WO2021143866A1 - Application de protéine de ramification photorespiratoire dans la régulation de caractéristiques de plantes - Google Patents

Application de protéine de ramification photorespiratoire dans la régulation de caractéristiques de plantes Download PDF

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WO2021143866A1
WO2021143866A1 PCT/CN2021/072225 CN2021072225W WO2021143866A1 WO 2021143866 A1 WO2021143866 A1 WO 2021143866A1 CN 2021072225 W CN2021072225 W CN 2021072225W WO 2021143866 A1 WO2021143866 A1 WO 2021143866A1
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plant
nucleic acid
protein
plants
acid construct
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Chinese (zh)
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李峰
彭新湘
林秀灵
沈博然
梁亚峰
孙洁
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山东舜丰生物科技有限公司
华南农业大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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    • 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
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    • C12Y102/03—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with oxygen as acceptor (1.2.3)
    • C12Y102/03004—Oxalate oxidase (1.2.3.4)
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    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to the field of biotechnology, in particular to the application of photorespiration branch proteins in regulating plant traits, and more specifically, to reagent combinations and fusion proteins for regulating plant agronomic traits, and their applications in regulating plant traits.
  • Photorespiration also known as the C2 cycle, refers to the process by which green tissues of plants absorb O 2 and release CO 2 by using light energy. Photorespiration requires the participation of chloroplasts, mitochondria, peroxisomes and cytoplasm, and depends on light and O 2. Both are indispensable. Photorespiration is the second largest metabolic flow in C3 plants after photosynthesis. Under normal environmental conditions, C3 plant photorespiration can consume 25-30% of its photosynthetic products, but under high temperature, drought, high light and other adversity conditions. The loss will be more serious. High photorespiration will not only reduce the photosynthetic efficiency of plants, but also the contribution of CO 2 to atmospheric carbon emissions cannot be underestimated.
  • CCMs CO 2 concentration mechanisms
  • the purpose of the present invention is to provide a new method for further regulating plant agronomic traits.
  • Another object of the present invention is to provide a new combination of agents, methods or fusion proteins for regulating plant agronomic traits, said plant traits including but not limited to: (i) photorespiration; and/or (ii) photosynthetic rate; And/or (iii) yield; and/or (iv) biomass; (v) chlorophyll content.
  • the reagent combination, method or fusion protein of the present invention can inhibit photorespiration, enhance photosynthesis, increase yield and biomass, and increase chlorophyll content.
  • the first aspect of the present invention provides a reagent combination, including:
  • P1 is the first promoter
  • Z1 is an optional coding sequence encoding a chloroplast signal peptide
  • Z2 is a coding sequence encoding a first photorespiration metabolic modification branch protein, and the first photorespiration metabolic modification branch protein is glycolate oxidase (GLO);
  • Z3 is a terminator
  • P2 is the second promoter
  • Z4 is an optional coding sequence encoding a chloroplast signal peptide
  • Z5 is a coding sequence encoding a second photorespiration metabolic modification branch protein, and the second photorespiration metabolic modification branch protein is oxalate oxidase (OXO);
  • Z6 is a terminator
  • P3 is the third promoter
  • Z7 is an optional coding sequence encoding a chloroplast signal peptide
  • Z8 is a coding sequence encoding a third photorespiration metabolic modification branch protein, and the third photorespiration metabolic modification branch protein is catalase (CATC);
  • Z9 is a terminator
  • the first carrier, the second carrier, and the third carrier are the same or different carriers.
  • first nucleic acid construct, the second nucleic acid construct, and the third nucleic acid construct are located in the same vector or different vectors.
  • the second aspect of the present invention provides a reagent combination, including:
  • P1 is the first promoter
  • Z1 is an optional coding sequence encoding a chloroplast signal peptide
  • Z2 is a coding sequence encoding a fusion protein selected from the group consisting of glycolate oxidase + oxalate oxidase, glycolate oxidase + catalase, oxalate oxidase + catalase, glycolate oxidase +oxalate oxidase + catalase, or a combination thereof;
  • Z3 is a terminator
  • P2 is the second promoter
  • Z4 is an optional coding sequence encoding a chloroplast signal peptide
  • Z5 is a coding sequence encoding a third element, which is an element other than the fusion protein, and the third element is one of glycolate oxidase, oxalate oxidase, and catalase ;
  • Z6 is a terminator
  • the second nucleic acid construct does not exist.
  • the fusion protein is selected from the group consisting of GLO+OXO, GLO+CATC, OXO+CATC, GLO+OXO+CATC, or a combination thereof.
  • the fusion protein is selected from the group consisting of OsGLO+OsOXO, OsGLO+KatE, OsOXO+KatE, OsGLO+OsOXO+KatE, or a combination thereof.
  • the fusion protein is selected from the group consisting of OsGLO+OsOXO, OsGLO+OsCATC, OsOXO+OsCATC, OsGLO+OsOXO+OsCATC, or a combination thereof.
  • the first promoter is selected from the group consisting of CaMV 35S, U6, U3, NOS, OCS, Rac1, UBi, Act1, Rss1, Adh1, TA29, or a combination thereof.
  • the second promoter is selected from the group consisting of CaMV 35S, U6, U3, NOS, OCS, Rac1, UBi, Act1, Rss1, Adh1, TA29, or a combination thereof.
  • the first carrier and the second carrier are the same or different carriers.
  • the nucleotide linking sequence is 1-200 nt.
  • nucleotide linking sequence does not affect the normal transcription and translation of each element.
  • the chloroplast signal peptide is selected from the group consisting of rbcS, TPC, RC1, PCS1, or a combination thereof.
  • the terminator is selected from the group consisting of NOS terminator, UBQ terminator, or a combination thereof.
  • first nucleic acid construct and the second nucleic acid construct are located in the same vector or different vectors.
  • the GLO, OXO or CATC are each independently derived from eukaryotes and/or prokaryotes.
  • the GLO, OXO or CATC are each independently derived from fungi (such as yeast), cocci, Enterobacter, Vibrio, anaerobic bacteria, mycobacteria, and animal-derived bacteria.
  • the GLO, OXO or CATC are each independently derived from plants and/or microorganisms.
  • the GLO, OXO or CATC are each independently derived from a monocotyledonous plant or a dicotyledonous plant.
  • the plant is selected from the following group: Cruciferae, Gramineae, Leguminosae, Solanaceae, Umbelliferae, Chenopodiaceae, Actinidiaceae, Moraceae, Malvaceae, Paeoniaceae, Rosaceae , Liliaceae, or a combination thereof.
  • the GLO, OXO or CATC are each independently derived from one or more plants selected from the group consisting of: Cruciferae, Gramineae, Leguminosae, Solanaceae, Umbelliferae, Chenopodiaceae , Actinidiaceae, Moraceae, Malvaceae, Paeoniaceae, Rosaceae, Liliaceae.
  • the GLO, OXO or CATC are each independently derived from one or more plants selected from the group consisting of Arabidopsis thaliana, potato, sweet potato, purple potato, yam, taro, cassava, chrysanthemum Potato, rice, wheat, barley, corn, sorghum, millet, soybean, peanut, millet, quinoa, tomato, tobacco, rape, cabbage, spinach, lettuce, cucumber, chrysanthemum, spinach, celery, lettuce, millet, peanut, Kiwi, cotton, strawberry, peony, perfume lily, tulip, mulberry, apple, pear, peach, cherry, pomegranate.
  • the GLO, OXO or CATC are each independently derived from one or more plants selected from the group consisting of Arabidopsis, wheat, barley, rice, potato, soybean, corn, tomato, sorghum ,Millet.
  • the CATC is derived from Escherichia coli.
  • the GLO is selected from the group consisting of: rice GLO3 gene (OsGLO3, accession number Os04g0623500), sorghum GLO3 gene (SbGLO3, accession number LOC8074729), millet GLO3 gene (SiGLO3, accession number GBYO01017938. 1), or a combination thereof.
  • the OXO is selected from the following group: OXO3 gene of rice (Os OXO3, accession number Os03g0693900), OXO gene of sorghum (SbOXO, accession number XP_002464052.1), OXO gene of maize (ZmOXO, accession number) No. NM_001147384.1), or a combination thereof.
  • the CATC is selected from the following group: CATC gene of rice (Os CATC, accession number Os03g0131200), CATC gene of sorghum (SbCAT, accession number XM_021448587.1), CATC gene of millet (SiCAT, accession number) No. XM_004985783.4), the KatE gene of Escherichia coli, or a combination thereof.
  • the glycolate oxidase includes OsGLO3.
  • the oxalate oxidase includes OsOXO3.
  • the catalase is selected from the group consisting of OsCATC, KatE, or a combination thereof.
  • the GLO includes wild-type GLO and mutant GLO.
  • the mutant type includes a mutant form in which the function of the encoded protein is not changed after mutation (that is, the function is the same or substantially the same as that of the wild-type encoded protein) and the function is enhanced.
  • polypeptide encoded by the mutant GLO gene is the same or substantially the same as the polypeptide encoded by the wild-type GLO gene.
  • the mutant GLO gene includes homology of ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95%, more preferably, ⁇ 98% compared with the wild-type GLO gene). % Or 99%) polynucleotides.
  • the mutant GLO gene is included in the 5'end and/or 3'end of the wild-type GLO gene, truncated or added 1-60 (preferably 1-30, more preferably 1 -10) nucleotide polynucleotides.
  • amino acid sequence of the GLO is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 1 is formed by substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and the GLO activity is derived from (i ) Derived polypeptide; or
  • the homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.:1 is ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95% or ⁇ 98%), a polypeptide having the GLO activity .
  • nucleotide sequence of the GLO gene encoding the GLO protein is selected from the following group:
  • the OXO includes wild-type OXO and mutant OXO.
  • the mutant type includes a mutant form in which the function of the encoded protein is not changed after mutation (that is, the function is the same or substantially the same as that of the wild-type encoded protein) and the function is enhanced.
  • polypeptide encoded by the mutant OXO gene is the same or substantially the same as the polypeptide encoded by the wild-type OXO gene.
  • the mutant OXO gene includes homology of ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95%, more preferably, ⁇ 98% compared with wild-type OXO gene). % Or 99%) polynucleotides.
  • the mutant OXO gene is included in the 5'end and/or 3'end of the wild-type OXO gene, truncated or added 1-60 (preferably 1-30, more preferably 1 -10) nucleotide polynucleotides.
  • amino acid sequence of the OXO is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 3 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and the OXO activity is derived from (i ) Derived polypeptide; or
  • the homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.: 3 is greater than or equal to 80% (preferably greater than or equal to 90%, more preferably greater than or equal to 95% or greater than or equal to 98%), a polypeptide having the OXO activity .
  • nucleotide sequence of the OXO gene encoding the OXO protein is selected from the following group:
  • the CATC includes wild-type CATC and mutant CATC.
  • the mutant type includes a mutant form in which the function of the encoded protein remains unchanged (that is, the function is the same or substantially the same as that of the wild-type encoded protein) and the function is enhanced after the mutation.
  • polypeptide encoded by the mutant CATC gene is the same or substantially the same as the polypeptide encoded by the wild-type CATC gene.
  • the mutant CATC gene includes homology ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95%, more preferably, ⁇ 98% compared with wild-type CATC gene). % Or 99%) polynucleotides.
  • the mutant CATC gene is included in the 5'end and/or 3'end of the wild-type CATC gene, truncated or added 1-60 (preferably 1-30, more preferably 1 -10) nucleotide polynucleotides.
  • amino acid sequence of the CATC is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 5 or 7 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, which has the CATC activity
  • amino acid sequence shown in SEQ ID NO.: 5 or 7 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, which has the CATC activity
  • Derived polypeptide or
  • the homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.: 5 or 7 is ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95% or ⁇ 98%), and has the CATC activity Of peptides.
  • nucleotide sequence of the CATC gene encoding the CATC protein is selected from the following group:
  • the vector is an expression vector that can be transfected or transformed into plant cells.
  • the vector is a plant expression vector.
  • the carrier is an Agrobacterium Ti carrier.
  • the vector is pCambia vector.
  • the vector is selected from the following group: pYL322d1, pYL322d2, pYL1305, pYLTAC380GW, or a combination thereof.
  • the carrier is circular or linear.
  • the third aspect of the present invention provides a kit containing the reagent combination according to the first aspect of the present invention or the second aspect of the present invention.
  • the kit further contains a label or instructions.
  • the fourth aspect of the present invention provides a method for regulating agronomic traits of plants, including the steps:
  • first nucleic acid construct or the first vector containing the first nucleic acid construct, the second nucleic acid construct or the second vector containing the second nucleic acid construct in the reagent combination can be introduced sequentially and simultaneously.
  • the "agronomic traits of the plant” include:
  • the "regulated agronomic traits of plants” includes:
  • the plant cell is derived from flower, leaf bud, embryo, suspension cell line, callus, or a combination thereof.
  • the callus is induced to form by plant cells selected from the group consisting of root, stem, leaf, flower, and/or seed cells.
  • the introduction is by Agrobacterium.
  • the introduction is by gene gun.
  • the fifth aspect of the present invention provides a method for preparing genetically engineered plant cells, including the steps:
  • the transfection adopts the Agrobacterium transformation method or the gene gun bombardment method.
  • the sixth aspect of the present invention provides a method for preparing genetically engineered plant cells, including the steps:
  • the seventh aspect of the present invention provides a method for preparing genetically engineered plants, including the steps:
  • the genetically engineered plant cell prepared by the method of the fifth aspect of the present invention or the sixth aspect of the present invention is regenerated into a plant body, thereby obtaining the genetically engineered plant.
  • the eighth aspect of the present invention provides a genetically engineered plant, which is prepared by the method described in the seventh aspect of the present invention.
  • the ninth aspect of the present invention provides a fusion protein, the structure of the fusion protein is shown in the following formula I or I':
  • A is the chloroplast signal peptide
  • GLO glycolate oxidase
  • OXO oxalate oxidase
  • CAC catalase
  • Each "-" is independently a connecting peptide or a peptide bond or a non-peptide bond.
  • the fusion protein is selected from the group consisting of GLO+OXO, GLO+CATC, OXO+CATC, GLO+OXO+CATC, or a combination thereof.
  • the fusion protein is selected from the group consisting of OsGLO+OsOXO, OsGLO+KatE, OsOXO+KatE, OsGLO+OsOXO+KatE, or a combination thereof.
  • the fusion protein is selected from the group consisting of OsGLO+OsOXO, OsGLO+OsCATC, OsOXO+OsCATC, OsGLO+OsOXO+OsCATC, or a combination thereof.
  • the glycolate oxidase includes OsGLO3.
  • the oxalate oxidase includes OsOXO3.
  • the catalase is selected from the group consisting of OsCATC, KatE, or a combination thereof.
  • the non-peptide bond includes PEG, polypropylene glycol (PPG), copolymer (ethylene/propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharide, dextran, polyvinyl alcohol , Polyvinylpyrrolidone, polyvinyl ether, polyacrylamide, polyacrylate, polycyanoacrylate, lipopolymer, chitin, hyaluronic acid, heparin or alkyl linker.
  • the chloroplast signal peptide is selected from the group consisting of rbcS, TPC, RC1, PCS1, or a combination thereof.
  • the connecting peptide has a length of 1-100 aa, preferably, 15-85 aa, more preferably, 25-70 aa, and even more preferably, 24-32 aa.
  • amino acid sequence of the connecting peptide is selected from the following group:
  • a polypeptide whose amino acid sequence is SEQ ID NO.: 9 or any one of 11-16;
  • the fusion protein has the amino acid sequence shown in SEQ ID NO.:8.
  • the tenth aspect of the present invention provides a polynucleotide which encodes the fusion protein according to the ninth aspect of the present invention.
  • the polynucleotide additionally contains auxiliary elements selected from the group consisting of secretory peptides, tag sequences (such as 6His), or a combination thereof on the flanks of the ORF of the fusion protein.
  • the polynucleotide is selected from the following group: DNA sequence, RNA sequence, or a combination thereof.
  • the eleventh aspect of the present invention provides a vector containing the polynucleotide according to the tenth aspect of the present invention.
  • the vector includes one or more promoters, which are operably linked to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, and selectable marker. , Nucleic acid restriction sites, and/or homologous recombination sites.
  • the vector is a plant expression vector.
  • the vector is an expression vector that can be transfected or transformed into plant cells.
  • the carrier is an Agrobacterium Ti carrier.
  • the vector is selected from the group consisting of pYL322d1, pYL322d2, pYL1305, pYLTAC380GW, or a combination thereof.
  • the construct is integrated into the T-DNA region of the vector.
  • the carrier is cyclic or linear.
  • the twelfth aspect of the present invention provides a genetically engineered cell, which contains the polynucleotide according to the tenth aspect of the present invention, or its genome integrates the polynucleotide according to the tenth aspect of the present invention.
  • the cell is a plant cell.
  • the plants include monocotyledonous plants and dicotyledonous plants.
  • the plants include herbaceous plants and woody plants.
  • the plant is selected from the group consisting of cruciferous plants, gramineous plants, leguminous plants, Solanaceae, Actinidiaceae, Moraceae, Malvaceae, Paeoniaceae, Rosaceae, Liliaceae, Or a combination.
  • the plant is selected from the group consisting of Arabidopsis thaliana, potato, sweet potato, purple sweet potato, rice, yam, taro, cassava, chrysanthemum, cabbage, soybean, tomato, corn, tobacco, wheat , Sorghum, barley, oats, millet, peanuts, kiwi, cotton, strawberry, peony, perfume lily, tulip, mulberry, apple, pear, peach, cherry, pomegranate, or a combination thereof.
  • the plant includes potato, sweet potato, purple potato, yam, taro, cassava, and chrysanthemum.
  • the genetically engineered cell is a method selected from the group consisting of introducing the polynucleotide of claim 10 into the cell: Agrobacterium transformation method, gene gun method, microinjection method, electric shock method , Ultrasonic method and polyethylene glycol (PEG)-mediated method.
  • the thirteenth aspect of the present invention provides a method for producing the fusion protein according to the ninth aspect of the present invention, which includes the steps:
  • the fusion protein is isolated.
  • the fourteenth aspect of the present invention provides a use of the fusion protein according to the ninth aspect of the present invention for regulating agronomic traits of plants or preparing a composition or preparation for regulating agronomic traits of plants, wherein the agronomic traits of the plant One or more selected from the following group:
  • the "regulated agronomic traits of plants” includes:
  • the composition includes an agricultural composition.
  • the plants include monocotyledonous plants and dicotyledonous plants.
  • the plants include herbaceous plants and woody plants.
  • the plant is selected from the group consisting of cruciferous plants, gramineous plants, leguminous plants, Solanaceae, Actinidiaceae, Moraceae, Malvaceae, Paeoniaceae, Rosaceae, Liliaceae, Or a combination.
  • the plant is selected from the group consisting of Arabidopsis thaliana, potato, sweet potato, purple sweet potato, rice, yam, taro, cassava, chrysanthemum, cabbage, soybean, tomato, corn, tobacco, wheat , Sorghum, barley, oats, millet, peanuts, kiwi, cotton, strawberry, peony, perfume lily, tulip, mulberry, apple, pear, peach, cherry, pomegranate, or a combination thereof.
  • the plant includes potato, sweet potato, purple potato, yam, taro, cassava, and chrysanthemum.
  • the fifteenth aspect of the present invention provides an agricultural preparation, including:
  • the expression vector includes a plant expression vector.
  • the vector is an expression vector that can be transfected or transformed into plant cells.
  • the carrier is an Agrobacterium Ti carrier.
  • the vector is selected from the following group: pYL322d1, pYL322d2, pYL1305, pYLTAC380GW, or a combination thereof.
  • the content of component (a) is 0.01%-99%, preferably, 1%-50%, more preferably, 10%-30%, based on the total amount of the agricultural preparation Recalculate.
  • the agricultural preparation also includes other substances that regulate agronomic traits.
  • the other substances for controlling agronomic traits are selected from the group consisting of photorespiration inhibitors, photosynthesis enhancers, or combinations thereof.
  • the photorespiration inhibitor is selected from the group consisting of carbon dioxide, glyoxylic acid, glutamic acid, aspartic acid, ⁇ -hydroxysulfonate, 2,3-epoxyacetic acid, and isotonic acid. Hydrazine, NaHSO 3 , or a combination thereof.
  • the photosynthesis enhancer is selected from the group consisting of potassium bicarbonate, sodium bisulfate, diethyl hexanoate, choline chloride, or a combination thereof.
  • the dosage form of the agricultural preparation is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, aerosol, or a combination thereof.
  • the sixteenth aspect of the present invention provides a method for regulating agronomic traits of plants, including:
  • the "regulated agronomic traits of plants” includes:
  • the application is selected from the group consisting of spraying, watering, drip irrigation, spraying, coating, injection, or a combination thereof.
  • the administration can be one-time administration, repeated administration or continuous administration.
  • the application dosage is 0.1g/mu-100g/mu, preferably 1g/mu-20g/mu, more preferably, 5g/mu-10g/mu.
  • the application time is before sowing, bud stage, and flowering stage.
  • the application method is to apply to plants, plant seeds, or the soil surrounding the plants or seeds.
  • the plants include monocotyledonous plants and dicotyledonous plants.
  • the plants include herbaceous plants and woody plants.
  • the plant is selected from the group consisting of cruciferous plants, gramineous plants, leguminous plants, solanaceae, moraceae, actinidiaceae, malvaceae, paeoniaceae, rosaceae, lily family, Or a combination.
  • the plant is selected from the group consisting of Arabidopsis thaliana, potato, sweet potato, purple potato,
  • the plant includes potato, sweet potato, purple potato, yam, taro, cassava, and chrysanthemum.
  • Figure 1 is the core part of the DI vector; among them, LoxP, 1L, and 2R are recombination sites; I-SceI and PI-SceI are homing endonuclease sites; MCS: multiple cloning sites.
  • Figure 2 is the core part of the Pubi-DI vector; among them, LoxP, 1L, and 2R are recombination sites; I-Sce I and PI-Sce I are homing endonuclease sites; pUbi: maize ubiquitin gene promoter; Tnos : Nos terminator; MCS: multiple cloning site.
  • Figure 3 is the core part of the 2 ⁇ P35s-DII vector; among them, LoxP, 2L, and 1R are recombination sites; I-SceI and PI-SceI are homing endonuclease sites; 2 ⁇ P35s: cauliflower mosaic virus 35S enhanced promoter; T35S: cauliflower mosaic virus 35S terminator; MCS: multiple cloning site.
  • Figure 4 is the core part of the pYL1305 vector; among them, LoxP and A2 are recombination sites; I-Sce I is classified as an endonuclease site; HPT, hygromycin resistance gene; LB, left border; RB, right border.
  • Figure 5 is the core part of the GOC-pYL1305 vector; among them, LoxP, 1L, and 2RL are recombination sites; I-Sce I and PI-Sce I are homing endonuclease sites; pUbi: maize ubiquitin gene promoter; Tnos : Nos terminator; 2 ⁇ P35s: cauliflower mosaic virus 35S enhanced promoter; T35S: cauliflower mosaic virus 35S terminator; MCS: multiple cloning site; HPT, hygromycin resistance gene; LB, left border; RB, right boundary.
  • Figure 6 shows the positive detection of transgenic plants
  • CK+ positive control
  • WT wild-type potato
  • GOC-1, GOC-2 are GOC-pBIA13 transgenic seedlings.
  • Figure 7 shows the determination of the GLO, KatE and OxXO enzyme activities of II-GOC plants (*p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001);
  • Panel a GLO enzyme activity determination of II-GOC plants
  • Panel b KatE enzyme activity determination of II-GOCGOC plants
  • Panel c OxO enzyme activity determination of II-GOCGOC plants.
  • Figure 9 shows the determination of the chlorophyll content of II-GOC plants (*p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001).
  • Figure a Determination of the fresh weight of individual aerial parts of II-GOC plants
  • Figure b Determination of the dry weight of individual aerial parts of II-GOC plants
  • Figure c Determination of the fresh weights of tubers per plant of II-GOC
  • Figure d II-GOC Determination of tuber dry weight per plant
  • Figure e Statistics of tuber numbers per plant of II-GOC.
  • Figure 11 shows the determination of the net photosynthetic rate and photorespiration rate index of II-GOC plants (*p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001).
  • Figure 12 shows the positive test result of the III-GOC transgenic vaccine.
  • Figure 13 shows the determination of the GLO, CAT and OxO enzyme activities of III-GOC plants (*p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001).
  • a figure the GLO enzyme activity measurement of III-GOC plants
  • b figure the CAT enzyme activity measurement of III-GOC plants
  • c figure the OxO enzyme activity measurement of III-GOC plants.
  • Figure 14 shows the determination of the chlorophyll content of III-GOC plants (*p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001).
  • the inventors unexpectedly found that the first nucleic acid construct with a specific structure, or the first vector containing the first acid construct, and the second nucleic acid construct or the second nucleic acid construct
  • the introduction of the second vector, the third nucleic acid construct, or the third vector containing the third nucleic acid construct into a plant can significantly regulate the agronomic traits of the plant, for example, (i) reducing the efficiency or rate of photorespiration; and/or (ii) Increase photosynthesis rate or efficiency; and/or (iii) increase yield; and/or (iv) increase biomass; and/or (v) increase chlorophyll content.
  • the present invention also obtains a new fusion protein.
  • the fusion protein of the present invention includes a specific chloroplast signal peptide and two or more photorespiration metabolic modification branch proteins.
  • the fusion protein of the present invention can significantly regulate the agronomy of plants. Traits.
  • the present invention also unexpectedly discovered that introducing a nucleic acid construct with a specific structure or a vector containing a nucleic acid construct with a specific structure into a plant cell can significantly regulate the agronomic traits of the plant. On this basis, the inventor completed the present invention.
  • the term "about” may refer to a value or composition within an acceptable error range of a specific value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “substantially consisting of” or “consisting of”.
  • Sequence identity is passed along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) ) Compare two aligned sequences and determine the number of positions where the same residue appears. Normally, this is expressed as a percentage.
  • a predetermined comparison window which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein
  • the present invention provides a reagent combination, which includes:
  • P1 is the first promoter
  • Z1 is an optional coding sequence encoding a chloroplast signal peptide
  • Z2 is a coding sequence encoding a first photorespiration metabolic modification branch protein, and the first photorespiration metabolic modification branch protein is glycolate oxidase (GLO);
  • Z3 is a terminator
  • P2 is the second promoter
  • Z4 is an optional coding sequence encoding a chloroplast signal peptide
  • Z5 is a coding sequence encoding a second photorespiration metabolic modification branch protein, and the second photorespiration metabolic modification branch protein is oxalate oxidase (OXO);
  • Z6 is the terminator
  • P3 is the third promoter
  • Z7 is an optional coding sequence encoding a chloroplast signal peptide
  • Z8 is a coding sequence encoding a third photorespiration metabolic modification branch protein, and the third photorespiration metabolic modification branch protein is catalase (CATC);
  • Z9 is a terminator
  • the present invention provides another reagent combination, which includes:
  • P1 is the first promoter
  • Z1 is an optional coding sequence encoding a chloroplast signal peptide
  • Z2 is a coding sequence encoding a fusion protein selected from the group consisting of glycolate oxidase + oxalate oxidase, glycolate oxidase + catalase, oxalate oxidase + catalase, glycolate oxidase +oxalate oxidase + catalase, or a combination thereof;
  • Z3 is a terminator
  • P2 is the second promoter
  • Z4 is an optional coding sequence encoding a chloroplast signal peptide
  • Z5 is a coding sequence encoding a third element, which is an element other than the fusion protein, and the third element is one of glycolate oxidase, oxalate oxidase, and catalase ;
  • Z6 is a terminator
  • the second nucleic acid construct does not exist.
  • the various elements used in the constructs of the present invention can be obtained by conventional methods, such as PCR methods, fully artificial chemical synthesis methods, and enzyme digestion methods, and then connected together by well-known DNA ligation techniques to form the constructs of the present invention. .
  • the vector of the present invention is transformed into plant cells so as to mediate the integration of the vector of the present invention into the chromosomes of the plant cells to produce genetically engineered plant cells.
  • the genetically engineered plant cell of the present invention is regenerated into a plant body, thereby obtaining a genetically engineered plant.
  • nucleic acid construct constructed by the present invention can be introduced into plant cells through conventional plant recombination technology (for example, Agrobacterium transformation technology), thereby obtaining the nucleic acid construct (or the vector carrying the nucleic acid construct) Or obtain a plant cell in which the nucleic acid construct is integrated in the genome.
  • plant recombination technology for example, Agrobacterium transformation technology
  • the chloroplast signal peptide becomes the chloroplast transit peptide, which can refer to the following amino acid sequence: it may exist at both ends of the target protein, preferably the N-terminus, which will guide the target protein to be positioned and transported into the chloroplast, and the protein may be mature
  • the chloroplast signal peptide is cleaved by signal peptidase after the completion of transport.
  • Chloroplast transit peptides usually contain about 20-150 amino acids, and these signal peptides have been observed to contain some common features.
  • the signal peptide contains very few negatively charged amino acids (such as aspartic acid, glutamic acid, asparagine or glutamine) (if any); the N-terminal region of the signal peptide lacks charged amino acids, glycine And proline; the central region of the signal peptide is likely to contain a very high proportion of basic or hydroxylated amino acids (such as serine and threonine); and the C-terminus of the signal peptide is likely to be rich in arginine and has a composition The ability of amphipathic ⁇ -sheet structure.
  • amino acids such as aspartic acid, glutamic acid, asparagine or glutamine
  • the chloroplast signal peptide of the present invention is a specific signal peptide, such as rbcS chloroplast signal peptide (StTP-58AA, NPTPC for short), PCS1, and TPC chloroplast signal peptide, which can significantly improve the efficiency of introduction.
  • rbcS chloroplast signal peptide StTP-58AA, NPTPC for short
  • PCS1 PCS1
  • TPC chloroplast signal peptide which can significantly improve the efficiency of introduction.
  • the branch proteins of photorespiration metabolism modification include glycolate oxidase (GLO), catalase (CAT), and oxalate oxidase (OXO).
  • GLO glycolate oxidase
  • CAT catalase
  • OXO oxalate oxidase
  • the three proteins form a metabolic pathway in the plant.
  • GLO oxidizes the photorespiration intermediate product glycolate to oxalate and hydrogen peroxide (H 2 O 2 ) with the participation of oxygen
  • CAT oxidizes hydrogen peroxide to water and oxygen
  • OXO with the participation of oxygen
  • Oxalic acid is oxidized to CO 2 , and the generated CO 2 directly enters the Calvin cycle.
  • the photorespiration branch occurs in the chloroplast, reduces the metabolic steps of glycolic acid entering the peroxidase and mitochondria, shortens the metabolic process, realizes the enrichment of CO 2 in the chloroplast, inhibits photorespiration, and enhances the photosynthetic efficiency.
  • the photorespiration metabolic modification branch protein described in the present invention can be derived from eukaryotes and prokaryotes.
  • the photorespiration metabolic modification branch protein includes two or more selected from the following group: (a) OsGLO3; (b) OsOXO3; (c) OsCATC or KatE (also known as EcCAT) ).
  • three photorespiration modification branches are constructed in plants, which are named I-GOC, II-GOC, and III-GOC, respectively.
  • I-GOC was constructed in rice, including three genes encoding OsGLO3, OsOXO3, and OsCAT protein; II-GOC was constructed in potato, including three genes encoding OsGLO3, OsOXO3, and KatE protein; III-GOC was constructed in potato , Including genes encoding the fusion protein (OsGLO3*OsCAT) and OsOXO3, respectively.
  • the photorespiration modification branch protected by the present invention is not limited to the type of GOC disclosed in the present invention.
  • the three genes constituting the modified branch can all be performed using homologous genes from different sources, with certain homology, and similar functions.
  • the three genes can be independently constructed in one or more vectors, or two fusions and the other can be constructed independently in one or more vectors. Among them, three gene fusions can also be constructed in one vector.
  • Various GOC branches improved based on the present invention are all included in the scope of the present invention, and their effects are basically equal to or better than those of the GOC branch disclosed in the present invention. Effect.
  • the GOC branch constructed by the present invention can be applied to plants, preferably, applied in C3 plants, more preferably applied in dicotyledonous plants, and more preferably applied in potato crops.
  • the "GOC branch”, “GOC photorespiration branch”, “photorespiration metabolic modification branch” and “photorespiration branch” in the present invention can be used interchangeably.
  • GLO protein As used herein, the terms "GLO protein”, “Glycolate oxidase” and “glycolate oxidase” are used interchangeably.
  • OXO protein As used herein, the terms "OXO protein”, “Oxalate oxidase gene” and “oxalate oxidase” are used interchangeably.
  • CATC protein As used herein, the terms “CATC protein”, “catalase”, and “catalase” are used interchangeably.
  • the present invention relates to a GLO protein and variants thereof.
  • the amino acid sequence of the GLO protein is shown in SEQ ID NO.:1.
  • the present invention also relates to an OXO protein and variants thereof.
  • the amino acid sequence of the OXO protein is shown in SEQ ID NO.: 3.
  • the present invention also relates to a CATC protein and variants thereof.
  • the amino acid sequence of the CATC protein is shown in SEQ ID NO.: 5 or 7.
  • the present invention also includes the sequence shown in SEQ ID NO. 1 or 3 or 5 or 7 of the present invention having 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) homologous polypeptides or proteins with the same or similar functions.
  • the "same or similar function” mainly refers to the function of regulating agronomic traits of plants, such as (i) reducing the efficiency or rate of photorespiration; and/or (ii) increasing the rate or efficiency of photosynthesis; and/or (iii) increasing yield ; And/or (iv) increase biomass; and/or (v) increase chlorophyll content.
  • the protein of the present invention can be a recombinant protein, a natural protein, or a synthetic protein.
  • the protein of the present invention can be a natural purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plants, insect and mammalian cells) using recombinant technology.
  • a prokaryotic or eukaryotic host for example, bacteria, yeast, higher plants, insect and mammalian cells
  • the protein of the present invention may be glycosylated or non-glycosylated.
  • the protein of the present invention may also include or not include the initial methionine residue.
  • the present invention also includes GLO protein, or OXO protein or CATC protein or GLO protein, or OXO protein or CATC protein fragments and the like having GLO protein, or OXO protein or CATC protein or its activity.
  • fragment and “analog” refer to a protein that substantially maintains the same biological function or activity as the natural GLO protein, or OXO protein, or CATC protein of the present invention.
  • the mutein fragment, derivative or analogue of the present invention may be (i) a mutein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acids
  • the residue may or may not be encoded by the genetic code, or (ii) a mutein with a substitution group in one or more amino acid residues, or (iii) a mature mutein and another compound (such as an extended mutein) Half-life compounds, such as polyethylene glycol) fused to form a mutant protein, or (iv) additional amino acid sequence fused to the mutant protein sequence to form a mutant protein (such as leader sequence or secretory sequence or used to purify the mutant protein)
  • the sequence or proprotein sequence, or the fusion protein formed with the antigen IgG fragment According to the teachings herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
  • conservatively substituted amino acids are preferably generated by amino acid substitution
  • substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Preferred substitution Ala(
  • the present invention also includes the natural GLO protein, or OXO protein or CATC protein of the present invention having 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most Preferably 98% or more, such as 99%) homologous polypeptides or proteins with the same or similar functions.
  • the protein variant can be obtained by substituting, deleting or adding at least one amino acid by several (usually 1-60, preferably 1-30, more preferably 1-20, most preferably 1-10). Derivative sequences, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • the function of the protein when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed, and the addition of one or several amino acids to the C-terminal and/or ⁇ terminal usually does not change the function of the protein.
  • the present invention includes that the difference between the natural GLO protein, or OXO protein or CATC protein analogue and the natural GLO protein, or OXO protein or CATC protein may be the difference in amino acid sequence, or the difference in the modified form that does not affect the sequence, or Both.
  • Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, site-directed mutagenesis or other known biological techniques.
  • Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins exemplified above.
  • Modified (usually without changing the primary structure) forms include: chemically derived forms of proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those that undergo glycosylation modifications during protein synthesis and processing. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). In addition, the mutant protein of the present invention can also be modified.
  • Modified (usually not changing the primary structure) forms include: in vivo or in vitro chemically derived forms of mutein such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modifications during the synthesis and processing of the mutant protein or during further processing steps. This modification can be accomplished by exposing the mutein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes mutant proteins that have been modified to increase their resistance to proteolysis or optimize their solubility.
  • the present invention also provides a polynucleotide sequence encoding GLO protein, or OXO protein or CATC protein.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the form of DNA includes: DNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded.
  • a polynucleotide encoding a mature polypeptide includes: a coding sequence that only encodes the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequences) of the mature polypeptide and non-coding sequences.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention.
  • the variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants.
  • These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the function of the encoded polypeptide. .
  • genes provided in the examples of the present invention are derived from rice and Escherichia coli, they are derived from other similar plants (especially plants belonging to the same family or genus as rice) or other similar bacteria that are different from those of the present invention.
  • the sequence preferably, the sequence is shown in SEQ ID NO.: 2 or 4 or 6 or 10.
  • the coding nucleic acid sequence of the present invention can be constructed by a method of synthesizing the nucleotide sequence in segments and then performing overlap extension PCR.
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotide of the present invention under stringent conditions (or stringent conditions).
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, and more Fortunately, hybridization occurs when more than 95%.
  • proteins and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, are purified to homogeneity.
  • the full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the method of using PCR technology to amplify DNA/RNA is preferably used to obtain the polynucleotide of the present invention.
  • the RACE method RACE-cDNA end rapid amplification method
  • the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein. And can be synthesized by conventional methods.
  • the amplified DNA/RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.
  • fusion protein of the present invention or “polypeptide” refers to the fusion protein of the present invention.
  • the structure of the fusion protein of the present invention is shown in the following formula I or I':
  • A is the chloroplast signal peptide
  • B is a photorespiration metabolic modification branch protein, which is selected from two or more of the following group: glycolate oxidase (GLO), oxalate oxidase (OXO), catalase (CATC); each "-" is independently a connecting peptide or a peptide bond or a non-peptide bond.
  • GLO glycolate oxidase
  • OXO oxalate oxidase
  • CAC catalase
  • the length of the connecting peptide has an effect on the activity of the fusion protein.
  • the preferred length of the connecting peptide is 1-100aa, preferably, 15-85aa, more preferably, 20-70aa, more preferably, 24- 32aa.
  • a preferred connecting peptide is shown in SEQ ID NO.: 9 or any one of the polypeptides 11-16.
  • the term "fusion protein” also includes the variant form shown in SEQ ID NO.: 8 that has the above-mentioned activity.
  • variant forms include (but are not limited to): 1-3 (usually 1-2, more preferably 1) amino acid deletion, insertion and/or substitution, and addition or addition at the C-terminus and/or N-terminus
  • One or several (usually 3 or less, preferably 2 or less, more preferably 1 or less) amino acids are deleted.
  • the term also includes the polypeptide of the present invention in monomeric and multimeric forms.
  • the term also includes linear and non-linear polypeptides (such as cyclic peptides).
  • the present invention also includes active fragments, derivatives and analogs of the above-mentioned fusion protein.
  • fragment refers to a polypeptide that substantially retains the function or activity of the fusion protein of the present invention.
  • polypeptide fragments, derivatives or analogues of the present invention can be (i) one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) in one or more A polypeptide with substitution groups in three amino acid residues, or (iii) a polypeptide formed by fusing an antigenic peptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide formed by fusion to this polypeptide sequence (a fusion protein formed by fusion with a leader sequence, a secretory sequence, or a tag sequence such as 6His). According to the teachings herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
  • a preferred type of active derivative means that compared with the amino acid sequence of Formula I, there are at most 3, preferably at most 2, and more preferably at most 1 amino acid replaced by an amino acid with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.
  • substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Preferred substitution Ala(
  • the present invention also provides analogs of the fusion protein of the present invention.
  • the difference between these analogs and the polypeptide shown in SEQ ID NO.: 8 may be the difference in the amino acid sequence, the difference in the modification form that does not affect the sequence, or both.
  • Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modified (usually not changing the primary structure) forms include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides produced by glycosylation modifications during the synthesis and processing of the polypeptide or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes polypeptides that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.
  • amino acid sequence of the fusion protein of the present invention is shown in SEQ ID NO.: 8.
  • the active substance of the present invention can be prepared into agricultural preparations by conventional methods, such as solutions, emulsions, suspensions, powders, foams, Pastes, granules, aerosols, natural and synthetic materials impregnated with active substances, microcapsules in polymers, coatings for seeds.
  • the active compound is mixed with extenders, which are liquid or liquefied gaseous or solid diluents or carriers, and optionally surfactants, emulsifiers and/or Dispersant and/or foam former.
  • extenders which are liquid or liquefied gaseous or solid diluents or carriers, and optionally surfactants, emulsifiers and/or Dispersant and/or foam former.
  • organic solvents can also be used as additives.
  • a liquid solvent When a liquid solvent is used as a diluent or carrier, it is basically suitable, such as: aromatic hydrocarbons, such as xylene, toluene or alkyl naphthalene; chlorinated aromatic or chlorinated aliphatic hydrocarbons, such as chlorobenzene, vinyl chloride Or dichloromethane; aliphatic hydrocarbons, such as cyclohexane or paraffin, such as mineral oil fractions; alcohols, such as ethanol or ethylene glycol and their ethers and lipids; ketones, such as acetone, methyl ethyl ketone, methyl isobutyl Base ketone or cyclohexanone; or uncommon polar solvents such as dimethylformamide and dimethyl sulfoxide, and water.
  • aromatic hydrocarbons such as xylene, toluene or alkyl naphthalene
  • chlorinated aromatic or chlorinated aliphatic hydrocarbons such as chlor
  • the diluent or carrier of liquefied gas refers to a liquid that will become a gas at normal temperature and pressure, such as aerosol propellants such as halogenated hydrocarbons and butane, propane, nitrogen and carbon dioxide.
  • the solid carrier can be ground natural minerals, such as kaolin, clay, talc, quartz, activated clay, montmorillonite, or diatomaceous earth, and ground synthetic minerals, such as highly dispersed silicic acid, alumina and silicate. .
  • the solid carrier for the particles is crushed and graded natural zircon, such as calcite, marble, pumice, sepiolite and dolomite, as well as particles synthesized from inorganic and organic coarse powder, and organic materials such as sawdust, coconut shell, Corn cobs and tobacco stalks, etc.
  • Nonionic and anionic emulsifiers can be used as emulsifiers and/or foam formers.
  • polyoxyethylene-fatty acid esters polyoxyethylene-fatty alcohol ethers, such as alkyl aryl polyglycol ethers, alkyl sulfonates, alkyl sulfates, aryl sulfonates and white Protein hydrolysate.
  • Dispersants include, for example, lignin sulfite waste liquor and methyl cellulose.
  • Binders such as carboxymethyl cellulose and natural and synthetic polymers in the form of powders, granules or emulsions such as gum arabic, polyvinyl alcohol and polyvinyl acetate can be used in the formulation.
  • Colorants such as inorganic dyes such as iron oxide, diamond oxide and Prussian blue; organic dyes such as organic dyes such as azo dyes or metal phthalocyanine dyes; and trace nutrients such as iron, manganese, boron, and copper , Cobalt, aluminum and zinc salts.
  • the "agricultural preparation” is usually an agricultural plant growth regulator, which contains the fusion protein of the present invention, or its encoding gene, or its expression vector as an active ingredient for regulating agronomic traits of plants; and agriculture Acceptable carrier.
  • the "agriculturally acceptable vector” is an agrochemically acceptable solvent or suspending agent used to deliver the fusion protein of the present invention, or its encoding gene, or its expression vector (vector) to plants. Or excipients.
  • the carrier can be liquid or solid.
  • the agriculturally acceptable carrier suitable for the present invention is selected from the group consisting of water, buffer, DMSO, surfactants such as Tween-20, or a combination thereof. Any agriculturally acceptable carrier known to those skilled in the art can be used in the present invention.
  • the agricultural preparations of the present invention can be made into a mixture with other substances regulating plant agronomic traits and present in their commercial preparations or in dosage forms prepared from these preparations.
  • These other substances regulating plant agronomic traits include (but are not limited to) ): photorespiration inhibitor, selected from: carbon dioxide, glyoxylic acid, glutamic acid, aspartic acid, ⁇ -hydroxysulfonate, 2,3-epoxyacetic acid, isoniazid, NaHSO3; photosynthesis enhancer , Selected from potassium bicarbonate, sodium bisulfate, diamine, choline chloride.
  • the dosage form of the agricultural preparation of the present invention can be various, as long as the dosage form can make the active ingredient reach the plant body effectively.
  • the preferred agricultural preparation is a spray Agent or solution formulation.
  • the agricultural preparations of the present invention usually contain 0.0001-99wt%, preferably 0.1-90wt% of the total weight of the agricultural preparations of the fusion protein of the present invention, or its coding gene, or its expression vector (vector).
  • concentration of the compound of the present invention in the commercial preparation or use dosage form can vary within a wide range.
  • concentration of the fusion protein of the present invention, or its encoding gene, or its expression vector (vector) in commercial preparations or dosage forms can range from 0.0000001-100% (g/v), preferably between 0.0001 and 1% (g/v) )between.
  • a method for regulating agronomic traits of plants specifically, applying the agricultural preparations of the invention to plants, thereby regulating the agronomic traits of plants, and the regulating and controlling the agronomic traits of plants includes:
  • Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the protein of the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • the promoter in the vector of the present invention can be a constitutive promoter, a tissue-specific promoter, or an inducible promoter. Preference is given to promoters suitable for use in plants.
  • the constitutive promoter of the present invention means that the gene expression level under its regulation is generally constant, and is not affected by time and space and external factors. It can be expressed in most plant tissues and there is no significant difference in the expression level of different tissue parts.
  • the constitutive promoters include the cauliflower mosaic virus (CaMV) 35S promoter, the ubiquitin 1 promoter (Ubi-1), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter, and the nopaline synthase promoter ( NOS), pEmu promoter, rubisco promoter, GRP1-8 promoter, octopine synthase (OCS), rice actin-1 promoter (Rac1), rice sucrose synthesis gene (Rss1) promoter, corn ethanol Hydrogenase gene Adh1 promoter, tobacco anther cell specific promoter TA29, and other transcription initiation regions from various plant genes known to those skilled in the art.
  • CaMV cauliflower mosaic virus
  • Ubi-1 the ubiquit
  • the tissue-specific promoter of the present invention means that the gene regulated by it is only expressed in certain specific parts or organs, and this specificity is usually based on specific tissue cells and chemical and physical signals. Localizing and expressing foreign genes in transgenic plants can not only reduce plant burdens and reduce the impact on crop agronomic traits, but also increase the concentration of foreign genes in specific parts and increase the effect of transgenes.
  • the construction vector of the photorespiration modification branch of the present invention can use tissue-specific promoters under necessary conditions to drive it to express efficiently in certain tissue parts, such as plant aerial parts, and further such parts as plant leaves, seeds, fruits, etc. .
  • the tissue-specific promoters include: fruit-specific promoters.
  • fruit-specific promoters mainly include E8, 2A11, 2A12, PG, MCP I, B33 and ACC oxidase promoters
  • stem and leaf specific expression promoters such as pyruvate orthophosphate dikinase (PPDK) promoter, light-harvesting chlorophyll a/b protein complex gene (CAB) promoter, ribulose diphosphate carboxylase small subunit gene (rbcS) promoter, etc.
  • PPDK pyruvate orthophosphate dikinase
  • CAB chlorophyll a/b protein complex gene
  • rbcS ribulose diphosphate carboxylase small subunit gene
  • the inducible promoter of the present invention means that under the stimulation of certain physical or chemical signals, the transcription level of the target gene driven by this type of promoter can be greatly increased.
  • the inducible promoters include: Adh1 promoter that can be induced by hypoxia or cold stress, Hsp70 promoter that can be induced by heat stress, PPDK promoter and PEP carboxylase (pepcarboxylase) that can be induced by light Promoters; chemically inducible promoters are also available, such as safener-induced In2-2 promoter, androgen-induced ERE promoter, and Axig1 promoter.
  • the present invention selects specific promoters suitable for plant cells, such as NOS promoter, 35S promoter, DD45 promoter, U6 promoter, Lat52 promoter, etc.
  • the proteins are usually connected by some flexible short peptides, namely Linker (connecting peptide sequence).
  • Linker connecting peptide sequence
  • the Linker can use XTEN.
  • the vector is not particularly limited, any binary vector is acceptable, not limited to the pCambia vector, nor is it limited to these two types of resistance.
  • Any vector that meets the following requirements can be used in the present invention: (1) It can be transformed into plants mediated by Agrobacterium; (2) allow RNA to be transcribed normally; (3) allow plants to acquire new traits.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • the vector is selected from the group consisting of pYL322d1, pYL322d2, pYL1305, pYLTAC380GW, or a combination thereof.
  • the above-mentioned vector is introduced into the plant recipient by a suitable method.
  • the introduction methods include but are not limited to: Agrobacterium transfection method, gene gun method, microinjection method, electric shock method, ultrasonic method and polyethylene glycol (PEG)-mediated method.
  • Recipient plants include, but are not limited to, Arabidopsis, potato, sweet potato, purple sweet potato, yam, taro, cassava, chrysanthemum, rice, wheat, barley, corn, sorghum, soybean, peanut, millet, quinoa, tomato, tobacco , Rape, cabbage, spinach, lettuce, cucumber, chrysanthemum, water spinach, celery, lettuce, millet, peanut, kiwi, cotton, strawberry, peony, perfume lily, tulip, mulberry, apple, pear, peach, cherry, pomegranate.
  • genetically engineered plants are obtained by the Agrobacterium soaking method.
  • the present invention finds for the first time that introducing a nucleic acid construct with a specific structure or a vector containing a nucleic acid construct with a specific structure into plant cells can significantly regulate the agronomic traits of the plant, such as (i) photosynthetic rate; and/or (ii) ) Biomass; and/or (iii) Chlorophyll.
  • the present invention found for the first time that the fusion protein of the present invention can significantly regulate agronomic traits of plants, such as (i) photosynthetic rate; and/or (ii) biomass; and/or (iii) chlorophyll.
  • the present invention uses all or part of the plant's own genes to reduce the possibility of biological rejection and has higher safety.
  • the present invention finds for the first time that the fusion protein of the present invention significantly improves photosynthetic rate, increases biomass, and increases chlorophyll.
  • This invention is of profound significance for the in-depth elucidation of the mechanism of high light efficiency, the production of crops and renewable energy, and even the reduction of CO 2 emissions.
  • the GOC photorespiration metabolic transformation branch can be transferred to different C3 plants for cultivation The kind that is more high-yield.
  • Japonica rice is a conventional commercial product.
  • the Pubi promoter sequence and Tnos terminator sequence are derived from the vector pYLCRISPR/Cas9Pubi-H (provided by researcher Liu Yaoguang from South China Agricultural University, Ma, X., Zhang, Q., et al. A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular plant 2015, 8:1274-1284).
  • 2 ⁇ P35s-DII for the vector pYL322d2 (provided by Researcher Liu Yaoguang, South China Agricultural University, Zhu, Q., Yu, S., et al. Development of "Purple Endosperm Rice” by Engineering Anthocyanin Biosynthesis in the Endosperm with High-Effency Transgene Stacking System. Molecular plant, 2017, 10(7): 918-929).
  • the modification method is to use conventional molecular cloning methods to insert the 2 ⁇ P35s promoter sequence and the T35S terminator sequence between SalI and Hind III between EcoRI and XhoI in the multiple cloning site of the vector pYL322d2.
  • the vector map is shown in Figure 3. Among them, the 2 ⁇ P35s promoter sequence and the T35S terminator sequence are derived from the vector pYLCRISPR/Cas9Pubi-H (provided by researcher Liu Yaoguang of South China Agricultural University).
  • the vector is modified from the commercial vector pCAMBIA1305.1.
  • the modification method is to use conventional molecular cloning methods to insert the loxP/Cre homologous recombination sequence into the Pst I in the multiple cloning site region of the pCAMBIA1305.1 vector.
  • the vector map is shown in the figure 4.
  • the loxP/Cre homologous recombination sequence is derived from the vector pYLTAC380GW.
  • pYLTAC380GW is in the literature "Zhu, Q., Yu, S., et al. Development of "Purple Endosperm Rice” by Engineering Anthocyanin Biosynthesis in the Endosperm with a High-Efficiency Transgene Stacking System. Molecularplant, 2007, 10(7) Disclosure in 918-929".
  • OsGLO, KatE and OsOXO are co-expressed to construct II-GOC photorespiration branch in rice.
  • Example 1.1 Obtaining TPC-OsGLO3, TPC-OsOXO3, TPC-KatE fusion protein expression genes
  • the amino acid sequences of OsGLO3, OsOXO3 and KatE are shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 7, respectively.
  • the primers were designed according to the cDNA sequences of OsGLO3 and TPC provided by NCBI (http://www.ncbi.nlm.nih.gov/).
  • the PCR amplified product was subjected to 1% agarose gel electrophoresis, and the DNA fragments of OsGLO3 (about 1100 bp) and TPC (about 150 bp) were recovered and purified; the recovered fragments were digested and consumed by BglII, and then used T4 Ligase is ligated and used as a template for the second round of PCR amplification under the guidance of primers TPC-1F and OsGLO3-R.
  • the amplified product was subjected to 1% agarose gel electrophoresis, and the DNA fragment (about 1300 bp) of TPC-OsGLO3 was recovered and purified, and the fragment was cloned into the pMD18-T vector to obtain the pMD18-TPC-OsGLO3 vector. Sequencing.
  • the primers were designed according to the cDNA sequence of OsOXO3 and TPC provided by NCBI (http://www.ncbi.nlm.nih.gov/).
  • the OsOXO3 and TPC genes were amplified by conventional methods under the guidance of primers OsOXO3-F and OsOXO3-R, and primers TPC-1F and TPC-2R.
  • the PCR amplified product was subjected to 1% agarose gel electrophoresis, and the DNA fragments of OsOXO3 (about 700 bp) and TPC (about 150 bp) were recovered and purified; the recovered fragments were digested and consumed by KpnI, and then used T4 Ligase is ligated and used as a template for the second round of PCR amplification under the guidance of primers TPC-1F and OsOXO3-R.
  • the amplified product was subjected to 1% agarose gel electrophoresis, and the DNA fragment (about 900bp) of TPC-OsOXO3 was recovered and purified, and the fragment was cloned into the pMD18-T vector (purchased from TAKARA) to obtain pMD18 -TPC-OsOXO3 vector, sequencing.
  • the primers were designed according to the cDNA sequence of katE provided by NCBI (http://www.ncbi.nlm.nih.gov/).
  • the KatE and TPC genes were amplified by conventional methods, respectively.
  • the PCR amplified products were subjected to 1% agarose gel electrophoresis, and the DNA fragments of KatE (about 1500bp) and TPC (about 150bp) were recovered and purified; the recovered fragments were digested with KpnI and consumed with T4 Ligase is ligated and used as a template to perform the second round of PCR amplification under the guidance of primers TPC-1F and KatE-R.
  • the amplified product was subjected to 1% agarose gel electrophoresis, and the DNA fragment (about 1700bp) of TPC-KatE was recovered and purified, and the fragment was cloned into the pMD18-T vector (purchased from TAKARA) to obtain pMD18 -TPC-KatE vector, sequencing.
  • the primers were designed according to the TPC-OsGLO3 fusion protein expression gene sequence, and the pMD18-TPC-OsGLO3 vector described in Example 1.1 was used as a template. Under the guidance of the primers TPC-OsGLO3-F and TPC-OsGLO3, TPC-OsGLO3 was amplified by conventional methods The gene was recovered by electrophoresis and cloned into the recombinant donor vector Pubi-DI (see Figure 2 for the vector map) between the PstI and BamHI restriction sites of the multiple cloning sites to obtain the vector Pubi-DI-TPC-OsGLO3.
  • Pubi-DI see Figure 2 for the vector map
  • TPC-OsOXO3 fusion protein expression gene sequence uses the pMD18-TPC-OsOXO3 vector described in Example 1.1 as a template, and use conventional methods for amplification under the guidance of the primers TPC-OsOXO3-F and TPC-OsOXO3-R
  • the TPC-OsOXO3 gene was recovered by electrophoresis and cloned into the plant transient expression vector 2 ⁇ P35s-DII between the EcoRI and BamHI restriction sites to obtain the vector 2 ⁇ P35s-DII-TPC-OsOXO3.
  • Design primers based on the 2 ⁇ P35s promoter sequence and T35s terminator sequence on the 2 ⁇ P35s-DII vector Using the above 2 ⁇ P35s-DII-TPC-OsOXO3 vector as the template, set the primers in the primers 2 ⁇ P35s-F and T35s-R. Under the guidance, the TPC-OsOXO3 gene expression cassette 2 ⁇ P35s-TPC-OsOXO3-T35s was amplified by conventional methods, and the expression cassette was cloned into the pMD18-T vector after electrophoresis recovery to obtain the vector pMD18-2 ⁇ P35s-TPC-OsOXO3-T35s , And sequenced.
  • Design primers according to the TPC-KatE fusion protein expression gene sequence use the pMD18-TPC-KatE vector described in Example 1.1 as a template, and use conventional methods to amplify under the guidance of primers TPC-KatE-F and TPC-KatE-R TPC-KatE gene, after electrophoresis recovery, clone the gene into the recombinant donor vector Pubi-DI (vector map is shown in Figure 2) between the HindIII and SpeI restriction sites of the multiple cloning site to obtain the vector Pubi-DI-TPC- KatE.
  • Pubi-DI vector map is shown in Figure 2
  • the TPC-KatE gene expression cassette Pubi-TPC-KatE-Tnos was amplified by conventional methods.
  • the expression cassette was cloned into the pMD18-T vector to obtain the vector pMD18-Pubi-TPC-KatE-Tnos, and sequenced.
  • pYL1305 Take Pubi-DI-TPC-OsGLO3, 2 ⁇ P35s-DII-TPC-OsOXO3 and Pubi-DI-TPC-KatE as the donor vector, pYL1305 (see Figure 4 for the vector map) as the acceptor vector, refer to the method of Lin et al. (Lin L, Liu Y G, Xu X, et al.. Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system. Proceedings of the National Academy of Sciences, 2003, 100(10): 5962-5967) After three rounds of recombination, the GOC photorespiration metabolic modification branch vector GOC-pYL1305 was finally obtained.
  • the vector map is shown in Figure 5.
  • the specific reorganization method is as follows:
  • the plasmid concentration is controlled at 100-200 ng/ ⁇ L.
  • the digested plasmid is transformed into E. coli TOP10 competent cells (purchased in Takara, Japan) and coated with Incubate overnight on LB plates containing 50 mg/L kanamycin.
  • upstream primers and sequencing primers are both I-CeuI-F(5′- CCAACTATAACGGTCCTAAGGTAGCG-3' (SEQ ID NO.: 35)), the downstream primers for detection are selected according to the direction of the target gene access to the donor, if the access direction of the target gene is the same as the labeling direction of MCS (upstream is the LoxP site), Use reverse primers that access the target gene, and vice versa.
  • rice glycolate oxidase OsGLO
  • E. coli catalase KatE
  • rice oxalate oxidase OsOXO
  • E35S-PCS1-DII using pBIA13-PCS1-eGFP plasmid as template, using primers EcoRI-PCS1-F and KpnI-PCS1-R to amplify the PCS1 gene sequence, using EcoRI and KpnI double enzyme digestion to construct in the vector
  • the primer sequence is as follows:
  • Kpn I-PCS1-R 5'-CCATGGTACCCATGGAGGCCTTGATTGA-3' (SEQ ID NO.: 24)
  • E35S-PCS1-OXO3--DII (2) Construction of E35S-PCS1-OXO3--DII: Using pBIA13-CPTPC-OsOXO3-eGFP plasmid as a template, using primers KpnI-OXO3-F and BamHI-OXO3-R to amplify the OsOXO3 gene sequence, using KpnI and BamHI double Restriction digestion is constructed on the vector E35S-PCS1-DII, and the primer sequence is as follows:
  • KpnI-OXO3-F 5’-CATGGGTACCATGGAGTACGGCTTCAAA-3’ (SEQ ID NO.: 25)
  • PTPC-KpnI-F TATA GGTACC ATGGCTTCCTCTGTTAT (SEQ ID NO.: 27)
  • NPTPC-EcoRV-R TCGA GATATC GCACCTAACTCTTCCACCAT (SEQ ID NO.: 28)
  • GLO3-EcoRV-F AGAT GATATC ATGGAGCTAATCACAAACGT (SEQ ID NO.: 29)
  • GLO3-SmaI-NR TAAT CCCGGG ATGGTGATGGTGATGATGCC (SEQ ID NO.: 30)
  • PTPC-KpnI-F TATA GGTACC ATGGCTTCCTCTGTTAT (SEQ ID NO.: 31)
  • NPTPC-SalI-R AGAT GTCGAC GCACCTAACTCTTCCACCAT (SEQ ID NO.:32)
  • EcCAT-SmaI-NR TAAT CCCGGG GGCAGGAATTTTGTCAATCT SEQ ID NO.: 34
  • pBIA13 As the acceptor vector, perform the first round of loxP/Cre homologous recombination with the donor vector XNOS-NPTPC-GLO3-DI; perform the second round of loxP/Cre with the donor vector E35S-PCS1-OXO3-DII Homologous recombination; perform the third round of loxP/Cre homologous recombination with the donor vector 35S-NPTPC-KatE-DI, and finally obtain the multi-gene expression vector XNosGLO3-E35S-OXO3-35SKatE-pBIA13 (name: II-GOC-pBIA13; Referred to as II-GOC).
  • the extracted Agrobacterium plasmid (colonies grown in step 2) DNA was heat-shocked to transform E. coli Top10 competent cells, and the bacterial solution was smeared on an LB plate (containing kanamycin) and cultured overnight. On the second day, a single colony was picked and shaken overnight, and sent to the sequencing company for sequencing to ensure that it was a positive transformant. After the positive transformant is determined, the positive transformant is shaken in LB solution (containing the corresponding antibiotics) overnight for about 20 hours, 500 ⁇ L of the bacterial solution is collected, and an equal volume of 30% glycerol is added to a 1.5mL centrifuge tube and stored at -80°C for later use. .
  • Potato seed potatoes are cleaned and cut, put into GA3 (2mg L -1 ) and soaked for 30 minutes, half buried in a sand tray to accelerate germination (maintain humidity), and take the buds for use after about 3 weeks. Remove the buds and rinse with water for 30 minutes, under aseptic conditions, 75% alcohol for 1 minute, 0.1% mercury liters for 8 minutes, then wash with sterile water 5 times, dry on sterile filter paper and cut into small sections Connect to MS medium and cultivate. Select the well-growing sterile seedlings and cut them according to one leaf and one section, and propagate on MS medium. During the rapid propagation of test-tube plantlets, materials containing symbiotic bacteria are gradually eliminated, and the obtained sterile plantlets can be used for test-tube potato induction.
  • Cut test tube potatoes Each tube can be cut into about 3 pieces. After receiving it on MS medium, a large amount of buds can be taken in about 10 days to rapidly multiply or induce test tube potatoes.
  • test tube seedlings stored for a long time can be multiplied every 20 days.
  • the top bud of each seedling has 2 leaves that can be used to continue reproduction.
  • the stem below is cut according to one leaf and one section and enters the tube for induction.
  • test tube plantlets one leaf and one section
  • tube tube induction medium MS medium with 80g L -1 sucrose and 2g L -1 activated carbon
  • test-tube potatoes can be harvested for potato genetic transformation.
  • the OD value of the bacteria liquid is about 0.5-0.7.
  • the bacteria liquid was transferred to a 50mL centrifuge tube, and the bacteria liquid was collected by centrifugation at 4000 rpm at 4°C for 6 min. Discard the supernatant, add 20 mL of liquid MS medium, pipette and mix well for later use.
  • MS medium Add a small amount of liquid MS medium to the petri dish, transfer the harvested tube potatoes to the petri dish, and slice them, with a thickness of about 1-2 mm. Transfer the sliced potato chips into a centrifuge tube containing the bacterial solution. Place the centrifuge tube horizontally on the ultra-clean workbench and let it stand for 10 minutes, shaking gently once or twice during the period. Discard the staining solution, transfer the potato chips to a petri dish lined with filter paper to absorb the staining solution and transfer it to P1 medium (MS medium, add 6-BA 0.5mg L-1+ZT2.0mg L-1+GA30. 2mg L-1+IAA 1.0mg L-1, sucrose 30g L-1).
  • the immersed material was cultured in the dark for 48 hours, and then transferred to P2 medium (P1 medium supplemented with 200mg L-1 cephalosporin, 75mg L-1 kanamycin, 30g sucrose L-1) for differentiation and screening culture.
  • P2 medium P1 medium supplemented with 200mg L-1 cephalosporin, 75mg L-1 kanamycin, 30g sucrose L-1
  • the lateral buds produced within 2 weeks need to be cut off in time. Change the medium every 14 days until the potato slices differentiate into buds, then cut off the young buds (1-2cm), and transfer to P3 medium (MS medium with 200mg L-1 cephalosporin Rooting screening, 50mg L-1 kanamycin, 30g sucrose L-1).
  • the rooted seedlings will be multiplied and then transplanted. Before transplanting, clean the root medium of the seedlings to prevent root rot due to the medium attached to the roots. After washing, transplant the seedlings into soil culture.
  • II-GOC-2 Eighteen seedlings of II-GOC transgenic plants were screened in the kanamycin sulfate selection medium, and they were planted outdoors for preliminary phenotypic observation, and II-GOC-1 with phenotype II-GOC transgenic lines was selected.
  • II-GOC-2 carries out NPTII gene amplification test.
  • the DNA of II-GOC potato transgenic seedlings was extracted by the CTAB method; the DNA was used as a template and specific primers were used to amplify the NPTII gene. As shown in Figure 6, the obtained II-GOC-1 and II-GOC-2 transgenic plants are all positive seedlings.
  • CK+ positive control
  • WT wild-type potato
  • II-GOC-1, II-GOC-2 are II-GOC-pBIA13 transgenic seedlings
  • the blank control is an equal volume of dH 2 O instead of glycolic acid.
  • the preparation of the crude enzyme solution is the same as above.
  • Phosphate buffer PBS 50mM, pH 7.5
  • CAT enzyme activity ( ⁇ mo H 2 O 2 min -1 mg -1 protein): that is, the number of ⁇ mol of H 2 O 2 produced per milligram of protein in one minute.
  • reaction temperature is 30°C.
  • the LI-6800 built-in program is automatically completed.
  • the light intensity gradient from high to low is 2200, 2000, 1800, 1600, 1400, 1200, 1000, 800, 600, 400, 300, 200, 150, 100, 80, 50, 20, 0, each light intensity is After staying for about 2-3 minutes, determine the net photosynthetic rate.
  • CO2 cylinders The use of CO2 cylinders is automatically completed by the LI-6800 built-in program. Set the parameters during the measurement: airflow 500 ⁇ mol s-1; temperature 25°C, CO2 concentration 400 ⁇ mol mol-1; light intensity 1600 ⁇ mol m-2s-1, light induction for 0.5h before measurement.
  • the CO2 concentration gradient from high to low is 1600, 1400, 1200, 1000, 800, 600, 400, 300, 200, 100, 80, 60, 40, 20 ⁇ mol mol-1, and the net is measured after 4-6 minutes of adaptation at each concentration. Photosynthetic rate.
  • this study measured the GLO, KatE and OxO enzyme activities of II-GOC plants.
  • the results are shown in Figure 7(ab), II-GOC-1 and II-GOC
  • the GLO enzyme activity of the -2 transgenic strain was increased by 29.1% and 23.9% (p ⁇ 0.05), respectively, compared with the wild type; the KatE enzyme activity was increased by 15.8% and 17.2% (p ⁇ 0.05), respectively, compared with the wild type. Since OXO enzyme activity cannot be detected in wild-type potato leaves, the OXO enzyme activity detected in transgenic plants should be caused by the expression of the introduced OsOXO3 gene.
  • the II-GOC branch can effectively promote the growth of potatoes.
  • This study further conducted statistical analysis on the biomass and yield of the II-GOC plants, and found that relative to the wild type, the fresh weight of the above-ground part of the individual plants of II-GOC-1 and II-GOC-2 The dry weight increased by 9.65-22.18% and 5.9-10.4%, respectively; correspondingly, the tuber fresh weight and dry weight per plant increased by 10.34-11.14% and 11.8-14.7%, respectively, with significant differences; the number of potatoes per plant was both The increase of 3-5 indicates that the introduction of the II-GOC branch has a similar effect, increasing potato biomass and yield ( Figure 10, ae)
  • the photosynthesis and photorespiration indexes of II-GOC transgenic plants were measured during the tuber expansion period.
  • the net photosynthetic rate of wild-type plants is 23.32 ⁇ 1.58 ⁇ mol CO 2 m -2 s -1 ; the photorespiration rate is 8.68 ⁇ 1.23 ⁇ mol CO 2 m -2 s -1 ;
  • II-GOC-1 net photosynthetic rate and II-GOC-2 plants were 24.96 ⁇ 1.65 ⁇ mol CO 2 m -2 s -1 and 26.05 ⁇ 2.00CO 2 m -2 s -1 is; light respiration rate were 8.11 ⁇ 0.65 ⁇ mol CO 2 m - 2 s -1 and 7.64 ⁇ 1.42 ⁇ mol CO 2 m -2 s -1 .
  • the photosynthetic rate of II-GOC plants increased by 7.02-11.68%; the photorespiration rate decreased by 6.59-12.01%.
  • the results of this study are the same as the previous ones. Similar to the report, the creation of the II-GOC photorespiration metabolism branch in potato chloroplasts can also effectively shunt photorespiration metabolism, inhibit photorespiration and effectively improve the photosynthetic efficiency of the plant.
  • the II-GOC photorespiration branch constructed by the present invention including OsGLO, OsOXO, and KatE can effectively play a role in potatoes, increase the intercellular CO 2 concentration of GOC plants, increase the photosynthetic rate, reduce the photorespiration rate, and increase plant height and stem Rough, increase chlorophyll content, increase biomass and yield.
  • the fusion protein (OsGLO3*OsCAT) and OsOXO3 are co-expressed to construct a III-GOC branch.
  • the amino acid sequences of OsGLO3, OsOXO3 and OsCAT are shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, respectively.
  • pBIA13 As the acceptor vector, perform the first round of loxP/Cre homologous recombination with NOS-StTP-80AA-GLO3CATC-DI, and perform the second round of loxP/Cre homologous recombination with the donor vector E35S-PCS1-OXO3-DII; Finally, a multi-gene expression vector 35SGLO3CATC-E35SOXO3-pBIA13 (named III-GOC-pBIA13; abbreviated (III-GOC) was obtained.
  • III-GOC transgenic seedlings were screened out in the kanamycin sulfate selection medium, planted outdoors for preliminary phenotypic observation, and the phenotype III-GOC transgenic strains III-GOC-5 and III- were screened out.
  • GOC-6 carries out NPTII gene amplification test.
  • the DNA of III-GOC potato transgenic seedlings was extracted by the CTAB method; the DNA was used as a template and specific primers were used to amplify the NPTII gene. As shown in Figure 12, III-GOC-5 and III-GOC-6 were obtained.
  • III-GOC-6 chlorophyll a, chlorophyll b, and total chlorophyll increased in a statistically significant difference ( Figure 14).
  • III-GOC modified branch constructed by the present invention comprising (OsGLO3*OsCAT) and OsOXO3 can effectively play a role in potatoes, increasing the chlorophyll content, plant biomass and yield.

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Abstract

Application de protéine de ramification photorespiratoire dans la régulation de caractéristiques de plantes. Plus particulièrement, l'invention concerne une combinaison de réactifs et une protéine de fusion. La combinaison de réactifs ou la protéine de fusion comprend une glycolate oxydase ou un acide nucléique de codage de cette dernière, et/ou une oxalate oxydase ou un acide nucléique de codage de cette dernière, et/ou une catalase ou un acide nucléique de codage de cette dernière. L'importation de la combinaison de réactifs ou de la protéine de fusion dans des cellules végétales peut réguler de manière significative les caractéristiques agronomiques de plantes.
PCT/CN2021/072225 2020-01-17 2021-01-15 Application de protéine de ramification photorespiratoire dans la régulation de caractéristiques de plantes WO2021143866A1 (fr)

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