# Patent

## Title
Generation of plants with altered protein, fiber, or oil content

## Abstract

The present invention is directed to plants that display an improved oil quantity phenotype or an improved meal quality phenotype due to altered expression of an IMQ nucleic acid. The invention is further directed to methods of generating plants with an improved oil quantity phenotype or improved meal quality phenotype.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This is a divisional of U.S. patent application Ser. No. 13/480,275, filed May 24, 2012; which is a divisional of U.S. patent application Ser. No. 13/221,790, filed Aug. 30, 2011, now U.S. Pat. No. 8,217,225, issued Jul. 10, 2012; which is a divisional of U.S. patent application Ser. No. 11/940,274, filed Nov. 14, 2007, now U.S. Pat. No. 8,034,993, issued Oct. 11, 2011; which claims the benefit of U.S. Provisional Application No. 60/866,056, filed Nov. 15, 2006; each of which is incorporated herein by reference in its entirety.


FIELD OF THE DISCLOSURE
The present disclosure is related to transgenic plants with altered oil, protein, and/or fiber content, as well as methods of making plants having altered oil, protein, and/or fiber content and producing oil from such plants.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN ASCII TEXT FILE
A Sequence Listing is submitted herewith as an ASCII compliant text file named “Sequence_Listing.txt”, created on Nov. 10, 2014, and having a size of 330,131 bytes, as permitted under 37 CFR 1.821(c). The material in the aforementioned file is hereby incorporated by reference in its entirety.
BACKGROUND
The ability to manipulate the composition of crop seeds, particularly the content and composition of seed oil and protein, as well as the available metabolizable energy (“AME”) in the seed meal in livestock, has important applications in the agricultural industries, relating both to processed food oils and to animal feeds. Seeds of agricultural crops contain a variety of valuable constituents, including oil, protein and starch. Industrial processing can separate some or all of these constituents for individual sale in specific applications. For instance, nearly 60% of the U.S. soybean crop is crushed by the soy processing industry. Soy processing yields purified oil, which is sold at high value, while the remaining seed meal is sold for livestock feed (U.S. Soybean Board, 2001 Soy Stats). Canola seed is also crushed to produce oil and the co-product canola meal (Canola Council of Canada). Canola meal contains a high percentage of protein and a good balance of amino acids but because it has a high fiber and phytate content, it is not readily digested by livestock (Slominski, B. A., et al., 1999 Proceedings of the 10th International Rapeseed Congress, Canberra, Australia) and has a lower value than soybean meal.
Over 55% of the corn produced in the U.S. is used as animal feed (Iowa Corn Growers Association). The value of the corn is directly related to its ability to be digested by livestock. Thus, it is desirable to maximize both oil content of seeds and the AME of meal. For processed oilseeds such as soy and canola, increasing the absolute oil content of the seed will increase the value of such grains, while increasing the AME of meal will increase its value. For processed corn, either an increase or a decrease in oil content may be desired, depending on how the other major constituents are to be used. Decreasing oil may improve the quality of isolated starch by reducing undesired flavors associated with oil oxidation. Alternatively, when the starch is used for ethanol production, where flavor is unimportant, increasing oil content may increase overall value.
In many feed grains, such as corn and wheat, it is desirable to increase seed oil content, because oil has higher energy content than other seed constituents such as carbohydrate. Oilseed processing, like most grain processing businesses, is a capital-intensive business; thus small shifts in the distribution of products from the low valued components to the high value oil component can have substantial economic impacts for grain processors. In addition, increasing the AME of meal by adjusting seed protein and fiber content and composition, without decreasing seed oil content, can increase the value of animal feed.
Biotechnological manipulation of oils has been shown to provide compositional alteration and improvement of oil yield. Compositional alterations include high oleic acid soybean and corn oil (U.S. Pat. Nos. 6,229,033 and 6,248,939), and laurate-containing seeds (U.S. Pat. No. 5,639,790), among others. Work in compositional alteration has predominantly focused on processed oilseeds, but has been readily extendable to non-oilseed crops, including corn. While there is considerable interest in increasing oil content, the only currently practiced biotechnology in this area is High-Oil Corn (HOC) technology (DuPont, U.S. Pat. No. 5,704,160). HOC employs high oil pollinators developed by classical selection breeding along with elite (male-sterile) hybrid females in a production system referred to as TopCross. The TopCross High Oil system raises harvested grain oil content in maize from about 3.5% to about 7%, improving the energy content of the grain.
While it has been fruitful, the HOC production system has inherent limitations. First, the system of having a low percentage of pollinators responsible for an entire field's seed set contains inherent risks, particularly in drought years. Second, oil content in current HOC fields has plateaued at about 9% oil. Finally, high-oil corn is not primarily a biochemical change, but rather an anatomical mutant (increased embryo size) that has the indirect result of increasing oil content. For these reasons, an alternative high oil strategy, particularly one that derives from an altered biochemical output, would be especially valuable.
Manipulation of seed composition has identified several components that improve the nutritive quality, digestibility, and AME in seed meal. Increasing the lysine content in canola and soybean (Falco et al., 1995 Bio/Technology 13:577-582) increases the availability of this essential amino acid and decreases the need for nutritional supplements. Soybean varieties with increased seed protein were shown to contain considerably more metabolizable energy than conventional varieties (Edwards et al., 1999, Poultry Sci. 79:525-527). Decreasing the phytate content of corn seed has been shown to increase the bioavailability of amino acids in animal feeds (Douglas et al., 2000, Poultry Sci. 79:1586-1591) and decreasing oligosaccharide content in soybean meal increases the metabolizable energy in the meal (Parsons et al., 2000, Poultry Sci. 79:1127-1131).
Soybean and canola are the most obvious target crops for the processed oil and seed meal markets since both crops are crushed for oil and the remaining meal sold for animal feed. A large body of commercial work (e.g., U.S. Pat. No. 5,952,544; PCT Application No. WO9411516) demonstrates that Arabidopsis is an excellent model for oil metabolism in these crops. Biochemical screens of seed oil composition have identified Arabidopsis genes for many critical biosynthetic enzymes and have led to identification of agronomically important gene orthologs. For instance, screens using chemically mutagenized populations have identified lipid mutants whose seeds display altered fatty acid composition (Lemieux et al., 1990, Theor. Appl. Genet. 80, 234-240; James and Dooner, 1990, Theor. Appl. Genet. 80, 241-245). T-DNA mutagenesis screens (Feldmann et al., 1989, Science 243: 1351-1354) that detected altered fatty acid composition identified the omega 3 desaturase (FADS) and delta-12 desaturase (FAD2) genes (U.S. Pat. No. 5,952,544; Yadav et al., 1993, Plant Physiol. 103, 467-476; Okuley et al., 1994, Plant Cell 6(1):147-158). A screen which focused on oil content rather than oil quality, analyzed chemically-induced mutants for wrinkled seeds or altered seed density, from which altered seed oil content was inferred (Focks and Benning, 1998, Plant Physiol. 118:91-101).
Another screen, designed to identify enzymes involved in production of very long chain fatty acids, identified a mutation in the gene encoding a diacylglycerol acyltransferase (DGAT) as being responsible for reduced triacyl glycerol accumulation in seeds (Katavic et al., 1995, Plant Physiol. 108(1):399-409). It was further shown that seed-specific over-expression of the DGAT cDNA was associated with increased seed oil content (Jako et al., 2001, Plant Physiol. 126(2):861-74). Arabidopsis is also a model for understanding the accumulation of seed components that affect meal quality. For example, Arabidopsis contains albumin and globulin seed storage proteins found in many dicotyledonous plants including canola and soybean (Shewry 1995, Plant Cell 7:945-956). The biochemical pathways for synthesizing components of fiber, such as cellulose and lignin, are conserved within the vascular plants, and mutants of Arabidopsis affecting these components have been isolated (reviewed in Chapel and Carpita 1998, Current Opinion in Plant Biology 1:179-185).
Activation tagging in plants refers to a method of generating random mutations by insertion of a heterologous nucleic acid construct comprising regulatory sequences (e.g., an enhancer) into a plant genome. The regulatory sequences can act to enhance transcription of one or more native plant genes; accordingly, activation tagging is a fruitful method for generating gain-of-function, generally dominant mutants (see, e.g., Hayashi et al., 1992, Science 258: 1350-1353; Weigel D et al., 2000, Plant Physiology, 122:1003-1013). The inserted construct provides a molecular tag for rapid identification of the native plant whose mis-expression causes the mutant phenotype. Activation tagging may also cause loss-of-function phenotypes. The insertion may result in disruption of a native plant gene, in which case the phenotype is generally recessive.
Activation tagging has been used in various species, including tobacco and Arabidopsis, to identify many different kinds of mutant phenotypes and the genes associated with these phenotypes (Wilson et al., 1996, Plant Cell 8: 659-671; Schaffer et al., 1998, Cell 93: 1219-1229; Fridborg et al., 1999, Plant Cell 11: 1019-1032; Kardailsky et al., 1999, Science 286: 1962-1965; and Christensen S et al., 1998, 9th International Conference on Arabidopsis Research, Univ. of Wisconsin-Madison, June 24-28, Abstract 165).
SUMMARY
Provided herein are transgenic plants having an Improved Seed Quality phenotype. Transgenic plants with an Improved Seed Quality phenotype may include an improved oil quantity and/or an improved meal quality. Transgenic plants with improved meal quality have an Improved Meal Quality (IMQ) phenotype and transgenic plants with improved oil quantity have an Improved Oil Quantity (IOQ) phenotype. The IMQ phenotype in a transgenic plant may include altered protein and/or fiber content in any part of the transgenic plant, for example in the seeds. The IOQ phenotype in a transgenic plant may include altered oil content in any part of the transgenic plant, for example in the seeds. In particular embodiments, a transgenic plant may include an IOQ phenotype and/or an IMQ phenotype. In some embodiments of a transgenic plant, the IMQ phenotype may be an increase in protein content in the seed and/or a decrease in the fiber content of the seed. In other embodiments of a transgenic plant, the IOQ phenotype is an increase in the oil content of the seed (a high oil phenotype). Also provided is seed meal derived from the seeds of transgenic plants, wherein the seeds have altered protein content and/or altered fiber content. Further provided is oil derived from the seeds of transgenic plants, wherein the seeds have altered oil content. Any of these changes can lead to an increase in the AME from the seed or seed meal from transgenic plants, relative to control, non-transgenic, or wild-type plants. Also provided herein is meal, feed, or food produced from any part of the transgenic plant with an IMQ phenotype and/or IOQ phenotype.
In certain embodiments, the disclosed transgenic plants comprise a transformation vector comprising an IMQ nucleotide sequence that encodes or is complementary to a sequence that encodes an “IMQ” polypeptide. In particular embodiments, expression of an IMQ polypeptide in a transgenic plant causes an altered oil content, an altered protein content, and/or an altered fiber content in the transgenic plant. In preferred embodiments, the transgenic plant is selected from the group consisting of plants of the Brassica species, including canola and rapeseed, soy, corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax, castor, peanut, wheat, oat and rice. Also provided is a method of producing oil or seed meal, comprising growing the transgenic plant and recovering oil and/or seed meal from said plant. The disclosure further provides feed, meal, grain, or seed comprising a nucleic acid sequence that encodes an IMQ polypeptide. The disclosure also provides feed, meal, grain, or seed comprising the IMQ polypeptide, or an ortholog thereof.
Examples of the disclosed transgenic plant are produced by a method that comprises introducing into progenitor cells of the plant a plant transformation vector comprising an IMQ nucleotide sequence that encodes, or is complementary to a sequence that encodes, an IMQ polypeptide, and growing the transformed progenitor cells to produce a transgenic plant, wherein the IMQ polynucleotide sequence is expressed, causing an IOQ phenotype and/or and IMQ phenotype in the transgenic plant. In some specific, non-limiting examples, the method produces transgenic plants wherein expression of the IMQ polypeptide causes a high (increased) oil, high (increased) protein, and/or low (decreased) fiber phenotype in the transgenic plant, relative to control, non-transgenic, or wild-type plants.
Additional methods are disclosed herein of generating a plant having an IMQ and/or an IOQ phenotype, wherein a plant is identified that has an allele in its IMQ nucleic acid sequence that results in an IMQ phenotype and/or an IOQ phenotype, compared to plants lacking the allele. The plant can generate progeny, wherein the progeny inherit the allele and have an IMQ phenotype and/or an IOQ phenotype. In some embodiments of the method, the method employs candidate gene/QTL methodology or TILLING methodology.
Also provided herein is a transgenic plant cell having an IMQ phenotype and/or an IOQ phenotype. The transgenic plant cell comprises a transformation vector comprising an IMQ nucleotide sequence that encodes or is complementary to a sequence that encodes an IMQ polypeptide. In preferred embodiments, the transgenic plant cell is selected from the group consisting of plants of the Brassica species, including canola and rapeseed, soy, corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax, castor, peanut, wheat, oat and rice. In other embodiments, the plant cell is a seed, pollen, propagule, or embryo cell. The disclosure also provides plant cells from a plant that is the direct progeny or the indirect progeny of a plant grown from said progenitor cells.


DETAILED DESCRIPTION
Terms
Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as they would to one skilled in the art of the present disclosure. Practitioners are particularly directed to Sambrook et al. (Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Press, Plainview, N.Y., 1989) and Ausubel F M et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1993) for definitions and terms of the art. It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary.
As used herein, the term “IMQ phenotype” refers to plants, or any part of a plant (for example, seeds, or meal produced from seeds), with an altered protein and/or fiber content (phenotype). As provided herein, altered protein and/or fiber content includes either an increased or decreased level of protein and/or fiber content in plants, seeds or seed meal. Any combination of these changes can lead to an IMQ phenotype. For example, in one specific non-limiting example, an IMQ phenotype can refer to increased protein and decreased fiber content. In another specific non-limiting example, an IMQ phenotype can refer to unchanged protein and decreased fiber content. In yet another specific non-limiting example, an IMQ phenotype can refer to increased protein and unchanged fiber content. It is also provided that any combination of these changes can lead to an increase in the AME (available metabolizable energy) from the seed or meal generated from the seed. An IMQ phenotype also includes an improved seed quality (ISQ) phenotype or an improved seed meal quality phenotype.
As used herein, the term “IOQ phenotype” refers to plants, or any part of a plant (for example, seeds), with an altered oil content (phenotype). As provided herein, altered oil content includes an increased, for example a high, oil content in plants or seeds. In some embodiments, a transgenic plant can express both an IOQ phenotype and an IMQ phenotype. In specific, non-limiting examples, a transgenic plant having a combination of an IOQ phenotype and an IMQ phenotype can lead to an increase in the AME (available metabolizable energy) from the seed or meal generated from the seed. An IOQ phenotype also includes an improved seed quality (ISQ) phenotype.
As used herein, the term “available metabolizable energy” (AME) refers to the amount of energy in the feed that is able to be extracted by digestion in an animal and is correlated with the amount of digestible protein and oil available in animal meal. AME is determined by estimating the amount of energy in the feed prior to feeding and measuring the amount of energy in the excreta of the animal following consumption of the feed. In one specific, non-limiting example, a transgenic plant with an increase in AME includes transgenic plants with altered seed protein and/or fiber content and without a decrease in seed oil content (seed oil content remains unchanged or is increased), resulting in an increase in the value of animal feed derived from the seed.
As used herein, the term “content” refers to the type and relative amount of, for instance, a seed or seed meal component.
As used herein, the term “fiber” refers to non-digestible components of the plant seed including cellular components such as cellulose, hemicellulose, pectin, lignin, and phenolics.
As used herein, the term “meal” refers to seed components remaining following the extraction of oil from the seed. Examples of components of meal include protein and fiber.
As used herein, the term “vector” refers to a nucleic acid construct designed for transfer between different host cells. An “expression vector” refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.
A “heterologous” nucleic acid construct or sequence has a portion of the sequence that is not native to the plant cell in which it is expressed. Heterologous, with respect to a control sequence refers to a control sequence (i.e. promoter or enhancer) that does not function in nature to regulate the same gene the expression of which it is currently regulating. Generally, heterologous nucleic acid sequences are not endogenous to the cell or part of the genome in which they are present, and have been added to the cell by infection, transfection, microinjection, electroporation, or the like. A “heterologous” nucleic acid construct may contain a control sequence/DNA coding sequence combination that is the same as, or different from, a control sequence/DNA coding sequence combination found in the native plant. Specific, non-limiting examples of a heterologous nucleic acid sequence include an IMQ nucleic acid sequence, or a fragment, derivative (variant), or ortholog thereof.
As used herein, the term “gene” means the segment of DNA involved in producing a polypeptide chain, which may or may not include regions preceding and following the coding region, e.g. 5′ untranslated (5′ UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons) and non-transcribed regulatory sequences.
As used herein, “recombinant” includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid sequence or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all as a result of deliberate human intervention.
As used herein, the term “gene expression” refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation; accordingly, “expression” may refer to either a polynucleotide or polypeptide sequence, or both. Sometimes, expression of a polynucleotide sequence will not lead to protein translation. “Over-expression” refers to increased expression of a polynucleotide and/or polypeptide sequence relative to its expression in a wild-type (or other reference [e.g., non-transgenic]) plant and may relate to a naturally-occurring or non-naturally occurring sequence. “Ectopic expression” refers to expression at a time, place, and/or increased level that does not naturally occur in the non-altered or wild-type plant. “Under-expression” refers to decreased expression of a polynucleotide and/or polypeptide sequence, generally of an endogenous gene, relative to its expression in a wild-type plant. The terms “mis-expression” and “altered expression” encompass over-expression, under-expression, and ectopic expression.
The term “introduced” in the context of inserting a nucleic acid sequence into a cell, includes “transfection,” “transformation,” and “transduction” and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell where the nucleic acid sequence may be incorporated into the genome of the cell (for example, chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (for example, transfected mRNA).
As used herein, a “plant cell” refers to any cell derived from a plant, including cells from undifferentiated tissue (e.g., callus), as well as from plant seeds, pollen, propagules, and embryos.
As used herein, the terms “native” and “wild-type” relative to a given plant trait or phenotype refers to the form in which that trait or phenotype is found in the same variety of plant in nature. In one embodiment, a wild-type plant is also a control plant. In another embodiment, a wild-type plant is a non-transgenic plant.
As used herein, the term “modified” regarding a plant trait, refers to a change in the phenotype of a transgenic plant (for example, a transgenic plant with any combination of an altered oil content, an altered protein content, and/or an altered fiber content) in any part of the transgenic plant, for example the seeds, relative to a similar non-transgenic plant. As used herein, the term “altered” refers to either an increase or a decrease of a plant trait or phenotype (for example, oil content, protein content, and/or fiber content) in a transgenic plant, relative to a similar non-transgenic plant. In one specific, non-limiting example, a transgenic plant with a modified trait includes a plant with an increased oil content, increased protein content, and/or decreased fiber content relative to a similar non-transgenic plant. In another specific, non-limiting example, a transgenic plant with a modified trait includes unchanged oil content, increased protein content, and/or decreased fiber content relative to a similar non-transgenic plant. In yet another specific, non-limiting example, a transgenic plant with a modified trait includes an increased oil content, increased protein content, and/or unchanged fiber content relative to a similar non-transgenic plant. Specific, non-limiting examples of a change in phenotype include an IMQ phenotype or an IOQ phenotype.
An “interesting phenotype (trait)” with reference to a transgenic plant refers to an observable or measurable phenotype demonstrated by a T1 and/or subsequent generation plant, which is not displayed by the corresponding non-transgenic plant (i.e., a genotypically similar plant that has been raised or assayed under similar conditions). An interesting phenotype may represent an improvement in the plant (for example, increased oil content, increased protein content, and/or decreased fiber content in seeds of the plant) or may provide a means to produce improvements in other plants. An “improvement” is a feature that may enhance the utility of a plant species or variety by providing the plant with a unique and/or novel phenotype or quality. Such transgenic plants may have an improved phenotype, such as an IMQ phenotype or an IOQ phenotype.
The phrase “altered oil content phenotype” refers to a measurable phenotype of a genetically modified (transgenic) plant, where the plant displays a statistically significant increase or decrease in overall oil content (i.e., the percentage of seed mass that is oil), as compared to the similar, but non-modified (non-transgenic) plant. A high oil phenotype refers to an increase in overall oil content. The phrase “altered protein content phenotype” refers to measurable phenotype of a genetically modified plant, where the plant displays a statistically significant increase or decrease in overall protein content (i.e., the percentage of seed mass that is protein), as compared to the similar, but non-modified plant. A high protein phenotype refers to an increase in overall protein content. The phrase “altered fiber content phenotype” refers to measurable phenotype of a genetically modified plant, where the plant displays a statistically significant increase or decrease in overall fiber content (i.e., the percentage of seed mass that is fiber), as compared to the similar, but non-modified plant. A low fiber phenotype refers to decrease in overall fiber content.
As used herein, a “mutant” polynucleotide sequence or gene differs from the corresponding wild-type polynucleotide sequence or gene either in terms of sequence or expression, where the difference contributes to a modified or altered plant phenotype or trait. Relative to a plant or plant line, the term “mutant” refers to a plant or plant line which has a modified or altered plant phenotype or trait, where the modified or altered phenotype or trait is associated with the modified or altered expression of a wild-type polynucleotide sequence or gene.
As used herein, the term “T1” refers to the generation of plants from the seed of T0 plants. The T1 generation is the first set of transformed plants that can be selected by application of a selection agent, e.g., an antibiotic or herbicide, for which the transgenic plant contains the corresponding resistance gene. The term “T2” refers to the generation of plants by self-fertilization of the flowers of T1 plants, previously selected as being transgenic. T3 plants are generated from T2 plants, etc. As used herein, the “direct progeny” of a given plant derives from the seed (or, sometimes, other tissue) of that plant and is in the immediately subsequent generation; for instance, for a given lineage, a T2 plant is the direct progeny of a T1 plant. The “indirect progeny” of a given plant derives from the seed (or other tissue) of the direct progeny of that plant, or from the seed (or other tissue) of subsequent generations in that lineage; for instance, a T3 plant is the indirect progeny of a T1 plant.
As used herein, the term “plant part” includes any plant organ or tissue, including, without limitation, seeds, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plant cells can be obtained from any plant organ or tissue and cultures prepared therefrom. Provided herein is a transgenic plant cell having an IMQ phenotype and/or an IOQ phenotype. The transgenic plant cell comprises a transformation vector comprising an IMQ nucleotide sequence that encodes or is complementary to a sequence that encodes an IMQ polypeptide. In preferred embodiments, the transgenic plant cell is selected from the group consisting of plants of the Brassica species, including canola and rapeseed, soy, corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax, castor, peanut, wheat, oat and rice. In other embodiments, the plant cell is a seed, pollen, propagule, or embryo cell. The disclosure also provides plant cells from a plant that is the direct progeny or the indirect progeny of a plant grown from said progenitor cells. The class of plants which can be used in the methods of the present invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
As used herein, “transgenic plant” includes a plant that comprises within its genome a heterologous polynucleotide. The heterologous polynucleotide can be either stably integrated into the genome, or can be extra-chromosomal. Preferably, the polynucleotide of the present invention is stably integrated into the genome such that the polynucleotide is passed on to successive generations. A plant cell, tissue, organ, or plant into which the heterologous polynucleotides have been introduced is considered “transformed,” “transfected,” or “transgenic.” Direct and indirect progeny of transformed plants or plant cells that also contain the heterologous polynucleotide are also considered transgenic.
Disclosed herein are transgenic plants having an Improved Seed Quality phenotype. Transgenic plants with an Improved Seed Quality phenotype may include an improved oil quantity and/or an improved meal quality. Transgenic plants with improved meal quality have an IMQ phenotype and transgenic plants with improved oil quantity have an IOQ phenotype. The IMQ phenotype in a transgenic plant may include altered protein and/or fiber content in any part of the transgenic plant, for example in the seeds. The IOQ phenotype in a transgenic plant may include altered oil content in any part of the transgenic plant, for example in the seeds. In particular embodiments, a transgenic plant may include an IOQ phenotype and/or an IMQ phenotype. In some embodiments of a transgenic plant, the IMQ phenotype may be an increase in protein content in the seed and/or a decrease in the fiber content of the seed. In other embodiments of a transgenic plant, the IOQ phenotype is an increase in the oil content of the seed (a high oil phenotype). Also provided is seed meal derived from the seeds of transgenic plants, wherein the seeds have altered protein content and/or altered fiber content. Further provided is oil derived from the seeds of transgenic plants, wherein the seeds have altered oil content. Any of these changes can lead to an increase in the AME from the seed or seed meal from transgenic plants, relative to control, non-transgenic, or wild-type plants. Also provided herein is meal, feed, or food produced from any part of the transgenic plant with an IMQ phenotype and/or IOQ phenotype.
In certain embodiments, the disclosed transgenic plants comprise a transformation vector comprising an IMQ nucleotide sequence that encodes or is complementary to a sequence that encodes an “IMQ” polypeptide. In particular embodiments, expression of an IMQ polypeptide in a transgenic plant causes an altered oil content, an altered protein content, and/or an altered fiber content in the transgenic plant. In preferred embodiments, the transgenic plant is selected from the group consisting of plants of the Brassica species, including canola and rapeseed, soy, corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax, castor, peanut, wheat, oat and rice. Also provided is a method of producing oil or seed meal, comprising growing the transgenic plant and recovering oil and/or seed meal from said plant. The disclosure further provides feed, meal, grain, or seed comprising a nucleic acid sequence that encodes an IMQ polypeptide. The disclosure also provides feed, meal, grain, or seed comprising the IMQ polypeptide, or an ortholog thereof.
Various methods for the introduction of a desired polynucleotide sequence encoding the desired protein into plant cells are available and known to those of skill in the art and include, but are not limited to: (1) physical methods such as microinjection, electroporation, and microprojectile mediated delivery (biolistics or gene gun technology); (2) virus mediated delivery methods; and (3) Agrobacterium-mediated transformation methods (see, for example, WO 2007/053482 and WO 2005/107437, which are incorporated herein by reference in their entirety).
The most commonly used methods for transformation of plant cells are the Agrobacterium-mediated DNA transfer process and the biolistics or microprojectile bombardment mediated process (i.e., the gene gun). Typically, nuclear transformation is desired but where it is desirable to specifically transform plastids, such as chloroplasts or amyloplasts, plant plastids may be transformed utilizing a microprojectile-mediated delivery of the desired polynucleotide.
Agrobacterium-mediated transformation is achieved through the use of a genetically engineered soil bacterium belonging to the genus Agrobacterium. A number of wild-type and disarmed strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes harboring Ti or Ri plasmids can be used for gene transfer into plants. Gene transfer is done via the transfer of a specific DNA known as “T-DNA” that can be genetically engineered to carry any desired piece of DNA into many plant species.
Agrobacterium-mediated genetic transformation of plants involves several steps. The first step, in which the virulent Agrobacterium and plant cells are first brought into contact with each other, is generally called “inoculation.” Following the inoculation, the Agrobacterium and plant cells/tissues are permitted to be grown together for a period of several hours to several days or more under conditions suitable for growth and T-DNA transfer. This step is termed “co-culture.” Following co-culture and T-DNA delivery, the plant cells are treated with bactericidal or bacteriostatic agents to kill the Agrobacterium remaining in contact with the explant and/or in the vessel containing the explant. If this is done in the absence of any selective agents to promote preferential growth of transgenic versus non-transgenic plant cells, then this is typically referred to as the “delay” step. If done in the presence of selective pressure favoring transgenic plant cells, then it is referred to as a “selection” step. When a “delay” is used, it is typically followed by one or more “selection” steps.
With respect to microprojectile bombardment (U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880, U.S. Pat. No. 5,610,042; and PCT Publication WO 95/06128; each of which is specifically incorporated herein by reference in its entirety), particles are coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, platinum, and preferably, gold.
An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is the Biolistics Particle Delivery System (BioRad, Hercules, Calif.), which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with monocot plant cells cultured in suspension.
Microprojectile bombardment techniques are widely applicable, and may be used to transform virtually any plant species. Examples of species that have been transformed by microprojectile bombardment include monocot species such as maize (PCT Publication No. WO 95/06128), barley, wheat (U.S. Pat. No. 5,563,055, incorporated herein by reference in its entirety), rice, oat, rye, sugarcane, and sorghum, as well as a number of dicots including tobacco, soybean (U.S. Pat. No. 5,322,783, incorporated herein by reference in its entirety), sunflower, peanut, cotton, tomato, and legumes in general (U.S. Pat. No. 5,563,055, incorporated herein by reference in its entirety).
To select or score for transformed plant cells regardless of transformation methodology, the DNA introduced into the cell contains a gene that functions in a regenerable plant tissue to produce a compound that confers upon the plant tissue resistance to an otherwise toxic compound. Genes of interest for use as a selectable, screenable, or scorable marker would include but are not limited to GUS, green fluorescent protein (GFP), luciferase (LUX), antibiotic or herbicide tolerance genes. Examples of antibiotic resistance genes include the penicillins, kanamycin (and neomycin, G418, bleomycin), methotrexate (and trimethoprim), chloramphenicol, and tetracycline. Polynucleotide molecules encoding proteins involved in herbicide tolerance are known in the art, and include, but are not limited to a polynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) described in U.S. Pat. No. 5,627,061, U.S. Pat. No. 5,633,435, and U.S. Pat. No. 6,040,497 and aroA described in U.S. Pat. No. 5,094,945 for glyphosate tolerance; a polynucleotide molecule encoding bromoxynil nitrilase (Bxn) described in U.S. Pat. No. 4,810,648 for Bromoxynil tolerance; a polynucleotide molecule encoding phytoene desaturase (crtI) described in Misawa et al. (Plant J. 4:833-840, 1993) and Misawa et al. (Plant J. 6:481-489, 1994) for norflurazon tolerance; a polynucleotide molecule encoding acetohydroxyacid synthase (AHAS, also known as ALS) described in Sathasiivan et al. (Nucl. Acids Res. 18:2188-2193, 1990) for tolerance to sulfonylurea herbicides; and the bar gene described in DeBlock et al. (EMBO J. 6:2513-2519, 1987) for glufosinate and bialaphos tolerance.
The regeneration, development, and cultivation of plants from various transformed explants are well documented in the art. This regeneration and growth process typically includes the steps of selecting transformed cells and culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil. Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay, may be cultured in media that supports regeneration of plants. Developing plantlets are transferred to soil less plant growth mix, and hardened off, prior to transfer to a greenhouse or growth chamber for maturation.
The present invention can be used with any transformable cell or tissue. By transformable as used herein is meant a cell or tissue that is capable of further propagation to give rise to a plant. Those of skill in the art recognize that a number of plant cells or tissues are transformable in which after insertion of exogenous DNA and appropriate culture conditions the plant cells or tissues can form into a differentiated plant. Tissue suitable for these purposes can include but is not limited to immature embryos, scutellar tissue, suspension cell cultures, immature inflorescence, shoot meristem, nodal explants, callus tissue, hypocotyl tissue, cotyledons, roots, and leaves.
Any suitable plant culture medium can be used. Examples of suitable media would include but are not limited to MS-based media (Murashige and Skoog, Physiol. Plant, 15:473-497, 1962) or N6-based media (Chu et al., Scientia Sinica 18:659, 1975) supplemented with additional plant growth regulators including but not limited to auxins, cytokinins, ABA, and gibberellins. Those of skill in the art are familiar with the variety of tissue culture media, which when supplemented appropriately, support plant tissue growth and development and are suitable for plant transformation and regeneration. These tissue culture media can either be purchased as a commercial preparation, or custom prepared and modified. Those of skill in the art are aware that media and media supplements such as nutrients and growth regulators for use in transformation and regeneration and other culture conditions such as light intensity during incubation, pH, and incubation temperatures that can be optimized for the particular variety of interest.
One of ordinary skill will appreciate that, after an expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
Identification of Plants with an Improved Oil Quantity Phenotype and/or Improved Meal Quality Phenotype
An Arabidopsis activation tagging screen (ACTTAG) was used to identify the association between 1) ACTTAG plant lines with an altered protein, fiber and/or oil content (phenotype, for example, see columns 4, 5 and 6, respectively, of Table 1, below) and 2) the nucleic acid sequences identified in column 3 of Tables 2 and 3, wherein each nucleic acid sequence is provided with a gene alias or an IMQ designation (IMQ#; see column 1 in Tables 1, 2, and 3). Briefly, and as further described in the Examples, a large number of Arabidopsis plants were mutated with the pSKI015 vector, which comprises a T-DNA from the Ti plasmid of Agrobacterium tumefaciens, a viral enhancer element, and a selectable marker gene (Weigel et al., 2000, Plant Physiology, 122:1003-1013). When the T-DNA inserts into the genome of transformed plants, the enhancer element can cause up-regulation of genes in the vicinity, generally within about nine kilobases (kb) of the enhancers. T1 plants were exposed to the selective agent in order to specifically recover transformed plants that expressed the selectable marker and therefore harbored T-DNA insertions. T1 plants were allowed to grow to maturity, self-fertilize and produce seed. T2 seed was harvested, labeled and stored. To amplify the seed stocks, about eighteen T2 were sown in soil and, after germination, exposed to the selective agent to recover transformed T2 plants. T3 seed from these plants was harvested and pooled. Oil, protein and fiber content of the seed were estimated using Near Infrared Spectroscopy (NIR) as described in the Examples.
Quantitative determination of fatty acid (FA) content (column 7, Table 1) in T2 seeds was performed using the following methods. A sample of 15 to 20 T2 seeds from each line tested. This sample generally contained plants with homozygous insertions, no insertions, and hemizygous insertions in a standard 1:1:2 ratios. The seed sample was massed on UMT-2 ultra-microbalance (Mettler-Toledo Co., Ohio, USA) and then transferred to a glass extraction vial. Lipids were extracted from the seeds and trans-esterified in 500 μl 2.5% H2SO4 in MeOH for 3 hours at 80° C., following the method of Browse et al. (Biochem J 235:25-31, 1986) with modifications. A known amount of heptadecanoic acid was included in the reaction as an internal standard. 750 μl of water and 400 μl of hexane were added to each vial, which was then shaken vigorously and allowed to phase separate. Reaction vials were loaded directly onto gas chromatography (GC) for analysis and the upper hexane phase was sampled by the autosampler. Gas chromatography with Flame Ionization detection was used to separate and quantify the fatty acid methyl esters. Agilent 6890 Plus GC's were used for separation with Agilent Innowax columns (30 m×0.25 mm ID, 250 um film thickness). The carrier gas was Hydrogen at a constant flow of 2.5 ml/minute. 1 μl of sample was injected in splitless mode (inlet temperature 220° C., Purge flow 15 ml/min at 1 minute). The oven was programmed for an initial temperature of 105° C., initial time 0.5 minutes, followed by a ramp of 60° C. per minute to 175° C., a 40° C./minute ramp to 260° C. with a final hold time of 2 minutes. Detection was by Flame Ionization (Temperature 275° C., Fuel flow 30.0 ml/min, Oxidizer 400.0 ml/min). Instrument control and data collection and analysis were monitored using the Millennium Chromatography Management System (Version 3.2, Waters Corporation, Milford, Mass.). Peaks were initially identified by comparison with standards. Integration and quantification were performed automatically, but all analyses were subsequently examined manually to verify correct peak identification and acceptable signal to noise ratio before inclusion of the derived results in the study.
The association of an IMQ nucleic acid sequence with an IMQ phenotype or an IOQ phenotype was discovered by analysis of the genomic DNA sequence flanking the T-DNA insertion in the ACTTAG line identified in column 3 of Table 1. An ACTTAG line is a family of plants derived from a single plant that was transformed with a T-DNA element containing four tandem copies of the CaMV 35S enhancers. Accordingly, the disclosed IMQ nucleic acid sequences and/or polypeptides may be employed in the development of transgenic plants having an improved seed quality phenotype, including an IMQ phenotype and/or an IOQ phenotype. IMQ nucleic acid sequences may be used in the generation of transgenic plants, such as oilseed crops, that provide improved oil yield from oilseed processing and result in an increase in the quantity of oil recovered from seeds of the transgenic plant. IMQ nucleic acid sequences may also be used in the generation of transgenic plants, such as feed grain crops, that provide an IMQ phenotype resulting in increased energy for animal feeding, for example, seeds or seed meal with an altered protein and/or fiber content, resulting in an increase in AME. IMQ nucleic acid sequences may further be used to increase the oil content of specialty oil crops, in order to augment yield and/or recovery of desired unusual fatty acids. Transgenic plants that have been genetically modified to express IMQ polypeptides can be used in the production of seeds, wherein the transgenic plants are grown, and oil and seed meal are obtained from plant parts (e.g. seed) using standard methods.
IMO Nucleic Acids and Polypeptides
The IMQ designation for each of the IMQ nucleic acid sequences discovered in the activation tagging screen described herein are listed in column 1 of Tables 1-3, below. The disclosed IMQ polypeptides are listed in column 5 of Table 2 and column 4 of Table 3. As used herein, the term “IMQ polypeptide” refers to any polypeptide that when expressed in a plant causes an IMQ phenotype and/or an IOQ phenotype in any part of the plant, for example the seeds. In one embodiment, an IMQ polypeptide refers to a full-length IMQ protein, or a fragment, derivative (variant), or ortholog thereof that is “functionally active,” such that the protein fragment, derivative, or ortholog exhibits one or more or the functional activities associated with one or more of the disclosed full-length IMQ polypeptides, for example, the amino acid sequences provided in the GenBank entry referenced in column 5 of Table 2, which correspond to the amino acid sequences set forth as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, or SEQ ID NO: 100, or an ortholog thereof. In one preferred embodiment, a functionally active IMQ polypeptide causes an IMQ phenotype and/or an IOQ phenotype in a transgenic plant. In another embodiment, a functionally active IMQ polypeptide causes an altered oil, protein, and/or fiber content phenotype (for example, an altered seed meal content phenotype) when mis-expressed in a plant. In other preferred embodiments, mis-expression of the IMQ polypeptide causes a high oil (such as, increased oil), high protein (such as, increased protein), and/or low fiber (such as, decreased fiber) phenotype in a plant. In another embodiment, mis-expression of the IMQ polypeptide causes an improved AME of meal. In yet another embodiment, a functionally active IMQ polypeptide can rescue defective (including deficient) endogenous IMQ activity when expressed in a plant or in plant cells; the rescuing polypeptide may be from the same or from a different species as the species with the defective polypeptide activity. The disclosure also provides feed, meal, grain, food, or seed comprising the IMQ polypeptide, or a fragment, derivative (variant), or ortholog thereof.
In another embodiment, a functionally active fragment of a full length IMQ polypeptide (for example, a functionally active fragment of a native polypeptide having the amino acid sequence set forth as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, or SEQ ID NO: 100, or a naturally occurring ortholog thereof) retains one or more of the biological properties associated with the full-length IMQ polypeptide, such as signaling activity, binding activity, catalytic activity, or cellular or extra-cellular localizing activity. An IMQ fragment preferably comprises an IMQ domain, such as a C- or N-terminal or catalytic domain, among others, and preferably comprises at least 10, preferably at least 20, more preferably at least 25, and most preferably at least 50 contiguous amino acids of an IMQ protein. Functional domains of IMQ genes are listed in column 8 of Table 2 and can be identified using the PFAM program (Bateman A et al., 1999, Nucleic Acids Res. 27:260-262) or INTERPRO (Mulder et al., 2003, Nucleic Acids Res. 31, 315-318) program. Functionally active variants of full-length IMQ polypeptides, or fragments thereof, include polypeptides with amino acid insertions, deletions, or substitutions that retain one of more of the biological properties associated with the full-length IMQ polypeptide. In some cases, variants are generated that change the post-translational processing of an IMQ polypeptide. For instance, variants may have altered protein transport or protein localization characteristics, or altered protein half-life, compared to the native polypeptide.
As used herein, the term “IMQ nucleic acid” refers to any polynucleotide that when expressed in a plant causes an IMQ phenotype and/or an IOQ phenotype in any part of the plant, for example the seeds. In one embodiment, an IMQ polynucleotide encompasses nucleic acids with the sequence provided in or complementary to the GenBank entry referenced in column 3 of Table 2, which correspond to nucleic acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73 SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, or SEQ ID NO: 99, as well as functionally active fragments, derivatives, or orthologs thereof. An IMQ nucleic acid of this disclosure may be DNA, derived from genomic DNA or cDNA, or RNA. Genomic sequences of the genes listed in Table 2 are known and available in public databases such as GenBank.
In one embodiment, a functionally active IMQ nucleic acid encodes or is complementary to a nucleic acid that encodes a functionally active IMQ polypeptide. A functionally active IMQ nucleic acid also includes genomic DNA that serves as a template for a primary RNA transcript (i.e., an mRNA precursor) that requires processing, such as splicing, before encoding the functionally active IMQ polypeptide. An IMQ nucleic acid can include other non-coding sequences, which may or may not be transcribed; such sequences include 5′ and 3′ UTRs, polyadenylation signals and regulatory sequences that control gene expression, among others, as are known in the art. Some polypeptides require processing events, such as proteolytic cleavage, covalent modification, etc., in order to become fully active. Accordingly, functionally active nucleic acids may encode the mature or the pre-processed IMQ polypeptide, or an intermediate form. An IMQ polynucleotide can also include heterologous coding sequences, for example, sequences that encode a marker included to facilitate the purification of the fused polypeptide, or a transformation marker. In another embodiment, a functionally active IMQ nucleic acid is capable of being used in the generation of loss-of-function IMQ phenotypes, for instance, via antisense suppression, co-suppression, etc. The disclosure also provides feed, meal, grain, food, or seed comprising a nucleic acid sequence that encodes an IMQ polypeptide.
In one preferred embodiment, an IMQ nucleic acid used in the disclosed methods comprises a nucleic acid sequence that encodes, or is complementary to a sequence that encodes, an IMQ polypeptide having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to a disclosed IMQ polypeptide sequence, for example the amino acid sequence set forth as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, or SEQ ID NO: 100.
In another embodiment, an IMQ polypeptide comprises a polypeptide sequence with at least 50% or 60% identity to a disclosed IMQ polypeptide sequence (for example, the amino acid sequence set forth as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, or SEQ ID NO: 100) and may have at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to a disclosed IMQ polypeptide sequence. In a further embodiment, an IMQ polypeptide comprises 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to a disclosed IMQ polypeptide sequence, and may include a conserved protein domain of the IMQ polypeptide (such as the protein domain(s) listed in column 8 of Table 2). In another embodiment, an IMQ polypeptide comprises a polypeptide sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to a functionally active fragment of the polypeptide referenced in column 5 of Table 2. In yet another embodiment, an IMQ polypeptide comprises a polypeptide sequence with at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity to the polypeptide sequence of the GenBank entry referenced in column 5 of Table 2 over its entire length and comprises a conserved protein domain(s) listed in column 8 of Table 2.
In another aspect, an IMQ polynucleotide sequence is at least 50% to 60% identical over its entire length to a disclosed IMQ nucleic acid sequence, such as the nucleic acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73 SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, or SEQ ID NO: 99, or nucleic acid sequences that are complementary to such an IMQ sequence, and may comprise at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to the disclosed IMQ sequence, or a functionally active fragment thereof, or complementary sequences. In another embodiment, a disclosed IMQ nucleic acid comprises a nucleic acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73 SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, or SEQ ID NO: 99, or nucleic acid sequences that are complementary to such an IMQ sequence, and nucleic acid sequences that have substantial sequence homology to a such IMQ sequences. As used herein, the phrase “substantial sequence homology” refers to those nucleic acid sequences that have slight or inconsequential sequence variations from such IMQ sequences, i.e., the sequences function in substantially the same manner and encode an IMQ polypeptide.
As used herein, “percent (%) sequence identity” with respect to a specified subject sequence, or a specified portion thereof, is defined as the percentage of nucleotides or amino acids in an identified sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol., 1990, 215:403-410) with search parameters set to default values. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. A “percent (%) identity value” is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported. “Percent (%) amino acid sequence similarity” is determined by performing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation. A conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected. Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine and glycine.
Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that selectively hybridize to the disclosed IMQ nucleic acid sequences (for example, the nucleic acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73 SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, or SEQ ID NO: 99). The stringency of hybridization can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. Conditions routinely used are well known (see, e.g., Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Press, Plainview, N.Y.).
In some embodiments, a nucleic acid molecule of the disclosure is capable of hybridizing to a nucleic acid molecule containing the disclosed nucleotide sequence under stringent hybridization conditions that are: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65° C. in a solution comprising 6× single strength citrate (SSC) (1×SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5×Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C. in a solution containing 6×SSC, 1×Denhardt's solution, 100 μg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C. for 1 h in a solution containing 0.1×SSC and 0.1% SDS (sodium dodecyl sulfate). In other embodiments, moderately stringent hybridization conditions are used that are: pretreatment of filters containing nucleic acid for 6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C. in a solution containing 2×SSC and 0.1% SDS. Alternatively, low stringency conditions can be used that comprise: incubation for 8 hours to overnight at 37° C. in a solution comprising 20% formamide, 5×SSC, 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1×SSC at about 37° C. for 1 hour.
As a result of the degeneracy of the genetic code, a number of polynucleotide sequences encoding an IMQ polypeptide can be produced. For example, codons may be selected to increase the rate at which expression of the polypeptide occurs in a particular host species, in accordance with the optimum codon usage dictated by the particular host organism (see, e.g., Nakamura et al., 1999, Nucleic Acids Res. 27:292). Such sequence variants may be used in the methods disclosed herein.
The disclosed methods may use orthologs of a disclosed Arabidopsis IMQ nucleic acid sequence. Representative putative orthologs of each of the disclosed Arabidopsis IMQ genes are identified in column 3 of Table 3, below. Methods of identifying the orthologs in other plant species are known in the art. In general, orthologs in different species retain the same function, due to presence of one or more protein motifs and/or 3-dimensional structures. In evolution, when a gene duplication event follows speciation, a single gene in one species, such as Arabidopsis, may correspond to multiple genes (paralogs) in another. As used herein, the term “orthologs” encompasses paralogs. When sequence data is available for a particular plant species, orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences. Sequences are assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen M A and Bork P, 1998, Proc. Natl. Acad. Sci., 95:5849-5856; Huynen M A et al., 2000, Genome Research, 10:1204-1210).
Programs for multiple sequence alignment, such as CLUSTAL (Thompson J D et al., 1994, Nucleic Acids Res. 22:4673-4680) may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees. In a phylogenetic tree representing multiple homologous sequences from diverse species (e.g., retrieved through BLAST analysis), orthologous sequences from two species generally appear closest on the tree with respect to all other sequences from these two species. Structural threading or other analysis of protein folding (e.g., using software by ProCeryon, Biosciences, Salzburg, Austria) may also identify potential orthologs. Nucleic acid hybridization methods may also be used to find orthologous genes and are preferred when sequence data are not available. Degenerate PCR and screening of cDNA or genomic DNA libraries are common methods for finding related gene sequences and are well known in the art (see, e.g., Sambrook, 1989, Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Press, Plainview, N.Y.; Dieffenbach and Dveksler, 1995, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY). For instance, methods for generating a cDNA library from the plant species of interest and probing the library with partially homologous gene probes are described in Sambrook et al. A highly conserved portion of the Arabidopsis IMQ coding sequence may be used as a probe. IMQ ortholog nucleic acids may hybridize to the nucleic acid of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73 SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, or SEQ ID NO: 99 under high, moderate, or low stringency conditions. After amplification or isolation of a segment of a putative ortholog, that segment may be cloned and sequenced by standard techniques and utilized as a probe to isolate a complete cDNA or genomic DNA clone.
Alternatively, it is possible to initiate an EST project to generate a database of sequence information for the plant species of interest. In another approach, antibodies that specifically bind known IMQ polypeptides are used for ortholog isolation (see, e.g., Harlow and Lane, 1988, 1999, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York). Western blot analysis can determine that an IMQ ortholog (i.e., a protein orthologous to a disclosed IMQ polypeptide) is present in a crude extract of a particular plant species. When reactivity is observed, the sequence encoding the candidate ortholog may be isolated by screening expression libraries representing the particular plant species. Expression libraries can be constructed in a variety of commercially available vectors, including lambda gtl1, as described in Sambrook, et al., 1989. Once the candidate ortholog(s) are identified by any of these means, candidate orthologous sequence are used as bait (the “query”) for the reverse BLAST against sequences from Arabidopsis or other species in which IMQ nucleic acid and/or polypeptide sequences have been identified.
IMQ nucleic acids and polypeptides may be obtained using any available method. For instance, techniques for isolating cDNA or genomic DNA sequences of interest by screening DNA libraries or by using polymerase chain reaction (PCR), as previously described, are well known in the art. Alternatively, nucleic acid sequence may be synthesized. Any known method, such as site directed mutagenesis (Kunkel T A et al., 1991, Methods Enzymol. 204:125-39), may be used to introduce desired changes into a cloned nucleic acid.
In general, the methods disclosed herein involve incorporating the desired form of the IMQ nucleic acid into a plant expression vector for transformation of plant cells, and the IMQ polypeptide is expressed in the host plant. Transformed plants and plant cells expressing an IMQ polypeptide express an IMQ phenotype and/or an IOQ phenotype and, in one specific, non-limiting example, may have high (increased) oil, high (increased) protein, and/or low (decreased) fiber content.
An “isolated” IMQ nucleic acid molecule is other than in the form or setting in which it is found in nature, and is identified and separated from least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the IMQ nucleic acid. However, an isolated IMQ nucleic acid molecule includes IMQ nucleic acid molecules contained in cells that ordinarily express IMQ where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
Generation of Genetically Modified Plants with an Improved Oil Quantity Phenotype and/or an Improved Meal Quality Phenotype
The disclosed IMQ nucleic acids and polypeptides may be used in the generation of transgenic plants having a modified or altered oil, protein, and/or fiber content phenotype. As used herein, an “altered oil content (phenotype)” may refer to altered oil content in any part of the plant. In a preferred embodiment, altered expression of the IMQ gene in a plant is used to generate plants with a high oil content (phenotype). As used herein, an “altered protein content (phenotype)” may refer to altered protein content in any part of the plant. In a preferred embodiment, altered expression of the IMQ gene in a plant is used to generate plants with a high (or increased) protein content (phenotype). As used herein, an “altered fiber content (phenotype)” may refer to altered fiber content in any part of the plant. In a preferred embodiment, altered expression of the IMQ gene in a plant is used to generate plants with a low (or decreased) fiber content (phenotype). The altered oil, protein, and/or fiber content is often observed in seeds. Examples of a transgenic plant include plants comprising a plant transformation vector with a nucleotide sequence that encodes or is complementary to a sequence that encodes an IMQ polypeptide having the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, or SEQ ID NO: 100, or an ortholog thereof.
Transgenic plants, such as corn, soybean and canola containing the disclosed nucleic acid sequences, can be used in the production of vegetable oil and meal. Vegetable oil is used in a variety of food products, while meal from seed is used as an animal feed. After harvesting seed from transgenic plants, the seed is cleaned to remove plant stalks and other material and then flaked in roller mills to break the hulls. The crushed seed is heated to 75-100° C. to denature hydrolytic enzymes, lyse the unbroken oil containing cells, and allow small oil droplets to coalesce. Most of the oil is then removed (and can be recovered) by pressing the seed material in a screw press. The remaining oil is removed from the presscake by extraction with and organic solvents, such as hexane. The solvent is removed from the meal by heating it to approximately 100° C. After drying, the meal is then granulated to a consistent form. The meal, containing the protein, digestible carbohydrate, and fiber of the seed, may be mixed with other materials prior to being used as an animal feed.
The methods described herein for generating transgenic plants are generally applicable to all plants. Although activation tagging and gene identification is carried out in Arabidopsis, the IMQ nucleic acid sequence (or an ortholog, variant or fragment thereof) may be expressed in any type of plant. In a preferred embodiment, oil-producing plants produce and store triacylglycerol in specific organs, primarily in seeds. Such species include soybean (Glycine max), rapeseed and canola (including Brassica napus, B. campestris), sunflower (Helianthus annus), cotton (Gossypium hirsutum), corn (Zea mays), cocoa (Theobroma cacao), safflower (Carthamus tinctorius), oil palm (Elaeis guineensis), coconut palm (Cocos nucifera), flax (Linum usitatissimum), castor (Ricinus communis), and peanut (Arachis hypogaea), as well as wheat, rice and oat. Fruit- and vegetable-bearing plants, grain-producing plants, nut-producing plants, rapid cycling Brassica species, alfalfa (Medicago sativa), tobacco (Nicotiana), turfgrass (Poaceae family), other forage crops, and wild species may also be a source of unique fatty acids. In other embodiments, any plant expressing the IMQ nucleic acid sequence can also express increased protein and/or decreased fiber content in a specific plant part or organ, such as in seeds.
The skilled artisan will recognize that a wide variety of transformation techniques exist in the art, and new techniques are continually becoming available. Any technique that is suitable for the target host plant can be employed within the scope of the present invention. For example, the constructs can be introduced in a variety of forms including, but not limited to, as a strand of DNA, in a plasmid, or in an artificial chromosome. The introduction of the constructs into the target plant cells can be accomplished by a variety of techniques, including, but not limited to, Agrobacterium-mediated transformation, electroporation, microinjection, microprojectile bombardment, calcium-phosphate-DNA co-precipitation, or liposome-mediated transformation of a heterologous nucleic acid. The transformation of the plant is preferably permanent, i.e. by integration of the introduced expression constructs into the host plant genome, so that the introduced constructs are passed onto successive plant generations. Depending upon the intended use, a heterologous nucleic acid construct comprising an IMQ polynucleotide may encode the entire protein or a biologically active portion thereof.
In one embodiment, binary Ti-based vector systems may be used to transfer polynucleotides. Standard Agrobacterium binary vectors are known to those of skill in the art, and many are commercially available (e.g., pBI121 Clontech Laboratories, Palo Alto, Calif.). A construct or vector may include a plant promoter to express the nucleic acid molecule of choice. In a preferred embodiment, the promoter is a plant promoter.
The optimal procedure for transformation of plants with Agrobacterium vectors will vary with the type of plant being transformed. Exemplary methods for Agrobacterium-mediated transformation include transformation of explants of hypocotyl, shoot tip, stem or leaf tissue, derived from sterile seedlings and/or plantlets. Such transformed plants may be reproduced sexually, or by cell or tissue culture. Agrobacterium transformation has been previously described for a large number of different types of plants and methods for such transformation may be found in the scientific literature. Of particular relevance are methods to transform commercially important crops, such as plants of the Brassica species, including canola and rapeseed, (De Block et al., 1989, Plant Physiol., 91:694-701), sunflower (Everett et al., 1987, Bio/Technology, 5:1201), soybean (Christou et al., 1989, Proc. Natl. Acad. Sci USA, 86:7500-7504; Kline et al., 1987, Nature, 327:70), wheat, rice and oat.
Expression (including transcription and translation) of an IMQ nucleic acid sequence may be regulated with respect to the level of expression, the tissue type(s) where expression takes place and/or developmental stage of expression. A number of heterologous regulatory sequences (e.g., promoters and enhancers) are available for controlling the expression of an IMQ nucleic acid. These include constitutive, inducible and regulatable promoters, as well as promoters and enhancers that control expression in a tissue- or temporal-specific manner. Exemplary constitutive promoters include the raspberry E4 promoter (U.S. Pat. Nos. 5,783,393 and 5,783,394), the nopaline synthase (NOS) promoter (Ebert et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:5745-5749, 1987), the octopine synthase (OCS) promoter (which is carried on tumor-inducing plasmids of Agrobacterium tumefaciens), the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Mol. Biol. 9:315-324, 1987) and the CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985 and Jones J D et al, 1992, Transgenic Res., 1:285-297), the figwort mosaic virus 35S-promoter (U.S. Pat. No. 5,378,619), the light-inducible promoter from the small subunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), the Adh promoter (Walker et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:6624-6628, 1987), the sucrose synthase promoter (Yang et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:4144-4148, 1990), the R gene complex promoter (Chandler et al., The Plant Cell 1:1175-1183, 1989), the chlorophyll a/b binding protein gene promoter, the CsVMV promoter (Verdaguer B et al., 1998, Plant Mol Biol., 37:1055-1067), and the melon actin promoter (published PCT application WO0056863). Exemplary tissue-specific promoters include the tomato E4 and E8 promoters (U.S. Pat. No. 5,859,330) and the tomato 2AII gene promoter (Van Haaren M J J et al., 1993, Plant Mol Bio., 21:625-640).
In one preferred embodiment, expression of the IMQ nucleic acid sequence is under control of regulatory sequences from genes whose expression is associated with early seed and/or embryo development. Indeed, in a preferred embodiment, the promoter used is a seed-enhanced promoter. Examples of such promoters include the 5′ regulatory regions from such genes as napin (Kridl et al., Seed Sci. Res. 1:209:219, 1991), globulin (Belanger and Kriz, Genet., 129: 863-872, 1991, GenBank Accession No. L22295), gamma zein Z 27 (Lopes et al., Mol Gen Genet., 247:603-613, 1995), L3 oleosin promoter (U.S. Pat. No. 6,433,252), phaseolin (Bustos et al., Plant Cell, 1(9):839-853, 1989), arcelin5 (U.S. Application No. 2003/0046727), a soybean 7S promoter, a 7Sα promoter (U.S. Application No. 2003/0093828), the soybean 7Sα′ beta conglycinin promoter, a 7S α′ promoter (Beachy et al., EMBO J., 4:3047, 1985; Schuler et al., Nucleic Acid Res., 10(24):8225-8244, 1982), soybean trypsin inhibitor (Riggs et al., Plant Cell 1(6):609-621, 1989), ACP (Baerson et al., Plant Mol. Biol., 22(2):255-267, 1993), stearoyl-ACP desaturase (Slocombe et al., Plant Physiol. 104(4):167-176, 1994), soybean a′ subunit of β-conglycinin (Chen et al., Proc. Natl. Acad. Sci. 83:8560-8564, 1986), Vicia faba USP (P-Vf.Usp, SEQ ID NO: 1, 2, and 3 in (U.S. Application No. 2003/229918) and Zea mays L3 oleosin promoter (Hong et al., Plant Mol. Biol., 34(3):549-555, 1997). Also included are the zeins, which are a group of storage proteins found in corn endosperm. Genomic clones for zein genes have been isolated (Pedersen et al., Cell, 29:1015-1026, 1982; and Russell et al., Transgenic Res. 6(2):157-168) and the promoters from these clones, including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD and genes, could also be used. Other promoters known to function, for example, in corn include the promoters for the following genes: waxy, Brittle, Shrunken 2, Branching enzymes I and II, starch synthases, debranching enzymes, oleosins, glutelins and sucrose synthases. Legume genes whose promoters are associated with early seed and embryo development include V. faba legumin (Baumlein et al., 1991, Mol. Gen. Genet. 225:121-8; Baumlein et al., 1992, Plant J. 2:233-9), V. faba usp (Fiedler et al., 1993, Plant Mol. Biol. 22:669-79), pea convicilin (Bown et al., 1988, Biochem. J. 251:717-26), pea lectin (dePater et al., 1993, Plant Cell 5:877-86), P. vulgaris beta phaseolin (Bustos et al., 1991, EMBO J. 10:1469-79), P. vulgaris DLEC2 and PHS [beta] (Bobb et al., 1997, Nucleic Acids Res. 25:641-7), and soybean beta-Conglycinin, 7S storage protein (Chamberland et al., 1992, Plant Mol. Biol. 19:937-49).
Cereal genes whose promoters are associated with early seed and embryo development include rice glutelin (“GluA-3,” Yoshihara and Takaiwa, 1996, Plant Cell Physiol. 37:107-11; “GluB-1,” Takaiwa et al., 1996, Plant Mol. Biol. 30:1207-21; Washida et al., 1999, Plant Mol. Biol. 40:1-12; “Gt3,” Leisy et al., 1990, Plant Mol. Biol. 14:41-50), rice prolamin (Zhou & Fan, 1993, Transgenic Res. 2:141-6), wheat prolamin (Hammond-Kosack et al., 1993, EMBO J. 12:545-54), maize zein (Z4, Matzke et al., 1990, Plant Mol. Biol. 14:323-32), and barley B-hordeins (Entwistle et al., 1991, Plant Mol. Biol. 17:1217-31).
Other genes whose promoters are associated with early seed and embryo development include oil palm GLO7A (7S globulin, Morcillo et al., 2001, Physiol. Plant 112:233-243), Brassica napus napin, 2S storage protein, and napA gene (Josefsson et al., 1987, J. Biol. Chem. 262:12196-201; Stalberg et al., 1993, Plant Mol. Biol. 1993 23:671-83; Ellerstrom et al., 1996, Plant Mol. Biol. 32:1019-27), Brassica napus oleosin (Keddie et al., 1994, Plant Mol. Biol. 24:327-40), Arabidopsis oleosin (Plant et al., 1994, Plant Mol. Biol. 25:193-205), Arabidopsis FAE1 (Rossak et al., 2001, Plant Mol. Biol. 46:717-25), Canavalia gladiata conA (Yamamoto et al., 1995, Plant Mol. Biol. 27:729-41), and Catharanthus roseus strictosidine synthase (Str, Ouwerkerk and Memelink, 1999, Mol. Gen. Genet. 261:635-43). In another preferred embodiment, regulatory sequences from genes expressed during oil biosynthesis are used (see, e.g., U.S. Pat. No. 5,952,544). Alternative promoters are from plant storage protein genes (Bevan et al., 1993, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 342:209-15). Additional promoters that may be utilized are described, for example, in U.S. Pat. Nos. 5,378,619; 5,391,725; 5,428,147; 5,447,858; 5,608,144; 5,608,144; 5,614,399; 5,633,441; 5,633,435; and 4,633,436.
In yet another aspect, in some cases it may be desirable to inhibit the expression of the endogenous IMQ nucleic acid sequence in a host cell. Exemplary methods for practicing this aspect of the invention include, but are not limited to antisense suppression (Smith, et al., 1988, Nature, 334:724-726; van der Krol et al., 1988, BioTechniques, 6:958-976); co-suppression (Napoli, et al., 1990, Plant Cell, 2:279-289); ribozymes (PCT Publication WO 97/10328); and combinations of sense and antisense (Waterhouse, et al., 1998, Proc. Natl. Acad. Sci. USA, 95:13959-13964). Methods for the suppression of endogenous sequences in a host cell typically employ the transcription or transcription and translation of at least a portion of the sequence to be suppressed. Such sequences may be homologous to coding as well as non-coding regions of the endogenous sequence. Antisense inhibition may use the entire cDNA sequence (Sheehy et al., 1988, Proc. Natl. Acad. Sci. USA, 85:8805-8809), a partial cDNA sequence including fragments of 5′ coding sequence, (Cannon et al., 1990, Plant Mol. Biol., 15:39-47), or 3′ non-coding sequences (Ch'ng et al., 1989, Proc. Natl. Acad. Sci. USA, 86:10006-10010). Cosuppression techniques may use the entire cDNA sequence (Napoli et al., 1990, Plant Cell, 2:279-289; van der Krol et al., 1990, Plant Cell, 2:291-299), or a partial cDNA sequence (Smith et al., 1990, Mol. Gen. Genetics, 224:477-481).
Standard molecular and genetic tests may be performed to further analyze the association between a nucleic acid sequence and an observed phenotype. Exemplary techniques are described below.
1. DNA/RNA Analysis
The stage- and tissue-specific gene expression patterns in mutant versus wild-type lines may be determined, for instance, by in situ hybridization. Analysis of the methylation status of the gene, especially flanking regulatory regions, may be performed. Other suitable techniques include over-expression, ectopic expression, expression in other plant species and gene knock-out (reverse genetics, targeted knock-out, viral induced gene silencing (VIGS; see, Baulcombe, Arch. Virol. Suppl. 15:189-201, 1999).
In a preferred application expression profiling, generally by microarray analysis, is used to simultaneously measure differences or induced changes in the expression of many different genes. Techniques for microarray analysis are well known in the art (Schena M et al., Science 1995 270:467-470; Baldwin et al., 1999, Cur. Opin. Plant Biol. 2(2):96-103; Dangond, Physiol Genomics (2000) 2:53-58; van Hal et al., J Biotechnol. (2000) 78:271-280; Richmond T and Somerville S, Curr. Opin. Plant Biol. 2000 3:108-116). Expression profiling of individual tagged lines may be performed. Such analysis can identify other genes that are coordinately regulated as a consequence of the over-expression of the gene of interest, which may help to place an unknown gene in a particular pathway.
2. Gene Product Analysis
Analysis of gene products may include recombinant protein expression, antisera production, immunolocalization, biochemical assays for catalytic or other activity, analysis of phosphorylation status, and analysis of interaction with other proteins via yeast two-hybrid assays.
3. Pathway Analysis
Pathway analysis may include placing a gene or gene product within a particular biochemical, metabolic or signaling pathway based on its mis-expression phenotype or by sequence homology with related genes. Alternatively, analysis may comprise genetic crosses with wild-type lines and other mutant lines (creating double mutants) to order the gene in a pathway, or determining the effect of a mutation on expression of downstream “reporter” genes in a pathway.
Generation of Mutated Plants with an Improved Oil Quantity Phenotype and/or Improved Meal Quality Phenotype
Additional methods are disclosed herein of generating a plant having an IMQ and/or an IOQ phenotype, wherein a plant is identified that has an allele in its IMQ nucleic acid sequence that results in an IMQ phenotype and/or an IOQ phenotype, compared to plants lacking the allele. The plant can generate progeny, wherein the progeny inherit the allele and have an IMQ phenotype and/or an IOQ phenotype. For example, provided herein is a method of identifying plants that have mutations in the endogenous IMQ nucleic acid sequence that confer an IMQ phenotype and/or an IOQ phenotype and generating progeny of these plants with an IMQ and/or IOQ phenotype that are not genetically modified. In some embodiments, the plants have an IMQ phenotype with an altered protein and/or fiber content or seed meal content, or an IOQ phenotype, with an altered oil content.
In one method, called “TILLING” (for targeting induced local lesions in genomes), mutations are induced in the seed of a plant of interest, for example, using EMS (ethylmethane sulfonate) treatment. The resulting plants are grown and self-fertilized, and the progeny are used to prepare DNA samples. PCR amplification and sequencing of the IMQ nucleic acid sequence is used to identify whether a mutated plant has a mutation in the IMQ nucleic acid sequence. Plants having IMQ mutations may then be tested for altered oil, protein, and/or fiber content, or alternatively, plants may be tested for altered oil, protein, and/or fiber content, and then PCR amplification and sequencing of the IMQ nucleic acid sequence is used to determine whether a plant having altered oil, protein, and/or fiber content has a mutated IMQ nucleic acid sequence. TILLING can identify mutations that may alter the expression of specific genes or the activity of proteins encoded by these genes (see Colbert et al., 2001, Plant Physiol. 126:480-484; McCallum et al., 2000, Nature Biotechnology 18:455-457).
In another method, a candidate gene/Quantitative Trait Locus (QTLs) approach can be used in a marker-assisted breeding program to identify alleles of or mutations in the IMQ nucleic acid sequence or orthologs of the IMQ nucleic acid sequence that may confer altered oil, protein, and/or fiber content (see Bert et al., Theor Appl Genet., 2003; 107(1):181-9; and Lionneton et al., Genome, 2002; 45(6):1203-15). Thus, in a further aspect of the disclosure, an IMQ nucleic acid is used to identify whether a plant having altered oil, protein, and/or fiber content has a mutation an endogenous IMQ nucleic acid sequence or has a particular allele that causes altered oil, protein, and/or fiber content.
While the disclosure has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the disclosure. All publications cited herein are expressly incorporated herein by reference for the purpose of describing and disclosing compositions and methodologies that might be used in connection with the disclosure. All cited patents, patent applications, and sequence information in referenced public databases are also incorporated by reference.
EXAMPLES
Example 1
Generation of Plants with an IMQ Phenotype and/or an TOO Phenotype by Transformation with an Activation Tagging Construct
This Example describes the generation of transgenic plants with altered oil, protein, and/or fiber content.
Mutants were generated using the activation tagging “ACTTAG” vector, pSKI015 (GI#6537289; Weigel D et al., 2000, Plant Physiology, 122:1003-1013). Standard methods were used for the generation of Arabidopsis transgenic plants, and were essentially as described in published application PCT WO0183697. Briefly, T0 Arabidopsis (Col-0) plants were transformed with Agrobacterium carrying the pSKI015 vector, which comprises T-DNA derived from the Agrobacterium Ti plasmid, an herbicide resistance selectable marker gene, and the 4× CaMV 35S enhancer element. Transgenic plants were selected at the T1 generation based on herbicide resistance. T2 seed (from T1 plants) was harvested and sown in soil. T2 plants were exposed to the herbicide to kill plants lacking the ACTTAG vector. T2 plants were grown to maturity, allowed to self-fertilize and set seed. T3 seed (from the T2 plants) was harvested in bulk for each line.
T3 seed was analyzed by Near Infrared Spectroscopy (NIR) at the time of harvest. NIR spectra were captured using a Bruker 22 near infrared spectrometer. Bruker Software was used to estimate total seed oil, total seed protein and total seed fiber content using data from NIR analysis and reference methods according to the manufacturer's instructions. Oil content predicting calibrations were developed following the general method of AOCS Procedure Am1-92, Official Methods and Recommended Practices of the American Oil Chemists Society, 5th Ed., AOCS, Champaign, Ill. A NIR protein content predicting calibration was developed using total nitrogen content data of seed samples following the general method of Dumas Procedure AOAC 968.06 (Official Methods of Analysis of AOAC International 17th Edition AOAC, Gaithersburg, Md.). A fiber content predicting calibration was developed by measuring crude fiber content in a set of seed samples. Fiber content of in a known mass of seed was determined using the method of Honig and Rackis, (1979, J. Agri. Food Chem., 27: 1262-1266). Digestible protein content of in a known mass of seed was determined by quantifying the individual amino acids liberated by an acid hydrolysis Steine and Moore (1958, Anal. Chem., 30:1185-1190). The quantification was performed by the Amino Quant (Agilent). The undigested protein remaining associated with the non-digestible fraction is measured by the same method described for the whole seed homogenate. Digestible protein content is determined by subtracting the amount of undigested protein associated with the non-digestible fraction from the total amount of protein in the seed sample.
Seed oil, protein, digestible protein and fiber values in 82,274 lines were determined by NIR spectroscopy and normalized to allow comparison of seed component values in plants grown at different times. Oil, protein and fiber values were normalized by calculating the average oil, protein and fiber values in seed from all plants planted on the same day (including a large number of other ACTTAG plants, including control, wild-type, or non-transgenic plants). The seed components for each line was expressed as a “percent relative value” which was calculated by dividing the component value for each line with the average component value for all lines planted on the same day (which should approximate the value in control, wild-type, or non-transgenic plants). The “percent relative protein” and “percent relative fiber” were calculated similarly.
Inverse PCR was used to recover genomic DNA flanking the T-DNA insertion. The PCR product was subjected to sequence analysis and placed on the genome using a basic BLASTN search and/or a search of the Arabidopsis Information Resource (TAIR) database (available at the publicly available website). Promoters within 9 kb of the enhancers in the ACTTAG element are considered to be within “activation space.” Genes with T-DNA inserts within coding sequences were not considered to be within “activation space.” The ACTTAG lines with the above average oil and protein values, and below average fiber values were identified and are listed in column 3 of Table 1.













TABLE 1








4.
5.







Relative 
Relative
6.






Seed
Seed
Relative



1. Gene

3. ACTTAG
Protein
Fiber
Seed Oil
7.


alias
2. Tair
Line
Content
Content
Content
GC FA





















IMQ42.3
At3g19870
W000093675
112.46%
94.21%
 95.25%



IMQ43.1
At3g20170
W000145325
114.17%
89.64%
 94.52%



IMQ43.2
At3g20180
W000145325
114.17%
89.64%
 94.52%



IMQ43.3
At3g20190
W000145325
114.17%
89.64%
 94.52%
97.33%


IMQ43.4
At3g20200
W000145325
114.17%
89.64%
 94.52%



IMQ43.5
At3g20210
W000145325
114.17%
89.64%
 94.52%



IMQ44.1
At3g21300
W000148155
114.84%
91.54%
 93.83%



IMQ44.2
At3g21310
W000148155
114.84%
91.54%
 93.83%



IMQ45.1
At3g24570
W000141484
113.59%
92.03%
 99.29%



IMQ45.2
At3g24580
W000141484
113.59%
92.03%
 99.29%



IMQ45.3
At3g24590
W000141484
113.59%
92.03%
 99.29%



IMQ45.4
At3g24600
W000141484
113.59%
92.03%
 99.29%



IMQ45.5
At3g24610
W000141484
113.59%
92.03%
 99.29%



IMQ46.1
At3g26900
W000109686
108.34%
92.65%
 97.24%



IMQ46.2
At3g26910
W000109686
108.34%
92.65%
 97.24%



IMQ46.2
At3g26910
W000109686
108.34%
92.65%
 97.24%



IMQ46.2
At3g26910
W000109686
108.34%
92.65%
 97.24%



IMQ46.3
At3g26920
W000109686
108.34%
92.65%
 97.24%



IMQ46.4
At3g26930
W000109686
108.34%
92.65%
 97.24%



IMQ47.1
At3g27770
W000194585
103.85%
81.80%
102.51%



IMQ47.2
At3g27785
W000194585
103.85%
81.80%
102.51%



IMQ48.1
At3g44300
W000160067
110.68%
95.29%
 92.96%



IMQ48.2
At3g44310
W000160067
110.68%
95.29%
 92.96%



IMQ48.2
At3g44310
W000160067
110.68%
95.29%
 92.96%



IMQ48.3
At3g44320
W000160067
110.68%
95.29%
 92.96%



IMQ49.1
At3g45060
W000185043
108.38%
88.28%
 94.42%



IMQ49.1
At3g45060
W000136337
124.01%
89.52%
 88.72%



IMQ49.2
At3g45070
W000185043
108.38%
88.28%
 94.42%



IMQ49.2
At3g45070
W000136337
124.01%
89.52%
 88.72%



IMQ49.3
At3g45080
W000185043
108.38%
88.28%
 94.42%



IMQ49.3
At3g45080
W000136337
124.01%
89.52%
 88.72%



IMQ49.4
At3g45090
W000185043
108.38%
88.28%
 94.42%



IMQ49.4
At3g45090
W000136337
124.01%
89.52%
 88.72%



IMQ49.4
At3g45090
W000185043
108.38%
88.28%
 94.42%



IMQ49.4
At3g45090
W000136337
124.01%
89.52%
 88.72%



IMQ49.5
At3g45100
W000136337
124.01%
89.52%
 88.72%



IMQ49.5
At3g45100
W000136337
124.01%
89.52%
 88.72%



IMQ49.5
At3g45100
W000136337
124.01%
89.52%
 88.72%



IMQ49.5
At3g45100
W000136337
124.01%
89.52%
 88.72%



IMQ49.6
At3g45110
W000136337
124.01%
89.52%
 88.72%



IMQ49.6
At3g45110
W000136337
124.01%
89.52%
 88.72%



IMQ50.1
At3g46510
W000185346
111.30%
89.94%
 94.03%



IMQ50.1
At3g46510
W000185346
111.30%
89.94%
 94.03%



IMQ50.2
At3g46520
W000185346
111.30%
89.94%
 94.03%



IMQ50.3
At3g46530
W000185346
111.30%
89.94%
 94.03%



IMQ50.4
At3g46540
W000185346
111.30%
89.94%
 94.03%



IMQ50.5
At3g46550
W000185346
111.30%
89.94%
 94.03%



IMQ50.6
At3g46560
W000185346
111.30%
89.94%
 94.03%



IMQ51.1 
At3g59410
W000148125
110.29%
91.22%
 96.30%
97.59%


IMQ51.1 
At3g59410
W000187558
103.08%
88.95%
 99.29%



IMQ51.2 
At3g59420
W000148125
110.29%
91.22%
 96.30%



IMQ51.2 
At3g59420
W000187558
103.08%
88.95%
 99.29%



IMQ51.3 
At3g59430
W000148125
110.29%
91.22%
 96.30%
97.59%


IMQ51.3 
At3g59430
W000187558
103.08%
88.95%
 99.29%



IMQ51.3 
At3g59430
W000148125
110.29%
91.22%
 96.30%



IMQ51.3 
At3g59430
W000187558
103.08%
88.95%
 99.29%



IMQ51.3 
At3g59430
W000148125
110.29%
91.22%
 96.30%



IMQ51.3 
At3g59430
W000187558
103.08%
88.95%
 99.29%



IMQ51.4 
At3g59440
W000148125
110.29%
91.22%
 96.30%



IMQ51.4 
At3g59440
W000187558
103.08%
88.95%
 99.29%



IMQ51.5 
At3g59450
W000148125
110.29%
91.22%
 96.30%



IMQ51.5 
At3g59450
W000187558
103.08%
88.95%
 99.29%



IMQ52.1
At4g02010
W000132781
119.33%
90.32%
 92.13%



IMQ52.2
At4g02020
W000132781
119.33%
90.32%
 92.13%



IMQ53.1
At4g03470
W000143264
127.95%
94.43%
 89.75%























TABLE 2











7. Putative





3. Nucleic

5.
6. 
biochemical



1. Gene

Acid seq.
4. SEQ 
Polypeptide
SEQ ID
function/protein
8. Conserved protein


alias
2. Tair
Gl#
ID NO
seq. Gl#
NO
name
domain







IMQ42.3
At3g19870
gi|30685527
SEQ ID 
gi|15230976
SEQ ID
unknown protein






NO: 1

NO: 2




IMQ43.1
At3g20170
gi|42565047
SEQ ID
gi|15231056
SEQ ID
unknown protein
IPR000225 Armadillo;





NO: 3

NO: 4

IPR000357 HEAT


IMQ43.2
At3g20180
gi|18402522
SEQ ID
gi|15231057
SEQ ID
unknown protein
IPR006121 Heavy metal





NO: 5

NO: 6

transport/detoxification









protein


IMQ43.3
At3g20190
gi|42565048
SEQ ID
gi|42565049
SEQ ID
ATP binding/kinase/
IPR001611 Leucine-rich





NO: 7

NO: 8
protein
repeat;








serine/threonine
IPR007090 Leucine-rich








kinase
repeat, plant specific;









IPR000719 Protein kinase;









IPR008271









Serine/threonine protein









kinase, active site


IMQ43.4
At3g20200
gi|30685667
SEQ ID
gi|30685668
SEQ ID
ATP binding/kinase/
IPR000719 Protein kinase;





NO: 9

NO: 10
protein kinase/protein
IPR008271








serine/threonine
Serine/threonine protein








kinase/protein-
kinase, active site;








tyrosine kinase
IPR006016 UspA


IMQ43.5
At3g20210
gi|30685671
SEQ ID
gi|15231080
SEQ ID
DELTA-VPE;
IPR001096 Peptidase





NO: 11

NO: 12
cysteine-type
C13, legumain








endopeptidase



IMQ44.1
At3g21300
gi|42565078
SEQ ID
gi|42565079
SEQ ID
RNA binding/RNA
IPR000051 SAM (and





NO: 13

NO: 14
methyltransferase
some other nucleotide)









binding motif;









IPR001566 23S rRNA









methyltransferase/RumA;









IPR002792









Deoxyribonuclease/rho









motif-related TRAM;









IPR007848









Methyltransferase small;









IPR010280 (Uracil-5)-









methyltransferase


IMQ44.2
At3g21310
gi|18402951
SEQ ID
gi|18402952
SEQ ID
unknown protein
IPR004949 Protein of





NO: 15

NO: 16

unknown function









DUF266, plant


IMQ45.1
At3g24570
gi|30687563
SEQ ID
gi|15230132
SEQ ID
unknown protein
IPR007248 Mpv17/PMP22





NO: 17

NO: 18




IMQ45.2
At3g24580
gi|18404244
SEQ ID
gi|15230133
SEQ ID
unknown protein
IPR006527 F-box protein





NO: 19

NO: 20

interaction domain;









IPR001810 Cyclin-like F-









box


IMQ45.3
At3g24590
gi|30687571
SEQ ID
gi|30687572
SEQ ID
peptidase/serine-type
IPR011056 Peptidase S24





NO: 21

NO: 22
peptidase
and S26, C-terminal









region;









IPR006198 Peptidase









S24, S26A and S26B;









IPR000223 Peptidase









S26A, signal peptidase I


IMQ45.4
At3g24600
gi|18404257
SEQ ID
gi|15230135
SEQ ID
unknown protein
IPR010847 Harpin-





NO: 23

NO: 24

induced 1


IMQ45.5
At3g24610
gi|18404260
SEQ ID
gi|15230136
SEQ ID
unknown protein
IPR001810 Cyclin-like F-





NO: 25

NO: 26

box;









IPR006652 Kelch repeat


IMQ46.1
At3g26900
gi|30688546
SEQ ID
gi|30688547
SEQ ID
ATP binding/
IPR000623 Shikimate





NO: 27

NO: 28
shikimate kinase
kinase


IMQ46.2
At3g26910
gi|79313785
SEQ ID
gi|79313786
SEQ ID
unknown protein
IPR006706 Extensin-like





NO: 29

NO: 30

region


IMQ46.2
At3g26910
gi|42565220
SEQ ID
gi|30688552
SEQ ID
unknown protein
IPR006706 Extensin-like





NO: 31

NO: 32

region


IMQ46.2
At3g26910
gi|30685726
SEQ ID
gi|30685727
SEQ ID
unknown protein
IPR006706 Extensin-like





NO: 33

NO: 34

region


IMQ46.3
At3g26920
gi|30688555
SEQ ID
gi|30688556
SEQ ID
unknown protein
IPR001810 Cyclin-like F-





NO: 35

NO: 36

box;









IPR001611 Leucine-rich









repeat


IMQ46.4
At3g26930
gi|42565221
SEQ ID
gi|42565222
SEQ ID
unknown protein
IPR006566 FBD;





NO: 37

NO: 38

IPR001611 Leucine-rich









repeat


IMQ47.1
At3g27770
gi|30688916
SEQ ID
gi|18405512
SEQ ID
unknown protein






NO: 39

NO: 40




IMQ47.2
At3g27785
gi|30688924
SEQ ID
gi|30688925
SEQ ID
MYB118; DNA
IPR001005 Myb, DNA-





NO: 41

NO: 42
binding/transcription
binding








factor



IMQ48.1
At3g44300
gi|30692061
SEQ ID
gi|15229932
SEQ ID
NIT2 (NITRILASE 2) 
IPR003010





NO: 43

NO: 44

Nitrilase/cyanide









hydratase and









apolipoprotein N-









acyltransferase;









IPR000132









Nitrilase/cyanide









hydratase


IMQ48.2
At3g44310
gi|30692071
SEQ ID
gi|30692072
SEQ ID
NIT1 (NITRILASE 1) 
IPR003010





NO: 45

NO: 46

Nitrilase/cyanide









hydratase and









apolipoprotein N-









acyltransferase;









IPR000132









Nitrilase/cyanide









hydratase


IMQ48.2
At3g44310
gi|30692066
SEQ ID
gi|30692067
SEQ ID
NIT1 (NITRILASE 1) 
IPR003010





NO: 47

NO: 48

Nitrilase/cyanide









hydratase and









apolipoprotein N-









acyltransferase;









IPR000132









Nitrilase/cyanide









hydratase


IMQ48.3
At3g44320
gi|30692076
SEQ ID
gi|15229936
SEQ ID
NIT3 (NITRILASE 3) 
IPR003010





NO: 49

NO: 50

Nitrilase/cyanide









hydratase and









apolipoprotein N-









acyltransferase;









IPR000132









Nitrilase/cyanide









hydratase


IMQ49.1
At3g45060
gi|42565547
SEQ ID
gi|15230589
SEQ ID
ATNRT2.6; nitrate
IPR007114 Major





NO: 51

NO: 52
transporter
facilitator superfamily;









IPR011701 Major









facilitator superfamily









MFS_1;









IPR004737 Nitrate









transporter


IMQ49.2
At3g45070
gi|30692514
SEQ ID
gi|15230602
SEQ ID
sulfotransferase
IPR000863





NO: 53

NO: 54

Sulfotransferase


IMQ49.3
At3g45080
gi|18407909
SEQ ID
gi|15230603
SEQ ID
sulfotransferase
IPR000863





NO: 55

NO: 56

Sulfotransferase


IMQ49.4
At3g45090
gi|42572582
SEQ ID
gi|42572583
SEQ ID
unknown protein
IPR001093 IMP





NO: 57

NO: 58

dehydrogenase/GMP









reductase;









IPR003593 AAA ATPase


IMQ49.4
At3g45090
gi|18407910
SEQ ID
gi|18407911
SEQ ID
unknown protein
IPR001093 IMP





NO: 59

NO: 60

dehydrogenase/GMP









reductase;









IPR003593 AAA ATPase


IMQ49.5
At3g45100
gi|30692534
SEQ ID
gi|30692535
SEQ ID
SETH2; transferase,
IPR001296 Glycosyl





NO: 61

NO: 62
transferring glycosyl 
transferase, group 1;








groups
IPR011835









Glycogen/starch









synthases, ADP-glucose









type


IMQ49.5
At3g45100
gi|30692528
SEQ ID
gi|18407913
SEQ ID
SETH2; transferase,
IPR001296 Glycosyl





NO: 63

NO: 64
transferring glycosyl
transferase, group 1;








groups
IPR011835









Glycogen/starch









synthases, ADP-glucose









type


IMQ49.6
At3g45110
gi|18407915
SEQ ID
gi|15230607
SEQ ID
unknown protein






NO: 65

NO: 66




IMQ50.1
At3g46510
gi|30692726
SEQ ID
gi|15231445
SEQ ID
ubiquitin-protein
IPR000225 Armadillo;





NO: 67

NO: 68
ligase
IPR003613 U box


IMQ50.1
At3g46510
gi|79527507
SEQ ID
gi|79527508
SEQ ID
ubiquitin-protein
IPR000225 Armadillo;





NO: 69

NO: 70
ligase
IPR003613 U box;









IPR001093 IMP









dehydrogenase/GMP









reductase


IMQ50.2
At3g46520
gi|30692727
SEQ ID
gi|15231447
SEQ ID
ACT12 (ACTIN-12);
IPR004000 Actin/actin-like





NO: 71

NO: 72
structural constituent









of cytoskeleton



IMQ50.3
At3g46530
gi|30692728
SEQ ID
gi|15231449
SEQ ID
RPP13
IPR002182 NB-ARC;





NO: 73

NO: 74
(RECOGNITION OF
IPR003593 AAA ATPase;








PERONOSPORA
IPR007111 NACHT








PARASITICA 13);
nucleoside triphosphatase








ATP binding



IMQ50.4
At3g46540
gi|30692729
SEQ ID
gi|15231451
SEQ ID
binding
IPR001026 Epsin, N-





NO: 75

NO: 76

terminal


IMQ50.5
At3g46550
gi|30692730
SEQ ID
gi|15231453
SEQ ID
SOS5 (SALT
IPR000782 Beta-Ig-





NO: 77

NO: 78
OVERLY SENSITIVE
H3/fasciclin








5)



IMQ50.6
At3g46560
gi|42565665
SEQ ID
gi|15231455
SEQ ID
TIM9; protein
IPR004217 Zinc finger,





NO: 79

NO: 80
translocase
Tim10/DDP-type


IMQ51.1
At3g59410
gi|30694991
SEQ ID
gi|30694992
SEQ ID
ATP binding/protein 
IPR011009 Protein kinase-





NO: 81

NO: 82
kinase/protein
like;








serine/threonine
IPR000719 Protein kinase;








kinase/protein-
IPR008271








tyrosine kinase/tRNA
Serine/threonine protein








ligase
kinase, active site;









IPR006575 RWD;









IPR004154 Anticodon-









binding;









IPR004516 Histidyl-tRNA









synthetase, class IIa


IMQ51.2
At3g59420
gi|30694994
SEQ ID
gi|15231681
SEQ ID
ACR4; kinase
IPR000719 Protein kinase;





NO: 83

NO: 84

IPR008271









Serine/threonine protein









kinase, active site;









IPR001368









TNFR/CD27/30/40/95









cysteine-rich region;









IPR009091 Regulator of









chromosome









condensation/beta-









lactamase-inhibitor protein









II


IMQ51.3
At3g59430
gi|79315716
SEQ ID
gi|79315717
SEQ ID
unknown protein






NO: 85

NO: 86




IMQ51.3
At3g59430
gi|42572730
SEQ ID
gi|42572731
SEQ ID
unknown protein






NO: 87

NO: 88




IMQ51.3
At3g59430
gi|42566058
SEQ ID
gi|15231683
SEQ ID
unknown protein






NO: 89

NO: 90




IMQ51.4
At3g59440
gi|30694998
SEQ ID
gi|15231685
SEQ ID
calcium ion binding
IPR002048 Calcium-





NO: 91

NO: 92

binding EF-hand


IMQ51.5
At3g59450
gi|18411175
SEQ ID
gi|15231687
SEQ ID
calcium ion binding
IPR002048 Calcium-





NO: 93

NO: 94

binding EF-hand


IMQ52.1
At4g02010
gi|30679030
SEQ ID
gi|30679031
SEQ ID
kinase
IPR011009 Protein kinase-





NO: 95

NO: 96

like;









IPR000719 Protein kinase;









IPR008271









Serine/threonine protein









kinase, active site


IMQ52.2
At4g02020
gi|30679033
SEQ ID
gi|18411808
SEQ ID
EZA1; transcription 
IPR001214 Nuclear





NO: 97

NO: 98
factor
protein SET


IMQ53.1
At4g03470
gi|18412273
SEQ ID
gi|15236312
SEQ ID
protein binding
IPR002110 Ankyrin





NO: 99

NO: 100




















TABLE 3











5. Orthologous Genes: Nucleic Acid/Polypeptide seq. Gl#













1. Gene

3. Nucleic Acid 
4. Polypeptide
Nucleic Acid
Polypeptide 



alias
2. Tair
seq. Gl#
seq. Gl#
Gl#
Gl#
Species





IMQ42.3
At3g19870
gi|30685527
gi|15230976
gi|50916119
gi|50916120
Oryza sativa (japonica cultivar-group)






gi|66811721
gi|66811722
Dictyostelium discoideum






gi|47227243
gi|47227414
Tetraodon nigroviridis


IMQ43.1
At3g20170
gi|42565047
gi|15231056
gi|18406655
gi|18406656
Arabidopsis thaliana






gi|30695181
gi|15232303
Arabidopsis thaliana






gi|50303104
gi|50303105
Kluyveromyces lactis NRRL Y-1140


IMQ43.2
At3g20180
gi|18402522
gi|15231057
gi|50253060
gi|50253093
Oryza sativa (japonica cultivar-group)






gi|50253060
gi|50253092
Oryza sativa (japonica cultivar-group)






gi|50253060
gi|50253091
Oryza sativa (japonica cultivar-group)


IMQ43.3
At3g20190
gi|42565048
gi|42565049
gi|30694806
gi|30694807
Arabidopsis thaliana






gi|3015487
gi|3015488
Lycopersicon esculentum






gi|18421452
gi|15238494
Arabidopsis thaliana


IMQ43.4
At3g20200
gi|30685667
gi|30685668
gi|18394397
gi|15219360
Arabidopsis thaliana






gi|42563329
gi|30699374
Arabidopsis thaliana






gi|42567297
gi|42567298
Arabidopsis thaliana


IMQ43.5
At3g20210
gi|30685671
gi|15231080
gi|77799871
gi|34530959
Homo sapiens






gi|37542691
gi|37542692
Glycine max






gi|27544005
gi|27544006
Nicotiana tabacum


IMQ44.1
At3g21300
gi|42565078
gi|42565079
gi|34906469
gi|34906470
Oryza sativa (japonica cultivar-group)






gi|21672841
gi|21672850
Chlorobium tepidum TLS






gi|67938372
gi|67938422
Chlorobium phaeobacteroides BS1


IMQ44.2
At3g21310
gi|18402951
gi|18402952
gi|18403668
gi|15218028
Arabidopsis thaliana






gi|30683761
gi|15239155
Arabidopsis thaliana






gi|50540715
gi|50540730
Oryza sativa (japonica cultivar-group)


IMQ45.1
At3g24570
gi|30687563
gi|15230132
gi|34904255
gi|34904256
Oryza sativa (japonica cultivar-group)






gi|66816890
gi|66816891
Dictyostelium discoideum






gi|46107693
gi|46107694
Gibberella zeae PH-1


IMQ45.2
At3g24580
gi|18404244
gi|15230133
gi|18402249
gi|18402250
Arabidopsis thaliana






gi|18401581
gi|15229086
Arabidopsis thaliana






gi|42569113
gi|42569114
Arabidopsis thaliana


IMQ45.3
At3g24590
gi|30687571
gi|30687572
gi|45382012
gi|50251480
Oryza sativa (japonica cultivar-group)






gi|51535924
gi|51535944
Oryza sativa (japonica cultivar-group)






gi|50918990
gi|50918991
Oryza sativa (japonica cultivar-group)


IMQ45.4
At3g24600
gi|18404257
gi|15230135
gi|55741371
gi|55741372
Oryza sativa (japonica cultivar-group)






gi|34904259
gi|34904260
Oryza sativa (japonica cultivar-group)






gi|17385724
gi|56784375
Oryza sativa (japonica cultivar-group)


IMQ45.5
At3g24610
gi|18404260
gi|15230136
gi|18416176
gi|15236530
Arabidopsis thaliana






gi|18418316
gi|18418317
Arabidopsis thaliana






gi|18421782
gi|15240973
Arabidopsis thaliana


IMQ46.1
At3g26900
gi|30688546
gi|30688547
gi|55769656
gi|55769657
Oryza sativa (japonica cultivar-group)






gi|34894031
gi|34894032
Oryza sativa (japonica cultivar-group)






gi|50929064
gi|50929065
Oryza sativa (japonica cultivar-group)


IMQ46.2
At3g26910
gi|79313785
gi|79313786
gi|42565220
gi|30688552
Arabidopsis thaliana






gi|30693744
gi|30693745
Arabidopsis thaliana






gi|30693739
gi|30693740
Arabidopsis thaliana


IMQ46.2
At3g26910
gi|42565220
gi|30688552
gi|30693739
gi|30693740
Arabidopsis thaliana






gi|30693744
gi|30693745
Arabidopsis thaliana






gi|30685726
gi|30685727
Arabidopsis thaliana


IMQ46.2
At3g26910
gi|30685726
gi|30685727
gi|50923910
gi|50923911
Oryza sativa (japonica cultivar-group)






gi|42565220
gi|30688552
Arabidopsis thaliana






gi|52077544
gi|51536022
Oryza sativa (japonica cultivar-group)


IMQ46.3
At3g26920
gi|30688555
gi|30688556
gi|42565869
gi|42565870
Arabidopsis thaliana






gi|18408760
gi|15229075
Arabidopsis thaliana






gi|22328602
gi|22328603
Arabidopsis thaliana


IMQ46.4
At3g26930
gi|42565221
gi|42565222
gi|18405188
gi|15221971
Arabidopsis thaliana






gi|42565869
gi|42565870
Arabidopsis thaliana






gi|18408760
gi|15229075
Arabidopsis thaliana


IMQ47.1
At3g27770
gi|30688916
gi|18405512
gi|55770586
gi|55770587
Oryza sativa (japonica cultivar-group)






gi|42571426
gi|42571427
Arabidopsis thaliana






gi|42571424
gi|42571425
Arabidopsis thaliana






gi|42571428
gi|42571429
Arabidopsis thaliana






gi|30681842
gi|15220184
Arabidopsis thaliana






gi|42568719
gi|42568720
Arabidopsis thaliana


IMQ47.2
At3g27785
gi|30688924
gi|30688925
gi|18421976
gi|18421977
Arabidopsis thaliana






gi|30684524
gi|15234013
Arabidopsis thaliana






gi|77552765
gi|77553798
Oryza sativa (japonica cultivar-group)


IMQ48.1
At3g44300
gi|30692061
gi|15229932
gi|30692066
gi|30692067
Arabidopsis thaliana






gi|30692076
gi|15229936
Arabidopsis thaliana






gi|14211395
gi|14211396
Brassica napus


IMQ48.2
At3g44310
gi|30692071
gi|30692072
gi|30692066
gi|30692067
Arabidopsis thaliana






gi|30692061
gi|15229932
Arabidopsis thaliana






gi|30692076
gi|15229936
Arabidopsis thaliana






gi|42568008
gi|15242205
Arabidopsis thaliana


IMQ48.2
At3g44310
gi|30692066
gi|30692067
gi|30692061
gi|15229932
Arabidopsis thaliana






gi|30692076
gi|15229936
Arabidopsis thaliana






gi|14211395
gi|14211396
Brassica napus


IMQ48.3
At3g44320
gi|30692076
gi|15229936
gi|30692066
gi|30692067
Arabidopsis thaliana






gi|30692061
gi|15229932
Arabidopsis thaliana






gi|14211395
gi|14211396
Brassica napus


IMQ49.1
At3g45060
gi|42565547
gi|15230589
gi|18424396
gi|15239435
Arabidopsis thaliana






gi|48675348
gi|48675349
Prunus persica






gi|48675346
gi|48675347
Prunus persica


IMQ49.2
At3g45070
gi|30692514
gi|15230602
gi|18407909
gi|15230603
Arabidopsis thaliana






gi|30694345
gi|15239947
Arabidopsis thaliana






gi|30683282
gi|15222843
Arabidopsis thaliana


IMQ49.3
At3g45080
gi|18407909
gi|15230603
gi|30692514
gi|15230602
Arabidopsis thaliana






gi|30694345
gi|15239947
Arabidopsis thaliana






gi|30683282
gi|15222843
Arabidopsis thaliana


IMQ49.4
At3g45090
gi|42572582
gi|42572583
gi|18407910
gi|18407911
Arabidopsis thaliana






gi|30697427
gi|30697428
Arabidopsis thaliana






gi|40363800
gi|52076103
Oryza sativa (japonica cultivar-group)


IMQ49.4
At3g45090
gi|18407910
gi|18407911
gi|42572582
gi|42572583
Arabidopsis thaliana






gi|30697427
gi|30697428
Arabidopsis thaliana






gi|40363800
gi|52076103
Oryza sativa (japonica cultivar-group)


IMQ49.5
At3g45100
gi|30692534
gi|30692535
gi|30692528
gi|18407913
Arabidopsis thaliana






gi|50935714
gi|50935715
Oryza sativa (japonica cultivar-group)






gi|66810216
gi|66810217
Dictyostelium discoideum






gi|72083931
gi|72083932
Strongylocentrotus purpuratus


IMQ49.5
At3g45100
gi|30692528
gi|18407913
gi|30692534
gi|30692535
Arabidopsis thaliana






gi|50935714
gi|50935715
Oryza sativa (japonica cultivar-group)






gi|66810216
gi|66810217
Dictyostelium discoideum






gi|72083931
gi|72083932
Strongylocentrotus purpuratus


IMQ49.6
At3g45110
gi|18407915
gi|15230607
gi|48783781
gi|48784150
Burkholderia fungorum LB400






gi|18407923
gi|15230616
Arabidopsis thaliana






gi|48786263
gi|48786365
Burkholderia fungorum LB400


IMQ50.1
At3g46510
gi|30692726
gi|15231445
gi|30684026
gi|18401867
Arabidopsis thaliana






gi|51038702
gi|51038703
Oryza sativa (japonica cultivar-group)






gi|77552765
gi|77556856
Oryza sativa (japonica cultivar-group)


IMQ50.1
At3g46510
gi|79527507
gi|79527508
gi|50946934
gi|50946935
Oryza sativa (japonica cultivar-group)






gi|54291125
gi|54291136
Oryza sativa (japonica cultivar-group)






gi|30692726
gi|15231445
Arabidopsis thaliana


IMQ50.2
At3g46520
gi|30692727
gi|15231447
gi|30697210
gi|15238387
Arabidopsis thaliana






gi|3420238
gi|3420239
Gossypium hirsutum






gi|30693903
gi|18409908
Arabidopsis thaliana






gi|30687200
gi|30687201
Arabidopsis thaliana


IMQ50.3
At3g46530
gi|30692728
gi|15231449
gi|18408266
gi|15232624
Arabidopsis thaliana






gi|18408260
gi|15232622
Arabidopsis thaliana






gi|18406280
gi|15217954
Arabidopsis thaliana


IMQ50.4
At3g46540
gi|30692729
gi|15231451
gi|21104611
gi|56784669
Oryza sativa (japonica cultivar-group)






gi|34908157
gi|34908158
Oryza sativa (japonica cultivar-group)






gi|30680762
gi|22329424
Arabidopsis thaliana


IMQ50.5
At3g46550
gi|30692730
gi|15231453
gi|21104611
gi|56784671
Oryza sativa (japonica cultivar-group)






gi|34908161
gi|34908162
Oryza sativa (japonica cultivar-group)






gi|607773
gi|607774
Pinus taeda


IMQ50.6
At3g46560
gi|42565665
gi|15231455
gi|5107213
gi|5107214
Oryza sativa






gi|77552765
gi|77556866
Oryza sativa (japonica cultivar-group)






gi|5107211
gi|5107212
Mesembryanthemum crystallinum


IMQ51.1
At3g59410
gi|30694991
gi|30694992
gi|50925770
gi|50925771
Oryza sativa (japonica cultivar-group)






gi|74000019
gi|74000020
Canis familiaris






gi|50748343
gi|50748344
Gallus gallus


IMQ51.2
At3g59420
gi|30694994
gi|15231681
gi|40850572
gi|40850578
Musa acuminate






gi|50917202
gi|50917203
Oryza sativa (japonica cultivar-group)






gi|1597722
gi|1597723
Zea mays


IMQ51.3
At3g59430
gi|79315716
gi|79315717
gi|42572730
gi|42572731
Arabidopsis thaliana






gi|42566058
gi|15231683
Arabidopsis thaliana


IMQ51.3
At3g59430
gi|42572730
gi|42572731
gi|42566058
gi|15231683
Arabidopsis thaliana






gi|50725340
gi|50725357
Oryza sativa (japonica cultivar-group)






gi|74054322
gi|74054328
Streptococcus agalactiae






gi|56384961
gi|56384962
Streptococcus agalactiae


IMQ51.3
At3g59430
gi|42566058
gi|15231683
gi|42572730
gi|42572731
Arabidopsis thaliana






gi|50725340
gi|50725357
Oryza sativa (japonica cultivar-group)






gi|74054322
gi|74054328
Streptococcus agalactiae






gi|56384961
gi|56384962
Streptococcus agalactiae


IMQ51.4
At3g59440
gi|30694998
gi|15231685
gi|30689331
gi|18406202
Arabidopsis thaliana






gi|28301673
gi|28301674
Lotus corniculatus var. japonicus






gi|18397964
gi|15231470
Arabidopsis thaliana


IMQ51.5
At3g59450
gi|18411175
gi|15231687
gi|30694998
gi|15231685
Arabidopsis thaliana






gi|30689331
gi|18406202
Arabidopsis thaliana






gi|30679524
gi|15221358
Arabidopsis thaliana


IMQ52.1
At4g02010
gi|30679030
gi|30679031
gi|34902827
gi|34902828
Oryza sativa (japonica cultivar-group)






gi|6498456
gi|56783692
Oryza sativa (japonica cultivar-group)






gi|30696797
gi|22327898
Arabidopsis thaliana


IMQ52.2
At4g02020
gi|30679033
gi|18411808
gi|20152908
gi|20152909
Zea mays






gi|22535906
gi|22535907
Oryza sativa






gi|29565494
gi|29565495
Oryza sativa (indica cultivar-group)


IMQ53.1
At4g03470
gi|18412273
gi|15236312
gi|18412781 
gi|18412782
Arabidopsis thaliana






gi|42572834
gi|42572835
Arabidopsis thaliana






gi|42566786
gi|42566787
Arabidopsis thaliana






gi|30682836
gi|18414210
Arabidopsis thaliana









Example 2
Analysis of the Arabidopsis IMO Sequence
Sequence analyses were performed with BLAST (Altschul et al., 1990, J. Mol. Biol. 215:403-410), PFAM (Bateman et al., 1999, Nucleic Acids Res. 27:260-262), INTERPRO (Mulder et al. 2003 Nucleic Acids Res. 31, 315-318.), PSORT (Nakai K, and Horton P, 1999, Trends Biochem. Sci. 24:34-6), and/or CLUSTAL (Thompson J D et al., 1994, Nucleic Acids Res. 22:4673-4680). Conserved domains for each protein are listed in column 8 of Table 2.
Example 3
To test whether over-expression of the genes in Tables 1 and 2 alter the seed composition phenotype, protein, digestible protein, oil and fiber content in seeds from transgenic plants expressing these genes was compared with protein, digestible protein, oil and fiber content in seeds from non-transgenic control plants. To do this, the genes were cloned into plant transformation vectors behind the strong constitutive CsVMV promoter and the seed specific PRU promoter. These constructs were transformed into Arabidopsis plants using the floral dip method. The plant transformation vector contains a gene, which provides resistance to a toxic compound, and serves as a selectable marker. Seed from the transformed plants were plated on agar medium containing the toxic compound. After 7 days, transgenic plants were identified as healthy green plants and transplanted to soil. Non-transgenic control plants were germinated on agar medium, allowed to grow for 7 days and then transplanted to soil. Transgenic seedlings and non-transgenic control plants were transplanted to two inch pots that were placed in random positions in a 10 inch by 20 inch tray. The plants were grown to maturity, allowed to self-fertilize and set seed. Seed was harvested from each plant and its oil content estimated by Near Infrared (NIR) Spectroscopy using methods previously described. The effect of each construct on seed composition was examined in at least two experiments.
Table 4 lists constructs tested for causing a significant increase in oil, protein, digestible protein or a significant decrease in fiber were identified by a two-way Analysis of Variance (ANOVA) test at a p-value ≦0.05. ANOVA p-values for Protein, Oil, Digestible Protein and Fiber are listed in columns 4-7, respectively. Those with a significant p-value are listed in bold. The Average values for Protein, Oil, Digestible Protein and Fiber are listed in columns 8-11, respectively and were calculated by averaging the average values determined for the transgenic plants in each experiment.

















TABLE 4








4.
5.
6. ANOVA
7.


10.






ANOVA
ANOVA
Digestible 
ANOVA
8.
9.
Digestible 
11.


1. Gene
2. TAIR
3. Construct
Protein
Oil
Protein
Fiber
Protein 
Oil
Protein
Fiber

























IMQ43.3
At3g20190
CsVMV::At3g20190
0.659
0.336
0.005
0.002
100.6%
101.6%
101.9%
96.1%


IMQ43.3
At3g20190
Pru::At3g20190
0.868
0.709
0.005
0.009
100.3%
100.7%
102.3%
96.7%


IMQ47.1
At3g27770
Pru::At3g27770
0.404
0.143
0.033
0.004
99.2%
102.2%
101.1%
97.3%


IMQ49.1
At3g45060
CsVMV::At3g45060
0.441
0.623
0.016
0.004
101.4%
100.6%
102.6%
96.9%


IMQ49.1 
At3g45060
Pru::At3g45060
0.779
0.842
0.023
0.070
100.2%
100.9%
101.2%
98.6%


IMQ49.3
At3g45080
Pru::At3g45080
0.375
0.770
0.014
0.007
102.0%
99.6%
102.8%
97.6%


IMQ50.5
At3g46550
Pru::At3g46550
0.038
0.171
0.240
0.013
102.9%
102.6%
101.1%
97.1%


IMQ51.1
At3g59410
CsVMV::At3g59410
0.040
0.103
0.019
0.305
102.5%
97.3%
101.8%
98.7%


IMQ51.3
At3g59430
CsVMV::At3g59430
0.034
0.035
0.347
0.538
95.5%
104.2%
99.2%
99.4%


IMQ51.3 
At3g59430
Pru::At3g59430
0.911
0.038
0.239
0.017
100.4%
102.8%
101.3%
97.4%









Example 4
To test whether over-expression of the genes identified in Tables 1-4 alter the seed composition phenotype, protein, digestible protein, oil, and fiber content in seeds from transgenic plants expressing these genes is compared with protein, digestible protein, oil and fiber content in seeds from non-transgenic control plants. Any one of the genes identified in Tables 1-4 is used to transform Brassica napus (canola). To do this, the genes are cloned into plant transformation vectors behind the strong constitutive CsVMV promoter and the seed specific phaseolin promoter. These constructs (which include a gene encoding a selection agent) are transformed into canola plants.
Transformation of canola is accomplished via Agrobacterium-mediated transformation. Seeds are surface-sterilized with 10% commercial bleach for 10 minutes and rinsed 3 times with sterile distilled water. The seeds are then placed on one half concentration of MS basal medium (Murashige and Skoog, Physiol. Plant. 15:473-497, 1962) and maintained under growth regime set at 25° C., and a photoperiod of 16 hrs light/8 hrs dark.
Hypocotyl segments (3-5 mm) are excised from 5-7 day old seedlings and placed on callus induction medium K1D1 (MS medium with 1 mg/l kinetin and 1 mg/l 2,4-D) for 3 days as pre-treatment. The segments are then transferred into a petri plate, treated with Agrobacterium Z707S or LBA4404 strain containing pDAB721. The Agrobacterium is grown overnight at 28° C. in the dark on a shaker at 150 rpm and subsequently re-suspended in the culture medium.
After 30 minute treatment of the hypocotyl segments with Agrobacterium, these are placed back on the callus induction medium for 3 days. Following co-cultivation, the segments are placed on K1D1TC (callus induction medium containing 250 mg/l Carbenicillin and 300 mg/l Timentin) for one week of recovery. Alternately, the segments are placed directly on selection medium K1D1H1 (above medium with 1 mg/l selection agent, for example an herbicide). Carbenicillin and Timentin are antibiotics used to kill the Agrobacterium. The selection agent is used to allow the growth of the transformed cells.
Callus samples from independent events are tested by PCR. All the samples tested are positive for the presence of the transformed gene, whereas the non-transformed controls are negative. Callus samples are confirmed to express the appropriate protein as determined by ELISA.
Callused hypocotyl segments are then placed on B3Z1H1 (MS medium, 3 mg/l benzylamino purine, 1 mg/l Zeatin, 0.5 gm/l MES [2-(N-morpholino) ethane sulfonic acid], 5 mg/l silver nitrate, 1 mg/l selection agent, Carbenicillin and Timentin) shoot regeneration medium. After shoots start to regenerate (approximately 3 weeks), hypocotyl segments along with the shoots are transferred to B3Z1H3 medium (MS medium, 3 mg/l benzylamino purine, 1 mg/l Zeatin, 0.5 gm/l MES [2-(N-morpholino) ethane sulfonic acid], 5 mg/l silver nitrate, 3 mg/l selection agent, Carbenicillin and Timentin) for 3 weeks.
Shoots are excised from the hypocotyl segments and transferred to shoot elongation medium MESH10 (MS, 0.5 gm/l MES, 10 mg/l selection agent, Carbenicillin, Timentin) for 2-4 weeks. The elongated shoots are cultured for root induction on MSI.1 (MS with 0.1 mg/l Indolebutyric acid). Once the plants have a well-established root system, these are transplanted into soil. The plants are acclimated under controlled environmental conditions in the Conviron for 1-2 weeks before transfer to the greenhouse. The transformed T0 plants self-pollinate in the greenhouse to obtain T1 seed. Transgenic plants are selected at the T1 generation based on resistance to a selection agent. T2 seed (from T1 plants) is harvested and sown in soil. T2 plants are grown to maturity, allowed to self-fertilize and set seed. T3 seed (from the T2 plants) is harvested in bulk for each line. Seed oil, protein, digestible protein, and fiber values are measured as discussed in Example 1.

## Claims

1. A transgenic plant, comprising a heterologous nucleic acid that encodes a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 82, wherein the transgenic plant overexpresses the polypeptide and whereby the transgenic plant has increased protein content in seeds of the transgenic plant, relative to control plants.

2. The transgenic plant of claim 1, which is selected from the group consisting of plants of the Brassica species, soy, corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax, castor, peanut, wheat, oat, and rice.

3. The transgenic plant of claim 2, wherein the plant is canola or rapeseed.

4. A plant part obtained from the transgenic plant according to claim 1, wherein the plant part comprises the heterologous nucleic acid.

5. The plant part of claim 4, which is a transgenic seed, wherein the transgenic seed comprises the heterologous nucleic acid.

6. Meal, feed, or food produced from the transgenic seed of claim 5.

7. A method of producing meal, comprising growing the transgenic plant of claim 1, and recovering meal from the plant, thereby producing meal.

8. The method of claim 7, wherein the meal is produced from seeds of the plant.

9. A method of producing an improved meal quality phenotype in a plant, said method comprising:
a) introducing into progenitor cells of the plant a heterologous nucleic acid comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 82; and
b) growing the transformed progenitor cells to produce a transgenic plant, wherein the polypeptide is overexpressed, and the transgenic plant exhibits increased seed protein content relative to control plants,
thereby producing the improved meal quality phenotype in the plant.

10. A plant obtained by the method of claim 9.

11. The plant of claim 10, which is selected from the group consisting of plants of the Brassica species, soy, corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax, castor, peanut, wheat, oat, and rice.

12. The plant of claim 11, wherein the plant is canola or rapeseed.

13. The plant of claim 10, wherein the plant is selected from the group consisting of a plant grown from said progenitor cells, a plant that is the direct progeny of a plant grown from said progenitor cells, and a plant that is the indirect progeny of a plant grown from said progenitor cells.