CN114276426B

CN114276426B - Protein and biological material related to rice yield and application of protein and biological material in rice yield improvement

Info

Publication number: CN114276426B
Application number: CN202110738250.5A
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: 2021-06-30
Publication date: 2023-02-03
Anticipated expiration: 2041-06-30
Also published as: CN114276426A; CN113072630A

Abstract

The invention discloses a protein and a biological material related to rice yield and application of the protein and the biological material in improving the rice yield. The protein related to rice yield disclosed by the invention is OsDREB1C, the sequence of the protein is a sequence 1 in a sequence table, and the sequence of a coding gene of the protein is a sequence 2 in the sequence table. Experiments prove that the OsDREB1C and the related biological materials thereof can improve the photosynthetic 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 OsDREB1C and the related biological materials thereof have important biological significance and industrial value and good application prospect.

Description

Protein and biological material related to rice yield and application of protein and biological material in rice yield improvement

Technical Field

The invention relates to a protein and a biological material related to rice yield and application of the protein and the biological material in improving the rice yield in the field of biotechnology.

Background

With the population growth and economic development in the future, the food demand of China is still in a rapid growth trend. Under the conditions of continuous reduction of the cultivated land area and limited potential of grain planting area expansion, the continuous improvement of the crop yield per unit area in a large area becomes the only choice for guaranteeing the grain safety in China. Therefore, under the pressure of grain safety demand, high yield is a constantly pursued 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.

Heading stage is one of important agronomic traits of crops and determines season, regional adaptability and yield of rice. The proper heading period guarantees 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 "excellent but not early, early but not excellent" is a great problem in the cultivation of crop varieties.

In agricultural production, the application of nitrogen fertilizer in large quantities has always been one of the important measures for high yield of crops. The continuous large amount of nitrogen fertilizer input not only increases the planting cost, but also causes increasingly serious environmental pollution problems. 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. Exploring a regulation mechanism and a technical way for greatly increasing the yield of crops and efficiently utilizing resources is a preoccupation of sustainable development of agriculture in China.

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 yield of plants.

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

d1 Regulating plant yield;

d2 Preparing a product for regulating plant yield;

d3 Regulating plant photosynthesis and yield;

d4 Preparing products for regulating and controlling plant photosynthesis and yield;

d5 Regulating plant photosynthesis, nitrogen uptake or transport and yield;

d6 Preparing products for regulating and controlling plant photosynthesis, nitrogen absorption or transport and yield;

d7 Regulating plant photosynthesis, nitrogen content and yield;

d8 Preparing products for regulating and controlling plant photosynthesis, nitrogen content and yield;

d9 Regulating plant photosynthesis, nitrogen absorption or transport, flowering time and yield;

d10 Preparing products for regulating and controlling plant photosynthesis, nitrogen absorption or transport, flowering time and yield;

d11 Regulating plant photosynthesis, nitrogen content, flowering time and yield;

d12 Preparing products for regulating and controlling plant photosynthesis, nitrogen content, flowering time and yield;

d13 ) plant breeding;

the protein (the name of the protein is OsDREB 1C) is A1), A2), A3) or A4) as follows:

a1 Protein in which the amino acid sequence is sequence 1;

a2 Protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 1 in the sequence table;

a3 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 a label to the N-terminal or/and C-terminal of A1) or A2) or A3).

In order to facilitate the purification of the protein of A1), the amino terminus or the carboxy terminus of the protein consisting of the amino acid sequence shown in sequence 1 of the sequence listing may be attached with the tags shown in the following table.

Table (b): sequence of tags

Label (R)	Residue(s) of	Sequence of
			Poly-Arg	5-6 (generally 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) is a protein having an identity of 64% or more 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 in A2) can be artificially synthesized, or can 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 sequence 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 yield;

d2 Preparing a product for regulating plant yield;

d3 Regulating plant photosynthesis and yield;

d4 Preparing a product for regulating and controlling plant photosynthesis and yield;

d5 Regulating plant photosynthesis, nitrogen uptake or transport and yield;

d7 Regulating plant photosynthesis, nitrogen content and yield;

d9 Regulating plant photosynthesis, nitrogen uptake or transport, flowering time and yield;

d13 ) breeding plants;

the biological material is any one of the following B1) to B9):

b1 Nucleic acid molecule encoding OsDREB 1C;

b2 An expression cassette containing the nucleic acid molecule according to B1);

b3 A recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B2);

b4 A recombinant microorganism containing the nucleic acid molecule according to B1), or a recombinant microorganism containing the expression cassette according to B2), or a recombinant microorganism containing the recombinant vector according to B3);

b5 A transgenic plant cell line containing the nucleic acid molecule according to B1) or a transgenic plant cell line containing the expression cassette according to B2);

b6 A transgenic plant tissue containing the nucleic acid molecule according to B1) or a transgenic plant tissue containing the expression cassette according to B2);

b7 A transgenic plant organ containing the nucleic acid molecule according to B1) or a transgenic plant organ containing the expression cassette according to 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 containing a nucleic acid molecule according to B8).

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

b11 A cDNA molecule or DNA molecule with a coding sequence of a sequence 2 in a sequence table;

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

b13 3001-4131 of the sequence 3 in the sequence table;

b14 A cDNA molecule or DNA molecule having 73% or more identity with the nucleotide sequence defined in b 11) or b 12) or b 13) and encoding OsDREB 1C;

b15 A cDNA molecule or DNA molecule which hybridizes with the nucleotide sequence defined by b 11) or b 12) or b 13) or b 14) under strict conditions and codes for OsDREB 1C;

b2 The expression cassette is b 21) or b 22) or b 23) as follows:

b21 A DNA molecule shown in a sequence 3 in a sequence table;

b22 A DNA molecule with 73 percent or more than 73 percent of identity with the nucleotide sequence defined by the b 21) and with the same function;

b23 A DNA molecule which hybridizes with the nucleotide sequence defined by b 21) or b 22) under strict conditions and has the same function.

Wherein the nucleic acid molecule may be a DNA, such as a 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 a person 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 NaPO ₄ And 1mM EDTA, rinsed in2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ C, 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA mixed solution, 1 XSSC, 0.1%Rinsing in SDS; it can also be: SDS, 0.5M NaPO at 50 ℃ in 7% ₄ And 1mM EDTA, and rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ C, 7% SDS, 0.5M NaPO ₄ And 1mM EDTA, and rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: SDS, 0.5M NaPO at 50 ℃ in 7% ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: hybridizing in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washing the membrane once each with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; can also be: 2 XSSC, 0.1% SDS, hybridizing and washing the membrane at 68 ℃ 2 times, 5min each, in addition to hybridizing and washing the membrane at 68 ℃ 2 times, 15min each, in 0.5 XSSC, 0.1% SDS; can also be: 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, at 65 ℃.

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 (OSDREB 1C gene expression cassette) described in B2) refers to DNA capable of expressing OSDREB1C protein in a host cell, 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; a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN 2) 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); a seed-specific promoter, a promoter specific to a seed,such as the millet seed-specific promoter pF128 (CN 101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beach 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 herein 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 ) Nature313:810; rosenberg et al (1987) Gene, 56; guerineau et al (1991) mol.gen.genet, 262; probfoot (1991) Cell, 64; sanfacon et al Genes dev., 5; mogen et al (1990) Plant Cell, 2; munroe et al (1990) Gene, 91; ballad et al (1989) Nucleic Acids Res.17:7891; joshi et al (1987) Nucleic Acid Res, 15.

The recombinant vector containing the OSDREB1C gene expression box 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 polyadenylation signal can direct polyadenylic acid to the 3 'end of the mRNA precursor, and similar functions can be found in untranslated regions transcribed from the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein gene). 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 sources of the translational control signals and initiation codons are wide ranging from natural to synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate identification and screening of transgenic plant cells or plants, the plant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), marker genes for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, hph gene conferring resistance to the antibiotic hygromycin, and dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) which are expressible in plants, or marker genes for anti-chemical agents (e.g., herbicide resistant genes), mannose-6-phosphate isomerase genes providing the ability to metabolize mannose. From the safety of transgenic plants, the transformed plants can be screened directly in stress 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 may specifically be pBWA (V) HS-OsDREB1C. The described

pBWA (V) HS-OsDREB1C is a recombinant vector obtained by inserting OsDREB1C coding gene shown in sequence 2 in a sequence table at BsaI (Eco 31I) enzyme cutting site of pBWA (V) HS vector. The above-mentioned

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 may be a recombinant vector prepared using a crisper/cas9 system that can reduce the OSDREB1C content. The recombinant vector can express a sgRNA that targets the nucleic acid molecule of B1). The target sequence of the sgRNA can be 356 th to 374 th positions 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 EHA105.

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) a method for breeding a plant with increased yield, 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 increased yield;

x2) a method of breeding a plant with reduced yield comprising reducing OsDREB1C content or activity in a recipient plant, or inhibiting expression of an OsDREB1C coding gene in a recipient plant, to obtain a target plant with reduced yield as compared to said recipient plant;

x3) a method for cultivating a plant with enhanced photosynthesis and increased yield, comprising expressing OsDREB1C in a recipient plant, or increasing OsDREB1C content or activity in a recipient plant, to obtain a target plant with enhanced photosynthesis and increased yield;

x4) a method for cultivating a plant with enhanced photosynthesis, enhanced nitrogen uptake or transport capacity and increased yield comprising expressing OsDREB1C in a recipient plant or increasing OsDREB1C content or activity in a recipient plant to obtain a target plant with enhanced photosynthesis, enhanced nitrogen uptake or transport capacity and increased yield;

x5) a method for cultivating a plant with enhanced photosynthesis, increased nitrogen content, and increased yield comprising expressing OsDREB1C in a recipient plant or increasing OsDREB1C content or activity in a recipient plant to obtain a target plant with enhanced photosynthesis, increased nitrogen content, and increased yield;

x6) cultivating a plant having enhanced photosynthesis, enhanced nitrogen uptake or transport capacity, advanced flowering time and increased yield, comprising expressing OsDREB1C in a recipient plant, or increasing the content or activity of OsDREB1C in a recipient plant, to obtain a target plant having enhanced photosynthesis, enhanced nitrogen uptake or transport capacity, advanced flowering time and increased yield;

x7) A method for cultivating a plant having enhanced photosynthesis, increased nitrogen content, advanced flowering time, and increased yield, comprising expressing OsDREB1C in a recipient plant, or increasing the content or activity of OsDREB1C in a recipient plant, to obtain a target plant having enhanced photosynthesis, increased nitrogen content, advanced flowering time, and increased yield.

In the above method, X1) -X7) can be performed 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 the nucleic acid molecule of B1).

In the method, the coding gene of the OsDREB1C can be modified as follows and then introduced into a receptor plant to achieve better expression effect:

1) Modifying and optimizing according to actual needs to enable the gene to be efficiently expressed; for example, the codon of the coding gene of OsDREB1C of the present invention may be changed to conform to the preference of a recipient plant while maintaining the amino acid sequence thereof according to the preference of the recipient plant; 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; the promoters may include constitutive, inducible, temporal regulated, developmental regulated, chemical 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 shown 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 derived from CaMV, E9 derived 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 OsDREB1C. The recombinant expression vector can be specifically pBWA (V) HS-OsDREB1C.

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.

The plant of interest is understood to comprise not only the first generation plants in which the OsDREB1C protein or the gene encoding it is altered, but also the progeny thereof. 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 with any one of the following functions D1) -D6), which contains OsDREB1C or the biological material:

d1 Regulating plant yield;

d2 Regulating plant photosynthesis and yield;

d3 Regulating plant photosynthesis, nitrogen uptake or transport and yield;

d4 Regulating plant photosynthesis, nitrogen content and yield;

d5 Regulating plant photosynthesis, nitrogen absorption or transport, flowering time and yield;

d6 Regulate plant photosynthesis, nitrogen content, flowering time and yield.

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

m1) monocotyledonous or dicotyledonous plants;

m2) a graminaceous, cruciferous or leguminous plant;

m3) rice, wheat, maize, arabidopsis, oilseed rape or soybean.

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

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

the flowering time may be reflected in heading date;

the yield may be reflected in grain yield, aerial yield and/or harvest index.

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

The harvest index refers to the ratio of economic yield (grains, fruits, etc.) to biological yield of a plant at harvest.

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

Experiments prove that the OsDREB1C and related biological materials thereof can improve the photosynthetic efficiency of plants, promote nitrogen absorption and transportation, improve the nitrogen content in the plants and grains, promote early heading and improve the yield. 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 knockout rice material. (a) relative expression level of OsDREB1C gene; and (B) knocking out the gene editing sites of the rice by using the OsDREB1C gene.

FIG. 3 shows photosynthesis parameters of wild type and transgenic rice plants. A-photosynthetic daily variation; B-NPQ daily variation; c-light response curve; D-CO ₂ A response curve; e-maximum CO ₂ Carboxylation 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 part ¹⁵ The content of N; in B-root of ¹⁵ The content of N; c- ¹⁵ N absorption efficiency; d- ¹⁵ N 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.

FIG. 5 shows the detection result (A) of Bar gene in transgenic wheat and the detection result (B) of relative expression level of OsDREB1C gene in transgenic Arabidopsis.

FIG. 6 shows the phenotype of wild type and transgenic wheat. (A) Wild type wheat (Fielder) and transgenic wheat phenotypes. (B-D) heading time (B), photosynthetic rate (C), panicle number (D), thousand kernel weight (E) and individual grain yield (F) of wild type and transgenic lines.

FIG. 7 is a phenotypic characterization of wild-type and transgenic Arabidopsis thaliana. (A) Arabidopsis thaliana wild type Col-0 and transgenic line phenotype. (B-D) bolting time (B) of Arabidopsis wild type and transgenic lines, rosette leaf number (C) and overground fresh weight (D).

*P<0.05,**P<0.01。

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.

The pBWA (V) HS vector (ZHao et al, DEP1 is included in the regulated the carbon-nitro rubber floor to extract the tissue and quality in rice (Oriza sativa L.), PLOS ONE, march 11,2019, https:// doi.org/10.1371/j ournal.0213504) in the examples described below, which was publicly available from the applicant, was used only for repeating the experiments relevant to the present invention, and was not used for other purposes.

The psgR-Cas 9-Os-containing plasmids in the following examples (Humojiao, yangjia, chenCan, zhouhua, nivaan, wangxing, zmeiliang, cao Li Ming, huangwei. Oriented editing of the rice SD1 gene using the CRISPR/Cas9 system Chinese Rice science, 2018, 32 (3): 219-225.

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

This example provides a protein derived from rice plant of Nipponbare having functions of improving photosynthesis efficiency of rice, promoting nitrogen uptake, promoting pre-heading, and improving yield, which is named OsDREB1C, whose sequence is SEQ ID NO. 1, and in Nipponbare, whose coding gene sequence is SEQ ID NO. 2 and whose genome sequence is position 3001-4131 of SEQ ID NO. 3. In the sequence 3, the 1 st to 3000 th sites are the promoter of the OsDREB1C gene in the genomic DNA of Nipponbare.

Sequence alignment of rice OsDREB1C and homologous proteins in other plants shows that the identity of rice OsDREB1C and homologous proteins in other plants is 73.52%, 64.06%, 66.52%, 66.05% and 65.88% respectively (figure 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 (Eco 31I), the obtained enzyme digestion products are connected with a vector framework obtained by single enzyme digestion of pBWA (V) HS vector by BsaI (Eco 31I), and the obtained recombinant vector with correct sequence is marked as pBWA (V) HS-OsDREB1C. 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 (Eco 31I) enzyme cutting site of a pBWA (V) HS vector, and the pBWA (V) HS-OsDREB1C can overexpress a protein coded by the OsDREB1C gene (namely the OsDREB1C protein shown in the sequence 1) under the drive of a 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: design web site on-line (http://skl.scau.edu.cn/) The sgRNA and the primers were designed to determine the target sequence to be 5-. 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 framework 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, an OsU3 promoter drives sgRNA, osUBI promoter driving Cas9.

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

Sterilizing mature seeds of japonica rice Nipponbare of a rice variety, inducing to obtain embryonic callus, respectively introducing pBWA (V) HS-OsDREB1C and OsU3-sgRNA-OsUBI-Cas9-OsDREB1C obtained in the step 1 into agrobacterium EHA105, performing infection co-culture on the callus by using an agrobacterium-mediated rice genetic transformation method, obtaining transgenic plants by using resistance screening, wherein 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 method comprises the steps of using japonica rice variety Nipponbare (WT) as a reference, detecting OsDREB1C transgenic rice and the relative expression level of OsDREB1C gene in OsDREB1C knockout rice on RNA level by using a qRT-PCR method, wherein the used primers are as follows: 5' CATGATGATGCAGTACCAGGA-; the reference gene is rice Ubiqutin, and the reference gene primer is as follows: 5 'AAGAAGCTGAAGCATCCAGC-3', 5 'CCAGGACAAGATGATCTGCC-3'.

The results show that the relative expression level of the OsDREB1C gene in 3 lines (OE 1, OE2 and OE 5) of OsDREB1C transgenic rice is significantly higher than that of Wild Type (WT), and the three lines are all over-expressed OsDREB1C rice material (a in fig. 2). Base deletion or insertion exists in the OsDREB1C gene sequence in 3 lines (KO 1, KO2 and KO 3) of the OsDREB1C gene knockout rice material, so that frame shift mutation is caused, and the normal function of the OsDREB1C protein is lost (B in figure 2).

The PCR amplification and sequencing are carried out on 3 strains (KO 1, KO2 and KO 3) of the OsDREB1C gene knock-out rice material by utilizing a primer pair capable of amplifying a target sequence and upstream and downstream, and the result shows that the change conditions of the target sequences of the three strains are shown as 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 fine rice (WT), osDREB1C rice (OE 1/OE2/OE 5) over-expressed, and OsDREB1C knock-out rice (KO 1/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) ₂ A response curve. The measurement of the change of photosynthesis day is performed by selecting clear and cloudless weather, and is measured every 2-4 hours from 8. 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, CO ₂ The concentration was set at 400. Mu. Mol ^-1 Light intensity (PPFD) of 0 to 2000 [ mu ] mol m ^-2 s ^-1 。CO ₂ PPFD was set to 1200. Mu. Mol m in response curve measurement ^-2 s ^-1 ，CO ₂ The concentration is reduced from 400 to 50 mu mol ^-1 Then increasing from 400 to 1200 mu mol ^-1 . Photoresponse curve and CO ₂ The response curves were fitted using the Farquhar-von Cammer-Berry (FvCB) model and passed through CO ₂ The 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 with the OsDREB1C overexpression is obviously higher than that of the wild type in 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 and B in figure 3, and 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 CO ₂ The results of the response curves show that at light intensities below 200 μmol m ^-2 s ^-1 When the photosynthetic rate of the rice over-expressed OsDREB1C is higher than that of the wild rice, the net photosynthetic rate of the rice over-expressed OsDREB1C is higher than that of the wild rice, and the difference is gradually increased under high light intensity and is 2000 mu mol m ^-2 s ^-1 Under 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 2 ₂ The response curves were determined similarly to the photoresponse curves, in CO ₂ The concentration is more than 400 mu mol ^-1 Then, the photosynthetic rate of the rice over-expressing OsDREB1C was significantly increased compared to the wild type (D in FIG. 3, table 4), and the maximum carboxylation efficiency (V) was 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 comprehensive results show that the over-expression of the OsDREB1C gene in rice can simultaneously and obviously improve the light energy utilization efficiency and CO ₂ Assimilation ability.

TABLE 1 photosynthesis efficiency (. Mu. Mol CO) ₂ m ^-2 s ^-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 fine rice (WT), osDREB1C rice (OE 1/OE2/OE 5) over-expressed, and OsDREB1C knock-out rice (KO 1/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 ) ₂ SO ₄ And KNO ₃ ) 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 CaSO ₄ Soaking in deionized water for 1 min, sucking off residual water, and placing the root in a container containing 0.5mM K ¹⁵ NO ₃ Culturing in the nutrient solution of (4). After 3 hours, the seedling roots were again placed in 0.1mM CaSO ₄ Soaking 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 content of aerial parts and the content of roots were measured by IsoPrime 100 stable isotope ratio mass spectrometer (Elementar, germany) ¹⁵ N content, and nitrogen absorption efficiency and nitrogen transport efficiency were calculated. Nitrogen absorption efficiency = (of the above-ground part) ¹⁵ N content + in root ¹⁵ N content)/dry weight/3; nitrogen transport efficiency = above ground ¹⁵ N content/in root ¹⁵ And (4) N content.

Separately collecting mature rice plants to be detected 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 (0.5 mM (NH) ₄ ) ₂ SO ₄ ,1mM KNO ₃ ,0.54mM MgSO ₄ ·7H ₂ O,0.3mM CaCl ₂ ,0.18mM KH ₂ PO ₄ ,0.09mM K ₂ SO ₄ ,16μM Na ₂ SiO ₃ ·9H ₂ O,9.14μM MnCl ₂ ·4H ₂ O,46.2μM Na ₂ MoO ₄ ·2H ₂ O,0.76μM ZnSO ₄ ·7H ₂ O,0.32μM CuSO ₄ ·5H ₂ O,40μM Fe(II)-EDTA,pH＝5.8)。

Containing 0.5mM K ¹⁵ NO ₃ The nutrient solution of (1): 0.5M K ¹⁵ NO ₃ Diluting the mother liquor 1000 times, and adding into nitrogen-free Mucun nutrient solution (0.54 mM MgSO. Sub.zero) ₄ ·7H ₂ O,0.3mM CaCl ₂ ,0.18mM KH ₂ PO ₄ ,0.09mM K ₂ SO ₄ ,16μMNa ₂ SiO ₃ ·9H ₂ O,9.14μM MnCl ₂ ·4H ₂ O,46.2μM Na ₂ MoO ₄ ·2H ₂ O,0.76μM ZnSO ₄ ·7H ₂ O,0.32μMCuSO ₄ ·5H ₂ O,40μM Fe(II)-EDTA,pH＝5.8)。

The results show that the method has the advantages of high efficiency, ¹⁵ 3 hours after N treatment, in the aerial parts and roots of OsDREB1C overexpressing rice ¹⁵ The content of N is significantly higher than that of wild type (A and B in figure 4, table 6), mainly due to the fact that the nitrogen absorption efficiency (C in figure 4, table 6) of the root and the nitrogen transport efficiency (D in figure 4, table 6) from the root to the overground part are both significantly improved compared with the wild type, while the difference between OsDREB1C knockout rice and the wild type is not obvious except that ¹⁵ The 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 shows that the nitrogen content of the whole rice plant of the OsDREB1C overexpression rice is generally improved compared with that of the wild type (E in figure 4, table 7), wherein 50.3-66.4% of nitrogen is distributed into grains and is far higher than 41.1% of that of the wild type and 26-38.6% of that of OsDREB1C knockout rice (F in figure 4, table 7). While the nitrogen allocated to leaves and straws was reduced correspondingly, 21.1-29.7% and 12.5-19.9%, respectively, while the proportion in wild type was 42.66% and 16.28%, respectively, and 43.2-49.3% and 18.2-24.7% in OsDREB1C knockout rice (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 transportation 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 detection results of indicators of rice cultivated in the field

In table 7, WT indicates that the difference was significant (p < 0.05), and WT indicates that the difference was very significant (p < 0.01), compared to WT in the same year and same site.

5. Heading stage of OsDREB1C transgenic rice is advanced

Detecting the heading stage of the rice, wherein the rice to be detected: wild type Japanese fine rice (WT), osDREB1C rice (OE 1/OE2/OE 5) over-expressed, and OsDREB1C knock-out rice (KO 1/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: 3 cells are planted on each kind of rice to be tested, the rice to be tested is repeatedly and randomly arranged, the heading is recorded when 50% of ears of plants in the cells are exposed out of 1/2 of the sheaths of the blades of the sword-like leaves, and the heading period 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 gene 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 fine rice (WT), osDREB1C rice (OE 1/OE2/OE 5) over-expressed, and OsDREB1C knock-out rice (KO 1/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 overground part straws is as follows: 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 grain weight is the yield of single-plant grains after the single-plant rice ears of the rice are threshed and the grains are shrunken, and 20-30 single plants of each rice material are repeatedly taken for statistical analysis. And removing the border lines when the cell yield is counted, taking the weight of 30 rice seeds in the middle to measure the yield of one cell, and repeatedly using 3 cells for statistical analysis.

The rice quality is determined as follows: rice grains harvested in the Beijing field experiment of 2019 are naturally stored for 3 months and then used for rice quality analysis, and the determination method refers to an industry standard NY/T83-2017 rice quality determination method 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 yield of the single plant and the yield of the rice with OsDREB1C gene knockout 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 gene OsDREB1C is transferred into rice, so that the yield, rice quality and harvest index of the transgenic rice can be greatly improved, and the grain yield, rice quality and harvest index of the rice can be regulated and controlled by the OsDREB1C and the coding gene thereof.

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

Example 2 functional assay of OsDREB1C in wheat and Arabidopsis

1. Construction of recombinant vectors

Construction of wheat overexpression vector: the CDS full length of the OsDREB1C gene was amplified from a rice Nipponbare cDNA by PCR, and the resulting PCR-recovered product was ligated with a pWMB110 vector (Huiyun Liu, ke Wang, zimiao Jia, qiang Gong, zhishun Lin, lipu Du, xinwu Pei, xingguo Ye, efficiency indication of halogenated plants In which they were used for the expression of the TaMTL used an optimized Agrobacterium-mediated CRISPR system, journal of Experimental Botany, volume 71, issue 4,7February 2020, pages 1337-1349, https://////, anode/10.1093/jxb/erz) by an In-Fusion reaction system to obtain a double-digested vector, and the resulting vector was named as a pWMB110 recombinant vector, and BamHI 1 was ligated with the Sac-derived therefrom. The pWMB110-OsDREB1C is a recombinant vector obtained by replacing a DNA fragment between BamHI and SacI recognition sites of a pWMB110 vector with an OsDREB1C coding gene shown in a sequence 2 in a sequence table, and the pWMB110-OsDREB1C can express a protein coded by the OsDREB1C gene (namely the OsDREB1C protein shown in the sequence 1) under the drive of a UBI promoter.

The primer sequences used were as follows:

Fielder-OsDREB1C-OE-F：5′-CAGGTCGACTCTAGAGGATCCATGGAGTACTACTACTACTACGAGCAGGAG-3' (SEQ ID NO: 12 in the sequence Listing);

Fielder-OsDREB1C-OE-R：5′-CGATCGGGGAAATTCGAGCTCTCAGTAGCTCCAGAGTGTGAC-3' (SEQ ID NO: 13 in the sequence Listing).

Construction of Arabidopsis overexpression vector: CDS (containing no stop codon) of the OsDREB1C gene was amplified from a Nipponbare cDNA of rice by PCR, the resulting PCR-recovered product was ligated with Gateway system entry vector pENTR (ThermoFisher, K240020 SP), the resulting recombinant vector with the correct sequence was designated as pENTR-OsDREB1C, pENTR-OsDREB1C was ligated with pGWB5 vector (Nakagawa T, kurose T, hino T, tanaka K, kawamukai M, niwa Y, toyooka K, matsuoka K, jinbo T, kimura T.development of binding of tissue vectors, pGs, for purifying effect of fusion genes for plant transformation of plant Biocoding. Bioi Biocoding J. 104. JH. 104. For transforming plant, and the resulting recombinant vector was designated as pGEB 1C 5C-OsWB 5C 1C 11/10, and the correct sequence was transferred into pGEB 5 vector for recombination reaction. pGWB5-OsDREB1C can express the protein coded by the OsDREB1C gene (namely OsDREB1C protein shown in a sequence 1) under the drive of a CaMV 35S promoter.

The primer sequences used were as follows:

OsDREB1C-CDS-F:5 'CACCATGGAGTACAGCAGAG-3' (SEQ ID NO: 14 in the sequence listing);

OsDREB1C-CDS-R:5 'GTAGCTCCAGAGTGTGACGTC-3' (SEQ ID NO: 15 in the sequence table).

2. Construction of transgenic plants

And (2) disinfecting young embryos of a wheat variety Fielder, inducing to obtain embryonic callus, introducing pWMB110-OsDREB1C obtained in the step (1) into agrobacterium C58C1, carrying out infection co-culture on the callus by using an agrobacterium-mediated wheat genetic transformation method, and screening by using resistance to obtain a transgenic plant, wherein the screened transgenic plant is the OsDREB1C transgenic wheat material. Because the similarity of the OsDREB1C and the homologous gene of wheat is extremely high, the expression level of the OsDREB1C gene in rice cannot be detected by a qRT-PCR method. Therefore, the wild wheat Fielder is used as a reference, a PCR method is used for detecting whether the Bar gene of the vector exists in the transgenic wheat cDNA, and the used primers are as follows: 5 'CAGGAACCGCAGGAGTGGA-3' (SEQ ID NO: 16 in the sequence table), 5 'CCAGAAACCCCGTCATGCC-3' (SEQ ID NO: 17 in the sequence table). Electrophoresis detection results show that Bar genes exist in 3 lines (TaOE-5, taOE-8 and TaOE-9) of OsDREB1C transgenic wheat, but the Bar genes are not detected in wild types, which indicates that the three lines are all transgenic materials transferred into pWMB110-OsDREB1C vectors (A in figure 5).

And (2) introducing the pGWB5-OsDREB1C obtained in the step (1) into agrobacterium GV3101, transferring the Arabidopsis into an Arabidopsis wild type Col-0 by using an agrobacterium-mediated floral dip method, sowing the seeds into a resistance culture medium after harvesting, and screening to obtain a transgenic plant, namely the OsDREB1C transgenic Arabidopsis material. The relative expression level of the OsDREB1C gene in the OsDREB1C transgenic arabidopsis thaliana on the RNA level is detected by using arabidopsis thaliana wild type Col-0 as a control through a qRT-PCR method, and the used primers are as follows: 5' CATGATGATGCAGTACAGGA-; the reference gene is Arabidopsis thaliana Actin, and the reference gene primer is as follows: 5 'GCACCACCTGAAAGAAGTACA-3' (sequence 20 in sequence table) and 5 'CGATTCCTGGACCTGCCTCATC-3' (sequence 21 in sequence table). The results show that the relative expression levels of the OsDREB1C genes in 3 strains (AtOE-10, atOE-11 and AtOE-12) of OsDREB1C transgenic Arabidopsis are all significantly higher than that of the wild type (Col-0), and the three strains are all over-expressed OsDREB1 Arabidopsis material (B in figure 5).

3. Phenotype identification of OsDREB1C transgenic wheat

Detecting the phenotype of the greenhouse growing potted wheat, wherein the materials to be detected are as follows: wild type wheat (Fielder), over-expressed OsDREB1C wheat (TaOE-5, taOE-8 and TaOE-9). The greenhouse temperature is controlled at 22-24 deg.C, and the sunshine length is 12 hours light/12 hours dark.

Counting heading period of wild type and transgenic wheat, and measuring photosynthesis rate of flag leaf of wheat to be measured in heading period with LICOR-6400XT portable photosynthesis apparatus (LI-COR, USA), light intensity is set to 1000 μmol m ^-2 s ^-1 . After the wheat grains are mature, the grain number per ear, the thousand grain weight and the yield of single-plant grains are measured.

The results show (Table 11, FIG. 6) that transgenic wheat (TaOE-5, taOE-8 and TaOE-9) spiked earlier than wild type Fielder, while photosynthetic rates increased compared to wild type. The grain number per ear in the yield traits is also improved compared with that of the wild type, the thousand grain weight is obviously higher than that of the wild type, and finally the yield of a single plant is improved, so that the OsDREB1C also has the functions of heading in advance, improving the photosynthetic capacity and improving the yield in wheat. Heading date is the number of days elapsed from sowing to heading.

TABLE 11 heading date, photosynthetic rates and yield traits of wild-type and transgenic wheat

4. Phenotype identification of OsDREB1C transgenic arabidopsis

Detecting the phenotype of the greenhouse-grown arabidopsis, and detecting the materials: wild type Arabidopsis (Col-0), osDREB1C Arabidopsis (AtOE-10, atOE-11 and AtOE-12) were overexpressed. The culture was carried out for 2 weeks under short day (8 hours light/16 hours dark), and then transferred to long day (16 hours light/8 hours dark) culture.

And (4) counting bolting time, the number of rosette leaves and overground biomass (including fresh weight and dry weight) of the wild type and the transgenic arabidopsis thaliana. Bolting time is the number of days elapsed from sowing to stem elongation and flowering.

The results show (Table 12, FIG. 7) that transgenic Arabidopsis thaliana (AtOE-10, atOE-11 and AtOE-12) bolting earlier (1-3.9 days) than wild type Col-0, rosette leaves are 5-7 more than wild type in bolting, and the fresh weight and dry weight of the overground part of the rosette leaves are both improved compared with wild type, which shows that the over-expression OsDREB1C can also improve the biomass of dicotyledonous plant Arabidopsis thaliana and enables Arabidopsis thaliana to bolt earlier.

TABLE 12 phenotypic indices of wild-type and transgenic Arabidopsis

Arabidopsis thaliana	Bolting time (sky)	Number of rosette leaves	Fresh weight (gram)
				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

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 examples, it will be appreciated that the invention may 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> protein and biological material related to rice yield and application of protein and biological material in rice yield improvement

<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

1 5 10 15

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

20 25 30

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

<210> 2

<211> 645

<212> DNA

<213> Paddy rice (Oryza sativa L.)

<400> 2

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

gccggcggct acggcggcgg cgacgtcaca ctctggagct actga 645

<210> 3

<211> 4134

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 3

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

atgtaaaatt attttcagtt gattagtact caacaaggta tattgtgtca cgtagaccat 840

aagatattgt cttgtaggga tgtaagtgga caatctcact acccatataa agcctcactt 900

gccaattact ttctcaaaac tttaaaagaa aaaaaattaa ctagaacagg taaaagggaa 960

aaaaaatccc gcttgtccgc ttcccttggc atctggatgg tcacgtggca ggaaagaact 1020

tgtacagtag tagtacgtgc tagggtttga agtaggagcg cgtgtcaatc accaaaaaaa 1080

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

gctgcctgag agcaggtacc atagcatagc aggctacaaa ccagctacaa acatatttta 1440

aaaaaataaa taaggagaga gaatggtagc aggctacaga tttatagcca gctgtagcac 1500

ggacttcaag acacagtgtg tatgacagat gggaccatat attaatagtg taatatgtaa 1560

ctattatatg aatgagctat tatattggct atagatgaat ggtagttatt agttggctat 1620

actattgaac ttgccagaga ccaaccagct actccctcac gtgcggctgg attaaataaa 1680

cattaatcaa ttaattaatt atagagacaa tcagttgcca cttgccaatt aaggaagaag 1740

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

tactattata aaattgaaga tgtttttgcc ggtattctag tacatcatct gtgtatgagt 2100

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. Any of the following uses of the protein:

d1 Regulating plant yield;

d2 Preparing a product for regulating plant yield;

d3 Regulating plant photosynthesis and yield;

d5 Regulating plant photosynthesis, nitrogen uptake or transport and yield;

d6 Preparing products for regulating plant photosynthesis, nitrogen absorption or transport and yield;

d7 Regulating plant photosynthesis, nitrogen content and yield;

the protein is A1) or A2) as follows:

a1 Protein in which the amino acid sequence is sequence 1;

a2 A fusion protein obtained by attaching a tag to the N-terminus or/and C-terminus of A1).

2. Use of a biomaterial related to a protein as claimed in claim 1 in any one of the following applications:

d1 Regulating plant yield;

d2 Preparing a product for regulating plant yield;

d3 Regulating plant photosynthesis and yield;

d5 Regulating plant photosynthesis, nitrogen uptake or transport and yield;

d7 Regulating plant photosynthesis, nitrogen content and yield;

d10 Preparing products for regulating and controlling plant photosynthesis, nitrogen absorption or transport, flowering time and yield; d11 Regulating plant photosynthesis, nitrogen content, flowering time and yield;

the biological material 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 according to B1);

b8 A nucleic acid molecule for reducing the expression level of the protein of claim 1 or knocking out a gene encoding the protein of claim 1;

3. Use according to claim 2, characterized in that: b1 The nucleic acid molecule is b 11) or b 12) or b 13) as follows:

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

b13 3001-4131 of the sequence 3 in the sequence table;

b2 The expression cassette is a DNA molecule shown in a sequence 3 in a sequence table.

4. Any one of the following methods:

x1) cultivating a plant with increased yield, comprising expressing 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 plant of interest with increased yield;

x3) a method for growing plants with enhanced photosynthesis and increased yield, 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 a plant with enhanced photosynthesis and increased yield;

x4) a method for cultivating a plant with enhanced photosynthesis, enhanced nitrogen uptake or transport capacity and increased yield, comprising allowing the protein of claim 1 to be expressed in a recipient plant, or increasing the content or activity of the protein of claim 1 in the recipient plant, to obtain a plant of interest with enhanced photosynthesis, enhanced nitrogen uptake or transport capacity and increased yield;

x5) a method for cultivating a plant with enhanced photosynthesis, increased nitrogen content, and increased yield comprising expressing 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 plant of interest with enhanced photosynthesis, increased nitrogen content, and increased yield;

x6) A method for producing a plant having enhanced photosynthesis, enhanced nitrogen uptake or transport ability, advanced flowering time and increased yield, which comprises allowing the receptor plant to express the protein of claim 1 or increasing the content or activity of the protein of claim 1 in the receptor plant, to obtain a plant having enhanced photosynthesis, enhanced nitrogen uptake or transport ability, advanced flowering time and increased yield;

x7) A method for producing a plant having enhanced photosynthesis, increased nitrogen content, advanced flowering time and increased yield, which comprises expressing 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, thereby obtaining a plant having enhanced photosynthesis, increased nitrogen content, advanced flowering time and increased yield.

5. The method of claim 4, wherein: x1), X3) -X7) by introducing a gene encoding the protein of claim 1 into the recipient plant and allowing the gene to be expressed.

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

7. Use according to any one of claims 1 to 3, or a method according to any one of claims 4 to 6, wherein: the plant is a monocotyledon or a dicotyledon.

8. The use according to any one of claims 1 to 3, or the method according to any one of claims 4 to 6, wherein: the plant is a graminaceous plant, a cruciferous plant or a leguminous plant;

9. use according to any one of claims 1 to 3, or a method according to any one of claims 4 to 6, wherein: the plant is rice, wheat, corn, arabidopsis, rape or soybean.

10. Use according to any one of claims 1 to 3, or a method according to any one of claims 4 to 6, 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 intermediate is present at the heading stage;

the yield is reflected in grain yield, overground part yield and/or harvest index.