阅读说明：本技术 通过使用abc转运体序列来增加植物生长和产量 (Plant growth and yield enhancement by use of ABC transporter sequences ) 是由 M·贝其曼 于 2018-05-21 设计创作，主要内容包括：本文提供用于改善植物生长的组合物和方法。也提供编码ABC转运体蛋白的多核苷酸、包含ABC转运体蛋白的多肽、用于表达感兴趣的基因的表达构建体,包含所述多核苷酸、多肽和表达构建体的植物,以及制备转基因植物的方法；所述感兴趣的基因的表达可以改善农艺性质,包括但不限于作物产量、生物胁迫和非生物胁迫耐受性、早期萌发势。(Provided herein are compositions and methods for improving plant growth. Also provided are polynucleotides encoding ABC transporter proteins, polypeptides comprising ABC transporter proteins, expression constructs for expressing a gene of interest, plants comprising the polynucleotides, polypeptides and expression constructs, and methods of making transgenic plants; expression of the gene of interest may improve agronomic properties including, but not limited to, crop yield, biotic and abiotic stress tolerance, early vigour.)

1. A method for increasing crop yield, comprising transforming a plant with at least one ABC transporter protein coding sequence.

2. The method of claim 1, wherein the ABC transporter protein coding sequence comprises SEQ ID No. 1 or encodes a protein selected from the group consisting of SEQ ID No. 2 and 15-103.

3. A plant having stably introduced into its genome a promoter operably linked to an ABC transporter protein coding sequence that drives expression in a plant, wherein said promoter is heterologous to said ABC transporter protein coding sequence.

4. The plant of claim 3, wherein said ABC transporter protein coding sequence comprises SEQ ID NO 1 or encodes a protein selected from the group consisting of SEQ ID NO 2 and 15-103.

5. A transformed seed of the plant of any one of claims 3 to 4.

6. The plant of claim 3 or 4, wherein said plant is a monocot.

7. The plant of claim 3 or 4, wherein said plant is a dicot.

8. The method of claim 1 or 2, wherein the ABC transporter protein coding sequence is expressed from a reproductive regulatory promoter.

9. The method of claim 8, wherein the developmentally regulated promoter comprises SEQ ID NO 3 or 5.

10. The plant of claim 3 or 4, wherein said promoter driving expression in a plant is a developmentally regulated promoter.

11. The plant of claim 10, wherein said developmentally regulated promoter comprises SEQ ID NO 3 or 5.

12. A DNA construct comprising, in operative association:

a. a promoter which functions in plant cells, and

b. a nucleic acid sequence encoding an ABC transporter protein.

13. The DNA construct of claim 12, wherein the nucleic acid sequence encoding an ABC transporter protein comprises SEQ ID No. 1 or encodes a protein selected from the group consisting of SEQ ID NOs 2 and 15-103.

14. The DNA construct of claim 12 or 13, wherein the promoter functional in plant cells comprises SEQ ID NO 3 and 5.

15. The DNA construct of any one of claims 12 to 14 wherein the promoter is heterologous to the nucleic acid sequence encoding the ABC transporter protein.

Technical Field

The present invention relates to compositions and methods for increasing plant growth and yield by expressing an ABC transporter gene in a plant.

Background

The continued growth of the world's population and the shrinking supply of arable land available in agriculture has stimulated research and development of plants with higher biomass and yield. A common approach to crop and horticultural improvements is to use selective breeding techniques to identify plants with desired characteristics. However, such selective breeding techniques have various disadvantages, namely that they are often labour intensive and the resulting plants often contain heterologous genetic components which are not always capable of conferring the desired trait from the parent plant. Advances in molecular biology have provided means for precisely improving the germplasm of plants. Genetic engineering of plants allows one to isolate and manipulate genetic material (usually in the form of DNA or RNA) which is subsequently introduced into the plant. This technology enables the production of crops or plants with various improved economic, agronomic or horticultural traits.

Traits of interest include plant biomass and yield. Yield is generally defined as the measurable yield from the economic value of a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on factors such as the number and size of organs, plant architecture (e.g. number of shoots), seed production, leaf senescence etc. Root development, nutrient uptake, stress tolerance, rate of photosynthetic carbon assimilation and early vigour may also be important factors in determining yield. Therefore, optimizing the above factors can help to increase crop yield.

An increase in seed yield is a particularly important trait, since the seeds of many plants are important for human and animal consumption. Crops such as corn, rice, wheat, canola, and soybean account for more than half of the total human caloric intake, whether by direct consumption of the seeds themselves or by consumption of meat products based on processed seeds. They are also sources of sugars, oils and various metabolites used in industrial processes. Seeds contain the embryo (the origin of new shoots and roots) and the endosperm (the source of nutrients for embryo growth during germination and during early growth of seedlings). Seed development involves many genes and requires the transfer of metabolites from roots, leaves and stems into growing seeds. In particular, the endosperm assimilates the metabolic precursors of carbohydrates, oils and proteins and synthesizes them into storage macromolecules to fill the kernel. The increase in plant biomass is important for forage crops such as alfalfa, silage corn and hay. Many genes are involved in metabolic pathways that contribute to plant growth and development. Modulating the expression of one or more such genes in a plant may result in plants having better growth and development relative to control plants, but will also tend to result in plants having poorer growth and development relative to control plants. Thus, methods for improving plant growth and development are needed.

Disclosure of Invention

The present application provides compositions and methods for modulating gene expression in plants. The method increases plant growth and results in higher crop yield. The method comprises increasing expression of at least one ABC transporter gene in a plant of interest. The invention also includes constructs comprising a promoter operably linked to an ABC transporter coding sequence that drives expression in a plant cell. Compositions also include plants, plant seeds, plant organs, plant cells, and other plant parts that have increased expression of ABC transporter sequences. The present invention includes methods useful for increasing the expression of ABC transporter genes in plants. The ABC transporter gene may be a native sequence or may be a sequence that is heterologous to the plant of interest.

Embodiments of the invention include:

1. a method for increasing crop yield, comprising transforming a plant with at least one ABC transporter protein coding sequence.

2. The method of embodiment 1, wherein said ABC transporter protein coding sequence comprises SEQ ID NO 1 or encodes a protein selected from the group consisting of SEQ ID NO 2 and 15-103.

3. The method of embodiment 1, wherein said ABC transporter protein coding sequence encodes a protein having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO 2 and 15-103 and having ABC transporter function.

4. The method of embodiment 1, wherein said ABC transporter protein coding sequence encodes a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence positive rate relative to a sequence selected from the group consisting of SEQ id nos. 2 and 15-103 and ABC transporter function.

5. A plant having stably introduced into its genome a promoter operably linked to an ABC transporter protein coding sequence that drives expression in a plant, wherein said promoter is heterologous to said ABC transporter protein coding sequence.

6. The plant of embodiment 5, wherein said ABC transporter protein coding sequence comprises SEQ ID NO 1 or encodes a protein selected from the group consisting of SEQ ID NO 2 and 15-103.

7. A plant according to embodiment 5, wherein said ABC transporter protein coding sequence encodes a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs 2 and 15-103 and ABC transporter function.

8. A plant according to embodiment 5, wherein said ABC transporter protein coding sequence encodes a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence positive rate relative to a sequence selected from the group consisting of SEQ id nos. 2 and 15-103 and ABC transporter function.

9. A transformed seed of the plant according to any one of embodiments 5 to 8.

10. The plant according to any one of embodiments 5 to 8, wherein said plant is a monocotyledon.

11. The plant of embodiment 10, wherein the plant is from the genus zea, oryza, triticum, sorghum, secale, eleusina, setaria, saccharum, miscanthus, panicum, pennisetum, sarum, coco, pineapples, plantain, elaeis, avena or hordeum.

12. The plant of any one of embodiments 5 to 8, wherein the plant is a dicot.

13. The plant of embodiment 12, wherein the plant is from the genus glycine, brassica, medicago, helianthus, rubiaceae, nicotiana, solanum, gossypium, ipomoea, manihot, coffea, citrus, theobroma, camellia, avocado, ficus, guava, mangnolia, luteolin, papaya, anacardamom, macadamia, prunus, beta, populus, or eucalyptus.

14. The plant of any one of embodiments 5-8, wherein said plant exhibits increased growth relative to a control plant.

15. The plant of any one of embodiments 5-8, wherein said plant exhibits increased biomass yield relative to a control plant.

16. The plant of any one of embodiments 5-8, wherein said plant exhibits increased seed yield relative to control plants.

17. The method of any one of embodiments 1 to 4, wherein the ABC transporter protein coding sequence is expressed from a reproductive regulatory promoter.

18. The method of embodiment 17, wherein said developmentally regulated promoter comprises SEQ ID NO 3 or SEQ ID NO 5.

19. The method of any one of embodiments 1-18, further comprising transforming the plant with at least one additional protein coding sequence.

20. The method of embodiment 19, wherein the at least one additional protein coding sequence is selected from SEQ ID NOs 7 and 9, or encodes a protein having at least 90% identity to a sequence selected from SEQ ID NOs 8 and 10.

21. The method of embodiment 19 or 20, wherein said at least one additional protein encoding sequence encodes a protein selected from the group consisting of SEQ ID NOS: 8 and 10.

22. The plant of any one of embodiments 5-8, wherein said promoter that drives expression in a plant is a developmentally regulated promoter.

23. The plant of embodiment 22, wherein said developmentally regulated promoter comprises SEQ ID No. 3 or SEQ ID No. 5.

24. The plant of embodiment 5, having stably introduced into its genome a second promoter operably linked to a second protein coding sequence that drives expression in the plant, wherein the second promoter is heterologous to the second protein coding sequence.

25. The plant of embodiment 24, wherein said second protein coding sequence is selected from the group consisting of SEQ ID NOs 7 and 9, or encodes a protein having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs 8 and 10.

26. The plant of embodiment 24 or 25, wherein said second protein coding sequence encodes a protein selected from the group consisting of SEQ ID NOs 8 and 10.

27. A DNA construct comprising, in operative association:

a. a promoter which functions in plant cells, and

b. a nucleic acid sequence encoding an ABC transporter protein.

28. The DNA construct of embodiment 27, wherein the nucleic acid sequence encoding an ABC transporter protein comprises SEQ ID NO 1 or encodes a protein selected from the group consisting of SEQ ID NO 2 and 15-103.

29. The DNA construct of embodiment 27 or 28, wherein said nucleic acid sequence encoding an ABC transporter protein encodes a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs 2 and 15-103 and having ABC transporter function.

30. The DNA construct of embodiment 27 or 28, wherein said nucleic acid sequence encoding an ABC transporter protein encodes a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence positivity relative to a sequence selected from the group consisting of SEQ ID nos. 2 and 15-103 and ABC transporter function.

31. The DNA construct of embodiment 27 or 28, wherein the promoter functional in plant cells is selected from the group consisting of SEQ ID NO. 3 and 5.

32. The DNA construct of any one of embodiments 27 to 31, wherein the promoter is heterologous to the nucleic acid sequence encoding the ABC transporter protein.

33. A method for increasing crop yield, comprising modulating expression in a plant of at least one ABC transporter protein coding sequence.

34. The method of embodiment 33, wherein said modulation of expression comprises increasing expression of at least one ABC transporter protein coding sequence in a plant.

35. The method of embodiment 34, wherein said increasing expression comprises increasing activity of a native ABC transporter sequence in said plant or increasing activity of a native ABC transporter protein coding sequence in said plant.

36. A plant according to any one of embodiments 5 to 8, wherein said promoter that drives expression in a plant is active in leaf tissue.

37. The DNA construct of any one of embodiments 27 to 32, wherein the promoter that functions in plant cells is active in leaf tissue.

Detailed Description

The present application provides compositions and methods for increasing crop biomass and yield. The method comprises increasing expression of at least one ABC transporter gene in a plant of interest. Crop yield is an extremely complex trait that results from the growth of crop plants at all stages of their development as well as from plant resources allocated to harvestable parts of the plants. In some crops, including but not limited to corn and soybean, the major harvestable parts may include seeds, while the remaining biomass (e.g., leaves and stems) is used for secondary applications. In other crops, including but not limited to sugarcane and alfalfa, the major harvestable parts of a plant consist of the stem or the entire aerial parts of the plant. In other crops, including but not limited to potatoes and carrots, the major harvestable parts of the plant are located underground. Regardless of the harvested part of the crop plant, the accumulation of harvestable biomass is the result of the growth of the plant and the photosynthetic fixed carbon allocated to the harvested part of the plant. Plant growth can be controlled by modulating the expression of one or more plant genes. Such modulation may alter the function of one or more metabolic pathways, thereby contributing to plant growth and the accumulation of harvestable biomass.

The methods of the present invention encompass plant growth control for increasing yield by modulating expression of one or more genes encoding ABC transporter proteins. In a preferred embodiment, the expression of a gene encoding an ABC transporter protein is upregulated relative to the level of ABC transporter expression in control plants, such that ABC transporter expression is increased in harvestable biomass in plants relative to control plants. Any method for increasing the activity or expression of an ABC transporter protein coding sequence in a plant is encompassed by the present invention.

The compositions of the present invention include constructs comprising the coding sequence shown in SEQ ID NO. 1 or a coding sequence encoding a protein selected from the group consisting of SEQ ID NO. 2 and 15-103, or variants thereof, operably linked to a promoter functional in plant cells. "promoter" is intended to mean a DNA regulatory region capable of driving expression of a sequence in a plant or plant cell. It is recognized that where the ABC transporter protein sequences disclosed herein have been identified, it is within the level of skill in the art to isolate and identify other ABC transporter protein sequences and nucleotide sequences encoding ABC transporter protein sequences, e.g., by BLAST search, PCR assay, etc.

When the coding sequence of the present invention is assembled in a DNA construct such that the promoter is operably linked to the coding sequence of interest, it enables the ABC transporter protein to be expressed and accumulated in plant cells stably transformed with the DNA construct. "operatively connected" is intended to mean a functional connection between two or more elements. For example, the operative linkage between the promoter of the invention and the heterologous nucleotide of interest is a functional linkage that allows expression of the heterologous nucleotide sequence of interest. The operatively connected elements may or may not be contiguous. When used in reference to a junction of two protein coding regions, operably linked means that the coding regions are in the same reading frame. The cassette may additionally contain at least one further gene to be co-transformed into a plant. Alternatively, other genes may be provided in multiple expression cassettes or DNA constructs. The expression cassette may additionally contain a selectable marker gene.

In this manner, the nucleotide sequence encoding the ABC transporter protein of the present invention is provided in an expression cassette or expression construct, along with a promoter sequence of interest (typically a heterologous promoter sequence), for expression in a plant of interest. "heterologous promoter sequence" is intended to mean a sequence that is not naturally operably linked to an ABC transporter protein-encoding nucleotide sequence. When the ABC transporter protein-encoding nucleotide sequence and promoter sequence are heterologous to each other, the ABC transporter protein-encoding nucleotide sequence or heterologous promoter sequence may be homologous, native, heterologous, or foreign to the plant host. It will be appreciated that a promoter may also drive expression of its homologous or native nucleotide sequence. In this case, the transformed plant will have a phenotypic change.

Fragments and variants of the polynucleotide and amino acid sequences of the invention may also be expressed by a promoter operable in plant cells. By "fragment" is meant a portion of a polynucleotide or a portion of an amino acid sequence. "variant" is intended to mean substantially similar sequences. For polynucleotides, variants include: polynucleotides having deletions (i.e., truncations) at the 5 'and/or 3' end; a polynucleotide having a deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or a polynucleotide having a substitution of one or more nucleotides at one or more positions in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide includes a naturally occurring nucleotide sequence or amino acid sequence, respectively. In general, variants of a particular polynucleotide of the invention have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the particular polynucleotide as determined by the sequence alignment programs and parameters described elsewhere herein. Fragments and variants of the polynucleotides described herein may encode proteins that retain ABC transporter function.

"variant" amino acid or protein is intended to mean an amino acid or protein derived from a natural amino acid or protein by: deletion of one or more amino acids at the N-terminus and/or C-terminus of the native protein (so-called truncation); deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; 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, i.e., they continue to possess the desired biological activity of the native protein, such as hydrolysis of ATP and transport of inorganic ions. Biologically active variants of a native polypeptide have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence of the native sequence as determined by the sequence alignment programs and parameters described herein. In some embodiments, the variant polypeptide sequence comprises a conservative amino acid substitution. The sum of the number of such conservative amino acid substitutions and the number of amino acid identities divided by the total number of amino acids in the sequence of interest can be used to calculate sequence positivity. Sequence positive-value rate calculations were performed on the NCBI BLAST server, which is accessible on the world wide web via BLAST. Biologically active variants of a protein of the invention may differ from the 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.

Amino acids can be generally classified as aliphatic, hydroxyl-containing or sulfur/selenium-containing, cyclic, aromatic, basic, acidic, and amides thereof. Without being limited by theory, in some cases, conservative amino acid substitutions may be preferred over non-conservative amino acid substitutions for the production of variant protein sequences, as conservative substitutions may more readily allow the variant protein to retain its biological activity than non-conservative substitutions. Polynucleotides encoding polypeptides having one or more amino acid substitutions in the sequence fall within the scope of the invention. Table 1 below provides a list of examples of amino acids belonging to various classes.

Table 1: class of amino acids

Class of amino acids	Exemplary amino acids
		Aliphatic series	Gly,Ala,Val,Leu,Ile
Containing hydroxy groups or sulfur/selenium	Ser,Cys,Thr,Met,Sec
		In the form of a ring	Pro
Aromatic hydrocarbons	Phe,Tyr,Trp
		Basic property	His,Lys,Arg
Acids and amides thereof	Asp,Glu,Asn,Gln

Variant sequences can also be identified by analysis of existing databases of sequenced genomic data. In this way, the corresponding sequences can be identified and used in the methods of the invention.

Methods of comparing aligned sequences are well known in the art. Thus, a mathematical algorithm can be used to determine the percent sequence identity of any two sequences. Non-limiting examples of such mathematical algorithms are the algorithms of Myers and Miller (1988) CABIOS 4: 11-17; the local alignment algorithm of Smith et al (1981) adv.Appl.Math.2: 482; needleman and Wunsch (1970) J.Mol.biol.48: 443-; pearson and Lipman (1988) Proc. Natl. Acad. Sci.85: 2444-2448; karlin and Altschul (1990) Proc.Natl.Acad.Sci.USA 87: 2264-.

Computer implementations of these mathematical algorithms can be used for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligent genetics (Intelligenetics) Inc. of mountain View, Calif.); version 10 of the GCG Wisconsin Genetics software package (GCG Wisconsin Genetics software Package) (available from Accelrys Inc., No. 9685 of Stylordun, san Diego, Calif., USA) the ALIGN program (version 2.0) and GAP, BESTFIT, BLAST, FASTA and TFASTA. Alignments using these programs can be performed with default parameters. The CLUSTAL program has been developed by Higgins et al (1988) Gene 73:237-244 (1988); higgins et al (1989) CABIOS 5: 151-153; corpet et al (1988) Nucleic acids sRs.16: 10881-90; huang et al (1992) CABIOS 8: 155-65; and Pearson et al (1994) meth.mol.biol.24: 307-. The ALIGN program is based on the algorithm of Myers and Miller (1988) described above. For comparison of amino acid sequences, the ALIGN program can be used with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The BLAST program of Altschul et al (1990) J.mol.biol.215:403 is based on the algorithm of Karlin and Altschul (1990) described above. BLAST nucleotide searches (score 100, word length 12) can be performed using the BLASTN program to obtain nucleotide sequences homologous to the nucleotide sequences encoding the proteins of the present invention. BLAST protein searches (score 50, word length 3) can be performed using the BLASTX program to obtain amino acid sequences homologous to the proteins or polypeptides of the invention. For gap alignments to be used for comparison purposes, gapped BLAST can be utilized as described in Altschul et al (1997) Nucleic Acids Res.25:3389-3402 (in BLAST 2.0). Alternatively, an iterative search using PSI-BLAST (in BLAST 2.0) can be performed, which is used to detect near-far relationships between molecules. See Altschul et al (1997) supra. When BLAST, gapped BLAST, PSI-BLAST are used, default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See www.ncbi.nlna.nih.gov. The alignment may also be performed by manual inspection.

Such genes and coding regions may be codon optimized for expression in a plant of interest. A "codon-optimized gene" is a gene having a codon usage frequency designed to mimic the preferred codon usage frequency of the host cell. The nucleic acid molecule may be codon optimized in whole or in part. Because any one amino acid (except methionine and tryptophan) is encoded by multiple codons, the sequence of the nucleic acid molecule can be altered without changing the encoded amino acid. Codon optimization refers to the alteration of one or more codons at the nucleic acid level such that the amino acids are unchanged and expression is increased in a particular host organism. One skilled in the art will recognize that codon tables and other references providing preference information for various organisms are known in the art (see, e.g., Zhang et al (1991) Gene 105: 61-72; Murray et al (1989) Nucl. acids Res.17: 477-508). Methods for optimizing nucleotide sequences for expression in plants are provided, for example, in U.S. patent No. 6,015,891 and references cited therein, and WO 2012/142,371 and references cited therein.

The nucleotide sequences of the present invention may be used in recombinant polynucleotides. A "recombinant polynucleotide" comprises a combination of two or more chemically linked nucleic acid segments that are not directly joined in nature. By "directly joined" is meant that two nucleic acid segments are immediately adjacent and joined to each other by a chemical bond. In particular embodiments, the recombinant polynucleotide comprises the polynucleotide of interest or an active variant or fragment thereof such that another chemically linked nucleic acid segment is located 5', 3' or within the polynucleotide of interest. Alternatively, chemically linked nucleic acid segments of a recombinant polynucleotide may be formed by deletion of the sequence. The other chemically linked nucleic acid segment or sequences deleted to join linked nucleic acid segments can be of any length, including, for example, 1, 2,3, 4, 5,6, 7, 8, 9, 10, 15, 20, or more nucleotides. Disclosed herein are various methods for making such recombinant polynucleotides, including, for example, by chemical synthesis or by manipulation of isolated polynucleotide segments by genetic engineering techniques. In particular embodiments, a recombinant polynucleotide may comprise a recombinant DNA sequence or a recombinant RNA sequence. A "fragment of a recombinant polynucleotide" comprises at least one combination of two or more chemically linked amino acid segments that are not directly joined in nature.

By "altering" or "regulating" the expression level of a gene is meant that the expression of the gene is up-regulated or down-regulated. It will be appreciated that in some instances, plant growth and yield are increased by increasing the expression level, i.e., upregulating expression, of one or more genes encoding ABC transporter proteins. Also, in some cases, plant growth and yield are increased by decreasing the expression level, i.e., down-regulating expression, of one or more genes encoding ABC transporter proteins. Thus, the present invention encompasses the up-regulation or down-regulation of one or more genes encoding ABC transporter proteins. In addition, the method comprises up-regulating at least one gene encoding an ABC transporter protein and down-regulating at least one gene encoding a second ABC transporter protein in a plant of interest. Modulating the concentration and/or activity of at least one gene encoding an ABC transporter protein in a transgenic plant means that the concentration and/or activity is increased or decreased by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more relative to a native control plant, plant part or cell into which no sequence of the present invention has been introduced.

It will be appreciated that the level of expression of the gene encoding the ABC transporter protein of the present invention may be controlled by the use of one or more promoters that function in plant cells. The expression level of the gene encoding the ABC transporter protein of interest can be determined directly, for example, by detecting the level of ABC transporter gene transcript or encoded protein in the plant. Methods for such detection are well known in the art. For example, Northern blotting or quantitative reverse transcriptase-PCR (qRT-PCR) can be used to detect transcript levels, while Western blotting, ELISA assays, or enzymatic assays can be used to detect protein levels. ABC transporter function can be detected, for example, by the well-known ATPase assay (Glavinas et al 2008Expert Opinion on drug & diagnosis 4: 721-.

A "subject plant or plant cell" is a plant or plant cell that has been genetically altered (e.g., transformed) by the influence of a gene encoding an ABC transporter protein of interest, or a plant or plant cell derived from and comprising such an altered plant or cell. A "control" or "control plant cell" provides a reference point for measuring a phenotypic change of a subject plant or plant cell. Thus, depending on the method of the invention, the expression level of the gene encoding the ABC transporter protein of interest is higher or lower than the expression level in a control plant.

Control plants or plant cells may include, for example: (a) a wild-type plant or cell, i.e., the same genotype as the starting material used to produce the genetic alteration in the subject plant or cell; (b) plants or plant cells of the same genotype as the starting material but transformed with an empty construct (i.e., a construct that has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell that is a non-transformed segregant in the progeny of the subject plant or plant cell; (d) a plant or plant cell that is genetically identical to the subject plant or plant cell, but that is not exposed to conditions or stimuli that 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.

Although the present invention is described in terms of transformed plants, it is to be understood that the transformed organisms of the present invention also include plant cells, plant protoplasts, plant cell tissue cultures from regenerable plants, plant calli, plant clumps, and intact plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, shoots, fruits, kernels, ears, cobs, husks, stems, roots, root tips, anthers, and the like. Grain is intended to mean mature seed produced by commercial growers for purposes other than growth or reproduction of 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 polynucleotide.

To down-regulate expression of a gene encoding an ABC transporter protein of interest, an antisense construct can be constructed that is complementary to at least a portion of the messenger rna (mrna) of the sequence of the gene of interest, particularly the gene encoding the ABC transporter protein of interest. Antisense nucleotides are designed to hybridize to the corresponding mRNA. Modifications of the antisense sequence may be made as long as the sequence hybridizes to the corresponding mRNA and interferes with its expression. In this way, antisense constructs having 70%, preferably 80%, more preferably 85%, 90%, 95% or more sequence identity to the corresponding sequence to be silenced can be used. In addition, a portion of the antisense nucleotide can be used to interfere with expression of the target gene.

The polynucleotides of the invention can be used to isolate corresponding sequences from other plants. In this manner, such sequences can be identified based on their sequence homology or identity to the sequences set forth herein using methods such as PCR, hybridization, and the like. Sequences isolated based on their sequence identity relative to the complete sequences set forth herein, or variants and fragments thereof, are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. "ortholog" is intended to mean a gene derived from a common ancestral gene and present in a different species as a result of speciation. Genes present in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. The function of orthologs is generally highly conserved across species. Thus, isolated polynucleotides, or variants or fragments thereof, having transcriptional or enhancer activity and sharing at least 75% sequence identity with the sequences described herein are encompassed by the present invention.

Variant sequences can be isolated by PCR. Methods for designing PCR primers and PCR Cloning are well known in the art and are disclosed in Sambrook et al (1989) Molecular Cloning: A Laboratory Manual (Molecular Cloning: A Laboratory Manual) (second edition, Cold spring harbor Laboratory Press, Prone Wien, N.Y.). See also Innis et al, (1990) PCRProtocols: A Guide to Methods and Applications (PCR protocols: Guide for Methods and Applications) (American academy of academic Press, New York); innis and Gelfand, 1995 PCR Strategies (PCR Strategies) (academic Press, New York, USA); and Innis and Gelfand (1999) PCR Methods Manual (A handbook of PCR Methods) (American academic Press, New York).

Variant sequences can also be identified by analysis of existing databases of sequenced genomic data. In this manner, the corresponding sequences encoding ABC transporter proteins can be identified and used in the methods of the present invention. The variant sequences retain the biological activity of the ABC transporter protein (i.e., hydrolysis of ATP and transport of inorganic ions). The present invention shows, unexpectedly, that certain novel expression strategies for ABC transporter protein overexpression can lead to increased biomass and seed yields.

The expression cassette comprises in the 5'-3' direction of the transcript: transcription and translation initiation regions, polynucleotides encoding the ABC transporter proteins of the present invention, and transcription and translation termination regions (i.e., termination regions) that function in plants.

A variety of promoters may be used in the practice of the present invention. The polynucleotides encoding the ABC transporter proteins of the present invention may be expressed from promoters with constitutive expression profiles. Constitutive promoters include the CaMV 35S promoter (Odell et al (1985) Nature 313: 810-; 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) the or. 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.

The polynucleotides of the invention encoding the ABC transporter proteins of the invention may be expressed from a tissue-preferred promoter. Tissue-preferred promoters include Yamamoto et al (1997) Plant J.12(2): 255-265; kawamata et al (1997) Plant Cell physiol.38(7): 792-803; hansen et al (1997) mol.Gen Genet.254(3): 337-343; russell et al (1997) Transgenic Res.6(2): 157-168; rinehart et al (1996) Plant Physiol.112(3): 1331-1341; van Camp et al (1996) Plant Physiol.112(2):525 and 535; canevascini et al (1996) Plant Physiol.112(2): 513-; yamamoto et al (1994) Plant Cell physiol.35(5): 773-778; lam (1994) Results sheet cell Differ.20: 181-196; orozco et al (1993) Plant MolBiol.23(6): 1129-1138; matsuoka et al (1993) Proc Natl.Acad.Sci.USA90(20): 9586-9590; and Guevara-Garcia et al (1993) Plant J.4(3): 495-505. Leaf-preferred promoters are also known in the art. See, e.g., 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. USA90(20): 9586-9590.

For expression of a polynucleotide encoding an ABC transporter protein, a developmentally regulated promoter may be desirable. Such promoters may exhibit expression peaks at specific developmental stages. Such promoters have been described in the art, for example US 62/029,068; gan and Amasino (1995) Science 270: 1986-1988; rinehart et al (1996) plantaPhysiol 112: 1331-1341; Gray-Mitsumune et al (1999) Plant Mol Biol 39: 657-669; beaudoin and Rothstein (1997) Plant Mol Biol 33: 835-846; genschik et al (1994) Gene 148:195-202 et al.

For expression of a polynucleotide encoding an ABC transporter protein, a promoter that is induced upon application of a particular biotic and/or abiotic stress may be desirable. Such promoters are described in the art, for example, Yi et al (2010) Planta 232: 743-754; Yamaguchi-Shinozaki and Shinozaki (1993) Mol Gen Genet236: 331-; U.S. patent No.7,674,952; rerksiri et al (2013) Sci World J2013: ArticleiD 397401; khuran et al (2013) PLoS One 8: e 54418; tao et al (2015) Plant Mol Biol Rep 33:200-208 and the like.

For expression of a polynucleotide encoding an ABC transporter protein, a cell-preferred promoter may be desirable. Such promoters may preferentially drive expression of downstream genes in a particular cell species, such as mesophyll cells or bundle sheath cells. Such cell-preferred promoters have been described in the art, e.g., Viret et al (1994) Proc NatlAcad USA 91: 8577-8581; U.S. patent No. 8,455,718; U.S. patent No.7,642,347; sattarzadeh et al (2010) Plant Biotechnol J8: 112-; engelmann et al (2008) Plant Physiol 146: 1773-; matsuoka et al (1994) Plant J6: 311-319, etc.

It will be appreciated that a particular non-constitutive expression profile may provide an improved plant phenotype relative to constitutive expression of one or more genes of interest. For example, many plant genes are regulated by light conditions, the application of specific stresses, the diurnal cycle, or the developmental stage of the plant. These expression profiles may be important for the function of the gene or gene product in plants. One strategy that can be used to provide the desired expression profile is to use a synthetic promoter containing cis-regulatory elements that drive the desired expression levels at the desired time and location in the plant. Cis-regulatory elements useful for altering gene expression in plants have been described in the scientific literature (Vandepoele et al (2009) Plant Physiol 150: 535-546; Rushton et al (2002) Plant Cell 14: 749-762). Cis-regulatory elements can also be used to alter the promoter expression profile as described in Venter (2007) Trends Plant Sci 12: 118-124.

Plant terminators are known in the art and include plant terminators obtainable from the Ti plasmid of agrobacterium tumefaciens (a. tumefaciens), such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al (1991) mol.Gen.Genet.262: 141-144; propufoot (1991) Cell 64: 671-674; sanfacon et al (1991) Genes Dev.5: 141-149; mogen et al (1990) Plant Cell 2: 1261-; munroe et al (1990) Gene91: 151-158; ballas et al (1989) Nucleic Acids Res.17: 7891-7903; and Joshi et al (1987) Nucleic Acids Res.15: 9627-9639.

As shown, the nucleotides encoding the ABC transporter proteins of the present invention can be used in expression cassettes to transform plants of interest. Transformation protocols, and protocols for introducing polypeptide or polynucleotide sequences into plants, may vary depending on the species of plant or plant cell that is the target of transformation, i.e., monocots or dicots. The term "transformation" or "transforming" refers to any method for introducing a polypeptide or polynucleotide into a plant cell. Suitable Methods for introducing a polypeptide or polynucleotide into a Plant Cell include microinjection (Crossway et al (1986) Biotechnology 4:320-334), electroporation (Riggs et al (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski et al (1984) EMBO J.3:2717-2722), and projectile particle acceleration (see, e.g., U.S. Pat. No. 4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. No. 5,886,244; and U.S. Pat. No. 5,932,782; Tomes et al (1995) in Plant Cell, Tissue and Organ Culture Methods (Biotechnology et al; Verlag et al; published by Biotechnology et al; Biotechnology 926; Verlag et al; Verlag. Kouchi., U.S. 4,945; U.S. 923; Verlag. Pat. 3; Verlag. Skowski et al.); and Lec1 transformation (WO 00/28058). See also Weissinger et al (1988) Ann. Rev. Genet.22: 421-477; sanford et al (1987) molecular 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-; finer and McMullen (1991) In vitro cell Dev.biol.27P: 175-; singh et al (1998) the or. 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-; klein et al (1988) Biotechnology 6:559-563 (maize); U.S. patent No. 5,240,855; U.S. Pat. nos. 5,322,783; and U.S. patent No. 5,324,646; klein et al (1988) Plant Physiol.91:440-444 (maize); fromm et al (1990) Biotechnology 8: 833-; Hooykaas-Van Slogteren et al (1984) Nature (London) 311: 763-764; U.S. Pat. No. 5,736,369 (cereal); bytebier et al (1987) Proc.Natl.Acad.Sci.USA 84:5345-5349 (Liliaceae); de Wet et al (1985) in The Experimental management of Ovule Tissues (Experimental procedures for Ovule organization), Chapman et al, Inc. (Langmen, N.Y.), pp.197-209 (pollen); kaeppler et al (1990) Plant Cell Reports 9:415-418 and Kaeppler et al (1992) the or. 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 Biotechnology14:745-750 (maize, by Agrobacterium tumefaciens); all of these documents are incorporated herein by reference. "Stable transformation" is intended to mean that the nucleotide construct introduced into a plant is integrated into the genome of the plant and is capable of being inherited by its progeny.

The transformed cells can be grown into plants according to conventional means. See, e.g., McCormick et al (1986) plant cell Reports 5: 81-84. In this manner, the present invention provides transformed seeds (also referred to as "transgenic seeds") having stably introduced into their genome a polynucleotide of the present invention, e.g., an expression cassette of the present invention.

The present invention can be used to transform any plant species, including but not limited to monocots and dicots. Examples of plant species of interest include, but are not limited to: maize (Zea mays), brassica (e.g. brassica napus (b.napus), brassica rapa (b.rapa), brassica napus (b.juncea)), in particular oilseed rape seed species used as a source of rapeseed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), Sorghum (Sorghum bicolor), red-brown Sorghum (Sorghum vulgare)), millet (e.g. pearl millet (Pennisetum glaucum), millet (Panicum milinaceum), millet (Setaria italica), set millet (eleusioin coracanata), sunflower (Helianthus annuus), safflower (Carthamus sativus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana), potato (arabicum), sweet potato (Solanum), coffee (Gossypium), sweet potato (Gossypium barba), sweet potato (Gossypium), sweet potato (Gossypium barba), sweet potato (Gossypium) and sweet potato (Gossypium) are provided for example, Pineapple (Ananas comosus), Citrus trees (Citrus spp.), cacao (Theobroma cacao), tea (Camellia sinensis), rubber (Musa spp.), avocado (perseamerica), fig (Ficus Carica), guava (Psidium guajava), mango (mangiferica), olive (oleca europaea), papaya (Carica papaya), cashew (anacardium occidentale), Macadamia nut (Macadamia integrifolia), almond (Prunus amygdalus), beet (Beta vulgaris), sugarcane (Saccharum spp.), oil palm (elaeiss guineensis), poplar (populus (pouussp), Eucalyptus (Eucalyptus), Eucalyptus spp., Hordeum spp., barley (Avena sativa), and ornamental plants.

In one embodiment, a plant cell is transformed with a construct comprising a promoter operable in a plant cell operably linked to a coding sequence encoding an ABC transporter protein of the present invention. Regenerating the transformed plant cell to produce a transformed plant. These plants transformed with constructs comprising a functional promoter driving expression of an ABC transporter protein-encoding polynucleotide of the present invention have been shown to have increased plant yield, i.e., increased aboveground biomass and/or increased harvestable biomass and/or increased seed yield.

It has now been demonstrated that up-regulation of ABC transporters can increase plant yield, and other methods for increasing expression of endogenous ABC transporter sequences in plants of interest can also be employed. Expression of an ABC transporter gene present in a plant genome can be altered by inserting a transcriptional enhancer upstream of the ABC transporter gene present in the plant genome. This strategy would allow the expression of ABC transporter genes to maintain their normal developmental status while exhibiting elevated transcript levels. This strategy will be achieved by inserting an enhancer element upstream of the ABC transporter gene of interest using a meganuclease designed against the genomic sequence of interest. Alternatively, an enhancer element is inserted upstream of the ABC transporter gene of interest using a Cas9 endonuclease coupled to a guide rna (gRNA) designed for the genomic sequence of interest, or a Cpf1 endonuclease coupled to a gRNA designed for the genomic sequence of interest, or a Csm1 endonuclease coupled to a gRNA designed for the genomic sequence of interest. Alternatively, an inactivating endonuclease, such as an inactivated Cas9, Cpf1, or Csm1 endonuclease, fused to a transcriptional enhancer element is targeted to a genomic location near the transcription start site of the ABC transporter gene of interest, thereby modulating the expression of the ABC transporter gene of interest (Piatek et al (2015) Plant Biotechnol J13: 578-589).

Modulation of the expression of ABC transporter protein-encoding genes can be achieved by modulating the expression of endogenous sequences using precise genome editing techniques. In this manner, the nucleic acid sequence is inserted adjacent to the native plant sequence encoding the ABC transporter using methods known in the art. Such methods include, but are not limited to: meganucleases designed against Plant genomic sequences of interest (D' Halluin et al (2013) Plant Biotechnol J11: 933-941); CRISPR-Cas9, CRISPR-Cpf1, TALENs and other techniques for precise genome editing (Feng et al (2013) Cell Research23:1229-1232, Podevin et al (2013) Trends Biotechnology 31:375-383, Wei et al (2013) J Genomics 40:281-289, Zhang et al (2013) WO 2013/026740, Zetsche et al (2015) Cell 163:759-771, U.S. provisional patent application 62/295,325); argonaute Gracilaria protein-mediated DNA insertion (Gao et al (2016) Nat Biotechnol doi: 10.1038/nbt.3547); cre-lox site-specific recombination (Dale et al (1995) Plant J7: 649-659; Lyznik et al (2007) Transgenic Plant J1: 1-9; FLP-FRT recombination (Li et al (2009) Plant Physiol 151: 1087-1095); Bxb 1-mediated integration (Yau et al (2011) Plant J701: 147-166); zinc finger protein-mediated integration (Wright et al (2005) Plant J44: 693-705); Cai et al (2009) Plant Mol Biol 69: 699-709); and homologous recombination (Lieberman-Lazarovich and Levy (2011) Methods Mol Biol 701: 51-65; Puchta (2002) Plant Mol Biol 48: 173-182). The insertion of the nucleic acid sequence will be used to achieve the desired effect of over-expressing, reducing expression, and/or altering the expression profile of the ABC transporter gene.

Enhancers include any molecule capable of enhancing gene expression when inserted into the genome of a Plant, thus, enhancers may be inserted into the genomic upstream or downstream region of the ABC transporter sequence of interest to enhance expression, enhancers may be cis-acting and may be located anywhere in the genome relative to the gene whose expression is to be enhanced, for example, enhancers may be located within about 1Mbp, about 100kbp, about 50kbp, about 30kbp, about 20kbp, about 10kbp, about 5kbp, about 3kbp or about 1kbp of the coding sequence whose expression is enhanced, enhancers may also be located within about 1500bp of the gene whose expression is enhanced, or may be located directly adjacent to or within an intron of the gene whose expression is enhanced, enhancers for regulating the expression of endogenous genes encoding the ABC transporter proteins or homologues of the present invention include enhancer elements such as the CaMV 35S enhancer element, cytomegalovirus (cytomegalovirus) promoter element, 40 enhancer element, and enhancer element containing CMV enhancer elements such as the Cheryan enhancer element, enhancer element for regulating expression of an endogenous gene such as the maize gene promoter, the Biophyesk 19-13-gene promoter, enhancer (see examples of the introduction of the Plant, maize, rice Plant, rice, barley, rice.

Alteration of ABC transporter gene expression can also be achieved by modifying the DNA in such a way that the DNA sequence is not altered. Such changes may include modification of the ABC transporter gene of interest and/or the chromatin composition or structure of the DNA surrounding the ABC transporter gene. It is well known that such changes in chromatin composition or structure can affect gene transcription (Hirschhorn et al (1992) Genes and Dev 6: 2288-. Such changes may also include altering the methylation state of the ABC transporter gene of interest and/or the DNA surrounding the ABC transporter gene of interest. It is well known that such changes in DNA methylation can alter transcription (Hsieh (1994) Mol Cell Biol 14: 5487-5494). Targeting epigenomic editing has been shown to affect transcription of genes in a predictable manner (Hilton et al (2015)33: 510-517). It will be apparent to those skilled in the art that other similar alterations (collectively, "epigenetic alterations") can be applied to the DNA that modulates transcription of the ABC transporter gene of interest in order to achieve the desired effect of altering the ABC transporter gene expression profile.

Alteration of ABC transporter gene expression can also be achieved by altering gene expression using transposable element technology. It is understood that transposable elements can alter the expression of adjacent DNA (McGinnis et al (1983) Cell 34: 75-84). Alteration of the expression of a gene encoding an ABC transporter can be achieved by inserting a transposable element upstream of the ABC transporter gene of interest, resulting in altered expression of the gene.

Alteration of ABC transporter gene expression can also be achieved by expression of one or more transcription factors that modulate the expression of the ABC transporter gene of interest. It will be appreciated that alterations in the expression of a transcription factor may in turn alter the expression of the target gene for that transcription factor (Hiratsu et al (2003) Plant J34: 733-739). Alteration of ABC transporter gene expression can also be achieved by altering the expression of transcription factors known to interact with ABC transporter genes of interest (e.g., OCL 1; Javelle et al (2010) Plant Physiol 154: 273-286).

Alteration of ABC transporter gene expression can also be achieved by inserting a promoter upstream of the open reading frame encoding the native ABC transporter in the plant species of interest. This will be achieved by inserting the promoter of interest upstream of the open reading frame encoding the ABC transporter protein using a meganuclease designed against the genomic sequence of interest. This strategy is well known and it has been previously demonstrated that a transgene is inserted at a predetermined location in the cotton genome (D' Halluin et al (2013) Plant Biotechnol J11: 933-941). It will be apparent to those skilled in the art that other techniques can be employed to achieve similar results for insertion of genetic elements at predetermined genomic loci (e.g., CRISPR-Cas9, CRISPR-cpf1, CRISPR-Csm1, TALENs, and other techniques for precise editing of genomes) by causing double-strand breaks at the predetermined genomic loci and providing DNA templates suitable for insertion.

The following examples are provided by way of illustration and not by way of limitation. All publications and patent applications mentioned in this specification are indicative of the level of skill 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 apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Experimental part