WO2022188289A1 - OsDREB1C及其编码基因在提高水稻光合效率中的应用 - Google Patents

OsDREB1C及其编码基因在提高水稻光合效率中的应用 Download PDF

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WO2022188289A1
WO2022188289A1 PCT/CN2021/100545 CN2021100545W WO2022188289A1 WO 2022188289 A1 WO2022188289 A1 WO 2022188289A1 CN 2021100545 W CN2021100545 W CN 2021100545W WO 2022188289 A1 WO2022188289 A1 WO 2022188289A1
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plant
photosynthesis
transport
protein
flowering time
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French (fr)
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周文彬
李霞
魏少博
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中国农业科学院作物科学研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • 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
    • 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 to the application of OsDREB1C and its encoding gene in improving the photosynthetic efficiency of rice in the field of biotechnology.
  • Heading date is one of the important agronomic traits of crops, which determines the season, regional adaptability and yield of rice. A suitable heading date is the guarantee of stable and high yield of crops. Breeding new early-maturing and high-yielding varieties has always been one of the main directions of crop genetics and breeding research. "High-yielding but not early-maturing, early-maturing but not high-yielding” is the so-called “excellent but not early, early but not excellent” phenomenon, which is a major problem in the cultivation of crop varieties.
  • the technical problem to be solved by the present invention is how to improve the photosynthesis of plants.
  • the present invention firstly provides any of the following applications of a protein or a substance that regulates the activity or content of the protein:
  • Said protein (whose name is OsDREB1C) is the following A1), A2), A3) or A4):
  • amino acid sequence is the protein of sequence 1;
  • amino acid sequence shown in SEQ ID NO: 1 in the sequence listing has undergone the substitution and/or deletion and/or addition of one or several amino acid residues and has the same function;
  • A3 A protein derived from rice, millet, maize, sorghum, goat grass, wheat or Brachypodium bismuth and having 64% or more identity with sequence 1 and the same function as the protein described in A1);
  • A4 A fusion protein obtained by linking a tag to the N-terminus or/and C-terminus of A1) or A2) or A3).
  • amino-terminal or carboxyl-terminal of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing can be attached with the tags shown in the following table.
  • the protein in the above A2) is a protein having 64% or more identity to the amino acid sequence of the protein shown in SEQ ID NO: 1 and having the same function. Said having 64% or more identity is having 64%, having 75%, having 80%, having 85%, having 90%, having 95%, having 96%, having 97%, having 98% or having 99% % identity.
  • the protein in the above A2) can be artificially synthesized, or can be obtained by first synthesizing its encoding gene and then carrying out biological expression.
  • the coding gene of the protein in the above-mentioned A2) can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 2, and/or carrying out missense mutation of one or several base pairs, and/ Or the coding sequence of the tag shown in the above table is connected to its 5' end and/or 3' end.
  • the DNA molecule shown in SEQ ID NO: 2 encodes the protein shown in SEQ ID NO: 1.
  • the present invention also provides any of the following applications of the biomaterial related to OsDREB1C:
  • the biological material is any one of the following B1) to B9):
  • B2 an expression cassette containing the nucleic acid molecule of B1);
  • B3 a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2);
  • B4 a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);
  • B5 a transgenic plant cell line containing the nucleic acid molecule of B1), or a transgenic plant cell line containing the expression cassette of B2);
  • B6 a transgenic plant tissue containing the nucleic acid molecule of B1), or a transgenic plant tissue containing the expression cassette of B2);
  • B7 a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);
  • B9 An expression cassette, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ comprising the nucleic acid molecule of B8).
  • nucleic acid molecule of B1) may be as follows b11) or b12) or b13) or b14) or b15):
  • the coding sequence is the cDNA molecule or DNA molecule of sequence 2 in the sequence listing;
  • b14 has 73% or more identity to the nucleotide sequence defined by b11) or b12) or b13), and encodes a cDNA molecule or DNA molecule of OsDREB1C;
  • b15 hybridizes under stringent conditions to a nucleotide sequence defined in b11) or b12) or b13) or b14) and encodes a cDNA molecule or DNA molecule of OsDREB1C;
  • the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.
  • nucleotide sequence encoding the OsDREB1C protein of the present invention can easily mutate the nucleotide sequence encoding the OsDREB1C protein of the present invention using known methods, such as directed evolution and point mutation.
  • Those artificially modified nucleotides with 73% or higher identity to the nucleotide sequence of the OsDREB1C protein isolated by the present invention, as long as they encode the OsDREB1C protein and have the function of the OsDREB1C protein, are all derived from the nucleus of the present invention. nucleotide sequences and are equivalent to the sequences of the present invention.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes 73% or more, or 85% or more, or 90% or more, or 95% or more of the nucleotide sequence of the protein consisting of the amino acid sequence shown in the coding sequence 1 of the present invention. Nucleotide sequences of higher identity. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
  • the stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO 4 and 1 mM EDTA, 50°C, 2 ⁇ SSC, 0.1 Rinse in %SDS; also: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1mM EDTA, rinse at 50°C, 1 ⁇ SSC, 0.1% SDS; also: 50°C , hybridized in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1 mM EDTA, washed at 50°C, 0.5 ⁇ SSC, 0.1% SDS; also: 50°C, in 7% SDS, 0.5M NaPO 4 and Hybridize in a mixed solution of 1mM EDTA, rinse in 0.1 ⁇ SSC, 0.1% SDS at 50°C; alternatively: hybridize in a mixed solution of 7% SDS, 0.5M NaPO
  • the above-mentioned 73% or more identity may be 80%, 85%, 90% or more than 95% identity.
  • B2 described expression cassette (OsDREB1C gene expression cassette) containing the nucleic acid molecule of coding OsDREB1C protein, refers to the DNA that can express OsDREB1C protein in host cell, this DNA can not only include the start that starts OsDREB1C gene transcription It can also include a terminator that terminates transcription of the OsDREB1C gene. Further, the expression cassette may also include enhancer sequences. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development specific promoters, and inducible promoters.
  • promoters include, but are not limited to: the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiol acid S-methyl ester)); tomato Protease inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoter (US Pat. No. 5,187,267); tetracycline-inducible promoter (US Pat. No.
  • seed-specific promoters such as foxtail millet seed-specific promoter pF128 (CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (for example, promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety.
  • Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminators (see, eg: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al.
  • NOS terminator Agrobacterium nopaline synthase terminator
  • cauliflower mosaic virus CaMV 35S terminator tml terminator
  • pea rbcS E9 terminator nopaline and octopine Synthase terminators
  • the recombinant vector containing the OsDREB1C gene expression cassette can be constructed by using the existing expression vector.
  • the plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301 or pCAMBIA1391-Xb (CAMBIA company) and so on.
  • the plant expression vector may also contain the 3' untranslated region of the foreign gene, ie, containing the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression.
  • the poly(A) signal can guide the addition of poly(A) to the 3' end of the mRNA precursor, such as Agrobacterium crown gall-inducing (Ti) plasmid genes (such as nopaline synthase gene Nos), plant genes (such as soybean The untranslated regions transcribed from the 3' end of the storage protein gene) have similar functions.
  • enhancers can also be used, including translation enhancers or transcription enhancers.
  • the translation control signals and initiation codons can be derived from a wide variety of sources, either natural or synthetic.
  • the translation initiation region can be derived from a transcription initiation region or a structural gene.
  • the plant expression vector used can be processed, such as adding a gene (GUS gene, luciferase gene, luciferase gene) that can be expressed in plants encoding an enzyme that can produce color change or a luminescent compound.
  • marker genes for antibiotics such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene conferring resistance to methotrexate, the EPSPS gene conferring resistance to glyphosate
  • marker genes for chemical resistance such as herbicide resistance genes
  • mannose-6- which provides the ability to metabolize mannose Phosphoisomerase gene.
  • the vector may be a plasmid, cosmid, phage or viral vector.
  • the plasmid may specifically be a pBWA(V)HS vector or a psgR-Cas9-Os-containing plasmid.
  • the recombinant vector can specifically be pBWA(V)HS-OsDREB1C.
  • the pBWA(V)HS-OsDREB1C is a recombinant vector obtained by inserting the OsDREB1C encoding gene shown in SEQ ID NO: 2 in the sequence listing at the BsaI (Eco31I) restriction site of the pBWA(V)HS vector.
  • the pBWA(V)HS-OsDREB1C can overexpress the protein encoded by the OsDREB1C gene (ie, the OsDREB1C protein shown in sequence 1) under the drive of the CaMV 35S promoter.
  • the recombinant vector can be a recombinant vector prepared by using the crisper/cas9 system, which can reduce the content of OsDREB1C.
  • the recombinant vector can express sgRNA targeting the nucleic acid molecule of B1).
  • the target sequence of the sgRNA can be positions 356-374 of sequence 2 in the sequence listing.
  • the microorganisms may be yeast, bacteria, algae or fungi.
  • the bacteria can be Agrobacterium, such as Agrobacterium rhizogenes EHA105.
  • transgenic plant cell lines, transgenic plant tissues and transgenic plant organs may all include propagating material or may not include propagating material.
  • the present invention also provides any of the following methods:
  • X1 a method for cultivating photosynthesis-enhanced plants, comprising expressing OsDREB1C in a recipient plant, or increasing the content or activity of OsDREB1C in the recipient plant, to obtain a target plant with enhanced photosynthesis;
  • X2 A method for cultivating plants with enhanced photosynthesis and enhanced nitrogen absorption or transport capacity, comprising expressing OsDREB1C in recipient plants, or increasing the content or activity of OsDREB1C in recipient plants, to obtain plants with enhanced photosynthesis and enhanced nitrogen absorption or transport capabilities target plant;
  • X3 a method for cultivating plants with enhanced photosynthesis and increased nitrogen content, comprising expressing OsDREB1C in a recipient plant, or increasing the content or activity of OsDREB1C in the recipient plant, to obtain a target plant with enhanced photosynthesis and increased nitrogen content;
  • X4 a method for cultivating plants with enhanced photosynthesis and earlier flowering time, comprising expressing OsDREB1C in the recipient plant, or increasing the content or activity of OsDREB1C in the recipient plant, to obtain a target plant with enhanced photosynthesis and earlier flowering time;
  • a method for cultivating plants with enhanced photosynthesis, enhanced nitrogen absorption or transport capacity, and enhanced flowering time in advance comprising expressing OsDREB1C in recipient plants, or increasing the content or activity of OsDREB1C in recipient plants, to obtain enhanced photosynthesis, nitrogen absorption Or target plants with enhanced transport capacity and enhanced flowering time;
  • a method for cultivating plants with enhanced photosynthesis, increased nitrogen content and advanced flowering time comprising expressing OsDREB1C in a recipient plant, or increasing the content or activity of OsDREB1C in the recipient plant, resulting in enhanced photosynthesis, increased nitrogen content and flowering Plants of interest for which time is advanced.
  • the methods described in X1)-X6) can be achieved by introducing the encoding gene of OsDREB1C into the recipient plant and expressing the encoding gene.
  • the encoding gene can be the nucleic acid molecule of B1).
  • the encoding gene of OsDREB1C can be modified as follows first, and then introduced into the recipient plant to achieve better expression effect:
  • the promoters may include constitutive, inducible, time-sequential regulation, developmental regulation, chemical regulation, tissue-preferred and tissue-specific promoters ; the choice of promoter will vary with the temporal and spatial requirements of expression and will also depend on the target species; e.g. tissue- or organ-specific expression promoters, depending on what stage of development the receptor is desired; although the provenance of the source Many promoters for dicotyledonous plants are functional in monocotyledonous plants and vice versa, but ideally, a dicotyledonous promoter is chosen for expression in dicotyledonous plants and a monocotyledonous promoter for expression in monocots;
  • Linking with a suitable transcription terminator can also improve the expression efficiency of the gene of the present invention; for example, tml derived from CaMV, E9 derived from rbcS; any available terminator known to function in plants can be combined with The gene of the present invention is connected;
  • enhancer sequences such as intron sequences (e.g. from Adhl and bronzel) and viral leader sequences (e.g. from TMV, MCMV and AMV).
  • the OsDREB1C-encoding gene can be introduced into recipient plants using a recombinant expression vector containing the OsDREB1C-encoding gene.
  • the recombinant expression vector can specifically be the pBWA(V)HS-OsDREB1C.
  • the recombinant expression vector can be introduced into plant cells by using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation and other conventional biotechnology methods (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York). , pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition).).
  • the plant of interest is understood to include not only the first-generation plants in which the OsDREB1C protein or its encoding gene has been altered, but also its progeny.
  • the gene can be propagated in that species, and conventional breeding techniques can be used to transfer the gene into other varieties of the same species, including in particular commercial varieties.
  • the plants of interest include seeds, callus, whole plants and cells.
  • the present invention also provides a product having any of the following functions D1)-D6), the product containing OsDREB1C or the biological material:
  • the plant may be M1) or M2) or M3):
  • M2 grasses, crucifers or legumes
  • the photosynthesis can be embodied in photosynthetic rate, net photosynthetic rate, heat dissipation NPQ, maximum carboxylation efficiency and/or maximum electron transfer rate;
  • the nitrogen transport can be embodied in the transport from the roots to the shoot, or the transport into the grain;
  • the nitrogen content may be the nitrogen content in a plant or an organ
  • the flowering time can be reflected in the heading stage.
  • the organ may be the root, stem, leaf and/or grain of the plant.
  • the modulation can be enhancing or inhibiting, or promoting or inhibiting, or increasing or decreasing.
  • OsDREB1C or the biological material also belongs to the protection scope of the present invention.
  • Figure 1 shows the sequence alignment results.
  • Figure 2 shows the relative expression level detection of OsDREB1C gene in transgenic rice and the sequence detection results of the target region of gene knockout rice material.
  • A Relative expression level of OsDREB1C gene
  • B gene editing site of OsDREB1C gene knockout rice.
  • Figure 3 shows photosynthesis parameters of wild-type and transgenic rice plants.
  • a - diurnal variation of photosynthesis B - diurnal variation of NPQ;
  • C - light response curve D - CO 2 response curve;
  • E - maximum CO 2 carboxylation efficiency E - maximum electron transfer rate.
  • Figure 4 shows the detection results of nitrogen uptake and utilization of wild-type and transgenic rice plants.
  • A- 15 N content in shoots B- 15 N content in roots; C- 15 N uptake efficiency; D- 15 N transport efficiency from roots to shoots; E- nitrogen content in different tissues of rice; F- different tissues of rice distribution ratio of nitrogen.
  • E and F from top to bottom, the grains, stems and leaves are in order.
  • the pBWA(V)HS vector in the following examples (Zhao et al., DEP1is involved in regulating the carbon-nitrogen metabolic balance to affect grain yield and quality in rice (Oriza sativa L.), PLOS ONE, March 11, 2019 , https://doi.org/10.1371/journal.pone.0213504), the public can obtain the biological material from the applicant, and the biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.
  • Plasmids containing psgR-Cas9-Os in the following examples Hu Xuejiao, Yang Jia, Cheng Can, Zhou Jihua, Niu Fu'an, Wang Xinqi, Zhang Meiliang, Cao Liming, Chu Huangwei.
  • CRISPR/Cas9 system to edit the SD1 gene of rice.
  • the public can obtain the biological material from the applicant, and the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
  • Example 1 OsDREB1C has the effect of improving rice photosynthetic efficiency, promoting nitrogen absorption, promoting early heading and increasing yield
  • This example provides a protein derived from Nipponbare rice, which has the functions of improving rice photosynthesis efficiency, promoting nitrogen absorption, promoting early heading and improving yield.
  • the name of the protein is OsDREB1C, and its sequence is sequence 1 in the sequence table.
  • the coding gene sequence of OsDREB1C is sequence 2, and the genome sequence is 3001-4131 of sequence 3.
  • positions 1-3000 are the promoter of OsDREB1C gene in Nipponbare genomic DNA.
  • PCR product containing the full-length CDS of OsDREB1C gene was amplified from rice Nipponbare cDNA by PCR, and the obtained PCR product was digested with BsaI (Eco31I).
  • the pBWA(V)HS vector was linked to the backbone of the vector obtained by single digestion with BsaI(Eco31I), and the resulting recombinant vector with the correct sequence was denoted as pBWA(V)HS-OsDREB1C.
  • pBWA(V)HS-OsDREB1C is a recombinant vector obtained by inserting the OsDREB1C encoding gene shown in SEQ ID NO: 2 in the sequence table at the BsaI(Eco31I) restriction site of the pBWA(V)HS vector.
  • pBWA(V)HS-OsDREB1C can The protein encoded by the OsDREB1C gene (ie the OsDREB1C protein shown in sequence 1) was expressed under the drive of the CaMV 35S promoter.
  • the primer sequences used are as follows:
  • OsDREB1C-F 5'-CAGTGGTCTCACAACATGGAGTACTACGAGCAGGAGGAGT-3' (sequence 4 in the sequence listing);
  • OsDREB1C-R 5'-CAGTGGTCTCATACATCAGTAGCTCCAGAGTGTGACGTCG-3' (sequence 5 in the sequence listing).
  • sgRNA and primers were designed according to the online design website ( http://skl.scau.edu.cn/ ), and the target sequence was determined to be 5′-AGTCATGCCCGCACGACGC-3′ (No. 356-374 of sequence 2). bits).
  • the OsDREB1C-sgRNA-F and OsDREB1C-sgRNA-R were annealed, the resulting product was digested with BsaI, and the resulting digested product was connected to the vector skeleton obtained by digesting the psgR-Cas9-Os plasmid with BsaI, and the obtained
  • the recombinant vector with the correct sequence is the OsDREB1C knockout vector, denoted as OsU3-sgRNA-OsUBI-Cas9-OsDREB1C.
  • the OsU3 promoter drives sgRNA and the OsUBI promoter drives Cas9.
  • primer sequences used are as follows:
  • OsDREB1C-sgRNA-F 5'-TGTGTGGCGTCGTGCGGGCATGACT-3' (SEQ ID NO: 6 in the sequence listing);
  • OsDREB1C-sgRNA-R 5'-AAACAGTCATGCCCGCACGACGCCA-3' (SEQ ID NO: 7 in the sequence listing).
  • the mature seeds of the japonica rice variety Nipponbare were sterilized and induced to obtain embryogenic callus.
  • the pBWA(V)HS-OsDREB1C and OsU3-sgRNA-OsUBI-Cas9-OsDREB1C obtained in step 1 were introduced into Agrobacterium EHA105, respectively, and the Bacillus-mediated rice genetic transformation method infects and co-cultivates callus, and uses resistance screening to obtain transgenic plants.
  • the screened transgenic rice obtained from pBWA(V)HS-OsDREB1C is OsDREB1C transgenic rice.
  • the transgenic rice obtained by sgRNA-OsUBI-Cas9-OsDREB1C is the OsDREB1C knockout rice material.
  • the primers used were: 5′-CATGATGATGCAGTACCAGGA-3′ (Sequence 8 in the sequence listing), 5'-GATCATCAGTAGCTCCAGAGTG-3' (sequence 9 in the sequence listing);
  • the internal reference gene is rice Ubiqutin, and the primers for the internal reference gene are: 5'-AAGAAGCTGAAGCATCCAGC-3' (sequence 10 in the sequence listing), 5 '-CCAGGACAAGATGATCTGCC-3' (sequence 11 in the sequence listing).
  • the three lines (KO1, KO2 and KO3) of OsDREB1C knockout rice material were amplified and sequenced by PCR using primer pairs capable of amplifying the target sequence and its upstream and downstream. The results showed that the target sites of these three lines were The sequence changes are shown in B in Figure 2.
  • One nucleotide deletion occurred in KO1 and KO2 one nucleotide insertion occurred in KO3, and frameshift mutations occurred in the target genes of the three lines.
  • the rice to be tested wild-type Nipponbare rice (WT), OsDREB1C overexpressing rice (OE1/OE2/OE5), OsDREB1C knockout rice (KO1/KO2/KO3).
  • the flag leaves of the rice to be tested at the heading stage were measured with a LICOR-6400XT portable photosynthesis instrument (LI-COR, USA) to measure the photosynthetic diurnal variation, light response curve and CO 2 response curve, which reflect the photosynthetic capacity of plants.
  • the determination of the diurnal variation of photosynthesis was carried out in clear and cloudless weather, and the measurement was carried out every 2-4 hours from 8:00 to 16:00.
  • the rate of photosynthesis was measured with a LI-COR 6400XT portable photosynthesis instrument, and NPQ (non-photochemical quenching) was measured with a FluorPen FP100 (PSI, Czech Republic).
  • the CO 2 concentration was set to 400 ⁇ mol mol ⁇ 1 , and the light intensity (PPFD) was 0 to 2000 ⁇ mol m ⁇ 2 s ⁇ 1 .
  • the PPFD was set at 1200 ⁇ mol m ⁇ 2 s ⁇ 1 , and the CO 2 concentration was decreased from 400 to 50 ⁇ mol mol ⁇ 1 and then increased from 400 to 1200 ⁇ mol mol ⁇ 1 again.
  • the light response curve and CO 2 response curve were both fitted by the Farquhar-von Caemmerer-Berry (FvCB) model, and the maximum carboxylation efficiency (V cmax ) and the maximum electron transfer rate (J max ) were calculated from the CO 2 response curve .
  • FvCB Farquhar-von Caemmerer-Berry
  • the rice to be tested wild-type Nipponbare rice (WT), OsDREB1C overexpressing rice (OE1/OE2/OE5), OsDREB1C knockout rice (KO1/KO2/KO3).
  • the rice seedlings to be tested that were grown in nutrient solution hydroponics for 3 weeks in the greenhouse were placed in Kimura B solution (Kimura B solution) without nitrogen (without (NH 4 ) 2 SO 4 and KNO 3 ) in advance for nitrogen starvation treatment 3 sky.
  • Kimura B solution Kimura B solution
  • the roots of seedlings were completely immersed in 0.1 mM CaSO 4 solution (solvent is deionized water) for 1 minute, and then the residual water was absorbed, and the roots were placed in a nutrient solution containing 0.5 mM K 15 NO 3 for cultivation. After 3 hours, the roots of the seedlings were soaked in 0.1 mM CaSO 4 solution for 1 minute, and the residual water was absorbed.
  • the nutrient solution used is as follows:
  • the heading date of rice was detected, and the rice to be tested: wild-type Nipponbare rice (WT), OsDREB1C overexpressing rice (OE1/OE2/OE5), and OsDREB1C knockout rice (KO1/KO2/KO3).
  • each tested rice was counted under the Beijing field planting conditions.
  • the statistical method is as follows: each type of rice to be tested is planted in 3 plots, which are randomly arranged, and 50% of the plants in the plot are marked as heading when the ears of 50% of the plants are exposed from the flag leaf sheaths, and the heading period is the time from sowing to heading. days.
  • the aboveground dry weight and grain yield of rice were detected.
  • the rice to be tested wild-type Nipponbare rice (WT), OsDREB1C overexpressing rice (OE1/OE2/OE5), and OsDREB1C gene knockout rice (KO1/KO2/KO3).
  • each type of rice to be tested was planted in 3 replicate plots and randomly arranged. After the rice grains were mature, the grain yield per plant, the plot grain yield, and the dry weight of the aboveground straw were measured and calculated.
  • Harvest index is the ratio of rice grain yield per plant to above-ground biomass (the sum of above-ground straw dry weight and grain yield per plant).
  • the method for measuring the dry weight of aboveground straw is as follows: after the rice is mature, after removing the grains from the straw per plant, put it into a nylon mesh bag and dry it to constant weight at 80°C, and then weigh the sample. 20-30 replicates of each rice material were taken for statistical analysis.
  • the measurement method of grain yield is as follows: threshing and removing the shriveled grains per plant of rice is the grain yield per plant, and 20-30 single plants of each rice material are repeated for statistical analysis.
  • the side rows were removed, and the 30 rice plants in the middle were taken to measure the grain weight and counted as the yield of one plot, and 3 plots were repeated for statistical analysis.
  • the measurement method of rice quality is as follows: the rice grains harvested in the Beijing field experiment in 2019 were used for rice quality analysis after natural storage for 3 months. The results showed that the grain yield of rice overexpressing OsDREB1C was significantly higher than that of the wild type, and the difference was significant.
  • the brown rice rate, milled rice rate, and whole milled rice rate were all significantly higher than those of the wild type, and the chalkiness and chalky grain rate were reduced, which improved the appearance quality. , while the amylose and protein content had no significant effect.
  • the yield per plant and plot of OsDREB1C knockout rice were significantly lower than those of the wild type, and the above-ground biomass was increased, which directly led to a 22.4-37.7% reduction in the harvest index compared with the wild type.
  • the results indicated that OsDREB1C gene transfer into rice can greatly improve the yield, rice quality and harvest index of transgenic rice.
  • OsDREB1C and its encoded gene can regulate the grain yield, rice quality and harvest index of rice.
  • PCR was used to amplify the CDS full-length of OsDREB1C gene from rice Nipponbare cDNA, and the obtained PCR recovery product was combined with pWMB110 vector (Huiyun Liu, Ke Wang, Zimiao Jia, Qiang Gong,Zhishan Lin,Lipu Du,Xinwu Pei,Xingguo Ye,Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system,Journal of Experimental Botany,Volume 71,Issue 4,7 February 2020, Pages 1337–1349, https://doi.org/10.1093/jxb/erz529)
  • the backbone of the vector obtained by double digestion with BamHI and SacI was linked, and the resulting recombinant vector with the correct sequence was denoted as pWMB110-OsDREB1C.
  • pWMB110-OsDREB1C is a recombinant vector obtained by replacing the DNA fragment between the BamHI and SacI recognition sites of the pWMB110 vector with the OsDREB1C coding gene shown in SEQ ID NO: 2 in the sequence listing.
  • pWMB110-OsDREB1C can express the OsDREB1C gene under the drive of the UBI promoter.
  • the encoded protein ie, the OsDREB1C protein shown in SEQ ID NO: 1).
  • the primer sequences used are as follows:
  • Fielder-OsDREB1C-OE-F 5′-CAGGTCGACTCTAGA GGATCC ATGGAGTACTACGAGCAGGAG-3′ (sequence 12 in the sequence listing);
  • Fielder-OsDREB1C-OE-R 5'-CGATCGGGGAAATTC GAGCTC TCAGTAGCTCCAGAGTGTGAC-3' (sequence 13 in the Sequence Listing).
  • pGWBs gateway binary vectors
  • the CDS sequence of OsDREB1C was transferred into the final vector pGWB5, and the resulting recombinant vector with correct sequence was denoted as pGWB5-OsDREB1C.
  • pGWB5-OsDREB1C can express the protein encoded by the OsDREB1C gene (ie, the OsDREB1C protein shown in sequence 1) under the drive of the CaMV 35S promoter.
  • the primer sequences used are as follows:
  • OsDREB1C-CDS-F 5'-CACCATGGAGTACTACGAGCAGGAG-3' (sequence 14 in the sequence listing);
  • OsDREB1C-CDS-R 5'-GTAGCTCCAGGTGTGACGTC-3' (sequence 15 in the sequence listing).
  • Embryogenic callus was obtained by inducing young embryos of wheat cultivar Fielder after disinfection.
  • the pWMB110-OsDREB1C obtained in step 1 was introduced into Agrobacterium C58C1, and the callus was infected and co-cultured by Agrobacterium-mediated wheat genetic transformation. , using resistance screening to obtain transgenic plants, the screened OsDREB1C transgenic wheat material. Due to the high similarity between OsDREB1C and its own homologous genes in wheat, the expression level of OsDREB1C gene in rice could not be detected by qRT-PCR method. Therefore, using the wheat wild-type Fielder as a control, the PCR method was used to detect whether the Bar gene of the vector existed in the cDNA of transgenic wheat.
  • the primers used were: 5'-CAGGAACCGCAGGAGTGGA-3' (sequence 16 in the sequence table), 5'-CCAGAAACCCACGTCATGCC-3' (Sequence 17 in the Sequence Listing).
  • the results of electrophoresis showed that the Bar gene was present in all three lines (TaOE-5, TaOE-8 and TaOE-9) of OsDREB1C transgenic wheat, but not in the wild type, indicating that these three lines were all transgenic Transgenic material of the pWMB110-OsDREB1C vector (A in Figure 5).
  • the pGWB5-OsDREB1C obtained in step 1 was introduced into Agrobacterium GV3101, and transformed into Arabidopsis wild-type Col-0 by the Agrobacterium-mediated flower soaking method.
  • OsDREB1C transgenic Arabidopsis material For OsDREB1C transgenic Arabidopsis material.
  • Arabidopsis wild-type Col-0 as a control, the relative expression level of OsDREB1C gene at RNA level in OsDREB1C transgenic Arabidopsis was detected by qRT-PCR method.
  • the primers used were: 5′-CATGATGATGCAGTACCAGGA-3′ (Sequence Listing Middle sequence 18), 5'-GATCATCAGTAGCTCCAGAGTG-3' (sequence 19 in the sequence table); the internal reference gene is Arabidopsis Actin, and the internal reference gene primer is: 5'-GCACCACCTGAAAGGAAGTACA-3' (sequence 20 in the sequence table), 5' - CGATTCCTGGACCTGCCTCATC-3' (sequence 21 in the Sequence Listing).
  • the tested materials wild-type wheat (Fielder), overexpressing OsDREB1C wheat (TaOE-5, TaOE-8 and TaOE-9).
  • the greenhouse temperature was controlled at 22-24°C, and the sunshine length was 12 hours light/12 hours dark.
  • the heading dates of wild-type and transgenic wheat were counted, and the photosynthesis rate of the flag leaves of the tested wheat at the heading stage was measured with a LICOR-6400XT portable photosynthesis instrument (LI-COR, USA), and the light intensity was set to 1000 ⁇ mol m -2 s - 1 . After the wheat grains were mature, the number of grains per ear, 1000-grain weight and grain yield per plant were determined.
  • the materials to be tested were wild-type Arabidopsis (Col-0), overexpressing OsDREB1C Arabidopsis (AtOE-10, AtOE-11 and AtOE-12). Cultures were cultured under short-day (8 hours light/16 hours dark) for 2 weeks and then transferred to long day (16 hours light/8 hours dark) cultures.
  • Bolting time is the number of days from sowing to stem elongation and flowering.
  • the OsDREB1C and related biological materials of the present invention can improve the photosynthesis efficiency of plants, can promote nitrogen absorption and transport, increase nitrogen content in plants and grains, can promote early heading, and can also improve yield and quality.
  • the invention starts from the synergistic improvement of crop photosynthetic efficiency, nitrogen utilization efficiency and heading stage, realizes the synergistic improvement of nitrogen utilization efficiency while realizing a substantial increase in crop yield, and also provides a solution to the contradiction between high crop yield and early maturity. Therefore, the present invention can further greatly improve the crop yield potential and nitrogen fertilizer utilization efficiency, and realize "high yield and high efficiency";
  • the problems of early maturity and high yield and the "superparent late maturity" of inter-subspecies hybrid rice have important application potential.

Abstract

OsDREB1C的序列为序列表中序列1，其编码基因序列为序列表中序列2。实验证明，OsDREB1C及其相关的生物材料可以提高植物的光合作用效率，可以促进氮吸收与转运并提高植物及籽粒中的氮含量，可以促进提前抽穗，还可以提高产量和品质，OsDREB1C及其相关的生物材料具有重要的生物学意义和产业价值，应用前景广阔。

Description

OsDREB1C及其编码基因在提高水稻光合效率中的应用 技术领域
本发明涉及生物技术领域中，OsDREB1C及其编码基因在提高水稻光合效率中的应用。
背景技术
未来随着人口增长和经济发展，我国的粮食需求仍呈快速增长势头。在耕地面积持续减少和粮食种植面积扩大潜力有限的形势下，大面积提高作物单产来增加总产是保障我国粮食安全的唯一选择。因此，在粮食安全需求压力下，高产是农业生产不懈追求的目标。从二十世纪50年代中期开始兴起的“绿色革命”，通过作物品种遗传改良和栽培管理技术的提高，实现了作物产量潜力的大幅度提升。然而近年来，作物单产呈现停滞不前甚至下降的态势，如何提高作物单产需要新的策略和途径。
在农业生产中，通过施用氮肥一直是农作物增产的重要措施之一。持续大量的氮肥投入不仅增加了种植成本，还导致了气候变化、土壤酸化及水体富营养化等日益严重的环境污染问题。此外，大量施用氮肥会导致作物“贪青晚熟”现象，影响后茬作物播种；如何减少农业生产中的氮肥投入并持续提高作物产量，已成为当前我国农业可持续发展亟待解决的重大问题。探索作物大幅度增产和资源高效利用协同的调控机制与技术途径是我国农业可持续发展的当务之急。
抽穗期是作物重要的农艺性状之一，决定着水稻的季节、区域适应性以及产量。适宜的抽穗期是作物稳产高产的保障。选育早熟高产新品种一直是作物遗传育种研究的主攻方向之一。“高产不早熟、早熟不高产”即所谓“优而不早、早而不优”现象，是作物品种培育的重大难题。
目前研究人员从作物育种、栽培管理措施以及功能基因挖掘方面开展了相关探索和研究，也取得了一些重要进展，但针对上述作物生产中高产与资源高效、高产与早熟两大难题，尚未有有效解决方案。
发明公开
本发明所要解决的技术问题是如何提高植物的光合作用。
为解决上述技术问题，本发明首先提供了蛋白质或调控所述蛋白质活性或含量的物质的下述任一应用：
D1)调控植物光合作用；
D2)制备调控植物光合作用产品；
D3)调控植物光合作用与氮吸收或转运；
D4)制备调控植物光合作用与氮吸收或转运产品；
D5)调控植物光合作用与氮含量；
D6)制备调控植物光合作用与氮含量产品；
D7)调控植物光合作用与开花时间；
D8)制备调控植物光合作用与开花时间产品；
D9)调控植物光合作用、氮吸收或转运与开花时间；
D10)制备调控植物光合作用、氮吸收或转运与开花时间产品；
D11)调控植物光合作用、氮含量与开花时间；
D12)制备调控植物光合作用、氮含量与开花时间产品；
D13)植物育种；
所述蛋白质(其名称为OsDREB1C)为如下A1)、A2)、A3)或A4)：
A1)氨基酸序列是序列1的蛋白质；
A2)将序列表中序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质；
A3)来源于水稻、谷子、玉米、高粱、山羊草、小麦或二穗短柄草且与序列1具有64％或以上同一性并与A1)所述蛋白质具有相同功能的蛋白质；
A4)在A1)或A2)或A3)的N端或/和C端连接标签得到的融合蛋白质。
为了使A1)中的蛋白质便于纯化，可在由序列表中序列1所示的氨基酸序列组成的蛋白质的氨基末端或羧基末端连接上如下表所示的标签。
表：标签的序列
标签 残基 序列
Poly-Arg 5-6(通常为5个) RRRRR
Poly-His 2-10(通常为6个) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
上述A2)中的蛋白质，为与序列1所示蛋白质的氨基酸序列具有64％或64％以上同一性且具有相同功能的蛋白质。所述具有64％或64％以上同一性为具有 64％、具有75％、具有80％、具有85％、具有90％、具有95％、具有96％、具有97％、具有98％或具有99％的同一性。
上述A2)中的蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。
上述A2)中的蛋白质的编码基因可通过将序列2所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上上表所示的标签的编码序列得到。其中，序列2所示的DNA分子编码序列1所示的蛋白质。
本发明还提供了与OsDREB1C相关的生物材料的下述任一应用：
D1)调控植物光合作用；
D2)制备调控植物光合作用产品；
D3)调控植物光合作用与氮吸收或转运；
D4)制备调控植物光合作用与氮吸收或转运产品；
D5)调控植物光合作用与氮含量；
D6)制备调控植物光合作用与氮含量产品；
D7)调控植物光合作用与开花时间；
D8)制备调控植物光合作用与开花时间产品；
D9)调控植物光合作用、氮吸收或转运与开花时间；
D10)制备调控植物光合作用、氮吸收或转运与开花时间产品；
D11)调控植物光合作用、氮含量与开花时间；
D12)制备调控植物光合作用、氮含量与开花时间产品；
D13)植物育种；
所述生物材料为下述B1)至B9)中的任一种：
B1)编码OsDREB1C的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；
B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；
B5)含有B1)所述核酸分子的转基因植物细胞系、或含有B2)所述表达盒 的转基因植物细胞系；
B6)含有B1)所述核酸分子的转基因植物组织、或含有B2)所述表达盒的转基因植物组织；
B7)含有B1)所述核酸分子的转基因植物器官、或含有B2)所述表达盒的转基因植物器官；
B8)降低OsDREB1C表达量或敲除OsDREB1C编码基因的核酸分子；
B9)含有B8)所述核酸分子的表达盒、重组载体、重组微生物、转基因植物细胞系、转基因植物组织或转基因植物器官。
上述应用中，B1)所述核酸分子可为如下b11)或b12)或b13)或b14)或b15)：
b11)编码序列是序列表中序列2的cDNA分子或DNA分子；
b12)序列表中序列2所示的cDNA分子或DNA分子；
b13)序列表中序列3的第3001-4131位所示的DNA分子；
b14)与b11)或b12)或b13)限定的核苷酸序列具有73％或73％以上同一性，且编码OsDREB1C的cDNA分子或DNA分子；
b15)在严格条件下与b11)或b12)或b13)或b14)限定的核苷酸序列杂交，且编码OsDREB1C的cDNA分子或DNA分子；
B2)所述表达盒为如下b21)或b22)或b23)：
b21)序列表中序列3所示的DNA分子；
b22)与b21)限定的核苷酸序列具有73％或73％以上同一性，且具有相同功能的DNA分子；
b23)在严格条件下与b21)或b22)限定的核苷酸序列杂交，且具有相同功能的DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码OsDREB1C蛋白质的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明分离得到的OsDREB1C蛋白质的核苷酸序列73％或者更高同一性的核苷酸，只要编码OsDREB1C蛋白质且具有OsDREB1C蛋白质功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码序列1所示的氨基酸序列组成的蛋白质的核苷酸序列具有73％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述应用中，所述严格条件可为如下：50℃，在7％十二烷基硫酸钠(SDS)、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，2×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，0.5×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，0.1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在65℃，0.1×SSC，0.1％SDS中漂洗；也可为：在6×SSC，0.5％SDS的溶液中，在65℃下杂交,然后用2×SSC,0.1％SDS和1×SSC,0.1％SDS各洗膜一次；也可为：2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min；也可为：0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
上述73％或73％以上同一性，可为80％、85％、90％或95％以上的同一性。
上述应用中，B2)所述的含有编码OsDREB1C蛋白质的核酸分子的表达盒(OsDREB1C基因表达盒)，是指能够在宿主细胞中表达OsDREB1C蛋白质的DNA，该DNA不但可包括启动OsDREB1C基因转录的启动子，还可包括终止OsDREB1C基因转录的终止子。进一步，所述表达盒还可包括增强子序列。可用于本发明的启动子包括但不限于：组成型启动子，组织、器官和发育特异的启动子，和诱导型启动子。启动子的例子包括但不限于：花椰菜花叶病毒的组成型启动子35S；来自西红柿的创伤诱导型启动子，亮氨酸氨基肽酶("LAP",Chao等人(1999)Plant Physiol 120：979-992)；来自烟草的化学诱导型启动子，发病机理相关1(PR1)(由水杨酸和BTH(苯并噻二唑-7-硫代羟酸S-甲酯)诱导)；西红柿蛋白酶抑制剂II启动子(PIN2)或LAP启动子(均可用茉莉酮酸甲酯诱导)；热休克启动子(美国专利5，187，267)；四环素诱导型启 动子(美国专利5，057,422)；种子特异性启动子，如谷子种子特异性启动子pF128(CN101063139B(中国专利200710099169.7))，种子贮存蛋白质特异的启动子(例如，菜豆球蛋白、napin,oleosin和大豆beta conglycin的启动子(Beachy等人(1985)EMBO J.4：3047-3053))。它们可单独使用或与其它的植物启动子结合使用。此处引用的所有参考文献均全文引用。合适的转录终止子包括但不限于：农杆菌胭脂碱合成酶终止子(NOS终止子)、花椰菜花叶病毒CaMV 35S终止子、tml终止子、豌豆rbcS E9终止子和胭脂氨酸和章鱼氨酸合酶终止子(参见，例如：Odell等人(I 985)Nature 313:810；Rosenberg等人(1987)Gene,56:125；Guerineau等人(1991)Mol.Gen.Genet,262:141；Proudfoot(1991)Cell,64:671；Sanfacon等人Genes Dev.,5:141；Mogen等人(1990)Plant Cell,2:1261；Munroe等人(1990)Gene,91:151；Ballad等人(1989)Nucleic Acids Res.17:7891；Joshi等人(1987)Nucleic Acid Res.,15:9627)。
可用现有的表达载体构建含有所述OsDREB1C基因表达盒的重组载体。所述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等。如pAHC25、pBin438、pCAMBIA1302、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa、PSN1301或pCAMBIA1391-Xb(CAMBIA公司)等。所述植物表达载体还可包含外源基因的3′端非翻译区域，即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3′端，如农杆菌冠瘿瘤诱导(Ti)质粒基因(如胭脂碱合成酶基因Nos)、植物基因(如大豆贮存蛋白基因)3′端转录的非翻译区均具有类似功能。使用本发明的基因构建植物表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用植物表达载体进行加工，如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因(GUS基因、萤光素酶基因等)、抗生素的标记基因(如赋予对卡那霉素和相关抗生素抗性的nptII基因，赋予对除草剂膦丝菌素抗性的bar基因，赋予对抗生素潮霉素抗性的hph基因， 和赋予对氨甲喋呤抗性的dhfr基因，赋予对草甘磷抗性的EPSPS基因)或是抗化学试剂标记基因等(如抗除莠剂基因)、提供代谢甘露糖能力的甘露糖-6-磷酸异构酶基因。从转基因植物的安全性考虑，可不加任何选择性标记基因，直接以逆境筛选转化植株。
上述应用中，所述载体可为质粒、黏粒、噬菌体或病毒载体。所述质粒具体可为pBWA(V)HS载体或含psgR-Cas9-Os质粒。
B3)所述重组载体具体可为pBWA(V)HS-OsDREB1C。所述pBWA(V)HS-OsDREB1C为在pBWA(V)HS载体的BsaI(Eco31I)酶切位点处插入序列表中序列2所示的OsDREB1C编码基因得到的重组载体。所述pBWA(V)HS-OsDREB1C能在CaMV 35S启动子的驱动下过表达OsDREB1C基因所编码的蛋白质(即序列1所示的OsDREB1C蛋白质。
B8)所述重组载体可为利用crisper/cas9系统制备的可以降低OsDREB1C含量的重组载体。所述重组载体可表达靶向B1)所述核酸分子的sgRNA。所述sgRNA的靶序列可为序列表中序列2的第356-374位。
上述应用中，所述微生物可为酵母、细菌、藻或真菌。其中，细菌可为农杆菌，如发根农杆菌EHA105。
上述应用中，所述转基因植物细胞系、转基因植物组织和转基因植物器官均可包括繁殖材料，也可不包括繁殖材料。
本发明还提供了下述任一方法：
X1)培育光合作用增强植物的方法，包括使受体植物中表达OsDREB1C，或提高受体植物中OsDREB1C的含量或活性，得到光合作用增强的目的植物；
X2)培育光合作用增强且氮吸收或转运能力增强植物的方法，包括使受体植物中表达OsDREB1C，或提高受体植物中OsDREB1C的含量或活性，得到光合作用增强且氮吸收或转运能力增强的目的植物；
X3)培育光合作用增强且氮含量增加植物的方法，包括使受体植物中表达OsDREB1C，或提高受体植物中OsDREB1C的含量或活性，得到光合作用增强且氮含量增加的目的植物；
X4)培育光合作用增强且开花时间提前植物的方法，包括使受体植物中表达OsDREB1C，或提高受体植物中OsDREB1C的含量或活性，得到光合作用增强且开花时间提前的目的植物；
X5)培育光合作用增强、氮吸收或转运能力增强且开花时间提前增强植物的方法，包括使受体植物中表达OsDREB1C，或提高受体植物中OsDREB1C的含量或活性，得到光合作用增强、氮吸收或转运能力增强且开花时间提前增强的目的植物；
X6)培育光合作用增强、氮含量增加增加且开花时间提前植物的方法，包括使受体植物中表达OsDREB1C，或提高受体植物中OsDREB1C的含量或活性，得到光合作用增强、氮含量增加且开花时间提前增加的目的植物。
上述方法中，X1)-X6)所述方法可通过向所述受体植物中导入OsDREB1C的编码基因并使所述编码基因得到表达实现。
上述方法中，所述编码基因可为B1)所述核酸分子。
上述方法中，其中所述OsDREB1C的编码基因可先进行如下修饰，再导入受体植物中，以达到更好的表达效果：
1)根据实际需要进行修饰和优化，以使基因高效表达；例如，可根据受体植物所偏爱的密码子，在保持本发明所述OsDREB1C的编码基因的氨基酸序列的同时改变其密码子以符合植物偏爱性；优化过程中，最好能使优化后的编码序列中保持一定的GC含量，以最好地实现植物中导入基因的高水平表达，其中GC含量可为35％、多于45％、多于50％或多于约60％；
2)修饰邻近起始甲硫氨酸的基因序列，以使翻译有效起始；例如，利用在植物中已知的有效的序列进行修饰；
3)与各种植物表达的启动子连接，以利于其在植物中的表达；所述启动子可包括组成型、诱导型、时序调节、发育调节、化学调节、组织优选和组织特异性启动子；启动子的选择将随着表达时间和空间需要而变化，而且也取决于靶物种；例如组织或器官的特异性表达启动子，根据需要受体在发育的什么时期而定；尽管证明了来源于双子叶植物的许多启动子在单子叶植物中是可起作用的，反之亦然，但是理想地，选择双子叶植物启动子用于双子叶植物中的表达，单子叶植物的启动子用于单子叶植物中的表达；
4)与适合的转录终止子连接，也可以提高本发明基因的表达效率；例如来源于CaMV的tml，来源于rbcS的E9；任何已知在植物中起作用的可得到的终止子都可以与本发明基因进行连接；
5)引入增强子序列，如内含子序列(例如来源于Adhl和bronzel)和病 毒前导序列(例如来源于TMV,MCMV和AMV)。
所述OsDREB1C的编码基因可利用含有所述OsDREB1C的编码基因的重组表达载体导入受体植物。所述重组表达载体具体可为所述pBWA(V)HS-OsDREB1C。
所述重组表达载体可通过使用Ti质粒，植物病毒栽体，直接DNA转化，微注射，电穿孔等常规生物技术方法导入植物细胞(Weissbach,1998，Method for Plant Molecular Biology VIII,Academy Press,New York,pp.411-463；Geiserson and Corey,1998,Plant Molecular Biology(2nd Edition).)。
所述目的植物理解为不仅包含OsDREB1C蛋白或其编码基因被改变的第一代植物，也包括其子代。对于所述目的植物，可以在该物种中繁殖该基因，也可用常规育种技术将该基因转移进入相同物种的其它品种，特别包括商业品种中。所述目的植物包括种子、愈伤组织、完整植株和细胞。
本发明还提供了具有如下D1)-D6)中任一的功能的产品，所述产品含有OsDREB1C或所述生物材料：
D1)调控植物光合作用；
D2)调控植物光合作用与氮吸收或转运；
D3)调控植物光合作用与氮含量；
D4)调控植物光合作用与开花时间；
D5)调控植物光合作用、氮吸收或转运与开花时间；
D6)调控植物光合作用、氮含量与开花时间。
上文中，所述植物可为M1)或M2)或M3)：
M1)单子叶植物或双子叶植物；
M2)禾本科植物、十字花科植物或豆科植物；
M3)水稻、小麦、玉米、拟南芥、油菜或大豆。
上文中，所述光合作用可体现在光合速率、净光合速率、热耗散NPQ、最大羧化效率和/或最大电子传递速率上；
所述氮转运可体现在根部向地上部的转运，或向籽粒中的转运上；
所述氮含量可为植株或器官中氮含量；
所述开花时间可体现在抽穗期上。
所述器官可为所述植物的根、茎、叶片和/或籽粒。
所述调控可为增强或抑制，或促进或抑制，或提高或降低。
OsDREB1C或所述生物材料，也属于本发明的保护范围。
附图说明
图1为序列比对结果。
图2为转基因水稻中OsDREB1C基因的相对表达水平检测和基因敲除水稻材料目标区域的序列检测结果。(A)OsDREB1C基因的相对表达水平；(B)OsDREB1C基因敲除水稻的基因编辑位点。
图3为野生型和转基因水稻植株的光合作用参数。A-光合日变化；B-NPQ日变化；C-光响应曲线；D-CO 2响应曲线；E-最大CO 2羧化效率；F-最大电子传递速率。
图4为野生型和转基因水稻植株的氮素吸收利用检测结果。A-地上部 15N含量；B-根中 15N含量；C- 15N吸收效率；D- 15N由根向地上部的转运效率；E-水稻不同组织中氮含量；F-水稻不同组织中氮的分配比率。E、F的柱形图中，从上至下依次为籽粒、茎秆和叶片。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。下述实施例中，如无特殊说明，序列表中各核苷酸序列的第1位均为相应DNA/RNA的5′末端核苷酸，末位均为相应DNA/RNA的3′末端核苷酸。
下述实施例中的pBWA(V)HS载体(Zhao et al.,DEP1is involved in regulating the carbon-nitrogen metabolic balance to affect grain yield and quality in rice(Oriza sativa L.),PLOS ONE,March 11,2019,https://doi.org/10.1371/journal.pone.0213504)，公众可从申请人处获得该生物材料，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
下述实施例中的含psgR-Cas9-Os质粒(胡雪娇，杨佳，程灿，周继华，牛付安,王新其，张美良，曹黎明，储黄伟.利用CRISPR/Cas9系统定向编辑水稻SD1基因. 中国水稻科学，2018，32(3):219-225；Mao Y,Zhang H,Xu N,Zhang B,Gou F,Zhu J K.Application of the CRISPR-Cas system for efficient genome engineering in plants.Mol Plant,2013,6(6):2008-2011.)，公众可从申请人处获得该生物材料，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
实施例1、OsDREB1C具有提高水稻光合效率、促进氮吸收、促进提前抽穗和提高产量的作用
本实施例提供了一种来源于日本晴水稻的蛋白质，其具有提高水稻光合作用效率、促进氮吸收、促进提前抽穗和提高产量的功能，该蛋白质的名称为OsDREB1C，其序列为序列表中序列1，在日本晴中，OsDREB1C的编码基因序列为序列2，基因组序列为序列3的第3001-4131位。序列3中，第1-3000位为日本晴基因组DNA中OsDREB1C基因的启动子。
将水稻OsDREB1C与其它植物中同源蛋白的进行序列比对，发现其与谷子、玉米、高粱、山羊草、小麦、二穗短柄草的同一性分别为73.52％、64.06％、66.52％、66.05％、66.05％、65.88％(图1)。
1、重组载体的构建
过表达载体的构建：通过PCR从水稻日本晴cDNA中扩增得到含有OsDREB1C基因全长CDS的PCR产物，对所得到的PCR产物利用BsaI(Eco31I)进行单酶切，将所得到的酶切产物与pBWA(V)HS载体经过BsaI(Eco31I)单酶切得到的载体骨架相连，将得到的序列正确的重组载体记为pBWA(V)HS-OsDREB1C。pBWA(V)HS-OsDREB1C为在pBWA(V)HS载体的BsaI(Eco31I)酶切位点处插入序列表中序列2所示的OsDREB1C编码基因得到的重组载体，pBWA(V)HS-OsDREB1C能在CaMV 35S启动子的驱动下表达OsDREB1C基因所编码的蛋白质(即序列1所示的OsDREB1C蛋白质。
所用引物序列如下：
OsDREB1C-F：5′-CAGTGGTCTCACAACATGGAGTACTACGAGCAGGAGGAGT-3′(序列表中序列4)；
OsDREB1C-R：5′-CAGTGGTCTCATACATCAGTAGCTCCAGAGTGTGACGTCG-3′(序列表中序列5)。
基因敲除载体的构建：根据在线设计网站( http://skl.scau.edu.cn/)设计sgRNA及引物，确定靶点序列为5′-AGTCATGCCCGCACGACGC-3′(序列2的第356-374位)。将OsDREB1C-sgRNA-F与OsDREB1C-sgRNA-R进行退火，将得到的产物利用BsaI酶切，将得到的酶切产物与含psgR-Cas9-Os质粒经过BsaI酶切得到的载体骨架相连，得到的序列正确的重组载体即为OsDREB1C基因敲除载体，记为OsU3-sgRNA-OsUBI-Cas9-OsDREB1C，该重组载体中，OsU3启动子驱动sgRNA，OsUBI启动子驱动Cas9。
其中，所使用引物序列如下：
OsDREB1C-sgRNA-F：5′-TGTGTGGCGTCGTGCGGGCATGACT-3′(序列表中序列6)；
OsDREB1C-sgRNA-R：5′-AAACAGTCATGCCCGCACGACGCCA-3′(序列表中序列7)。
2、转基因植株的构建
将水稻粳稻品种日本晴的成熟种子消毒后诱导得到胚性愈伤组织，将步骤1得到的pBWA(V)HS-OsDREB1C和OsU3-sgRNA-OsUBI-Cas9-OsDREB1C分别导入农杆菌EHA105中后，利用农杆菌介导的水稻遗传转化方法对愈伤组织进行侵染共培养，利用抗性筛选得到转基因植株，筛选到的由pBWA(V)HS-OsDREB1C得到的转基因水稻即为OsDREB1C转基因水稻，由OsU3-sgRNA-OsUBI-Cas9-OsDREB1C得到的转基因水稻即为OsDREB1C基因敲除水稻材料。
利用水稻粳稻品种日本晴(WT)作为对照，用qRT-PCR方法检测OsDREB1C转基因水稻以及OsDREB1C基因敲除水稻中OsDREB1C基因在RNA水平上的的相对表达水平，所用引物为：5′-CATGATGATGCAGTACCAGGA-3′(序列表中序列8)，5′-GATCATCAGTAGCTCCAGAGTG-3′(序列表中序列9)；内参基因为水稻Ubiqutin，内参基因引物为：5′-AAGAAGCTGAAGCATCCAGC-3′(序列表中序列10)，5′-CCAGGACAAGATGATCTGCC-3′(序列表中序列11)。
结果显示，OsDREB1C转基因水稻的3个株系(OE1、OE2和OE5)中OsDREB1C基因的相对表达水平均显著高于野生型(WT)，这三个株系均为过表达OsDREB1C水稻材料(图2中A)。OsDREB1C基因敲除水稻材料的3个株系(KO1、KO2和KO3)中OsDREB1C基因序列中存在碱基缺失或插入，造成移码突变，导致失去OsDREB1C蛋白的正常功能(图2中B)。
利用能扩增靶点序列及其上下游的引物对对OsDREB1C基因敲除水稻材料的3个株系(KO1、KO2和KO3)进行PCR扩增并测序，结果显示这三个株系的靶点 序列的变化情况如图2中B所示，KO1、KO2均发生了一个核苷酸的缺失，KO3发生了一个核苷酸的插入，三个株系的目标基因均发生了移码突变。
3、转OsDREB1C基因水稻光合作用效率的提升
检测大田生长水稻的光合作用指标，待测水稻：野生型日本晴水稻(WT)，过表达OsDREB1C水稻(OE1/OE2/OE5)，OsDREB1C基因敲除水稻(KO1/KO2/KO3)。
将处于抽穗期的待测水稻剑叶，用LICOR-6400XT便携式光合仪(LI-COR，美国)分别测定反映植物光合能力的光合日变化、光响应曲线及CO 2响应曲线。光合日变化的测定选择晴朗无云的天气进行，从8:00-16:00每隔2-4小时测定一次。其中光合作用速率用LI-COR 6400XT便携式光合仪测定，NPQ(非光化学淬灭)用FluorPen FP100(PSI，捷克)测定，测定NPQ前叶片需暗适应15-20分钟。在光响应曲线的测定中，CO 2浓度设定为400μmol mol -1，光强(PPFD)为0到2000μmol m -2s -1。CO 2响应曲线测定中，PPFD设定为1200μmol m -2s -1，CO 2浓度从400降至50μmol mol -1然后从400再升至1200μmol mol -1。光响应曲线和CO 2响应曲线均采用Farquhar-von Caemmerer-Berry(FvCB)模型进行拟合，并通过CO 2响应曲线计算得出最大羧化效率(V cmax)及最大电子传递速率(J max)。
结果表明，过表达OsDREB1C水稻的光合效率(也即光合作用效率)在白天均显著高于野生型，在中午光强最高时差异达到最大，并达到显著水平，可较野生型提高29.5-43.3％，同时用于消耗过多吸收光能的热耗散NPQ显著低于野生型(图3中A、B，表1和表2)，差异均达到显著水平，说明更多的光能用于参与光合作用。进一步测定光响应曲线和CO 2响应曲线的结果表明，在光强低于200μmol m -2s -1时，过表达OsDREB1C水稻和野生型水稻的净光合速率之间无较大差异，而在高光强下差异逐渐增大，在2000μmol m -2s -1光强下，过表达OsDREB1C水稻的光合速率较野生型提高18.4-27.6％(图3中C，表3)，差异达到显著水平。CO 2响应曲线的测定结果与光响应曲线相似，在CO 2浓度大于400μmol mol -1时，过表达OsDREB1C水稻的光合速率较野生型有显著的提高(图3中D，表4)，进一步计算得到最大羧化效率(V cmax)及最大电子传递速率(J max)均显著高于野生型(图3中E、F，表5)。而OsDREB1C基因敲除水稻的光合作用相关参数均低于野生型或与之相近。综合以上结果表明，在水稻中过表达OsDREB1C基因可同时显著提高光能利用效率以及CO 2同化能力。
表1、光合作用效率(μmol CO 2m -2s -1)的检测结果
时间 WT OE1 OE2 OE5 KO1 KO2 KO3
8:00 21.59±1.42 22.78±1.08 25.51±1.76** 25.53±0.80** 18.44±0.80 19.35±1.49* 20.41±0.70
10:00 20.78±2.53 23.94±1.41* 26.26±0.49** 27.18±1.69** 21.88±1.42 20.49±2.43 19.74±1.83
13:00 14.07±0.95 20.16±0.80** 20.12±0.76** 18.22±0.63** 13.77±1.19 12.81±1.31 13.41±2.29
16:00 13.44±1.59 18.08±1.79** 19.03±2.09** 16.78±1.57** 14.00±2.61* 11.44±0.79* 11.55±0.91*
表1中，与同年份同地点WT相比*表示差异达到显著水平(p＜0.05)，**表示差异达到极显著水平(p＜0.01)。
表2、NPQ的检测结果
时间 WT OE1 OE2 OE5 KO1 KO2 KO3
8:00 3.12±0.76 0.25±0.13** 1.03±0.74** 0.89±0.58** 1.60±0.25* 2.96±0.32 2.50±0.57
10:00 3.97±0.28 1.36±0.64** 2.05±0.43** 1.21±0.74** 2.85±0.21** 3.78±0.53 3.75±0.54
12:00 3.45±0.07 1.83±0.23** 2.26±0.33** 1.72±0.25** 4.23±0.47* 3.67±0.44 3.66±0.80
14:00 3.62±0.20 1.58±0.80* 1.65±1.13* 1.98±1.05* 3.32±0.45 4.23±0.72 3.95±1.19
16:00 2.70±0.24 0.23±0.12** 0.95±0.73* 0.76±0.65** 2.65±0.66 2.82±0.67 2.82±0.31
18:00 0.28±0.33 0.06±0.07 0.01±0.02 0.45±0.51 1.63±0.24** 1.62±0.49 1.28±0.43*
表2中，与同年份同地点WT相比，*表示差异达到显著水平(p＜0.05)，**表示差异达到极显著水平(p＜0.01)。
Figure PCTCN2021100545-appb-000001
Figure PCTCN2021100545-appb-000002
表5、最大羧化效率(V cmax)及最大电子传递速率(J max)的结果
Figure PCTCN2021100545-appb-000003
表5中，与同年份同地点WT相比，*表示差异达到显著水平(p＜0.05)，**表示差异达到极显著水平(p＜0.01)。
4、转OsDREB1C基因水稻氮素利用效率的提高
检测水稻的氮利用效率，待测水稻：野生型日本晴水稻(WT)，过表达OsDREB1C水稻(OE1/OE2/OE5)，OsDREB1C基因敲除水稻(KO1/KO2/KO3)。
温室里营养液水培生长3周的待测水稻幼苗，提前置于不含氮(无(NH 4) 2SO 4和KNO 3)的木村B营养液(Kimura B solution)中进行氮饥饿处理3天。氮饥饿处理后将幼苗根部完全浸入0.1mM CaSO 4溶液(溶剂为去离子水)中浸泡1分钟后吸干残余水分，将根部置于含0.5mM K 15NO 3的营养液中培养。3小时后，将幼苗根部再次置于0.1mM CaSO 4溶液中浸泡1分钟后吸干残余水分，分别收取地上部和根部后于70℃烘干3天至恒重，并记录样品干重。样品研磨粉碎后，用IsoPrime 100稳定同位素比例质谱仪(Elementar，德国)分别测定地上部和根中的 15N含量，并计算氮吸收效率和氮转运效率。氮吸收效率＝(地上部的 15N含量+根中的 15N含量)/根干重/3；氮转运效率＝地上部的 15N含量/根中的 15N含量。
另取在北京田间生长的成熟待测水稻植株，分别收获单株叶片、秸杆及籽粒，置于105℃杀青30分钟，70℃烘干3天。样品研磨粉碎后，用IsoPrime 100 稳定同位素比例质谱仪(Elementar，德国)分别测定氮含量，并计算氮在不同部位的分配比例。
所用营养液如下：
营养液：木村B营养液(0.5mM(NH 4) 2SO 4,1mM KNO 3,0.54mM MgSO 4·7H 2O,0.3mM CaCl 2,0.18mM KH 2PO 4,0.09mM K 2SO 4,16μM Na 2SiO 3·9H 2O,9.14μM MnCl 2·4H 2O,46.2μM Na 2MoO 4·2H 2O,0.76μM ZnSO 4·7H 2O,0.32μM CuSO 4·5H 2O,40μM Fe(II)-EDTA,pH＝5.8)。
含0.5mM K 15NO 3的营养液：0.5M K 15NO 3母液稀释1000倍加入不含氮的木村营养液中(0.54mM MgSO 4·7H 2O,0.3mM CaCl 2,0.18mM KH 2PO 4,0.09mM K 2SO 4,16μM Na 2SiO 3·9H 2O,9.14μM MnCl 2·4H 2O,46.2μM Na 2MoO 4·2H 2O,0.76μM ZnSO 4·7H 2O,0.32μM CuSO 4·5H 2O,40μM Fe(II)-EDTA,pH＝5.8)。
结果表明， 15N处理3小时后，过表达OsDREB1C水稻的地上部和根部中 15N的含量显著高于野生型(图4中A、B，表6)，主要归因于其根部的氮素吸收效率(图4中C，表6)以及氮素由根部到地上部的转运效率(图4中D，表6)均较野生型有显著地提升，而OsDREB1C基因敲除水稻与野生型的差异不明显，除 15N的吸收效率较野生型降低外，其余指标均与野生型相近。进一步分析田间生长水稻植株在不同组织的氮素分配结果表明，过表达OsDREB1C水稻整株的氮素含量与野生型相比总体有所提高(图4中E，表7)，其中50.3-66.4％的氮分配至籽粒中，远高于野生型的41.1％和OsDREB1C基因敲除水稻的26-38.6％(图4中F，表7)。同时分配至叶片和秸杆的氮相应减少，分别为21.1-29.7％和12.5-19.9％，而野生型中的比例分别为42.66％和16.28％，OsDREB1C基因敲除水稻中为43.2-49.3％和18.2-24.7％(图4中F)。综合以上结果表明，在水稻中过表达OsDREB1C基因可显著提高植株对氮素的吸收和转运效率，并将更多的氮素分配至籽粒中。
表6、温室中培养的水稻的指标检测结果
Figure PCTCN2021100545-appb-000004
Figure PCTCN2021100545-appb-000005
表6中，与同地点同处理WT相比，*表示差异达到显著水平(p＜0.05)，**表示差异达到极显著水平(p＜0.01)。
表7、大田中培养的水稻的指标检测结果
Figure PCTCN2021100545-appb-000006
表7中，与同年份同地点WT相比，*表示差异达到显著水平(p＜0.05)，**表示差异达到极显著水平(p＜0.01)。
5、转OsDREB1C基因水稻的抽穗期提前
检测水稻的抽穗期，待测水稻：野生型日本晴水稻(WT)，过表达OsDREB1C水稻(OE1/OE2/OE5)，OsDREB1C基因敲除水稻(KO1/KO2/KO3)。
在北京田间种植条件下分别统计各待测水稻的抽穗期。统计方法为：每种待测水稻均种植3个小区重复，随机排列，以小区内50％植株的穗由剑叶叶鞘露出1/2时记为抽穗，抽穗期为从播种到抽穗所经历的天数。
结果表明(表8)，过表达OsDREB1C水稻的抽穗期较野生型大幅提前，2018年提前13-17天，2019年提前17-19天，而OsDREB1C基因敲除水稻的抽穗期分别较野生型延迟6-8天和3-5天，水稻籽粒灌浆成熟期也相应的提前或者推迟。说明，OsDREB1C及其编码基因可以调控水稻的抽穗期。
表8、野生型和转基因水稻在北京田间条件下的抽穗期(天)
水稻 2018年 2019年
WT 117±1 120.6±0.5
OE1 99.3±0.5** 103±1**
OE2 103.3±0.5** 101.3±0.5**
OE5 101.6±0.5** 103±1**
KO1 123.7±0.58** 124.7±0.58**
KO2 123.3±0.58** 124±0**
KO3 124.7±0.58** 123.7±0.58**
表8中，**表示与同年份同地点WT相比，差异达到极显著水平(p＜0.01)。
6、转基因水稻在大田的产量、品质及收获指数大幅提高
检测水稻的地上部干重和籽粒产量，待测水稻：野生型日本晴水稻(WT)，过表达OsDREB1C水稻(OE1/OE2/OE5)，OsDREB1C基因敲除水稻(KO1/KO2/KO3)。
在北京和海南两地进行的大田实验，每种待测水稻均种植3个小区重复，随机排列，在水稻籽粒成熟后测量单株籽粒产量，小区籽粒产量，地上部秸杆干重，并计算收获指数，收获指数为水稻单株籽粒产量与地上部生物量(地上部秸杆干重和单株籽粒产量之和)的比值。
地上部秸杆干重的测量方法如下：水稻成熟后，单株秸秆去除籽粒后，装入尼龙网袋中于80℃烘干至恒重，对样品进行称重。每个水稻材料取20-30个单株重复用于统计分析。
籽粒产量的测量方法如下：水稻单株稻穗脱粒并去除瘪粒后称籽粒重量即为单株籽粒产量，每个水稻材料取20-30个单株重复用于统计分析。统计小区产量时去除边行，取中间的30株水稻测定籽粒重量计为一个小区产量，3个小 区重复用于统计分析。
稻米品质的测定方法如下：取2019年北京田间实验收获的水稻籽粒自然储藏3个月后用于稻米品质分析，测定方法参照农业部行业标准NY/T 83-2017米质测定方法。结果表明，过表达OsDREB1C水稻的籽粒产量较野生型大幅提升，差异达到显著水平，同时其地上部秸杆重量显著低于野生型，使得收获指数较野生型显著提高。
由产量结果来看(表9)，在北京和海南，转基因水稻其单株产量分别较野生型提高45.1-67.6％和7.8-16％，小区产量提高41.3-68.3％和11.9-27.4％。同时，地上部秸秆较野生型降低12.1-24.7％和17.5-25.8％，最终导致转基因水稻的收获指数(HI)较野生型显著提升，在北京HI的增幅可达10.3-55.7％。同时转基因水稻的稻米品质也较野生型有不同程度提高(表10)，其中糙米率、精米率、整精米率均较野生型显著增加，垩白度和垩白粒率降低，改善了外观品质，而直链淀粉和蛋白含量则无显著影响。OsDREB1C基因敲除水稻的单株产量和小区产量均较野生型显著降低，同时地上部生物量增加，直接导致收获指数较野生型大幅降低22.4-37.7％。说明在水稻中转入OsDREB1C基因后，可大幅提高转基因水稻的产量、稻米品质以及收获指数，OsDREB1C及其编码基因可以调控水稻的籽粒产量、稻米品质和收获指数。
各指标的结果如表9和表10所示。
Figure PCTCN2021100545-appb-000007
Figure PCTCN2021100545-appb-000008
实施例2、OsDREB1C在小麦和拟南芥中的功能检测
1、重组载体的构建
小麦过表达载体的构建：通过PCR从水稻日本晴cDNA中扩增得到OsDREB1C基因的CDS全长，通过In-Fusion反应体系将所得到的PCR回收产物与pWMB110载体(Huiyun Liu,Ke Wang,Zimiao Jia,Qiang Gong,Zhishan Lin,Lipu Du,Xinwu Pei,Xingguo Ye,Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system,Journal of Experimental Botany,Volume 71,Issue 4,7 February 2020,Pages 1337–1349,https://doi.org/10.1093/jxb/erz529)经过BamHI和SacI双酶切得到的载体骨架相连，将得到的序列正确的重组载体记为pWMB110-OsDREB1C。pWMB110-OsDREB1C为将pWMB110载体的BamHI和SacI识别位点间的DNA片段替换为序列表中序列2所示的OsDREB1C编码基因得到的重组载体，pWMB110-OsDREB1C能在UBI启动子的驱动下表达OsDREB1C基因所编码的蛋白质(即序列1所示的OsDREB1C蛋白质)。
所用引物序列如下：
Fielder-OsDREB1C-OE-F：5′-CAGGTCGACTCTAGA GGATCCATGGAGTACTACGAGCAGGAG-3′(序列表中序列12)；
Fielder-OsDREB1C-OE-R：5′-CGATCGGGGAAATTC GAGCTCTCAGTAGCTCCAGAGTGTGAC-3′(序列表中序列13)。
拟南芥过表达载体的构建：通过PCR从水稻日本晴cDNA中扩增得到OsDREB1C基因的CDS(不包含终止密码子)，将所得到的PCR回收产物与Gateway系统入门载体pENTR(ThermoFisher，K240020SP)相连，将得到的序列正确的重组载体记为pENTR-OsDREB1C，将pENTR-OsDREB1C与pGWB5载体(Nakagawa T,Kurose T,Hino T,Tanaka K,Kawamukai M,Niwa Y,Toyooka K,Matsuoka K,Jinbo T,Kimura T.Development of series of gateway binary vectors,pGWBs,for realizing efficient construction of fusion genes for plant transformation.J Biosci Bioeng.2007Jul；104(1):34-41.doi:10.1263/jbb.104.34.)进行LR反应将OsDREB1C的CDS序列转移至终载体pGWB5中，所得到的序列正确的重组载体记为pGWB5-OsDREB1C。 pGWB5-OsDREB1C能在CaMV 35S启动子的驱动下表达OsDREB1C基因所编码的蛋白质(即序列1所示的OsDREB1C蛋白质)。
所用引物序列如下：
OsDREB1C-CDS-F：5′-CACCATGGAGTACTACGAGCAGGAG-3′(序列表中序列14)；
OsDREB1C-CDS-R：5′-GTAGCTCCAGAGTGTGACGTC-3′(序列表中序列15)。
2、转基因植株的构建
将小麦品种Fielder的幼胚消毒后诱导得到胚性愈伤组织，将步骤1得到的pWMB110-OsDREB1C导入农杆菌C58C1中，利用农杆菌介导的小麦遗传转化方法对愈伤组织进行侵染共培养，利用抗性筛选得到转基因植株，筛选到的即为OsDREB1C转基因小麦材料。因OsDREB1C与小麦自身的同源基因相似度极高，无法用qRT-PCR方法检测OsDREB1C基因在水稻中的表达水平。因此利用小麦野生型Fielder作为对照，用PCR方法检测转基因小麦cDNA中是否存在载体的Bar基因，所用引物为：5′-CAGGAACCGCAGGAGTGGA-3′(序列表中序列16)，5′-CCAGAAACCCACGTCATGCC-3′(序列表中序列17)。电泳检测结果显示，OsDREB1C转基因小麦的3个株系(TaOE-5、TaOE-8和TaOE-9)中均存在Bar基因，而野生型中未检出，说明这三个株系均为转入pWMB110-OsDREB1C载体的转基因材料(图5中A)。
将步骤1得到的pGWB5-OsDREB1C导入农杆菌GV3101中，利用农杆菌介导的浸花法转入拟南芥野生型Col-0，种子收获后播入抗性培养基进行筛选得到转基因植株，即为OsDREB1C转基因拟南芥材料。利用拟南芥野生型Col-0作为对照，用qRT-PCR方法检测OsDREB1C转基因拟南芥中OsDREB1C基因在RNA水平上的的相对表达水平，所用引物为：5′-CATGATGATGCAGTACCAGGA-3′(序列表中序列18)，5′-GATCATCAGTAGCTCCAGAGTG-3′(序列表中序列19)；内参基因为拟南芥Actin，内参基因引物为：5′-GCACCACCTGAAAGGAAGTACA-3′(序列表中序列20)，5′-CGATTCCTGGACCTGCCTCATC-3′(序列表中序列21)。结果显示，OsDREB1C转基因拟南芥的3个株系(AtOE-10、AtOE-11和AtOE-12)中OsDREB1C基因的相对表达水平均显著高于野生型(Col-0)，这三个株系均为 过表达OsDREB1拟南芥材料(图5中B)。
3、转OsDREB1C基因小麦的表型鉴定
检测温室生长盆栽小麦的表型，待测材料：野生型小麦(Fielder)，过表达OsDREB1C小麦(TaOE-5、TaOE-8和TaOE-9)。温室温度控制在22-24℃，日照长度为12小时光照/12小时黑暗。
统计野生型及转基因小麦的抽穗期，并用LICOR-6400XT便携式光合仪(LI-COR，美国)测定处于抽穗期的待测小麦旗叶的光合作用速率，光强设定为1000μmol m -2s -1。小麦籽粒成熟后，测定穗粒数、千粒重及单株籽粒产量。
结果表明(表11、图6)，转基因小麦(TaOE-5、TaOE-8和TaOE-9)比野生型Fielder提前抽穗，同时光合速率较野生型提高。产量性状中穗粒数也较野生型有所提高，千粒重显著高于野生型，最终导致单株产量提高，表明OsDREB1C在小麦中同样具有提前抽穗、提高光合能力以及提高产量的功能。抽穗期为从播种到抽穗所经历的天数。
表11、野生型和转基因小麦的抽穗期、光合速率及产量性状
Figure PCTCN2021100545-appb-000009
4、转OsDREB1C基因拟南芥的表型鉴定
检测温室生长拟南芥的表型，待测材料：野生型拟南芥(Col-0)，过表达OsDREB1C拟南芥(AtOE-10、AtOE-11和AtOE-12)。在短日照(8小时光照/16小时黑暗)下培养2周，然后转移到长日照(16小时光照/8小时黑暗)培养。
统计野生型及转基因拟南芥的抽薹时间、抽薹时莲座叶数目以及地上部生物量(包括鲜重和干重)。抽薹时间为从播种到茎伸长并开花所经历的天数。
结果表明(表12、图7)，转基因拟南芥(AtOE-10、AtOE-11和AtOE-12)比野生型Col-0提前抽薹(1-3.9天)，抽薹时莲座叶较野生型多5-7片，且其 地上部鲜重和干重均较野生型提高，表明过表达OsDREB1C同样能够提高双子叶植物拟南芥的生物量，并使拟南芥提前抽薹。
表12、野生型和转基因拟南芥的表型指标
拟南芥 抽薹时间(天) 莲座叶数目 鲜重(克)
Col-0 38±0.94 17.6±0.97 0.81±0.08
AtOE-10 35.1±0.99 23.1±1.91 0.93±0.12
AtOE-11 34.1±0.74 24.6±1.26 1.01±0.14
AtOE-12 37±0.82 22.6±1.35 1.10±0.20
工业应用
实验证明，本发明的OsDREB1C及其相关的生物材料可以提高植物的光合作用效率，可以促进氮吸收与转运并提高植物及籽粒中的氮含量，可以促进提前抽穗，还可以提高产量和品质。本发明从协同提高作物光合效率、氮素利用效率和抽穗期出发，在实现作物产量大幅提升的同时实现了氮素利用效率的协同提升，另外还为作物高产和早熟矛盾提供了解决方案。因此，本发明对于进一步大幅提高作物产量潜力和氮肥利用效率，实现“高产高效”；另一方面，高产早熟矛盾的解决对于对解决直播稻和粮经、粮菜、粮油连作稻、双季稻的早熟丰产以及亚种间杂交稻“超亲晚熟”等问题具有重要的应用潜力。

Claims (18)

  1. 蛋白质或调控所述蛋白质活性或含量的物质的下述任一应用：
    D1)调控植物光合作用；
    D2)制备调控植物光合作用产品；
    D3)调控植物光合作用与氮吸收或转运；
    D4)制备调控植物光合作用与氮吸收或转运产品；
    D5)调控植物光合作用与氮含量；
    D6)制备调控植物光合作用与氮含量产品；
    D7)调控植物光合作用与开花时间；
    D8)制备调控植物光合作用与开花时间产品；
    D9)调控植物光合作用、氮吸收或转运与开花时间；
    D10)制备调控植物光合作用、氮吸收或转运与开花时间产品；
    D11)调控植物光合作用、氮含量与开花时间；
    D12)制备调控植物光合作用、氮含量与开花时间产品；
    D13)植物育种；
    所述蛋白质为如下A1)、A2)、A3)或A4)：
    A1)氨基酸序列是序列1的蛋白质；
    A2)将序列表中序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质；
    A3)来源于水稻、玉米、高粱、大豆、油菜、拟南芥、谷子、山羊草、二穗短柄草或小麦且与序列1具有64％或以上同一性并与A1)所述蛋白质具有相同功能的蛋白质；
    A4)在A1)或A2)或A3)的N端或/和C端连接标签得到的融合蛋白质。
  2. 根据权利要求1所述的应用，其特征在于：所述植物为M1)或M2)或M3)：
    M1)单子叶植物或双子叶植物；
    M2)禾本科植物、十字花科植物或豆科植物；
    M3)水稻、小麦、玉米、拟南芥、油菜或大豆。
  3. 根据权利要求1或2所述的应用，其特征在于：所述光合作用体现在光 合速率、净光合速率、热耗散NPQ、最大羧化效率和/或最大电子传递速率上；
    所述转运体现在根部向地上部的转运，或向籽粒中的转运上；
    所述氮含量为植株或器官中氮含量；
    所述开花时间体现在抽穗期上。
  4. 与权利要求1中所述蛋白质相关的生物材料的下述任一应用：
    D1)调控植物光合作用；
    D2)制备调控植物光合作用产品；
    D3)调控植物光合作用与氮吸收或转运；
    D4)制备调控植物光合作用与氮吸收或转运产品；
    D5)调控植物光合作用与氮含量；
    D6)制备调控植物光合作用与氮含量产品；
    D7)调控植物光合作用与开花时间；
    D8)制备调控植物光合作用与开花时间产品；
    D9)调控植物光合作用、氮吸收或转运与开花时间；
    D10)制备调控植物光合作用、氮吸收或转运与开花时间产品；
    D11)调控植物光合作用、氮含量与开花时间；
    D12)制备调控植物光合作用、氮含量与开花时间产品；
    D13)植物育种；
    所述生物材料为下述B1)至B9)中的任一种：
    B1)编码权利要求1中所述蛋白质的核酸分子；
    B2)含有B1)所述核酸分子的表达盒；
    B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；
    B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；
    B5)含有B1)所述核酸分子的转基因植物细胞系、或含有B2)所述表达盒的转基因植物细胞系；
    B6)含有B1)所述核酸分子的转基因植物组织、或含有B2)所述表达盒的转基因植物组织；
    B7)含有B1)所述核酸分子的转基因植物器官、或含有B2)所述表达盒的 转基因植物器官；
    B8)降低权利要求1中所述蛋白质表达量或敲除权利要求1中所述蛋白质编码基因的核酸分子；
    B9)含有B8)所述核酸分子的表达盒、重组载体、重组微生物、转基因植物细胞系、转基因植物组织或转基因植物器官。
  5. 根据权利要求4所述的应用，其特征在于：B1)所述核酸分子为如下b11)或b12)或b13)或b14)或b15)：
    b11)编码序列是序列表中序列2的cDNA分子或DNA分子；
    b12)序列表中序列2所示的cDNA分子或DNA分子；
    b13)序列表中序列3的第3001-4131位所示的DNA分子；
    b14)与b11)或b12)或b13)限定的核苷酸序列具有73％或73％以上同一性，且编码权利要求1中所述蛋白质的cDNA分子或DNA分子；
    b15)在严格条件下与b11)或b12)或b13)或b14)限定的核苷酸序列杂交，且编码权利要求1中所述蛋白质的cDNA分子或DNA分子；
    B2)所述表达盒为如下b21)或b22)或b23)：
    b21)序列表中序列3所示的DNA分子；
    b22)与b21)限定的核苷酸序列具有73％或73％以上同一性，且具有相同功能的DNA分子；
    b23)在严格条件下与b21)或b22)限定的核苷酸序列杂交，且具有相同功能的DNA分子。
  6. 根据权利要求4或5所述的应用，其特征在于：所述植物为M1)或M2)或M3)：
    M1)单子叶植物或双子叶植物；
    M2)禾本科植物、十字花科植物或豆科植物；
    M3)水稻、小麦、玉米、拟南芥、油菜或大豆。
  7. 根据权利要求4或5所述的应用，其特征在于：所述光合作用体现在光合速率、净光合速率、热耗散NPQ、最大羧化效率和/或最大电子传递速率上；
    所述转运体现在根部向地上部的转运，或向籽粒中的转运上；
    所述氮含量为植株或器官中氮含量；
    所述开花时间体现在抽穗期上。
  8. 根据权利要求4或5所述的应用，其特征在于：所述植物为M1)或M2)或M3)：
    M1)单子叶植物或双子叶植物；
    M2)禾本科植物、十字花科植物或豆科植物；
    M3)水稻、小麦、玉米、拟南芥、油菜或大豆；
    所述光合作用体现在光合速率、净光合速率、热耗散NPQ、最大羧化效率和/或最大电子传递速率上；
    所述转运体现在根部向地上部的转运，或向籽粒中的转运上；
    所述氮含量为植株或器官中氮含量；
    所述开花时间体现在抽穗期上。
  9. 下述任一方法：
    X1)培育光合作用增强植物的方法，包括使受体植物中表达权利要求1中所述蛋白质，或提高受体植物中权利要求1中所述蛋白质的含量或活性，得到光合作用增强的目的植物；
    X2)培育光合作用增强且氮吸收或转运能力增强植物的方法，包括使受体植物中表达权利要求1中所述蛋白质，或提高受体植物中权利要求1中所述蛋白质的含量或活性，得到光合作用增强且氮吸收或转运能力增强的目的植物；
    X3)培育光合作用增强且氮含量增加植物的方法，包括使受体植物中表达权利要求1中所述蛋白质，或提高受体植物中权利要求1中所述蛋白质的含量或活性，得到光合作用增强且氮含量增加的目的植物；
    X4)培育光合作用增强且开花时间提前植物的方法，包括使受体植物中表达权利要求1中所述蛋白质，或提高受体植物中权利要求1中所述蛋白质的含量或活性，得到光合作用增强且开花时间提前的目的植物；
    X5)培育光合作用增强、氮吸收或转运能力增强且开花时间提前增强植物的方法，包括使受体植物中表达权利要求1中所述蛋白质，或提高受体植物中权利要求1中所述蛋白质的含量或活性，得到光合作用增强、氮吸收或转运能力增强且开花时间提前增强的目的植物；
    X6)培育光合作用增强、氮含量增加增加且开花时间提前植物的方法，包括使受体植物中表达权利要求1中所述蛋白质，或提高受体植物中权利要求1中所述蛋白质的含量或活性，得到光合作用增强、氮含量增加且开花时间提前 增加的目的植物。
  10. 根据权利要求9所述的方法，其特征在于：X1)-X6)所述方法通过向所述受体植物中导入权利要求1中所述蛋白质的编码基因并使所述编码基因得到表达实现。
  11. 根据权利要求10所述的方法，其特征在于：所述编码基因为权利要求4或5中B1)所述核酸分子。
  12. 根据权利要求9-11中任一所述的方法，其特征在于：所述植物为M1)或M2)或M3)：
    M1)单子叶植物或双子叶植物；
    M2)禾本科植物、十字花科植物或豆科植物；
    M3)水稻、小麦、玉米、拟南芥、油菜或大豆。
  13. 根据权利要求9-11中任一所述的方法，其特征在于：所述光合作用体现在光合速率、净光合速率、热耗散NPQ、最大羧化效率和/或最大电子传递速率上；
    所述转运体现在根部向地上部的转运，或向籽粒中的转运上；
    所述氮含量为植株或器官中氮含量；
    所述开花时间体现在抽穗期上。
  14. 根据权利要求9-11中任一所述的方法，其特征在于：所述植物为M1)或M2)或M3)：
    M1)单子叶植物或双子叶植物；
    M2)禾本科植物、十字花科植物或豆科植物；
    M3)水稻、小麦、玉米、拟南芥、油菜或大豆；
    所述光合作用体现在光合速率、净光合速率、热耗散NPQ、最大羧化效率和/或最大电子传递速率上；
    所述转运体现在根部向地上部的转运，或向籽粒中的转运上；
    所述氮含量为植株或器官中氮含量；
    所述开花时间体现在抽穗期上。
  15. 具有如下D1)-D6)中任一的功能的产品，含有权利要求1中所述蛋白质或权利要求4或5中所述生物材料：
    D1)调控植物光合作用；
    D2)调控植物光合作用与氮吸收或转运；
    D3)调控植物光合作用与氮含量；
    D4)调控植物光合作用与开花时间；
    D5)调控植物光合作用、氮吸收或转运与开花时间；
    D6)调控植物光合作用、氮含量与开花时间。
  16. 根据权利要求15所述的产品，其特征在于：所述植物为M1)或M2)或M3)：
    M1)单子叶植物或双子叶植物；
    M2)禾本科植物、十字花科植物或豆科植物；
    M3)水稻、小麦、玉米、拟南芥、油菜或大豆。
  17. 根据权利要求15或16所述的产品，其特征在于：所述光合作用体现在光合速率、净光合速率、热耗散NPQ、最大羧化效率和/或最大电子传递速率上；
    所述转运体现在根部向地上部的转运，或向籽粒中的转运上；
    所述氮含量为植株或器官中氮含量；
    所述开花时间体现在抽穗期上。
  18. 权利要求1中所述蛋白质或权利要求4或5中所述生物材料。
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