CN113072631A

CN113072631A - OsDREB1C and application of coding gene thereof in improving rice photosynthetic efficiency

Info

Publication number: CN113072631A
Application number: CN202110259881.9A
Authority: CN
Inventors: 周文彬; 李霞; 魏少博
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2021-03-10
Filing date: 2021-03-10
Publication date: 2021-07-06
Also published as: CN114276425B; CN114276425A; WO2022188289A1

Abstract

The invention discloses an OsDREB1C and application of a coding gene thereof in improving rice photosynthetic efficiency. The sequence of the OsDREB1C is the sequence 1 in the sequence table, and the sequence of the coding gene is the sequence 2 in the sequence table. Experiments prove that the OsDREB1C and the related biological materials thereof can improve the photosynthesis efficiency of plants, promote nitrogen absorption and transportation, improve the nitrogen content in the plants and grains, promote early heading, and improve the yield and the quality, and the OsDREB1C and the related biological materials thereof have important biological significance and industrial value and wide application prospect.

Description

OsDREB1C and application of coding gene thereof in improving rice photosynthetic efficiency

Technical Field

The invention relates to the field of biotechnology, and discloses application of OsDREB1C and a coding gene thereof in improving rice photosynthetic efficiency.

Background

With the population growth and economic development in the future, the food demand in China still has a rapid growth tendency. Under the conditions of continuous reduction of the cultivated land area and limited potential of grain planting area expansion, increasing the total yield by increasing the yield per crop in a large area is the only option for guaranteeing the grain safety in China. Therefore, under the pressure of grain safety demand, high yield is a constantly sought goal in agricultural production. The green revolution started from the middle of the 50 s in the twentieth century realizes the great improvement of the yield potential of crops by the genetic improvement of crop varieties and the improvement of cultivation management technology. However, in recent years, the yield per crop is in a situation of standing still or even descending, and a new strategy and approach are needed for improving the yield per crop.

In agricultural production, the application of nitrogen fertilizer has always been one of the important measures for increasing the yield of crops. The continuous large amount of nitrogen fertilizer is input, so that the planting cost is increased, and increasingly serious environmental pollution problems such as climate change, soil acidification, water eutrophication and the like are caused. In addition, the phenomenon of 'green and late maturity' of crops can be caused by applying a large amount of nitrogen fertilizer, and the sowing of later crops is influenced; how to reduce the nitrogen fertilizer input in agricultural production and continuously improve the crop yield becomes a major problem to be urgently solved in the agricultural sustainable development of China at present. The urgent need for sustainable development of agriculture in China is to explore a regulation mechanism and a technical way for the synergy of great crop yield increase and efficient resource utilization.

Heading stage is one of important agronomic traits of crops and determines season, regional adaptability and yield of rice. The proper heading period is the guarantee of stable yield and high yield of crops. The breeding of early-maturing high-yield new varieties is always one of the main aspects of crop genetic breeding research. The phenomenon of high yield without precocity and precocity without high yield, namely the phenomenon of 'good but not early, early but not good', is a great problem in the cultivation of crop varieties.

At present, researchers develop related exploration and research from the aspects of crop breeding, cultivation management measures and functional gene mining, and also make some important progress, but no effective solution is provided aiming at the two problems of high yield and high resource efficiency, high yield and early maturity in crop production.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the photosynthesis of plants.

In order to solve the technical problems, the invention firstly provides any one of the following applications of the protein or the substance for regulating the activity or the content of the protein:

D1) regulating plant photosynthesis;

D2) preparing a product for regulating and controlling plant photosynthesis;

D3) regulating and controlling plant photosynthesis and nitrogen absorption or transport;

D4) preparing a product for regulating and controlling plant photosynthesis and nitrogen absorption or transport;

D5) regulating and controlling photosynthesis and nitrogen content of the plant;

D6) preparing a product for regulating and controlling plant photosynthesis and nitrogen content;

D7) regulating and controlling photosynthesis and flowering time of plants;

D8) preparing products for regulating and controlling plant photosynthesis and flowering time;

D9) regulating and controlling plant photosynthesis, nitrogen absorption or transport and flowering time;

D10) preparing products for regulating and controlling plant photosynthesis, nitrogen absorption or transport and flowering time;

D11) regulating and controlling photosynthesis, nitrogen content and flowering time of the plant;

D12) preparing products for regulating and controlling plant photosynthesis, nitrogen content and flowering time;

D13) plant breeding;

the protein (named as OsDREB1C) is A1), A2), A3) or A4 as follows:

A1) the amino acid sequence is the protein of sequence 1;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1 in the sequence table and has the same function;

A3) a protein derived from rice, millet, corn, sorghum, aegilops tauschii, wheat or brachypodium distachyon and having 64% or more identity to sequence 1 and the same function as the protein of A1);

A4) a fusion protein obtained by connecting labels at the N terminal or/and the C terminal of A1) or A2) or A3).

In order to facilitate the purification of the protein of A1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in sequence 1 in the sequence listing may be labeled as shown in the following table.

Table: sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein in A2) above 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. The having 64% or greater than 64% 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 of A2) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of A2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in SEQ ID No. 2, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching the coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. Wherein, the DNA molecule shown in sequence 2 encodes the protein shown in sequence 1.

The invention also provides any one of the following applications of the biological material related to OsDREB 1C:

D1) regulating plant photosynthesis;

D2) preparing a product for regulating and controlling plant photosynthesis;

D7) regulating and controlling photosynthesis and flowering time of plants;

D13) plant breeding;

the biomaterial is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding OsDREB 1C;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising 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);

B8) nucleic acid molecules for reducing the expression level of OsDREB1C or knocking out the coding gene of OsDREB 1C;

B9) an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ comprising the nucleic acid molecule according to B8).

In the above application, the nucleic acid molecule of B1) may be B11) or B12) or B13) or B14) or B15):

b11) the coding sequence is cDNA molecule or DNA molecule of sequence 2 in the sequence table;

b12) a cDNA molecule or a DNA molecule shown in a sequence 2 in a sequence table;

b13) a DNA molecule shown in the 3001-4131 site of the sequence 3 in the sequence table;

b14) a cDNA molecule or a DNA molecule having 73% or more identity with the nucleotide sequence defined in b11) or b12) or b13) and encoding OsDREB 1C;

b15) a cDNA molecule or a DNA molecule which hybridizes with the nucleotide sequence defined by b11) or b12) or b13) or b14) under strict conditions and codes for OsDREB 1C;

B2) the expression cassette is b21) or b22) or b23) as follows:

b21) DNA molecule shown in sequence 3 in the sequence table;

b22) DNA molecule with 73% or above 73% identity with the nucleotide sequence defined by b21) and with the same function;

b23) a DNA molecule which is hybridized with the nucleotide sequence defined by b21) or b22) under strict conditions and has the same function.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding OSDREB1C protein of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 73% or more identity to the nucleotide sequence of OSDREB1C protein isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode OSDREB1C protein and have the function of OSDREB1C protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 73% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 1 of the present invention. Identity can be assessed visually or by 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.

In the above application, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; and alsoCan be as follows: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: hybridization in a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; can also be: hybridization and washing of membranes 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of membranes 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; can also be: 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ and washing the membrane.

The identity of 73% or more may be 80%, 85%, 90% or 95% or more.

In the above applications, the expression cassette containing a nucleic acid molecule encoding OSDREB1C protein (OSDREB1C gene expression cassette) described in B2) refers to DNA capable of expressing OSDREB1C protein in host cells, and the DNA may include not only a promoter for initiating transcription of OSDREB1C gene, but also a terminator for terminating transcription of OSDREB1C gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter of cauliflower mosaic virus 35S; 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-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., phaseolin, napin, ol)The promoters of eosin 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, e.g., Odell et al (I)⁹⁸⁵) 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 (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

The recombinant vector containing the OSDREB1C gene expression cassette can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301, or pCAMBIA1391-Xb (CAMBIA Corp.), etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid can be a pBWA (V) HS vector or a plasmid containing psgR-Cas 9-Os.

B3) The recombinant vector can be pBWA (V) HS-OsDREB 1C. The pBWA (V) HS-OsDREB1C is a recombinant vector obtained by inserting an OsDREB1C coding gene shown in a sequence 2 in a sequence table into a BsaI (Eco31I) enzyme cutting site of a pBWA (V) HS vector. The pBWA (V) HS-OsDREB1C can over-express the protein coded by OsDREB1C gene (namely OsDREB1C protein shown in sequence 1) under the drive of CaMV 35S promoter.

B8) The recombinant vector can be a recombinant vector which is prepared by utilizing a crisper/cas9 system and can reduce the content of OSDREB 1C. The recombinant vector can express a sgRNA targeted to B1) the nucleic acid molecule. The target sequence of the sgRNA can be the 356-374 position of the sequence 2 in the sequence table.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacteria can be Agrobacterium, such as Agrobacterium rhizogenes EHA 105.

In the above application, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation material.

The invention also provides any one of the following methods:

x1), comprising expressing OsDREB1C in a recipient plant, or increasing OsDREB1C content or activity in the recipient plant, to obtain a target plant with enhanced photosynthesis;

x2) a method for cultivating a plant with enhanced photosynthesis and enhanced nitrogen absorption or transport capacity, 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 enhanced nitrogen absorption or transport capacity;

x3) cultivating photosynthesis-enhanced and nitrogen-content-enhanced plants, comprising expressing OsDREB1C in a recipient plant, or increasing OsDREB1C content or activity in the recipient plant, to obtain photosynthesis-enhanced and nitrogen-content-enhanced target plants;

x4) cultivating photosynthesis enhancing and flowering time advancing plant, comprising expressing OsDREB1C in a recipient plant, or increasing OsDREB1C content or activity in the recipient plant, to obtain photosynthesis enhancing and flowering time advancing target plant;

x5) cultivating a plant with enhanced photosynthesis, enhanced nitrogen uptake or transport capacity and advanced flowering time, comprising expressing OsDREB1C in a recipient plant, or increasing OsDREB1C content or activity in the recipient plant, to obtain a target plant with enhanced photosynthesis, enhanced nitrogen uptake or transport capacity and advanced flowering time;

x6) method for cultivating photosynthesis-enhancing, nitrogen-content-increasing and flowering-time-advancing plants, comprising expressing OsDREB1C in a recipient plant, or increasing OsDREB1C content or activity in a recipient plant, to obtain a target plant with photosynthesis-enhancing, nitrogen-content-increasing and flowering-time-advancing effects.

X1) -X6) in the above method, can be carried out by introducing a coding gene of OsDREB1C into the recipient plant and allowing the coding gene to be expressed.

In the above method, the encoding gene may be B1).

In the above method, the coding gene of OsDREB1C may be modified as follows, and then introduced into the recipient plant, so as to achieve better expression effect:

1) modifying and optimizing according to actual needs to enable the gene to be efficiently expressed; for example, according to the codon preferred by the recipient plant, the codon can be changed to conform to the plant preference while maintaining the amino acid sequence of the coding gene of OsDREB1C of the present invention; during the optimization, it is desirable to maintain a GC content in the optimized coding sequence to best achieve high expression levels of the introduced gene in plants, wherein the GC content can be 35%, more than 45%, more than 50%, or more than about 60%;

2) modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

3) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

4) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

5) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

The coding gene of OsDREB1C can be introduced into a receptor plant by using a recombinant expression vector containing the coding gene of OsDREB 1C. The recombinant expression vector can be specifically pBWA (V) HS-OsDREB 1C.

The recombinant expression vector can be introduced into Plant cells by using conventional biotechnological methods such as Ti plasmid, Plant virus vector, direct DNA transformation, microinjection, electroporation, etc. (Weissbach,1998, Method for Plant Molecular Biology VIII, academic 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 plant in which the OsDREB1C protein or the gene encoding it is altered, but also its progeny. For the plant of interest, the gene may be propagated in the species, or transferred into other varieties of the same species, including commercial varieties in particular, using conventional breeding techniques. The plant of interest includes seeds, callus, whole plants and cells.

The invention also provides a product having the function of any one of D1) -D6) as follows, the product containing OsDREB1C or the biomaterial:

D1) regulating plant photosynthesis;

D2) regulating and controlling plant photosynthesis and nitrogen absorption or transport;

D3) regulating and controlling photosynthesis and nitrogen content of the plant;

D4) regulating and controlling photosynthesis and flowering time of plants;

D5) regulating and controlling plant photosynthesis, nitrogen absorption or transport and flowering time;

D6) regulating and controlling plant photosynthesis, nitrogen content and flowering time.

Above, the plant may be M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) rice.

Above, the photosynthesis may be embodied in photosynthetic rate, net photosynthetic rate, heat dissipation NPQ, maximum carboxylation efficiency and/or maximum electron transfer rate;

the nitrogen transport may be embodied in transport from the root to the aerial part, or into the kernel;

the nitrogen content can be the nitrogen content in a plant or organ;

the flowering time may be reflected in heading date.

The organ may be a root, stem, leaf and/or grain of the plant.

The modulation may be enhancement or inhibition, or promotion or inhibition, or enhancement or reduction.

OsDREB1C or the biological material also belongs to the protection scope of the invention.

Experiments prove that the OsDREB1C and related biological materials thereof can improve the photosynthesis efficiency of plants, promote nitrogen absorption and transportation, improve the nitrogen content in the plants and seeds, promote early heading, and improve the yield and the quality. The method starts from the synergistic improvement of the photosynthetic efficiency, the nitrogen utilization efficiency and the heading stage of the crops, realizes the synergistic improvement of the nitrogen utilization efficiency while realizing the great improvement of the crop yield, and provides a solution for the contradiction between the high crop yield and the early maturity. Therefore, the invention further greatly improves the crop yield potential and the nitrogen fertilizer utilization efficiency, and realizes high yield and high efficiency; on the other hand, the solution of the contradiction of high yield and early maturing has important application potential for solving the problems of early maturing and high yield of direct seeding rice, grain and longitude, grain and vegetable, grain and oil continuous cropping rice and double cropping rice, super-parent and late maturing of inter-subspecies hybrid rice and the like.

Drawings

FIG. 1 shows the alignment of the sequences.

FIG. 2 shows the results of the detection of the relative expression level of OsDREB1C gene in transgenic rice and the detection of the sequence of the target region of gene-knocked-out rice material. (A) Relative expression level of OsDREB1C gene; (B) the OsDREB1C gene knocks out the gene editing site of rice.

FIG. 3 shows photosynthesis parameters of wild type and transgenic rice plants. A-photosynthetic daily change; B-NPQ daily change; c-light response curve; D-CO₂A response curve; e-maximum CO₂Carboxylation efficiency; f-maximum electron transfer rate.

FIG. 4 shows the results of nitrogen uptake and utilization measurements of wild-type and transgenic rice plants. A-the above-ground part¹⁵The content of N; in B-root¹⁵Content of N；C-¹⁵N absorption efficiency; d-¹⁵N transport efficiency from root to overground part; e-nitrogen content in different tissues of rice; f-distribution ratio of nitrogen in different tissues of rice. E. In the column diagram of F, there are seeds, stalks and leaves in sequence from top to bottom.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

pBWA (V) HS vector (Zhao et al, DEP1 is included in the transformed vector and quality in rice (Oriza sativa L.), PLOS ONE, March 11,2019, https:// doi.org/10.1371/journal.po.0213504) in the following examples, which was obtained by the public from the applicant and was used only for the repetition of the relevant experiments of the present invention and was not used for other purposes.

The plasmid containing psgR-Cas9-Os in the following examples (Humojiao, Yangjia, Chenglan, Zhouhua, nivaan, Wangxing, Zmeiliang, Cao dao, Huangwei. oriented editing of the rice SD1 gene by using CRISPR/Cas9 System Chinese Rice science, 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 effective genetic engineering in plants, 2013,6(6):2008-2011.) was publicly available from the applicant, and was only used for repeating experiments related to the present invention, and was not used for other purposes.

Example 1 OsDREB1C has effects of improving rice photosynthetic efficiency, promoting nitrogen absorption, promoting pre-heading and increasing yield

The embodiment provides a protein derived from Nipponbare rice, which has the functions of improving the photosynthesis efficiency of the rice, promoting nitrogen absorption, promoting early heading and improving yield, and is named as OsDREB1C, the sequence of the protein is shown as sequence 1 in a sequence table, in Nipponbare, the coding gene sequence of OsDREB1C is shown as sequence 2, and the genome sequence is shown as position 3001-4131 of sequence 3. In the sequence 3, the 1 st to 3000 th sites are the promoter of the OsDREB1C gene in the Nipponbare genome DNA.

Sequence alignment of rice OsDREB1C with homologous proteins in other plants revealed that the identities of rice with millet, corn, sorghum, aegilops tauschii, wheat, brachypodium distachyon were 73.52%, 64.06%, 66.52%, 66.05%, 65.88%, respectively (fig. 1).

1. Construction of recombinant vectors

Construction of overexpression vectors: PCR products containing OsDREB1C gene full-length CDS are obtained by PCR amplification from rice Nipponbare cDNA, single enzyme digestion is carried out on the obtained PCR products by BsaI (Eco31I), the obtained enzyme digestion products are connected with a vector framework obtained by single enzyme digestion of pBWA (V) HS vector by BsaI (Eco31I), and the obtained recombinant vector with correct sequence is marked as pBWA (V) HS-OsDREB 1C. pBWA (V) HS-OsDREB1C is a recombinant vector obtained by inserting OsDREB1C coding gene shown in sequence 2 in a sequence table into BsaI (Eco31I) enzyme cutting site of pBWA (V) HS vector, and pBWA (V) HS-OsDREB1C can over-express protein coded by OsDREB1C gene (namely OsDREB1C protein shown in sequence 1) under the drive of CaMV 35S promoter.

The primer sequences used were as follows:

OsDREB1C-F：5′-CAGTGGTCTCACAACATGGAGTACTACGAGCAGGAGGAGT-3′；

OsDREB1C-R：5′-CAGTGGTCTCATACATCAGTAGCTCCAGAGTGTGACGTCG-3′。

construction of a gene knockout vector: the sgRNA and primers were designed based on the online design website (http:// skl. scau. edu. cn /), and the target sequence was determined to be 5'-AGTCATGCCCGCACGACGC-3' (356 nd 374 th position of sequence 2). Annealing OsDREB1C-sgRNA-F and OsDREB1C-sgRNA-R, carrying out enzyme digestion on the obtained product by using BsaI, connecting the obtained enzyme digestion product with a vector skeleton obtained by carrying out enzyme digestion on a plasmid containing psgR-Cas9-Os by using BsaI, and obtaining a recombinant vector with a correct sequence, namely an OsDREB1C gene knockout vector, which is marked as OsU3-sgRNA-OsUBI-Cas9-OsDREB1C, wherein in the recombinant vector, a OsU3 promoter drives sgRNA, and an OsUBI promoter drives Cas 9.

Wherein, the primer sequences used are as follows:

OsDREB1C-sgRNA-F：5′-TGTGTGGCGTCGTGCGGGCATGACT-3′；

OsDREB1C-sgRNA-R：5′-AAACAGTCATGCCCGCACGACGCCA-3′。

2. construction of transgenic plants

Mature seeds of japonica rice variety Nipponbare are disinfected and induced to obtain embryogenic callus, pBWA (V) HS-OsDREB1C and OsU3-sgRNA-OsUBI-Cas9-OsDREB1C obtained in the step 1 are respectively introduced into agrobacterium EHA105, the callus is infected and co-cultured by an agrobacterium-mediated rice genetic transformation method, transgenic plants are obtained by resistance screening, the screened transgenic rice obtained by pBWA (V) HS-OsDREB1C is OsDREB1C transgenic rice, and the screened transgenic rice obtained by OsU3-sgRNA-OsUBI-Cas9-OsDREB1C is OsDREB1C gene knockout rice material.

The relative expression level of OsDREB1C transgenic rice and OsDREB1C gene knockout rice OsDREB1C gene on RNA level is detected by using japonica rice variety Nipponbare (WT) of rice as a control and using qRT-PCR method, and the used primers are as follows: 5'-CATGATGATGCAGTACCAGGA-3', 5'-GATCATCAGTAGCTCCAGAGTG-3', respectively; the reference gene is rice Ubiqutin, and the reference gene primer is as follows: 5'-AAGAAGCTGAAGCATCCAGC-3', 5'-CCAGGACAAGATGATCTGCC-3' are provided.

The results show that the relative expression levels of the OsDREB1C genes in 3 lines (OE1, OE2 and OE5) of OsDREB1C transgenic rice are all significantly higher than that of Wild Type (WT), and the three lines are all over-expressed OsDREB1C rice materials (a in fig. 2). The OsDREB1C gene sequence of 3 strains (KO1, KO2 and KO3) of OsDREB1C gene knockout rice materials has base deletion or insertion, so that frame shift mutation is caused, and the normal function of OsDREB1C protein is lost (B in figure 2).

The results of PCR amplification and sequencing of 3 strains (KO1, KO2 and KO3) of OsDREB1C gene knock-out rice material by using primer pairs capable of amplifying target sequences and upstream and downstream thereof show that the target sequences of the three strains are changed as shown in B in figure 2, wherein KO1 and KO2 are both subjected to one nucleotide deletion, KO3 is subjected to one nucleotide insertion, and target genes of the three strains are subjected to frame shift mutation.

3. Improvement of photosynthesis efficiency of OsDREB1C transgenic rice

Detecting the photosynthesis index of rice growing in a field, wherein the rice to be detected: wild type Japanese clear rice (WT), OsDREB1C over-expressed rice (OE1/OE2/OE5), OsDREB1C knock-out rice (KO1/KO2/KO 3).

Respectively measuring the photosynthetic daily change, photoresponse curve and CO of the rice sword leaves to be measured in heading stage by using LICOR-6400XT portable photosynthetic apparatus (LI-COR, USA)₂A response curve. The measurement of the change of the photosynthesis day is carried out by selecting the sunny and cloudy weather, and the measurement is carried out once every 2 to 4 hours from 8:00 to 16: 00. Wherein the photosynthesis rate is measured by LI-COR 6400XT portable photosynthesizer, NPQ (non-photochemical quenching) is measured by FluorPen FP100(PSI, Czech), and the dark adaptation of the leaf before NPQ is measured for 15-20 min. In the determination of the photoresponse curve, CO₂The concentration was set at 400. mu. mol^-1Light intensity (PPFD) of 0 to 2000 [ mu ] mol m^-2s^-1。CO₂In response Curve determination, PPFD was set to 1200. mu. mol m^-2s^-1，CO₂The concentration is reduced from 400 to 50 mu mol^-1Then increasing from 400 to 1200 mu mol^-1. Photoresponse curve and CO₂The response curves were fitted using the Farquhar-von Cammer-Berry (FvCB) model and passed through CO₂The maximum carboxylation efficiency (V) is calculated by a response curve_cmax) And maximum electron transfer rate (J)_max)。

The results show that the photosynthetic efficiency (i.e. the photosynthetic efficiency) of the rice over-expressing OsDREB1C is obviously higher than that of the wild type in the daytime, the difference reaches the maximum when the light intensity is highest at noon and reaches a significant level, the difference can be improved by 29.5-43.3% compared with that of the wild type, meanwhile, the heat dissipation NPQ for consuming excessive absorption light energy is obviously lower than that of the wild type (A, B in figure 3, tables 1 and 2), and the difference reaches a significant level, which indicates that more light energy is used for participating in photosynthesis. Further determination of photoresponse curves and CO₂The results of the response curves show that the intensity is below 200. mu. mol m^-2s^-1When the photosynthetic rate of the rice over-expressed OsDREB1C is higher than that of the wild rice, the difference is gradually increased under high light intensity and is 2000 mu mol m^-2s^-1Under the light intensity, the photosynthetic rate of the rice over-expressing OsDREB1C is improved by 18.4-27.6% compared with that of the wild type (C in figure 3, table 3), and the difference reaches a significant level. CO 2₂The response curves were determined similarly to the photoresponse curves, in CO₂The concentration is more than 400 mu mol^-1Then, the photosynthetic rate of the rice over-expressing OsDREB1C is obviously improved compared with that of the wild type (D in figure 3, table 4), and the maximum carboxylation efficiency (V) is further calculated_cmax) And maximum electron transfer rate (J)_max) All were significantly higher than wild type (E, F in fig. 3, table 5). The photosynthesis related parameters of the OsDREB1C gene knockout rice are lower than or similar to those of the wild rice. The results are combined to show that the over-expression of the OsDREB1C gene in rice can simultaneously and obviously improve the light energy utilization efficiency and CO₂Assimilation ability.

TABLE 1 photosynthesis efficiency (. mu. mol CO)₂ m^-2s^-1) Result of detection of

Time

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*

In table 1, the difference was significant (p < 0.05) compared to WT at the same site in the same year, and the difference was very significant (p < 0.01).

TABLE 2 detection results of NPQ

Time

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*

In table 2, the difference was shown to reach a significant level (p < 0.05) and the difference was shown to reach a very significant level (p < 0.01) compared to WT at the same site in the same year.

TABLE 5 maximum carboxylation efficiency (V)_cmax) And maximum electron transfer rate (J)_max) As a result of (A)

In table 5, the difference was significant (p < 0.05) and the difference was very significant (p < 0.01) compared to WT at the same site in the same year.

4. Improvement of nitrogen utilization efficiency of OsDREB1C transgenic rice

Detecting the nitrogen utilization efficiency of the rice, wherein the rice to be detected: wild type Japanese clear rice (WT), OsDREB1C over-expressed rice (OE1/OE2/OE5), OsDREB1C knock-out rice (KO1/KO2/KO 3).

Culturing rice seedling to be tested with nutrient solution in greenhouse for 3 weeks, and placing in advance nitrogen-free (NH-free)₄)₂SO₄And KNO₃) The Mumura B nutrient solution (Kimura B solution) was subjected to nitrogen starvation treatment for 3 days. After nitrogen starvation treatment, the roots of the seedlings were completely immersed in 0.1mM CaSO₄Soaking in deionized water for 1 min, sucking off residual water, and placing the root in a container containing 0.5mM K¹⁵NO₃Culturing in the nutrient solution of (4). After 3 hours, the seedling roots were again placed in 0.1mM CaSO₄Soaking in the solution for 1 min, sucking off residual water, collecting overground part and root part, oven drying at 70 deg.C for 3 days to constant weight, and recording sample dry weight. After the sample was ground and pulverized, the aerial parts and the roots were measured by an IsoPrime 100 stable isotope ratio mass spectrometer (Elementar, Germany)¹⁵N content, and nitrogen absorption efficiency and nitrogen transport efficiency were calculated. Efficiency of nitrogen absorption (of the aerial part)¹⁵N content + in root¹⁵N content)/dry weight/3; nitrogen transport efficiency of the aerial part¹⁵N content/in root¹⁵And (4) N content.

Separately collecting mature rice plants to be tested growing in Beijing field, respectively harvesting single leaf, straw and seed, deactivating enzyme at 105 deg.C for 30 min, and oven drying at 70 deg.C for 3 days. After the sample was ground and pulverized, the nitrogen content was measured by an IsoPrime 100 stable isotope ratio mass spectrometer (Elementar, germany), and the distribution ratio of nitrogen at different sites was calculated.

The nutrient solution used was as follows:

nutrient solution: mucun B nutrient solution (0.5mM (NH)₄)₂SO₄,1mM KNO₃,0.54mM MgSO₄·7H₂O,0.3mM CaCl₂,0.18mM KH₂PO₄,0.09mM K₂SO₄,16μM Na₂SiO₃·9H₂O,9.14μM MnCl₂·4H₂O,46.2μM Na₂MoO₄·2H₂O,0.76μM ZnSO₄·7H₂O,0.32μM CuSO₄·5H₂O,40μM Fe(II)-EDTA,pH＝5.8)。

Containing 0.5mM K¹⁵NO₃The nutrient solution of (1): 0.5M K¹⁵NO₃Diluting the mother liquor 1000 times, and adding into nitrogen-free Mucun nutrient solution (0.54mM MgSO. sub.zero)₄·7H₂O,0.3mM CaCl₂,0.18mM KH₂PO₄,0.09mM K₂SO₄,16μM Na₂SiO₃·9H₂O,9.14μM MnCl₂·4H₂O,46.2μM Na₂MoO₄·2H₂O,0.76μM ZnSO₄·7H₂O,0.32μM CuSO₄·5H₂O,40μM Fe(II)-EDTA,pH＝5.8)。

The results show that the method has the advantages of high yield,¹⁵3 hours after N treatment, in the aerial parts and roots of OsDREB1C overexpressing rice¹⁵The content of N is significantly higher than that of the wild type (A, B in figure 4, table 6), mainly due to the fact that the nitrogen absorption efficiency (C in figure 4, table 6) of roots and the nitrogen transfer efficiency (D in figure 4, table 6) from the roots to the overground part of the wild type are significantly improved, and the difference between the OsDREB1C knockout rice and the wild type is not obvious except that¹⁵The absorption efficiency of N is lower than that of wild type, and other indexes are similar to those of wild type. Further analysis of nitrogen distribution results of rice plants grown in the field in different tissues showed that the nitrogen content of the whole rice plant of OsDREB1C over-expressed was overall improved compared with the wild type (E in FIG. 4, Table 7), wherein 50.3-66.4% of nitrogen was distributed to the seeds, which was much higher than 41.1% of the wild type and 26-38.6% of OsDREB1C knockout rice (F in FIG. 4, Table 7). The nitrogen distributed to the leaves and the straws is reduced correspondingly and respectively accounts for 21.1 to 29.7 percent and 12.5 to 19.9 percentWhile the proportion in the wild type was 42.66% and 16.28%, respectively, in OsDREB1C knock-out rice 43.2-49.3% and 18.2-24.7% (F in FIG. 4). The results are combined to show that the over-expression of the OsDREB1C gene in the rice can obviously improve the absorption and transport efficiency of the plant to nitrogen, and more nitrogen is distributed to grains.

TABLE 6 index test results of rice cultivated in greenhouse

In table 6, differences reached significant levels (p < 0.05) and differences reached very significant levels (p < 0.01) compared to the same-site treated WTs.

TABLE 7 index test results of rice cultured in the field

In table 7, the difference was shown to reach a significant level (p < 0.05) and the difference was shown to reach a very significant level (p < 0.01) compared to WT at the same site in the same year.

5. Heading period of OsDREB1C transgenic rice is advanced

Detecting the heading stage of the rice, wherein the rice to be detected: wild type Japanese clear rice (WT), OsDREB1C over-expressed rice (OE1/OE2/OE5), OsDREB1C knock-out rice (KO1/KO2/KO 3).

And respectively counting the heading period of each rice to be detected under the Beijing field planting condition. The statistical method comprises the following steps: planting 3 cells of each rice to be tested, repeating the planting in the cells, randomly arranging the planted cells, recording the emergence time when 50% of ears of plants in the cells are exposed 1/2 from scabs of the sword-like leaves as heading, and the heading time is the days from sowing to heading.

The results show that (table 8), the heading stage of the rice with the OsDREB1C overexpression is greatly advanced compared with that of the wild type, 13-17 days are advanced in 2018, 17-19 days are advanced in 2019, the heading stage of the rice with the OsDREB1C gene knockout is respectively delayed by 6-8 days and 3-5 days compared with that of the wild type, and the filling and maturing stage of rice grains is correspondingly advanced or delayed. The OsDREB1C and the coding gene thereof can regulate the heading stage of rice.

TABLE 8 heading date (days) for wild-type and transgenic rice under Beijing field conditions

Rice (Oryza sativa L.) with improved resistance to stress	2018 years old	2019
			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**

In table 8, indicates that the difference reached a very significant level (p < 0.01) compared to WT at the same site in the same year.

6. The yield, quality and harvest index of the transgenic rice in the field are greatly improved

Detecting the dry weight of the overground part and the yield of grains of the rice, wherein the rice to be detected: wild type Japanese clear rice (WT), OsDREB1C over-expressed rice (OE1/OE2/OE5), OsDREB1C knock-out rice (KO1/KO2/KO 3).

In field experiments carried out in Beijing and Hainan, 3 cells are planted in each kind of rice to be tested, the rice to be tested is repeatedly and randomly arranged, the yield of single-plant seeds, the yield of the seeds in the cells and the dry weight of the straws on the overground part are measured after the rice seeds are mature, and a harvest index is calculated, wherein the harvest index is the ratio of the yield of the single-plant seeds of the rice to the biomass on the overground part (the sum of the dry weight of the straws on the overground part and the yield of the single-plant seeds).

The method for measuring the dry weight of the straws on the overground part comprises the following steps: after the rice is mature, removing seeds from single straws, putting the straws into a nylon mesh bag, drying the straws at the temperature of 80 ℃ to constant weight, and weighing the sample. 20-30 individual replicates per rice material were taken for statistical analysis.

The measurement method of the grain yield is as follows: the weight of the seeds is called as the yield of the seeds of the single plant after the ears of the rice are threshed and the grains are shrunken, and 20-30 single plants of each rice material are repeatedly used for statistical analysis. And removing the border row when the cell yield is counted, taking the weight of the kernel measured by 30 rice plants in the middle as the cell yield, and repeatedly using 3 cells for counting analysis.

The rice quality is determined as follows: rice grains harvested by the Beijing field experiment in 2019 are naturally stored for 3 months and then used for rice quality analysis, and the determination method refers to the rice quality determination method of industry standard NY/T83-2017 of Ministry of agriculture. The result shows that the grain yield of the rice over-expressing OsDREB1C is greatly improved compared with that of the wild type, the difference reaches a remarkable level, and meanwhile, the weight of the overground part straw is remarkably lower than that of the wild type, so that the harvest index is remarkably improved compared with that of the wild type.

From the yield results (Table 9), in Beijing and Hainan, the yield of the transgenic rice is respectively increased by 45.1-67.6% and 7.8-16% compared with the wild type, and the yield of the transgenic rice is increased by 41.3-68.3% and 11.9-27.4% compared with the wild type. Meanwhile, the overground part straws are reduced by 12.1-24.7% and 17.5-25.8% compared with wild type, and finally, the Harvest Index (HI) of the transgenic rice is obviously improved compared with the wild type, and the amplification of the HI in Beijing can reach 10.3-55.7%. Meanwhile, the rice quality of the transgenic rice is improved to a different extent compared with the wild type (Table 10), wherein the brown rice rate, the polished rice rate and the whole polished rice rate are obviously increased compared with the wild type, the chalky degree and the chalky grain rate are reduced, the appearance quality is improved, and the amylose content and the protein content are not obviously influenced. The single-plant yield and the cell yield of the OsDREB1C gene knockout rice are both obviously reduced compared with the wild type, and the biomass of the overground part is increased, so that the harvest index is directly reduced by 22.4-37.7% compared with the wild type. The results show that after the OsDREB1C gene is transferred into the rice, the yield, the rice quality and the harvest index of the transgenic rice can be greatly improved, and the OsDREB1C and the coding gene thereof can regulate and control the grain yield, the rice quality and the harvest index of the rice.

The results of the indices are shown in tables 9 and 10.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of crop science of Chinese academy of agricultural sciences

<120> OsDREB1C and application of coding gene thereof in improving rice photosynthetic efficiency

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 214

<212> PRT

<213> Rice (Oryza sativa L.)

<400> 1

Met Glu Tyr Tyr Glu Gln Glu Glu Tyr Ala Thr Val Thr Ser Ala Pro

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Pro Lys Arg Pro Ala Gly Arg Thr Lys Phe Arg Glu Thr Arg His Pro

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Val Tyr Arg Gly Val Arg Arg Arg Gly Pro Ala Gly Arg Trp Val Cys

35 40 45

Glu Val Arg Glu Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe

50 55 60

Ala Thr Ala Glu Ala Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala

65 70 75 80

Leu Arg Gly Arg Gly Ala Cys Leu Asn Phe Ala Asp Ser Ala Arg Leu

85 90 95

Leu Arg Val Asp Pro Ala Thr Leu Ala Thr Pro Asp Asp Ile Arg Arg

100 105 110

Ala Ala Ile Glu Leu Ala Glu Ser Cys Pro His Asp Ala Ala Ala Ala

115 120 125

Ala Ala Ser Ser Ser Ala Ala Ala Val Glu Ala Ser Ala Ala Ala Ala

130 135 140

Pro Ala Met Met Met Gln Tyr Gln Asp Asp Met Ala Ala Thr Pro Ser

145 150 155 160

Ser Tyr Asp Tyr Ala Tyr Tyr Gly Asn Met Asp Phe Asp Gln Pro Ser

165 170 175

Tyr Tyr Tyr Asp Gly Met Gly Gly Gly Gly Glu Tyr Gln Ser Trp Gln

180 185 190

Met Asp Gly Asp Asp Asp Gly Gly Ala Gly Gly Tyr Gly Gly Gly Asp

195 200 205

Val Thr Leu Trp Ser Tyr

210

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<213> Rice (Oryza sativa L.)

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atggagtact acgagcagga ggagtacgcg acggtgacgt cggcgccgcc gaagcggccg 60

gcggggagga ccaagttcag ggagacgagg cacccggtgt accgcggcgt gcggcggcgg 120

gggcccgcgg ggcggtgggt gtgcgaggtc agggagccca acaagaagtc ccgcatctgg 180

ctcggcacct tcgccaccgc cgaggccgcc gcgcgcgccc acgacgtcgc cgcgctcgcc 240

ctccgcggcc gcggcgcgtg cctcaacttc gccgactcgg cccgcctcct ccgcgtcgac 300

ccggccaccc tcgccacccc cgacgacatc cgccgcgccg ccatcgagct cgccgagtca 360

tgcccgcacg acgccgccgc cgccgccgcc tccagctccg ccgccgccgt cgaggcctcc 420

gccgccgccg cgcccgccat gatgatgcag taccaggacg acatggcggc gacgccgtcc 480

agctacgact acgcgtacta cggcaacatg gacttcgacc agccgtccta ctactacgac 540

gggatgggcg gcggcggcga gtaccagagc tggcagatgg acggcgacga cgatggtggc 600

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<213> Rice (Oryza sativa L.)

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cccgtaatat tgattttgat tttttcttat gttgtttgac cattcgtctt aatcaagaaa 60

ctttgaaatt attatttatt ttatttatat cttactttat tatccaaagt actttaagca 120

taatttttta ttttttatat ttgcacaaat tttttgaata agaagagtgg tcaaacagtg 180

taaaaaaagt caaaatcact tatattatgg gatggaggga gtatgtgttt acctaaagta 240

gtactctatt catattattt actaggaaca aaaaaatgcc gtcgactttt tattaacatt 300

tgatcaatcg tcttattcaa aatttatgtg taaatataaa agtatttatg tcatgcttta 360

aaaacatttg aagataaatc aagtcataat aaaataaatg ataaaataag acgaatggtc 420

aagcgttgta aagttaacgg cgttatatat taaaatacga agagagtact gcatcagtat 480

catactagaa gaagtaatac cagtactaaa cagctttgat attttcgcta gtagcccgca 540

tatatcctca gttaatttgt tgctttagtt aaattgtgtt aagagtgaat tgcaccttga 600

tctgagtatc tttttttttg ttatcgtagt tttactttag atcacctctt aattcatggt 660

ttttacttta aaactaggga atgttacatt ttctagaata gatggtccaa aatgaagctc 720

tggaataaaa tatagcccaa tgtataattc attaataaga atagtagtat aaaaatctcc 780

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aagatattgt cttgtaggga tgtaagtgga caatctcact acccatataa agcctcactt 900

gccaattact ttctcaaaac tttaaaagaa aaaaaattaa ctagaacagg taaaagggaa 960

aaaaaatccc gcttgtccgc ttcccttggc atctggatgg tcacgtggca ggaaagaact 1020

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aaaaacaaaa aaaggcacag cagcgagtgg aaaggagtcg atgggatttt atgcccaaaa 1140

ccgcgagaga tccaccgctg tttgatgggt tcgttgggac ttgggaagga atccatcggc 1200

gaatcgccgc gcggctccga tccggggcgc aagcgcaagg ccaccggccc acgtactcca 1260

ctactcctac tccgtagaga ggcgacgcgt ttgcgcgggc ggcgccgcgg ccgcccaacc 1320

caaccgtcgc tgcctccgtc tccgtagtcc gtactgcctg tgtctgcctc tgctactgct 1380

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ctattatatg aatgagctat tatattggct atagatgaat ggtagttatt agttggctat 1620

actattgaac ttgccagaga ccaaccagct actccctcac gtgcggctgg attaaataaa 1680

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gcataagtac tgtagcactg cagccgcaca ctccacgtct ccattgggag gaaatggaaa 1800

atagtttttc ttttttttaa aaaaaaatag aaaatagtgc tccttccgtt ttatattata 1860

aatcgtttga tatttttctt agtcaaaatt tattaggttt gatcaaatat atagaaaaaa 1920

agaaacatgt acaacatcaa atttcattaa atttaacatt gtatatattt taatataatg 1980

tttattttat attaaaaaac attactatat tttgcttaaa gtttgactta aaaaaatctc 2040

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cggtttttaa gtttgtttgc ttttgaaaat atatatccgt atttgagttg gtttgtaaga 2160

tcgttcactt ttgtatgata caaaaggaat catataagaa atctgtttaa aataactcac 2220

atgctaaatt gagacgatcg gattccaaac tttagctcat gattttctaa aaatatatat 2280

atccaagtga actcccacag tgaattttat cttaactaaa ctatataatt aaaatagatt 2340

tcacccgttg caacgcacga tattttttct agtactccct ccgtttcata atgtaagact 2400

tcaaacattg gttatattca tatatgtgtt aatgaatcta gacacacata tatatctaga 2460

ttcattaaca tctatatgaa tatggacaat actagaaaga cttacattat aaaacagagg 2520

aagtaactca taaatacgga acgaaggggt agaaatatcc gcatctcata cacacacaga 2580

agtggtcagc cgccgcccaa aagcttgcct ttgtcgccat ctccacgtgg ccacccccat 2640

atactattgc ttaacgctgt cacctcaccc tctcgcggtt tgagttttcc acttccacgg 2700

ttccaccccg acacctagcg aagtactcgt agtaatccga atcccaccgt tccatccgtc 2760

gcggacgcca cgctctcggc tctcagctca cgtgacgtca accccgccaa aacgcgttat 2820

tgccgtagta ctacgcctct tcttccacct ccatctcccc ctccgacatc tccagccaat 2880

tccagctcag ctctcgccgc cctcccctct cccgccacgt gcgcgccgcc cctcctccaa 2940

atctcctttt cttttccttt tctataaata aatcaaaatt cacacaccaa atcctatata 3000

aacttcttcc tctccatccc cttcccgctc aaactcaaac accaacacct tcttcctctt 3060

cttcttcttc cagcagcagc aacacacact actgacatgg agtactacga gcaggaggag 3120

tacgcgacgg tgacgtcggc gccgccgaag cggccggcgg ggaggaccaa gttcagggag 3180

acgaggcacc cggtgtaccg cggcgtgcgg cggcgggggc ccgcggggcg gtgggtgtgc 3240

gaggtcaggg agcccaacaa gaagtcccgc atctggctcg gcaccttcgc caccgccgag 3300

gccgccgcgc gcgcccacga cgtcgccgcg ctcgccctcc gcggccgcgg cgcgtgcctc 3360

aacttcgccg actcggcccg cctcctccgc gtcgacccgg ccaccctcgc cacccccgac 3420

gacatccgcc gcgccgccat cgagctcgcc gagtcatgcc cgcacgacgc cgccgccgcc 3480

gccgcctcca gctccgccgc cgccgtcgag gcctccgccg ccgccgcgcc cgccatgatg 3540

atgcagtacc aggacgacat ggcggcgacg ccgtccagct acgactacgc gtactacggc 3600

aacatggact tcgaccagcc gtcctactac tacgacggga tgggcggcgg cggcgagtac 3660

cagagctggc agatggacgg cgacgacgat ggtggcgccg gcggctacgg cggcggcgac 3720

gtcacactct ggagctactg atgatcgcga gttggagcta gcagttttga gctcaaccag 3780

ctttgctcct cctatacagc taaatactgt aggagaaatt aatggagatt ttttccttct 3840

ttattttttt tatatttttt ccaagataaa atggctgagc tggtagggag ttctagaagg 3900

aggaaataaa gattacgaga tttgtaatac tactacgagg gacagcatgc aaaaaagaag 3960

tataattgct atgctctctc tctctctctc tctcttttgt gtggagtgga ataaaatgcc 4020

agctctgctt atggtcagat tttcactttt ttttggagtg ttaagacaaa tgaaagctcc 4080

agataaatta gtgagtgcta ctgcttatct gaataaatga gatcattcat catc 4134

Claims

1. The use of a protein or a substance which modulates the activity or content of said protein, as defined in any one of the following:

D1) regulating plant photosynthesis;

D2) preparing a product for regulating and controlling plant photosynthesis;

D7) regulating and controlling photosynthesis and flowering time of plants;

D13) plant breeding;

the protein is A1), A2), A3) or A4) as follows:

A1) the amino acid sequence is the protein of sequence 1;

A3) a protein derived from rice, maize, sorghum, soybean, canola, arabidopsis thaliana, millet, aegilops tauschii, brachypodium distachyon or wheat and having 64% or more identity to sequence 1 and having the same function as the protein of A1);

2. Use of a biological material related to a protein according to claim 1, wherein the biological material is selected from the group consisting of:

D1) regulating plant photosynthesis;

D2) preparing a product for regulating and controlling plant photosynthesis;

D7) regulating and controlling photosynthesis and flowering time of plants;

D13) plant breeding;

the biomaterial is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B8) a nucleic acid molecule which reduces the expression level of the protein of claim 1 or knockdown the gene encoding the protein of claim 1;

3. Use according to claim 2, characterized in that: B1) the nucleic acid molecule is b11) or b12) or b13) or b14) or b15) as follows:

b14) a cDNA or DNA molecule having 73% or more identity with the nucleotide sequence defined in b11) or b12) or b13) and encoding the protein of claim 1;

b15) a cDNA molecule or a DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b11) or b12) or b13) or b14) and encodes the protein of claim 1;

B2) the expression cassette is b21) or b22) or b23) as follows:

b21) DNA molecule shown in sequence 3 in the sequence table;

4. Any one of the following methods:

x1) a method for producing a photosynthesis enhancing plant, comprising allowing a plant of interest to express the protein of claim 1 in a recipient plant, or increasing the content or activity of the protein of claim 1 in a recipient plant, to obtain a photosynthesis enhancing plant;

x2) a method for cultivating plants with enhanced photosynthesis and enhanced nitrogen uptake or transport capacity, comprising allowing the recipient plant to express the protein of claim 1 or increasing the content or activity of the protein of claim 1 in the recipient plant to obtain plants with enhanced photosynthesis and enhanced nitrogen uptake or transport capacity;

x3) a method for cultivating plants with enhanced photosynthesis and increased nitrogen content, which comprises allowing the receptor plants to express the protein of claim 1 or increasing the content or activity of the protein of claim 1 in the receptor plants, to obtain plants with enhanced photosynthesis and increased nitrogen content;

x4) a method for cultivating photosynthesis-enhanced flowering time-advanced plants, which comprises allowing a recipient plant to express the protein of claim 1, or increasing the content or activity of the protein of claim 1 in the recipient plant, to obtain photosynthesis-enhanced flowering time-advanced plants;

x5) A method for cultivating a plant with enhanced photosynthesis, enhanced nitrogen uptake or transport capacity and advanced flowering time, which comprises allowing a recipient plant to express the protein of claim 1 or increasing the content or activity of the protein of claim 1 in the recipient plant to obtain a target plant with enhanced photosynthesis, enhanced nitrogen uptake or transport capacity and advanced flowering time;

x6) A method for cultivating plants with enhanced photosynthesis, increased nitrogen content and advanced flowering time, which comprises allowing the recipient plant to express the protein of claim 1 or increasing the content or activity of the protein of claim 1 in the recipient plant, thereby obtaining a target plant with enhanced photosynthesis, increased nitrogen content and advanced flowering time.

5. The method of claim 4, wherein: x1) -X6) by introducing a gene encoding the protein of claim 1 into the recipient plant and allowing the encoding gene to be expressed.

6. The method of claim 5, wherein: the coding gene is the nucleic acid molecule of B1) in claim 2 or 3.

7. A product having the function of any one of D1) -D6) comprising the protein of claim 1 or the biomaterial of claim 2 or 3:

D1) regulating plant photosynthesis;

D4) regulating and controlling photosynthesis and flowering time of plants;

8. Use according to any one of claims 1 to 3, or a method according to any one of claims 4 to 6, or a product according to claim 7, wherein: the plant is M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) rice.

9. Use according to any one of claims 1 to 3, or a method according to any one of claims 4 to 6, or a product according to claim 7, or a use, method or product according to claim 8, wherein: said photosynthesis being manifested in a photosynthetic rate, a net photosynthetic rate, a heat dissipation NPQ, a maximum carboxylation efficiency and/or a maximum electron transfer rate;

the transport is embodied in a transport of the root to the overground part, or into the kernel;

the nitrogen content is the nitrogen content in the plant or organ;

the flowering time is now at heading.

10. A protein according to claim 1 or a biomaterial according to claim 2 or 3.