WO2022188288A1 - Protein related to rice nitrogen absorption and transformation, encoding gene thereof and application thereof - Google Patents

Abstract

Protein OsDREB1C related to rice nitrogen absorption and transformation, the sequence of which is sequence 1 in the sequence listing and the encoding gene sequence is sequence 2 in the sequence listing. Experiments prove that OsDREB1C and biomaterials related thereto can improve the photosynthetic efficiency of plants, facilitate nitrogen absorption and transportation, enhance the nitrogen content in plants and grains, facilitate early heading and improve the yield. OsDREB1C and the biomaterial related thereto have important biological significance and industrial value, as well as good application prospects.

Description

Proteins related to nitrogen uptake and transformation in rice and their encoding genes and applications technical field

The present invention relates to a protein related to nitrogen absorption and transformation of rice and its encoding gene and application in the field of biotechnology.

Background technique

In agricultural production, applying a large amount of nitrogen fertilizer has always been one of the important measures for high crop yield. The continuous large amount of nitrogen fertilizer input not only increases the cost of cultivation, but also leads to increasingly serious environmental pollution problems. How to reduce nitrogen fertilizer input in agricultural production and continuously improve crop yield has become a major problem to be solved urgently for the sustainable development of agriculture in my country. It is imperative to explore the regulation mechanism and technical approach for the substantial increase in crop yield and the efficient utilization of resources in my country's agricultural sustainable development.

Heading date is one of the important agronomic traits of crops, which determines the season, regional adaptability and yield of rice. A suitable heading date is the guarantee of stable and high yield of crops. Breeding new early-maturing and high-yielding varieties has always been one of the main directions of crop genetics and breeding research. "High-yielding but not early-maturing, early-maturing but not high-yielding" is the so-called "excellent but not early, early but not excellent" phenomenon, which is a major problem encountered in the cultivation of crop varieties.

In the future, with population growth and economic development, my country's food demand will continue to grow rapidly. Under the situation that the arable land area continues to decrease and the potential for the expansion of grain planting area is limited, the continuous increase of crop yield on a large area has become the only option to ensure food security in my country. Therefore, under the pressure of food security demand, high yield is the unremitting goal of agricultural production. The "green revolution" that began in the mid-1950s has achieved a substantial increase in crop yield potential through genetic improvement of crop varieties and improvements in cultivation and management techniques. However, in recent years, crop yields have shown a stagnant or even declining trend. How to improve crop yields requires new strategies and approaches.

At present, researchers have carried out related exploration and research in crop breeding, cultivation management measures and functional gene mining, and have also made some important progress. solution.

Invention Disclosure

The technical problem to be solved by the present invention is how to improve the nitrogen absorption and transport capacity of plants and increase the nitrogen content of plants.

In order to solve the above-mentioned technical problems, the present invention firstly provides any of the following applications of a protein or a substance that regulates the activity or content of the protein:

D1) regulating plant nitrogen absorption or transport;

D2) prepare a product that regulates nitrogen absorption or transport of plants;

D3) regulating the nitrogen content of plants;

D4) preparing a product for regulating and controlling plant nitrogen content;

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

D6) prepare a product that regulates plant nitrogen absorption or transport and flowering time;

D7) regulating plant nitrogen content and flowering time;

D8) prepare a product that regulates plant nitrogen content and flowering time;

D9) regulating plant nitrogen absorption or transport and yield;

D10) preparation and regulation of plant nitrogen absorption or transport and yield products;

D11) Regulate nitrogen content and yield of plants;

D12) preparation and regulation of plant nitrogen content and yield products;

D13) regulating plant nitrogen absorption or transport, flowering time and yield;

D14) preparation and regulation of plant nitrogen absorption or transport, flowering time and yield products;

D15) regulating plant nitrogen content, flowering time and yield;

D16) preparation and regulation of plant nitrogen content, flowering time and yield products;

D17) Plant breeding;

Said protein (whose name is OsDREB1C) is the following A1), A2), A3) or A4):

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

A2) The amino acid sequence shown in SEQ ID NO: 1 in the sequence listing has undergone the substitution and/or deletion and/or addition of one or several amino acid residues and has the same function;

A3) A protein derived from rice, millet, maize, sorghum, goat grass, wheat or Brachypodium bismuth and having 64% or more identity with sequence 1 and the same function as the protein described in A1);

A4) A fusion protein obtained by linking a tag to the N-terminus or/and C-terminus of A1) or A2) or A3).

In order to facilitate the purification of the protein in A1), the amino-terminal or carboxyl-terminal of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing can be attached with the tags shown in the following table.

Table: Sequence of tags

Label	Residues	sequence
Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
FLAG	8	DYKDDDDK

Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

The protein in the above A2) is a protein having 64% or more identity to the amino acid sequence of the protein shown in SEQ ID NO: 1 and having the same function. Said having 64% or more identity is having 64%, having 75%, having 80%, having 85%, having 90%, having 95%, having 96%, having 97%, having 98% or having 99% % identity.

The protein in the above A2) can be artificially synthesized, or can be obtained by first synthesizing its encoding gene and then carrying out biological expression.

The coding gene of the protein in the above-mentioned A2) can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 2, and/or carrying out missense mutation of one or several base pairs, and/ Or the coding sequence of the tag shown in the above table is connected to its 5' end and/or 3' end. Wherein, the DNA molecule shown in SEQ ID NO: 2 encodes the protein shown in SEQ ID NO: 1.

The present invention also provides any of the following applications of the biomaterial related to OsDREB1C:

D1) regulating plant nitrogen absorption or transport;

D2) prepare a product that regulates nitrogen absorption or transport of plants;

D3) regulating the nitrogen content of plants;

D4) preparing a product for regulating and controlling plant nitrogen content;

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

D6) prepare a product that regulates plant nitrogen absorption or transport and flowering time;

D7) regulating plant nitrogen content and flowering time;

D8) prepare a product that regulates plant nitrogen content and flowering time;

D9) regulating plant nitrogen absorption or transport and yield;

D10) preparation and regulation of plant nitrogen absorption or transport and yield products;

D11) Regulate nitrogen content and yield of plants;

D12) preparation and regulation of plant nitrogen content and yield products;

D13) regulating plant nitrogen absorption or transport, flowering time and yield;

D14) preparation and regulation of plant nitrogen absorption or transport, flowering time and yield products;

D15) regulating plant nitrogen content, flowering time and yield;

D16) preparation and regulation of plant nitrogen content, flowering time and yield products;

D17) Plant breeding;

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

B1) a nucleic acid molecule encoding OsDREB1C;

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

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

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

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

B6) a transgenic plant tissue containing the nucleic acid molecule of B1), or a transgenic plant tissue containing the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

B8) reducing the expression of OsDREB1C or knocking out the nucleic acid molecule of the gene encoding OsDREB1C;

B9) An expression cassette, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ comprising the nucleic acid molecule of B8).

In the above application, the nucleic acid molecule of B1) may be as follows b11) or b12) or b13) or b14) or b15):

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

b12) the cDNA molecule or DNA molecule shown in sequence 2 in the sequence listing;

b13) DNA molecules shown in positions 3001-4131 of sequence 3 in the sequence listing;

b14) has 73% or more identity to the nucleotide sequence defined by b11) or b12) or b13), and encodes a cDNA molecule or DNA molecule of OsDREB1C;

b15) hybridizes under stringent conditions to a nucleotide sequence defined in b11) or b12) or b13) or b14) and encodes a cDNA molecule or DNA molecule of OsDREB1C;

B2) The expression cassette is the following b21) or b22) or b23):

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

b22) A DNA molecule having 73% or more identity with the nucleotide sequence defined in b21) and having the same function;

b23) DNA molecules that hybridize under stringent conditions to the nucleotide sequences defined in b21) or b22) and have the same function.

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

Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the OsDREB1C protein of the present invention using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides with 73% or higher identity to the nucleotide sequence of the OsDREB1C protein isolated by the present invention, as long as they encode the OsDREB1C protein and have the function of the OsDREB1C protein, are all derived from the nucleus of the present invention. nucleotide sequences and are equivalent to the sequences of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 73% or more, or 85% or more, or 90% or more, or 95% or more of the nucleotide sequence of the protein consisting of the amino acid sequence shown in the coding sequence 1 of the present invention. Nucleotide sequences of higher identity. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above application, the stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ and 1 mM EDTA, 50°C, 2×SSC, 0.1 Rinse in %SDS; also: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA, rinse at 50°C, 1×SSC, 0.1% SDS; also: 50°C , hybridized in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1 mM EDTA, washed at 50°C, 0.5×SSC, 0.1% SDS; also: 50°C, in 7% SDS, 0.5M NaPO ₄ and Hybridize in a mixed solution of 1mM EDTA, rinse in 0.1×SSC, 0.1% SDS at 50°C; alternatively: hybridize in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA at 50°C, at 65°C , washed in 0.1×SSC, 0.1% SDS; also can be: in 6×SSC, 0.5%SDS solution, hybridize at 65℃, then use 2×SSC, 0.1%SDS and 1×SSC, 0.1%SDS Each membrane is washed once; it can also be: hybridized in 2×SSC, 0.1% SDS solution at 68°C and washed twice for 5 min each, and then in 0.5×SSC, 0.1% SDS solution at 68°C Hybridize and wash the membrane twice at ℃ for 15 min each time; or in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS, hybridize and wash the membrane at 65°C.

The above-mentioned 73% or more identity may be 80%, 85%, 90% or more than 95% identity.

In above-mentioned application, B2) described expression cassette (OsDREB1C gene expression cassette) containing the nucleic acid molecule of coding OsDREB1C protein, refers to the DNA that can express OsDREB1C protein in host cell, this DNA can not only include the start that starts OsDREB1C gene transcription It can also include a terminator that terminates transcription of the OsDREB1C gene. Further, the expression cassette may also include enhancer sequences. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiol acid S-methyl ester)); tomato Protease inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoter (US Pat. No. 5,187,267); tetracycline-inducible promoter (US Pat. No. 5,057,422) seed-specific promoters, such as foxtail millet seed-specific promoter pF128 (CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (for example, promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminators (see, eg: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; ^Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (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 Acids Res., 15:9627).

The recombinant vector containing the OsDREB1C gene expression cassette can be constructed by using the existing expression vector. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301 or pCAMBIA1391-Xb (CAMBIA company) and so on. The plant expression vector may also contain the 3' untranslated region of the foreign gene, ie, containing the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The poly(A) signal can guide the addition of poly(A) to the 3' end of the mRNA precursor, such as Agrobacterium crown gall-inducing (Ti) plasmid genes (such as nopaline synthase gene Nos), plant genes (such as soybean The untranslated regions transcribed from the 3' end of the storage protein gene) have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc., but must be associated with the coding. The reading frames of the sequences are identical to ensure correct translation of the entire sequence. The translation control signals and initiation codons can be derived from a wide variety of sources, either natural or synthetic. The translation initiation region can be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding a gene (GUS gene, luciferase gene, luciferase gene) that can be expressed in plants encoding an enzyme that can produce color change or a luminescent compound. Gene, etc.), marker genes for antibiotics (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene conferring resistance to methotrexate, the EPSPS gene conferring resistance to glyphosate) or marker genes for chemical resistance (such as herbicide resistance genes), mannose-6- which provides the ability to metabolize mannose Phosphoisomerase gene. Considering the safety of transgenic plants, the transformed plants can be directly screened under stress without adding any selectable marker gene.

In the above applications, the vector may be a plasmid, cosmid, phage or viral vector. The plasmid may specifically be a pBWA(V)HS vector or a psgR-Cas9-Os-containing plasmid.

B3) The recombinant vector can specifically be pBWA(V)HS-OsDREB1C. The pBWA(V)HS-OsDREB1C is a recombinant vector obtained by inserting the OsDREB1C encoding gene shown in SEQ ID NO: 2 in the sequence listing at the BsaI (Eco31I) restriction site of the pBWA(V)HS vector. The pBWA(V)HS-OsDREB1C can overexpress the protein encoded by the OsDREB1C gene (ie, the OsDREB1C protein shown in sequence 1) under the drive of the CaMV 35S promoter.

B8) The recombinant vector can be a recombinant vector prepared by using the crisper/cas9 system, which can reduce the content of OsDREB1C. The recombinant vector can express sgRNA targeting the nucleic acid molecule of B1). The target sequence of the sgRNA can be positions 356-374 of sequence 2 in the sequence listing.

In the above applications, the microorganisms may be yeast, bacteria, algae or fungi. Wherein, the bacteria can be Agrobacterium, such as Agrobacterium rhizogenes EHA105.

In the above applications, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs may all include propagating material or may not include propagating material.

The present invention also provides any of the following methods:

X1) A method for cultivating plants with 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 nitrogen absorption or transport capacity;

X2) a method for cultivating plants with increased nitrogen content, comprising expressing OsDREB1C in a recipient plant, or increasing the content or activity of OsDREB1C in the recipient plant, to obtain a target plant with increased nitrogen content;

X3) A method for cultivating plants with enhanced nitrogen absorption or transport capacity and earlier flowering time, comprising expressing OsDREB1C in recipient plants, or increasing the content or activity of OsDREB1C in recipient plants, to obtain plants with enhanced nitrogen absorption or transport capabilities and earlier flowering time target plant;

X4) a method for cultivating plants with increased nitrogen content and earlier flowering time, comprising expressing OsDREB1C in the recipient plant, or increasing the content or activity of OsDREB1C in the recipient plant, to obtain a target plant with increased nitrogen content and earlier flowering time;

X5) A method for cultivating plants with enhanced nitrogen absorption or transport capacity and 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 enhanced nitrogen absorption or transport capacity and increased yield ;

X6) a method for cultivating plants with increased nitrogen content and 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 nitrogen content and increased yield;

X7) A method for cultivating plants with enhanced nitrogen absorption or transport capacity, advanced flowering time and increased yield, comprising expressing OsDREB1C in recipient plants, or increasing the content or activity of OsDREB1C in recipient plants, to obtain enhanced nitrogen absorption or transport capacity, flowering Plants of interest with advanced timing and increased yields;

X8) A method for cultivating plants with increased nitrogen content, earlier flowering time and increased yield, comprising expressing OsDREB1C in a recipient plant, or increasing the content or activity of OsDREB1C in the recipient plant, to obtain a plant with increased nitrogen content, earlier flowering time and increased yield target plant.

In the above methods, the methods described in X1)-X8) can be achieved by introducing the encoding gene of OsDREB1C into the recipient plant and expressing the encoding gene.

In the above method, the encoding gene can be the nucleic acid molecule of B1).

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

1) Carry out modification and optimization according to actual needs, so that the gene can be expressed efficiently; for example, according to the codon preferred by the recipient plant, while maintaining the amino acid sequence of the coding gene of OsDREB1C of the present invention, its codons can be changed to meet the Plant preference; in the optimization process, it is best to keep a certain GC content in the optimized coding sequence to best achieve high-level expression of the introduced gene in plants, where the GC content can be 35% or more than 45% , more than 50% or more than about 60%;

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

3) Link with various plant-expressed promoters to facilitate their expression in plants; the promoters may include constitutive, inducible, time-sequential regulation, developmental regulation, chemical regulation, tissue-preferred and tissue-specific promoters ; the choice of promoter will vary with the temporal and spatial requirements of expression and will also depend on the target species; e.g. tissue- or organ-specific expression promoters, depending on what stage of development the receptor is desired; although the provenance of the source Many promoters for dicotyledonous plants are functional in monocotyledonous plants and vice versa, but ideally, a dicotyledonous promoter is chosen for expression in dicotyledonous plants and a monocotyledonous promoter for expression in monocots;

4) Linking with a suitable transcription terminator can also improve the expression efficiency of the gene of the present invention; for example, tml derived from CaMV, E9 derived from rbcS; any available terminator known to function in plants can be combined with The gene of the present invention is connected;

5) Introduction of enhancer sequences such as intron sequences (eg from Adhl and bronzel) and viral leader sequences (eg from TMV, MCMV and AMV).

The OsDREB1C-encoding gene can be introduced into recipient plants using a recombinant expression vector containing the OsDREB1C-encoding gene. The recombinant expression vector can specifically be the pBWA(V)HS-OsDREB1C.

The recombinant expression vector can be introduced into plant cells by using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation and other conventional biotechnology methods (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York). , pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition).).

The plant of interest is understood to include not only the first-generation plants in which the OsDREB1C protein or its encoding gene has been altered, but also its progeny. For the plant of interest, the gene can be propagated in that species, and conventional breeding techniques can be used to transfer the gene into other varieties of the same species, including in particular commercial varieties. The plants of interest include seeds, callus, whole plants and cells.

The present invention also provides a product having any of the following functions D1)-D8), the product containing OsDREB1C or the biological material:

D1) regulating plant nitrogen absorption or transport;

D2) regulating the nitrogen content of plants;

D3) regulating plant nitrogen absorption or transport and flowering time;

D4) regulating plant nitrogen content and flowering time;

D5) regulating plant nitrogen absorption or transport and yield;

D6) Regulate nitrogen content and yield of plants;

D7) regulating plant nitrogen absorption or transport, flowering time and yield;

D8) Regulate plant nitrogen content, flowering time and yield.

In the above, the plant may be M1) or M2) or M3):

M1) monocots or dicots;

M2) grasses, crucifers or legumes;

M3) Rice, wheat, corn, Arabidopsis, rape or soybean.

In the above, the transport can be embodied in the transport of the roots to the shoot, or the transport into the grain;

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

The yield can be expressed in grain yield, shoot yield and/or harvest index;

The flowering time can be reflected in the heading stage.

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

The harvest index refers to the ratio of economic yield (grain, fruit, etc.) to biological yield when a plant is harvested.

The modulation can be enhancing or inhibiting, or promoting or inhibiting, or increasing or decreasing.

Description of drawings

Figure 1 shows the sequence alignment results.

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

Figure 3 shows photosynthesis parameters of wild-type and transgenic rice plants. A - diurnal variation of photosynthesis; B - diurnal variation of NPQ; C - light response curve; D - CO ₂ response curve; E - maximum CO ₂ carboxylation efficiency; F - maximum electron transfer rate.

Figure 4 shows the detection results of nitrogen uptake and utilization of wild-type and transgenic rice plants. A- ¹⁵ N content in shoots; B- ¹⁵ N content in roots; C- ¹⁵ N uptake efficiency; D- ¹⁵ N transport efficiency from roots to shoots; E- nitrogen content in different tissues of rice; F- different tissues of rice distribution ratio of nitrogen. In the column charts of E and F, from top to bottom, the grains, stems and leaves are in order.

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

Figure 6 shows the phenotypes of wild type and transgenic wheat. (A) Wild-type wheat (Fielder) and transgenic wheat phenotypes. (B-D) Heading time (B), photosynthetic rate (C), number of grains per ear (D), 1000-grain weight (E), and grain yield per plant (F) for wild-type and transgenic lines.

Figure 7 is a phenotypic profile of wild-type and transgenic Arabidopsis. (A) Arabidopsis wild-type Col-0 and transgenic line phenotypes. (B-D) Bolting time (B), number of rosette leaves (C), and fresh shoot weight (D) of Arabidopsis wild-type and transgenic lines.

*P<0.05, **P<0.01.

Best way to implement your invention

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product specification. Materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged. In the following examples, unless otherwise specified, the first 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 nucleus of the corresponding DNA/RNA. Glycosides.

The pBWA(V)HS vector in the following examples (Zhao et al., DEP1is involved in regulating the carbon-nitrogen metabolic balance to affect grain yield and quality in rice (Oriza sativa L.), PLOS ONE, March 11, 2019 , https://doi.org/10.1371/journal.pone.0213504), the public can obtain the biological material from the applicant, and the biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

Plasmids containing psgR-Cas9-Os in the following examples (Hu Xuejiao, Yang Jia, Cheng Can, Zhou Jihua, Niu Fuan, Wang Xinqi, Zhang Meiliang, Cao Liming, Chu Huangwei. Using CRISPR/Cas9 system to edit the SD1 gene of rice. China 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 efficient genome engineering in plants. Mol Plant, 2013, 6( 6): 2008-2011.), the public can obtain the biological material from the applicant, and the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.

Example 1. OsDREB1C has the effect of improving rice photosynthetic efficiency, promoting nitrogen absorption, promoting early heading and increasing yield

This example provides a protein derived from Nipponbare rice, which has the functions of improving rice photosynthesis efficiency, promoting nitrogen absorption, promoting early heading and improving yield. The name of the protein is OsDREB1C, and its sequence is sequence 1 in the sequence table. , in Nipponbare, the coding gene sequence of OsDREB1C is sequence 2, and the genome sequence is 3001-4131 of sequence 3. In sequence 3, positions 1-3000 are the promoter of OsDREB1C gene in Nipponbare genomic DNA.

The sequence alignment of rice OsDREB1C with homologous proteins in other plants found that its identities with millet, maize, sorghum, goat grass, wheat, and Brachypodium were 73.52%, 64.06%, 66.52%, 66.05%, respectively. %, 66.05%, 65.88% (Figure 1).

1. Construction of recombinant vector

Construction of overexpression vector: PCR product containing the full-length CDS of OsDREB1C gene was amplified from rice Nipponbare cDNA by PCR, and the obtained PCR product was digested with BsaI (Eco31I). The pBWA(V)HS vector was linked to the backbone of the vector obtained by single digestion with BsaI(Eco31I), and the resulting recombinant vector with the correct sequence was denoted as pBWA(V)HS-OsDREB1C. pBWA(V)HS-OsDREB1C is a recombinant vector obtained by inserting the OsDREB1C encoding gene shown in SEQ ID NO: 2 in the sequence table at the BsaI(Eco31I) restriction site of the pBWA(V)HS vector. pBWA(V)HS-OsDREB1C can The protein encoded by the OsDREB1C gene (ie the OsDREB1C protein shown in sequence 1) was expressed under the drive of the CaMV 35S promoter.

The primer sequences used are as follows:

OsDREB1C-F: 5'-CAGTGGTCTCACAACATGGAGTACTACGAGCAGGAGGAGT-3' (sequence 4 in the sequence listing);

OsDREB1C-R: 5'-CAGTGGTCTCATACATCAGTAGCTCCAGAGTGTGACGTCG-3' (sequence 5 in the sequence listing).

Construction of gene knockout vector: sgRNA and primers were designed according to the online design website ( http://skl.scau.edu.cn/ ), and the target sequence was determined to be 5′-AGTCATGCCCGCACGACGC-3′ (No. 356-374 of sequence 2). bits). The OsDREB1C-sgRNA-F and OsDREB1C-sgRNA-R were annealed, the resulting product was digested with BsaI, and the resulting digested product was connected to the vector skeleton obtained by digesting the psgR-Cas9-Os plasmid with BsaI, and the obtained The recombinant vector with the correct sequence is the OsDREB1C knockout vector, denoted as OsU3-sgRNA-OsUBI-Cas9-OsDREB1C. In this recombinant vector, the OsU3 promoter drives sgRNA and the OsUBI promoter drives Cas9.

Among them, the primer sequences used are as follows:

OsDREB1C-sgRNA-F: 5'-TGTGTGGCGTCGTGCGGGCATGACT-3' (SEQ ID NO: 6 in the sequence listing);

OsDREB1C-sgRNA-R: 5'-AAACAGTCATGCCCGCACGACGCCA-3' (SEQ ID NO: 7 in the sequence listing).

2. Construction of transgenic plants

The mature seeds of the japonica rice variety Nipponbare were sterilized and induced to obtain embryogenic callus. The pBWA(V)HS-OsDREB1C and OsU3-sgRNA-OsUBI-Cas9-OsDREB1C obtained in step 1 were introduced into Agrobacterium EHA105, respectively, and the Bacillus-mediated rice genetic transformation method infects and co-cultivates callus, and uses resistance screening to obtain transgenic plants. The screened transgenic rice obtained from pBWA(V)HS-OsDREB1C is OsDREB1C transgenic rice. The transgenic rice obtained by sgRNA-OsUBI-Cas9-OsDREB1C is the OsDREB1C knockout rice material.

Using the japonica variety Nipponbare (WT) as a control, the relative expression level of OsDREB1C gene at RNA level in OsDREB1C transgenic rice and OsDREB1C gene knockout rice was detected by qRT-PCR method. The primers used were: 5′-CATGATGATGCAGTACCAGGA-3′ (Sequence 8 in the sequence listing), 5'-GATCATCAGTAGCTCCAGAGTG-3' (sequence 9 in the sequence listing); the internal reference gene is rice Ubiqutin, and the primers for the internal reference gene are: 5'-AAGAAGCTGAAGCATCCAGC-3' (sequence 10 in the sequence listing), 5 '-CCAGGACAAGATGATCTGCC-3' (sequence 11 in the sequence listing).

The results showed that the relative expression levels of OsDREB1C gene in three lines (OE1, OE2 and OE5) of OsDREB1C transgenic rice were significantly higher than those of wild type (WT), and these three lines were all OsDREB1C-overexpressing rice materials (Fig. 2). middle A). There are base deletions or insertions in the OsDREB1C gene sequence in the three lines (KO1, KO2 and KO3) of the OsDREB1C gene knockout rice material, resulting in frameshift mutations, resulting in the loss of the normal function of the OsDREB1C protein (Figure 2B).

The three lines (KO1, KO2 and KO3) of OsDREB1C knockout rice material were amplified and sequenced by PCR using primer pairs capable of amplifying the target sequence and its upstream and downstream. The results showed that the target sites of these three lines were The sequence changes are shown in B in Figure 2. One nucleotide deletion occurred in KO1 and KO2, one nucleotide insertion occurred in KO3, and frameshift mutations occurred in the target genes of the three lines.

3. Enhancement of photosynthesis efficiency in OsDREB1C transgenic rice

To detect the photosynthesis indicators of rice grown in the field, the rice to be tested: wild-type Nipponbare rice (WT), OsDREB1C overexpressing rice (OE1/OE2/OE5), OsDREB1C knockout rice (KO1/KO2/KO3).

The flag leaves of the rice to be tested at the heading stage were measured with a LICOR-6400XT portable photosynthesis instrument (LI-COR, USA) to measure the photosynthetic diurnal variation, light response curve and CO ₂ response curve reflecting plant photosynthetic capacity. The determination of the diurnal variation of photosynthesis was carried out in clear and cloudless weather, and the measurement was carried out every 2-4 hours from 8:00 to 16:00. The rate of photosynthesis was measured with a LI-COR 6400XT portable photosynthesis instrument, and NPQ (non-photochemical quenching) was measured with a FluorPen FP100 (PSI, Czech Republic). In the measurement of the light response curve, the CO ₂ concentration was set to 400 μmol mol ⁻¹ , and the light intensity (PPFD) was 0 to 2000 μmol m ⁻² s ⁻¹ . In the CO ₂ response curve determination, the PPFD was set at 1200 μmol m ⁻² s ⁻¹ , and the CO ₂ concentration was decreased from 400 to 50 μmol mol ⁻¹ and then increased from 400 to 1200 μmol mol ⁻¹ again. The light response curve and CO ₂ response curve were both fitted by the Farquhar-von Caemmerer-Berry (FvCB) model, and the maximum carboxylation efficiency (V _cmax ) and the maximum electron transfer rate (J _max ) were calculated from the CO ₂ response curve .

The results showed that the photosynthetic efficiency (ie, photosynthetic efficiency) of rice overexpressing OsDREB1C was significantly higher than that of the wild type during the day, and the difference reached the maximum at noon when the light intensity was the highest, and reached a significant level, which could be increased by 29.5-43.3% compared with the wild type. , while the heat dissipation NPQ used to consume excessive absorbed light energy is significantly lower than that of the wild type (A, B in Figure 3, Table 1 and Table 2), and the difference reaches a significant level, indicating that more light energy is used to participate in photosynthesis. The results of further determination of the light response curve and CO ₂ response curve showed that when the light intensity was lower than 200 μmol m ^-2 s ^-1 , there was no significant difference between the net photosynthetic rate of OsDREB1C-overexpressing rice and wild-type rice, but at high light intensity. Under the light intensity of 2000 μmol m ^-2 s ^-1 , the photosynthetic rate of rice overexpressing OsDREB1C increased by 18.4-27.6% compared with wild type (C in Figure 3, Table 3), and the difference reached a significant level. The measured results of the CO ₂ response curve were similar to the light response curve. When the CO ₂ concentration was greater than 400 μmol mol ^-1 , the photosynthetic rate of rice overexpressing OsDREB1C was significantly increased compared with the wild type (D in Figure 3, Table 4). Further calculation The obtained maximum carboxylation efficiency (V _cmax ) and maximum electron transfer rate (J _max ) were significantly higher than those of the wild type (E, F in Figure 3, Table 5). However, the photosynthesis-related parameters of OsDREB1C knockout rice were lower than or similar to those of wild type. Taken together, the above results show that overexpression of OsDREB1C gene in rice can significantly improve both light energy use efficiency and CO ₂ assimilation ability.

Table 1. Detection results of photosynthesis efficiency (μmol CO ₂ m ^-2 s ^-1 )

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, compared with WT in the same year and the same place, * means that the difference reaches a significant level (p<0.05), and ** means that the difference reaches a very significant level (p<0.01).

Table 2. Test 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, compared with WT in the same year and the same place, * indicates that the difference reaches a significant level (p<0.05), and ** indicates that the difference reaches a very significant level (p<0.01).

Table 5. Results of maximum carboxylation efficiency (V _cmax ) and maximum electron transfer rate (J _max )

In Table 5, compared with WT in the same year and place, * indicates that the difference reaches a significant level (p<0.05), and ** indicates that the difference reaches a very significant level (p<0.01).

4. Improvement of nitrogen use efficiency in OsDREB1C transgenic rice

To test the nitrogen use efficiency of rice, the rice to be tested: wild-type Nipponbare rice (WT), OsDREB1C overexpressing rice (OE1/OE2/OE5), OsDREB1C knockout rice (KO1/KO2/KO3).

The rice seedlings to be tested that were grown in nutrient solution hydroponics for 3 weeks in the greenhouse were placed in Kimura B solution (Kimura B solution) without nitrogen (without (NH ₄ ) ₂ SO ₄ and KNO ₃ ) in advance for nitrogen starvation treatment 3 sky. After nitrogen starvation treatment, the roots of seedlings were completely immersed in 0.1 mM CaSO ₄ solution (solvent is deionized water) for 1 minute, and then the residual water was absorbed, and the roots were placed in a nutrient solution containing 0.5 mM K ¹⁵ NO ₃ for cultivation. After 3 hours, the roots of the seedlings were soaked in 0.1 mM CaSO ₄ solution for 1 minute, and the residual water was absorbed. The shoots and roots were collected and dried at 70 °C for 3 days to constant weight, and the dry weight of the samples was recorded. After the samples were ground and pulverized, the content of ¹⁵ N in shoots and roots were measured with an IsoPrime 100 stable isotope ratio mass spectrometer (Elementar, Germany), respectively, and the nitrogen absorption efficiency and nitrogen transport efficiency were calculated. Nitrogen absorption efficiency=( ^15N content in shoots+ ^15N content in roots)/root dry weight/3; nitrogen transport efficiency= ^15N content in shoots/ ^15N content in roots.

In addition, mature rice plants to be tested grown in the Beijing field were harvested, and individual leaves, straws and grains were harvested, and were placed at 105 °C for 30 minutes, and dried at 70 °C for 3 days. After the samples were 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 in different parts was calculated.

The nutrient solution used is as follows:

Nutrient solution: Kimura B nutrient solution (0.5mM (NH ₄ ) ₂ SO ₄ , 1 mM KNO ₃ , 0.54 mM MgSO ₄ ·7H ₂ O, 0.3 mM CaCl ₂ , 0.18 mM KH ₂ PO ₄ , 0.09 mM K ₂ SO ₄ , ₁₆ μM Na2SiO3 _{· 9H2O} , 9.14 μM MnCl2 _· 4H2O, 46.2 μM Na2MoO4 _· 2H2O, 0.76 μM ZnSO4 _{· 7H2O} , 0.32 μM _{CuSO4 · 5H2O} , _{40 μM} Fe (II)-EDTA, pH=5.8).

Nutrient solution containing 0.5mM K ¹⁵ NO ₃ : 0.5M K ¹⁵ NO ₃ stock solution was diluted 1000 times and added to Kimura nutrient solution without nitrogen (0.54 mM MgSO ₄ ·7H ₂ O, 0.3 mM CaCl ₂ , 0.18 mM KH ₂ PO _{4 )} , 0.09mM K ₂ SO ₄ , 16 μM Na ₂ SiO ₃ ·9H ₂ O, 9.14 μM MnCl ₂ ·4H ₂ O, 46.2 μM Na ₂ MoO ₄ ·2H ₂ O, 0.76 μM ZnSO ₄ ·7H ₂ O, 0.32 μM CuSO _4.5H2O , 40 μM Fe( _II )-EDTA, pH=5.8).

The results showed that after ¹⁵ N treatment for 3 hours, the content of ¹⁵ N in the shoots and roots of OsDREB1C overexpressing rice was significantly higher than that of the wild type (A and B in Figure 4, Table 6), mainly due to the nitrogen in the roots. The absorption efficiency (C in Figure 4, Table 6) and the transport efficiency of nitrogen from roots to shoots (D in Figure 4, Table 6) were significantly improved compared with the wild-type, while the OsDREB1C gene knockout rice was similar to the wild-type. The difference was not obvious, except that the absorption efficiency of ¹⁵ N was lower than that of the wild type, other indicators were similar to the wild type. Further analysis of nitrogen distribution in different tissues of field-grown rice plants showed that the nitrogen content of rice plants overexpressing OsDREB1C was generally increased compared with wild type (E in Figure 4, Table 7), of which 50.3-66.4% The distribution of nitrogen into the grain was much higher than that of 41.1% of wild-type and 26-38.6% of OsDREB1C knockout rice (Fig. 4F, Table 7). Nitrogen allocated to both leaves and straw was correspondingly reduced by 21.1-29.7% and 12.5-19.9%, respectively, compared with 42.66% and 16.28% in wild-type and 43.2-49.3% in OsDREB1C knockout rice and 18.2-24.7% (F in Figure 4). Taken together, the above results show that overexpression of OsDREB1C gene in rice can significantly improve the nitrogen uptake and transport efficiency of plants, and distribute more nitrogen into grains.

Table 6. The index detection results of rice cultivated in the greenhouse

In Table 6, compared with WT in the same location and treatment, * indicates that the difference reaches a significant level (p<0.05), and ** indicates that the difference reaches a very significant level (p<0.01).

Table 7. The index detection results of rice cultivated in the field

In Table 7, compared with WT in the same year and place, * indicates that the difference reaches a significant level (p<0.05), and ** indicates that the difference reaches a very significant level (p<0.01).

5. Advance heading date of OsDREB1C transgenic rice

The heading date of rice was detected, and the rice to be tested: wild-type Nipponbare rice (WT), OsDREB1C overexpressing rice (OE1/OE2/OE5), and OsDREB1C knockout rice (KO1/KO2/KO3).

The heading date of each tested rice was counted under the Beijing field planting conditions. The statistical method is as follows: each type of rice to be tested is planted in 3 plots, which are randomly arranged, and 50% of the plants in the plot are marked as heading when the ears of 50% of the plants are exposed from the flag leaf sheaths, and the heading period is the time from sowing to heading. days.

The results showed (Table 8) that the heading date of OsDREB1C-overexpressing rice was significantly earlier than that of the wild type, 13-17 days earlier in 2018 and 17-19 days earlier in 2019, while the heading date of OsDREB1C knockout rice was later than that of the wild type, respectively. 6-8 days and 3-5 days, the grain filling maturity of rice is correspondingly advanced or delayed. This indicated that OsDREB1C and its encoding gene could regulate the heading date of rice.

Table 8. Heading period (days) of wild-type and transgenic rice under field conditions in Beijing

rice	2018	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 compared with WT in the same year and location, the difference reached a very significant level (p<0.01).

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

The aboveground dry weight and grain yield of rice were detected. The rice to be tested: wild-type Nipponbare rice (WT), OsDREB1C overexpressing rice (OE1/OE2/OE5), and OsDREB1C gene knockout rice (KO1/KO2/KO3).

In the field experiments conducted in Beijing and Hainan, each type of rice to be tested was planted in 3 replicate plots and randomly arranged. After the rice grains were mature, the grain yield per plant, the plot grain yield, and the dry weight of the aboveground straw were measured and calculated. Harvest index, the harvest index is the ratio of rice grain yield per plant to above-ground biomass (the sum of above-ground straw dry weight and grain yield per plant).

The method for measuring the dry weight of aboveground straw is as follows: after the rice is mature, after removing the grains from the straw per plant, put it into a nylon mesh bag and dry it to constant weight at 80°C, and then weigh the sample. 20-30 replicates of each rice material were taken for statistical analysis.

The measurement method of grain yield is as follows: threshing and removing the shriveled grains per plant of rice is the grain yield per plant, and 20-30 single plants of each rice material are repeated for statistical analysis. When calculating the yield of the plot, the side row was removed, and the 30 rice plants in the middle were taken to measure the grain weight as the yield of one plot, and 3 plots were repeated for statistical analysis.

The measurement method of rice quality is as follows: the rice grains harvested in the Beijing field experiment in 2019 were used for rice quality analysis after natural storage for 3 months. The results showed that the grain yield of rice overexpressing OsDREB1C was significantly higher than that of the wild type, and the difference was significant.

Judging from the yield results (Table 9), in Beijing and Hainan, the yield per plant of transgenic rice was increased by 45.1-67.6% and 7.8-16%, and the yield of the plot was increased by 41.3-68.3% and 11.9-27.4%, respectively, compared with the wild type. At the same time, the aboveground straw was reduced by 12.1-24.7% and 17.5-25.8% compared with the wild type, which eventually led to a significant increase in the harvest index (HI) of the transgenic rice compared with the wild type, and the increase of HI in Beijing could reach 10.3-55.7%. At the same time, the rice quality of transgenic rice was also improved to varying degrees compared with the wild type (Table 10). The brown rice rate, milled rice rate, and whole milled rice rate were all significantly higher than those of the wild type, and the chalkiness and chalky grain rate were reduced, which improved the appearance quality. , while the amylose and protein content had no significant effect. The yield per plant and plot of OsDREB1C knockout rice were significantly lower than those of the wild type, and the above-ground biomass was increased, which directly led to a 22.4-37.7% reduction in the harvest index compared with the wild type. The results indicated that OsDREB1C gene transfer into rice can greatly improve the yield, rice quality and harvest index of transgenic rice. OsDREB1C and its encoded gene can regulate the grain yield, rice quality and harvest index of rice.

The results of each index are shown in Table 9 and Table 10.

Example 2. Functional detection of OsDREB1C in wheat and Arabidopsis

1. Construction of recombinant vector

Construction of wheat overexpression vector: PCR was used to amplify the CDS full-length of OsDREB1C gene from rice Nipponbare cDNA, and the obtained PCR recovery product was combined with pWMB110 vector (Huiyun Liu, Ke Wang, Zimiao Jia, Qiang Gong,Zhishan Lin,Lipu Du,Xinwu Pei,Xingguo Ye,Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system,Journal of Experimental Botany,Volume 71,Issue 4,7 February 2020, Pages 1337–1349, https://doi.org/10.1093/jxb/erz529) The backbone of the vector obtained by double digestion with BamHI and SacI was linked, and the resulting recombinant vector with the correct sequence was denoted as pWMB110-OsDREB1C. pWMB110-OsDREB1C is a recombinant vector obtained by replacing the DNA fragment between the BamHI and SacI recognition sites of the pWMB110 vector with the OsDREB1C coding gene shown in SEQ ID NO: 2 in the sequence listing. pWMB110-OsDREB1C can express the OsDREB1C gene under the drive of the UBI promoter. The encoded protein (ie, the OsDREB1C protein shown in SEQ ID NO: 1).

The primer sequences used are as follows:

Fielder-OsDREB1C-OE-F: 5′-CAGGTCGACTCTAGA GGATCC ATGGAGTACTACGAGCAGGAG-3′ (sequence 12 in the sequence listing);

Fielder-OsDREB1C-OE-R: 5'-CGATCGGGGAAATTC GAGCTC TCAGTAGCTCCAGAGTGTGAC-3' (sequence 13 in the Sequence Listing).

Construction of Arabidopsis overexpression vector: The CDS of OsDREB1C gene (without stop codon) was amplified from rice Nipponbare cDNA by PCR, and the PCR recovery product obtained was connected to Gateway system entry vector pENTR (ThermoFisher, K240020SP) , the obtained recombinant vector with the correct sequence is recorded as pENTR-OsDREB1C, and pENTR-OsDREB1C and pGWB5 vector (Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, Toyooka K, Matsuoka K, Jinbo T, Kimura T. Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng. 2007Jul;104(1):34-41.doi:10.1263/jbb.104.34.) for LR In the reaction, the CDS sequence of OsDREB1C was transferred into the final vector pGWB5, and the resulting recombinant vector with correct sequence was denoted as pGWB5-OsDREB1C. pGWB5-OsDREB1C can express the protein encoded by the OsDREB1C gene (ie, the OsDREB1C protein shown in sequence 1) under the drive of the CaMV 35S promoter.

The primer sequences used are as follows:

OsDREB1C-CDS-F: 5'-CACCATGGAGTACTACGAGCAGGAG-3' (sequence 14 in the sequence listing);

OsDREB1C-CDS-R: 5'-GTAGCTCCAGGTGTGACGTC-3' (sequence 15 in the sequence listing).

2. Construction of transgenic plants

Embryogenic callus was obtained by inducing young embryos of wheat cultivar Fielder after disinfection. The pWMB110-OsDREB1C obtained in step 1 was introduced into Agrobacterium C58C1, and the callus was infected and co-cultured by Agrobacterium-mediated wheat genetic transformation. , using resistance screening to obtain transgenic plants, the screened OsDREB1C transgenic wheat material. Due to the high similarity between OsDREB1C and its own homologous genes in wheat, the expression level of OsDREB1C gene in rice could not be detected by qRT-PCR method. Therefore, using the wheat wild-type Fielder as a control, the PCR method was used to detect whether the Bar gene of the vector existed in the cDNA of transgenic wheat. The primers used were: 5'-CAGGAACCGCAGGAGTGGA-3' (sequence 16 in the sequence table), 5'-CCAGAAACCCACGTCATGCC-3' (Sequence 17 in the Sequence Listing). The results of electrophoresis showed that the Bar gene was present in all three lines (TaOE-5, TaOE-8 and TaOE-9) of OsDREB1C transgenic wheat, but not in the wild type, indicating that these three lines were all transgenic Transgenic material of the pWMB110-OsDREB1C vector (A in Figure 5).

The pGWB5-OsDREB1C obtained in step 1 was introduced into Agrobacterium GV3101, and transformed into Arabidopsis wild-type Col-0 by the Agrobacterium-mediated flower soaking method. For OsDREB1C transgenic Arabidopsis material. Using Arabidopsis wild-type Col-0 as a control, the relative expression level of OsDREB1C gene at RNA level in OsDREB1C transgenic Arabidopsis was detected by qRT-PCR method. The primers used were: 5′-CATGATGATGCAGTACCAGGA-3′ (Sequence Listing Middle sequence 18), 5'-GATCATCAGTAGCTCCAGAGTG-3' (sequence 19 in the sequence table); the internal reference gene is Arabidopsis Actin, and the internal reference gene primer is: 5'-GCACCACCTGAAAGGAAGTACA-3' (sequence 20 in the sequence table), 5' - CGATTCCTGGACCTGCCTCATC-3' (sequence 21 in the Sequence Listing). The results showed that the relative expression levels of OsDREB1C gene in three lines (AtOE-10, AtOE-11 and AtOE-12) of OsDREB1C transgenic Arabidopsis were significantly higher than those of wild type (Col-0). All were Arabidopsis materials overexpressing OsDREB1 (B in Figure 5).

3. Phenotypic identification of OsDREB1C transgenic wheat

To detect the phenotype of greenhouse grown potted wheat, the tested materials: wild-type wheat (Fielder), overexpressing OsDREB1C wheat (TaOE-5, TaOE-8 and TaOE-9). The greenhouse temperature was controlled at 22-24°C, and the sunshine length was 12 hours light/12 hours dark.

The heading dates of wild-type and transgenic wheat were counted, and the photosynthesis rate of the flag leaves of the tested wheat at the heading stage was measured with a LICOR-6400XT portable photosynthesis instrument (LI-COR, USA), and the light intensity was set to 1000 μmol m ^-2 s ^{- 1} . After the wheat grains were mature, the number of grains per ear, 1000-grain weight and grain yield per plant were determined.

The results showed (Table 11, Figure 6) that the transgenic wheat (TaOE-5, TaOE-8 and TaOE-9) headed earlier than the wild-type Fielder, and the photosynthetic rate was higher than that of the wild-type. Among the yield traits, the number of grains per spike was also higher than that of the wild type, and the 1000-grain weight was significantly higher than that of the wild type, which ultimately led to an increase in yield per plant, indicating that OsDREB1C also had the functions of early heading, photosynthetic capacity and yield enhancement in wheat. Heading period is the number of days from sowing to heading.

Table 11. Heading date, photosynthetic rate and yield traits of wild-type and transgenic wheat

4. Phenotypic identification of OsDREB1C transgenic Arabidopsis

To detect the phenotype of Arabidopsis grown in greenhouse, the materials to be tested: wild-type Arabidopsis (Col-0), overexpressing OsDREB1C Arabidopsis (AtOE-10, AtOE-11 and AtOE-12). Cultures were cultured under short-day (8 hours light/16 hours dark) for 2 weeks and then transferred to long day (16 hours light/8 hours dark) cultures.

The bolting time, the number of rosette leaves and the above-ground biomass (including fresh and dry weights) of wild-type and transgenic Arabidopsis were counted. Bolting time is the number of days from sowing to stem elongation and flowering.

The results showed (Table 12, Figure 7) that the transgenic Arabidopsis (AtOE-10, AtOE-11 and AtOE-12) bolted earlier (1-3.9 days) than the wild type Col-0, and had more rosette leaves than the wild type. 5-7 pieces, and the fresh weight and dry weight of the shoots were higher than those of the wild type, indicating that overexpression of OsDREB1C can also increase the biomass of the dicotyledonous plant Arabidopsis thaliana, and make Arabidopsis thaliana bolt earlier.

Table 12. Phenotypic indicators of wild-type and transgenic Arabidopsis

Arabidopsis	Bolting time (days)	Number of rosette leaves	Fresh weight (g)
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. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has been given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.

Industrial application

Experiments have proved that the OsDREB1C and related biological materials of the present invention can improve the photosynthetic efficiency of plants, can promote nitrogen absorption and transport, increase nitrogen content in plants and grains, can promote early heading, and can also increase yield. The invention starts from the synergistic improvement of crop photosynthetic efficiency, nitrogen utilization efficiency and heading stage, realizes the synergistic improvement of nitrogen utilization efficiency while realizing a substantial increase in crop yield, and also provides a solution to the contradiction between high crop yield and early maturity. Therefore, the present invention can further greatly improve the crop yield potential and nitrogen fertilizer utilization efficiency, and realize "high yield and high efficiency"; The problems of early maturity and high yield and the "superparent late maturity" of inter-subspecies hybrid rice have important application potential.

Claims (17)

1. Any of the following uses of a protein or a substance that modulates the activity or content of said protein:

   D1) regulating plant nitrogen absorption or transport;

   D2) prepare a product that regulates nitrogen absorption or transport of plants;

   D3) regulating the nitrogen content of plants;

   D4) preparing a product for regulating and controlling plant nitrogen content;

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

   D6) prepare a product that regulates plant nitrogen absorption or transport and flowering time;

   D7) regulating plant nitrogen content and flowering time;

   D8) prepare a product that regulates plant nitrogen content and flowering time;

   D9) regulating plant nitrogen absorption or transport and yield;

   D10) preparation and regulation of plant nitrogen absorption or transport and yield products;

   D11) Regulate nitrogen content and yield of plants;

   D12) preparation and regulation of plant nitrogen content and yield products;

   D13) regulating plant nitrogen absorption or transport, flowering time and yield;

   D14) preparation and regulation of plant nitrogen absorption or transport, flowering time and yield products;

   D15) regulating plant nitrogen content, flowering time and yield;

   D16) preparation and regulation of plant nitrogen content, flowering time and yield products;

   D17) Plant breeding;

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

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

   A2) The amino acid sequence shown in SEQ ID NO: 1 in the sequence listing has undergone the substitution and/or deletion and/or addition of one or several amino acid residues and has the same function;

   A3) is derived from rice, corn, sorghum, soybean, rape, Arabidopsis, millet, goat grass, Brachypodium or wheat and has 64% or more identity to Sequence 1 and is identical to the protein described in A1) functional protein;

   A4) A fusion protein obtained by linking a tag to the N-terminus or/and C-terminus of A1) or A2) or A3).
2. Application according to claim 1, is characterized in that: described plant is M1) or M2) or M3):

   M1) monocots or dicots;

   M2) grasses, crucifers or legumes;

   M3) Rice, wheat, corn, Arabidopsis, rape or soybean.
3. The application according to claim 1 or 2, characterized in that: the transporter is embodied in the transport from the root to the shoot, or the transport to the grain;

   The nitrogen content is the nitrogen content in plants or organs;

   The flowering time is reflected in the heading stage;

   The yield is expressed in grain yield, shoot yield and/or harvest index.
4. Any of the following uses of the biomaterial associated with the protein of claim 1:

   D1) regulating plant nitrogen absorption or transport;

   D2) prepare a product that regulates nitrogen absorption or transport of plants;

   D3) regulating the nitrogen content of plants;

   D4) preparing a product for regulating and controlling plant nitrogen content;

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

   D6) prepare a product that regulates plant nitrogen absorption or transport and flowering time;

   D7) regulating plant nitrogen content and flowering time;

   D8) prepare a product that regulates plant nitrogen content and flowering time;

   D9) regulating plant nitrogen absorption or transport and yield;

   D10) preparation and regulation of plant nitrogen absorption or transport and yield products;

   D11) Regulate nitrogen content and yield of plants;

   D12) preparation and regulation of plant nitrogen content and yield products;

   D13) regulating plant nitrogen absorption or transport, flowering time and yield;

   D14) preparation and regulation of plant nitrogen absorption or transport, flowering time and yield products;

   D15) regulating plant nitrogen content, flowering time and yield;

   D16) preparation and regulation of plant nitrogen content, flowering time and yield products;

   D17) Plant breeding;

   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 containing the nucleic acid molecule of B1);

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

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

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

   B6) a transgenic plant tissue containing the nucleic acid molecule of B1), or a transgenic plant tissue containing the expression cassette of B2);

   B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

   B8) reducing the protein expression level of claim 1 or knocking out the nucleic acid molecule of the protein-coding gene described in claim 1;

   B9) An expression cassette, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ comprising the nucleic acid molecule of B8).
5. The application according to claim 4, wherein: the nucleic acid molecule of B1) is the following b11) or b12) or b13) or b14) or b15):

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

   b12) the cDNA molecule or DNA molecule shown in sequence 2 in the sequence listing;

   b13) DNA molecules shown in positions 3001-4131 of sequence 3 in the sequence listing;

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

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

   B2) The expression cassette is the following b21) or b22) or b23):

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

   b22) A DNA molecule having 73% or more identity with the nucleotide sequence defined in b21) and having the same function;

   b23) DNA molecules that hybridize under stringent conditions to the nucleotide sequences defined in b21) or b22) and have the same function.
6. The application according to claim 4 or 5, characterized in that: the transporter is embodied in the transport from the root to the shoot, or the transport to the grain;

   The nitrogen content is the nitrogen content in plants or organs;

   The flowering time is reflected in the heading stage;

   The yield is expressed in grain yield, shoot yield and/or harvest index.
7. Application according to claim 4 or 5, is characterized in that: described plant is M1) or M2) or M3):

   M1) monocots or dicots;

   M2) grasses, crucifers or legumes;

   M3) Rice, wheat, corn, Arabidopsis, rape or soybean.
8. The application according to claim 4 or 5, characterized in that: the transporter is embodied in the transport from the root to the shoot, or the transport to the grain;

   The nitrogen content is the nitrogen content in plants or organs;

   The flowering time is reflected in the heading stage;

   The yield is reflected in grain yield, shoot yield and/or harvest index;

   The plant is M1) or M2) or M3):

   M1) monocots or dicots;

   M2) grasses, crucifers or legumes;

   M3) Rice, wheat, corn, Arabidopsis, rape or soybean.
9. Either of the following methods:

   X1) A method for cultivating plants with enhanced nitrogen uptake or transport capacity, comprising expressing the protein of claim 1 in the recipient plant, or increasing the content or activity of the protein of claim 1 in the recipient plant to obtain nitrogen uptake or Target plants with enhanced transport capacity;

   X2) A method for cultivating plants with increased nitrogen content, comprising expressing the protein according to claim 1 in the recipient plant, or increasing the content or activity of the protein according to claim 1 in the recipient plant to obtain a target plant with increased nitrogen content ;

   X3) a method for cultivating plants with enhanced nitrogen uptake or transport ability and advanced flowering time, comprising expressing the protein of claim 1 in the recipient plant, or increasing the content or activity of the protein of claim 1 in the recipient plant, Obtain target plants with enhanced nitrogen absorption or transport capacity and earlier flowering time;

   X4) A method for cultivating plants with increased nitrogen content and advanced flowering time, comprising expressing the protein of claim 1 in the recipient plant, or increasing the content or activity of the protein of claim 1 in the recipient plant to obtain a nitrogen content Plants of interest with increased and earlier flowering time;

   X5) A method for cultivating plants with enhanced nitrogen absorption or transport capacity and increased yield, comprising expressing the protein of claim 1 in the recipient plant, or increasing the content or activity of the protein of claim 1 in the recipient plant, to obtain Plants of interest with enhanced nitrogen uptake or transport capacity and increased yield;

   X6) A method of cultivating plants with 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 the recipient plant, resulting in increased nitrogen content and target plants with increased yield;

   X7) A method for cultivating plants with enhanced nitrogen absorption or transport capacity, advanced flowering time and increased yield, comprising expressing the protein of claim 1 in the recipient plant, or increasing the content of the protein of claim 1 in the recipient plant or activity to obtain target plants with enhanced nitrogen absorption or transport capacity, earlier flowering time and increased yield;

   X8) A method for cultivating plants with increased nitrogen content, advanced flowering time 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 the recipient plant, A desired plant with increased nitrogen content, earlier flowering time and increased yield is obtained.
10. The method according to claim 9, characterized in that: the method X1)-X8) is implemented by introducing the gene encoding the protein of claim 1 into the recipient plant and expressing the encoding gene.
11. The method according to claim 10, wherein the encoding gene is the nucleic acid molecule described in B1) of claim 4 or 5.
12. The method according to any one of claims 9-11, wherein the plant is M1) or M2) or M3):

    M1) monocots or dicots;

    M2) grasses, crucifers or legumes;

    M3) Rice, wheat, corn, Arabidopsis, rape or soybean.
13. The method according to any one of claims 9-11, wherein the transport is embodied in the transport from the root to the shoot, or the transport to the grain;

    The nitrogen content is the nitrogen content in plants or organs;

    The flowering time is reflected in the heading stage;

    The yield can be expressed in grain yield, shoot yield and/or harvest index.
14. The method according to any one of claims 9-11, wherein the plant is M1) or M2) or M3):

    M1) monocots or dicots;

    M2) grasses, crucifers or legumes;

    M3) Rice, wheat, corn, Arabidopsis, rape or soybean;

    The transport is embodied in the transport of the roots to the shoot, or the transport into the grain;

    The nitrogen content is the nitrogen content in plants or organs;

    The flowering time is reflected in the heading stage;

    The yield can be expressed in grain yield, shoot yield and/or harvest index.
15. A product having the function of any of the following D1)-D8), containing the protein described in claim 1 or the biological material described in claim 4 or 5:

    D1) regulating plant nitrogen absorption or transport;

    D2) regulating the nitrogen content of plants;

    D3) regulating plant nitrogen absorption or transport and flowering time;

    D4) regulating plant nitrogen content and flowering time;

    D5) regulating plant nitrogen absorption or transport and yield;

    D6) Regulate nitrogen content and yield of plants;

    D7) regulating plant nitrogen absorption or transport, flowering time and yield;

    D8) Regulate plant nitrogen content, flowering time and yield.
16. product according to claim 15, is characterized in that: described plant is M1) or M2) or M3):

    M1) monocots or dicots;

    M2) grasses, crucifers or legumes;

    M3) Rice, wheat, corn, Arabidopsis, rape or soybean.
17. The product according to claim 15 or 16, characterized in that: the transporter is embodied in the transport from the root to the shoot, or the transport to the grain;

    The nitrogen content is the nitrogen content in plants or organs;

    The flowering time is reflected in the heading stage;

    The yield can be expressed in grain yield, shoot yield and/or harvest index.

Images