUCG

54/55/56





GAGAACAAGGA
GAGAACAAG
UCAGCGACC




 


0077


AAGTGTCCTGGG
GUGUCCUGGG
GAACUCAAGC

57/58/59





CTTGAGTTCCA
CUUGAGUUC
CCAGGACAC




 


0078
ise1c.pk003.f7
Juvenile
AAGAAGAAGCTC
GAAGAAGCUC
CACGUGGAGG

60/61/62




hormone
CTCCACGTGTT
CUCCACGUG
AGCUUCUUC






query







 


0079


AAGGTCGCTGAC
GGUCGCUGAC
CUUGUUCUCG

63/64/65





GAGAACAAGGA
GAGAACAAG
UCAGCGACC




 


0080


AATGTCCTGGGG
UGUCCUGGGG
GAAACUCAGC
+
66/67/68





CTGAGTTTCAA
CUGAGUUUC
CCCAGGACA




 


0081
ise1c.pk005.a15
Juvenile
AAGAATAAGCTC
GAAUAAGCUC
CACGUGGAGG

69/70/71




hormone
CTCCACGTGTT
CUCCACGUG
AGCUUAUUC






query







 


0082


AATTTGTCGAGG
UUUGUCGAGG
AUAGGGUCUC

72/73/74





AGACCCTATTG
AGACCCUAU
CUCGACAAA




 


0083


AAGTTCGCGTTC
GUUCGCGUUC
UUCAAGAGUG
NT
75/76/77





ACTCTTGAAGA
ACUCUUGAA
AACGCGAAC




 


0084
ise1c.pk006.d24
Juvenile
AACTGCCCCTTA
CUGCCCCUUA
AGAUGAGGUU
+
78/79/80




hormone
ACCTCATCTAT
ACCUCAUCU
AAGGGGCAG






query







 


0085


AATCACGCTGAA
UCACGCUGAA
UACAGUGGUU

81/82/83





ACCACTGTATA
ACCACUGUA
UCAGCGUGA




 


0086
ise2c.pk009.i4
Juvenile
AAAATATGGCGC
AAUAUGGCGC
ACAAUAGGCG

84/85/86




hormone
GCCTATTGTTT
GCCUAUUGU
CGCCAUAUU






query







 


0087


AACGTTCTCGGT
CGUUCUCGGU
CAGUGAAAGA

87/88/89





CTTTCACTGCT
CUUUCACUG
CCGAGAACG




 


0088


AAGTCATCGTTC
GUCAUCGUUC
GUAGACUUGG

90/91/92





CAAGTCTACCT
CAAGUCUAC
AACGAUGAC




 


0089
ise2c.pk001.d19
vacuolar
AACCCCTTGAAT
CCCCUUGAAU
GACCUUAACA
+
93/94/95




query
GTTAAGGTCGG
GUUAAGGUC
UUCAAGGGG




 


0090


AAGTACACCATG
GUACACCAUG
UACUUGCAAC

96/97/98





TTGCAAGTATG
UUGCAAGUA
AUGGUGUAC




 


0091


AACGTGTCCATG
CGUGUCCAUG
GUCAGCCAUC
+
 99/100/101





ATGGCTGACTC
AUGGCUGAC
AUGGACACG




 


0092
ise2c.pk001.e14
vacuolar
AAACCTACAAAA
ACCUACAAAA
UUUCGGCCAU

102/103/104




query
TGGCCGAAAAC
UGGCCGAAA
UUUGUAGGU




 


0093


AATCTACGGACC
UCUACGGACC
CCAAAGAAGG

105/106/107





CTTCTTTGGAG
CUUCUUUGG
GUCCGUAGA




 


0094
ise2c.pk001.f20
vacuolar
AACTCTGACGTC
CUCUGACGUC
GUAGAUGAUG

108/109/110




query
ATCATCTACGT
AUCAUCUAC
ACGUCAGAG




 


0095


AAGTGCTTGGGT
GUGCUUGGGU
GUCGGGGUUA

111/112/113





AACCCCGACAG
AACCCCGAC
CCCAAGCAC




 


0096


AACTGGCTCATC
CUGGCUCAUC
GCUGUAGGAG

114/115/116





TCCTACAGCAA
UCCUACAGC
AUGAGCCAG




 


0097
ise2c.pk010.h3
cadherin
AAACAGTGCGTC
ACAGUGCGUC
AUAUAUUACG

117/118/119




query
GTAATATATTC
GUAAUAUAU
ACGCACUGU




 


0098


AAGGCACATGGT
GGCACAUGGU
CAGUGAAGGA

120/121/122





CCTTCACTGAT
CCUUCACUG
CCAUGUGCC




 


0099


AACACCATGACC
CACCAUGACC
GUACACGAGG

123/124/125





CTCGTGTACAA
CUCGUGUAC
GUCAUGGUG




 


0100
ise2c.pk007.k24
cuticle
AACGAGGCCGGA
CGAGGCCGGA
CUUAAGAGAU

457/458/459




protein
TCTCTTAAGCA
UCUCUUAAG
CCGGCCUCG




 


0101


AACTTCACACAT
CUUCACACAU
UGUCUAGUUA

460/461/462





AACTAGACAAA
AACUAGACA
UGUGUGAAG




 


0102


AATGCGTGGCGA
UUAGAAAUUA
CUGGGCUUAU

463/464/465





TTTCAAACTTA
UAAGCCCAG
AAUUUCUAA




 


0103
ise2c.pk011.a10
cuticle
AAAAAACACAGA
AAAACACAGA
UGAACGUGGU

126/127/128




protein
CCACGTTCACA
CCACGUUCA
CUGUGUUUU




 


0104


AATCGATGGTGG
UCGAUGGUGG
CGAAUAACAC
+
129/130/131





TGTTATTCGCT
UGUUAUUCG
CACCAUCGA




 


0105
ise2c.pk011.h12
cuticle
AAAGAAAATGCT
AGAAAAUGCU
GUAACGCGUA

132/133/134




protein
ACGCGTTACGA
ACGCGUUAC
GCAUUUUCU




 


0106


AACCCTTGGACA
CCCUUGGACA
UUCCAGUAGU

135/136/137





CTACTGGAAGA
CUACUGGAA
GUCCAAGGG




 


0107


AAGGATCCTATG
GGAUCCUAUG
CCUGGUACAC

138/139/140





TGTACCAGGTT
UGUACCAGG
AUAGGAUCC




 


0108
ise2c.pk001.d22
translation
AAACTCGGCACA
ACUCGGCACA
AUUGUGUUGU
+
141/142/143




initiation
CAACACAATGG
CAACACAAU
GUGCCGAGU






factor







 


0109


AATACGAAGATA
UACGAAGAUA
AAGGGCAGAU
+
144/145/146





TCTGCCCTTCC
UCUGCCCUU
AUCUUCGUA




 


0110


AATCAACAGCTC
UCAACAGCUC
UUUAUGUAAG
+
147/148/149





TTACATAAATG
UUACAUAAA
AGCUGUUGA




 


0111
ise2c.pk001.d9
translation
AAAGAAGATCAG
AGAAGAUCAG
GCCAAUCUUC

150/151/152




initiation
AAGATTGGCCG
AAGAUUGGC
UGAUCUUCU






factor







 


0112


AAAAGCCGTCTG
AAGCCGUCUG
GUUGGAUAGC

153/154/155





CTATCCAACAA
CUAUCCAAC
AGACGGCUU




 


0113


AATGCTAAATGC
UGCUAAAUGC
GCAAGCAUGG
+
156/157/158





CATGCTTGCAT
CAUGCUUGC
CAUUUAGCA




 


0114
ise2c.pk001.i23
translation
AAGATCAGAAGA
GAUCAGAAGA
UCCGGCCAAU
+
159/160/161




initiation
TTGGCCGGAAG
UUGGCCGGA
CUUCUGAUC






factor







 


0115


AATTCTTCAGCA
UUCUUCAGCA
GUAUCGAUUU
NT
162/163/164





AATCGATACCA
AAUCGAUAC
GCUGAAGAA




 


0116


AAATGCTGTCAA
AUGCUGUCAA
UAAAUCCUCU

165/166/167





GAGGATTTAAA
GAGGAUUUA
UGACAGCAU




 


0117
ise2c.pk001.l24
translation
AAGCTCGAGACT
GCUCGAGACU
UCAAGAGCAA
+
168/169/170




initiation
TGCTCTTGATG
UGCUCUUGA
GUCUCGAGC






factor







 


0118


AACTGTTAGCTC
CUGUUAGCUC
GCAGACCUUG
+
171/172/173





AAGGTCTGCTA
AAGGUCUGC
AGCUAACAG




 


0119


AAGACTTTCTAT
GACUUUCUAU
CAAAUUCUGA

174/175/176





CAGAATTTGCG
CAGAAUUUG
UAGAAAGUC




 


0120
ise2c.pk005.b9
translation
AAACTTAATCAT
ACUUAAUCAU
UCGUCGUCCA

177/178/179




initiation
GGACGACGACA
GGACGACGA
UGAUUAAGU






factor







 


0121


AAAGAAGAAGAA
AGAAGAAGAA
CCCUUCUUCU
+
180/181/182





GAAGAAGGGAG
GAAGAAGGG
UCUUCUUCU




 


0122


AAGATCAAGAGA
GAUCAAGAGA
CCUCGACAUU
+
183/184/185





ATGTCGAGGAT
AUGUCGAGG
CUCUUGAUC




 


0123
ise2c.pk002.m10
SAR1
AAAATCGTCGGT
AAUCGUCGGU
GUCGCUAAAA
+
186/187/188





TTTAGCGACGT
UUUAGCGAC
CCGACGAUU




 


0124


AACTGTCAATAG
CUGUCAAUAG
GCAUACUGCC
+
189/190/191





GCAGTATGCGT
GCAGUAUGC
UAUUGACAG




 


0125


AACCTGTACCAA
CCUGUACCAA
AGUGGUCUGU
+
192/193/194





CAGACCACTGG
CAGACCACU
UGGUACAGG




 


0126
ise2c.pk001.c14
Elongation
AACCAAAAATGG
CCAAAAAUGG
UUUCCUUGCC
+
195/196/197




factor
GCAAGGAAAAG
GCAAGGAAA
CAUUUUUGG




 


0127


AACGTGGTATCA
CGUGGUAUCA
UAUCGAUGGU
+
198/199/200





CCATCGATATT
CCAUCGAUA
GAUACCACG




 


0128


AACAAAATGGAC
CAAAAUGGAC
CUCAGUGGAG
+
201/202/203





TCCACTGAGCC
UCCACUGAG
UCCAUUUUG




 


0129
ise2c.pk001.d16
Elongation
AATCCGTGACTA
UCCGUGACUA
AUUUUUGGUU
+
204/205/206




factor
ACCAAAAATGG
ACCAAAAAU
AGUCACGGA




 


0130


AACATTGTCGTC
CAUUGUCGUC
GUGUCCAAUG
+
207/208/209





ATTGGACACGT
AUUGGACAC
ACGACAAUG




 


0131
ise2c.pk005.h3
phosphooligo-
AATTTGTGAGAC
UUUGUGAGAC
CGGCCACCAG

421/422/423




saccharide 
TGGTGGCCGAA
UGGUGGCCG
UCUCACAAA






...







 


0132


AATCTGATTGTA
UCUGAUUGUA
GGGGGCGAAU

424/425/426





TTCGCCCCCTC
UUCGCCCCC
ACAAUCAGA




 


0133


AACACTCTAGTT
CACUCUAGUU
AAUAGGCAGA

427/428/429





CTGCCTATTCT
CUGCCUAUU
ACUAGAGUG




 


0134
ise2c.pk001.d21
myosin
AACACACATCAC
CACACAUCAC
UCCGCCAUUG

430/431/432





AATGGCGGATA
AAUGGCGGA
UGAUGUGUG




 


0135


AAGGATGGCATC
GGAUGGCAUC
CUUGCCGAUG

433/434/435





ATCGGCAAGAA
AUCGGCAAG
AUGCCAUCC




 


0136


AAAGGCTTCATC
AGGCUUCAUC
CGCGGUGUCG

436/437/438





GACACCGCGAA
GACACCGCG
AUGAAGCCU




 


0137
ise2c.pk001.j9
myosin
AAACTCCAATTA
ACUCCAAUUA
AGUAGGUUAU

210/211/212





TAACCTACTAG
UAACCUACU
AAUUGGAGU




 


0138


AAGTACAAGGAT
GUACAAGGAU
GCCGAUCAGA

213/214/215





CTGATCGGCAA
CUGAUCGGC
UCCUUGUAC




 


0139


AAGACTTTCTTC
GACUUUCUUC
GGGCCACAUG

216/217/218





ATGTGGCCCAT
AUGUGGCCC
AAGAAAGUC




 


0140
ise2c.pk002.f12
myosin
AAACAAAGTATC
ACAAAGUAUC
GGUGUAGGCG

439/440/441





GCCTACACCGC
GCCUACACC
AUACUUUGU




 


0141


AATAGCGTCGAT
UAGCGUCGAU
UCGUUGAAGA

442/443/444





CTTCAACGACT
CUUCAACGA
UCGACGCUA




 


0142
ise2c.pk001.b14
potassium
AACTCATAGAGC
CUCAUAGAGC
ACACAUCAAG

219/220/221




channel
TTGATGTGTGG
UUGAUGUGU
CUCUAUGAG






amino acid









transporter







 


0143


AAGATGTGGATG
GAUGUGGAUG
CAGUGACGUC
+
221/223/224





ACGTCACTGGT
ACGUCACUG
AUCCACAUC




 


0144


AACCTTCCTGAT
CCUUCCUGAU
CAGAAGAGAA
+
225/226/227





TCTCTTCTGTG
UCUCUUCUG
UCAGGAAGG




 


0145
ise2c.pk003.f2
potassium
AACAGTGCTTGT
CAGUGCUUGU
UCACUUAUCA
+
228/229/230




inwardly
GATAAGTGAAC
GAUAAGUGA
CAAGCACUG






rectifier 









. . .







 


0146


AAGTTAATGGTG
GUUAAUGGUG
GAGGGCAGUC
+
231/232/233





ACTGCCCTCGA
ACUGCCCUC
ACCAUUAAC




 


0147


AATAAAGCGATG
UAAAGCGAUG
CUAUGGGGUC
+
234/235/236





ACCCCATAGGA
ACCCCAUAG
AUCGCUUUA




 


0148
ise2c.pk005.l20
amino acid
AAACGGTACTGC
ACGGUACUGC
CUUUUUGCUG
+
237/238/239




transporter
AGCAAAAAGAC
AGCAAAAAG
CAGUACCGU




 


0149


AAGCTGCATACT
GCUGCAUACU
GAGCCAAGAA
+
240/241/242





TCTTGGCTCTC
UCUUGGCUC
GUAUGCAGC




 


0150


AAATGTTTACAG
AUGUUUACAG
AUCGCGUCUC
+
243/244/245





AGACGCGATGA
AGACGCGAU
UGUAAACAU




 


0151
ise2c.pk001.d1
tubulin
AACGTCGATCTT
CGUCGAUCUU
GAACUCGGUA
+
246/247/248





ACCGAGTTCCA
ACCGAGUUC
AGAUCGACG




 


0152
ise2c.pk001.k6
tubulin
AATTCAAAATGC
UUCAAAAUGC
UGCACUCACG

249/250/251





GTGAGTGCATC
GUGAGUGCA
CAUUUUGAA




 


0153


AAATCGTAGACC
AUCGUAGACC
CGAGGACUAG
+
252/253/254





TAGTCCTCGAC
UAGUCCUCG
GUCUACGAU




 


0154
ise2c.pk001.l2
tubulin
AAACTCAATTCA
ACUCAAUUCA
CACGCAUUUU
+
255/256/257





AAATGCGTGAG
AAAUGCGUG
GAAUUGAGU




 


0155


AACTTATCACTG
CUUAUCACUG
CUUCCUUACC

258/259/260





GTAAGGAAGAT
GUAAGGAAG
AGUGAUAAG




 


0156
ise2c.pk002.b4
ubiquitin
AAGAGTTACGAA
GAGUUACGAA
UGGUGACGGU
+
261/262/263





CCGTCACCATA
CCGUCACCA
UCGUAACUC




 


0157


AAACTTAGTCCG
ACUUAGUCCG
UUCAUUAUCC
+
264/265/266





GATAATGAACC
GAUAAUGAAt
GGACUAAGU




 


0158


AAGGCGATGTAC
GGCGAUGUAC
CAGGUUCUCG
+
267/268/269





GAGAACCTGTT
GAGAACCUG
UACAUCGCC




 


0159
ise2c.pk001.j16
small
AACGACAAGATG
CGACAAGAUG
CUCCUUCAGC
+
270/271/272




nuclear
CTGAAGGAGAC
CUGAAGGAG
AUCUUGUCG






ribonucleo-









protein







 


0160
ise2c.pk006.h23 
small
AAGATAAAGGTC
GAUAAAGGUC
UCCACACGCG
+





nuclear
GCGTGTGGACC
GCGUGUGGA
ACCUUUAUC






ribonucleo- 









protein







 


0161


AATGTCAAGACT
UGUCAAGACU
GUUUGGAUCA
+
276/277/278





GATCCAAACAC
GAUCCAAAC
GUCUUGACA




 


0162


AACATTCGAGTC
CAUUCGAGUC
ACCUGUUCAG
+
279/280/281





TGAACAGGTGG
UGAACAGGUt
ACUCGAAUG





(Note: the sense RNA primer sequence and the antisense RNA primer sequences shown in table 8 were generated with 2 thymine residues at the 3′ end.)






















TABLE 9














SEQ ID











NO











Target











region/








30
15
8
sense/


Sample
seq id
gene id
Target sequence
forward
reverse
ppm
ppm
ppm
antisense







0075


AAGGTCGCTGACGA
GGUCGCUGACG
CUUGUUCUCGUCA



51/52/53





GAACAAGGA
AGAACAAG
GCGACC






 


0076


AAGTGTCCTGGGCTT
GUGUCCUGGGC
GAACUCAAGCCCA
+


54/55/56





GAGTTCCA
UUGAGUUC
GGACAC






 


0077
ise1c.pk003.f7
Juvenile
AAGAAGAAGCTCCT
GAAGAAGCUCC
CACGUGGAGGAGC



57/58/59




hormone
CCACGTGTT
UCCACGUG
UUCUUC








query









 


0078


AAGGTCGCTGACGA
GGUCGCUGACG
CUUGUUCUCGUCA



60/61/62





GAACAAGGA
AGAACAAG
GCGACC






 


0079


AATGTCCTGGGGCT
UGUCCUGGGGC
GAAACUCAGCCCC



63/64/65





GAGTTTCAA
UGAGUUUC
AGGACA






 


0080
ise1c.pk005.a15
Juvenile
AAGAATAAGCTCCT
GAAUAAGCUCC
CACGUGGAGGAGC
+
+

66/67/68




hormone 
CCACGTGTT
UCCACGUG
UUAUUC








query









 


0081


AATTTGTCGAGGAG
UUUGUCGAGGA
AUAGGGUCUCCUC



69/70/71





ACCCTATTG
GACCCUAU
GACAAA






 


0082


AAGTTCGCGTTCACT
GUUCGCGUUCA
UUCAAGAGUGAA
NT


72/73/74





CTTGAAGA
CUCUUGAA
CGCGAAC






 


0083
ise1c.pk006.d24
Juvenile
AACTGCCCCTTAACC
CUGCCCCUUAAC
AGAUGAGGUUAA



75/76/77




hormone 
TCATCTAT
CUCAUCUtt
GGGGCAG








query









 


0084


AATCACGCTGAAAC
UCACGCUGAAA
UACAGUGGUUUC
+


78/79/80





CACTGTATA
CCACUGUAtt
AGCGUGA






 


0085
ise2c.pk009.i4
Juvenile
AAAATATGGCGCGC
AAUAUGGCGCG
ACAAUAGGCGCGC



81/82/83




hormone 
CTATTGTTT
CCUAUUGU
CAUAUU








query









 


0086


AACGTTCTCGGTCTT
CGUUCUCGGUC
CAGUGAAAGACCG



84/85/86





TCACTGCT
UUUCACUG
AGAACG






 


0087


AAGTCATCGTTCCAA
GUCAUCGUUCC
GUAGACUUGGAA
+


87/88/89





GTCTACCT
AAGUCUAC
CGAUGAC






 


0088
ise2c.pk001.d19
vacuolar
AACCCCTTGAATGTT
CCCCUUGAAUG
GACCUUAACAUUC
+


90/91/92




query 
AAGGTCGG
UUAAGGUC
AAGGGG






 


0089


AAGTACACCATGTT
GUACACCAUGU
UACUUGCAACAUG



93/94/95





GCAAGTATG
UGCAAGUA
GUGUAC






 


0090


AACGTGTCCATGAT
CGUGUCCAUGA
GUCAGCCAUCAUG
NT


96/97/98





GGCTGACTC
UGGCUGAC
GACACG






 


0091
ise2c.pk001.e14
vacuolar
AAACCTACAAAATG
ACCUACAAAAU
UUUCGGCCAUUUU



 99/100/101




query 
GCCGAAAAC
GGCCGAAA
GUAGGU






 


0092


AATCTACGGACCCTT
UCUACGGACCC
CCAAAGAAGGGUC
+


102/103/104





CTTTGGAG
UUCUUUGG
CGUAGA






 


0093
ise2c.pk001.f20
vacuolar
AACTCTGACGTCATC
CUCUGACGUCA
GUAGAUGAUGAC



105/106/107




query 
ATCTACGT
UCAUCUAC
GUCAGAG






 


0094


AAGTGCTTGGGTAA
GUGCUUGGGUA
GUCGGGGUUACCC



108/109/110





CCCCGACAG
ACCCCGAC
AAGCAC






 


0095


AACTGGCTCATCTCC
CUGGCUCAUCU
GCUGUAGGAGAU



111/112/113





TACAGCAA
CCUACAGC
GAGCCAG






 


0096
ise2c.pk010.h3
cadherin
AAACAGTGCGTCGT
ACAGUGCGUCG
AUAUAUUACGAC



114/115/116




query 
AATATATTC
UAAUAUAU
GCACUGU






 


0097


AAGGCACATGGTCC
GGCACAUGGUC
CAGUGAAGGACCA



117/118/119





TTCACTGAT
CUUCACUG
UGUGCC






 


0098


AACACCATGACCCT
CACCAUGACCCU
GUACACGAGGGUC
NT


120/121/122





CGTGTACAA
CGUGUAC
AUGGUG






 


0099
ise2c.pk007.k24
cuticle
AACGAGGCCGGATC
CGAGGCCGGAU
CUUAAGAGAUCCG



123/124/125




protein
TCTTAAGCA
CUCUUAAG
GCCUCG






 


0100


AACTTCACACATAA
CUUCACACAUA
UGUCUAGUUAUG



457/458/459





CTAGACAAA
ACUAGACA
UGUGAAG






 


0101


AATGCGTGGCGATTT
UUAGAAAUUAU
CUGGGCUUAUAA



460/461/462





CAAACTTA
AAGCCCAG
UUUCUAA






 


0102
ise2c.pk011.a10
cuticle 
AAAAAACACAGACC
AAAACACAGAC
UGAACGUGGUCU



463/464/465




protein
ACGTTCACA
CACGUUCA
GUGUUUU






 


0103


AATCGATGGTGGTG
UCGAUGGUGGU
CGAAUAACACCAC
+


126/127/128





TTATTCGCT
GUUAUUCG
CAUCGA






 


0104
ise2c.pk011.h12
cuticle 
AAAGAAAATGCTAC
AGAAAAUGCUA
GUAACGCGUAGCA



129/130/131




protein
GCGTTACGA
CGCGUUAC
UUUUCU






 


0105


AACCCTTGGACACT
CCCUUGGACAC
UUCCAGUAGUGUC
+


132/133/134





ACTGGAAGA
UACUGGAA
CAAGGG






 


0106


AAGGATCCTATGTGT
GGAUCCUAUGU
CCUGGUACACAUA
NT


135/136/137





ACCAGGTT
GUACCAGG
GGAUCC






 


0107
ise2c.pk001.d22
translation
AAACTCGGCACACA
ACUCGGCACAC
AUUGUGUUGUGU



138/139/140




initiation 
ACACAATGG
AACACAAU
GCCGAGU








factor









 


0108


AATACGAAGATATC
UACGAAGAUAU
AAGGGCAGAUAU
+


141/142/143





TGCCCTTCC
CUGCCCUU
CUUCGUA






 


0109


AATCAACAGCTCTTA
UCAACAGCUCU
UUUAUGUAAGAG
+


144/145/146





CATAAATG
UACAUAAA
CUGUUGA






 


0110
ise2c.pk001.d9
translation
AAAGAAGATCAGAA
AGAAGAUCAGA
GCCAAUCUUCUGA



147/148/149




initiation 
GATTGGCCG
AGAUUGGC
UCUUCU








factor









 


0111


AAAAGCCGTCTGCT
AAGCCGUCUGC
GUUGGAUAGCAG



150/151/152





ATCCAACAA
UAUCCAAC
ACGGCUU






 


0112


AATGCTAAATGCCA
UGCUAAAUGCC
GCAAGCAUGGCAU
+


153/154/155





TGCTTGCAT
AUGCUUGC
UUAGCA






 


0113
ise2c.pk001.i23
translation
AAGATCAGAAGATT
GAUCAGAAGAU
UCCGGCCAAUCUU
+


156/157/158




initiation 
GGCCGGAAG
UGGCCGGA
CUGAUC








factor









 


0114


AATTCTTCAGCAAAT
UUCUUCAGCAA
GUAUCGAUUUGC
NT


159/160/161





CGATACCA
AUCGAUAC
UGAAGAA






 


0115


AAATGCTGTCAAGA
AUGCUGUCAAG
UAAAUCCUCUUGA
+


162/163/164





GGATTTAAA
AGGAUUUA
CAGCAU






 


0116
ise2c.pk001.i24
translation
AAGCTCGAGACTTG
GCUCGAGACUU
UCAAGAGCAAGUC
+


165/166/167




initiation 
CTCTTGATG
GCUCUUGA
UCGAGC








factor









 


0117


AACTGTTAGCTCAA
CUGUUAGCUCA
GCAGACCUUGAGC
+


168/169/170





GGTCTGCTA
AGGUCUGC
UAACAG






 


0118


AAGACTTTCTATCAG
GACUUUCUAUC
CAAAUUCUGAUA



171/172/173





AATTTGCG
AGAAUUUG
GAAAGUC






 


0119
ise2c.pk005.b9
translation
AAACTTAATCATGG
ACUUAAUCAUG
UCGUCGUCCAUGA



174/175/176




initiation 
ACGACGACA
GACGACGA
UUAAGU








factor









 


0120


AAAGAAGAAGAAGA
AGAAGAAGAAG
CCCUUCUUCUUCU
+


177/178/179





AGAAGGGAG
AAGAAGGG
UCUUCU






 


0121


AAGATCAAGAGAAT
GAUCAAGAGAA
CCUCGACAUUCUC
+
+
+
180/181/182





GTCGAGGAT
UGUCGAGG
UUGAUC






 


0122


AAAATCGTCGGTTTT
AAUCGUCGGUU
GUCGCUAAAACCG
NT


183/184/185





AGCGACGT
UUAGCGAC
ACGAUU






 


0123
ise2c.pk002.m10
SAR1
AACTGTCAATAGGC
CUGUCAAUAGG
GCAUACUGCCUAU
+


186/187/188





AGTATGCGT
CAGUAUGC
UGACAG






 


0124


AACCTGTACCAACA
CCUGUACCAAC
AGUGGUCUGUUG
+


189/190/191





GACCACTGG
AGACCACU
GUACAGG






 


0125
ise2c.pk001.c14
Elongation
AACCAAAAATGGGC
CCAAAAAUGGG
UUUCCUUGCCCAU
+


192/193/194




factor
AAGGAAAAG
CAAGGAAA
UUUUGG






 


0126


AACGTGGTATCACC
CGUGGUAUCAC
UAUCGAUGGUGA
+


195/196/197





ATCGATATT
CAUCGAUA
UACCACG






 


0127


AACAAAATGGACTC
CAAAAUGGACU
CUCAGUGGAGUCC
+


198/199/200





CACTGAGCC
CCACUGAG
AUUUUG






 


0128
ise2c.pk001.d16
Elongation
AATCCGTGACTAAC
UCCGUGACUAA
AUUUUUGGUUAG
+


201/202/203




factor
CAAAAATGG
CCAAAAAU
UCACGGA






 


0129


AACATTGTCGTCATT
CAUUGUCGUCA
GUGUCCAAUGACG



204/205/206





GGACACGT
UUGGACAC
ACAAUG






 


0130
ise2c.pk005.h3
phosphooligo-
AATTTGTGAGACTG
UUUGUGAGACU
CGGCCACCAGUCU
NT


207/208/209




saccharide
GTGGCCGAA
GGUGGCCG
CACAAA








. . .









 


0131


AATCTGATTGTATTC
UCUGAUUGUAU
GGGGGCGAAUAC



421/422/423





GCCCCCTC
UCGCCCCC
AAUCAGA






 


0132


AACACTCTAGTTCTG
CACUCUAGUUC
AAUAGGCAGAAC



424/425/426





CCTATTCT
UGCCUAUU
UAGAGUG






 


0133
ise2c.pk001.d21
myosin
AACACACATCACAA
CACACAUCACA
UCCGCCAUUGUGA



427/428/429





TGGCGGATA
AUGGCGGA
UGUGUG






 


0134


AAGGATGGCATCAT
GGAUGGCAUCA
CUUGCCGAUGAUG



430/431/432





CGGCAAGAA
UCGGCAAG
CCAUCC






 


0135


AAAGGCTTCATCGA
AGGCUUCAUCG
CGCGGUGUCGAUG



433/434/435





CACCGCGAA
ACACCGCG
AAGCCU






 


0136
ise2c.pk001.j9
myosin
AAACTCCAATTATA
ACUCCAAUUAU
AGUAGGUUAUAA



436/437/438





ACCTACTAG
AACCUACU
UUGGAGU






 


0137


AAGTACAAGGATCT
GUACAAGGAUC
GCCGAUCAGAUCC



210/211/212





GATCGGCAA
UGAUCGGC
UUGUAC






 


0138


AAGACTTTCTTCATG
GACUUUCUUCA
GGGCCACAUGAAG
NT


213/214/215





TGGCCCAT
UGUGGCCC
AAAGUC






 


0139
ise2c.pk002.f12
myosin
AAACAAAGTATCGC
ACAAAGUAUCG
GGUGUAGGCGAU



216/217/218





CTACACCGC
CCUACACC
ACUUUGU






 


0140


AATAGCGTCGATCTT
UAGCGUCGAUC
UCGUUGAAGAUC



439/440/441





CAACGACT
UUCAACGA
GACGCUA






 


0141
ise2c.pk001.b14
potassium
AACTCATAGAGCTT
CUCAUAGAGCU
ACACAUCAAGCUC



442/443/444




channel 
GATGTGTGG
UGAUGUGU
UAUGAG








amino acid 











transporter









 


0142


AAGATGTGGATGAC
GAUGUGGAUGA
CAGUGACGUCAUC



219/220/221





GTCACTGGT
CGUCACUG
CACAUC






 


0143


AACCTTCCTGATTCT
CCUUCCUGAUU
CAGAAGAGAAUC
+


222/223/224





CTTCTGTG
CUCUUCUG
AGGAAGG






 


0144
ise2c.pk003.f2
potassium
AACAGTGCTTGTGAT
CAGUGCUUGUG
UCACUUAUCACAA
+
+

225/226/227




inwardly
AAGTGAAC
AUAAGUGA
GCACUG








rectifier











. . .









 


0145


AAGTTAATGGTGAC
GUUAAUGGUGA
GAGGGCAGUCACC
+


228/229/230





TGCCCTCGA
CUGCCCUC
AUUAAC






 


0146


AATAAAGCGATGAC
UAAAGCGAUGA
CUAUGGGGUCAUC
NT


231/232/233





CCCATAGGA
CCCCAUAG
GCUUUA






 


0147
ise2c.pk005.l20
amino acid
AAACGGTACTGCAG
ACGGUACUGCA
CUUUUUGCUGCAG
+


234/235/236




transporter
CAAAAAGAC
GCAAAAAG
UACCGU






 


0148


AAGCTGCATACTTCT
GCUGCAUACUU
GAGCCAAGAAGU



237/238/239





TGGCTCTC
CUUGGCUC
AUGCAGC






 


0149


AAATGTTTACAGAG
AUGUUUACAGA
AUCGCGUCUCUGU
+


240/241/242





ACGCGATGA
GACGCGAU
AAACAU






 


0150
ise2c.pk001.d1
tubulin
AACGTCGATCTTACC
CGUCGAUCUUA
GAACUCGGUAAG
+
+

243/244/245





GAGTTCCA
CCGAGUUC
AUCGACG






 


0151
ise2c.pk001.k6
tubulin
AATTCAAAATGCGT
UUCAAAAUGCG
UGCACUCACGCAU
+


246/247/248





GAGTGCATC
UGAGUGCA
UUUGAA






 


0152


AAATCGTAGACCTA
AUCGUAGACCU
CGAGGACUAGGUC
+
+

249/250/251





GTCCTCGAC
AGUCCUCG
UACGAU






 


0153
ise2c.pk001.l2
tubulin
AAACTCAATTCAAA
ACUCAAUUCAA
CACGCAUUUUGAA
+
+

252/253/254





ATGCGTGAG
AAUGCGUG
UUGAGU






 


0154


AACTTATCACTGGTA
CUUAUCACUGG
CUUCCUUACCAGU
NT


255/256/257





AGGAAGAT
UAAGGAAG
GAUAAG






 


0155
ise2c.pk002.b4
ubiquitin
AAGAGTTACGAACC
GAGUUACGAAC
UGGUGACGGUUC
+


258/259/260





GTCACCATA
CGUCACCA
GUAACUC






 


0156


AAACTTAGTCCGGA
ACUUAGUCCGG
UUCAUUAUCCGGA
+
+

261/262/263





TAATGAACC
AUAAUGAA
CUAAGU






 


0157


AAGGCGATGTACGA
GGCGAUGUACG
CAGGUUCUCGUAC
+


264/265/266





GAACCTGTT
AGAACCUG
AUCGCC






 


0158
ise2c.pk001.j16
small 
AACGACAAGATGCT
CGACAAGAUGC
CUCCUUCAGCAUC
+


267/268/269




nuclear
GAAGGAGAC
UGAAGGAG
UUGUCG








ribonucleo-











protein









 


0159
ise2c.pk006.h23
small 
AAGATAAAGGTCGC
GAUAAAGGUCG
UCCACACGCGACC
+


270/271/272




nuclear
GTGTGGACC
CGUGUGGA
UUUAUC








ribonucleo-











protein









 


0160


AATGTCAAGACTGA
UGUCAAGACUG
GUUUGGAUCAGU
+


273/274/275





TCCAAACAC
AUCCAAAC
CUUGACA






 


0161


AACATTCGAGTCTG
CAUUCGAGUCU
ACCUGUUCAGACU
+
+

276/277/278





AACAGGTGG
GAACAGGU
CGAAUG





(Note: the sense RNA primer sequence and the antisense RNA primer sequences shown in table 9 were generated with 2 thymine residuesat the 3′ end.)


























TABLE 10









Injection
Droplet
Topical
Topical
Topical
Topical
Topical
Topical








Mortality
Feeding
assay 1
Assay 2
Assay 3
Assay 4
Assay 4
Assay 4
Top. 5
Top. 5


well
seq i.d.
midgut
gene id
(%)
Result
30 ppm
30 ppm
30 ppm
30 ppm
15 ppm
8 ppm
15 ppm
8 ppm




























77
ise1c.pk002.m13
no
Juvenile
NT
NT
NT
−
−
+


+






hormone





query


81
ise1c.pk005.a15
no
Juvenile
NT
NT
NT
−
−
+
+

+





hormone





query


114
ise2c.pk001.i23
no
translation
NT
NT
NT
+
+
+


+





initiation





factor


122
ise2c.pk005.b9
Yes
translation
NT
NT
NT
+
+
+
+
+
+
+





initiation





factor


143
ise2c.pk001.b14
no
potassium
NT
NT
NT
−
+



+





channel ami-





acid





transporter


145
ise2c.pk003.f2
Yes
potassium
NT
NT
NT
+
+
+
+

+





inwardly





rectifier . . .


146
ise2c.pk003.f2
Yes
potassium
NT
NT
NT
+
+
+


+





inwardly





rectifier . . .


149
ise2c.pk005.l20
Yes
hypothetical
NT
NT
NT
+
+



+





sodium





dependent





transport


151
ise2c.pk001.d1
No
alpha tubulin
NT
NT
NT
+
+
+
+

+


154
ise2c.pk001.l2
No
alpha tubulin
NT
NT
NT
+
+
+
+

+


157
ise2c.pk002.b4
Yes
Probable
NT
NT
NT
+
+
+
+

+





ubiquitin--





protein





ligase


158
ise2c.pk002.b4
Yes
Probable
NT
NT
NT
+
+
−


+





ubiquitin--





protein





ligase


162
ise2c.pk006.h23
Yes
RNA-
NT
NT
NT
+
+
+
+

+





binding





protein squid






















TABLE 11





DsRNA #
gene id
seqID
Comment
8 ppm
4 ppm
2 ppm







0163
pre-mRNA-binding protein
ise2c.pk006.m8
Plate 2 A1





0164
pre-mRNA-binding protein
ise2c.pk006.m8
Plate 2 B1


0165
pre-mRNA-binding protein
ise2c.pk006.m8
Plate 2 C1


0166
pre-mRNA-binding protein
ise2c.pk006.m8
Plate 2 D1
S


0167
pre-mRNA-binding protein
ise2c.pk006.m8
Plate 2 E1
S


0168
chymotrypsin-like; protease
ise2c.pk001.a23
Plate 2 F1
S


0169
chymotrypsin-like; protease
ise2c.pk001.a23
Plate 2 G1


0170
chymotrypsin-like; protease
ise2c.pk001.a23
Plate 2 H1


0171
chymotrypsin-like; protease
ise2c.pk001.a23
Plate 2 A2
S


0172
chymotrypsin-like; protease
ise2c.pk001.a23
Plate 2 B2
S


0173
chymotrypsinogen; protease
ise2c.pk001.a7
Plate 2 C2
S


0174
chymotrypsinogen; protease
ise2c.pk001.a7
Plate 2 D2


0175
chymotrypsinogen; protease
ise2c.pk001.a7
Plate 2 E2


0176
chymotrypsinogen; protease
ise2c.pk001.a7
Plate 2 F2


0177
chymotrypsinogen; protease
ise2c.pk001.a7
Plate 2 G2


0178
actin-depolymerizing
ise2c.pk004.c4
Plate 2 H2


0179
actin-depolymerizing
ise2c.pk004.c4
Plate 2 A3


0180
actin-depolymerizing
ise2c.pk004.c4
Plate 2 B3


0181
actin-depolymerizing
ise2c.pk004.c4
Plate 2 C3


0182
actin-depolymerizing
ise2c.pk004.c4
Plate 2 D3


0183
actin depolymerizing factor
ise2c.pk004.l4
Plate 2 E3


0184
actin depolymerizing factor
ise2c.pk004.l4
Plate 2 F3


0185
actin depolymerizing factor
ise2c.pk004.l4
Plate 2 G3


0186
actin depolymerizing factor
ise2c.pk004.l4
Plate 2 H3


0187
actin depolymerizing factor
ise2c.pk004.l4
Plate 2 A4


0188
dismutase; superoxide
ise2c.pk004.n19
Plate 2 B4


0189
dismutase; superoxide
ise2c.pk004.n19
Plate 2 C4


0190
dismutase; superoxide
ise2c.pk004.n19
Plate 2 D4


0191
dismutase; superoxide
ise2c.pk004.n19
Plate 2 E4


0192
dismutase; superoxide
ise2c.pk004.n19
Plate 2 F4


0193
superoxide dismutase
ise2c.pk005.f21
Plate 2 G4


0194
superoxide dismutase
ise2c.pk005.f21
Plate 2 H4


0195
superoxide dismutase
ise2c.pk005.f21
Plate 2 A5


0196
adenylate kinase isozyme 3
ise2c.pk010.h5
Plate 2 B5


0197
adenylate kinase isozyme 3
ise2c.pk010.h5
Plate 2 C5


0198
adenylate kinase isozyme 3
ise2c.pk010.h5
Plate 2 D5


0199
adenylate kinase isozyme 3
ise2c.pk010.h5
Plate 2 E5


0200
adenylate kinase isozyme 3
ise2c.pk010.h5
Plate 2 F5


0201
ecdysone oxidase
ise2c.pk001.c18
Plate 2 G5


0202
ecdysone oxidase
ise2c.pk001.c18
Plate 2 H5


0203
ecdysone oxidase
ise2c.pk001.c18
Plate 2 A6


0204
innexin-2
ise2c.pk004.p1
Plate 2 B6


0205
innexin-2
ise2c.pk004.p1
Plate 2 C6


0206
innexin-2
ise2c.pk004.p1
Plate 2 D6


0207
innexin-2
ise2c.pk004.p1
Plate 2 E6


0208
innexin-2
ise2c.pk004.p1
Plate 2 F6






















TABLE 12





DsRNA#
gene id
seqID
Comment
16 ppm
8 ppm
4 ppm







0163
pre-mRNA-binding protein
ise2c.pk006.m8
Plate 2 A1
S




0164
pre-mRNA-binding protein
ise2c.pk006.m8
Plate 2 B1
S


0165
pre-mRNA-binding protein
ise2c.pk006.m8
Plate 2 C1
S


0166
pre-mRNA-binding protein
ise2c.pk006.m8
Plate 2 D1
S


0167
pre-mRNA-binding protein
ise2c.pk006.m8
Plate 2 E1
S


0168
chymotrypsin-like; protease
ise2c.pk001.a23
Plate 2 F1
ss
S


0169
chymotrypsin-like; protease
ise2c.pk001.a23
Plate 2 G1
ss


0170
chymotrypsin-like; protease
ise2c.pk001.a23
Plate 2 H1


0171
chymotrypsin-like; protease
ise2c.pk001.a23
Plate 2 A2


0172
chymotrypsin-like; protease
ise2c.pk001.a23
Plate 2 B2


0173
chymotrypsinogen; protease
ise2c.pk001.a7
Plate 2 C2
ss


0174
chymotrypsinogen; protease
ise2c.pk001.a7
Plate 2 D2
S


0175
chymotrypsinogen; protease
ise2c.pk001.a7
Plate 2 E2
S


0176
chymotrypsinogen; protease
ise2c.pk001.a7
Plate 2 F2
S


0177
chymotrypsinogen; protease
ise2c.pk001.a7
Plate 2 G2
S


0178
actin-depolymerizing
ise2c.pk004.c4
Plate 2 H2


0179
actin-depolymerizing
ise2c.pk004.c4
Plate 2 A3
ss


0180
actin-depolymerizing
ise2c.pk004.c4
Plate 2 B3
s


0181
actin-depolymerizing
ise2c.pk004.c4
Plate 2 C3


0182
actin-depolymerizing
ise2c.pk004.c4
Plate 2 D3


0183
actin depolymerizing factor
ise2c.pk004.l4
Plate 2 E3


0184
actin depolymerizing factor
ise2c.pk004.l4
Plate 2 F3


0185
actin depolymerizing factor
ise2c.pk004.l4
Plate 2 G3


0186
actin depolymerizing factor
ise2c.pk004.l4
Plate 2 H3


0187
actin depolymerizing factor
ise2c.pk004.l4
Plate 2 A4


0188
dismutase; superoxide
ise2c.pk004.n19
Plate 2 B4


0189
dismutase; superoxide
ise2c.pk004.n19
Plate 2 C4
s


0190
dismutase; superoxide
ise2c.pk004.n19
Plate 2 D4


0191
dismutase; superoxide
ise2c.pk004.n19
Plate 2 E4


0192
dismutase; superoxide
ise2c.pk004.n19
Plate 2 F4
s


0193
superoxide dismutase
ise2c.pk005.f21
Plate 2 G4


0194
superoxide dismutase
ise2c.pk005.f21
Plate 2 H4


0195
superoxide dismutase
ise2c.pk005.f21
Plate 2 A5


0196
adenylate kinase isozyme 3
ise2c.pk010.h5
Plate 2 B5


0197
adenylate kinase isozyme 3
ise2c.pk010.h5
Plate 2 C5


0198
adenylate kinase isozyme 3
ise2c.pk010.h5
Plate 2 D5


0199
adenylate kinase isozyme 3
ise2c.pk010.h5
Plate 2 E5


0200
adenylate kinase isozyme 3
ise2c.pk010.h5
Plate 2 F5


0201
ecdysone oxidase
ise2c.pk001.c18
Plate 2 G5


0202
ecdysone oxidase
ise2c.pk001.c18
Plate 2 H5


0203
ecdysone oxidase
ise2c.pk001.c18
Plate 2 A6


0204
innexin-2
ise2c.pk004.p1
Plate 2 B6


0205
innexin-2
ise2c.pk004.p1
Plate 2 C6


0206
innexin-2
ise2c.pk004.p1
Plate 2 D6


0207
innexin-2
ise2c.pk004.p1
Plate 2 E6


0208
innexin-2
ise2c.pk004.p1
Plate 2 F6
















TABLE 13







Summary of FAW droplet feeding data for the first set of synthetic dsRNA primers















Assay #4



Assay #1
Assay #2
Assay #3
(table 8)














(table 6)
(table 7)
(table 8)
30
15
8



30 ppm
30 ppm
30 ppm
ppm
ppm
ppm



















0075
ise1c.pk002.m13
Juvenile hormone query
−
−
−
NT
−
−


0076


NT
−
−
−
−
−


0077


NT
−
−
+
−
−


0078
ise1c.pk003.f7
Juvenile hormone query
NT
−
−
−
−
−


0079


NT
−
−
−
−
−


0080


NT
−
+
−
−
−


0081
ise1c.pk005.a15
Juvenile hormone query
NT
−
−
+
+
−


0082


NT
−
−
−
−
−


0083


+
+
NT
NT
−
−


0084
ise1c.pk006.d24
Juvenile hormone query
NT
−
+
−
−
−


0085


+
−
−
+
−
−


0086
ise2c.pk009.i4
Juvenile hormone query
−
−
−
−
−
−


0087


NT
−
−
−
−
−


0088


+
−
−
+
−
−


0089
ise2c.pk001.d19
vacuolar query
+
−
+
+
−
−


0090


NT
−
−
−
−
−


0091


+
−
+
NT
−
−


0092
ise2c.pk001.e14
vacuolar query
NT
−
−
−
−
−


0093


NT
−
−
+
−
−


0094
ise2c.pk001.f20
vacuolar query
+
−
−
−
−
−


0095


+
−
−
−
−
−


0096


NT
−
−
−
−
−


0097
ise2c.pk010.h3
cadherin query
NT
−
−
−
−
−


0098


NT
+
−
−
−
−


0099


NT
−
−
NT
−
−


0100
ise2c.pk007.k24
cuticle protein
NT
−
−
−
−
−


0101


NT
−
−
−
−
−


0102


NT
−
−
−
−
−


0103
ise2c.pk011.a10
cuticle protein
NT
+
−
−
−
−


0104


NT
+
+
+
−
−


0105
ise2c.pk011.h12
cuticle protein
NT
−
−
−
−
−


0106


NT
+
−
+
−
−


0107


+
−
−
NT
−
−


0108
ise2c.pk001.d22
translation initiation factor
NT
−
+
−
−
−


0109


NT
+
+
+
−
−


0110


NT
−
+
+
−
−


0111
ise2c.pk001.d9
translation initiation factor
NT
−
−
−
−
−


0112


NT
+
−
−
−
−


0113


NT
−
+
+
−
−


0114
ise2c.pk001.i23
translation initiation factor
NT
+
+
+
−
−


0115


−
−
NT
NT
−
−


0116


NT
−
−
+
−
−


0117
ise2c.pk001.l24
translation initiation factor
NT
−
+
+
−
−


0118


NT
−
+
+
−
−


0119


NT
+
−
−
−
−


0120
ise2c.pk005.b9
translation initiation factor
NT
−
−
−
−
−


0121


NT
+
+
+
−
−


0122


NT
+
+
+
+
+


0123
ise2c.pk002.m10
SAR1
−
−
+
NT
−
−


0124


NT
−
+
+
−
−


0125


NT
+
+
+
−
−


0126
ise2c.pk001.c14
Elongation factor
NT
+
+
+
−
−


0127


NT
+
+
+
−
−


0128


NT
−
+
+
−
−


0129
ise2c.pk001.d16
Elongation factor
NT
+
+
+
−
−


0130


NT
−
+
−
−
−


0131
ise2c.pk005.h3
phosphooligosaccharide . . .
−
−
−
NT
−
−


0132


NT
−
−
−
−
−


0133


NT
−
−
−
−
−


0134
ise2c.pk001.d21
myosin
NT
−
−
−
−
−


0135


NT
−
−
−
−
−


0136


NT
−
−
−
−
−


0137
ise2c.pk001.j9
myosin
NT
−
−
−
−
−


0138


NT
+
−
−
−
−


0139


−
−
−
NT
−
−


0140
ise2c.pk002.f12
myosin
NT
−
−
−
−
−


0141


NT
−
−
−
−
−


0142
ise2c.pk001.b14
potassium channel amino acid
NT
−
−
−
−
−




transporter


0143


NT
−
+
−
−
−


0144


NT
−
+
+
−
−


0145
ise2c.pk003.f2
potassium inwardly rectifier . . .
NT
+
+
+
+
−


0146


NT
+
+
+
−
−


0147


+
+
+
NT
−
−


0148
ise2c.pk005.l20
amino acid transporter
NT
+
+
+
−
−


0149


NT
+
+
−
−
−


0150


NT
−
+
+
−
−


0151
ise2c.pk001.d1
tubulin
NT
+
+
+
+
−


0152
ise2c.pk001.k6
tubulin
NT
−
−
+
−
−


0153


NT
+
+
+
+
−


0154
ise2c.pk001.l2
tubulin
NT
+
+
+
+
−


0155


−
−
−
NT
−
−


0156
ise2c.pk002.b4
ubiquitin
NT
−
+
+
−
−


0157


NT
+
+
+
+
−


0158


NT
+
+
+
−
−


0159
ise2c.pk001.j16
small nuclear ribonucleoprotein
NT
+
+
+
−
−


0160
ise2c.pk006.h23
small nuclear ribonucleoprotein
NT
−
+
+
−
−


0161


NT
−
+
+
−
−


0162


NT
+
+
+
+
−









Example 2
Transformation of Maize
Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing the silencing element of the invention operably linked to either a tissue specific, tissue selective, or constitutive promoter and the selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37), which confers resistance to the herbicide Bialaphos. In one embodiment, the constructs will have 2 identical 2-300 bp segments of the target gene in opposite orientations with an “intron” segment between them acting as a hairpin loop. Such a construct can be linked to the dMMB promoter. Alternatively, the selectable marker gene is provided on a separate plasmid. Transformation is performed as follows. Media recipes follow below.
Preparation of Target Tissue
The ears are husked and surface sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5 cm target zone in preparation for bombardment.
A plasmid vector comprising the silencing element of interest operably linked to either the tissue specific, tissue selective, or constitutive promoter is made. This plasmid DNA plus plasmid DNA containing a PAT selectable marker is precipitated onto 1.1 μm (average diameter) tungsten pellets using a CaCl2 precipitation procedure as follows: 100 μl prepared tungsten particles in water; 10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA); 100 μl 2.5 M CaCl2; and, 10 μl 0.1 M spermidine.
Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 10 μl spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
The sample plates are bombarded at level #4 in a particle gun. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
Following bombardment, the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5′′ pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity.
Plants are monitored and scored for the appropriate marker, such as the control of Lepidoptera and have insecticidal activity. For example, a FAW feeding assay could be preformed. In such assays, leaf discs from the transgenic plant are excised using a 1 cm cork borer or leaf punch. Six leaf discs are prepared for each plant. The leaves are placed in a 24 well microtiter plate on top of 500 ul of 0.8% agar. Each leaf disc is infested with 2 neonate Fall armyworm and the plate is then sealed with mylar. A small ventilation hole is made for each well and the plates are then stored in a 28 C growth chamber. The assay is scored for mortality, stunting, and leaf consumption at 96 hours.
Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added after sterilizing the medium and cooling to room temperature).
Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCl, 0.10 g/l pyridoxine HCl, and 0.40 g/l glycine brought to volume with polished D-I H2O) (Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume with polished D-I H2O after adjusting to pH 5.6); 3.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 1.0 mg/l indoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing the medium and cooling to 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCl, 0.10 g/l pyridoxine HCl, and 0.40 g/l glycine brought to volume with polished D-I H2O), 0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-I H2O after adjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing to volume with polished D-I H2O), sterilized and cooled to 60° C.
Example 3
Agrobacterium-Mediated Transformation of Maize
For Agrobacterium-mediated transformation of maize with a silencing element of the invention, the method of Zhao is employed (U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326; the contents of which are hereby incorporated by reference). Such as a construct can comprise 2 identical 2-300 bp segments of the target gene in opposite orientations with an “intron” segment between them acting as a hairpin loop. Such a construct can be linked to the dMMB promoter. Briefly, immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring the polynucleotide comprising the silencing element to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step the immature embryos are immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos are co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). The immature embryos are cultured on solid medium following the infection step. Following this co-cultivation period an optional “resting” step is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). The immature embryos are cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step). The immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus is then regenerated into plants (step 5: the regeneration step), and calli grown on selective medium are cultured on solid medium to regenerate the plants.
Example 4
Soybean Embryo Transformation
Culture Conditions
Soybean embryogenic suspension cultures (cv. Jack) are maintained in 35 ml liquid medium SB196 (see recipes below) on rotary shaker, 150 rpm, 26° C. with cool white fluorescent lights on 16:8 hr day/night photoperiod at light intensity of 60-85 μE/m2/s. Cultures are subcultured every 7 days to two weeks by inoculating approximately 35 mg of tissue into 35 ml of fresh liquid SB196 (the preferred subculture interval is every 7 days).
Soybean embryogenic suspension cultures are transformed with the plasmids and DNA fragments described in the examples above by the method of particle gun bombardment (Klein et al. (1987) Nature, 327:70).
Soybean Embryogenic Suspension Culture Initiation
Soybean cultures are initiated twice each month with 5-7 days between each initiation.
Pods with immature seeds from available soybean plants 45-55 days after planting are picked, removed from their shells and placed into a sterilized magenta box. The soybean seeds are sterilized by shaking them for 15 minutes in a 5% Clorox solution with 1 drop of ivory soap (95 ml of autoclaved distilled water plus 5 ml Clorox and 1 drop of soap). Mix well. Seeds are rinsed using 2 l-liter bottles of sterile distilled water and those less than 4 mm are placed on individual microscope slides. The small end of the seed are cut and the cotyledons pressed out of the seed coat. Cotyledons are transferred to plates containing SB1 medium (25-30 cotyledons per plate). Plates are wrapped with fiber tape and stored for 8 weeks. After this time secondary embryos are cut and placed into SB 196 liquid media for 7 days.
Preparation of DNA for Bombardment
Either an intact plasmid or a DNA plasmid fragment containing the genes of interest and the selectable marker gene are used for bombardment. Plasmid DNA for bombardment are routinely prepared and purified using the method described in the PromegaTM Protocols and Applications Guide, Second Edition (page 106). Fragments of the plasmids carrying the silencing element of interest are obtained by gel isolation of double digested plasmids. In each case, 100 ug of plasmid DNA is digested in 0.5 ml of the specific enzyme mix that is appropriate for the plasmid of interest. The resulting DNA fragments are separated by gel electrophoresis on 1% SeaPlaque GTG agarose (BioWhitaker Molecular Applications) and the DNA fragments containing silencing element of interest are cut from the agarose gel. DNA is purified from the agarose using the GELase digesting enzyme following the manufacturer's protocol.
A 50 μl aliquot of sterile distilled water containing 3 mg of gold particles (3 mg gold) is added to 5 μl of a 1 μg/l DNA solution (either intact plasmid or DNA fragment prepared as described above), 50 μl 2.5 M CaCl2 and 20 μl of 0.1 M spermidine. The mixture is shaken 3 min on level 3 of a vortex shaker and spun for 10 sec in a bench microfuge. After a wash with 400 μl 100% ethanol the pellet is suspended by sonication in 40 μl of 100% ethanol. Five μl of DNA suspension is dispensed to each flying disk of the Biolistic PDS1000/HE instrument disk. Each 5 μl aliquot contains approximately 0.375 mg gold per bombardment (i.e. per disk).
Tissue Preparation and Bombardment with DNA
Approximately 150-200 mg of 7 day old embryonic suspension cultures are placed in an empty, sterile 60×15 mm petri dish and the dish covered with plastic mesh. Tissue is bombarded 1 or 2 shots per plate with membrane rupture pressure set at 1100 PSI and the chamber evacuated to a vacuum of 27-28 inches of mercury. Tissue is placed approximately 3.5 inches from the retaining/stopping screen.
Selection of Transformed Embryos
Transformed embryos were selected either using hygromycin (when the hygromycin phosphotransferase, HPT, gene was used as the selectable marker) or chlorsulfuron (when the acetolactate synthase, ALS, gene was used as the selectable marker).
Hygromycin (HPT) Selection
Following bombardment, the tissue is placed into fresh SB196 media and cultured as described above. Six days post-bombardment, the SB196 is exchanged with fresh SB196 containing a selection agent of 30 mg/L hygromycin. The selection media is refreshed weekly. Four to six weeks post selection, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates to generate new, clonally propagated, transformed embryogenic suspension cultures.
Chlorsulfuron (ALS) Selection
Following bombardment, the tissue is divided between 2 flasks with fresh SB196 media and cultured as described above. Six to seven days post-bombardment, the SB196 is exchanged with fresh SB196 containing selection agent of 100 ng/ml Chlorsulfuron. The selection media is refreshed weekly. Four to six weeks post selection, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates containing SB196 to generate new, clonally propagated, transformed embryogenic suspension cultures.
Regeneration of Soybean Somatic Embryos into Plants
In order to obtain whole plants from embryogenic suspension cultures, the tissue must be regenerated.
Embryo Maturation
Embryos are cultured for 4-6 weeks at 26° C. in SB196 under cool white fluorescent (Phillips cool white Econowatt F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) on a 16:8 hr photoperiod with light intensity of 90-120 uE/m2s. After this time embryo clusters are removed to a solid agar media, SB166, for 1-2 weeks. Clusters are then subcultured to medium SB103 for 3 weeks. During this period, individual embryos can be removed from the clusters and screened for the appropriate marker or the ability of the plant, when injected with the silencing elements, to control the Lepidoptera.
Embryo Desiccation and Germination
Matured individual embryos are desiccated by placing them into an empty, small petri dish (35×10 mm) for approximately 4-7 days. The plates are sealed with fiber tape (creating a small humidity chamber). Desiccated embryos are planted into SB71-4 medium where they were left to germinate under the same culture conditions described above. Germinated plantlets are removed from germination medium and rinsed thoroughly with water and then planted in Redi-Earth in 24-cell pack tray, covered with clear plastic dome. After 2 weeks the dome is removed and plants hardened off for a further week. If plantlets looked hardy they are transplanted to 10′′ pot of Redi-Earth with up to 3 plantlets per pot.










Media Recipes







SB 196 - FN Lite liquid proliferation medium (per liter) -














MS FeEDTA - 100x Stock 1
10
ml



MS Sulfate - 100x Stock 2
10
ml



FN Lite Halides - 100x Stock 3
10
ml



FN Lite P, B, Mo - 100x Stock 4
10
ml



B5 vitamins (1 ml/L)
1.0
ml



2,4-D (10 mg/L final concentration)
1.0
ml



KNO3
2.83
gm



(NH4)2 SO 4
0.463
gm



Asparagine
1.0
gm



Sucrose (1%)
10
gm



pH 5.8











FN Lite Stock Solutions










Stock #

1000 ml
500 ml





1
MS Fe EDTA 100x Stock



Na2 EDTA*
3.724 g 
1.862 g 



FeSO4—7H2O
2.784 g 
1.392 g 


2
MS Sulfate 100x stock



MgSO4—7H2O
37.0 g
18.5 g



MnSO4—H2O
1.69 g
0.845 g 



ZnSO4—7H2O
0.86 g
0.43 g



CuSO4—5H2O
0.0025 g 
0.00125 g  


3
FN Lite Halides 100x Stock



CaCl2—2H2O
30.0 g
15.0 g



KI
0.083 g 
0.0715 g 



CoCl2—6H2O
0.0025 g 
0.00125 g  


4
FN Lite P, B, Mo 100x Stock



KH2PO4
18.5 g
9.25 g



H3BO3
0.62 g
0.31 g



Na2MoO4—2H2O
0.025 g 
0.0125 g 





*Add first, dissolve in dark bottle while stirring






SB1 solid medium (per liter) comprises: 1 pkg. MS salts (Gibco/BRL—Cat#11117-066); 1 ml B5 vitamins 1000× stock; 31.5 g sucrose; 2 ml 2,4-D (20 mg/L final concentration); pH 5.7; and, 8 g TC agar.
SB 166 solid medium (per liter) comprises: 1 pkg. MS salts (Gibco/BRL—Cat#11117-066); 1 ml B5 vitamins 1000× stock; 60 g maltose; 750 mg MgCl2 hexahydrate; 5 g activated charcoal; pH 5.7; and, 2 g gelrite.
SB 103 solid medium (per liter) comprises: 1 pkg. MS salts (Gibco/BRL—Cat#11117-066); 1 ml B5 vitamins 1000× stock; 60 g maltose; 750 mg MgCl2 hexahydrate; pH 5.7; and, 2 g gelrite.
SB 71-4 solid medium (per liter) comprises: 1 bottle Gamborg's B5 salts w/sucrose (Gibco/BRL—Cat#21153-036); pH 5.7; and, 5 g TC agar.
2,4-D stock is obtained premade from Phytotech cat# D 295—concentration is 1 mg/ml.
B5 Vitamins Stock (per 100 ml) which is stored in aliquots at −20 C comprises: 10 g myo-inositol; 100 mg nicotinic acid; 100 mg pyridoxine HCl; and, 1 g thiamine. If the solution does not dissolve quickly enough, apply a low level of heat via the hot stir plate. Chlorsulfuron Stock comprises 1 mg/ml in 0.01 N Ammonium Hydroxide
The article “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one or more element.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

## Claims

1. A plant cell having stably incorporated into its genome a heterologous polynucleotide encoding a double stranded RNA, wherein said double stranded RNA comprises a sense and an antisense strand, wherein the antisense strand comprises at least 20 consecutive nucleotides fully complementary to the target gene sequence set forth in SEQ ID NO: 6, and wherein the sense strand is fully complementary to the antisense strand,
wherein said double stranded RNA, when ingested by a pest from the Lepidoptera order, controls the pest from the Lepidoptera order.

2. The plant cell of claim 1, wherein said pest comprises Spodoptera frugiperda.

3. The plant cell of claim 1, wherein said double stranded RNA comprises
a) the sense and antisense sequence of the sequence set forth in SEQ ID NO: 93, 96, or 99; or
b) the sequences set forth in SEQ ID NO: 94, and 95, 97, and 98, or 100 and 101.

4. The plant cell of claim 1, wherein said double stranded RNA comprises a hairpin RNA.

5. The plant cell of claim 4, wherein said double stranded RNA comprises a first segment, a second segment, and a third segment, wherein
a) said first segment comprises at least 20 consecutive nucleotides fully complementary to the target sequence set forth in SEQ ID NO: 6;
b) said second segment comprises a loop of sufficient length to allow the double stranded RNA to be transcribed as a hairpin RNA; and,
c) said third segment comprises at least about 20 consecutive nucleotides fully complementary to the first segment.

6. The plant cell of claim 1, wherein said polynucleotide is operably linked to a heterologous promoter.

7. The plant cell of claim 1, wherein said plant cell has stably incorporated into its genome a second polynucleotide comprising a suppressor enhancer element comprising the target pest sequence or an active variant or fragment thereof, wherein the combined expression of the double stranded RNA and the suppressor enhancer element increases the concentration of an inhibitory RNAi specific for the pest target sequence in said plant cell.

8. The plant cell of claim 1, wherein said plant cell is from a monocot.

9. The plant cell of claim 8, wherein said monocot is maize, barley, millet, wheat or rice.

10. The plant cell of claim 1, wherein said plant cell is from a dicot.

11. The plant cell of claim 10, wherein said dicot is soybean, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, or cotton.

12. A plant or plant part comprising a plant cell of claim 1.

13. The plant or plant part of claim 7, wherein the combined expression of said double stranded RNA and the suppressor enhancer element increases the concentration of an inhibitory RNA specific for the pest target sequence in the phloem of said plant or plant part.

14. A transgenic seed from the plant of claim 12.

15. A method for controlling Lepidoptera comprising feeding to a Lepidoptera a composition comprising a double stranded RNA, wherein the double stranded RNA comprises a sense and an antisense strand, wherein the antisense strand comprises at least 20 consecutive nucleotides fully complementary to the target gene sequence set forth in SEQ ID NO: 6, and wherein the sense strand is fully complementary to the antisense strand,
wherein said double stranded RNA, when ingested by said Lepidoptera controls the Lepidoptera.

16. The method of claim 15, wherein said composition comprises a plant or plant part having stably incorporated into its genome a polynucleotide encoding said double stranded RNA.

17. The method of claim 15, wherein said pest comprises Spodoptera frugiperda.

18. The method of claim 15, wherein said double stranded RNA comprises:
a) the sense and antisense sequence of the sequence set forth in SEQ ID NO: 93, 96, or 99; or
b) the sequences set forth in SEQ ID NO: 94 and 95, 97 and 98, or 100 and 101.

19. The method of claim 15, wherein said double stranded RNA comprises a hairpin RNA.

20. The method of claim 19 wherein said double stranded RNA comprises a first segment, a second segment, and a third segment, wherein
a) said first segment comprises at least 20 consecutive nucleotides fully complementary to the target polynucleotide;
b) said second segment comprises a loop of sufficient length to allow the double stranded RNA to be transcribed as a hairpin RNA; and,
c) said third segment comprises at least 20 consecutive nucleotides fully complementary to the first segment.

21. The method of claim 16, wherein said plant or plant part has stably incorporated into its genome a second polynucleotide comprising a suppressor enhancer element comprising the target pest sequence or an active variant or fragment thereof, wherein the combined expression of the double stranded RNA and the suppressor enhancer element increases the concentration of an inhibitory RNAi specific for the pest target sequence in said plant cell.

22. The method of claim 15, wherein said plant is a monocot.

23. The method of claim 22, wherein said monocot is maize, barley, millet, wheat or rice.

24. The method of claim 15, wherein said plant is a dicot.

25. The method of claim 24, wherein said plant is soybean, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, or cotton.