# Patent

## Title
Maize multidrug resistance-associated protein polynucleotides and methods of use

## Abstract

Compositions and methods are provided for modulating the level of phytate in plants. More specifically, the invention relates to methods of modulating the level of phytate utilizing nucleic acids comprising multidrug resistance-associated protein (MRP) nucleotide sequences to modulate the expression of MRP(s) in a plant of interest. The compositions and methods of the invention find use in agriculture for improving the nutritional quality of food and feed by reducing the levels of phytate and/or increasing the levels of non-phytate phosphorus in food and feed. The invention also finds use in reducing the environmental impact of animal waste.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 11/133,075, filed May 19, 2005, which claims the benefit of U.S. Provisional Application No. 60/572,704, filed May 20, 2004, the contents of which are hereby incorporated by reference in their entirety.


INCORPORATION OF SEQUENCE LISTING
The application contains a sequence listing, a paper copy of which is filed concurrently herewith via EFS Web. The CRF copy of the sequence listing filed on May 19, 2005 has been transferred from the parent application (U.S. application Ser. No. 11/133,075). The content of the attached paper copy of the listing and the CRF copy transferred from U.S. application Ser. No. 11/133,075 are the same. The sequence listing is hereby incorporated in its entirety into the instant specification.
FIELD OF THE INVENTION
The present invention relates to the field of animal nutrition. Specifically, the present invention relates to the identification and use of genes encoding enzymes involved in the metabolism of phytate in plants and the use of these genes and mutants thereof to reduce the levels of phytate, and/or increase the levels of non-phytate phosphorus in food or feed.
BACKGROUND OF THE INVENTION
The role of phosphorous in animal nutrition is well recognized. Phosphorus is a critical component of the skeleton, nucleic acids, cell membranes and some vitamins. Though phosphorous is essential for the health of animals, not all phosphorous in feed is bioavailable.
Phytates are the major form of phosphorous in seeds. For example, phytate represents about 60-80% of total phosphorous in corn and soybean. When seed-based diets are fed to non-ruminants, the consumed phytic acid forms salts with several important mineral nutrients, such as potassium, calcium, and iron, and also binds proteins in the intestinal tract. These phytate complexes cannot be metabolized by monogastric animals and are excreted, effectively acting as anti-nutritional factors by reducing the bioavailability of dietary phosphorous and minerals. Phytate-bound phosphorous in animal excreta also has a negative environmental impact, contributing to surface and ground water pollution.
There have been two major approaches to reducing the negative nutritional and environmental impacts of phytate in seed. The first involves post-harvest interventions, which increase the cost and processing time of feed. Post-harvest processing technologies remove phytic acid by fermentation or by the addition of compounds, such as phytases.
The second is a genetic approach. One genetic approach involves developing crop germplasm with heritable reductions in seed phytic acid. While some variability for phytic acid was observed, there was no change in non-phytate phosphorous. Further, only 2% of the observed variation in phytic acid was heritable, whereas 98% of the variation was attributed to environmental factors. Another genetic approach involves selecting low phytate lines from a mutagenized population to produce germplasm. Most mutant lines exhibit a loss of function and are presumably blocked in the phytic acid biosynthetic pathway; therefore, low phytic acid accumulation will likely be a recessive trait. In certain cases, this approach has revealed that homozygosity for substantially reduced phytate can be lethal. Another genetic approach is transgenic technology, which has been used to increase phytase levels in plants. These transgenic plant tissues or seed have been used as dietary supplements.
The biosynthetic route leading to phytate is complex and not completely understood, and it has been proposed that the production of phytic acid occurs by one of two possible pathways. One possible pathway involves the sequential phosphorylation of Ins(3)P or myo-inositol, leading to the production of phytic acid. Another possible pathway involves hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C, followed by the phosphorylation of Ins(1,4,5)P3 by inositol phosphate kinases. In developing plant seeds, accumulating evidence favors the sequential phosphorylation pathway. Such evidence includes studies of the Lpa2 gene, a gene encoding a maize inositol phosphate kinase which has multiple kinase activities. The Lpa2 gene has been cloned, and the lpa2 mutation has been shown to impair phytic acid synthesis. Mutant lpa2 seeds accumulate myo-inositol and inositol phosphate intermediates.
The maize low phytic acid 1 mutant (lpa1) was isolated from an EMS-mutagenized population in the early 1990s by USDA scientists. However, the original lpa1-1 allele was previously known to have a phenotype of up to 15% loss of seed dry weight, which could translate into a yield drag if the lpa1-1 mutant was used in product development. Since the discovery of lpa1, the gene responsible for the lpa1 mutation has been sought for two reasons: 1) the mutant has a phenotype of low phytic acid and high available phosphorus in grain which makes it useful in animal feeding and phosphorus waste management; and 2) the lpa1 mutant does not accumulate myo-inositol phosphate intermediates, indicating that mutation in this locus impairs a critical step in the phytic acid biosynthesis pathway which was previously uncharacterized.
Based on the foregoing, there exists the need to improve the nutritional content of plants, particularly corn and soybean, by increasing non-phytate phosphorous and reducing seed phytate. Accordingly, it is desirable to isolate and characterize the Lpa1 gene in order to place the expression of this gene under tight control so as to produce plants which have reduced seed phytate and increased non-phytate phosphorus.
SUMMARY OF THE INVENTION
Compositions and methods are provided for modulating the level of phytate in plants. More specifically, the invention relates to methods of modulating the level of phytate utilizing Lpa1 (ZmMRP3) nucleic acids to produce transformed plants that exhibit decreased expression of at least one multidrug resistance-associated protein (MRP). The compositions and methods of the invention find use in agriculture for improving the nutritional quality of food and feed by reducing the levels of phytate and/or increasing the levels of non-phytate phosphorus in food and feed. Thus, the invention finds use in producing food and feed products as well as in reducing the environmental impact of animal waste. Also provided are compositions and methods for producing MRP proteins.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B: Alignment of ZmMRP3 (SEQ ID NO: 3) with Pfam consensus sequences for ABC transporter (“ABC_tran”; SEQ ID NO: 62) and ABC transporter transmembrane (“ABC_membrane”; SEQ ID NO: 63) region.
FIG. 2: Diagram of ZmMRP3 and rice OsMRP13 gene structure.
FIG. 3: Phylogenetic comparison of maize, rice and Arabidopsis MRP genes, showing that maize ZmMRP3, rice OsMRP13 and Arabidopsis AtMRP5 are closely related.
FIGS. 4A, 4B, 4C, 4D, 4E: cDNA sequence alignment of the maize Lpa1 gene (SEQ ID NO: 2) and its rice homolog OsMRP13 (SEQ ID NO: 6).
FIGS. 5A, 5B, 5C: Protein Sequence alignment of maize Lpa1 (ZmMRP3; SEQ ID NO: 3) with rice and Arabidopsis homologs OsMRP13 (SEQ ID NO: 7) and AtMRP5 (SEQ ID NO: 9). Matches to the consensus are indicated by bold type; conservative changes are indicated by underlined text.
FIG. 6: Diagram of sample constructs. These sample constructs illustrate various configurations that can be used in expression cassettes for use in inhibition of expression, for example, for use in hairpin RNA interference. Sample construct 1 shows a single promoter and fully or partially complementary sequences of “region 1” and “region 2.” Sample construct 2 illustrates a configuration of two sets of fully or partially complementary sequences. In this sample construct, “region 1” is fully or partially complementary to “region 2” and “region 3” is fully or partially complementary to “region 4.” Sample construct 3 illustrates yet another configuration of two sets of fully or partially complementary sequences; here, too, “region 1” is fully or partially complementary to “region 2” and “region 3” is fully or partially complementary to “region 4.”



DETAILED DESCRIPTION OF THE INVENTION
The invention is drawn to compositions and methods for modulating the level of phytate in plants. Compositions of the invention comprise multidrug resistance-associated proteins (“MRPs”) of the invention (i.e., proteins that have multidrug resistance-associated protein activity (“MRP activity”)), polynucleotides that encode them, and associated noncoding regions as well as fragments and variants of the exemplary disclosed sequences. For example, the disclosed Lpa1 polypeptides having amino acid sequences set forth in SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15 are MRPs and therefore have multidrug resistance-associated protein (“MRP”) activity. In particular, the present invention provides for isolated polynucleotides comprising nucleotide sequences set forth in SEQ ID NOs: 1, 2, 4, 6, 8, 10, 12, and 14, or encoding the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15, and fragments and variants thereof. In addition, the invention provides polynucleotides comprising the complements of these nucleotide sequences. Also provided are polypeptides comprising the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15, polypeptides comprising the conserved domains set forth in SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, and 24, fragments and variants thereof, and nucleotide sequences encoding these polypeptides. Compositions of the invention also include polynucleotides comprising at least a portion of the promoter sequence set forth in nucleotides 1 to 3134 of SEQ ID NO: 1 as well as polynucleotides comprising other noncoding regions
Thus, the compositions of the invention comprise isolated nucleic acids that encode MRP proteins (e.g., Lpa1), fragments and variants thereof, cassettes comprising polynucleotides of the invention, and isolated MRP proteins. The compositions also include nucleic acids comprising nucleotide sequences which are the complement, or antisense, of these MRP nucleotide sequences. The invention further provides plants and microorganisms transformed with these novel nucleic acids as well as methods involving the use of such nucleic acids, proteins, and transformed plants in producing food (including food products) and feed with reduced phytate and/or increased non-phytate phosphorus levels. In some embodiments, the transformed plants of the invention and food and feed produced therefrom have improved nutritional quality due to increased availability (bioavailability) of nutrients including, for example, zinc and iron.
In some embodiments, MRP activity is reduced or eliminated by transforming a maize plant cell with an expression cassette that expresses a polynucleotide that inhibits the expression of an MRP enzyme such as, for example, an Lpa1 polypeptide. The polynucleotide may inhibit the expression of one or more MRPs directly, by preventing translation of the MRP messenger RNA, or indirectly, by encoding a polypeptide that inhibits the transcription or translation of a maize gene encoding an MRP. Methods for inhibiting or eliminating the expression of a gene in a plant are well known in the art, and any such method may be used in the present invention to inhibit the expression of one or more maize MRPs. Because MRP activity is difficult to measure directly, a decrease in MRP activity can be measured by a decreased level of phytate in a plant or plant part. See, e.g., the working examples in the Experimental section.
In accordance with the present invention, the expression of an MRP protein is inhibited if the transcript or protein level of the MRP is statistically lower than the transcript or protein level of the same MRP in a plant that has not been genetically modified or mutagenized to inhibit the expression of that MRP. In particular embodiments of the invention, the transcript or protein level of the MRP in a modified plant according to the invention is less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the protein level of the same MRP in a plant that is not a mutant or that has not been genetically modified to inhibit the expression of that MRP. The expression level of the MRP may be measured directly, for example, by assaying for the level of MRP expressed in the cell or plant, or indirectly, for example, by measuring the amount of phytate in the cell or plant. The activity of an MRP protein is “eliminated” according to the invention when it is not detectable by at least one assay method.
In other embodiments of the invention, the activity of one or more MRPs is reduced or eliminated by transforming a plant cell with an expression cassette comprising a polynucleotide encoding a polypeptide that inhibits the activity of one or more MRPs. The activity of an MRP is inhibited according to the present invention if the activity of that MRP in the transformed plant or cell is statistically lower than the activity of that MRP in a plant that has not been genetically modified to inhibit the activity of at least one MRP. In particular embodiments of the invention, an MRP activity of a modified plant according to the invention is less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of that MRP activity in an appropriate control plant that has not been genetically modified to inhibit the expression of that MRP. Changes in MRP activity may be inferred, for example, by alterations in phytate content of a transformed plant or plant cell.
In other embodiments, the activity of an MRP may be reduced or eliminated by disrupting the gene encoding the MRP. The invention encompasses mutagenized plants that carry at least one mutation in an MRP gene, wherein the at least one mutation reduces expression of an MRP gene or inhibits the activity of an MRP.
Thus, many methods may be used to reduce or eliminate the activity of an MRP. More than one method may be used to reduce the activity of a single plant MRP. In addition, combinations of methods may be employed to reduce or eliminate the activity of two or more different MRPs. Non-limiting examples of methods of reducing or eliminating the expression of a plant MRP are given below.
In some embodiments of the present invention, a plant cell is transformed with an expression cassette that is capable of producing a polynucleotide that inhibits the expression of an MRP. The term “expression” as used herein refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product. For example, for the purposes of the present invention, an expression cassette capable of expressing a polynucleotide that inhibits the expression of at least one maize MRP is an expression cassette capable of producing an RNA molecule that inhibits the transcription and/or translation of at least one maize MRP.
“Expression” generally refers to the transcription and/or translation of a coding region of a DNA molecule, messenger RNA, or other nucleic acid molecule to produce the encoded protein or polypeptide. In other contexts, “expression” refers to the transcription of RNA from an expression cassette, such as, for example, the transcription of a hairpin construct from an expression cassette for use in hpRNA interference.
“Coding region” refers to the portion of a messenger RNA (or the corresponding portion of another nucleic acid molecule such as a DNA molecule) which encodes a protein or polypeptide. “Noncoding region” refers to all portions of a messenger RNA or other nucleic acid molecule that are not a coding region, including, for example, the promoter region, 5′ untranslated region (“UTR”), and/or 3′ UTR.
Some examples of polynucleotides and methods that inhibit the expression of an MRP are given below. While specific examples are given below, a variety of methods are known in the art by which it is possible to inhibit expression. While the invention is not bound by any particular theory of operation or mechanism of action, the invention provides the exemplary nucleotide and protein sequences disclosed herein and thereby provides a variety of methods by which expression can be inhibited. For example, fragments of noncoding region can be used to make constructs that inhibit expression of an MRP; such fragments can include portions of the promoter region or portions of the 3′ noncoding region (i.e., the 3′ UTR).
In some embodiments of the invention, inhibition of the expression of an MRP may be obtained by sense suppression or cosuppression. For cosuppression, an expression cassette is designed to express an RNA molecule corresponding to all or part of a messenger RNA encoding an MRP in the “sense” orientation. Overexpression of the RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the cosuppression expression cassette are screened to identify those that show suitable inhibition of MRP expression.
The polynucleotide used for cosuppression or other methods to inhibit expression may correspond to all or part of the sequence encoding the MRP, all or part of the 5′ and/or 3′ untranslated region of an MRP transcript, or all or part of both the coding region and the untranslated regions of a transcript encoding MRP. A polynucleotide used for cosuppression or other gene silencing methods may share 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 85%, 80%, or less sequence identity with the target sequence. When portions of the polynucleotides are used to disrupt the expression of the target gene, generally, sequences of at least 15, 20, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 900 nucleotides or 1 kb or greater may be used. In some embodiments where the polynucleotide comprises all or part of the coding region for the MRP, the expression cassette is designed to eliminate the start codon of the polynucleotide so that no protein product will be transcribed. In this manner, an expression cassette may cause permanent modification of the coding and/or noncoding region of an endogenous gene.
Thus, in some embodiments, for example, the polynucleotide used for cosuppression or another method to inhibit expression will comprise a sequence selected from a particular region of the coding and/or noncoding region. That is, the polynucleotide will comprise a sequence or the complement of a sequence selected from the region between nucleotides 1 and 5139 of the sequence set forth in SEQ ID NO: 2, or selected from the region with a first endpoint at nucleotide 1, 150, 250, 400, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, 3100, 3250, 3400, 3550, 3700, 3850, 4000, 4150, 4300, 4450, 4600, 4750, 4900, 5050, or 5139 and a second endpoint at nucleotide 244, 400, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, 3100, 3250, 3400, 3550, 3700, 3850, 4000, 4150, 4300, 4450, 4600, 4750, 4900, 5050, or 5139. As discussed elsewhere herein, fragments and/or variants of the exemplary disclosed sequences may also be used.
In some embodiments, for example, the polynucleotide will comprise a sequence or the complement of a sequence selected from the region between nucleotides 1 and 3134 of the sequence set forth in SEQ ID NO:1, or selected from the region with a first endpoint at nucleotide 1, 150, 400, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, or 3134, and a second endpoint at nucleotide 1, 150, 400, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, or 3134. Where a noncoding region is used for cosuppression or other method to inhibit expression, it may be advantageous to use a noncoding region that comprises CpG islands (see, e.g., Tariq et al. (2004) Trends Genet. 20: 244-251). As discussed elsewhere herein, variants and/or fragments of the exemplary disclosed sequences may also be used.
In some embodiments, for example, the polynucleotide will comprise a sequence or the complement of a sequence selected from the region between nucleotides 1 and 5123 of the sequence set forth in SEQ ID NO:6, or selected from the region with a first endpoint at nucleotide 1, 150, 300, 450, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, 3100, 3250, 3400, 3550, 3700, 3850, 4000, 4150, 4300, 4450, 4600, 4750, 4900, or 5123, and a second endpoint at nucleotide 1, 150, 300, 450, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, 3100, 3250, 3400, 3550, 3700, 3850, 4000, 4150, 4300, 4450, 4600, 4750, 4900, or 5123. As discussed elsewhere herein, variants and/or fragments of the exemplary disclosed sequences may also be used.
In some embodiments, for example, the polynucleotide will comprise a sequence or the complement of a sequence selected from the region between nucleotides 1 and 1350 of the sequence set forth in SEQ ID NO:10, or selected from the region with a first endpoint at nucleotide 1, 150, 300, 450, 550, 700, 850, 1000, 1150, 1300, or 1350, and a second endpoint at nucleotide 1, 150, 300, 450, 550, 700, 850, 1000, 1150, 1300, or 1350. As discussed elsewhere herein, variants and/or fragments of the exemplary disclosed sequences may also be used.
In some embodiments, for example, the polynucleotide will comprise a sequence or the complement of a sequence selected from the region between nucleotides 1 and 465 of the sequence set forth in SEQ ID NO:12, or selected from the region with a first endpoint at nucleotide 1, 150, 300, 450, or 465, and a second endpoint at nucleotide 1, 150, 300, 450, or 465. As discussed elsewhere herein, variants and/or fragments of the exemplary disclosed sequences may also be used.
In some embodiments, for example, the polynucleotide will comprise a sequence or the complement of a sequence selected from the region between nucleotides 1 and 556 of the sequence set forth in SEQ ID NO:71, or selected from the region with a first endpoint at nucleotide 1, 150, 300, 450, or 556, and a second endpoint at nucleotide 1, 150, 300, 450, or 556. As discussed elsewhere herein, variants and/or fragments of the exemplary disclosed sequences may also be used.
Cosuppression may be used to inhibit the expression of plant genes to produce plants having undetectable protein levels for the proteins encoded by these genes. See, for example, Broin et al. (2002) Plant Cell 14: 1417-1432. Cosuppression may also be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Pat. No. 5,942,657. Methods for using cosuppression to inhibit the expression of endogenous genes in plants are described in Flavell et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3490-3496; Jorgensen et al. (1996) Plant Mol. Biol. 31: 957-973; Johansen and Carrington (2001) Plant Physiol. 126: 930-938; Broin et al. (2002) Plant Cell 14: 1417-1432; Stoutjesdijk et al (2002) Plant Physiol. 129: 1723-1731; Yu et al. (2003) Phytochemistry 63: 753-763; and U.S. Pat. Nos. 5,034,323; 5,283,184; and 5,942,657; each of which is herein incorporated by reference. The efficiency of cosuppression may be increased by including a poly-dT region in the expression cassette at a position 3′ to the sense sequence and 5′ of the polyadenylation signal. See, e.g., U.S. Patent Publication No. 20020048814, herein incorporated by reference. Typically, such a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, for example, greater than about 65%, 80%, 85%, 90%, 95%, or more sequence identity. See, U.S. Pat. Nos. 5,283,184 and 5,034,323, herein incorporated by reference.
In some embodiments of the invention, inhibition of the expression of the MRP may be obtained by antisense suppression. For antisense suppression, the expression cassette is designed to express an RNA molecule complementary to all or part of a messenger RNA comprising a region encoding the MRP. Overexpression of the antisense RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the antisense suppression expression cassette are screened to identify those that show the greatest inhibition of MRP expression.
The polynucleotide for use in antisense suppression may correspond to all or part of the complement of the sequence encoding the MRP, all or part of the complement of the 5′ and/or 3′ untranslated region of the MRP transcript, or all or part of the complement of both the coding sequence and the untranslated regions of a transcript encoding the MRP. In addition, the antisense polynucleotide may be fully complementary (i.e., 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target sequence. That is, an antisense polynucleotide may share 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 85%, 80%, or less sequence identity with the target sequence. Antisense suppression may be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Pat. No. 5,942,657. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 450, 500, or 550 nucleotides or greater may be used.
Methods for using antisense suppression to inhibit the expression of endogenous genes in plants are described, for example, in Liu et at (2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829 and 5,942,657, each of which is herein incorporated by reference. Efficiency of antisense suppression may be increased by including a poly-dT region in the expression cassette at a position 3′ to the antisense sequence and 5′ of the polyadenylation signal. See, U.S. Patent Publication No. 20020048814, herein incorporated by reference.
In some embodiments of the invention, inhibition of the expression of an MRP may be obtained by double-stranded RNA (dsRNA) interference. For dsRNA interference, a sense RNA molecule like that described above for cosuppression and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule are expressed in the same cell, resulting in inhibition of the expression of the corresponding endogenous messenger RNA.
Expression of the sense and antisense molecules can be accomplished by designing the expression cassette to comprise both a sense sequence and an antisense sequence. Alternatively, separate expression cassettes may be used for the sense and antisense sequences. Multiple plant lines transformed with the dsRNA interference expression cassette or expression cassettes are then screened to identify plant lines that show the greatest inhibition of MRP expression. Methods for using dsRNA interference to inhibit the expression of endogenous plant genes are described in Waterhouse et al. (1998) Proc. Natl. Acad. Sci. USA 95: 13959-13964, Liu et al. (2002) Plant Physiol. 129: 1732-1743, and WO 99/49029, WO 99/53050, WO 99/61631, and WO 00/49035; each of which is herein incorporated by reference.
In some embodiments of the invention, inhibition of the expression of one or more MRPs may be obtained by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference. These methods are highly efficient at inhibiting the expression of endogenous genes. See, Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4: 29-38 and the references cited therein. These methods can make use of either coding region sequences or promoter or regulatory region sequences.
For hpRNA interference, the expression cassette is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single-stranded loop or “spacer” region and a base-paired stem. In some embodiments, the base-paired stem region comprises a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the gene whose expression is to be inhibited, and an antisense sequence that is fully or partially complementary to the sense sequence. The antisense sequence may be located “upstream” of the sense sequence (i.e., the antisense sequence may be closer to the promoter driving expression of the hairpin RNA than the sense sequence). In some embodiments, the base-paired stem region comprises a first portion of a noncoding region such as a promoter and a second portion of the noncoding region that is in inverted orientation relative to the first portion and that is fully or partially complementary to the first portion. In some embodiments, the base-paired stem region comprises a first portion and a second portion which are fully or partially complementary to each other but which comprise both coding and noncoding regions.
In some embodiments, the expression cassette comprises more than one base-paired “stem” region; that is, the expression cassette comprises sequences from different coding and/or noncoding regions which have the potential to form more than one base-paired “stem” region, for example, as diagrammed in FIG. 6 (construct 2 and construct 3). Where more than one base-paired “stem” region is present in an expression cassette, the “stem” regions may flank one another as diagrammed in FIG. 6 (construct 3) or may be in some other configuration (for example, as diagrammed in FIG. 6 (construct 2)). That is, for example, an expression cassette may comprise more than one combination of promoter and complementary sequences as shown in FIG. 6 (construct 1), and each such combination may be driven by a separate promoter. One of skill will be able to create and test a variety of configurations to determine the optimal construct for use in this or any other method for inhibition of expression.
Thus, the base-paired stem region of the molecule generally determines the specificity of the RNA interference. The sense sequence and the antisense sequence (or first and second portion of the noncoding region) are generally of similar lengths but may differ in length. Thus, these sequences may be portions or fragments of at least 10, 19, 20, 30, 50, 70, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 500, 600, 700, 800, 900 nucleotides in length, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb in length. The loop region of the expression cassette may vary in length. Thus, the loop region may be at least 100, 200, 300, 400, 500, 600, 700, 800, 900 nucleotides in length, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb in length. In some embodiments, the loop region comprises an intron such as, for example, the Adh1 intron.
hpRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants. See, for example, Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97: 4985-4990; Stoutjesdijk et al. (2002) Plant Physiol. 129: 1723-1731; and Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4: 29-38. Methods for using hpRNA interference to inhibit or silence the expression of genes are described, for example, in Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97: 4985-4990; Stoutjesdijk et al. (2002) Plant Physiol. 129: 1723-1731; Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4: 29-38; Pandolfini et al. BMC Biotechnology 3: 7, and U.S. Patent Publication No. 20030175965; each of which is herein incorporated by reference. A transient assay for the efficiency of hpRNA constructs to silence gene expression in vivo has been described by Panstruga et al. (2003) Mol. Biol. Rep. 30: 135-140, herein incorporated by reference.
For ihpRNA, the interfering molecules have the same general structure as for hpRNA (including the same sizes of sense sequences and antisense sequences), but the RNA molecule additionally comprises an intron in the loop or “spacer” region that is capable of being spliced in the cell in which the ihpRNA is expressed. The use of an intron minimizes the size of the loop in the hairpin RNA molecule following splicing, and this increases the efficiency of interference. See, for example, Smith et al. (2000) Nature 407: 319-320 (which demonstrated 100% suppression of endogenous gene expression using ihpRNA-mediated interference). Methods for using ihpRNA interference to inhibit the expression of endogenous plant genes are described, for example, in Smith et al. (2000) Nature 407: 319-320; Wesley et al. (2001) Plant J. 27: 581-590; Wang and Waterhouse (2001) Curr. Opin. Plant Biol. 5: 146-150; Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4: 29-38; Helliwell and Waterhouse (2003) Methods 30: 289-295, and U.S. Patent Publication No. 20030180945, each of which is herein incorporated by reference.
The expression cassette for hpRNA interference may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA. In this embodiment, the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene. Thus, in this embodiment, it is the loop region that determines the specificity of the RNA interference. See, for example, WO 02/00904, herein incorporated by reference.
Transcriptional gene silencing (TGS) may be accomplished through use of hpRNA constructs wherein the inverted repeat of the hairpin shares sequence identity with the promoter region of a gene to be silenced. Processing of the hpRNA into short RNAs which can interact with the homologous promoter region may trigger degradation or methylation to result in silencing (Aufsatz et al. (2002) Proc. Nat'l. Acad. Sci. USA 99 (Suppl. 4): 16499-16506; Mette et al. (2000) EMBO J. 19(19): 5194-5201). As the invention is not bound by a particular mechanism or mode of operation, a decrease in expression may also be achieved by other mechanisms.
Amplicon expression cassettes comprise a plant virus-derived sequence that contains all or part of the target gene but generally not all of the genes of the native virus. The viral sequences present in the transcription product of the expression cassette allow the transcription product to direct its own replication. The transcripts produced by the amplicon may be either sense or antisense relative to the target sequence (i.e., the messenger RNA for MRP). Methods of using amplicons to inhibit the expression of endogenous plant genes are described, for example, in Angell and Baulcombe (1997) EMBO J. 16: 3675-3684, Angell and Baulcombe (1999) Plant J. 20: 357-362, and U.S. Pat. No. 6,646,805, each of which is herein incorporated by reference.
In some embodiments, the polynucleotide expressed by the expression cassette of the invention is catalytic RNA or has ribozyme activity specific for the messenger RNA of MRP. Thus, the polynucleotide causes the degradation of the endogenous messenger RNA, resulting in reduced expression of the MRP. This method is described, for example, in U.S. Pat. No. 4,987,071, herein incorporated by reference.
In some embodiments of the invention, inhibition of the expression of one or more MRPs may be obtained by RNA interference by expression of a gene encoding a micro RNA (miRNA). miRNAs are regulatory agents consisting of about 22 ribonucleotides. miRNAs are highly efficient at inhibiting the expression of endogenous genes. See, for example Javier et al. (2003) Nature 425: 257-263, herein incorporated by reference.
For miRNA interference, the expression cassette is designed to express an RNA molecule that is modeled on an endogenous miRNA gene. The miRNA gene encodes an RNA that forms a hairpin structure containing a 22-nucleotide sequence that is complementary to another endogenous gene (target sequence). For suppression of MRP expression, the 22-nucleotide sequence is selected from an MRP transcript sequence and contains 22 nucleotides of said MRP sequence in sense orientation and 21 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence. miRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants.
In one embodiment, the polynucleotide encodes a zinc finger protein that binds to a gene encoding an MRP resulting in reduced expression of the gene. In particular embodiments, the zinc finger protein binds to a regulatory region of an MRP gene. In other embodiments, the zinc finger protein binds to a messenger RNA encoding an MRP and prevents its translation. Methods of selecting sites for targeting by zinc finger proteins have been described, for example, in U.S. Pat. No. 6,453,242, and methods for using zinc finger proteins to inhibit the expression of genes in plants are described, for example, in U.S. Patent Publication No. 20030037355; each of which is herein incorporated by reference.
In some embodiments of the invention, the polynucleotide encodes an antibody that binds to at least one maize MRP and reduces the phytate level of the plant. In another embodiment, the binding of the antibody results in increased turnover of the antibody-MRP complex by cellular quality control mechanisms. The expression of antibodies in plant cells and the inhibition of molecular pathways by expression and binding of antibodies to proteins in plant cells are well known in the art. See, for example, Conrad and Sonnewald (2003) Nature Biotech. 21: 35-36, incorporated herein by reference. In other embodiments of the invention, the polynucleotide encodes a polypeptide that specifically inhibits the MRP activity of a maize MRP, i.e., an MRP inhibitor.
In some embodiments of the present invention, the activity of an MRP is reduced or eliminated by disrupting the gene encoding the MRP. The gene encoding the MRP may be disrupted by any method known in the art. For example, in one embodiment, the gene is disrupted by transposon tagging. In another embodiment, the gene is disrupted by mutagenizing maize plants using random or targeted mutagenesis, and selecting for plants that have reduced MRP activity.
In one embodiment of the invention, transposon tagging is used to reduce or eliminate the activity of one or more MRPs. Transposon tagging comprises inserting a transposon within an endogenous MRP gene to reduce or eliminate expression of the MRP. “MRP gene” is intended to mean the gene that encodes an MRP protein according to the invention.
In this embodiment, the expression of one or more MRPs is reduced or eliminated by inserting a transposon within a regulatory region or coding region of the gene encoding the MRP. A transposon that is within an exon, intron, 5′ or 3′ untranslated sequence, a promoter, or any other regulatory sequence of an MRP gene may be used to reduce or eliminate the expression and/or activity of the encoded MRP.
Methods for the transposon tagging of specific genes in plants are well known in the art. See, for example, Maes et al. (1999) Trends Plant Sci. 4: 90-96; Dharmapuri and Sonti (1999) FEMS Microbiol. Lett. 179: 53-59; Meissner et al. (2000) Plant J. 22: 265-274; Phogat et al. (2000) J. Biosci. 25: 57-63; Walbot (2000) Curr. Opin. Plant Biol. 2: 103-107; Gai et al. (2000) Nucleic Acids Res. 28: 94-96; Fitzmaurice et al. (1999) Genetics 153: 1919-1928. In addition, the TUSC process for selecting Mu insertions in selected genes has been described in Bensen et al. (1995) Plant Cell 7: 75-84; Mena et al. (1996) Science 274: 1537-1540; and U.S. Pat. No. 5,962,764; each of which is herein incorporated by reference.
Additional methods for decreasing or eliminating the expression of endogenous genes in plants are also known in the art and can be similarly applied to the instant invention. These methods include other forms of mutagenesis, such as ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines in which the endogenous gene has been deleted. For examples of these methods see Ohshima et al. (1998) Virology 243: 472-481; Okubara et al. (1994) Genetics 137: 867-874; and Quesada et al. (2000) Genetics 154: 421-436; each of which is herein incorporated by reference. In addition, a fast and automatable method for screening for chemically induced mutations, TILLING (Targeting Induced Local Lesions In Genomes), using denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the instant invention. See McCallum et al. (2000) Nat. Biotechnol. 18: 455-457, herein incorporated by reference.
Mutations that impact gene expression or that interfere with the function of the encoded protein are well known in the art. Insertional mutations in gene exons usually result in null-mutants. Mutations in conserved residues are particularly effective in inhibiting the MRP activity of the encoded protein. Conserved residues of plant MRPs suitable for mutagenesis with the goal to eliminate MRP activity are described herein, for example in the conserved domains set forth in SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24. Such mutants can be isolated according to well-known procedures, and mutations in different MRP loci can be stacked by genetic crossing. See, for example, Gruis et al. (2002) Plant Cell 14: 2863-2882.
In another embodiment of this invention, dominant mutants can be used to trigger RNA silencing due to gene inversion and recombination of a duplicated gene locus. See, for example, Kusaba et al. (2003) Plant Cell 15: 1455-1467.
The invention encompasses additional methods for reducing or eliminating the activity of one or more MRPs. Examples of other methods for altering or mutating a genomic nucleotide sequence in a plant are known in the art and include, but are not limited to, the use of chimeric vectors, chimeric mutational vectors, chimeric repair vectors, mixed-duplex oligonucleotides, self-complementary oligonucleotides, and recombinogenic oligonucleobases. Such vectors and methods of use are known in the art. See, for example, U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984; each of which are herein incorporated by reference. See also, WO 98/49350, WO 99/07865, WO 99/25821, and Beetham et al. (1999) Proc. Natl. Acad. Sci. USA 96: 8774-8778; each of which is herein incorporated by reference. Other methods of suppressing expression of a gene involve promoter-based silencing. See, for example, Mette et al. (2000) EMBO J. 19: 5194-5201; Sijen et al. (2001) Curr. Biol. 11: 436-440; Jones et al. (2001) Curr. Biol. 11: 747-757.
Where polynucleotides are used to decrease or inhibit MRP activity, it is recognized that modifications of the exemplary sequences disclosed herein may be made as long as the sequences act to decrease or inhibit expression of the corresponding mRNA. Thus, for example, polynucleotides having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the exemplary sequences disclosed herein may be used. Furthermore, portions or fragments of the exemplary sequences or portions or fragments of polynucleotides sharing a particular percent sequence identity to the exemplary sequences may be used to disrupt the expression of the target gene. Generally, fragments or sequences of at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 250, 260, 280, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more contiguous nucleotides, or greater may be used. It is recognized that in particular embodiments, the complementary sequence of such sequences may be used. For example, hairpin constructs comprise both a sense sequence fragment and a complementary, or antisense, sequence fragment corresponding to the gene of interest. Antisense constructs may share less than 100% sequence identity with the gene of interest, and may comprise portions or fragments of the gene of interest, so long as the object of the embodiment is achieved, i.e., so long as expression of the gene of interest is decreased.
Accordingly, the methods of the invention include methods for modulating the levels of endogenous transcription and/or gene expression by transforming plants with antisense or sense constructs to produce plants with reduced levels of phytate. In some embodiments, such modifications will alter the amino acid sequence of the proteins encoded by the genomic sequence as to reduce or eliminate the activity of a particular endogenous gene, such as MRP, in a plant or part thereof, for example, in a seed.
Furthermore, it is recognized that the methods of the invention may employ a nucleotide construct that is capable of directing, in a transformed plant, the expression of at least one protein, or the transcription of at least one RNA, such as, for example, an antisense RNA that is complementary to at least a portion of an mRNA. Typically such a nucleotide construct is comprised of a coding sequence for a protein or an RNA operably linked to 5′ and 3′ transcriptional regulatory regions. Alternatively, it is also recognized that the methods of the invention may employ a nucleotide construct that is not capable of directing, in a transformed plant, the expression of a protein or transcription of an RNA.
In addition, it is recognized that methods of the present invention do not depend on the incorporation of the entire nucleotide construct into the genome, only that the plant or cell thereof is altered as a result of the introduction of the nucleotide construct into a cell. In one embodiment of the invention, the genome may be altered following the introduction of the nucleotide construct into a cell. For example, the nucleotide construct, or any part thereof, may incorporate into the genome of the plant. Alterations to the genome of the present invention include, but are not limited to, additions, deletions, and substitutions of nucleotides in the genome. While the methods of the present invention do not depend on additions, deletions, or substitutions of any particular number of nucleotides, it is recognized that such additions, deletions, or substitutions comprise at least one nucleotide.
The use of the term “nucleotide constructs” herein is not intended to limit the present invention to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides, comprised of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides may also be employed in the methods disclosed herein. Thus, the nucleotide constructs of the present invention encompass all nucleotide constructs that can be employed in the methods of the present invention for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs of the invention also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
The invention encompasses isolated or substantially purified nucleic acid or protein compositions. An “isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the nucleic acid molecule or protein as found in its naturally occurring environment. Thus, an isolated or purified nucleic acid molecule or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, an “isolated” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
By “modulating” or “modulate” as used herein is intended that the level or amount of a product is increased or decreased in accordance with the goal of the particular embodiment. For example, if a particular embodiment were useful for producing purified MRP enzyme, it would be desirable to increase the amount of MRP protein produced. As another example, if a particular embodiment were useful for decreasing the amount of phytate in a transgenic plant, it would be desirable to decrease the amount of MRP protein expressed by the plant.
The article “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one or more element.
Throughout the specification the word “comprising,” or variations such as “comprises,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Fragments and/or variants of the disclosed polynucleotides and proteins encoded thereby are also encompassed by the present invention. By “fragment” is intended a portion of the polynucleotide or a portion of the nucleotide sequence and hence protein encoded thereby, if any. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein and hence have MRP activity. Alternatively, fragments of a nucleotide sequence that are useful as hybridization probes or in sense or antisense suppression generally do not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence may range in length from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the proteins of the invention.
A fragment of an MRP nucleotide sequence that encodes a biologically active portion of an MRP protein of the invention will encode at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500 contiguous amino acids, or up to the total number of amino acids present in a full-length MRP protein of the invention (for example, 1510 amino acids for SEQ ID NO: 3). Fragments of an MRP nucleotide sequence that are useful in non-coding embodiments, for example, as PCR primers or for sense or antisense suppression, generally need not encode a biologically active portion of an MRP protein. A fragment of an MRP polypeptide of the invention will contain at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500 contiguous amino acids, or up to the total number of amino acids present in a full-length MRP protein of the invention (for example, 1510 amino acids for SEQ ID NO: 3).
Thus, a fragment of an MRP nucleotide sequence may encode a biologically active portion of an MRP protein, or it may be a fragment that can be used, for example, as a hybridization probe or in sense or antisense suppression using methods disclosed herein and known in the art. A biologically active portion of an MRP protein can be prepared by isolating a portion of one of the MRP polynucleotides of the invention, expressing the encoded portion of the MRP protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the MRP protein. Nucleic acid molecules that are fragments or portions of an MRP polynucleotide comprise at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 3,000, 4,000, or 5,000 contiguous nucleotides, or up to the number of nucleotides present in a full-length MRP polynucleotide disclosed herein (for example, 5139 nucleotides for SEQ ID NO: 2).
“Variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition at one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polypeptide or polynucleotide comprises a naturally occurring amino acid sequence or nucleotide sequence. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the MRP polypeptides of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically-derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode an MRP protein of the invention. Generally, variants of a particular polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.
Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, an isolated polynucleotide that encodes a polypeptide with a given percent sequence identity to the polypeptide of SEQ ID NO: 3 are disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. Sequences of the invention may be variants or fragments of an exemplary polynucleotide sequence, or they may be both a variant and a fragment of an exemplary sequence.
“Variant” protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, MRP activity as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native MRP protein of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. Sequences of the invention may be variants or fragments of an exemplary protein sequence, or they may be both a variant and a fragment of an exemplary sequence.
The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the MRP proteins can be prepared by the creation of mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-492; Kunkel et al. (1987) Methods in Enzymol. 154: 367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Nat'l. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be made.
Thus, the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired MRP activity. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.
The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by the methods used in Example 1 and references cited therein as well as by other assays known in the art.
Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different MRP coding sequences can be manipulated to create a new MRP possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the MRP gene of the invention and other known MRP genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased Km in the case of an enzyme. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751; Stemmer (1994) Nature 370: 389-391; Crameri et al. (1997) Nature Biotech. 15: 436-438; Moore et al. (1997) J. Mol. Biol. 272: 336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94: 4504-4509; Crameri et al. (1998) Nature 391: 288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.
The present invention further provides a method for modulating (i.e., increasing or decreasing) the concentration or composition of the polypeptides of the claimed invention in a plant or part thereof. Modulation can be effected by increasing or decreasing the concentration and/or the composition (i.e., the ratio of the polypeptides of the claimed invention) in a plant.
In some embodiments, the method comprises transforming a plant cell with a cassette comprising a polynucleotide of the invention to obtain a transformed plant cell, growing the transformed plant cell under conditions allowing expression of the polynucleotide in the plant cell in an amount sufficient to modulate concentration and/or composition of the corresponding protein in the plant cell. In some embodiments, the method comprises utilizing the polynucleotides of the invention to create a deletion or inactivation of the native gene. Thus, a deletion may constitute a functional deletion, i.e., the creation of a “null” mutant, or it may constitute removal of part or all of the coding region of the native gene. Methods for creating null mutants are well-known in the art and include, for example, chimeraplasty as discussed elsewhere herein.
In some embodiments, the content and/or composition of polypeptides of the present invention in a plant may be modulated by altering, in vivo or in vitro, the promoter of a non-isolated gene of the present invention to up- or down-regulate gene expression. In some embodiments, the coding regions of native genes of the present invention can be altered via substitution, addition, insertion, or deletion to decrease activity of the encoded enzyme. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868. One method of down-regulation of the protein involves using PEST sequences that provide a target for degradation of the protein.
In addition to sense and antisense suppression, catalytic RNA molecules or ribozymes can also be used to inhibit expression of plant genes. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al. (1988) Nature 334: 585-591.
A variety of cross-linking agents, alkylating agents and radical-generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect, and/or cleave nucleic acids. For example, Vlassov et al. (1986) Nucl. Acids Res. 14: 4065-4076 describes covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to target sequences. Similar work is reported in Knorre et al. (1985) Biochimie 67: 785-789. Others have also showed sequence-specific cleavage of single-stranded DNA mediated by incorporation of a modified nucleotide which was capable of activating cleavage (Iverson and Dervan (1987) J. Am. Chem. Soc. 109: 1241-1243). Meyer et al. ((1989) J. Am. Chem. Soc. 111: 8517-8519) demonstrated covalent crosslinking to a target nucleotide using an alkylating agent complementary to the single-stranded target nucleotide sequence. Lee et al. ((1988) Biochemistry 27: 3197-3203) disclosed a photoactivated crosslinking to single-stranded oligonucleotides mediated by psoralen. Home et al. ((1990) J. Am. Chem. Soc. 112: 2435-2437) used crosslinking with triple-helix-forming probes. Webb and Matteucci ((1986) J. Am. Chem. Soc. 108: 2764-2765) and Feteritz et al. ((1991) J. Am. Chem. Soc. 113: 4000) used N4, N4-ethanocytosine as an alkylating agent to crosslink to single-stranded oligonucleotides. In addition, various compounds to bind, detect, label, and/or cleave nucleic acids are known in the art. See, for example, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648; and 5,681,941. Such embodiments are collectively referred to herein as “chemical destruction.”
In some embodiments, an isolated nucleic acid (e.g., a vector) comprising a promoter sequence is transfected into a plant cell. Subsequently, a plant cell comprising the promoter operably linked to a nucleic acid or polynucleotide comprising a nucleotide sequence of the present invention is selected for by means known to those of skill in the art such as, but not limited to, Southern blot, DNA sequencing, or PCR analysis using primers specific to the promoter and to the gene and detecting amplicons produced therefrom. A plant or plant part altered or modified by the foregoing embodiments is grown under plant-forming conditions for a time sufficient to modulate the concentration and/or composition of polypeptides of the present invention in the plant. Plant-forming conditions are well known in the art.
In general, when an endogenous polypeptide is modulated using the methods of the invention, the content of the polypeptide in a plant or part or cell thereof is increased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more relative to a native control plant, plant part, or cell lacking the aforementioned cassette. Modulation in the present invention may occur during and/or subsequent to growth of the plant to the desired stage of development. Modulating nucleic acid expression temporally and/or in particular tissues can be controlled by employing the appropriate promoter operably linked to a polynucleotide of the present invention in, for example, sense or antisense orientation.
A transformed plant or transformed plant cell of the invention is one in which genetic alteration, such as transformation, has been effected as to a gene of interest, or is a plant or plant cell which is descended from a plant or cell so altered and which comprises the alteration. A “control” or “control plant” or “control plant cell” provides a reference point for measuring changes in phenotype of the subject plant or plant cell. A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.
The polynucleotides of the invention can be used to isolate corresponding sequences from other organisms, particularly other plants. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire MRP sequences set forth herein or to variants and fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. “Orthologs” is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, isolated sequences that encode an MRP protein or have Lpa1 promoter activity and which hybridize under stringent conditions to the Lpa1 sequences disclosed herein, or to variants or fragments thereof, are encompassed by the present invention.
In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.
In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other nucleic acids comprising corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the MRP sequences of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
For example, the entire MRP sequences disclosed herein, or one or more portions thereof, may be used as probes capable of specifically hybridizing to corresponding MRP sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among MRP sequences and are at least about 10, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 60, 70, 80, 90, or more nucleotides in length. Such probes may be used to amplify corresponding MRP sequences from a chosen plant by PCR. This technique may be used to isolate additional coding sequences from a desired plant or as a diagnostic assay to determine the presence of coding sequences in a plant. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 or 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4, 8, or 12 hours.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, “% form” is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm, of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). The duration of the wash time will be at least a length of time sufficient to reach equilibrium, for example, 4 hours, 8 hours, or 12 hours.
The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, and (d) “percentage of sequence identity.”
(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
(b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, or 100 nucleotides in length, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4: 11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453; the search-for-local-alignment-method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877.
Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73: 237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153; Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang et al. (1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24: 307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al (1990) J. Mol. Biol. 215: 403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See http://www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3 and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2; and the BLOSUM62 scoring matrix or any equivalent program thereof. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.
GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
(c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
(d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
The use of the term “polynucleotide” is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
The MRP polynucleotide of the invention can be provided in expression cassettes for expression in the plant of interest. The cassette will include any necessary 5′ and 3′ regulatory sequences operably linked to an MRP polynucleotide of the invention. “Operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, “operably linked” is intended to mean that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the MRP polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes. If protein expression is desired, the cassette may be referred to as a protein expression cassette and will include in the 5′-3′ direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), an MRP nucleotide sequence of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in plants.
The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the MRP polynucleotide of the invention may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the MRP polynucleotide of the invention may be heterologous to the host cell or to each other. As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from that from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form, or the promoter is not the native promoter for the operably linked polynucleotide.
While it may be optimal to express the sequences using heterologous promoters, the native promoter sequences (e.g., the promoter sequence set forth in SEQ ID NO: 1) may be used. Such constructs can change expression levels of MRP in the plant or plant cell. Thus, the phenotype of the plant or plant cell can be altered. The promoter sequence set forth in SEQ ID NO:1 contains a putative TATA box from nucleotides 2464 to 2470; the 5′ UTR may contain an intron.
In an expression cassette, the termination region may be native with the transcriptional initiation region, may be native with the operably linked nucleotide sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the nucleotide sequence of interest, the plant host, or any combination thereof). Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15: 9627-9639.
Where appropriate, the polynucleotides may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17: 477-498, herein incorporated by reference.
Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell, and the sequence may be modified to avoid predicted hairpin secondary mRNA structures.
The expression cassettes may additionally contain 5′ leader sequences in the cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2): 233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154: 9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353: 90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81: 382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968.
The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85: 610-9 and Fetter et al. (2004) Plant Cell 16: 215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117: 943-54 and Kato et al. (2002) Plant Physiol 129: 913-42), and yellow florescent protein (PhiYFPTM from Evrogen; see Bolte et al. (2004) J. Cell Science 117: 943-54).
See generally, Yarranton (1992) Curr. Opin. Biotech. 3: 506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6314-6318; Yao et al. (1992) Cell 71: 63-72; Reznikoff (1992) Mol. Microbiol. 6: 2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48: 555-566; Brown et al. (1987) Cell 49: 603-612; Figge et al. (1988) Cell 52: 713-722; Deuschle et al. (1989) Proc. Natl. Acad. Aci. USA 86: 5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86: 2549-2553; Deuschle et al. (1990) Science 248: 480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90: 1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10: 3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89: 3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88: 5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19: 4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89: 5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36: 913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334: 721-724. Such disclosures are herein incorporated by reference.
The above list of selectable marker genes is not meant to be limiting. Any suitable selectable marker gene can be used in the present invention, and one of skill in the art will be able to determine which selectable marker gene is suitable for a particular application.
In preparing the cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.
A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, or other promoters. Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313: 810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18: 675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J. 3: 2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.
Chemical-regulated promoters can be used to modulate the transcription and/or expression of a particular nucleotide sequence in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J. 14(2): 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.
Tissue-preferred promoters can be utilized to target enhanced MRP transcription and/or expression within a particular plant tissue. Tissue-preferred promoters include those described in Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al. (1997) Mol. Gen. Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2): 525-535; Canevascini et al. (1996) Plant Physiol. 112(2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20): 9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3): 495-505. Such promoters can be modified, if necessary, for weak expression.
Leaf-preferred promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kwon et al. (1994) Plant Physiol. 105: 357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Gotor et al. (1993) Plant J. 3: 509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20): 9586-9590.
Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2): 207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10): 1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3): 433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1): 11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7): 633-641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-specific promoter activity was preserved. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1): 69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri et al. (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2′ gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see EMBO J. 8(2): 343-350). The TR1′ gene, fused to nptII (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4): 759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4): 681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.
“Seed-preferred” promoters include both “seed-specific” promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seed-germinating” promoters (those promoters active during seed germination). See Thompson et al. (1989) BioEssays 10: 108, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphate synthase); oleosin; and celA (cellulose synthase) (see WO 00/11177 and U.S. Pat. No. 6,225,529, herein incorporated by reference). Gamma-zein is a preferred endosperm-specific promoter. Globulin (Glb-1) is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, where seed-preferred promoters from end1 and end2 genes are disclosed; herein incorporated by reference.
Where low level transcription or expression is desired, weak promoters will be used. Generally, by “weak promoter” is intended a promoter that drives transcription and/or expression of a coding sequence at a low level. By low level is intended at levels of about 1/1000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts. Alternatively, it is recognized that weak promoters also encompasses promoters that are expressed in only a few cells and not in others to give a total low level of transcription and/or expression. Where a promoter is expressed at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease transcription and/or expression levels.
Such weak constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), the core 35S CaMV promoter, and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. See also, U.S. Pat. No. 6,177,611, herein incorporated by reference.
In one embodiment, the polynucleotides of interest are targeted to the chloroplast for expression. In this manner, where the nucleic acid of interest is not directly inserted into the chloroplast, the expression cassette will additionally contain a nucleic acid encoding a transit peptide to direct the gene product of interest to the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196: 1414-1421; and Shah et al. (1986) Science 233: 478-481.
Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5): 3335-3342); 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al. (1990) J. Bioenerg. Biomemb. 22(6): 789-810); tryptophan synthase (Zhao et al. (1995) J. Biol. Chem. 270(11): 6081-6087); plastocyanin (Lawrence et al. (1997) J. Biol. Chem. 272(33): 20357-20363); chorismate synthase (Schmidt et al. (1993) J. Biol. Chem. 268(36): 27447-27457); and the light harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al. (1988) J. Biol. Chem. 263: 14996-14999). See also Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196: 1414-1421; and Shah et al. (1986) Science 233: 478-481.
Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87: 8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90: 913-917; Svab and Maliga (1993) EMBO J. 12: 601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91: 7301-7305.
The polynucleotides of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the polynucleotides of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831, herein incorporated by reference.
In specific embodiments, the MRP sequences of the invention can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the MRP protein or variants and fragments thereof directly into the plant or the introduction of an MRP transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol. Gen. Genet. 202: 179-185; Nomura et al. (1986) Plant Sci. 44: 53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107: 775-784, all of which are herein incorporated by reference. Alternatively, the MRP polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, the transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use particles coated with polyethylimine (PEI; Sigma # P3143).
Thus, transgenic plants having low phytic acid content and high levels of bioavailable phosphorus can be generated by reducing or inhibiting MRP gene expression in a plant. For example, the transgenic plant can contain a transgene comprising an inverted repeat of Lpa1 that suppresses endogenous Lpa1 gene expression. In this manner, transgenic plants having the low phytic acid phenotype of lpa1 mutant plants can be generated. The transgenic plant can contain an MRP suppressor sequence alone or an MRP suppressor sequence can be “stacked” with one or more polynucleotides of interest, including, for example, one or more polynucleotides that can affect phytic acid levels or that provide another desirable phenotype to the transgenic plant. For example, such a transgene can be “stacked” with similar constructs involving one or more additional inositol phosphate kinase genes such as ITPK-5 (inositol 1,3,4-trisphosphate 5/6 kinase; e.g., SEQ ID NO: 65; see also WO 03/027243), IPPK (inositol polyphosphate kinase; e.g., SEQ ID NO: 64; see also WO 02/049324), and/or a myo-inositol-1 phosphate synthase gene (milps; see U.S. Pat. Nos. 6,197,561 and 6,291,224; e.g., milps-3 (SEQ ID NO: 25)). With such “stacked” transgenes, even greater reduction in phytic acid content of a plant can be achieved, thereby making more phosphorus bioavailable.
Thus, in certain embodiments the nucleic acid sequences of the present invention can be “stacked” with any combination of nucleic acids of interest in order to create plants with a desired phenotype. By “stacked” or “stacking” is intended that a plant of interest contains one or more nucleic acids collectively comprising multiple nucleotide sequences so that the transcription and/or expression of multiple genes are altered in the plant. For example, antisense nucleic acids of the present invention may be stacked with other nucleic acids which comprise a sense or antisense nucleotide sequence of at least one of ITPK-5 (e.g., SEQ ID NO: 65) and/or inositol polyphosphate kinase (IPPK; e.g., SEQ ID NO: 64), or other genes implicated in phytic acid metabolic pathways such as Lpa3 or myo-inositol kinase (see, e.g., copending application entitled, “Plant Myo-Inositol Kinase Polynucleotides and Methods of Use, Appl. No. 60/573,000, filed May 20, 2004; SEQ ID NO: 68); Lpa2 (see U.S. Pat. Nos. 5,689,054 and 6,111,168); myo-inositol 1-phosphate synthase (milps; e.g., SEQ ID NO: 25), myo-inositol monophosphatase (IMP) (see WO 99/05298 and U.S. application Ser. No. 10/042,465, filed Jan. 9, 2002); IP2K (e.g., SEQ ID NO: 67); and the like. The addition of such nucleic acids could enhance the reduction of phytic acid and InsP intermediates, thereby providing a plant with more bioavailable phosphate and/or reduced phytate. The nucleic acids of the present invention can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations. For example, in some embodiments, a phytase gene (e.g., SEQ ID NO: 66) is stacked with an lpa1 mutant so that phytase is expressed at high levels in the transgenic plant. Phytase genes are known in the art. See, for example, Maugenest et al. (1999) Plant Mol. Biol. 39: 503-514; Maugenest et al. (1997) Biochem. J. 322: 511-517; WO 200183763; WO200200890.
An MRP polynucleotide also can be stacked with any other polynucleotide(s) to produce plants having a variety of desired trait combinations including, for example, traits desirable for animal feed such as high oil genes (see, e.g., U.S. Pat. No. 6,232,529, which is incorporated herein by reference); balanced amino acids (e.g., hordothionins; see U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409, each of which is incorporated herein by reference); barley high lysine (Williamson et al. (1987) Eur. J. Biochem. 165: 99-106 and WO 98/20122); high methionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261: 6279; Kirihara et al. (1988) Gene 71: 359; and Musumura et al. (1989) Plant Mol. Biol. 12: 123); increased digestibility (e.g., modified storage proteins) and thioredoxins (U.S. Pat. No. 7,009,087).
An MRP polynucleotide also can be stacked with one or more polynucleotides encoding a desirable trait such as a polynucleotide that confers, for example, insect, disease or herbicide resistance (e.g., Bacillus thuringiensis toxic proteins; U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et al. (1986) Gene 48: 109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24: 825); fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence and disease resistance genes (Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432; Mindrinos et al. (1994) Cell 78: 1089); acetolactate synthase mutants that lead to herbicide resistance such as the S4 and/or Hra mutations; inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., the bar gene); and glyphosate (e.g., the EPSPS gene and the GAT gene; see, for example, U.S. Publication No. 20040082770 and WO 03/092360). Additional polynucleotides that can be stacked with a MRP polynucleotide include, for example, those encoding traits desirable for processing or process products such as modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516); modified starches (e.g., ADPG pyrophosphorylases, starch synthases, starch branching enzymes, and starch debranching enzymes); and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321). An MRP polynucleotide of the invention also can be stacked with one or more polynucleotides that provide desirable agronomic traits such as male sterility (e.g., U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g., WO 99/61619; WO 00/17364; WO 99/25821). Other desirable traits that are known in the art include high oil content; increased digestibility; balanced amino acid content; and high energy content. Such traits may refer to properties of both seed and non-seed plant tissues, or to food or feed prepared from plants or seeds having such traits; such food or feed will have improved quality.
These stacked combinations can be created by any method including but not limited to cross breeding plants by any conventional or TopCross methodology, or genetic transformation. In this regard, it is understood that transformed plants of the invention include a plant that contains a sequence of the invention that was introduced into that plant via breeding of a transformed ancestor plant. If traits are stacked by genetically transforming the plants, the nucleic acids of interest can be combined at any time and in any order. More generally, where any method requires more than one step to be performed, it is understood that steps may be performed in any order that accomplishes the desired end result. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of cassettes suitable for transformation. For example, if two sequences will be introduced, the two sequences can be contained in separate cassettes (trans) or contained on the same transformation cassette (cis). Transcription and/or expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other cassettes to generate the desired combination of traits in the plant. Alternatively, traits may be stacked by transforming different plants to obtain those traits; the transformed plants may then be crossed together and progeny may be selected which contains all of the desired traits.
Stacking may also be performed with fragments of a particular gene or nucleic acid. In such embodiments, a plants is transformed with at least one fragment and the resulting transformed plant is crossed with another transformed plant; progeny of this cross may then be selected which contain the fragment in addition to other transgenes, including, for example, other fragments. These fragments may then be recombined or otherwise reassembled within the progeny plant, for example, using site-specific recombination systems known in the art. Such stacking techniques could be used to provide any property associated with fragments, including, for example, hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference.
It is understood that in some embodiments the nucleic acids to be stacked with MRP can also be designed to reduce or eliminate the expression of a particular protein, as described in detail herein for MRP. Thus, the methods described herein with regard to the reduction or elimination of expression of MRP are equally applicable to other nucleic acids and nucleotide sequences of interest, such as, for example, IPPK, ITPK-5, and milps, examples of which are known in the art and which are expected to exist in most varieties of plants. Accordingly, the descriptions herein of MRP fragments, variants, and other nucleic acids and nucleotide sequences apply equally to other nucleic acids and nucleotide sequences of interest such as milps (e.g., SEQ ID NO: 25), IPPK (e.g., SEQ ID NO: 64), ITPK-5 (e.g., SEQ ID NO: 65), IP2K (e.g., SEQ ID NO:67), and Lpa3 or MIK (myo-inositol kinase; e.g., SEQ ID NO: 68). For example, an antisense construct could be designed for milps comprising a nucleotide sequence that shared 90% sequence identity to the complement of SEQ ID NO: 25 or was at least a 19-nucleotide fragment of the complement of SEQ ID NO: 25.
Transformation protocols as well as protocols for introducing polypeptides or polynucleotides into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides or polynucleotides into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4: 320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83: 5602-5606, Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al., U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3: 2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No. 5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6: 923-926); and Lec1 transformation (WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22: 421-477; Sanford et al. (1987) Particulate Science and Technology 5: 27-37 (onion); Christou et al. (1988) Plant Physiol. 87: 671-674 (soybean); McCabe et al. (1988) Bio/Technology 6: 923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96: 319-324 (soybean); Datta et al. (1990) Biotechnology 8: 736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85: 4305-4309 (maize); Klein et al. (1988) Biotechnology 6: 559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol. 91: 440-444 (maize); Fromm et al. (1990) Biotechnology 8: 833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311: 763-764; Bowen et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9: 415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84: 560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12: 250-255 and Christou and Ford (1995) Annals of Botany 75: 407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.
The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5: 81-84. These plants may then be grown and either pollinated with the same transformed strain or different strains; the resulting progeny having the desired phenotypic characteristic can then be identified. Two or more generations may be grown to ensure that the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure that stable transformants exhibiting the desired phenotypic characteristic have been achieved. In this manner, the present invention provides transformed seed (also referred to as “transgenic seed”) having a nucleotide construct of the invention, for example, a cassette of the invention, stably incorporated into their genome.
As used herein, the term “plant” includes plant cells, plant protoplasts, plant cell tissue cultures from which maize plant can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides.
The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica spp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.
Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.
Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). In specific embodiments, plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments, corn and soybean plants are optimal, and in yet other embodiments corn plants are optimal.
Other plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
The methods of the invention involve introducing a polypeptide or polynucleotide into a plant. “Introducing” is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a sequence into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide or polypeptides into plants are known in the art, including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
“Stable transformation” is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof “Transient transformation” is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or that a polypeptide is introduced into a plant.
Thus, it is recognized that methods of the present invention do not depend on the incorporation of an entire nucleotide construct into the genome, only that the plant or cell thereof is altered as a result of the introduction of a nucleotide construct or polypeptide into a cell. In one embodiment of the invention, the genome may be altered following the introduction of a nucleotide construct into a cell. For example, the nucleotide construct, or any part thereof, may incorporate into the genome of the plant. Alterations to the genome of the present invention include, but are not limited to, additions, deletions, and substitutions of nucleotides in the genome. While the methods of the present invention do not depend on additions, deletions, or substitutions of any particular number of nucleotides, it is recognized that such additions, deletions, or substitutions comprise at least one nucleotide.
In other embodiments, the polynucleotides of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the invention within a viral DNA or RNA molecule. It is recognized that an MRP of the invention may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing nucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367; 5,316,931, and Porta et al. (1996) Molecular Biotechnology 5: 209-221; herein incorporated by reference.
The use of the term polynucleotides herein is not intended to limit the present invention to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides, comprised of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides may also be employed in the methods disclosed herein. Thus, the nucleotide constructs of the present invention encompass all nucleotide constructs that can be employed in the methods of the present invention for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs of the invention also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
The promoter nucleotide sequences and methods disclosed herein are useful in regulating expression of any heterologous nucleotide sequence in a host plant in order to vary the phenotype of a plant. Because the Lpa1 promoter provides weak constitutive expression of operably linked coding regions, the Lpa1 promoter finds particular use in altering gene expression in various tissues.
Various changes in phenotype are of interest including modifying the fatty acid composition in seeds, altering the amino acid content of seeds, altering a seed's pathogen defense mechanism, and the like. These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in embryos. Alternatively, the results can be achieved by providing for a reduction of expression of one or more endogenous products, particularly enzymes or cofactors in the seed. These changes result in a change in phenotype of the transformed plant.
Genes of interest are reflective of the commercial markets and interests of those involved in the development of the crop. Crops and markets of interest change, and as developing nations open up world markets, new crops and technologies will emerge also. In addition, as our understanding of agronomic traits and characteristics such as yield and heterosis increase, the choice of genes for transformation will change accordingly. General categories of genes of interest include, for example, those genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include genes encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products. Genes of interest include, generally, those involved in oil, starch, carbohydrate, or nutrient metabolism as well as those affecting kernel size, sucrose loading, and the like.
Agronomically important traits such as oil, starch, and protein content can be genetically altered by genetic engineering in addition to using traditional breeding methods. Modifications include increasing content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur, providing essential amino acids, and also modification of starch. Hordothionin protein modifications are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389, herein incorporated by reference. Another example is lysine and/or sulfur rich seed protein encoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016, and the chymotrypsin inhibitor from barley, described in Williamson et al. (1987) Eur. J. Biochem. 165: 99-106, the disclosures of which are herein incorporated by reference.
Derivatives of the coding sequences can be made by site-directed mutagenesis to increase the level of preselected amino acids in the encoded polypeptide. For example, the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor, U.S. application Ser. No. 08/740,682, filed Nov. 1, 1996, and WO 98/20133, the disclosures of which are herein incorporated by reference. Other proteins include methionine-rich plant proteins such as from sunflower seed (Lilley et al. (1989) Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed. Applewhite (American Oil Chemists Society, Champaign, Ill.), pp. 497-502); corn (Pedersen et al. (1986) J. Biol. Chem. 261: 6279; Kirihara et al. (1988) Gene 71: 359); and rice (Musumura et al. (1989) Plant Mol. Biol. 12: 123). Other agronomically important genes encode latex, Floury 2, growth factors, seed storage factors, and transcription factors.
Insect resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48: 109, and the like.
Genes encoding disease resistance traits include detoxification genes, such as against fumonisin (U.S. Pat. No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432; and Mindrinos et al. (1994) Cell 78: 1089); and the like.
Herbicide resistance traits may include genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance, in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides that act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS-gene mutants encode resistance to the herbicide chlorsulfuron. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development.
The quality of grain is reflected in traits such as, for example, levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, and levels of cellulose. In corn, modified hordothionin proteins are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.
Commercial traits can also be encoded on a gene or genes that could increase for example, starch for ethanol production, or provide expression of proteins. Another important commercial use of transformed plants is the production of polymers and bioplastics such as described in U.S. Pat. No. 5,602,321. Genes such as β-Ketothiolase, PHBase (polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase (see Schubert et al. (1988) J. Bacteriol. 170: 5837-5847) facilitate expression of polyhyroxyalkanoates (PHAs).
Exogenous products include plant enzymes and products as well as those from other sources including procaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones, and the like. The level of proteins, particularly modified proteins having improved amino acid distribution to improve the nutrient value of the plant, can be increased. This is achieved by the expression of such proteins having enhanced amino acid content.
Some chemicals can inhibit MRP protein transport activity. For example, the sulfonylurea glibenclamide can inhibit the glucuronide transport activity of Arabidopsis AtMRP5 and can affect its function in guard cells (Gaedeke et al. (2001) EMBO J. 20: 1875-1887; Lee et al. (2004) Plant Physiol. 134: 528-538). It is expected that glibenclamide would also inhibit maize MRP3 transport activity and thus would produce a low phytic acid phenotype.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
EXPERIMENTAL
Example 1
Identification and Characterization of Maize Low Phytic Acid (Lpa) Mutant Plants
A collection of indexed mutagenized F2 families derived from several Mu active stocks (Bensen et al. (1995) Plant Cell 7: 75-84) was screened for seeds having high inorganic phosphate content using a rapid Pi assay as described below. Candidates identified as producing high-Pi seed were crossed with suitable maize and the progeny examined to confirm the mutations and to determine whether the mutations were allelic to the previously identified lpa1 mutant (referred to herein as “lpa1-1”; see U.S. Pat. No. 5,689,054; Raboy et al. (2000) Plant Physiol. 124: 355-68). Several of these lpa lines were allelic to the earlier-identified lpa1 mutant, and these Mu-insertion alleles of the lpa1 mutant were used to clone the gene responsible for the lpa1 mutation. Segregation populations were created by crossing heterozygous line PV03 57 C-05 (carrying Mu-tagged lpa1) with homozygous line GP24L3 (carrying EMS allele lpa1-1). F1 plants were self-pollinated to produce F2 seeds. The phenotype of F1 plants was determined by analyzing F2 seed Pi and phytic acid. Genomic DNA was extracted from leaves of individual F1 plants and used for PCR analysis as further described in Example° 2.
Inorganic Phosphate (Pi) Assay
A rapid test was used to assay inorganic phosphate content in kernels. Individual kernels were placed in a 25-well plastic tray and crushed at 2000 psi using a hydraulic press. Two milliliters of 1N H2SO4 was added to each sample. The samples were incubated at room temperature for two hours, after which four milliliters of 0.42% ammonium molybdate-1N H2SO4:10% ascorbic acid (6:1) was added to each sample. Increased Pi content was signaled by the development of blue color within about 20 minutes. Positive controls included lpa2 mutant kernels, and negative controls included wild-type kernels.
Determination of Phytic Acid and Inorganic Phosphate Content
Dry, mature seeds were assayed for phytic acid and Pi content using modifications of the methods described by Haug and Lantzsch ((1983) J. Sci. Food Agric. 34: 1423-1426, entitled “Sensitive method for the rapid determination of phytate in cereals and cereal products”) and Chen et al. ((1956) Anal. Chem. 28: 1756-1758, entitled “Microdetermination of phosphorus”). Single kernels were ground using a Geno/Grinder2000TM grinder (Sepx CertiPrep®, Metuchen, N.J.). Samples of 25 to 35 mg were placed into 1.5 ml Eppendorf® tubes and 1 ml of 0.4 N HCl was added to the tubes, which were then shaken on a gyratory shaker at room temperature for 3.5 hours. The tubes were then centrifuged at 3,900 g for 15 minutes. Supernatants were transferred into fresh tubes and used for both phytic acid and Pi determinations; measurements were performed in duplicate.
For the phytic acid assay, 35 μl of each extract was placed into wells of a 96-well microtiter plate and then 35 μl of distilled H2O and 140 μl of 0.02% ammonium iron (III) sulphate-0.2 N HCl were added to each sample. The microtiter plate was covered with a rubber lid and heated in a thermal cycler at 99° C. for 30 minutes, then cooled to 4° C. and kept on an ice water bath for 15 minutes, and then left at room temperature for 20 minutes. The plate was then sealed with sticky foil and centrifuged at 3,900 g at 24° C. for 30 minutes. Eighty μl of each supernatant was placed into wells of a fresh 96-well plate. For absorbance measurements, 120 μl of 1% 2,2′-bipyridine-1% thioglycolic acid solution (10 g 2,2′-bipyridine (Merck Art. 3098), 10 ml thioglycolic acid (Merck Art. 300) in ddw to 1 liter) was added to each well and absorbance was recorded at 519 nm using a VERSAmaxTM microplate reader (Molecular Devices®, Sunnyvale, Calif.). Phytic acid content is presented as phytic acid phosphorus (PAP). Authentic phytic acid (Sigma®, P-7660) served as a standard. This phytic acid assay also measured InsP5 and InsP4 present in the samples.
Phytic acid was also assayed according to modifications of the methods described by Latta & Eskin (1980) (J. Agric Food Chem. 28: 1313-1315) and Vaintraub & Lapteva (1988) (Analytical Biochemistry 175: 227-230). For this assay, 25 μl of extract was placed into wells of a 96-well microtiter plate; then 275 μl of a solution of 36.3 mM NaOH and 100 μl of Wade reagent (0.3% sulfosalicylic acid in 0.03% FeCl3.6H2O) was added to each well. The samples were mixed and centrifuged at 39,000 g at 24° C. for 10 minutes. An aliquot of supernatant (200 μl) from each well was transferred into a new 96-well plate, and absorbance was recorded at 500 nm using a VERSAmaxTM microplate reader (Molecular Devices®, Sunnyvale, Calif.).
To determine Pi, 200 μl of each extract was placed into wells of a 96 well microtiter plate. 100 μl of 30% aqueous trichloroacetic acid was then added to each sample and the plates were shaken and then centrifuged at 3,900 g for 10 minutes. Fifty μl of each supernatant was transferred into a fresh plate and 100 μl of 0.42% ammonium molybdate-1N H2SO4: 10% ascorbic acid (7:1) was added to each sample. The plates were incubated at 37° C. for 30 minutes and then absorbance was measured at 800 nm. Potassium phosphate was used as a standard. Pi content was presented as inorganic phosphate phosphorus.
Determination of Seed Myo-Inositol
Myo-inositol was quantified in dry, mature seeds and excised embryos. Tissue was ground as described above and mixed thoroughly. 100 milligram samples were placed into 7 ml scintillation vials and 1 ml of 50% aqueous ethanol was added to each sample. The vials were then shaken on a gyratory shaker at room temperature for 1 hour. Extracts were decanted through a 0.45 μm nylon syringe filter attached to a 1 ml syringe barrel. Residues were re-extracted with 1 ml fresh 50% aqueous ethanol and the second extracts were filtered as before. The two filtrates were combined in a 10×75 mm glass tube and evaporated to dryness in a SpeedVac® microcentrifuge (Savant). The myo-inositol derivative was produced by redissolving the residues in 50 μl of pyridine and 50 μl of trimethylsilyl-imidazole:trimethylchlorosilane (100:1) (Tacke and Casper (1996) J. AOAC Int. 79: 472-475). Precipitate appearing at this stage indicates that the silylation reaction did not work properly. The tubes were capped and incubated at 60° C. for 15 minutes. One milliliter of 2,2,4-trimethylpentane and 0.5 milliliters of distilled water were added to each sample. The samples were then vortexed and centrifuged at 1,000 g for 5 minutes. The upper organic layers were transferred with Pasteur pipettes into 2 milliliter glass autosampler vials and crimp capped.
Myo-inositol was quantified as a hexa-trimethylsilyl ether derivative using an Agilent Technologies® model 5890 gas chromatograph coupled with an Agilent Technologies® model 5972 mass spectrometer. Measurements were performed in triplicate. One μl samples were introduced in the splitless mode onto a 30 m×0.25 mm i.d.×0.25 μm film thickness 5MS column (Agilent Technologies®). The initial oven temperature of 70° C. was held for 2 minutes, then increased at 25° C. per minute to 170° C., then increased at 5° C. per minute to 215° C., and finally increased at 25° C. per minute to 250° C. and then held for 5 minutes. The inlet and transfer line temperatures were 250° C. Helium at a constant flow of 1 ml per minute was used as the carrier gas. Electron impact mass spectra from m/z 50-560 were acquired at −70 eV after a 5-minute solvent delay. The myo-inositol derivative was well resolved from other peaks in the total ion chromatograms. Authentic myo-inositol standards in aqueous solutions were dried, derivatized, and analyzed at the same time. Regression coefficients of four-point calibration curves were typically 0.999-1.000.
Determination of Seed Inositol Phosphates
The presence of significant amounts of inositol phosphates in mature seeds was determined by HPLC according to the Dionex Application Note AN65, “Analysis of inositol phosphates” (Dionex Corporation®, Sunnyvale, Calif.). Tissue was ground and mixed as described above. 500 mg samples were placed into 20 ml scintillation vials and 5 ml of 0.4 M HCl was added to the samples. The samples were shaken on a gyratory shaker at room temperature for 2 hours and then allowed to sit at 4° C. overnight. Extracts were centrifuged at 1,000 g for 10 min and filtered through a 0.45 μm nylon syringe filter attached to a 5 ml syringe barrel. Just prior to HPLC analysis, 600 μl aliquots of each sample were clarified by passage through a 0.22 μm centrifugal filter. A Dionex Corporation® DX 500 HPLC with a Dionex Corporation® model AS3500 autosampler was used. 25 μl samples were introduced onto a Dionex Corporation® 4×250 mm OmniPacTM PAX-100 column; Dionex Corporation® 4×50 mm OmniPacTM PAX-100 guard and ATC-1 anion trap columns also were used. Inositol phosphates were eluted at 1 ml/min with the following mobile phase gradient: 68% A (distilled water)/30% B (200 mM NaOH) for 4.0 min; 39% A/59% B at 4.1 through 15.0 min; return to initial conditions at 15.1 min. The mobile phase contained 2% C (50% aqueous isopropanol) at all times to maintain column performance. A Dionex Corporation® conductivity detector module II was used with a Dionex Corporation® ASRS-Ultra II anion self-regenerating suppressor set up in the external water mode and operated with a current of 300 mA. Although quantitative standards were available, InsP3, InsP4 and InsP5 were partially but clearly resolved from each other and InsP6.
The results of the above assays demonstrated that the lpa1 mutant maize plants have a phenotype of reduced phytic acid and increased Pi in seeds, but lpa1 seeds do not accumulate inositol phosphate intermediates.
Example 2
Isolation and Characterization of Maize MRP3 (Lpa1) Gene
Initially, a PCR-based method was used in an effort to clone the lpa1 gene, but this effort was unsuccessful. However, a Mu-insertion site in a transcriptional activator gene was identified, and co-segregation analysis indicated that this Mu-insertion site was very closely linked to the Lpa1 locus. This marker, designated “TAP,” was used for map-based cloning of the Lpa1 gene.
The PCR protocol used to identify the TAP marker is known as SAIFF: Selected Amplification of Insertion Flanking Fragments. First, genomic DNA was prepared from 5-8 plants of individual lines which were Mu+ and Mu−. The genomic DNA was digested with BfaI or MseI, neither of which cuts the Mu TIR (Terminal Inverted Repeat). The restriction ends generated by BfaI and MseI are the same and are compatible with the Mse/Bfa adaptor.

    
    
        10×RL buffer: 2.5 μl
        BSA: 0.25 μl
        DNA: 0.3-0.5 μg
        Enzymes: 1 μl
        Water: bring to 25 μl
    
    


This mixture was incubated at 37° C. for 3 to 6 hours and then denatured at 65° C. for 20 minutes. Adaptors were then ligated to the digested DNA by adding 5 μl of adaptor mixture to each reaction:

    
    
        100 mM rATP: 0.3 μl
        10×RL buffer: 0.5 μl
        40 uM Adaptor: 1 μl
        T4 ligase: 1 μl (3 U/μl)
        Water: bring to 5 μl
    
    


This mixture was then incubated at 4° C. overnight. The ligation reaction was purified with a PCR Purification Kit (Qiagen®) to remove excess adaptors, and the reaction was brought to a final volume of 50 μl in water or elution buffer.
Control PCR was performed to check the digestion and ligation. Either regular Taq enzyme or another non-hot start DNA polymerase was used for the control PCR. 1 μl of the purified ligation reaction was used as the template in a 10 μl PCR reaction. The primer used was the adaptor primer (MspExt18 or the nested MseInt18 primer). DMSO was added to the mixture to a final level of 5%. The PCR conditions were 94° C. 2 min; 35 cycles of 94° C. 30 sec, 55° C. 30 sec, and 72° C. 2 min 30 sec; and a final extension at 72° C. for 7 min. The reaction was then run on a 1% agarose gel and the amplification reaction visualized. Non-specific adaptor-to-adaptor amplification should occur, and there should be a nice smear on the gel ranging in size from 300 bp to 3 kb.
1 μl of the purified ligation reaction was then used as the template in a 10 μl PCR reaction using Hot StartTM DNA polymerase (Qiagen®). Primers MuExt22D and MspExt18 were added to a final concentration of 0.3-0.5 μM. DMSO was added to a final level of 5%. PCR conditions were 95° C. 15 min, 20 cycles of 94° C. 30 sec, 55° C. 30 sec, and 72° C. 2 min 30 sec, followed by a final extension at 72° C. for 7 min. The reaction was then diluted 1:10 with water.
Nested (2nd round) PCR was performed with Ex Taq DNA polymerase, but any robust enzyme could be used. 1 μl of the Mu+ and Mu− pools was used as template in a 10 μl reaction. The primers were MuInt19 and Adaptor nested primers (+2 selective primers, 0.3-0.5 μM final concentration). DMSO was added to a final level of 5%. “Touchdown” PCR conditions were: 95° C. 2 min, 11 cycles of 94° C. 30 sec, (65° C.-0.8° C./cycle) for 30 sec, and 72° C. 2 min 30 sec, followed by 24 cycles of 94° C. 30 sec, 56° C. 30 sec, and 72° C. 2 min 30 sec, with a final extension at 72° C. for 7 min. PCR reactions were electrophoresed on a 1.5% agarose gel and examined to identify bands which were present in the Mu+ pool but absent in the Mu− pool.
The second-round (nested) PCR was then repeated using as template first round PCR reactions from individual plants to confirm the co-segregation. DNA fragments that were present in all Mu+ individuals and absent in all Mu− individuals were isolated from the gel and purified. The purified DNA was cloned into a vector such as TOPO TA or pGEM-T Easy according to the manufacturer's instructions (InvitrogenTM, Carlsbad, Calif.; Promega®, Madison, Wis.).
Clones were screened with PCR to identify correctly-cloned inserts for each fragment of interest. White colonies (8) were selected and resuspended in 40 μl water; the remainder of the colony was streaked on selective media (LB+Amp) for later recovery. 1 μl of the resuspended colonies were used as the template in a 10 μl PCR reaction. PCR conditions were the same as described above for nested PCR, and one positive clone was selected for each fragment.
Cultures of bacteria carrying the selected clone were grown in liquid selective media (LB+Amp). Plasmid minipreps were performed using a Spin Column Miniprep Kit (Qiagen®). The final volume was brought to 50 μl with elution buffer, and the minipreps were checked by digesting 2 μl of plasmid DNA with EcoRI. The DNA was then sequenced to confirm that each plasmid contained the MuTIR (53 bp including the MuInt19 site). The sequence of the fragment was then used to design a fragment-specific primer to pair with MuInt19 or MuExt22D, and co-segregation analysis was performed using PCR on DNA from all individuals in the segregation population.
BfaI and MseI share the same adaptor:







MseI/BfaI adaptor-lower:








5′-TACTCAGGACTCATCGACCGT-3′
(SEQ ID NO: 26)


 







MseI/BfaI adaptor-upper:








5′-GTGAACGGTCGATGAGTCCTGAG-3′
(SEQ ID NO: 27)






Adaptors were made by mixing these two oligonucleotides, denaturing at 95° C. for 5 minutes, and then cooling the mixture down slowly to room temperature. The adaptor is designed in such a way that the original restriction sites are not restored after the ligation.









Adaptor Ext 18 primer (MspExt18):










5′-GTGAACGGTCGATGAGTC-3′
(SEQ ID NO: 28)


 









MseI/BfaI adp Int18 primer (MseInt18):










5′-GTCGATGAGTCCTGAGTA-3′
(SEQ ID NO: 29)






BfaI+2 selective primers (16):











BfaIntGAA:
GATGAGTCCTGAGTAGAA
(SEQ ID NO: 30)


 



BfaIntGAC:
GATGAGTCCTGAGTAGAC
(SEQ ID NO: 31)


 



BfaIntGAG:
GATGAGTCCTGAGTAGAG
(SEQ ID NO: 32)


 



BfaIntGAT:
GATGAGTCCTGAGTAGAT
(SEQ ID NO: 33)


 



BfaIntGCA:
GATGAGTCCTGAGTAGCA
(SEQ ID NO: 34)


 



BfaIntGCC:
GATGAGTCCTGAGTAGCC
(SEQ ID NO: 35)


 



BfaIntGCG:
GATGAGTCCTGAGTAGCG
(SEQ ID NO: 36)


 



BfaIntGCT:
GATGAGTCCTGAGTAGCT
(SEQ ID NO: 37)


 



BfaIntGGA:
GATGAGTCCTGAGTAGGA
(SEQ ID NO: 38)


 



BfaIntGGC:
GATGAGTCCTGAGTAGGC
(SEQ ID NO: 39)


 



BfaIntGGG:
GATGAGTCCTGAGTAGGG
(SEQ ID NO: 40)


 



BfaIntGGT:
GATGAGTCCTGAGTAGGT
(SEQ ID NO: 41)


 



BfaIntGTA:
GATGAGTCCTGAGTAGTA
(SEQ ID NO: 42)


 



BfaIntGTC:
GATGAGTCCTGAGTAGTC
(SEQ ID NO: 43)


 



BfaIntGTG:
GATGAGTCCTGAGTAGTG
(SEQ ID NO: 44)


 



BfaIntGTT:
GATGAGTCCTGAGTAGTT
(SEQ ID NO: 45)






MseI+2 selective primers (16):









MseIntAAA:
CGATGAGTCCTGAGTAAAA
(SEQ ID NO: 46)


 


MseIntAAC:
CGATGAGTCCTGAGTAAAC
(SEQ ID NO: 47)


 


MseIntAAG:
CGATGAGTCCTGAGTAAAG
(SEQ ID NO: 48)


 


MseIntAAT:
CGATGAGTCCTGAGTAAAT
(SEQ ID NO: 49)


 


MseIntACA:
CGATGAGTCCTGAGTAACA
(SEQ ID NO: 50)


 


MseIntACC:
GATGAGTCCTGAGTAACC
(SEQ ID NO: 51)


 


MseIntACG:
GATGAGTCCTGAGTAACG
(SEQ ID NO: 52)


 


MseIntACT:
GATGAGTCCTGAGTAACT
(SEQ ID NO: 53)


 


MseIntAGA:
CGATGAGTCCTGAGTAAGA
(SEQ ID NO: 54)


 


MseIntAGC:
GATGAGTCCTGAGTAAGC
(SEQ ID NO: 55)


 


MseIntAGG:
GATGAGTCCTGAGTAAGG
(SEQ ID NO: 56)


 


MseIntAGT:
CGATGAGTCCTGAGTAAGT
(SEQ ID NO: 57)


 


MseIntATA:
CGATGAGTCCTGAGTAATA
(SEQ ID NO: 58)


 


MseIntATC:
GATGAGTCCTGAGTAATC
(SEQ ID NO: 59)


 


MseIntATG:
GATGAGTCCTGAGTAATG
(SEQ ID NO: 60)


 


MseIntATT:
CGATGAGTCCTGAGTAATT
(SEQ ID NO: 61)






10×RL Buffer:

    
    
        100 mM Tris-HCl, pH 7.5
        100 mM MgOAc,
        500 mM KOAc,
        50 mM DTT
    
    


Map-based cloning requires a high-resolution genetic map and a physical map around the locus of interest. Using the TAP marker, which was closely linked to the Lpa1 locus, the inventors identified a BAC contig containing about 120 BAC clones from a proprietary BAC library. PCR markers were developed based on BAC-end sequences and EST sequences, and the segregating populations of individuals described above were also used for genetic mapping. Individual F1 seeds were phenotyped by measuring Pi and phytic acid content. DNA was extracted from the individual F1 seeds with the Qiagen® Genomic DNA Purification Kit. Individuals were genotyped using PCR carried out according to the instructions of the Expand High Fidelity PCR system (Roche®). 792 individuals were analyzed to construct a fine map around the Lpa1 locus.
Based on the genetic map and the BAC physical map, the inventors identified two over-lapping BACs which cover the Lpa1 locus. The two BACs, b149a.i9 and b156a.m1, were sequenced. Open reading frames in each BAC were identified by using the Fgenesh computer program and BLAST searching against maize EST databases. BAC b149a.i9 is 140 kb in length and has several ORFs predicted by Fgenesh. Only two ORFs were found to have corresponding ESTs. One of the ORFs encodes an MRP ABC transporter protein. Gene-specific primers were synthesized from these two ORFs and used to search for the Mu insertion in the lpa1 mutant Mu-insertion alleles. A Mu insertion was found in the MRP ABC transporter gene in lpa1 allele PV03 56 C-05. A Mu insertion was also found in the same gene for eight other lpa1 alleles. Mu is inserted in Exon 1 at nucleotide 585 in Mu82978.17; at nucleotide 874 in PV03 57 C-3; and in Exon 11 at nucleotide 6069 in Mu82911.08. The remaining 6 alleles all have the same Mu insertion site as Mu82978.17. The MRP gene was also sequenced from four lpa1 EMS alleles. In two alleles (91286 and 94580), a stop codon was introduced in place of codons encoding R and Q at amino acids 371 and 595, respectively. In allele 91281, E was changed to L at amino acid 680, while in the original lpa1-1 allele, A was mutated to V at amino acid 1432.
The maize MRP ABC transporter gene was designated ZmMRP3 (Zea mays multidrug resistance-associated protein 3), or Lpa1 (low phytic acid). The MRP group of the ABC transporter family includes many proteins which are involved in diverse cellular responses. MRPs can transport a great range of substances. Some of the MRPs also have regulatory activity on other transporters or channel proteins. This maize MRP (ZmMRP3) is the first MRP shown to play a role in phytic acid metabolism and cellular function, and provides a new way in which phytic acid and available phosphorus content of plant seeds may be manipulated. Previously, the phytic acid biosynthesis pathway was altered by manipulating genes encoding the enzymes that convert glucose 6-P to phytic acid. In contrast, while the invention is not bound by a particular mechanism of operation, MRP is a transporter and/or transporter regulator. Thus, altering MRP expression and/or functionality in transgenic plants would be expected to have minimal effects on InsP intermediates of phytic acid biosynthesis pathway.
During the course of this study, the inventors determined that knockout lpa1 alleles are lethal when they are homozygous. Because the Lpa1 gene has now been cloned and further characterized as disclosed herein, it is now possible to make transgenic plants with Lpa1 expression constructs under tight control. An advantage of using Lpa1 is that it could be used to develop the low phytic acid trait without changing the composition of myo-inositol phosphate intermediates. In addition, a suppression of Lpa1 expression that was limited to suppression in developing embryos could produce transgenic plants having low phytic acid and high available phosphorus in seeds with minimal impact on agronomic performance.
Thus, SEQ ID NO:1 sets forth the genomic sequence of ZmMRP3 (Lpa1), SEQ ID NO:2 sets forth the deduced cDNA sequence, and SEQ ID NO: 3 sets forth the deduced amino acid sequence of the ZmMRP3 (Lpa1) protein. The Lpa1 protein contains 1510 amino acids and has a calculated molecular weight of about 166.8 kiloDaltons and a pI of about 8.44.
Zm-MRP3 Protein Structure
The Lpa1 polypeptide was identified as an ABC transporter, as it contains consensus features of the ABC transporter family of proteins. ABC transporters are a large family of proteins found in bacteria, fungi, plants and animals. In coupling to ATP hydrolysis, the ABC transporter transports a great variety of substrates across the plasma membrane and various intracellular membranes. Among the substrates known to be transported by ABC transporters are sugars, amino acids, inorganic acids, lipids, peptides, heavy metal ions, glutathione conjugates, alkaloids, and secondary metabolites.
The member of the ABC superfamily can be divided into several subfamilies based on phylogenic pathways and structural features. The names used to define the subfamilies are historic and related to the function of drug resistance, although many members are not involved in drug transport. The three best characterized subfamilies are the pleiotropic drug resistance protein (PDR), multidrug resistance protein (MDR), and multidrug resistance-associated protein (MRP). The maize Lpa1 is a MRP ABC transporter. Previously, two MRP genes, ZmMRP1 and ZmMRP2, have been cloned from maize and their function is not known. The Lpa1 gene differs from those two ZmMRPs and thus was designated ZmMRP3.
FIG. 1A and FIG. 1B show a comparison of the Lpa1 polypeptide with Pfam consensus sequences for the ABC transporter (“ABC_tran”; Pfam Accession No. PF00005; SEQ ID NO: 62) and the ABC transporter transmembrane region (“ABC_membrane”; Pfam Accession No. PF00664; SEQ ID NO: 63). All ABC proteins consist of one or two copies of a modular structure which has two basic structural elements: an integral transmembrane domain (TMD) and a cytosolic ATP-binding domain (also known as nucleotide binding fold, or NBF). The NBF is involved in binding ATP and it contains a Walker A box, an ABC signature motif, and a Walker B box. The Walker A and B boxes also are found in other nucleotide-binding proteins, such as P-, F- and V-ATPase, G-proteins and adenylate kinase. The ABC signature motif, however, is unique to the NBFs of ABC transporters.
The members of the MRP subfamily of ABC transporters have two copies of the modular structure (see FIG. 1). Maize ZmMRP3 contains about 10 transmembrane spans in the first copy and 4 in the second copy. Two ATP-binding domains of ZmMRP3 are located at amino acids 631-843 and amino acids 1267-1450, respectively. Within the ATP-binding domains, a Walker A box is at amino acids 664-672 (GVIGSGKSS; SEQ ID NO: 18) and amino acids 1301-1309 (GRTGSGKST; SEQ ID NO: 19), an ABC signature motif is at amino acids 754-765 (LSGGQKQRVQLA; SEQ ID NO: 20) and amino acids 1404-1415 (WSVGQRQLIALG; SEQ ID NO: 21), and a Walker B box is at amino acids 774-779 (IYLLDD; SEQ ID NO: 22) and amino acids 1424-1428 (ILVLD; SEQ ID NO: 23). The second ATP-binding domain of ZmMRP3 is followed by a C1 domain with a motif of IAHRI (SEQ ID NO: 24) from amino acids 1458-1462.
The MRP gene was amplified from different maize lines by PCR and sequenced. This revealed a variant Lpa1 polypeptide (SEQ ID NO: 5) which differs from Lpa1 at positions 3, 17, and 61. This variant polypeptide is encoded by the cDNA set forth in SEQ ID NO: 4.
Example 3
Identification of Lpa1 Homologs in Other Plants
Database searches identified similar proteins from other plants which were not previously known to have a role in phytic acid metabolism as discussed herein. Accordingly, the invention additionally provides Lpa1 plant proteins and proteins comprising Lpa1 consensus sequences and domains as well as polynucleotides encoding them.
The maize MRP3 (Lpa1) gene is located on the short arm of chromosome 1 and consists of 11 exons and 10 introns. It is well known that there is significant conservation of gene content and gene order among the genomes of the plant family Gramineae. Previously, extensive studies have been focused on comparison of rice and maize gene linkage blocks and a comparative map established. Using the Lpa1 locus and its surrounding sequences, the inventors found the corresponding region in rice on chromosome 3 and identified an MRP gene in this region. Although twelve rice MRP genes had been annotated previously (Jasinski et al. (2003) Plant Physiol. 131: 1169-77), this annotation did not include this MRP on chromosome 3, which we designated OsMRP13 (SEQ ID NO: 6). OsMRP13 has the same number of exons and introns as the maize Lpa1 gene ZmMRP3 and encodes a protein of 1505 amino acids (SEQ ID NO: 7). The maize MzMRP3 and rice OsMRP13 genes share 83% nucleotide sequence identity and the encoded proteins share 91% amino acid sequence identity (see FIGS. 4 and 5). The two genes also share similar structures (see FIG. 2). The inventors conducted a LynxTM study to determine the expression patterns of the rice gene. LynxTM gene expression profiling technology utilizes massively parallel signature sequence (MPSS; see Brenner et al. (2000) Nature Biotechnology 18: 630-634; Brenner et al. (2000) Proc. Nat'l. Acad. Sci. USA 97: 1665-1670). MPSS generates 17-mer sequence tags of millions of cDNA molecules, which are cloned on microbeads. The technique provides an unprecedented depth and sensitivity of mRNA detection, including messages expressed at very low levels. The LynxTM database search revealed that the rice gene OsMRP13 is expressed in developing seeds but has lower levels of expression in other tissues. It is very likely that the rice OsMRP13 has the same function as the maize Lpa1 gene in phytic acid metabolism in developing seeds.
Arabidopsis has 14 known MRP genes (AtMRP15 is a pseudogene). The inventors discovered that AtMRP5 has the same exon/intron organization as the maize ZmMRP3 gene, and that the sizes of corresponding exons and introns also are similar. The maize ZmMRP3 and Arabidopsis AtMRP5 share 62% nucleotide sequence identity and 67% amino acid sequence identity. Among the 14 known Arabidopsis MRPs, AtMRP5 shares the highest level of sequence identity with ZmMRP3. A LynxTM study was performed on AtMRP5 and confirmed that AtMRP5 is expressed in Arabidopsis seeds. It remains to be determined whether Arabidopsis AtMRP5 has the same function as maize ZmMRP3 in phytic acid metabolism.
A soybean homolog of maize ZmMRP3 also was identified by searching a soybean EST database. The inventors conducted a LynxTM study to characterize the expression of the soybean gene (corresponding to the sequence set forth in SEQ ID NO: 10). The LynxTM study revealed that the soybean gene is expressed in developing seeds but has lower levels of expression in other tissues. A study of EST distribution in various plant tissues also indicated that the soybean gene expression is seed-preferred.
Example 4
Stacking Lpa1 with Other Inositol Phosphate Kinase Genes
By “stacking” (i.e., transforming a plant with) constructs designed to reduce or eliminate the expression of Lpa1 and other proteins, it is expected that the reduction of phytic acid and increase in available phosphorus will be enhanced in comparison to plants transformed with constructs designed to reduce or eliminate the expression of Lpa1 alone. Accordingly, expression cassettes are prepared making use of inverted repeat constructs known as Inverted Repeats Without Terminators, or “IRNTs.” The first and second portion of such constructs self-hybridize to produce a hairpin structure which can suppress expression of the relevant endogenous gene. Each expression cassette contains an IRNT (“Lpa1 IRNT”) that can suppress endogenous Lpa1 gene expression. This Lpa1 IRNT includes two portions of an Lpa1 inverted repeat surrounding the Adh1 gene intron. Other expression cassettes contain an additional IRNT that can suppress expression of IPPK, ITPK-5, myo-inositol kinase (MIK), IP2K, phytase, and MI1PS3, respectively. “Glb1” indicates the globulin 1 promoter, and “Ole” indicates the oleosin promoter. Each expression cassette is provided in a plasmid which contains additional useful features for transformation and expression in plants. Lpa1 constructs can also be stacked with constructs designed to increase the expression of other proteins, such as, for example, phytase.
The plasmids are inserted into Agrobacterium vectors and used to transform maize cells. Sample protocols for creation of Agrobacterium strains harboring a plasmid are described, for example, in Lin (1995) in Methods in Molecular Biology, ed. Nickoloff, J. A. (Humana Press, Totowa, N.J.). Successful transformation can be verified by restriction analysis of the plasmid after transformation back into E. coli DH5α cells. The Agrobacterium is used to transform a host plant such as maize, and the resulting transgenic plants are screened for transformation and for phytic acid phenotype as described in detail above.
In some embodiments, the Lpa1 gene is mutated and the mutated Lpa1 gene is over-expressed in order to generate transgenic plants with dominant phenotype of reduced Lpa1 activity. For example, the mutation found in EMS-generated allele lpa1-1 is A1432V (i.e., the alanine at position 1432 is changed to valine). This mutation can be introduced into a polynucleotide by PCR-based mutagenesis in which a primer is synthesized with an altered nucleotide corresponding to the desired change. The resulting PCR product is then ligated with other fragments to make a full-length mutated Lpa1 gene carrying the lpa1-1 mutation. A transformation construct consisting of the mutated Lpa1 gene driven by the oleosin promoter could be used to produce transgenic plants having the dominant phenotype of reduced Lpa1 activity; these plants would yield grain with reduced phytate and increased available phosphorus.
Total knockout of the Lpa1 gene (for example, in Mutator-insertion alleles) is lethal. It is believed that the lethality of an Lpa1 knockout could be rescued by overexpressing phytase in a plant lacking Lpa1 activity.
Plants with Lpa1 constructs or mutations can then be crossed with plants containing other constructs to obtain progeny containing multiple constructs. Thus, for example, a plant with an Lpa1 construct can be crossed with a plant containing an Lpa3 construct; progeny containing both the Lpa1 and the Lpa3 construct may then be obtained.
Example 5
Production of Lpa1 Transgenic Plants Using Agrobacterium-mediated Transformation
For Agrobacterium-mediated transformation of maize with an Lpa1 construct of the invention, preferably the method of Zhao is employed (U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326; the contents of which are hereby incorporated by reference). Briefly, immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring the Lpa1 construct to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step the immature embryos are preferably immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos are co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). Preferably the immature embryos are cultured on solid medium following the infection step. Following this co-cultivation period, an optional “resting” step is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). Preferably the immature embryos are cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step). Preferably, the immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus is then regenerated into plants (step 5: the regeneration step), and preferably calli grown on selective medium are cultured on solid medium to regenerate the plants.
Bombardment and Culture Media
Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (Sigma® C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000× Sigma®-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 2.0 g/l GelriteTM (added after bringing to volume with D-I H2O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 4.0 g/l N6 basal salts (Sigma® C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000× Sigma®-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 3.0 g/l GelriteTM (added after bringing to volume with D-I H2O); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added after sterilizing the medium and cooling to room temperature).
Plant regeneration medium (288J) comprises 4.3 g/l MS salts (Gibco® 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H2O) (Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume with polished D-I H2O after adjusting to pH 5.6); 3.0 g/l GelriteTM (added after bringing to volume with D-I H2O); and 1.0 mg/l indoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing the medium and cooling to 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (Gibco® 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H2O), 0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-I H2O after adjusting pH to 5.6); and 6 g/l Bacto-agar (added after bringing to volume with polished D-I H2O), sterilized and cooled to 60° C.
Example 6
Production of Lpa1 Transgenic Plants Using Soybean Embryo Transformation
Soybean embryos are bombarded with a plasmid containing an Lpa1 construct as follows. To induce somatic embryos, cotyledons 3-5 mm in length dissected from surface-sterilized, immature seeds of the soybean cultivar A2872 are cultured in the light or dark at 26° C. on an appropriate agar medium for six to ten weeks. Somatic embryos producing secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos that multiplied as early, globular-staged embryos, the suspensions are maintained as described below.
Soybean embryogenic suspension cultures can maintained in 35 ml liquid media on a rotary shaker at 150 rpm at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 ml of liquid medium.
Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont Biolistic PDS1000/HE instrument (helium retrofit) can be used for these transformations.
A selectable marker gene that can be used to facilitate soybean transformation is a transgene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313: 810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188), and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The expression cassette comprising the Lpa1 construct operably linked to the CaMV 35S promoter can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.
To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (in order): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl2 (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μl 70% ethanol and resuspended in 40 μl of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five microliters of the DNA-coated gold particles are then loaded on each macro carrier disk.
Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm Petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi, and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.
Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post-bombardment with fresh media containing 50 mg/ml hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post-bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
Example 7
Production of Lpa1 Transgenic Plants Using Brassica napus Seed Transformation
Brassica napus seeds are transformed using a transformation and regeneration protocol modified from Mehra-Palta et al. (1991), “Genetic Transformation of Brassica napus and Brassica rapa,” in Proc. 8th GCIRC Congr., ed. McGregor (University Extension Press, Saskatoon, Sask., Canada), pp. 1108-1115 and Stewart et al. (1996), “Rapid DNA Extraction From Plants,” in Fingerprinting Methods Based on Arbitrarily Primed PCR, Micheli and Bova, eds. (Springer, Berlin), pp. 25-28. See Cardoza and Stewart (2003) Plant Cell Rep. 21: 599-604.
Seeds are surface-sterilized for 5 minutes with 10% sodium hypochlorite with 0.1% TweenTM added as a surfactant, rinsed for one minute with 95% ethanol, and then washed thoroughly with sterile distilled water. Seeds are germinated on MS basal medium (Murashige and Skoog (1962) Physiol. Plant 15: 473-497) containing 20 g/liter sucrose and 2 g/liter GelriteTM. Hypocotyls are excised from 8- to 10-day-old seedlings, cut into 1-cm pieces, and preconditioned for 72 hours on MS medium supplemented with 1 mg/liter 2,4-D (2,4-dichlorophenoxy acetic acid) and containing 30 g/liter sucrose and 2 g/liter GelriteTM.
Agrobacterium containing a plasmid comprising an Lpa1 construct of the invention is grown overnight in liquid LB medium to an OD600 of 0.8, pelleted by centrifugation, and resuspended in liquid callus induction medium containing acetosyringone at a final concentration of 0.05 mM. Agrobacterium is then cocultivated with the preconditioned hypocotyl segments for 48 hours on MS medium with 1 mg/liter 2,4-D. After the cocultivation period, explants are transferred to MS medium containing 1 mg/liter 2,4-D, 400 mg/liter timentin, and 200 mg/liter kanamycin to select for transformed cells. After 2 weeks, in order to promote organogenesis, the explants are transferred to MS medium containing 4 mg/liter BAP (6-benzylaminopurine), 2 mg/liter zeatin, 5 mg/liter silver nitrate, antibiotics selective for the transformation construct, 30 g/liter sucrose, and 2 g/liter GelriteTM. After an additional 2 weeks, in order to promote shoot development, tissue is transferred to MS medium containing 3 mg/liter BAP, 2 mg/liter zeatin, antibiotics, 30 g/liter sucrose, and 2 g/liter GelriteTM. Shoots that develop are transferred for elongation to MS medium containing 0.05 mg/liter BAP, 30 g/liter sucrose, antibiotics, and 3 g/liter GelriteTM. Elongated shoots are then transferred to root development medium containing half-strength MS salts, 10 mg/liter sucrose, 3 g/liter GelriteTM, 5 mg/liter IBA (indole-3-butyric acid), and antibiotics. All cultures are maintained at 25° C.+/−2° C. in a 16-hour light/8-hour dark photoperiod regime with light supplied by cool white daylight fluorescent lights. The rooted shoots are transferred to soil and grown under the same photoperiod regime at 20° C. in a plant growth chamber.
Transformation of plants with the Lpa1 construct is confirmed using PCR of DNA extracted from putative transgenic plants.
Example 8
Variants of Lpa1
A. Variant Nucleotide Sequences of Lpa1 (SEQ ID NO: 2) that do not Alter the Encoded Amino Acid Sequence
The Lpa1 nucleotide sequence set forth in SEQ ID NO: 2 is used to generate variant nucleotide sequences having the nucleotide sequence of the open reading frame with about 70%, 76%, 81%, 86%, 92%, and 97% nucleotide sequence identity when compared to the starting unaltered ORF nucleotide sequence of SEQ ID NO: 2. In some embodiments, these functional variants are generated using a standard codon table. In these embodiments, while the nucleotide sequence of the variant is altered, the amino acid sequence encoded by the open reading frame does not change.
B. Variant Amino Acid Sequences of Lpa1
Variant amino acid sequences of Lpa1 are generated. In this example, one amino acid is altered. Specifically, the open reading frame set forth in SEQ ID NO: 2 is reviewed to determined the appropriate amino acid alteration. The selection of the amino acid to change is made by consulting the protein alignment (with the other homologs or orthologs and other gene family members from various species). See FIGS. 1, 4, and 5. An amino acid is selected that is deemed not to be under high selection pressure (not highly conserved) and which is rather easily substituted by an amino acid with similar chemical characteristics (i.e., similar functional side-chain). Using the alignments set forth in FIGS. 1, 4, and 5, an appropriate amino acid can be changed. Variants having about 70%, 75%, 80%, 85%, 90%, 95%, and 97% nucleic acid sequence identity to SEQ ID NO: 2 are generated using this method.
C. Additional Variant Amino Acid Sequences of Lpa1
In this example, artificial protein sequences are created having about 80%, 85%, 90%, 95%, and 97% identity relative to the reference protein sequence. This latter effort requires identifying conserved and variable regions from the alignments set forth in FIGS. 1, 4, and 5 and then the judicious application of an amino acid substitutions table. These parts will be discussed in more detail below.
Largely, the determination of which amino acid sequences are altered is made based on the conserved regions among MRPs. See FIGS. 1, 4, and 5. It is recognized that conservative substitutions can be made in the conserved regions below without altering function. In addition, one of skill will understand that functional variants of the Lpa1 sequence of the invention can have minor non-conserved amino acid alterations in the conserved domain.
Artificial protein sequences are then created that are different from the original in the intervals of 80-85%, 85-90%, 90-95%, and 95-100% identity. Midpoints of these intervals are targeted, with liberal latitude of plus or minus 1%, 2%, or 3%, for example. The amino acids substitutions will be effected by a custom Perl script. The substitution table is provided below in Table 1.







TABLE 1







Substitution Table













Strongly Similar
Rank of




Amino
and Optimal
Order to



Acid
Substitution
Change
Comment
















I
L, V
1
50:50 substitution



L
I, V
2
50:50 substitution



V
I, L
3
50:50 substitution



A
G
4



G
A
5



D
E
6



E
D
7



W
Y
8



Y
W
9



S
T
10



T
S
11



K
R
12



R
K
13



N
Q
14



Q
N
15



F
Y
16



M
L
17
First methionine






cannot change



H

Na
No good substitutes



C

Na
No good substitutes



P

Na
No good substitutes










First, any conserved amino acids in the protein that should not be changed is identified and “marked off” for insulation from the substitution. The start methionine will of course be added to this list automatically. Next, the changes are made.
H, C, and P are not changed in any circumstance. The changes will occur with isoleucine first, sweeping N-terminal to C-terminal, then leucine, and so on down the list until the desired target of percent change is reached. Interim number substitutions can be made so as not to cause reversal of changes. The list is ordered 1-17, so start with as many isoleucine changes as needed before leucine, and so on down to methionine. Clearly, many amino acids will in this manner not need to be changed. Changes between L, I, and V will involve a 50:50 substitution of the two alternate optimal substitutions.
The variant amino acid sequences are written as output. Perl script is used to calculate the percent identities. Using this procedure, variants of Lpa1 are generated having about 80%, 85%, 90%, and 95% amino acid identity to the starting unaltered ORF nucleotide sequence of SEQ ID NO: 2.
Example 9
Pedigree Breeding
Pedigree breeding starts with the crossing of two genotypes, such as a transformed (i.e., transgenic) inbred line and one other elite inbred line having one or more desirable characteristics that is lacking or which complements the first transgenic inbred line. If the two original parents do not provide all the desired characteristics, other sources can be included in the breeding population. In the pedigree method, superior segregating plants are selfed and selected in successive filial generations. In the succeeding filial generations the heterozygous condition gives way to homogeneous lines as a result of self-pollination and selection. Typically in the pedigree method of breeding, five or more successive filial generations of selfing and selection are practiced: F1→F2; F2→F3; F3→F4; F4→F5, etc. After a sufficient amount of inbreeding, successive filial generations will serve to increase seed of the developed inbred. Preferably, the inbred line comprises homozygous alleles at about 95% or more of its loci.
In addition to being used to create a backcross conversion, backcrossing can also be used in combination with pedigree breeding to modify a transgenic inbred line and a hybrid that is made using the transgenic inbred line. Backcrossing can be used to transfer one or more specifically desirable traits from one line, the donor parent, to an inbred called the recurrent parent, which has overall good agronomic characteristics yet lacks that desirable trait or traits.
Therefore, an embodiment of this invention is a method of making a backcross conversion of a maize transgenic inbred line containing an Lpa1 construct, comprising the steps of crossing a plant of an elite maize inbred line with a donor plant comprising a mutant gene or transgene conferring a desired trait, selecting an F1 progeny plant comprising the mutant gene or transgene conferring the desired trait, and backcrossing the selected F1 progeny plant to a plant of the elite maize inbred line. This method may further comprise the step of obtaining a molecular marker profile of the elite maize inbred line and using the molecular marker profile to select for a progeny plant with the desired trait and the molecular marker profile of the maize elite inbred line. In the same manner, this method may be used to produce an F1 hybrid seed by adding a final step of crossing the desired trait conversion of the elite maize inbred line with a different maize plant to make F1 hybrid maize seed comprising a mutant gene or transgene conferring the desired trait.
Recurrent Selection and Mass Selection
Recurrent selection is a method used in a plant breeding program to improve a population of plants. The method entails individual plants cross-pollinating with each other to form progeny. The progeny are grown and superior progeny are selected by any number of selection methods, which include individual plant, half-sib progeny, full-sib progeny, selfed progeny and topeross yield evaluation. The selected progeny are cross-pollinated with each other to form progeny for another population. This population is planted and again superior plants are selected to cross-pollinate with each other. Recurrent selection is a cyclical process and therefore can be repeated as many times as desired. The objective of recurrent selection is to improve the traits of a population. The improved population can then be used as a source of breeding material to obtain inbred lines to be used in hybrids or used as parents for a synthetic cultivar. A synthetic cultivar is the resultant progeny formed by the intercrossing of several selected inbreds.
Mass selection is a useful technique when used in conjunction with molecular marker enhanced selection. In mass selection seeds from individuals are selected based on phenotype and/or genotype. These selected seeds are then bulked and used to grow the next generation. Bulk selection requires growing a population of plants in a bulk plot, allowing the plants to self-pollinate, harvesting the seed in bulk and then using a sample of the seed harvested in bulk to plant the next generation. Instead of self-pollination, directed pollination could be used as part of the breeding program.
Mutation Breeding
Mutation breeding is one of many methods that could be used to introduce new traits into a particular maize inbred line. Mutations that occur spontaneously or are artificially induced can be useful sources of variability for a plant breeder. The goal of artificial mutagenesis is to increase the rate of mutation for a desired characteristic. Mutation rates can be increased by many different means. Such means include: temperature; long-term seed storage; tissue culture conditions; radiation such as X-rays, Gamma rays (e.g., cobalt 60 or cesium 137), neutrons, (product of nuclear fission by uranium 235 in an atomic reactor), Beta radiation (emitted from radioisotopes such as phosphorus 32 or carbon 14), or ultraviolet radiation (preferably from 2500 to 2900 nm); genetic means such as transposable elements or DNA damage repair mutations; chemical mutagens (such as base analogues (5-bromo-uracil); and related compounds (8-ethoxy caffeine), antibiotics (streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, or acridines. Once a desired trait is observed through mutagenesis the trait may then be incorporated into existing germplasm by traditional breeding techniques, such as backcrossing. Details of mutation breeding can be found in Fehr (1993) “Principals of Cultivar Development” (Macmillan Publishing Company), the disclosure of which is incorporated herein by reference. In addition, mutations created in other lines may be used to produce a backcross conversion of a transgenic elite line that comprises such mutation.
Example 10
Gene Silencing with the Lpa1 Promoter
The promoter of a target gene (e.g., Lpa1) is inactivated by introducing into a plant an expression cassette comprising a promoter and an inverted repeat of fragments of the Lpa1 promoter. For example, an expression cassette may be created that comprises the Ole promoter operably linked to an inverted repeat comprising fragments of the Lpa1 promoter that are approximately 200 bp in length and that are separated by the Adh1 intron. The Lpa1 promoter fragments may be selected from a portion of the promoter which is rich in CpG islands, such as, for example, the 3′ portion of the Lpa1 promoter. The sequence of the Lpa1 promoter is set forth in nucleotides 1-3134 of SEQ ID NO: 1. The expression cassette is used to transform a plant, which is then assayed for lack of expression of the Lpa1 gene. While the invention is not bound by any particular mechanism of operation, the method is thought to produce a small RNA molecule which recognizes the native promoter of the target gene and leads to methylation and inactivation (i.e., gene silencing) of the native promoter. Consequently, the gene associated with the promoter is not expressed. This trait is heritable and cosegregates with the transgenic construct.
Example 11
Construction of an Lpa1 Silencing Plasmid Driven by KT13
An expression cassette was prepared making use of an inverted repeat construct known as Inverted Repeats Without Terminators, or “IRNTs.” The first and second portion of such a construct hybridize to each other to produce a hairpin structure which can suppress expression of the corresponding endogenous gene (e.g., Lpa1). In this Lpa1 IRNT, the first and second portions are separated by a “spacer” portion.
To make the spacer DNA, a polynucleotide fragment encoding part of the soybean Fad2-1 and soybean Fad2-2 proteins (Heppard et al. (1996) Plant Physiol. 110: 311-9) was produced as follows. First, a recombinant DNA fragment (“KSFad2-hybrid”, set forth in SEQ ID NO: 72) was produced that contained a polynucleotide fragment of about 890 nucleotides comprising about 470 nucleotides from the soybean Fad2-2 gene and about 420 nucleotides from the soybean Fad2-1 gene. This KSFad2-hybrid recombinant DNA fragment was constructed by PCR amplification as follows. A DNA fragment of approximately 0.47 kb was obtained by PCR amplification using primers KS1 (SEQ ID NO: 73) and KS2 (SEQ ID NO: 74) from a template of genomic DNA purified from leaves of Glycine max cv. Jack. An approximately 0.42 kb DNA fragment was obtained from the same template by PCR amplification using primers KS3 (SEQ ID NO: 75) and KS4 (SEQ ID NO: 76). The 0.47 kb DNA fragment and 0.42 kb DNA fragment were gel-purified using GeneClean® (Qbiogene, Irvine Calif.), and then were mixed together and used as a template for PCR amplification with primers KS1 and KS4 to yield an approximately 890 bp fragment (“KSFad2-hybrid”, set forth in SEQ ID NO: 72) that was cloned into the commercially available plasmid pGEM-T Easy (Promega, Madison, Wis.).
The KSFad2 hybrid fragment was then modified to contain additional restriction enzyme recognition sites, as follows. The KSFad2 hybrid fragment named “KSFad2-hybrid” was re-amplified by standard PCR methods using Pfu Turbo DNA polymerase (Stratagene®, La Jolla, Calif.), a plasmid containing KSFad2-hybrid as DNA template, and the following primer sets. The oligonucleotide primers (SEQ ID NO: 77 and SEQ ID NO: 78) were designed to add a BsiWI restriction endonuclease to the 5′ end of the amplified fragment and to add an AvrII site to its 3′ end. The resulting DNA “spacer” sequence comprising about 470 nucleotides from the soybean Fad2-2 gene and 418 nucleotides from the soybean Fad2-1 is shown in SEQ ID NO: 79.
To prepare the first and second portions of the inverted repeat constructs, a polynucleotide fragment encoding part of the soybean Lpa1 protein (Lpa1, SEQ ID NO: 10) was amplified by standard PCR methods using Pfu Turbo® DNA polymerase (Stratagene®, La Jolla, Calif.) and the following primer sets. Lpa1 oligonucleotide primers (SEQ ID NO: 69 and SEQ ID NO: 70) were designed to add NotI and SalI restriction endonuclease sites at the 5′ end of the amplified fragment and BsiWI and AvrII restriction endonuclease sites at the 3′ end of the amplified fragment as well as a stop codon (TAA) at its 3′ end. The DNA sequence comprising the 556 bp polynucleotide from soybean Lpa1 is set forth in SEQ ID NO: 71.
Preparation of Expression Cassette
An expression cassette was constructed comprising the Lpa1 “IRNTs” operably linked to the strong seed-specific promoter KTI3 (Jofuku et al. (1989) Plant Cell 1: 1079-1093).
A plasmid derived from pKS121 was used to construct the expression cassette. Plasmid pKS121 was described in PCT Pub. No. WO 02/00904; this plasmid contains the KTI3 promoter/NotI/Kti3 3′ terminator fragment. For use in the present expression cassette, the plasmid pKS121 was engineered to contain a second hygromycin phosphotransferase gene with a 35S-CaMV promoter. The plasmid was then digested with the restriction enzymes NotI and SalI and the digest was run on a 0.8% TAE-agarose gel to isolate and purify a 7350 bp DNA fragment using the Qiagen® gel extraction kit.
In order to insert the inverted repeat constructs and the spacer region into this plasmid, several polynucleotide fragments were prepared. Aliquots of the polynucleotide fragment comprising the 556 bp polynucleotide from soybean Lpa1 (SEQ ID NO: 71) were digested with two separate sets of restriction enzymes. First, an aliquot of the amplified Lpa1 fragment was digested with NotI and BsiWI and run on a 0.8% TAE-agarose gel to isolate a 566 bp DNA fragment, which was purified using the Qiagen® gel extraction kit. A separate aliquot of the amplified Lpa1 fragment was digested with SalI and AvrII and run on a 0.8% TAE-agarose gel to isolate a 579 bp DNA fragment, which was also purified using the Qiagen® gel extraction kit. Furthermore, the amplified polynucleotide comprising the DNA “spacer” sequence (SEQ ID NO: 79) was digested with BsiWI and AvrII, run on a 0.8% TAE-agarose gel and a 901 bp DNA fragment was purified using the Qiagen® gel extraction kit.
To assemble the expression cassette comprising the Lpa1 “IRNTs” operably linked to the strong seed-specific promoter KTI3, all four isolated and purified fragments described above were ligated together. The ligation mixture was transformed into E. coli and transformed colonies were selected on hygromycin. Hygromycin-resistant colonies were selected and grown overnight in LB media with appropriate antibiotic selection. Proper construction of the expression cassette was confirmed by isolating DNA from these bacterial cultures using a Qiagen® miniprep kit according to the manufacturer's protocol and then analyzing with appropriate restriction digests.
Example 12
Production of High Pi Lpa1 Transgenic Soybean Somatic Embryos
The expression cassettes comprising the Lpa1 “IRNTs” operably linked to the strong seed-specific promoter KTI3 (described in Example 11) was transformed into soybean embryogenic suspension cultures using the protocol described in Example 6. Individual immature soybean embryos were then dried down by transferring them into an empty small Petri dish that was seated on top of a 10-cm Petri dish containing some agar gel to allow slow dry down. This process is intended to mimic the last stages of soybean seed development, and dried-down embryos are capable of producing plants when transferred to soil or soil-less media. Storage products produced by embryos at this stage are similar in composition to storage products produced by zygotic embryos at a similar stage of development and most importantly the storage product profile is predictive of plants derived from a somatic embryo line (see PCT Pub. No. WO 94/11516).
Determination Inorganic Phosphate Content
Somatic soybean embryos were assayed for Pi (inorganic phosphate) content using modifications of Chen et al. ((1956) Anal. Chem. 28: 1756-1758). Single embryos were weighed and placed into 1.2 ml deep-well tubes of a 96 well rack (Corning® Incorporated). Metal balls were then added to the tubes and the samples were ground using a Geno/Grinder2000TM grinder (Sepx CertiPrep®, Metuchen, N.J.). Then 150 μl water was added to each tube and the rack was shaken for 5 minutes and centrifuged at 3,000 g for 5 minutes. The pellet was resuspended and the complete slurry was transferred (without the metal balls) to a new set of into 1.2 ml deep-well tubes of a 96 well rack. The original tubes (still containing the metal balls) were washed with an additional 150 μl water and then shaken for 5 minutes and centrifuged at 2,500 g for 5 minutes. This solution was then pooled with the complete slurry in the new tubes, and 75 μl of 2N HCl was added to each tube. The tubes were incubated for 2 hours at room temperature. Thereafter, 188 μl of 30% aqueous trichloroacetic acid was added to each sample, and the samples were mixed and centrifuged at 2,500 g for 10 minutes. The supernatants were transferred into fresh tubes and used for Pi determinations; measurements were performed in duplicate.
To determine Pi, 100 μl of each supernatant was placed into a well of a 96 well microtiter plate and 100 μl of a mixture of 0.42% ammonium molybdate-1N H2SO4: 10% ascorbic acid (7:1) was added to each sample. The plates were incubated at 37° C. for 30 minutes and absorbance was measured at 800 nm; sodium phosphate (NaH2PO4) was used as a standard. Table 2 shows data comparing the Pi content of transgenic soybean lines transformed with pJMS33 (described in Example 11) to wild type somatic embryos. Multiple events were generated expressing the Lpa1 IRNT described in Example 11. Ten out of twenty lines analyzed (50%) showed an increased Pi content when compared to wild-type somatic embryos, ranging from 3.5-fold higher than wild type to nearly 8-fold higher than wild type.







TABLE 2







Pi content of somatic soybean embryos from different transgenic


events expressing the Lpa1 IRNT (as % of wild type (wt) content)










Event
Pi (% of wt)







Wild type
100



embryo



4-3
755



3-1
464



4-2
350



7-7
432



1-2
496



7-1
520



8-2
759



7-6
381



4-1
543



8-3
478










Example 13
Transgenic Maize Seeds have Reduced Phytic Acid and Increased Pi Content
Two expression cassettes were constructed to provide cosuppression of an MRP. These expression cassettes (designated plasmids P36 and P94) were made using MRP polynucleotide fragments. Each construct contained an inverted repeat of an MRP polynucleotide such that the first and second portions self-hybridized to produce a hairpin structure that can suppress expression of the relevant endogenous gene (e.g., maize Lpa1). Between the two fragments of the inverted repeat was an intron that helped to form the loop portion in the hairpin structure. Transcription was driven by the oleosin promoter in plasmid P36 and by the Glb1 promoter in plasmid P94; neither construct had a terminator. In both plasmids P36 and P94, the intron used was the Adh1 intron (GenBank Accession No. X04050), although other introns may also be used.
The plasmids were used to produce transgenic maize using protocols described in Example 1. Transgenic T1 seeds were screened for elevated Pi content using a rapid Pi assay, and quantitative analysis of phytic acid and Pi were also performed. The results of these assays demonstrated that cosuppression of MRP expression resulted in a decrease in phytic acid content and an increase in Pi in the transgenic seeds (see Table 3).







TABLE 3







Maize plants transformed with MRP expression constructs produced


transgenic seeds with reduced phytic acid and increased Pi content.















Wt K
CS K



Plasmid #/
Wt K Pi
CS K Pi
PAP
PAP


Event
(mg/g)
(mg/g)
(mg/g)
(mg/g)
PA reduction





Plasmid 36







1
0.31
1.17
2.76
0.72
74%


2
0.18
1.05
2.75
0.73
74%


3
0.27
0.99
2.53
0.99
61%


4
0.26
1.21
2.43
0.84
66%


5
0.43
1.12
2.15
0.85
60%


6
0.31
1.20
2.41
0.79
67%


7
0.34
1.06
2.59
0.61
77%


8
0.26
1.15
2.60
0.57
78%


9
0.21
1.09
2.61
0.70
73%


10 
0.31
1.26
2.55
0.82
68%


11 
0.19
1.08
2.46
0.66
73%


12 
0.32
1.09
2.50
0.78
69%


Plasmid 94


1
0.14
1.01
3.47
2.29
34%


2
0.12
1.37
3.10
1.12
64%


3
0.16
1.44
3.09
1.00
68%


4
0.10
1.20
3.44
1.75
49%


5
0.24
1.25
3.04
1.53
50%


6
0.24
1.47
2.67
0.98
63%


7
0.21
1.46
2.98
1.11
63%


8
0.18
1.17
3.00
1.76
41%





Wt K = wild-type kernels in a segregation ear;


CS K = cosuppression kernels in a segregation ear;


Pi = inorganic phosphate phosphorus;


PAP = phytic acid phosphorus;


PA = phytic acid






As indicated in the table legend, “Wt K” were kernels in a segregation ear without the MRP transgene and “CS K” were the kernels in the same segregation ear that did contain the MRP transgene. The PAP values in Table 3 were measured according to modifications of the methods described by Latta & Eskin (1980) J. Agric Food Chem. 28: 1313-1315 and Vaintraub & Lapteva (1988) Analytical Biochemistry 175: 227-230; see Example 1 for detail.
Example 14
Production of Transgenic Sorghum 
The promoter construct prepared in Example 10 is used to transform sorghum according to the teachings of U.S. Pat. No. 6,369,298. Briefly, a culture of Agrobacterium is transformed with a vector comprising an expression cassette containing the promoter construct prepared in Example 10. The vector also comprises a T-DNA region into which the promoter construct is inserted. General molecular techniques used in the invention are provided, for example, by Sambrook et al. (eds.) Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Immature sorghum embryos are obtained from the fertilized reproductive organs of a mature sorghum plant. Immature embryos are aseptically isolated from the developing kernel at about 5 days to about 12 days after pollination and held in sterile medium until use; generally, the embryos are about 0.8 to about 1.5 mm in size.
The Agrobacterium-mediated transformation process of the invention can be broken into several steps. The basic steps include: an infection step (step 1); a co-cultivation step (step 2); an optional resting step (step 3); a selection step (step 4); and a regeneration step (step 5). In the infection step, the embryos are isolated and the cells contacted with the suspension of Agrobacterium. 
The concentration of Agrobacterium used in the infection step and co-cultivation step can affect the transformation frequency. Very high concentrations of Agrobacterium may damage the tissue to be transformed, such as the immature embryos, and result in a reduced callus response. The concentration of Agrobacterium used will vary depending on the Agrobacterium strain utilized, the tissue being transformed, the sorghum genotype being transformed, and the like. Generally a concentration range of about 0.5×109 cfu/ml to 1×109 cfu/ml will be used.
The embryos are incubated with the suspension of Agrobacterium about 5 minutes to about 8 minutes. This incubation or infection step takes place in a liquid solution that includes the major inorganic salts and vitamins of N6 medium (referred to as “N6 salts,” or medium containing about 463.0 mg/l ammonium sulfate; about 1.6 mg/l boric acid; about 125 mg/l calcium chloride anhydrous; about 37.25 mg/l Na2-EDTA; about 27.8 mg/l ferrous sulfate.7H2O; about 90.37 mg/l magnesium sulfate; about 3.33 mg/l manganese sulfate H2O; about 0.8 mg/l potassium iodide; about 2,830 mg/l potassium nitrate; about 400 mg/l potassium phosphate monobasic; and about 1.5 mg/l zinc sulfate.7H2O.
In addition, the media in the infection step generally excludes AgNO3. AgNO3 is generally included in the co-cultivation, resting (when used) and selection steps when N6 media is used. In the co-cultivation step, the immature embryos are co-cultivated with the Agrobacterium on a solid medium. The embryos are positioned axis-down on the solid medium and the medium can include AgNO3 at a range of about 0.85 to 8.5 mg/l. The embryos are co-cultivated with the Agrobacterium for about 3-10 days.
Following the co-cultivation step, the transformed cells may be subjected to an optional resting step. Where no resting step is used, an extended co-cultivation step may utilized to provide a period of culture time prior to the addition of a selective agent. For the resting step, the transformed cells are transferred to a second medium containing an antibiotic capable of inhibiting the growth of Agrobacterium. This resting phase is performed in the absence of any selective pressures on the plant cells to permit preferential initiation and growth of callus from the transformed cells containing the heterologous nucleic acid. The antibiotic added to inhibit Agrobacterium growth may be any suitable antibiotic; such antibiotics are known in the art and include Cefotaxime, timetin, vancomycin, carbenicillin, and the like. Concentrations of the antibiotic will vary according to what is standard for each antibiotic, and those of ordinary skill in the art will recognize this and be able to optimize the antibiotic concentration for a particular transformation protocol without undue experimentation. The resting phase cultures are preferably allowed to rest in the dark at 28° C. for about 5 to about 8 days. Any of the media known in the art can be utilized for the resting step.
Following the co-cultivation step, or following the resting step, where it is used, the transformed plant cells are exposed to selective pressure to select for those cells that have received and are expressing polypeptide from the heterologous nucleic acid introduced by Agrobacterium. Where the cells are embryos, the embryos are transferred to plates with solid medium that includes both an antibiotic to inhibit growth of the Agrobacterium and a selection agent. The agent used to select for transformants will select for preferential growth of explants containing at least one selectable marker insert positioned within the superbinary vector and delivered by the Agrobacterium. Generally, any of the media known in the art suitable for the culture of sorghum can be used in the selection step, such as media containing N6 salts or MS salts. During selection, the embryos are cultured until callus formation is observed. Typically, calli grown on selection medium are allowed to grow to a size of about 1.5 to about 2 cm in diameter.
After the calli have reached the appropriate size, the calli are cultured on regeneration medium in the dark for several weeks to allow the somatic embryos to mature, generally about 1 to 3 weeks. Preferred regeneration media includes media containing MS salts. The calli are then cultured on rooting medium in a light/dark cycle until shoots and roots develop. Methods for plant regeneration are known in the art (see, e.g., Kamo et al. (1985) Bot. Gaz. 146(3): 327-334; West et al. (1993) Plant Cell 5:1361-1369; and Duncan et al. (1985) Planta 165: 322-332).
Small plantlets are then transferred to tubes containing rooting medium and allowed to grow and develop more roots for approximately another week. The plants are then transplanted to soil mixture in pots in the greenhouse.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claim(s).

## Claims

1. An expression cassette comprising a nucleic acid molecule which comprises a promoter that drives expression in a plant cell and is operably linked to:
a) a first nucleotide sequence having the nucleotide sequence as set forth in SEQ ID NO: 71;
b) a spacer nucleotide sequence; and
c) a second nucleotide sequence that is fully complementary to the first nucleotide sequence;
wherein a), b) and c) are arranged in a 5′ to 3′ direction, respectively; and wherein expression of said nucleic acid molecule in a soybean plant transformed with said expression cassette results in a hairpin RNA structure which decreases the expression of an endogenous multidrug resistance-associated protein (MRP) in said soybean plant and thereby reducing phytate levels of said transformed soybean plant compared to a control soybean plant lacking said expression cassette.

2. A soybean plant stably transformed with a heterologous nucleic acid molecule which comprises a promoter active in said soybean plant and is operably linked to:
a) a first nucleotide sequence having at least 500 contiguous nucleotides of the nucleotide sequence as set forth in SEQ ID NO: 10;
b) a spacer nucleotide sequence; and
c) a second nucleotide sequence that is fully complementary to the first nucleotide sequence;
wherein a), b) and c) are arranged in a 5′ to 3′ direction, respectively; and wherein expression of said nucleic acid molecule in said soybean plant results in a hairpin RNA structure which decreases the expression of an endogenous multidrug resistance-associated protein (MRP) in said transformed soybean plant and thereby reducing phytate levels of said transformed soybean plant compared to a control soybean plant lacking said heterologous nucleic acid molecule.

3. The soybean plant of claim 2, wherein said first nucleotide sequence is the nucleotide sequence as set forth in SEQ ID NO: 71.

4. A transformed seed obtained from the soybean plant of claim 2, and wherein said seed comprises said heterologous nucleic acid molecule.

5. A transformed seed obtained from the soybean plant of claim 3, and wherein said seed comprises said heterologous nucleic acid molecule.

6. A method of producing a transformed soybean plant having reduced levels of phytate, said method comprising the steps of:
i) transforming soybean plant cells with an expression cassette comprising a nucleic acid molecule which comprises a promoter that drives expression in a plant cell and is operably linked to:
a) a first nucleotide sequence having at least 500 contiguous nucleotides of the nucleotide sequence as set forth in SEQ ID NO: 10;
b) a spacer nucleotide sequence; and
c) a second nucleotide sequence that is fully complementary to the first nucleotide sequence;
wherein a), b) and c) are arranged in a 5′ to 3′ direction, respectively; and

ii) regenerating a transformed soybean plant from the transformed soybean plant cells of step i);
and wherein expression of said nucleic acid molecule in the transformed soybean plant of step ii) results in a hairpin RNA structure which decreases the expression of an endogenous multidrug resistance-associated protein (MRP) in the transformed soybean plant of step ii) and thereby reducing phytate levels in the transformed soybean plant compared to a control soybean plant lacking said expression cassette.

7. The method of claim 6, wherein said first nucleotide sequence is set forth in SEQ ID NO: 71.