WO2018009632A1 - Activase rubisco mutée - Google Patents

Activase rubisco mutée Download PDF

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WO2018009632A1
WO2018009632A1 PCT/US2017/040859 US2017040859W WO2018009632A1 WO 2018009632 A1 WO2018009632 A1 WO 2018009632A1 US 2017040859 W US2017040859 W US 2017040859W WO 2018009632 A1 WO2018009632 A1 WO 2018009632A1
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rca
isoform
plant
altered
sequence
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PCT/US2017/040859
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Steven C. Huber
Sang Yeol Kim
Martin H. SPALDING
David A. Wright
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The United States Of America, As Represented By The Secretary Of Agriculture
Iowa State University Research Foundation, Inc.
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Publication of WO2018009632A1 publication Critical patent/WO2018009632A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8269—Photosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01039—Ribulose-bisphosphate carboxylase (4.1.1.39)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates generally to a mutated ribulose 1 ,5-bisphosphate carboxylase/oxygenase (RuBisCO) activase (also referred to as RCA), the genomic sequences that encode the mutated RCA that is truncated, and genetically altered plants that produce the mutated RCA that is truncated.
  • RuBisCO mutated ribulose 1 ,5-bisphosphate carboxylase/oxygenase
  • RuBisCO catalyzes the first and rate-limiting step in photosynthetic carbon fixation, the transfer of atmospheric CO 2 to ribulose- 1, 5 -bisphosphate. As such, it is the major enzyme that removes CO 2 from the atmosphere, and significant variation in kinetic properties of this enzyme are found among various phylogenetic groups.
  • RuBisCO homologues are available in the public literature (e.g., over 1,000 different RuBisCO homologues are listed in GenBank alone), and the crystal structure of RuBisCO has been solved for several variants of the protein.
  • RuBisCO contains two competing enzymatic activities: an oxygenase and a carboxylase activity.
  • the oxygenation reaction catalyzed by RuBisCO is a "wasteful" process since it competes with and significantly reduces the net amount of carbon fixed.
  • its catalytic cycling rate (kcat) at about 3 reactions per second, for the enzymes from higher plants, is relatively slow.
  • RuBisCO accounts for more than 35% of leaf total soluble proteins. Increasing RuBisCO 's catalytic efficiency would proportionally increase the rate of photosynthesis and, in turn, increase plant productivity.
  • RuBisCO activase through ATP hydrolysis and highly specific interaction with RuBisCO causes a conformational change to the RuBisCO protein that allows release of inhibitory sugar phosphates tightly bound at RuBisCO's active sites, thereby activating the enzyme.
  • a mathematical model indicates that RCA limits non-steady-state photosynthesis in plants receiving fluctuating light (e.g., light flecks formed in a canopy), even at moderate temperatures (Mott K A and Woodrow I E, (2000) J Exp Botany 51:399-406).
  • the present invention meets these and other needs and provides such improvements and opportunities.
  • carboxylase/oxygenase activase (RCA) a-isoform also referred to as an "altered" RCA a- isoform having reduced light sensitive inhibitory activity compared to light sensitive inhibitory activity in a wild-type RCA a-isoform. It is another object of this invention that the mutated RCA a-isoform has reduced amount of redox activity at its carboxy terminus (or no redox activity at its carboxy terminus) compared to the amount of redox activity a wild-type RCA ⁇ -isoform has its carboxy terminus.
  • the altered RCA ⁇ -isoform has less than two redox-sensitive cysteines at its redox active site (one or none cysteines) whereas the wild-type RCA ⁇ -isoform has two redox-sensitive cysteines within said redox active site.
  • the altered RCA ⁇ -isoform has three or more redox-sensitive
  • the altered RCA a- isoform having less than two redox-sensitive cysteines at its redox active site (one or none
  • cysteines is a rice altered RCA ⁇ -isoform and has at least one carboxy terminal amino acid
  • the altered RCA ⁇ -isoform having three or more redox-sensitive cysteines at its redox active site is a rice altered RCA ⁇ -isoform and has at least one carboxy terminal amino acid sequence set forth in SEQ ID NOs: 6, 12, 22, 27, and 29. It is further object of this invention to have an altered (or mutated) plant containing one or more of these altered RCA a-isoforms. It is another object of this invention that these altered RCA a-isoforms have a reduced amount of light sensitive inhibition of RuBisCO activity compared to wild-type RCA's amount of light sensitive inhibition of RuBisCO activity. It is another object of this invention that these mutated RCA a- isoforms have increased activation rate of RuBisCO compared to wild-type RCA's activation rate of RuBisCO.
  • the altered RCA ⁇ -isoform causes the increase the agronomic phenotype compared to the agronomic phenotype level in a wild-type plant containing a wild-type RCA a-isoform, and the agronomic phenotype can be increased plant growth, leaf width, seed yield per plant,
  • photosynthetic induction rate RCA ⁇ -isoform activation rate, RuBisCO activation rate, or a combination thereof.
  • an altered seed from this altered plant such that the altered seed contains altered plant cells that produce one or more of these altered RCA ⁇ -isoforms.
  • an altered plant cell from this altered plant such that the altered plant cell produces at least one of the altered RCA a- isoforms.
  • the altered plant is an altered rice, and that at least one altered RCA ⁇ -isoform has a carboxy terminal amino acid sequence that can be SEQ ID NOs: 4, 6, 8, 10, 12, 15, 17, 19, 22, 24, 27, 29, or a combination thereof.
  • the agronomic phenotype can be increased plant growth, leaf width, seed yield per plant, photosynthetic induction rate, RCA a-isoform activation rate, RuBisCO activation rate, or a combination thereof.
  • the altering step involves transforming the wild-type plant cell with an expression vector containing a promoter operably linked to DNA encoding Cas9 and a promoter operably linked to DNA encoding an appropriate sgRNA having a sequence that targets a genomic sequence prior to or at RCA a-isoform's carboxy terminus redox active site.
  • the DNA alteration occurs in the RCA a- isoform gene's exon that encodes the redox-sensitive cysteines at the carboxy terminus of the protein.
  • RNAi to silence the production of RCA a-isoform by transforming a wild-type plant cell with an expression vector containing a promoter operably linked to DNA encodes at least 19 nucleotides that is the reverse complement to the same number of nucleotides of RCA ⁇ -isoform within the protein's carboxy terminus.
  • the expression vector could contain a promoter operably linked to DNA that encodes at least 19 nucleotides of the sense coding RCA ⁇ -isoform within the protein's carboxy terminus and the complementary anti-sense nucleotides and a linker between the sense and anti-sense sequences.
  • one of the parent plants contains at least one altered RCA -isoform gene that encodes the altered RCA a-isoform lacking redox activity at its carboxy terminus and that this altered RCA ⁇ -isoform gene is present in the progeny seeds.
  • the agronomic trait can be improved plant growth, leaf width, seed yield per plant, photosynthetic induction rate, RCA a-isoform activation rate, RuBisCO activation rate, or a combination thereof.
  • RCA a- isoform C-terminal extension contains two redox-sensitive cysteines responsible for inactivation of RCA in the dark or low light. In other plants, this activity may be in a separate RuBisCO alpha gene.
  • reduction of this redox activity or inhibition of this activity can be caused by disrupting RCA a-isoform or its formation of the activation complex with RCA ⁇ - isoform, a decrease in the expression of RCA -isoform gene through the use of co-suppression, antisense, or RNA silencing or interference, or through gene editing to form indels that disrupt the carboxy-terminal region of the RCA ⁇ -isoform gene.
  • Another object of this invention is polynucleotides that encode altered RCA a-isoforms lacking the redox-sensitive cysteines at the protein's carboxy terminus.
  • one can reduce RCA ⁇ -isoform activity by (a) introducing into a wild- type plant cell at least one expression vector containing a promoter operably linked to a polynucleotide that encodes one or more RCA ⁇ -isoform regulatory inhibition sequences or a subsequence thereof, (b) selecting transformed plant cells that express the polynucleotide encoding the inhibition sequence, and (c) growing the selected transformed plant cells into transformed plant where the produced polynucleotide encoding the RCA ⁇ -isoform regulatory inhibition sequence modulates (or decreases) the activity of one or more wild-type RCA ⁇ -isoform regulatory sequences compared to a corresponding wild-type plant.
  • the expression vector can be introduced by techniques including, but not limited to, electroporation, micro-projectile bombardment, Agrobacterium-mediated transfer, and the like.
  • FIG. 1 contains the DNA and amino acid sequences of wild-type O. sativa cultivar Kitaake RCA a-isoform's carboxy terminus (as encoded in exon 7); SEQ ID NOs: 1 and 2 respectively.
  • FIG. 2 shows the DNA and amino acid sequences (SEQ ID NO: 3 and 4, respectively) of an altered O. sativa cultivar Kitaake RCA ⁇ -isoform's carboxy terminus (plant line 2-1) containing 147 bp deletion in intron 6 and exon 7.
  • FIG. 3 shows the 3 ' DNA and amino acid carboxy terminal sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant lines 8-1 and 8-2.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosome 1 has 4 bp deletion (SEQ ID NO: 5 and 6), and chromosome 2 has 1 bp insertion (SEQ ID NO: 7 and 8).
  • FIG. 4 shows the 3 ' DNA and amino acid carboxy terminus sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant line 8-3.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosome 1 has 2 bp deletion (SEQ ID NO: 9 and 10), and chromosome 2 has 6 bp deletion and 2 bp insertion (SEQ ID NO: 11 and 12).
  • FIG. 5 shows the 3 ' DNA and amino acid carboxy terminus sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant lines 8-4 and 8-5.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosome 1 has 1 bp insertion (SEQ ID NO: 13 and 8) and chromosome 2 has 1 bp insertion (SEQ ID NO: 14 and 15).
  • FIG. 6 shows the 3 ' DNA and amino acid carboxy terminus sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant line 8-6.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosome 1 has 4 bp deletion (SEQ ID NO: 5 and 6) and chromosome 2 has 2 bp deletion and 3 bp insertion (SEQ ID NO: 16 and 17).
  • FIG. 7 shows the 3 ' DNA and amino acid carboxy terminus sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant lines 8-7 and 8-8.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosome 1 has 4 bp deletion (SEQ ID NO: 5 and 6) and chromosome 2 has 1 bp insertion (SEQ ID NO: 7 and 8).
  • FIG. 8 shows the 3 ' DNA and amino acid carboxy terminus sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant lines 20-1, 20-2, and 20-3.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosome 1 has 1 bp insertion (SEQ ID NO: 7 and 8) and chromosome 2 has 5 bp deletion and 3 bp insertion (SEQ ID NO: 18 and 19).
  • FIG. 9 shows the 3 ' DNA and amino acid carboxy terminus sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant line 1.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosome 1 has 4 bp deletion (SEQ ID NO: 5 and 6) and chromosome 2 has 1 bp insertion (SEQ ID NO: 14 and 15).
  • FIG. 10 shows the 3' DNA and amino acid carboxy terminus sequences for the altered RCA - isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant lines 6-1 and 6-2.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosome 1 has 1 bp insertion (SEQ ID NO: 14 and 15) and chromosome 2 has 1 bp insertion (SEQ ID NO: 13 and 8).
  • FIG. 11 shows the 3 ' DNA and amino acid carboxy terminus sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant lines 6-3 and 6-4.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosome 1 has 4 bp deletion (SEQ ID NO: 5 and 6) and chromosome 2 has 1 bp insertion (SEQ ID NO: 7 and 8).
  • FIG. 12 shows the 3' DNA and amino acid carboxy terminus sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant line 8-10.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosome 1 has 4 bp deletion (SEQ ID NO: 5 and 6) and chromosome 2 has 2 bp deletion (SEQ ID NO: 20 and 19).
  • FIG. 13 shows the 3' DNA and amino acid carboxy terminus sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant line 9-1.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosomes 1 and 2 have 4 bp deletion (SEQ ID NO: 5 and 6).
  • FIG. 14 shows the 3' DNA and amino acid carboxy terminus sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant line 9-2.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosome 1 has 1 bp deletion (SEQ ID NO: 21 and 22) and chromosome 2 has 5 bp deletion (SEQ ID NO: 23 and 24).
  • FIG. 15 shows the 3' DNA and amino acid carboxy terminus sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant line 9-3.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosome 1 has 1 bp deletion (SEQ ID NO: 21 and 22) and chromosome 2 has 1 bp deletion (SEQ ID NO: 25 and 15).
  • FIG. 16 shows the 3' DNA and amino acid carboxy terminus sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant lines 13-1 and 13-2.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosomes 1 and 2 have 1 bp deletion (SEQ ID NO: 21 and 22).
  • FIG. 17 shows the 3' DNA and amino acid carboxy terminus sequences for the altered RCA - isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant line 16.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosomes 1 and 2 have 1 bp deletion (SEQ ID NO: 21 and 22).
  • FIG. 18 shows the 3 ' DNA and amino acid carboxy terminus sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant line 17.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosome 1 has 1 bp deletion (SEQ ID NO: 21 and 22) and chromosome 2 has 9 bp deletion (SEQ ID NO: 26 and 27).
  • FIG. 19 shows the 3' DNA and amino acid carboxy terminus sequences for the altered RCA a- isoform exon 7 in genetically modified O. sativa cultivar Kitaake plant lines 21-1 and 21-2.
  • the DNA sequences start with the last nucleotide of intron 6.
  • Chromosome 1 has 16 bp deletion (SEQ ID NO: 28 and 29) and chromosome 2 has 4 bp deletion (SEQ ID NO: 5 and 6).
  • FIG. 20A shows an immunoblot demonstrating removal of the long, redox-regulated RCA a- isoform from the transgenic rice lines 6-3, 8-4 and 9-3 but the presence of RCA ⁇ -isoform.
  • the staining of the RuBisCO large isoform (RbcL) with coomassie brilliant blue (CBB) in the lower gel shows equal loading of the lanes.
  • FIG. 20B compares the growth of the transgenic rice line 9-3 compared to wild-type (WT) rice during reproductive development.
  • WT wild-type
  • FIG. 20C shows that the transgenic rice lines 6-3, 8-4 and 9- 3 have higher CO 2 assimilation rate following an increase in light from low to high irradiance, referred to as photosynthetic induction, compared to the wild-type (WT) rice plant.
  • FIG. 20D shows that the transgenic rice lines 6-3, 8-4 and 9-3 have increased seed yield per plant compared to wild-type rice plants, while stover amounts are relatively equal.
  • FIG. 21 shows an alignment of RCA a-isoform carboxy terminal amino acids from nineteen economically important plants, demonstrating the presence of two cysteine amino acids (in bold and underlined) at the carboxy terminus of the RCA a-isoform which are involved in the redox regulation of the protein.
  • RuBisCO activase (also called “RCA") is needed to activate the primary photosynthetic carbon assimilation enzyme, RuBisCO.
  • RCA protein is a complex of two related proteins, the alpha- and beta-isoforms, which form a heteromeric complex involved in RuBisCO activation.
  • the ⁇ -isoform is essentially identical in sequence to the shorter ⁇ -isoform, except that the carboxy-terminus of the ⁇ -isoform is encoded in exons 7a and 7b, while the carboxy-terminus of the ⁇ -isoform is encoded only in exon 7a.
  • Exon 7b encodes the two cysteine amino acids that appear in wild-type a-isoform.
  • exons 7a and 7b are also referred to as "exon 7".
  • Altered RCA ⁇ -isoform lacking one or both cysteine amino acids at the carboxy terminus of the protein results in an increase in altered RCA's activity, compared to wild-type RCA's activity.
  • the increase in altered RCA's activity may result in an increased activation state for RuBisCO, which allows plants to take better advantage of intermittent high light conditions (generally found in the lower leaves in canopies of crop plants) and quicker photosynthetic induction in varying light conditions. This results in the genetically altered plants growing better in alternating low-light/high-light conditions as well as non- fluctuating light conditions.
  • This invention involves modifying RCA's ⁇ -isoform gene sequence via one or more truncations, deletions, and/or insertions in the exon that encodes RCA's ⁇ -isoform redox-sensitive cysteines, thereby altering the amino acid sequence encoded in that exon (which is at the gene's 3' end).
  • RCA's activity By eliminating one or both cysteine amino acids in the RCA's ⁇ -isoform carboxy terminus, one can alter RCA's activity and thus change the phenotype of the modified plant (i.e, RCA -isoform activation, RuBisCO activation, increased growth, plant height, leaf width, panicle number, and seed yield per plant).
  • RCA's a- isoform redox-sensitive cysteines are encoded in exon 7b.
  • mutated RCA containing with an ⁇ -isoform with a carboxy terminal mutation results in plants that have increased growth, height, leaf width, panicle number, RCA ⁇ -isoform activation, RuBisCO activation, and seed yield per plant under constant light during the photoperiod compared to those phenotypes in wild-type plants growing under similar conditions.
  • the DNA sequence of RuBisCO activase a-isoform in O. sativa cultivar Kitaake is identical to the DNA sequence in GenBank Accession XR_001541378 for O. sativa Japonica.
  • the DNA and amino acid sequences of RuBisCO activase ⁇ -isoform in O. sativa cultivar Kitaake is identical to the DNA and amino acid sequences in GenBank Accession XM_015761411 for O. sativa Japonica.
  • the DNA and amino acid sequences of the wild-type O. sativa cultivar Kitaake exons 7a and 7b are in SEQ ID NO: 1 and 2, respectively; see FIG. 1.
  • Some plants have the a- and ⁇ -isoforms of RCA encoded in separate genes (unlike rice where there is one RCA gene that is alternatively spliced to form two transcripts). It is also possible to refer to "chromosome 1" as “transcript 1” and “chromosome 2” as “transcript 2” because they arise from same gene ⁇ RCA a- isoform) even though the gene on each chromosome can have different mutations/alterations and thus some of the encoded RCA a-isoforms have different amino acid sequences. See FIGs. 3-19 and Table 1, infra. Some plants have undergone polyploidy events, so that genes are present in multiple copies, as is the case for soybean and its RCA genes.
  • RCA genes may have copies of RCA genes on more than one chromosome. Regardless of the number of RCA genes in a plant's gene, the RCA genes can be altered in a similar manner as described in the examples below and produce genetically altered plants with the described phenotype.
  • the DNA and amino acid sequences of the mutated RCA a-isoform exon 7 in the transformed rice plants discussed herein, as well as other important sequences are in Table 1. See also FIGs. 1-19 for the RCA a-isoform carboxy terminal sequences for wild-type rice and transgenic rice lines. Note that the DNA sequences for transgenic rice lines in FIGs. 1 and 3-19 include the last nucleotide of intron 6. FIG. 2 contains a longer portion of intron 6 than the other figures.
  • Tr. line 8-3 (FIG. 4)
  • Tr. lines 8-4 & 8-5 (FIG. 5)
  • Tr. line 8-6 (FIG. 6)
  • Tr. lines 8-7 & 8-8 (FIG. 7)
  • Tr. line 1 (FIG. 9)
  • Tr. lines 6-1 & 6-2 (FIG. 10)
  • Tr. lines 6-3 & 6-4 (FIG. 11)
  • Tr. line 9-1 (FIG. 13)
  • Tr. lines 13-1 & 13-2 (FIG. 16)
  • Tr. line 16 (FIG. 17)
  • Tr. lines 21-1 & 21-2 (FIG. 19)
  • SEQ ID NOs: 37, 38, 39, 40, and 42 lists the carboxy terminal amino acid sequences of five different mutated RCA a genes containing mutations in exon 7. These sequences are discussed infra in the examples. They are similar to some of the amino acid sequences listed in Table 1, supra, except that SEQ ID NOs: 37, 38, 39, 40, and 42 have an initial glycine (G) amino acid which arises from the codon spanning intron 6 and intron 7, whereas the amino acid sequences listed in Table 1 start with the first amino acid for which its codon is completely in exon 7.
  • G initial glycine
  • the carboxy terminal amino acid sequence of RCA a-isoform is very similar in the following economically important plants (which is not an exhaustive list): Brassica napus (rapeseed; SEQ ID NO: 45), Camelina sativa (SEQ ID NO: 46), Arachis duranensis (SEQ ID NO: 47), Vigna angularis (red mung bean; SEQ ID NO: 48), Cucumis melo (muskmelon; SEQ ID NO: 49), Cucumis sativus (cucumber; SEQ ID NO: 50), Fragaria vesca subsp.
  • vesca wild strawberry; SEQ ID NO: 51
  • Gossypium hirsutum upland cotton; SEQ ID NO: 52
  • Theobroma cacao cacao; SEQ ID NO: 53
  • Morus notabilis mulberry; SEQ ID NO: 54
  • Prunus mume plum; SEQ ID NO: 55
  • This alignment indicates that the deletion of one or both of these redox-sensitive cysteine amino acids in the RCA ⁇ -isoform for these economically important plants, as well as other plants with a similar RCA a-isoform, would result in the modified plants having the same altered phenotype that is described herein.
  • the sequences in FIG. 21 are obtained from public databases which have the full-length amino acid and DNA sequences for RCA ⁇ -isoform of those plants, and other plants. One can altered the exon that encodes these redox-sensitive cysteines to generate an altered RCA -isoform gene that encodes an altered RCA ⁇ -isoform with the properties described herein.
  • Methods are provided to reduce or eliminate the regulatory activity of RuBisCO activase a- isoform in one embodiment by transforming a plant cell with an expression cassette that expresses a polynucleotide that reduces the gene expression, mRNA production, and/or translation of the RCA a- isoform polypeptide.
  • the polynucleotide may reduce the expression of the RCA ⁇ -isoform polypeptide directly, by preventing transcription or translation of the RCA ⁇ -isoform mRNA, or indirectly, by encoding a polypeptide that reduces the transcription or translation of a RCA ⁇ -isoform gene or mRNA.
  • Methods for reducing 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 disclosure to reduce the expression of RCA a-isoform polypeptide.
  • specific mutations are introduced into the portion of a cell's genome which encodes RCA ⁇ -isoform to prevent transcription or translation of a "wild-type” functional ⁇ -isoform having "wild-type” activity.
  • mutations or changes in the DNA encoding RCA ⁇ -isoform (exon 7 in rice or the appropriate exon in other plants) can occur through gene editing or site directed mutagenesis.
  • the mutation is introduced in the appropriate exon of RCA a-isoform gene which results in the translation of an altered RCA a-isoform protein lacking one or both cysteine amino acids at the carboxy terminus that results in improved plant performance and seed yield, as the result of the altered RCA having a higher continuous activation state compared to wild-type RCA's activation state.
  • the expression of a RCA ⁇ -isoform polypeptide is reduced if the amount of the RCA a-isoform polypeptide is less than 100%, 99% 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1% of the amount of the same RCA ⁇ -isoform polypeptide in a control (wild-type) plant.
  • the amount of the RCA ⁇ -isoform polypeptide in a modified plant is less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than
  • the RCA ⁇ -isoform polynucleotide expression level and/or amount of polypeptide and/or enzymatic activity may be reduced or altered such that the reduction/alteration is phenotypically sufficient to provide increase in plant biomass, yield seed per plant, and/or RuBisCO's activation status.
  • the level or activity of one or more RCA ⁇ -isoform polynucleotides, polypeptides or enzymes may be impacted.
  • the expression level of the RCA ⁇ -isoform polypeptide may be measured directly, for example, by assaying for the quantity of RCA ⁇ -isoform polypeptide expressed in the plant cell or plant, or indirectly, for example, by measuring the RCA activity in the plant cell or plant or by measuring the phenotypic changes in the plant or by measuring RuBisCO levels or activation status. Methods for performing such assays are described elsewhere herein.
  • the activity of the RCA ⁇ -isoform polypeptide is reduced or eliminated by transforming a plant cell with an expression cassette containing a polynucleotide encoding a polypeptide that inhibits the activity of a RCA ⁇ -isoform polypeptide.
  • the activity of a RCA ⁇ -isoform polypeptide is reduced if the RCA a-isoform polypeptide's activity is less than 100%, 99% 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1% of the activity of the same RCA ⁇ -isoform polypeptide in a control plant.
  • the RCA a-isoform's activity in a modified plant is less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less than 5% of the RCA a-isoform's activity in a control plant.
  • the RCA ⁇ -isoform polypeptide's activity is "eliminated" when it is not detectable by the assay methods described elsewhere herein. Methods of determining the alteration of activity of a RCA a- isoform polypeptide are described elsewhere herein.
  • RCA ⁇ -isoform polypeptide's activity may be reduced or eliminated by mutating, disrupting, or excising at least a part of the RCA gene encoding the RCA ⁇ -isoform polypeptide, and, in one embodiment, RCA gene's exon that encodes the carboxy terminal redox-sensitive cysteines.
  • Mutagenized plants that carry mutations in RCA a-isoform genes (in one or more chromosomes) also result in reduced production of wild-type RCA a-isoform protein and/or reduced activity of the altered RCA a-isoform.
  • many methods may be used to reduce or eliminate the activity of a wild-type RCA a- isoform protein.
  • One or more methods may be used to reduce the activity of a single RCA a-isoform polypeptide.
  • One or more methods may be used to reduce the activity of multiple RCA a-isoform polypeptides.
  • nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the lUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms defined below are more fully defined by reference to the specification as a whole.
  • amplified is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template.
  • Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, Persing, et al, eds., American Society for Microbiology, Washington, DC (1993).
  • the product of amplification is termed an amplicon.
  • the term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations” and represent one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • AUG which is ordinarily the only codon for methionine; one exception is Micrococcus rubens, for which GTG is the methionine codon (Ishizuka, et ah, (1993) J. Gen. Microbiol. 139:425-32) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid, which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence and incorporated herein by reference. See Table 2.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" when the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered.
  • 1, 2, 3, 4, 5, 7 or 10 alterations can be made.
  • Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived.
  • substrate specificity, enzyme activity, or ligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80% or 90%, preferably 60-90% of the native protein for its native substrate.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • consisting essentially of means the inclusion of additional sequences to an object polynucleotide where the additional sequences do not selectively hybridize, under stringent hybridization conditions, to the same cDNA as the polynucleotide and where the hybridization conditions include a wash step in 0.1X SSC and 0.1% sodium dodecyl sulfate at 65°C.
  • nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA).
  • non-translated sequences e.g., introns
  • the information by which a protein is encoded is specified by the use of codons.
  • amino acid sequence is encoded by the nucleic acid using the "universal" genetic code.
  • variants of the universal code such as is present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum (Yamao, et al., (1985) Proc. Natl. Acad. Sci. USA 82:2306-9), or the ciliate Macronucleus, may be used when the nucleic acid is expressed using these organisms.
  • nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledonous plants or dicotyledonous plants as these preferences have been shown to differ (Murray, et al., (1989) Nucleic Acids Res. 17:477-98).
  • the rice preferred codon for a particular amino acid might be derived from known gene sequences from rice.
  • heterologous in reference to a nucleic acid is a nucleic acid 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.
  • a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived or, if from the same species, one or both are substantially modified from their original form.
  • a heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
  • host cell is meant a cell, which comprises a heterologous nucleic acid sequence of the invention, which contains a vector and supports the replication and/or expression of the expression vector.
  • Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, plant, amphibian, or mammalian cells.
  • host cells are monocotyledonous or dicotyledonous plant cells, including, but not limited to, maize, sorghum, sunflower, soybean, wheat, alfalfa, rice, cotton, canola, lawn grass, barley, millet, and tomato.
  • the monocotyledonous host cell is a rice host cell.
  • hybridization complex includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.
  • the term "introduced” in the context of inserting a nucleic acid into a cell means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • isolated or “isolated nucleic acid” or “isolated protein” refer to material, such as a nucleic acid or a protein, which is substantially or essentially free from components which normally accompany or interact with it as found in its naturally occurring environment.
  • isolated material optionally comprises material not found with the material in its natural environment.
  • Nucleic acids which are “isolated”, as defined herein, are also referred to as “heterologous" nucleic acids.
  • RCA a-isoform nucleic acid means a nucleic acid comprising a polynucleotide ("RCA a-isoform polynucleotide”) encoding a full length or partial length RCA ⁇ -isoform polypeptide with RCA a-isoform activity as defined herein.
  • nucleic acid includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).
  • nucleic acid library is meant a collection of isolated DNA or R A molecules, which comprise and substantially represent the entire transcribed fraction of a genome of a specified organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, (1987) Guide To Molecular Cloning Techniques, from the series Methods in Enzymology, vol. 152, Academic Press, Inc., San Diego, CA; Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual, 2 nd ed., vols. 1-3; and Current Protocols in Molecular Biology, Ausubel, et al, eds, Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994 Supplement).
  • operably linked includes reference to a functional linkage between a first sequence, such as a promoter, and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA corresponding to the second sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
  • plant includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same.
  • Plant cell as used herein includes, without limitation, cells in or from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
  • the class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants including species from the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Bro
  • yield may include reference to bushels per acre of a grain crop at harvest, as adjusted for grain moisture (15% typically for maize, for example), and/or the volume of biomass generated (for forage crops such as alfalfa, and plant root size for multiple crops).
  • Grain moisture is measured in the grain at harvest.
  • the adjusted test weight of grain is determined to be the weight in pounds per bushel, adjusted for grain moisture level at harvest. Biomass is measured as the weight of harvestable plant material generated.
  • polynucleotide includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s).
  • a polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof.
  • DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
  • polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • promoter includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • a "plant promoter” is a promoter capable of initiating transcription in plant cells.
  • Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples are promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibres, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as "tissue- preferred.”
  • a "cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves.
  • An “inducible” or “regulatable” promoter is a promoter which is under environmental control.
  • inducible promoters examples include anaerobic conditions or the presence of light.
  • Another type of promoter is a developmentally regulated promoter, for example, a promoter that drives expression during pollen development.
  • Tissue preferred, cell type specific, developmentally regulated, and inducible promoters constitute the class of "non-constitutive" promoters.
  • a “constitutive” promoter is a promoter, which is active under most environmental conditions.
  • the terms "RuBisCO activase alpha-isoform polypeptide", "RCA a-isoform”, “RCA a-isoform protein”, and similar variations refer to the same protein/polypeptide.
  • the term is also inclusive of fragments, variants, homologs, alleles or precursors (e.g., preproproteins or proproteins) thereof that retain the function of the regulatory/inactivation of RCA in the dark or in low light. In rice, this is the carboxy-terminal extension found only in the alpha-isoform, specifically the two redox-sensitive cysteine residues.
  • the terms "RuBisCO activase alpha nucleic acid”, “RCA a-isoform polynucleotide”, “RCA a-isoform gene” and similar variations means a polynucleotide encoding a RCA ⁇ -isoform polypeptide.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, organism, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells may express genes/polynucleotides that are not found within the native (non-recombinant or wild-type) form of the cell or express native genes in an otherwise abnormal amount - over-expressed, under-expressed or not expressed at all - compared to the non-recombinant or wild-type cell or organism.
  • the term "recombinant” as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
  • a "recombinant expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements, which permit
  • the recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
  • the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed, and a promoter.
  • amino acid residue or “amino acid residue” or “amino acid” are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein”).
  • the amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
  • carboxy-terminus, carboxyl-terminal, C-terminal, carboxy-terminus, C-terminus, and similar phrases refer to the portion of the protein and/or polypeptide distal to the amino terminus.
  • the amino terminus is translated from mRNA first and the carboxy-terminus is translated from mRNA last.
  • sequences include reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids.
  • Selectively hybridizing sequences typically have about at least 40% sequence identity, preferably 60-90% sequence identity, and most preferably 100% sequence identity (i.e., complementary) with each other.
  • stringent conditions or “stringent hybridization conditions” include reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than 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 can be identified which can be up to 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
  • the probe is approximately 500 nucleotides in length, but can vary greatly in length from less than 500 nucleotides to equal to the entire length of the target sequence.
  • 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 or Denhardt's.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX 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. IX SSC at 60 to 65°C.
  • T m 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 T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T m is reduced by about 1°C for each 1% of mismatching; thus, T m , 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 T m can be decreased 10°C.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) 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 (T m );
  • moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10°C lower than the thermal melting point (T m ); 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 (T m ).
  • T m thermal melting point
  • T m thermal melting point
  • high stringency is defined as hybridization in 4X SSC, 5X Denhardt's (5 g Ficoll, 5 g polyvinypyrrohdone, 5 g bovine serum albumin in 500ml of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65°C, and a wash in 0.1X SSC, 0.1% SDS at 65°C.
  • transgenic plant includes reference to a plant, which comprises within its genome a heterologous polynucleotide.
  • the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
  • transgenic “transformed”, “transformation”, and “transfection” are similar in meaning to "recombinant”.
  • Transformation", “transgenic”, and “transfection” refer to the transfer of a
  • polynucleotide into a host organism or into a cell.
  • Such a transfer of polynucleotides can result in genetically stable inheritance of the polynucleotides or in the polynucleotides remaining extra- chromosomally (not integrated into the chromosome of the cell).
  • Genetically stable inheritance may potentially require the transgenic organism or cell to be subjected for a period of time to one or more conditions which require the transcription of some or all of the transferred polynucleotide in order for the transgenic organism or cell to live and/or grow.
  • Polynucleotides that are transformed into a cell but are not integrated into the host's chromosome remain as an expression vector within the cell.
  • a genetically altered organism or altered organism is any organism with any changes to its genetic material, whether in the nucleus or cytoplasm (organelle).
  • a genetically altered organism can be a recombinant or transformed organism.
  • a genetically altered organism can also be an organism that was subjected to one or more mutagens or the progeny of an organism that was subjected to one or more mutagens and has mutations in its DNA caused by the one or more mutagens, as compared to the wild-type organism ⁇ i.e., organism not subjected to the mutagens).
  • an organism that has been bred to incorporate a mutation into its genetic material is a genetically altered organism.
  • vector includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.
  • sequence relationships between two or more nucleic acids or polynucleotides or polypeptides are used to describe the sequence relationships between two or more nucleic acids or polynucleotides or polypeptides: (a) “reference sequence,” (b) “comparison window,” (c) “sequence identity,” (d) “percentage of sequence identity,” and (e) “substantial identity.”
  • 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.
  • comparison window means includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of 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.
  • the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, and 50, 100 or longer.
  • the BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences.
  • GAP uses the algorithm of Needleman and Wunsch, supra, 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 Wisconsin Genetics Software Package are 8 and 2, respectively.
  • the gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100.
  • the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, and 40, 50 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 Wisconsin Genetics Software Package is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc.
  • sequence identity/similarity values refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul, et al, (1997) Nucleic Acids Res. 25:3389-402).
  • BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar.
  • a number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Claverie and States, (1993) Comput. Chem. 17:191-201) low-complexity filters can be employed alone or in combination.
  • sequence identity in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences, which are the same when aligned for maximum correspondence over a specified comparison window.
  • sequence identity 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. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • Sequences which 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., according to the algorithm of Meyers and Miller, (1988) Computer Applic. Biol. Sci. 4:11-17, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).
  • 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.
  • substantially identical of polynucleotide sequences means that a polynucleotide comprises a sequence that has between 50-100% sequence identity, preferably at least 50% sequence identity, preferably at least 60% sequence identity, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • sequence identity preferably at least 50% sequence identity, preferably at least 60% sequence identity, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95%.
  • nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions.
  • the degeneracy of the genetic code allows for many amino acids substitutions that lead to variety in the nucleotide sequence that code for the same amino acid, hence it is possible that the DNA sequence could code for the same polypeptide but not hybridize to each other under stringent conditions. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • One indication that two nucleic acid sequences are substantially identical is that the polypeptide, which the first nucleic acid encodes, is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • substantially identical in the context of a peptide indicates that a peptide comprises a sequence with between 55-100% sequence identity to a reference sequence preferably at least 55% sequence identity, preferably 60% preferably 70%, more preferably 80%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, supra.
  • An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
  • a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
  • a peptide can be substantially identical to a second peptide when they differ by a non-conservative change if the epitope that the antibody recognizes is substantially identical.
  • Peptides which are "substantially similar" share sequences as noted above, except that residue positions which are not identical may differ by conservative amino acid changes. Reducing the Regulatory Activity of an RCA a-isoform Polypeptide
  • a plant is transformed with an expression cassette that is capable of expressing a polynucleotide that inhibits the expression of a RCA ⁇ -isoform polypeptide of the invention.
  • expression refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product.
  • an expression cassette capable of expressing a polynucleotide that inhibits the expression of at least one RCA ⁇ -isoform polypeptide is an expression cassette capable of producing an RNA molecule that inhibits the transcription and/or translation of at least one RCA ⁇ -isoform polypeptide of the invention.
  • the "expression” or “production” of a protein or polypeptide from a DNA molecule refers to the transcription and translation of the coding sequence to produce the protein or polypeptide
  • the "expression” or “production” of a protein or polypeptide from an RNA molecule refers to the translation of the RNA coding sequence to produce the protein or polypeptide.
  • inhibition of the expression of a RCA a-isoform polypeptide may be obtained by sense suppression or cosuppression.
  • an expression cassette is designed to express an RNA molecule corresponding to all or part of exon that encodes the carboxy terminal redox-sensitive cysteines of a messenger RNA encoding a RCA ⁇ -isoform polypeptide that in the "sense" orientation.
  • Over-expression 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 the greatest inhibition of RCA ⁇ -isoform polypeptide expression.
  • the polynucleotide used for cosuppression may correspond to all or part of the coding sequences for RCA -isoform' s exon which encodes the carboxy terminal redox sensitive cysteines , all or part of the 5' and/or 3' untranslated region of a RCA ⁇ -isoform polypeptide transcript, or all or part of both the coding sequence and the untranslated regions of a transcript encoding a RCA ⁇ -isoform polypeptide.
  • the expression cassette is designed to eliminate the start codon of the polynucleotide so that no protein product will be translated.
  • 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. Patent 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.
  • nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, optimally greater than about 65% sequence identity, more optimally greater than about 85% sequence identity, most optimally greater than about 95% sequence identity. See U.S. Patent Nos. 5,283,184 and 5,034,323.
  • inhibition of the expression of the RCA a-isoform polypeptide may be obtained by antisense suppression.
  • the expression cassette is designed to express an RNA molecule complementary to all or part of exon which encodes the carboxy terminal redox-sensitive cysteines in a messenger RNA encoding the RCA a-isoform polypeptide. Over- expression of the antisense RNA molecule can result in reduced expression of the native gene.
  • the polynucleotide for use in antisense suppression may correspond to all or part of the complement of the exon sequence which encodes the the carboxy terminal redox-sensitive cysteines in RCA ⁇ -isoform polypeptide, all or part of the complement of the 5' and/or 3' untranslated region of the RCA ⁇ -isoform RCA a-isoform transcript, or all or part of the complement of both the coding sequence and the untranslated regions of a transcript encoding the RCA ⁇ -isoform polypeptide.
  • 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.
  • Antisense suppression may be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Patent No. 5,942,657.
  • portions of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, 300, 400, 450, 500, 550, or greater may be used.
  • inhibition of the expression of a RCA a-isoform polypeptide may be obtained by double-stranded RNA (dsRNA) interference.
  • 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.
  • the dsRNA would contain at least 19 contiguous nucleotides present in the RCA -isoform exon 7b portion of gene (for rice) or at least 19 contiguous in the exon that encodes the carboxy terminal redox-sensitive cysteines for any plant's RCA -isoform.
  • 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 RCA ⁇ -isoform polypeptide 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.
  • inhibition of the expression of a RCA a-isoform polypeptide may be obtained by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference.
  • hpRNA hairpin RNA
  • ihpRNA intron-containing hairpin RNA
  • 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 region and a base- paired stem.
  • 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 base-paired stem region may correspond to a portion of a promoter sequence controlling expression of the gene to be inhibited.
  • the dsRNA would contain at least 19 contiguous nucleotides present in the exon that encodes the carboxy terminal redox-sensitive cysteines for any plant's RCA a-isoform.
  • the base-paired stem region of the molecule generally determines the specificity of the RNA interference.
  • hpR A 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.
  • the interfering molecules have the same general structure as for hpRNA, but the RNA molecule additionally comprises an intron 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. In fact, Smith, et al. , show 100% suppression of endogenous gene expression using ihpRNA-mediated interference.
  • 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.
  • 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.
  • it is the loop region that determines the specificity of the RNA interference.
  • 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 the RCA a-isoform polypeptide).
  • the transcript targets the RCA exon 7b portion of gene (for rice) or, for other types of plants, the exon that encodes the carboxy terminal redox-sensitive cysteines for a plant's RCA a-isoform.
  • the polynucleotide expressed by the expression cassette of the invention is catalytic RNA or has ribozyme activity specific for the messenger RNA of the RCA a-isoform polypeptide.
  • the polynucleotide causes the degradation of the endogenous messenger RNA, resulting in reduced expression of the RCA ⁇ -isoform polypeptide. This method is described, for example, in U.S. Patent No. 4,987,071.
  • polypeptide 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. miRNA are highly efficient at inhibiting the expression of endogenous genes. See, for example Javier, et al., (2003) Nature 425:257-263.
  • 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).
  • target sequence an endogenous gene
  • the 22-nucleotide sequence is selected from a RCA ⁇ -isoform transcript sequence and contains 22 nucleotides of said RCA ⁇ -isoform 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.
  • the miRNA would target the sequences of the exon that encodes the carboxy terminal redox-sensitive cysteines for any plant's RCA -isoform.
  • the polynucleotide encodes a zinc finger protein that binds to a gene encoding a RCA ⁇ -isoform polypeptide, resulting in reduced expression of the gene.
  • the zinc finger protein binds to a regulatory region of a RCA ⁇ -isoform gene.
  • the zinc finger protein binds to a messenger RNA encoding a RCA ⁇ -isoform polypeptide and prevents its translation.
  • TALE and dCas9 or dCfpl could also be used, as well as any DNA binding domain that could bind the promoter. If a repressor protein, such as KRAB or 3xSRDX, is fused to any of these DNA binding domains, then repression is enhanced.
  • a repressor protein such as KRAB or 3xSRDX
  • the polynucleotide encodes an antibody that binds to at least one RCA ⁇ -isoform polypeptide, and reduces the activity of the RCA ⁇ -isoform polypeptide.
  • the binding of the antibody results in increased turnover of the antibody-RCA a- isoform complex by cellular quality control mechanisms.
  • the activity of a RCA ⁇ -isoform polypeptide may be reduced or eliminated by disrupting the gene encoding the RCA ⁇ -isoform polypeptide.
  • the gene sequence encoding the RCA a-isoform polypeptide 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 plants using random or targeted mutagenesis, and selecting for plants that have desired traits.
  • transposon tagging is used to reduce or eliminate the RCA ⁇ -isoform activity of one or more RCA ⁇ -isoform polypeptides.
  • Transposon tagging comprises inserting a transposon within an endogenous RCA ⁇ -isoform gene to reduce or eliminate expression of the RCA a- isoform polypeptide.
  • "RuBisCO activase alpha-isoform gene” and RCA ⁇ -isoform gene” is intended to mean the gene that encodes a RCA ⁇ -isoform polypeptide.
  • the expression of one or more RCA a-isoform polypeptides is reduced or eliminated by inserting a transposon within a regulatory region or coding region of the gene encoding the RCA ⁇ -isoform polypeptide.
  • a transposon that is within an exon, intron, 5' or 3' untranslated sequence, a promoter, or any other regulatory sequence of a RCA ⁇ -isoform gene may be used to reduce or eliminate the expression and/or activity of the encoded RCA ⁇ -isoform polypeptide.
  • the transposon would target exon 7b of the RCA gene in rice.
  • the transposon would target the exon that encodes the carboxy terminal redox-sensitive cysteines for that plant's RCA a-isoform.
  • one would have an active or activated DNA transposon in the area of rice's RCA gene's exon 7b. The DNA transposon can then move into exon 7b.
  • 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.
  • one or more or all of the genetic modifications is a mutation of an endogenous gene which partially or completely inactivates the gene, such as a point mutation, an insertion, or a deletion (or a combination of one or more thereof).
  • a genetic modification is usually a mutation introduced to the genome.
  • the point mutation may be a premature stop codon, a splice site mutation, a frame shift mutation or an amino acid substitution mutation that reduces activity of the gene or the encoded polypeptide.
  • the deletion may be of one or more nucleotides within a transcribed exon or promoter of the gene, or extend across or into more than one exon, or extend to deletion of the entire gene.
  • the deletion is introduced by use of ZF, TALEN or CRISPR technologies.
  • one or more or all of the genetic modifications is an exogenous polynucleotide encoding an RNA molecule which inhibits expression of the endogenous gene, wherein the exogenous polynucleotide is operably linked to a promoter which is capable of directing expression of the polynucleotide in the plant, or part thereof.
  • Genome editing uses engineered nucleases such as RNA guided DNA endonucleases or nucleases composed of sequence specific DNA binding domains fused to a non-specific DNA cleavage module.
  • engineered nucleases enable efficient and precise genetic modifications by inducing targeted DNA double stranded breaks that stimulate the cell's endogenous cellular DNA repair mechanisms to repair the induced break.
  • Such mechanisms include, for example, error prone non-homologous end joining (NHEJ) and homology directed repair (HDR).
  • HDR can lead to the introduction of single or multiple transgenes to correct or replace existing genes.
  • NHEJ-mediated repair yields small insertion or deletion mutations of the target that cause gene disruption.
  • Engineered nucleases useful in the methods of the present invention include zinc finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALEN) and CRISPR/Cas9 type nucleases.
  • ZFNs zinc finger nucleases
  • TALEN transcription activator-like effector nucleases
  • CRISPR/Cas9 type nucleases typically nuclease encoded genes are delivered into cells by plasmid DNA, viral vectors or in vitro transcribed mRNA.
  • a zinc finger nuclease comprises a DNA-binding domain and a DNA-cleavage domain, wherein the DNA binding domain is comprised of at least one zinc finger and is operatively linked to a DNA-cleavage domain.
  • the zinc finger DNA-binding domain is at the N-terminus of the protein and the DNA-cleavage domain is located at the C-terminus of said protein.
  • a ZFN must have at least one zinc finger.
  • a ZFN would have at least three zinc fingers in order to have sufficient specificity to be useful for targeted genetic recombination in a host cell or organism.
  • a ZFN having more than three zinc fingers would have progressively greater specificity with each additional zinc finger.
  • the zinc finger domain can be derived from any class or type of zinc finger.
  • the zinc finger domain comprises the Cis2His2 type of zinc finger that is very generally represented, for example, by the zinc finger transcription factors TFIIIA or Spl.
  • the zinc finger domain comprises three Cis2His2 type zinc fingers.
  • the DNA recognition and/or the binding specificity of a ZFN can be altered in order to accomplish targeted genetic recombination at any chosen site in cellular DNA. Such modification can be accomplished using known molecular biology and/or chemical synthesis techniques. (See, for example, Bibikova et al., 2002).
  • the ZFN DNA-cleavage domain is derived from a class of non-specific DNA cleavage domains, for example the DNA-cleavage domain of a Type II restriction enzyme such as Fold (Kim et al., 1996).
  • Other useful endonucleases may include, for example, Hhal, Hindlll, Nod, BbvCI, EcoRI, Bgll, and AlwI.
  • a transcription activator-like (TAL) effector nuclease comprises a TAL effector DNA binding domain and an endonuclease domain.
  • TAL effectors are proteins of plant pathogenic bacteria that are injected by the pathogen into the plant cell, where they travel to the nucleus and function as transcription factors to turn on specific plant genes.
  • the primary amino acid sequence of a TAL effector dictates the nucleotide sequence to which it binds.
  • target sites can be predicted for TAL effectors, and TAL effectors can be engineered and generated for the purpose of binding to particular nucleotide sequences.
  • Fused to the TAL effector-encoding nucleic acid sequences are sequences encoding a nuclease or a portion of a nuclease, typically a nonspecific cleavage domain from a type II restriction endonuclease such as Fokl (Kim et al., 1996).
  • Other useful endonucleases may include, for example, Hhal, Hindlll, Nod, BbvCI, EcoRI, Bgll, and Ahvl. The fact that some endonucleases (e.g., Fokl) only function as dimers can be capitalized upon to enhance the target specificity of the TAL effector.
  • each Fokl monomer can be fused to a TAL effector sequence that recognizes a different DNA target sequence, and only when the two recognition sites are in close proximity do the inactive monomers come together to create a functional enzyme.
  • a highly site-specific restriction enzyme can be created.
  • a sequence-specific TALEN can recognize a particular sequence within a preselected target nucleotide sequence present in a cell.
  • a target nucleotide sequence can be scanned for nuclease recognition sites, and a particular nuclease can be selected based on the target sequence.
  • a TALEN can be engineered to target a particular cellular sequence.
  • CRISPR clustered regulatory interspaced short palindromic repeats
  • CRISPR systems rely on CRISPR RNA (crRNA) and transactivating chimeric RNA (tracrRNA) for sequence-specific cleavage of DNA.
  • CRISPR RNA CRISPR RNA
  • tracrRNA transactivating chimeric RNA
  • Cas9 serves as an RNA-guided DNA endonuclease that cleaves DNA upon crRNA-tracrRNA target recognition.
  • CRISPR RNA base pairs with tracrRNA to form a two-RNA structure that guides the Cas9 endonuclease to complementary DNA sites for cleavage.
  • the CRISPR system can be portable to plant cells by co-delivery of plasmids expressing the Cas endonuclease and the necessary crRNA components.
  • the Cas endonuclease may be converted into a nickase to provide additional control over the mechanism of DNA repair (Cong, et ai, Science
  • CRISPRs are typically short partially palindromic sequences of 24-40 bp containing inner and terminal inverted repeats of up to 11 bp. Although isolated elements have been detected, they are generally arranged in clusters (up to about 20 or more per genome) of repeated units spaced by unique intervening 20-58 bp sequences. CRISPRs are generally homogenous within a given genome with most of them being identical. However, there are examples of heterogeneity in, for example, the Archaea (Mojica, et ai, Mol. Microbiol. 36(l):244-6 2000).
  • indels of the RCA a-isoform gene have been created. These genomic mutations have been shown to reduce accumulation of the alpha-isoform and hence concomitant low light inhibition of RuBisCO activase.
  • the indels are made in the area surrounding and including exon 7, which encodes the carboxy-end extension of the alpha spliced version of the RCA protein in rice.
  • genomic changes in and around exon 7 include indels that result in the production of an altered RCA ⁇ -isoform protein having an amino acid sequence at the carboxy end of the protein being one or more of the sequences in SEQ ID NOs: 4, 6, 8, 10, 12, 15, 17, 19, 22, 24, 27, and/or 29.
  • the invention includes polynucleotide sequences which encode part or all of altered RCA a indel proteins
  • the polynucleotide sequences encode these specific amino acid sequences listed in the above mentioned SEQ ID NOs.
  • the invention also includes modified RCA proteins and their variants.
  • the invention also includes vectors, plant cells, tissues, plant lines, varieties, and hybrids which include the same as well as their use in breeding.
  • TILLING or “Targeting Induced Local Lesions IN Genomics” refers to a mutagenesis technology useful to generate and/or identify and to eventually isolate mutagenised variants of a particular nucleic acid with modulated expression and/or activity (McCallum, et al, (2000), Plant Physiology 123:439-442; McCallum, et al, (2000) Nature Biotechnology 18:455-457 and Colbert, et al, (2001) Plant Physiology 126:480-484).
  • TILLING combines high density point mutations with rapid sensitive detection of the mutations.
  • EMS ethylmethanesulfonate
  • Ml ethylmethanesulfonate
  • TILLING also allows selection of plants carrying mutant variants. These mutant variants may exhibit modified expression, either in strength or in location or in timing (if the mutations affect the promoter, for example). These mutant variants may exhibit higher or lower activity than that exhibited by the gene in its natural form.
  • TILLING combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei and Koncz, (1992) In Methods in Arabidopsis Research, Koncz, et al., eds. Singapore, World Scientific Publishing Co, pp. 16-82; Feldmann, et al., (1994) In Arabidopsis.
  • mutagenic methods can also be employed to introduce mutations in a disclosed gene.
  • Methods for introducing genetic mutations into plant genes and selecting plants with desired traits are well known.
  • seeds or other plant material can be treated with a mutagenic chemical substance, according to standard techniques.
  • chemical substances include, but are not limited to, the following: diethyl sulfate, ethylene imine, and N-nitroso-N-ethylurea.
  • ionizing radiation from sources such as X-rays or gamma rays can be used.
  • 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.
  • 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.
  • 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 activity of the encoded protein. conserveed residues of plant RCA a-isoform polypeptides suitable for mutagenesis with the goal to eliminate RCA a- isoform activity have been described. Such mutants can be isolated according to well-known procedures, and mutations in different RCA ⁇ -isoform loci can be stacked by genetic crossing. See, for example, Gruis, et al, (2002) Plant Cell 14:2863-2882.
  • 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 RCA ⁇ -isoform polypeptides.
  • methods for altering or mutating a genomic nucleotide sequence in a plant include, but are not limited to, the use of R A:DNA vectors, R A:DNA mutational vectors, R A:DNA repair vectors, mixed-duplex oligonucleotides, self-complementary R A:DNA oligonucleotides, and recombinogenic
  • oligonucleobases Such vectors and methods of use are known in the art. See, for example, U.S. Patent Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984. See also, WO 98/49350, WO 99/07865, WO 99/25821, and Beetham, et al, (1999) Proc. Natl. Acad. Sci. USA 96:8774-8778. Plant Transformation Methods
  • Numerous methods for introducing foreign genes into plants are known and can be used to insert a RCA a-isoform polynucleotide into a plant host, including biological and physical plant transformation protocols. See, e.g., Miki et al., "Procedure for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). The methods chosen vary with the host plant, and include chemical transfection methods such as calcium phosphate, microorganism-mediated gene transfer such as Agrobacterium
  • the isolated polynucleotides or polypeptides may be introduced into the plant by one or more techniques typically used for direct delivery into cells. Such protocols may vary depending on the type of organism, cell, plant or plant cell, i.e. monocot or dicot, targeted for gene modification. Suitable methods of transforming plant cells include microinjection (Crossway, et al, (1986) Biotechniques 4:320-334; and U.S. Patent 6,300,543), electroporation (Riggs, et al, (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, direct gene transfer (Paszkowski et al, (1984) EMBO J.
  • Agrobacterium mediated maize transformation U.S. Patent 5,981,840
  • silicon carbide whisker methods Fine, et al, (1994) Plant J. 6:941-948
  • laser methods Guo, et al, (1995) Physiologia Plantarum 93: 19-24
  • sonication methods Boo, et al, (1997) Ultrasound in Medicine & Biology 23:953-959; Finer and Finer, (2000) Lett Appl Microbiol. 30:406-10; Amoah, et al, (2001) J Exp Bot 52: 1135-42);
  • A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria, which genetically transform plant cells.
  • the Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of plants. See, e.g., Kado, (1991) Crit. Rev. Plant Sci. 10: 1. Descriptions of the Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided in Gruber, et al, supra; Miki, et al, supra; and Moloney, et al. , ( 1989) Plant Cell Reports 8 :238.
  • the gene can be inserted into the T-DNA region of a Ti or Ri plasmid derived from A. tumefaciens or A. rhizogenes, respectively.
  • expression cassettes can be constructed as above, using these plasmids.
  • Many control sequences are known which when coupled to a heterologous coding sequence and transformed into a host organism show fidelity in gene expression with respect to tissue/organ specificity of the original coding sequence. See, e.g., Benfey and Chua, (1989) Science 244: 174-81.
  • Particularly suitable control sequences for use in these plasmids are promoters for constitutive leaf-specific expression of the gene in the various target plants.
  • NOS nopaline synthase gene
  • the NOS promoter and terminator are present in the plasmid pARC2, available from the American Type Culture Collection and designated ATCC 67238. If such a system is used, the virulence ⁇ vir) gene from either the Ti or Ri plasmid must also be present, either along with the T-DNA portion, or via a binary system where the vir gene is present on a separate vector.
  • Such systems, vectors for use therein, and methods of transforming plant cells are described in U.S. Patent No. 4,658,082; U.S. Patent Application No. 913,914, filed Oct. 1, 1986, as referenced in U.S. Patent No. 5,262,306; and Simpson, et al, (1986) Plant Mol. Biol. 6:403-15 (also referenced in the '306 patent).
  • these plasmids can be placed into A. rhizogenes or A. tumefaciens and these vectors used to transform cells of plant species, which are ordinarily susceptible to Fusarium or
  • Alternaria infection Several other transgenic plants are also contemplated by the present invention including but not limited to soybean, corn, sorghum, alfalfa, rice, clover, cabbage, banana, coffee, celery, tobacco, cowpea, cotton, melon and pepper.
  • A. tumefaciens or A. rhizogenes will depend on the plant being transformed thereby.
  • A. tumefaciens is an useful organism for transformation. Most dicotyledonous plants, some gymnosperms, and a few monocotyledonous plants (e.g., certain members of the Liliales and Arales) are susceptible to infection with A. tumefaciens.
  • A. tumefaciens is an useful organism for transformation. Most dicotyledonous plants, some gymnosperms, and a few monocotyledonous plants (e.g., certain members of the Liliales and Arales) are susceptible to infection with A. tumefaciens
  • rhizogenes also has a wide host range, embracing most dicots and some gymnosperms, which includes members of the Leguminosae, Compositae, and Chenopodiaceae. Monocot plants can now be transformed with some success.
  • European Patent Application No. 604 662 Al discloses a method for transforming monocots using Agrobacterium.
  • European Application No. 672 752 Al discloses a method for transforming monocots with Agrobacterium using the scutellum of immature embryos. Ishida, et al. , discuss a method for transforming maize by exposing immature embryos to A. tumefaciens (Nature Biotechnology 14:745-50 (1996)).
  • these cells can be used to regenerate transgenic plants.
  • whole plants can be infected with these vectors by wounding the plant and then introducing the vector into the wound site. Any part of the plant can be wounded, including leaves, stems and roots.
  • plant tissue in the form of an explant, such as cotyledonary tissue or leaf disks, can be inoculated with these vectors, and cultured under conditions, which promote plant regeneration. Roots or shoots transformed by inoculation of plant tissue with A. rhizogenes or A.
  • tumefaciens containing the gene coding for the fumonisin degradation enzyme, can be used as a source of plant tissue to regenerate fumonisin-resistant transgenic plants, either via somatic embryogenesis or organogenesis. Examples of such methods for regenerating plant tissue are disclosed in Shahin, (1985) Theor. Appl. Genet. 69:235-40; U.S. Patent No. 4,658,082; Simpson, et al., supra; and U.S. Patent Application Nos. 913,913 and 913,914, both filed Oct. 1, 1986, as referenced in U.S. Patent No. 5,262,306.
  • Agrobacterium- ediated transformation Despite the fact that the host range for Agrobacterium- ediated transformation is broad, some major cereal crop species and gymnosperms have generally been recalcitrant to this mode of gene transfer, even though some success has recently been achieved in rice (Hiei, et al., (1994) The Plant Journal 6:271-82).
  • Several methods of plant transformation collectively referred to as direct gene transfer, have been developed as an alternative to Agrobacterium- ediated transformation.
  • a generally applicable method of plant transformation is microprojectile-mediated transformation, where DNA is carried on the surface of microprojectiles measuring about 1 to 4 ⁇ .
  • the expression vector is introduced into plant tissues with a biolistic device that accelerates the
  • Another method for physical delivery of DNA to plants is sonication of target cells as described in Zang, et al., (1991) BioTechnology 9:996.
  • liposome or spheroplast fusions have been used to introduce expression vectors into plants. See, e.g., Deshayes, et al., (1985) EMBO J. 4:2731 ; and Christou, et al., (1987) Proc. Natl. Acad. Sci. USA 84:3962.
  • Direct uptake of DNA into protoplasts using CaCb precipitation, polyvinyl alcohol, or poly-L-ornithine has also been reported. See, e.g., Hain, et al., (1985) Mol. Gen. Genet. 199: 161 ; and Draper, et al., (1982) Plant Cell Physiol. 23:451.
  • 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.
  • nucleic acid sequences of the present invention can be used in combination ("stacked") with other polynucleotide sequences of interest in order to create plants with a desired phenotype.
  • the combinations generated can include multiple copies of any one or more of the polynucleotides of interest.
  • the polynucleotides of the present invention may be stacked with any gene or combination of genes to produce plants with a variety of desired trait combinations, including but not limited to traits desirable for animal feed such as high oil genes (e.g., U.S. Patent No. 6,232,529);
  • balanced amino acids e.g., hordothionins (U.S. Patent Nos. 5,990,389; 5,885,801 ; 5,885,802; and 5,703,049); barley high lysine (Williamson, et al , (1987) Eur. J. Biochem. 165:99-106; and WO
  • polynucleotides of the present invention can also be stacked with traits desirable for insect, disease or herbicide resistance (e.g., Bacillus thuringiensis toxic proteins (U.S. Patent Nos.
  • ALS acetolactate synthase
  • ALS acetolactate synthase
  • inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene); and glyphosate resistance (EPSPS gene)
  • traits desirable for processing or process products such as high oil (e.g., U.S. Patent No. 6,232,529 ); modified oils (e.g., fatty acid desaturase genes (U.S. Patent No.
  • modified starches e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)
  • polymers or bioplastics e.g., U.S. Patent No. 5.602,321 ; ⁇ - ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert, et al. , (1988) J. Bacteriol. 170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)).
  • polynucleotides of the present invention could also combine with polynucleotides affecting agronomic traits such as male sterility (e.g., see U.S. Patent 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).
  • agronomic traits such as male sterility (e.g., see U.S. Patent 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).
  • sequences of interest improve plant growth and/or crop yields.
  • sequences of interest include agronomically important genes that result in improved primary or lateral root systems.
  • genes include, but are not limited to, nutrient/water transporters and growth induces.
  • genes include but are not limited to, maize plasma membrane H + -ATPase (MHA2) (Frias, et al.
  • AKT1 a component of the potassium uptake apparatus in Arabidopsis, (Spalding, et al , (1999) J Gen Physiol 113:909-18); RML genes which activate cell division cycle in the root apical cells (Cheng, et al , (1995) Plant Physiol 108:881); maize glutamine synthetase genes (Sukanya, et al , (1994) Plant Mol Biol 26: 1935-46) and hemoglobin (Duff, et al , (1997) J. Biol. Chem 27: 16749-16752, Arredondo-Peter, et al , (1997) Plant Physiol.
  • sequence of interest may also be useful in expressing antisense nucleotide sequences of genes that that negatively affects root development. Methods of Use for RCA a-isoform polynucleotide, expression cassettes, and additional polynucleotides
  • nucleotides, expression cassettes and methods disclosed herein are useful in modulating RCA activity and thus RuBisCO activity.
  • the increase in RuBisCO activity alters the plant's phenotype, as discussed above and in the examples below.
  • the present invention provides, inter alia, isolated nucleic acids of RNA, DNA, homologs, paralogs and orthologs and/or chimeras thereof, comprising a RCA a-isoform polynucleotide. This includes naturally occurring as well as synthetic variants and homologs of the sequences.
  • homologous sequences can be derived from any plant including monocots and dicots and in particular agriculturally important plant species, including but not limited to, crops such as soybean, wheat, corn (maize), potato, cotton, rape, oilseed rape (including canola), sunflower, alfalfa, clover, sugarcane, and turf; or fruits and vegetables, such as banana, blackberry, blueberry, strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn, tobacco, tomato, tomatillo, watermelon, rosaceous fruits (such as apple, peach, pear, cherry and plum) and vegetable brassicas (such as broccoli, cabbage
  • Other crops including fruits and vegetables, whose phenotype can be changed and which comprise homologous sequences include barley; rye; millet; sorghum; currant; avocado; citrus fruits such as oranges, lemons, grapefruit and tangerines, artichoke, cherries; nuts such as the walnut and peanut; endive; leek; roots such as arrowroot, beet, cassava, turnip, radish, yam, and sweet potato; and beans.
  • the homologous sequences may also be derived from woody species, such pine, poplar and eucalyptus, or mint or other labiates.
  • homologous sequences may be derived from plants that are evolutionarily-related to crop plants, but which may not have yet been used as crop plants. Examples include deadly nightshade (Atropa belladona), related to tomato; jimson weed ⁇ Datura strommium), related to peyote; and teosinte (Zea species), related to corn (maize). See FIG. 21 for the homologous carboxy terminal RCA ⁇ -isoform amino acid sequences for various economically important plants.
  • Homologous sequences as described above can comprise orthologous or paralogous sequences.
  • orthologs and paralogs are evolutionarily related genes that have similar sequence and similar functions. Orthologs are structurally related genes in different species that are derived by a speciation event. Paralogs are structurally related genes within a single species that are derived by a duplication event.
  • gene duplication may result in two copies of a particular gene, giving rise to two or more genes with similar sequence and often similar function known as paralogs.
  • a paralog is therefore a similar gene formed by duplication within the same species.
  • Paralogs typically cluster together or in the same clade (a group of similar genes) when a gene family phylogeny is analyzed using programs such as CLUSTAL (Thompson et al. (1994) Nucleic Acids Res. 22: 4673-4680; Higgins et al. (1996) Methods Enzymol. 266: 383-402). Groups of similar genes can also be identified with pair- wise BLAST analysis (Feng and Doolittle (1987) J. Mol. Evol. 25: 351-360).
  • Orthologous genes from different organisms have highly conserved functions, and very often essentially identical functions (Lee et al. (2002) Genome Res. 12: 493-502; Remm et al. (2001) J. Mol. Biol. 314: 1041-1052). Paralogous genes, which have diverged through gene duplication, may retain similar functions of the encoded proteins. In such cases, paralogs can be used interchangeably with respect to certain embodiments of the instant invention (for example, transgenic expression of a coding sequence).
  • the RCA a-isoform nucleotide sequences are used to generate variant nucleotide sequences having the nucleotide sequence of the 5'-untranslated region, 3 '-untranslated region, or promoter region that is approximately 70%, 75%, and 80%, 85%, 90% and 95% identical to the original nucleotide sequence. These variants are then associated with natural variation in the germplasm for component traits. The associated variants are used as marker haplotypes to select for the desirable traits.
  • Variant amino acid sequences of the RCA a-isoform polypeptides are generated.
  • one amino acid is altered.
  • the open reading frames are reviewed to determine the appropriate amino acid alteration.
  • the selection of the amino acid to change is made by consulting the protein alignment (with the other orthologs and other gene family members from various species).
  • 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).
  • an appropriate amino acid can be changed. Once the targeted amino acid is identified, the procedure outlined herein is followed.
  • Variants having about 70%, 75%, 80%, 85%, 90% and 95% nucleic acid sequence identity are generated using this method.
  • the associated variants are used as marker haplotypes to select for the desirable traits.
  • the present invention also includes polynucleotides optimized for expression in different organisms. For example, for expression of the polynucleotide in a particular plant, the sequence can be altered to account for specific codon.
  • the isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques, or combinations thereof.
  • the polynucleotides of the present invention will be cloned, amplified, or otherwise constructed from a fungus or bacteria.
  • the nucleic acids may conveniently comprise sequences in addition to a polynucleotide of the present invention.
  • a multi-cloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in isolation of the polynucleotide.
  • translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention.
  • a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention.
  • the nucleic acid of the present invention - excluding the polynucleotide sequence - is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention. Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell.
  • the length of a nucleic acid of the present invention less the length of its polynucleotide of the present invention is less than 20 kilobase pairs, often less than 15 kb, and frequently less than 10 kb.
  • nucleic acids include such vectors as: M13, lambda ZAP Express, lambda ZAP II, lambda gtlO, lambda gtl 1, pBK-CMV, pBK-RSV, pBluescript II, lambda DASH II, lambda EMBL 3, lambda EMBL 4, pWE15, SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS+/-, pSG5, pBK, pCR-Script, pET, pSPUTK, p3'SS, pGEM, pSK+/-, pGEX, pSPORTI and II, pOPRSVI CAT, pOPI3 CAT, pXTl, pSG5, pPbac, pMbac, pMClneo, pOG44, pOG45,
  • Optional vectors for the present invention include but are not limited to, lambda ZAP II, and pGEX.
  • pGEX a description of various nucleic acids see, e.g., Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, CA); and, Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, IL).
  • the isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang, et al., (1979) Meth. Enzymol.
  • This may be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
  • a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
  • RNA Ribonucleic Acids Res. 13:7375.
  • Positive sequence motifs include translational initiation consensus sequences (Kozak, (1987) Nucleic Acids Res.15:8125) and the 5 ⁇ G> 7 methyl GpppG RNA cap structure (Drummond, et al., (1985) Nucleic Acids Res. 13:7375).
  • Negative elements include stable intramolecular 5' UTR stem-loop structures (Muesing, et al, (1987) Cell 48:691) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5' UTR (Kozak, supra, Rao, et al, (1988) Mol. and Cell. Biol. 8:284). Accordingly, the present invention provides 5' and/or 3' UTR regions for modulation of translation of heterologous coding sequences.
  • polypeptide-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage.
  • Altered codon usage can be employed to alter translational efficiency and/or to optimize the coding sequence for expression in a desired host or to optimize the codon usage in a heterologous sequence for expression in rice.
  • Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as "Codon Preference" available from the University of Wisconsin Genetics Computer Group. See, Devereaux, et al, (1984) Nucleic Acids Res. 12:387-395); or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.).
  • the present invention provides a codon usage frequency characteristic of the coding region of at least one of the polynucleotides of the present invention.
  • the number of polynucleotides (3 nucleotides per amino acid) that can be used to determine a codon usage frequency can be any integer from 3 to the number of polynucleotides of the present invention as provided herein.
  • the polynucleotides will be full-length sequences.
  • An exemplary number of sequences for statistical analysis can be at least 1, 5, 10, 20, 50 or 100.
  • the present invention provides methods for sequence shuffling using polynucleotides of the present invention, and compositions resulting therefrom. Sequence shuffling is described in PCT
  • sequence shuffling provides a means for generating libraries of polynucleotides having a desired characteristic, which can be selected or screened for.
  • Libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides, which comprise sequence regions, which have substantial sequence identity and can be homologously recombined in vitro or in vivo.
  • the population of sequence-recombined polynucleotides comprises a subpopulation of polynucleotides which possess desired or advantageous characteristics and which can be selected by a suitable selection or screening method.
  • the characteristics can be any property or attribute capable of being selected for or detected in a screening system, and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription, RNA processing, RNA stability, chromatin conformation, translation, or other expression property of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any feature which confers a selectable or detectable property.
  • the selected characteristic will be an altered K m and/or Kcat over the wild-type protein as provided herein.
  • a protein or polynucleotide generated from sequence shuffling will have a ligand binding affinity greater than the non-shuffled wild- type polynucleotide.
  • a protein or polynucleotide generated from sequence shuffling will have an altered pH optimum as compared to the non-shuffled wild-type polynucleotide. The increase in such properties can be at least 110%, 120%, 130%, 140% or greater than 150% of the wild- type value.
  • the present disclosure further provides recombinant expression cassettes comprising a nucleic acid of the present disclosure, which is designed for reducing the activity of RCA a.
  • a nucleic acid sequence coding for the desired polynucleotide of the present disclosure for example a cDNA or a genomic sequence encoding a polypeptide long enough to code for an active protein of the present disclosure, can be used to construct a recombinant expression cassette which can be introduced into the desired host cell.
  • a recombinant expression cassette will typically comprise a polynucleotide of the present disclosure operably linked to transcriptional initiation regulatory sequences which will direct the transcription of the polynucleotide in the intended host cell, such as tissues of a transformed plant.
  • plant expression vectors may include (1) a cloned plant gene under the
  • Such plant expression vectors may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue- specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site and/or a polyadenylation signal.
  • a promoter regulatory region e.g., one conferring inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue- specific/selective expression
  • a transcription initiation start site e.g., one conferring inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue- specific/selective expression
  • a transcription initiation start site e.g., one conferring inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue- specific/selective expression
  • RNA processing signal e.g., RNA processing signal
  • a plant promoter fragment can be employed which will direct expression of a polynucleotide of the present disclosure in essentially all tissues of a regenerated plant.
  • Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation.
  • Examples of constitutive promoters include the ⁇ - or 2'-promoter derived from T-DNA of Agrobacterium tumefaciens, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No.
  • the Nos promoter the RuBisCO promoter, the GRP1- 8 promoter, the 35S promoter from cauliflower mosaic virus (CaMV), as described in Odell, et al., (1985) Nature 313:810-2; rice actin (McElroy, et al, (1990) Plant Cell 163-171); ubiquitin (Christensen, et al, (1992) Plant Mol. Biol. 12:619-632 and Christensen, et al, (1992) Plant Mol. Biol. 18:675-89); pEMU (Last, et al, (1991) Theor. Appl. Genet.
  • CaMV cauliflower mosaic virus
  • MAS MAS
  • H3 histone MAS
  • ALS promoter as described in WO 1996/30530 and other transcription initiation regions from various plant genes known to those of skill.
  • ubiquitin is one useful promoter for expression in monocot plants.
  • the plant promoter can direct expression of a polynucleotide of the present disclosure in a specific tissue or may be otherwise under more precise environmental or developmental control.
  • Such promoters may be "inducible" promoters.
  • Environmental conditions that may affect transcription by inducible promoters include pathogen attack, anaerobic conditions or the presence of light.
  • inducible promoters are the Adhl promoter, which is inducible by hypoxia or cold stress, the Hsp70 promoter, which is inducible by heat stress and the PPDK promoter, which is inducible by light.
  • Diurnal promoters that are active at different times during the circadian rhythm are also known (U.S. Patent Application Publication No. 2011/0167517).
  • promoters under developmental control include promoters that initiate transcription only, or preferentially, in certain tissues, such as leaves, roots, fruit, seeds or flowers (tissue-specific promoters).
  • tissue-specific promoters The operation of a promoter may also vary depending on its location in the genome. Thus, an inducible promoter may become fully or partially constitutive in certain locations.
  • polypeptide expression it is generally desirable to include a polyadenylation region at the 3 '-end of a polynucleotide coding region.
  • the polyadenylation region can be derived from a variety of plant genes, or from T-DNA.
  • the 3' end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or alternatively from any other eukaryotic gene.
  • regulatory elements include, but are not limited to, 3' termination and/or polyadenylation regions such as those of the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan, et al., (1983) Nucleic Acids Res. 12:369-85); the potato proteinase inhibitor II (PINII) gene (Keil, et al, (1986) Nucleic Acids Res. 14:5641-50 and An, et al, (1989) Plant Cell 1: 115- 22) and the CaMV 19S gene (Mogen, et al, (1990) Plant Cell 2: 1261-72).
  • PINII potato proteinase inhibitor II
  • An intron sequence can be added to the 5' untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol.
  • Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg, (1988) Mol. Cell Biol. 8:4395-4405; Callis, et al, (1987) Genes Dev. 1 : 1183-200).
  • Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit.
  • Use of maize introns Adhl-S intron 1, 2 and 6, the Bronze- 1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, eds., Springer, New York (1994).
  • Plant signal sequences including, but not limited to, signal-peptide encoding DNA/RNA sequences which target proteins to the extracellular matrix of the plant cell (Dratewka-Kos, et al., (1989) J. Biol. Chem. 264:4896-900), such as the Nicotiana plumbaginifolia extension gene (DeLoose, et al., (1991) Gene 99:95-100); signal peptides which target proteins to the vacuole, such as the sweet potato sporamin gene (Matsuka, et al., (1991) Proc. Natl. Acad. Sci.
  • the vector comprising the sequences from a polynucleotide of the present disclosure will typically comprise a marker gene, which confers a selectable phenotype on plant cells.
  • the selectable marker gene may encode antibiotic resistance, with suitable genes including genes coding for resistance to the antibiotic spectinomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance.
  • SPT streptomycin phosphotransferase
  • NPTII neomycin phosphotransferase
  • HPT hygromycin phosphotransferase
  • the bar gene encodes resistance to the herbicide basta and the ALS gene encodes resistance to the herbicide chlorsulfuron.
  • Constructs described herein may comprise a polynucleotide of interest encoding a reporter or marker product.
  • suitable reporter polynucleotides known in the art can be found in, for example, Jefferson, et al., (1991) in Plant Molecular Biology Manual, ed. Gelvin, et al., (Kluwer Academic Publishers), pp. 1-33; DeWet, et al. (1987) Mol. Cell. Biol. 7:725-737; Goff, et al, (1990) EMBO J. 9:2517-2522; Kain, et al, (1995) Bio Techniques 19:650-655 and Chiu, et al, (1996) Current Biology 6:325-330.
  • the polynucleotide of interest encodes a selectable reporter.
  • selectable reporter can include polynucleotides that confer antibiotic resistance or resistance to herbicides.
  • suitable selectable marker polynucleotides include, but are not limited to, genes encoding resistance to chloramphenicol, methotrexate, hygromycin, streptomycin, spectinomycin, bleomycin, sulfonamide, bromoxynil, glyphosate and phosphinothricin.
  • the expression cassettes disclosed herein comprise a polynucleotide of interest encoding scorable or screenable markers, where presence of the polynucleotide produces a measurable product.
  • examples include a ⁇ -glucuronidase, or uidA gene (GUS), which encodes an enzyme for which various chromogenic substrates are known (for example, U.S. Pat. Nos. 5,268,463 and 5,599,670); chloramphenicol acetyl transferase and alkaline phosphatase.
  • anthocyanin/flavonoid polynucleotides including, for example, a R-locus polynucleotide, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues, the genes which control biosynthesis of flavonoid pigments, such as the maize CI and C2, the B gene, the pi gene and the bronze locus genes, among others.
  • markers encoded by polynucleotides of interest include the cyan fluorescent protein (CYP) gene, the yellow fluorescent protein gene, a lux gene, which encodes a luciferase, the presence of which may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry, a green fluorescent protein (GFP) and
  • DsRed2 (Clontechniques, 2001) where plant cells transformed with the marker gene are red in color, and thus visually selectable.
  • Additional examples include a p-lactamase gene encoding an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin), a xylE gene encoding a catechol dioxygenase that can convert chromogenic catechols, an a-amylase gene and a tyrosinase gene encoding an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form the easily detectable compound melanin.
  • PADAC chromogenic cephalosporin
  • a xylE gene encoding a catechol dioxygenase that can convert chromogenic catechols
  • an a-amylase gene e.g., a tyrosinase gene
  • 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 ⁇ - 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
  • GFP green fluorescent protein
  • selectable marker genes are not meant to be limiting. Any selectable marker gene can be used in the compositions and methods disclosed herein.
  • Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers, et al., (1987) Meth. Enzymol. 153:253-77. These vectors are plant integrating vectors in that on transformation, the vectors integrate a portion of vector DNA into the genome of the host plant.
  • Ti tumor-inducing
  • Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl, et al., (1987) Gene 61 : 1-11 and Berger, et al, (1989) Proc. Natl. Acad. Sci. USA, 86:8402-6.
  • Another useful vector herein is plasmid pBHOl .2 that is available from CLONTECH Laboratories, Inc. (Palo Alto, Calif).
  • Prokaryotic cells may be used as hosts for expression. Prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the ⁇ -lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et ai, (1977) Nature 198: 1056), the tryptophan (trp) promoter system (Goeddel, et ai, (1980) Nucleic Acids Res.
  • promoters for transcription initiation optionally with an operator, along with ribosome binding site sequences
  • promoters include such commonly used promoters as the ⁇ -lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et ai,
  • Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA. Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva, et ai, (1983) Gene
  • the pGEX-4T-l plasmid vector from Pharmacia is one E. coli expression vector for the present invention.
  • eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art. As explained briefly below, the present invention can be expressed in these eukaryotic systems. In some embodiments, transformed/transfected plant cells, as discussed infra, are employed as expression systems for production of the proteins of the instant invention.
  • Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g.,
  • Suitable vectors usually have expression control sequences, such as promoters, including 3- phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.
  • a protein of the present invention once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lysates or the pellets.
  • the monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.
  • the sequences encoding proteins of the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used.
  • a number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21, and CHO cell lines.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HAS tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen, et al., (1986) Immunol. Rev. 89:49), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences.
  • Other animal cells useful for production of proteins of the present invention are available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (7 th ed., 1992).
  • Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus.
  • suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth, and Drosophila cell lines such as a Schneider cell line (see, e.g., Schneider, (1987) J. Embryol. Exp. Morphol. 27:353-65).
  • polyadenlyation or transcription terminator sequences are typically incorporated into the vector.
  • An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included.
  • An example of a splicing sequence is the VP1 intron from SV40 (Sprague et al., J. Virol. 45:773-81 (1983)).
  • gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type- vectors (Saveria-Campo, "Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector," in DNA Cloning: A Practical Approach, vol. II, Glover, ed., IRL Press, Arlington, VA, pp. 213-38 (1985)).
  • the RCA a-isoform gene placed in the appropriate plant expression vector can be used to transform plant cells.
  • the polypeptide can then be isolated from plant callus or the transformed cells can be used to regenerate transgenic plants.
  • Such transgenic plants can be harvested, and the appropriate tissues (seed or leaves, for example) can be subjected to large scale protein extraction and purification techniques.
  • Additional, agronomically important traits such as oil, starch, and protein content can be genetically altered in addition to using traditional breeding methods and are contemplated herein along with the RCA a-isoform modifications.
  • 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. Patent Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.
  • Another example is lysine and/or sulfur rich seed protein encoded by the soybean 2S albumin described in U.S. Patent No. 5,850,016, and the chymotrypsin inhibitor from barley, described in Williamson, et al, (1987) Eur. J. Biochem. 165:99-106.
  • Derivatives of the coding sequences can be made by site-directed mutagenesis to increase the level of preselected amino acids in the encoded polypeptide.
  • the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor (see, U.S. Patent
  • 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, Illinois), 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).
  • sunflower seed Liilley, 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, Illinois), pp. 497-502)
  • corn Pedersen, et al., (1986) J. Biol. Chem. 261
  • 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. Patent Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;
  • Genes encoding disease resistance traits include detoxification genes, such as against fumonosin (U.S. Patent No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones, et al, (1994)
  • 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 nptll gene encodes resistance to the antibiotics kanamycin and geneticin
  • the ALS-gene mutants encode resistance to the herbicide chlorsulfuron.
  • Sterility genes can also be encoded in an expression cassette and provide an alternative to physical detasseling. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as QM, described in U.S. Patent No. 5,583,210. 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 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. Patent Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.
  • PHAs polyhyroxyalkanoates
  • Exogenous products include plant enzymes and products as well as those from other sources including prokaryotes 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.
  • Hybrid seed production requires elimination or inactivation of pollen produced by the female parent. Incomplete removal or inactivation of the pollen provides the potential for selfing, raising the risk that inadvertently self-pollinated seed will unintentionally be harvested and packaged with hybrid seed.
  • the selfed plants can be identified and selected; the selfed plants are genetically equivalent to the female inbred line used to produce the hybrid.
  • the selfed plants are identified and selected based on their decreased vigor relative to the hybrid plants. For example, female selfed plants of e are identified by their less vigorous appearance for vegetative and/or reproductive
  • Selfed lines also can be identified using molecular marker analyses (see, e.g., Smith and Wych, (1995) Seed Sci. Technol. 14: 1-8). Using such methods, the homozygosity of the self-pollinated line can be verified by analyzing allelic composition at various loci in the genome.
  • hybrid plants are important and valuable field crops, plant breeders are continually working to develop high-yielding hybrids that are agronomically sound based on stable inbred lines.
  • the availability of such hybrids allows a maximum amount of crop to be produced with the inputs used, while minimizing susceptibility to pests and environmental stresses.
  • the plant breeder must develop superior inbred parental lines for producing hybrids by identifying and selecting genetically unique individuals that occur in a segregating population.
  • the present disclosure contributes to this goal, for example by providing plants that, when crossed, generate male sterile progeny, which can be used as female parental plants for generating hybrid plants.
  • the RCA modulated plants of the disclosure may be used in a plant breeding program.
  • the goal of plant breeding is to combine, in a single variety or hybrid, various desirable traits.
  • these traits may include, for example, resistance to diseases and insects, tolerance to heat and drought, tolerance to chilling or freezing, reduced time to crop maturity, greater yield and better agronomic quality.
  • uniformity of plant characteristics such as germination and stand establishment, growth rate, maturity and plant and ear height is desirable.
  • Traditional plant breeding is an important tool in developing new and improved commercial crops.
  • This disclosure encompasses methods for producing a plant by crossing a first parent plant with a second parent plant wherein one or both of the parent plants is a transformed plant displaying a phenotype as described herein..
  • Plant breeding techniques known in the art and used in a plant breeding program include, but are not limited to, recurrent selection, bulk selection, mass selection, backcrossing, pedigree breeding, open pollination breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection, doubled haploids and transformation. Often combinations of these techniques are used.
  • a genetic trait which has been engineered into a particular plant using gene editing or transformation techniques can be moved into another line using traditional breeding techniques that are well known in the plant breeding arts. For example, a backcrossing approach is commonly used to move a modified plant to an elite inbred line and the resulting progeny would then comprise the modification. Also, if an inbred line was used for the transformation or editing, then those plants could be crossed to a different inbred in order to produce a hybrid plant. As used herein, "crossing" can refer to a simple X by Y cross or the process of backcrossing, depending on the context.
  • the development of a hybrid in a plant breeding program involves three steps: (1) the selection of plants from various germplasm pools for initial breeding crosses; (2) the selfing of the selected plants from the breeding crosses for several generations to produce a series of inbred lines, which, while different from each other, breed true and are highly homozygous and (3) crossing the selected inbred lines with different inbred lines to produce the hybrids.
  • the vigor of the lines decreases. Vigor is restored when two different inbred lines are crossed to produce the hybrid.
  • An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid created by crossing a defined pair of inbreds will always be the same.
  • Transgenic plants of the present disclosure may be used to produce, e.g., a single cross hybrid, a three-way hybrid or a double cross hybrid.
  • a single cross hybrid is produced when two inbred lines are crossed to produce the Fl progeny.
  • a double cross hybrid is produced from four inbred lines crossed in pairs (A x B and C x D) and then the two Fl hybrids are crossed again (A x B) times (C x D).
  • a three-way cross hybrid is produced from three inbred lines where two of the inbred lines are crossed (A x B) and then the resulting Fl hybrid is crossed with the third inbred (A x B) x C.
  • Much of the hybrid vigor and uniformity exhibited by Fl hybrids is lost in the next generation (F2). Consequently, seed produced by hybrids is consumed rather than planted.
  • Example 1 Generation of transgenic rice having altered RCA a with altered DNA and amino acid sequence in exon 7
  • Bacterial strains were grown in LB liquid or on agar plates with appropriate antibiotics for plasmid selection. All cloning was performed using restriction endonucleases and T4 DNA ligase unless indicated otherwise and transformations were performed by electroporation into E. coli strain DH10B.
  • the CRISPR/Cas9 construct used to make targeted mutations in exon 7 of rice RCA a-isoform gene. The goal was to modify the following sequence in exon 7: 5'-
  • the oligo pairs for the BtgZI sites were AEP1650 (5 ' -TGTTGGGCAAGGAGCACAGCAAGC-3 ' SEQ ID NO: 30) and AEP1651 (5 '-AAACGCTTGCTGTGCTCCTTGCCC-3 ' SEQ ID NO: 31); and the oligo pairs for the Bsal sites were AEP1652 (5 '-GTGTGGGCAAGGAGCACAGCAAGC-3 ' SEQ ID NO: 32)and AEP1651 (5 ' -AAACGCTTGCTGTGCTCCTTGCCC-3 ' SEQ ID NO: 31).
  • ISU containing a duel U6 sgRNA cassette without targeting oligos and a kanamycin selection cassette, was cut open at BtgZI, then the phosphorylated and annealed oligo pair AEP1650 (SEQ ID NO: 30) and AEP 1651 (SEQ ID NO : 31 ) was ligated in to create plasmid pLW 1 , which now contains a complete single U6 sgRNA.
  • pLWl was cut at Bsal, then the phosphorylated and annealed oligo pair AEP 1651 (SEQ ID NO: 31) and AEP1652 (SEQ ID NO: 32) was ligated in to create pLW4, containing two complete U6 sgRNAs that target SEQ ID NO: 43 of exon 7 of rice RCA. Note that the same site is targeted by both oligo pairs.
  • pBY02:gamma CAS9-ccdB (Bing Yang lab) was joined to pLW4 using Gateway cloning to create pLW6.
  • pLW6 is 16,351 bp and contains the rice RCA exon 7 U6 sgRNA targeting cassette followed by a maize ubiquitin promoter with a rice optimized CAS9 gene and a NOS terminator, a bacterial kanamycin resistance selection cassette and a 2x35 S promoter with a hygromycin resistance gene and a 35S terminator for selection of rice transformants. Note that because both pLW4 and pBY02:gamma CAS9-ccdB are kanamycin resistant, the bacterial activity of the 2x35S hygromycin resistance gene cassette was used for selection during Gateway cloning. pLW6 was transformed by electroporation into Agrobacterium tumefaciens strain EHA105 for rice transformation.
  • the U6 promoters expresses the sgRNAs containing the sequence 5'- GGGCAAGGAGCACAGCAAGC-3 ' (SEQ ID NO: 44) and the ubiquitin promoter expressed the Cas9 RNA, which was translated into Cas9 protein.
  • the Cas9 protein binds the sgRNA then the complex binds the chromosome target sequence 5 ' -GGGC AAGGAGC ACAGC AAGC AGG-3 ' (SEQ ID NO: 43) because of the presence of the AGG PAM sequence, which in turn triggers the nuclease activity.
  • Nuclease activity causes a chromosome break in the target sequence (SEQ ID NO: 43) at or near a position six nucleotides from the 3 ' end of the target sequence.
  • chromosome break After a chromosome break is detected by the cell, the break is repaired, typically through nonhomologous end-joining where DNA at the break point is removed then a repair is made by rejoining the DNA using micro-homology at the remaining ends.
  • This action typically causes small deletions, insertions or base changes at the target site resulting in mutation of existing sequence that may cause frameshifts, additions, deletions or residue changes in the open reading frame of a coding sequence, thereby resulting in alteration of encoded protein as seen in FIGs. 2-19 depicting the sequence of various obtained rice mutants.
  • A. tumefaciens strain EHA105 containing pLW6 was grown in liquid callus induction media containing 200 micromolar acetosyringone to an OD600 of 0.1 to 0.2.
  • A. tumefaciens strain EHA105 and rice calli were incubated together for 30 minutes then calli were blotted dry on sterile Whatman paper.
  • Calli and A. tumefaciens strain EHA105 were co-cultivated for 3 days at 22°C in light on callus induction media plus 200 ⁇ acetosyringone.
  • calli were then washed in 50 ml sterile selection media containing 400 ⁇ g/ml carbenicillin until the solution was mostly clear, then calli were blotted dry and placed on selection media containing 50 ⁇ g/ml hygromycin at 30°C with continuous light for 3 weeks. Calli were moved to fresh selection media with hygromycin and incubated at 30°C with continuous light for 3 more weeks. Resistant calli were picked off the main callus bodies and transferred to selection media with hygromycin then incubated at 30°C with continuous light until they were 2-3 mm in size. 2-3 mm calli were placed on regeneration media with hygromycin and grown at 30°C in continuous light until they turned green (approximately 4 weeks).
  • Rice plants were grown either individually in pots or together in large trays using field soil or Berger brand BM custom potting mix containing peatmoss, perlite and bark. Plants were submerged in water 2 to 3 cm over the top of the soil or potting mix and supplemented with Everris water soluble fertilizer diluted to 5 ml/L from a 37 g/L stock and Sprint 330 chelated iron diluted to 5 ml/L in water from a 119 g/L stock. Plants were watered with 1 L of fertilizer and iron chelate twice per week and water was maintained at 2 to 3 cm over the soil or potting mix. Plant growth was at 28°C and approximately 160 ⁇ -Einsteins of light with a 16 hour day length.
  • Mutation in exon 7 of the rice RCA gene was detected by PCR followed by T7 endonuclease assays and sequence verification for positive events. Mutations are expected to occur at the CRISPR/Cas9 target site by non-homologous end-joining (NHEJ), which is expected to create insertions, deletions or base changes at the target site. These changes are detected by T7 endonuclease using annealed wild type and mutant PCR products, which results base mismatches and subsequent restriction of the PCR product into smaller fragments by T7 endonuclease. Restriction digestion is identified on electrophoresis agarose gels. Mutant identity is confirmed by DNA sequence analysis.
  • NHEJ non-homologous end-joining
  • Primers AEP1722 (5 ' -CTACTATATCTTGTCTGCATTTTCTC-3 ' SEQ ID NO: 33) and AEP1701 (5 ' -CCATGAATGTCAC ATGTGAATT AG-3 ' SEQ ID NO: 34) were used to generate an approximate 590 bp PCR product directly from rice leaves as instructed by the Phire PCR kit (Thermo kit #F-130-WH). Briefly, PCR products for the known wild-type rice sample and a transgenic mutated rice samples were mixed then melted and annealed through a temperature range of 100°C, down to 25°C, over a 12 minute period in a PCR machine.
  • T7 assay positive control 20 ⁇ ⁇ PCR reactions were performed for each plant along with positive and negative contamination controls from the kit, positive and negative wild type plant controls and a T7 assay positive control for a mutant and wild type DNA samples.
  • the T7 controls used mutant and wildtype Chlamydomonas reinhardtii Arg7 gene sequences from plasmids pDW2638 and pJD67 respectively using primers AEP976 (5 ' -TGCTCTACTACTGTTCCTGGCTAC-3 ' SEQ ID NO: 35) and AEP1236 (5 '-CGCAGGTGTCTGACCGCGACTTTG-3 ' SEQ ID NO: 36) to yield an approximate 337 bp PCR product.
  • Two ⁇ _- of each PCR reaction were run on a 2.5% agarose gel to check PCR product quality for each sample.
  • T7 assays were performed by mixing 3.5 ⁇ _- of wild type and 3.5 ⁇ _- of experimental plant PCR product with 13 ⁇ _- ddH20, then the mixture was heated to 100°C and cooled in 10°C steps for 1 minute per step down to 20°C. After cooling, 0.2 ⁇ _ of T7 endonuclease (NEB#M0302L - 10,000 units per ml) was added to each tube, and these were incubated at 37°C for 2 hours.
  • the T7 control was a mixture of 3.5 ⁇ _- mutant and 3.5 ⁇ _- wild-type Arg7 PCR product in 13 ⁇ _- ddH20 products, and that 7 ⁇ _- of wild-type rice PCR product mixed with 13 ⁇ _- ddH20 was used as a negative control.
  • Controls were treated in the same manner as experimentals. After digestion, 4 ⁇ _- of SUDS was added to each reaction, and the reactions were run on a 2.5% agarose gel. Positive reactions were indicated by the appearance of approximate 100 and a 200 bp band in the T7 positive control, no lower bands in the wild-type rice control and approximate 470 and 120 bp bands in experimentals lanes.
  • FIGs. 2-19 provide the DNA and amino acid sequences of the mutated RCA a gene exon 7 in chromosomes 1 and 2 for the genetically altered rice plant lines indicated on each figure, and made using the above protocols.
  • underlined amino acids are from the wild-type exon 7 coding sequence;
  • the other amino acids arise from the genetic change caused by
  • CRISPR/Cas9 CRISPR/Cas9; (iii) an asterisk is a stop codon, and (iv) lower case nucleotides are nucleotides that are inserted into exon 7 (except for the last nucleotide of intron 6).
  • the indicated inserted cytosine nucleotide could be a guanine nucleotide instead in chromosome 1 ; and in chromosome 2 the indicated 5 bp deletion with insertion AGT nucleotides could also be viewed as a 3 bp deletion with 1 bp insertion.
  • Example 2 Phenotypic changes in the transgenic rice plants with altered RCA a DNA & amino acid sequences
  • Plant growth Wild-type (Kitake) and CRISPR driven transgenics were germinated on the wet paper and transferred to individual pot using custom mix soil (Sungro Horticulture). Fertilizer containing Fe, N, P and K was provided once a week. The plants were grown in HL (continuous high light for 16 hrs) or LL-HL (1 hr high light and 1 hr low light for 16 hrs) with 25°C and 80% humidity. The intensities of high and low light are 40 and 400 (umolm ' V 1 ), respectively.
  • transgenic rice lines 6-3 (96 ⁇ 2.2 cm), 8-4 (96 ⁇ 4.1 cm) and 9-3 (98.7 ⁇ 2.6 cm) were considerably taller.
  • the length of individual leaves was not increased, but leaves were wider in the transgenic rice lines than in the wild-type rice.
  • Wild-type rice leaves were 1.07 ⁇ 0.05 cm wide, and the transgenic rice lines' leaves were approximately 20% wider: transgenic rice line 6-3 leaves were 1.30 ⁇ 0.08 wide; transgenic rice line 8-4 leaves were 1.37 ⁇ 0.10; and transgenic rice line 9-3 leaves were 1.33 ⁇ 0.05.
  • the transgenic rice plants had a greater total leaf area to assimilate atmospheric CO 2 for growth and seed production.
  • Seed yield per plant For the total seed number, the 14 weeks old plants were selected and counted. At maturity, the three transgenic lines produced more seeds per plant compared to amount of seed produced by wild-type cultivar, indicating that the editing of the RCA gene's sequence to remove the di-sulfide bond at the carboxyl terminus of the a-isoform directly impacts seed production (see FIG. 20D). Interestingly, stover yield at maturity was not affected by the editing of the RCA gene's sequence.
  • the wild-type rice RCA a-isoform carboxy terminal amino acid sequence in chromosome 1 is GQGAQQAGNLPVPEGCTDPVAKNFDPTARSDDGSCLYTF* (SEQ ID NO: 37; the cysteine amino acids are underlined), and the RCA ⁇ -isoform carboxy terminal amino acid sequence in chromosome 2 is GSAPSS (SEQ ID NO: 38).
  • SEQ ID NO: 37 is the same as SEQ ID NO: 2, except that SEQ ID NO: 45 lists a glycine (G) as the initial amino acid, and this glycine arises from the codon that is generated during the splicing between exon 6 and exon 7.
  • Altered rice plant line 6-3 has a 4 bp deletion in RCA exon 7 in chromosome 1, which frame shifts to a longer open reading frame (ORF) than the original, non-mutated ORF, and chromosome 2 has a 1 bp deletion and frame shifts to a stop codon (termination) prior to the first cysteine in exon 7.
  • ORF open reading frame
  • chromosome 2 has a 1 bp deletion and frame shifts to a stop codon (termination) prior to the first cysteine in exon 7.
  • the altered RCA ⁇ -isoform carboxy terminal amino acid sequence from the RCA gene in chromosome 1 is
  • SEQ ID NO: 39 is the same as SEQ ID NO: 6 except that SEQ ID NO: 39 lists a glycine (G) as the initial amino acid, and this glycine arises from the codon that is generated during the splicing between exon 6 and exon 7.
  • G glycine
  • SEQ ID NO: 40 is the same as SEQ ID NO: 8 except that SEQ ID NO: 40 lists a glycine (G) as the initial amino acid, and this glycine arises from the codon that is generated during the splicing between exon 6 and exon 7.
  • transformed rice plant lines 8-1, 8-2, 8-4, 8-5, 8-7, 8-8, 20-1, 20-2, 20-3, 6-1, 6-4 which all have a mutation the RCA gene exon 7 in chromosomes 1 and/or 2 that encode an RCA ⁇ -isoform with the same carboxy terminal amino acids.
  • Altered rice plant line 8-4 has a 1 bp insertion in the mutated RCA exon 7 gene on both chromosomes 1 and 2, which results in frame shifts to a stop codon (termination) before the first cysteine encoded within exon 7.
  • the altered RCA a-isoform carboxy terminal amino acid sequence encoded by the mutated RCA exon 7 gene on chromosome 1 is GQGAQHSR* (SEQ ID NO: 41).
  • the altered RCA a- isoform carboxy terminal amino acid sequence for rice plant line 8-4 encoded by the mutated RCA exon 7 gene on chromosome 2 is also GQGAQQSR* (SEQ ID NO: 41).
  • SEQ ID NO: 41 is the same as SEQ ID NO: 15 except that SEQ ID NO: 41 lists a glycine (G) as the initial amino acid, and this glycine arises from the codon that is generated during the splicing between exon 6 and exon 7.
  • Altered rice plant line 9-3 has a 1 bp deletion in the mutated RCA exon 7 gene on chromosome 1 which results in a frame shift to the longer ORF than the original, non-mutated ORF and a 1 bp insertion in the mutated RCA exon 7 gene on chromosome 2 which results in a frame shift to a stop codon before the first cysteine in exon 7.
  • the altered RCA ⁇ -isoform carboxy terminal amino acid sequence encoded by the mutated RCA exon 7 gene on chromosome 1 in altered rice plant line 9-3 is
  • SEQ ID NO: 42 is the same as SEQ ID NO: 22 except that SEQ ID NO: 42 lists a glycine (G) as the initial amino acid, and this glycine arises from the codon that is generated during the splicing between exon 6 and exon 7. See also Table 1 supra, and the applicable figures for transformed rice plant lines 9-2, 13-1, 13-2, 16, and 17 which all have a mutation the RCA gene exon 7 in chromosomes 1 and/or 2 that encode an RCA ⁇ -isoform with the same carboxy terminal amino acids.
  • G glycine
  • the altered RCA ⁇ -isoform carboxy terminal amino acid sequence encoded by the mutated RCA a gene exon 7 on Chromosome 2 is GQGAQQSR* (SEQ ID NO: 41) which is discussed supra.
  • redox-sensitive cysteines One can alter RCA -isoform genomic sequences in these plants, and other plants, to remove one or both cysteines involved in the redox regulation of RuBisCO's activity, thereby increasing RuBisCO's activity state and generating altered plants with the desired agronomic phenotypes described herein for the altered rice.
  • the transformed plant cells are selected for those cells that produce the selection marker and/or Cas9 and the desired sgRNA sequence. The selected transformed plant cells.
  • the transformed plant cells can be induced to grow into transformed plants and assess for an increase in at least one agronomic phenotype described herein (e.g. , RCA ⁇ -isoform activation, RuBisCO activation, plant growth, leaf width, seed yield per plant, and photosynthetic induction rate) when grown under constant light during the photoperiod compared to the level of the agronomic phenotype in the wild-type plant.
  • agronomic phenotype described herein e.g. , RCA ⁇ -isoform activation, RuBisCO activation, plant growth, leaf width, seed yield per plant, and photosynthetic induction rate
  • These transformed plants will have an altered RCA ⁇ -isoform with an altered carboxy terminal amino acid sequence, a loss of redox activity, and increased activation of RuBisCO.
  • RCA ⁇ -isoform mRNA and RCA ⁇ -isoform mRNA are compared and found to be nearly identical, except for the absence of a 85 bp segment from the a-isoform mRNA resulting from alternative splicing. Presence of the 85 bp region in ⁇ -isoform mRNA ensures that exon 7a is encoded, and the resulting protein is the ⁇ -isoform. The absence of the 85 bp region in a- isoform mRNA ensures that exon 7b is encoded, and the resulting protein is the a-isoform.
  • This 36 bp sequence is placed in an expression vector such that two copies, in opposite orientation (sense and anti-sense sequence), with a spacer sequence placed between the sense and anti- sense sequence so that a hairpin loop can be founded and the sense sequence and anti-sense sequence can bind, forming dsRNA.
  • the expression vector contains a left border sequence, CaMV35S promoter driving expression of the hygB resistance gene with a 35S terminator, a rice UBI promoter, EcoRI restriction endonuclease site, the sense target sequence (SEQ ID NO: 64), BamHI restriction endonuclease site, a generic spacer sequence with a chloramphenicol resistance gene for E.
  • Agrobacterium T-DNA vector One such example of the expression vector is pHellsgate 8.0 plasmid.
  • the completed Agrobacterium T-DNA vector is electroporated into Agrobacterium tumefaciens strain EHA105 by electroporation and selected on LB media supplemented with 100 ⁇ g/ml of streptomycin for T-DNA vector selection.
  • Agrobacterium colonies are grown at 30°C then isolated and grown for vector verification via restriction digestion and running on a gel.
  • Kitaake rice callus is generated on callus induction media then infected with the EHA105
  • Agrobacterium tumefacians strain transformed with the vector Infected callus are grown on co-cultivation media then selected on selection media supplemented with hygromycin B at 50 ⁇ g/ml. Once callus are selected, regeneration media supplemented with 50 ⁇ g/ml hygromycin B is used to produce rice shoots followed by rooting on root generation media again supplemented with 50 ⁇ g/ml hygromycin B.
  • Specific protocol details are described in Cheng, et al. 2009 and also described above in Example 1.
  • RNAi transgene is confirmed RT-PCR, and then the RCA a-isoform gene expression is analyzed by RT-PCR.
  • RCA a-isoform gene silencing or reduction of gene expression is also verified by examining the genetically altered plants for morphological changes described above (broader leaves, larger size, increased seed yield/plant) and by examining RCA ⁇ -isoform protein expression using antibodies in a Western blot assay.
  • the genetically altered rice are distinguished from wild-type rice via Western blot because the genetically altered rice produce RCA ⁇ -isoform only whereas the wild-type rice produce RCA ⁇ -isoform and RCA ⁇ -isoform.

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Abstract

L'invention concerne une isoforme alpha modifiée de la RuBisCO activase (RCA) dépourvue d'activité redox au niveau de son extrémité carboxy, ainsi que des plantes génétiquement modifiées qui codent pour une isoforme alpha modifiée de la RCA et dont l'ADN est modifié au niveau de son extrémité 3' (ou au niveau de l'exon qui code pour les cystéines sensibles à l'oxydo-réduction qui sont présentes à l'extrémité carboxy de la protéine) de telle sorte que l'isoforme alpha de la RCA pour laquelle il code soit dépourvue d'activité redox au niveau de son extrémité carboxy. Les plantes génétiquement modifiées qui produisent l'isoforme alpha de la RCA dépourvue d'activité redox au niveau de son extrémité carboxy présentent une augmentation pour au moins un phénotype à valeur agronomique. L'invention concerne également des méthodes permettant de réaliser la modification génétique dans des plantes, et des plantes génétiquement modifiées.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020002152A3 (fr) * 2018-06-27 2020-02-13 Basf Se Rubisco activase thermostable et utilisations associées
CN113943744A (zh) * 2021-11-02 2022-01-18 云南农业大学 叶斑艺兰花rca基因的应用及其载体构建方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040241860A1 (en) * 1998-06-19 2004-12-02 Advanta Technology Limited Method of breeding glyphosate resistant plants
US20140182015A1 (en) * 2006-12-21 2014-06-26 Basf Plant Science Gmbh Plants Having Enhanced Yield-Related Traits and a Method for Making the Same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040241860A1 (en) * 1998-06-19 2004-12-02 Advanta Technology Limited Method of breeding glyphosate resistant plants
US20140182015A1 (en) * 2006-12-21 2014-06-26 Basf Plant Science Gmbh Plants Having Enhanced Yield-Related Traits and a Method for Making the Same

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE NCBI [O] 5 May 2011 (2011-05-05), XP055454735, Database accession no. ABG22615.1 *
MORENO, JOAQUIN ET AL.: "Redox modulation of Rubisco conformation and activity through its cysteine residues", JOURNAL OF EXPERIMENTAL BOTANY, vol. 59, no. 7, 22 January 2008 (2008-01-22), pages 1605 - 1614, XP055454731 *
WANG, DAFU ET AL.: "Increased sensitivity of oxidized large isoform of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activase to ADP inhibition is due to an interaction between its carboxyl extension and nucleotide-binding pocket", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 281, no. 35, 1 September 2006 (2006-09-01), pages 25241 - 25249, XP055454729 *
ZHANG, NING ET AL.: "Light modulation of Rubisco in Arabidopsis requires a capacity for redox regulation of the larger Rubisco activase isoform", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 99, no. 5, 5 March 2002 (2002-03-05), pages 3330 - 3334, XP002433768 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020002152A3 (fr) * 2018-06-27 2020-02-13 Basf Se Rubisco activase thermostable et utilisations associées
CN113943744A (zh) * 2021-11-02 2022-01-18 云南农业大学 叶斑艺兰花rca基因的应用及其载体构建方法

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