Application of glutamine synthetase and its gene BnaGLN1;2a in nitrogen-efficient breeding of rapeseed

Abstract

The present invention provides the use of glutamine synthetase and its gene, BnaGLN1;2a, in nitrogen-efficient rapeseed breeding. Glutamine synthetase and related biomaterials can be used in any of the following ways: improving nitrogen absorption and / or utilization efficiency in rapeseed; preparing products that improve nitrogen absorption and / or utilization efficiency in rapeseed; increasing the nitrogen content of rapeseed; preparing products that improve and regulate the nitrogen content of rapeseed; increasing the biomass and / or yield of rapeseed; and preparing products that increase the biomass and / or yield of rapeseed.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of glutamine synthetase and its gene BnaGLN1;2a in rapeseed nitrogen high-efficiency breeding. Background Art

[0002] Nitrogen, as a component of amino acids, nucleic acids, chlorophyll and other important substances in life activities, is a large-scale nutrient element necessary for crop growth and development, and plays an important role in ensuring crop yield and quality. As one of the main oil crops in my country, rapeseed requires a large supply of nitrogen fertilizer during its production process, and its utilization rate is relatively low. In addition to increasing agricultural production costs, it also causes serious environmental pollution and waste of resources. Therefore, it is necessary to fundamentally solve the problem of low nitrogen utilization rate of rapeseed in my country, fully tap its genetic potential for nitrogen utilization, and cultivate nitrogen-efficient varieties, which are currently difficult problems to be solved. Rapeseed is a nitrogen-loving crop, and its nitrogen efficiency is mainly divided into nitrogen absorption efficiency and nitrogen utilization efficiency, and it has a relatively complex gene regulatory network. Summary of the Invention

[0003] The inventors' resequencing and transcriptome analysis of high- and low-nitrogen-efficiency rapeseed varieties revealed significant expression differences in the gene encoding glutamine synthetase between these two varieties, suggesting distinct regulatory differences in nitrogen efficiency between these varieties. Therefore, to address the issues presented in the prior art, the present invention provides a glutamine synthetase and its gene, BnaGLN1;2a, for use in nitrogen-efficient rapeseed breeding. This glutamine synthetase can improve nitrogen absorption and utilization efficiency, and the superior haplotype of the BnaGLN1;2a gene can be used as a molecular marker for screening nitrogen-efficient rapeseed varieties.

[0004] The technical solution of the present invention to solve the above technical problems is as follows:

[0005] In a first aspect, the present invention provides any of the following uses of glutamine synthetase:

[0006] A1) improving nitrogen absorption and / or utilization efficiency in rapeseed;

[0007] A2) preparing a product that improves nitrogen absorption and / or utilization efficiency of rapeseed;

[0008] A3) Increase the nitrogen content of rapeseed;

[0009] A4) preparing a product for increasing the nitrogen content of rapeseed;

[0010] A5) increasing the biomass and / or yield of rapeseed;

[0011] A6) preparing products that increase the biomass and / or yield of rapeseed;

[0012] The amino acid sequence of the glutamine synthetase is shown in SEQ ID NO: 1.

[0013] In a second aspect, the present invention provides any of the following uses of the biomaterial related to the above-mentioned glutamine synthetase:

[0014] A1) improving nitrogen absorption and / or utilization efficiency in rapeseed;

[0015] A2) preparing a product for improving nitrogen absorption and / or utilization efficiency in rapeseed;

[0016] A3) Increase the nitrogen content of rapeseed;

[0017] A4) preparing a product for increasing the nitrogen content of rapeseed;

[0018] A5) increasing the biomass and / or yield of rapeseed;

[0019] A6) preparing products that increase the biomass and / or yield of rapeseed;

[0020] The biological material is any one of the following:

[0021] B1) a nucleic acid molecule encoding the glutamine synthetase according to claim 1;

[0022] B2) a recombinant vector containing the nucleic acid molecule described in B1);

[0023] B3) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the recombinant vector described in B2);

[0024] B4) a transgenic rapeseed cell line containing the nucleic acid molecule described in B1);

[0025] B5) transgenic rapeseed tissue containing the nucleic acid molecule described in B1);

[0026] B6) Transgenic rapeseed organs containing the nucleic acid molecule described in B1).

[0027] According to the above scheme, the nucleotide sequence of the nucleic acid molecule in B1) is shown as SEQ ID NO: 2.

[0028] In a third aspect, the present invention provides any of the following methods:

[0029] D1) A method for cultivating rapeseed with improved nitrogen absorption and / or nitrogen utilization efficiency, comprising expressing the glutamine synthetase of claim 1 in rapeseed, or increasing the content of the glutamine synthetase of claim 1 in rapeseed, thereby improving nitrogen absorption and / or nitrogen utilization efficiency in rapeseed;

[0030] D2) A method for cultivating rapeseed with increased nitrogen content, comprising expressing the glutamine synthetase of claim 1 in rapeseed, or increasing the content of the glutamine synthetase of claim 1 in rapeseed, thereby increasing the nitrogen content in the rapeseed;

[0031] D3) A method for cultivating rapeseed with increased biomass and / or yield, comprising expressing the glutamine synthetase according to claim 1 in the rapeseed, or increasing the content of the glutamine synthetase according to claim 1 in the rapeseed, thereby increasing the nitrogen content in the rapeseed.

[0032] According to the above scheme, the methods D1) to D3) are achieved by introducing the above-mentioned glutamine synthetase encoding gene into rapeseed and expressing the encoding gene.

[0033] According to the above scheme, the coding gene is a nucleic acid molecule encoding the above glutamine synthetase.

[0034] According to the above scheme, the methods described in D1)-D3) are specifically: inserting the nucleic acid molecule shown in SEQ ID NO: 2 into an overexpression vector to construct a recombinant expression vector, and then using a transformation method to transform the target fragment into rapeseed to cultivate transgenic plants.

[0035] According to the above scheme, the transformation method is Agrobacterium transformation, and the recombinant expression vector is transferred into Agrobacterium and transformed into rapeseed.

[0036] According to the above scheme, the Agrobacterium is Agrobacterium GV3101.

[0037] According to the above scheme, the bacterial solution of Agrobacterium GV3101 containing the recombinant expression vector was used to infect the hypocotyls of rapeseed.

[0038] According to the above scheme, the method for constructing the recombinant expression vector is as follows: the amplified target fragment is fused with the PMDC83-GFP vector by seamless cloning technology to form 35S-BnaGLN1; 2a-GFP (the restriction enzyme cutting site is KpnⅠ).

[0039] In a fourth aspect, the present invention provides the use of the above-mentioned glutamine synthetase gene in rapeseed nitrogen high-efficiency breeding.

[0040] According to the above scheme, the glutamine synthetase gene has four haplotypes, including Hap1, Hap2, Hap3 and Hap4, whose nucleotide sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The haplotypes all include 8 significant SNPs. Based on the rapeseed reference genome version 4.1, the 8 SNP sites are located at sites 6637711, 6637782, 6638039, 6638041, 6638119, 6639122, 6639192 and 6639500 on chromosome 2, respectively. Among them, Hap4 with the sequence of SEQ ID NO: 6 is its excellent haplotype.

[0041] In a fifth aspect, the present invention provides a method for screening nitrogen-efficient rapeseed varieties, which detects the expression level of the glutamine synthetase gene BnaGLN1;2a;

[0042] Alternatively, the glutamine synthetase gene BnaGLN1;2a is amplified and its haplotype type is determined. If it is the excellent haplotype shown in SEQ ID NO:6, the rapeseed is a nitrogen-efficient variety.

[0043] The beneficial effects of the present invention are:

[0044] The application of glutamine synthetase and its gene BnaGLN1;2a in the breeding of rapeseed for nitrogen-efficient growth. Overexpression of this glutamine synthetase significantly increased plant biomass compared to the wild type, and promoted nitrate absorption and reduction, as well as ammonium assimilation.

[0045] The BnaGLN1;2a gene sequence exhibits significant variation in natural rapeseed populations, with chlorate-sensitive genotypes expressing significantly higher levels than insensitive genotypes. Under high-nitrogen conditions, chlorate-sensitive genotypes with high BnaGLN1;2a expression levels exhibit significant advantages over insensitive genotypes in yield, nitrogen efficiency, and agronomic traits. Therefore, the superior haplotypes of the BnaGLN1;2a gene provided in this application can be used as molecular markers for the screening and breeding of nitrogen-efficient rapeseed varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1The expression pattern analysis of the gene BnaGLN1;2a provided in Example 1 of the present invention; wherein A is the relative mRNA expression result of the gene BnaGLN1;2a in rapeseed after 7 days of normal nitrate and low nitrate concentration treatment; B is the relative mRNA expression result of the gene BnaGLN1;2a in rapeseed at different time points after normal nitrate and low nitrate concentration treatment; C is the subcellular localization analysis of the rapeseed gene BnaGLN1;2a in tobacco epidermal cells and Arabidopsis protoplasts; D is the tissue localization analysis of the rapeseed gene BnaGLN1;2a heterologously expressed in Arabidopsis;

[0047] Figure 2 Analysis of the growth phenotype of the gene BnaGLN1;2a provided in an embodiment of the present invention after overexpression in rapeseed and treatment with different nitrate concentrations; wherein A and B are identification of the gene BnaGLN1;2a overexpression material, C is the growth phenotype of the rapeseed wild type and the gene BnaGLN1;2a overexpression material under normal and low nitrate conditions; D is the aboveground biomass of the rapeseed wild type and the gene BnaGLN1;2a overexpression material under normal and low nitrate conditions; E is the root biomass of the rapeseed wild type and the gene BnaGLN1;2a overexpression material under normal and low nitrate conditions;

[0048] Figure 3 Analysis of nitrogen-related indicators of the gene BnaGLN1;2a provided in an embodiment of the present invention after overexpression in rapeseed and treatment with different nitrate concentrations; wherein, A and D are the aboveground nitrogen concentration and content of the rapeseed wild type and gene BnaGLN1;2a overexpressing materials under normal and low nitrate conditions; B and E are the root nitrogen concentration and content of the rapeseed wild type and gene BnaGLN1;2a overexpressing materials under normal and low nitrate conditions; C is the nitrogen transport coefficient analysis of the rapeseed wild type and gene BnaGLN1;2a overexpressing materials under normal and low nitrate conditions; F is the nitrogen absorption efficiency analysis of the rapeseed wild type and gene BnaGLN1;2a overexpressing materials under normal and low nitrate conditions;

[0049] Figure 4 This is an analysis of nitrogen metabolism-related indicators in rapeseed overexpressing the gene BnaGLN1;2a provided in an embodiment of the present invention and treated with different nitrate concentrations. Figures A and D show the glutamine synthetase activity in the shoots and roots of wild-type rapeseed and BnaGLN1;2a overexpressing materials under normal and low nitrate conditions; Figures B and E show the free ammonium concentration in the shoots and roots of wild-type rapeseed and BnaGLN1;2a overexpressing materials under normal and low nitrate conditions; and Figures C and F show the glutamate concentration in the shoots and roots of wild-type rapeseed and BnaGLN1;2a overexpressing materials under normal and low nitrate conditions.

[0050] Figure 5 The present invention provides an embodiment of the gene BnaGLN1; 2a overexpressed in rapeseed and treated with different nitrate concentrations. A and B are analyses of free nitrate concentrations in the aboveground and root systems of the rapeseed wild type and the gene BnaGLN1; 2a overexpressing materials under normal and low nitrate conditions; C and D are analyses of nitrate reductase activity in the aboveground and root systems of the rapeseed wild type and the gene BnaGLN1; 2a overexpressing materials under normal and low nitrate conditions; E is a determination of nitrate absorption rates of the rapeseed wild type and the gene BnaGLN1; 2a overexpressing materials under normal and low nitrate conditions; F is an analysis of the expression levels of nitrate transporter and nitrate reductase-related coding genes in the aboveground and root systems of the rapeseed wild type and the gene BnaGLN1; 2a overexpressing materials under normal and low nitrate conditions;

[0051] Figure 6 Root proteome analysis of different extreme chlorate-sensitive varieties provided in the embodiments of the present invention; wherein A is the PCA analysis of the proteome of extreme chlorate-sensitive varieties under normal and low nitrate conditions; B is the statistics of differential proteins; C is the KEGG enrichment analysis of up-regulated differential proteins; D is the expression abundance of nitrate transport and utilization-related proteins among the differential proteins;

[0052] Figure 7 The results of genetic variation analysis of the gene BnaGLN1;2a in 505 natural rapeseed varieties provided by the present invention are shown. Among them, AD represents the candidate gene association analysis of the gene BnaGLN1;2a in 505 resequenced natural rapeseed varieties; E represents the haplotype analysis of the gene BnaGLN1;2a; E represents the expression level analysis of the gene BnaGLN1;2a in the aerial parts of extremely chlorate-sensitive varieties; F represents the expression level analysis of the gene BnaGLN1;2a in the roots of extremely chlorate-sensitive varieties;

[0053] Figure 8 The field performance of the chlorate-sensitive varieties provided in Example 5 of the present invention at different nitrogen concentrations under 210 kg / ha N conditions is shown;

[0054] Figure 9 The field performance of the chlorate-sensitive varieties provided in Example 5 of the present invention under different nitrogen concentrations at 70 kg / ha N is shown. DETAILED DESCRIPTION

[0055] The principles and features of the present invention are described below with reference to the accompanying drawings and specific embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0056] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0057] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. Reagents not specifically described in detail herein are conventional reagents and are commercially available; methods not specifically described in detail are conventional experimental methods and are known in the art.

[0058] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0059] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0060] In this application, the term "gene" refers to a nucleic acid fragment that expresses a specific protein or functional RNA molecule, which may contain regulatory sequences preceding the coding sequence (5' non-coding region) and following the coding sequence (3' non-coding region).

[0061] In this application, the term "expression vector" refers to a vector that adds expression elements (such as promoter, RBS, terminator, etc.) to the basic skeleton (empty skeleton) of a cloning vector to enable the expression of the target gene.

[0062] In this application, the term "infusion seamless cloning" refers to an enzyme-free ligation technique used in expression vector construction. It stems primarily from the discovery of infusion enzymes, which can recognize any 16 bases between the 5' and 3' ends of linearized DNA fragments, forming sticky ends. After target plasmids have been linearized by enzyme digestion or PCR, they can also be recognized by the infusion enzyme. Construction of the vector is completed by simply annealing the sticky ends formed by the vector and gene.

[0063] In this application, the term "nucleic acid molecule" refers to a polymer of RNA or DNA, which is single-stranded or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. A nucleic acid molecule in the form of a DNA polymer can be composed of one or more segments of cDNA, genomic DNA or synthetic DNA.

[0064] The embodiment provides a glutamine synthetase from rapeseed, the amino acid sequence of which is shown in SEQ ID NO: 1, the CDS sequence of which is shown in SEQ ID NO: 2, the encoding gene of which is BnaGLN1; 2a, located on chromosome A02 of rapeseed, and the ID number in NCBI is XP_009127474.

[0065] SEQ ID NO: 1

[0066] MSLLTDLVNLDLSDNTEKIIAEYIWVGGSGMDMRSKARTLPGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDTYTPAGEPIPTNKRHAAAQIFSNPDVVAEVPWYGIEQEYTLLQKDVNWPVGWPIGGFPGPQGPYYCSVGADKSFGRDIVDAHYKA CLYAGINISGINGEVMPGQWEFQVGPSVGISAADEVWIARFILERITEIAGVVVSFDPKPIPGDWNGAGAHTNYSTKSMREEGGYEIIKKAIDKLGLRHKEHISAYGEGNERRLTGHHETADINTFKWGVANRGASIRVGRDTEKEGKGYFEDRRPASNMDPYTVTSMIAETTLLWNP

[0067] The gene names of the glutamine synthetase and its encoding gene BnaGLN1;2a provided by the present invention are both derived from the rapeseed database (https: / / yanglab.hzau.edu.cn / ), specifically from the wild-type rapeseed Westar10.

[0068] In order to determine the effects of BnaGLN1;2a and its encoded glutamine synthetase on rapeseed nitrogen efficiency and its role in genetic breeding, the inventors overexpressed BnaGLN1;2a. Compared with the wild type, the absorption, reduction and ammonium assimilation of nitrate in the overexpressing plants were improved, and the nitrogen content, biomass and rapeseed yield were increased.

[0069] Therefore, the inventors provide any of the following uses of the above-mentioned glutamine synthetase or a substance that regulates the activity or content of the glutamine synthetase:

[0070] A1) improving nitrogen absorption and / or utilization efficiency in rapeseed;

[0071] A2) preparing a product that improves nitrogen absorption and / or utilization efficiency of rapeseed;

[0072] A3) Increase the nitrogen content of rapeseed;

[0073] A4) preparing a product for increasing the nitrogen content of rapeseed;

[0074] A5) increasing the biomass and / or yield of rapeseed;

[0075] A6) preparing products that increase the biomass and / or yield of rapeseed;

[0076] The above-mentioned glutamine synthetase can be a protein with an amino acid sequence as shown in SEQ ID NO: 1, or a protein with the same function as the sequence shown in SEQ ID NO: 1 after one or more amino acid residues are substituted and / or deleted and / or added, or a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the above-mentioned protein.

[0077] The above-mentioned proteins with the same function after substitution and / or deletion and / or addition of one or more amino acid residues can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0078] The above-mentioned protein having the same function after substitution and / or deletion and / or addition of one or more amino acid residues can be obtained by deleting the codons for one or more amino acid residues in the DNA sequence shown in SEQ ID NO: 2, and / or performing missense mutations of one or more base pairs, and / or ligating a tag sequence to its 5' end and / or 3' end. The DNA sequence shown in SEQ ID NO: 2 encodes the protein shown in SEQ ID NO: 1.

[0079] The present invention also provides any of the following applications of the biomaterial related to the above-mentioned glutamine synthetase:

[0080] A1) improving nitrogen absorption and / or utilization efficiency in rapeseed;

[0081] A2) preparing a product for improving nitrogen absorption and / or utilization efficiency in rapeseed;

[0082] A3) Increase the nitrogen content of rapeseed;

[0083] A4) preparing a product for increasing the nitrogen content of rapeseed;

[0084] A5) increasing the biomass and / or yield of rapeseed;

[0085] A6) preparing products that increase the biomass and / or yield of rapeseed;

[0086] The biological material is any one of the following:

[0087] B1) a nucleic acid molecule encoding the glutamine synthetase according to claim 1;

[0088] B2) a recombinant vector containing the nucleic acid molecule described in B1);

[0089] B3) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the recombinant vector described in B2);

[0090] B4) a transgenic rapeseed cell line containing the nucleic acid molecule described in B1);

[0091] B5) transgenic rapeseed tissue containing the nucleic acid molecule described in B1);

[0092] B6) Transgenic rapeseed organs containing the nucleic acid molecule described in B1).

[0093] According to the above scheme, the nucleic acid molecule in B1) is a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 2, or a DNA molecule having a nucleotide sequence of more than 85% identity with the nucleotide sequence shown in SEQ ID NO: 2 and encoding glutamine synthetase, or a DNA molecule that hybridizes with the above nucleotide sequence under stringent conditions and encodes glutamine synthetase.

[0094] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA, or may be RNA, such as mRNA or hnRNA.

[0095] Those skilled in the art can easily mutate the nucleotide sequence encoding the glutamine synthetase of the present invention using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotides that have 85% or higher identity with the nucleotide sequence of the glutamine synthetase isolated from the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention, as long as they encode glutamine synthetase and have the function of glutamine synthetase.

[0096] As used herein, the term "identity" refers to sequence similarity to a naturally occurring nucleic acid sequence. Identity includes nucleotide sequences that are 85% or greater, or 90% or greater, or 95% or greater identical to the glutamine synthetase encoding nucleotide sequence of the present invention. Identity can be assessed visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences.

[0097] Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue-, organ- and development-specific promoters, and inducible promoters.

[0098] The promoter used in the examples of the present invention is the constitutive promoter 35S of cauliflower mosaic virus.

[0099] An existing expression vector can be used to construct a recombinant vector containing the BnaGLN1;2a gene expression cassette. The vector can be a plasmid, cosmid, phage or viral vector.

[0100] The vector used in the embodiment of the present invention is PMDC83-GFP vector, and the constructed recombinant expression vector is PMDC83-BnaGLN1;2a-GFP.

[0101] 35S-BnaGLN1;2a-GFP is a recombinant expression vector obtained by inserting the gene encoding glutamine synthetase shown in SEQ ID NO: 2 into the KpnⅠ restriction site of PMDC83-GFP vector.

[0102] PMDC83-BnaGLN1;2a-GFP can overexpress glutamine synthetase encoded by the BnaGLN1;2a gene under the drive of the 35S promoter.

[0103] The present invention also provides any of the following methods:

[0104] D1) A method for cultivating rapeseed with improved nitrogen absorption and / or nitrogen utilization efficiency, comprising expressing the glutamine synthetase of claim 1 in rapeseed, or increasing the content of the glutamine synthetase of claim 1 in rapeseed, thereby improving nitrogen absorption and / or nitrogen utilization efficiency in rapeseed;

[0105] D2) A method for cultivating rapeseed with increased nitrogen content, comprising expressing the glutamine synthetase of claim 1 in rapeseed, or increasing the content of the glutamine synthetase of claim 1 in rapeseed, thereby increasing the nitrogen content in the rapeseed;

[0106] D3) A method for cultivating rapeseed with increased biomass and / or yield, comprising expressing the glutamine synthetase according to claim 1 in the rapeseed, or increasing the content of the glutamine synthetase according to claim 1 in the rapeseed, thereby increasing the nitrogen content in the rapeseed.

[0107] In some specific embodiments, the methods D1) to D3) are achieved by introducing the above-mentioned glutamine synthetase encoding gene into rapeseed and expressing the encoding gene.

[0108] In some specific embodiments, the encoding gene is a nucleic acid molecule encoding the above-mentioned glutamine synthetase.

[0109] In some specific embodiments, the gene encoding the glutamine synthetase can be modified as follows before being introduced into rapeseed to achieve better expression effects: for example, codon optimization can be performed, or the gene sequence adjacent to the start methionine can be modified to enable efficient translation initiation, and an enhancer sequence can be introduced.

[0110] The gene encoding the glutamine synthetase can be introduced into rapeseed using a recombinant expression vector containing the gene encoding the glutamine synthetase. Specifically, the recombinant expression vector can be the 35S-BnaGLN1;2a-GFP.

[0111] In some specific embodiments, the methods D1)-D3) are specifically: inserting the nucleic acid molecule shown in SEQ ID NO: 2 into an overexpression vector to construct a recombinant expression vector, and then using a transformation method to transform the target fragment into rapeseed to cultivate transgenic plants.

[0112] In some specific embodiments, the transformation method is Agrobacterium transformation, in which the recombinant expression vector is transferred into Agrobacterium and then transformed into rapeseed.

[0113] In some specific embodiments, the Agrobacterium is Agrobacterium GV3101.

[0114] In some specific embodiments, the bacterial solution of Agrobacterium GV3101 containing the recombinant expression vector is used to infect the hypocotyls of rapeseed.

[0115] In order to explore the nitrogen utilization efficiency of rapeseed, proteomic analysis was performed on the roots of chlorate-sensitive and chlorate-insensitive genotypes. Chlorate is an analog of nitrate and shares the same absorption and utilization pathway with nitrate. Therefore, varieties that absorb more chlorate have higher absorption and utilization than nitrate, that is, nitrate-sensitive varieties.

[0116] The BnaGLN1;2a sequence shows significant variation in natural rapeseed populations. Proteomic analysis of the roots of chlorate-sensitive and chlorate-insensitive genotypes showed that the expression level of BnaGLN1;2a in chlorate-sensitive genotypes was significantly higher than that in insensitive genotypes. Under high nitrogen conditions, chlorate-sensitive genotypes with high BnaGLN1;2a expression levels had obvious advantages over insensitive genotypes in yield, nitrogen efficiency and agronomic traits. Therefore, the superior haplotype of BnaGLN1;2a can be used as a molecular auxiliary marker for the breeding of nitrogen-efficient rapeseed varieties.

[0117] The above-mentioned glutamine synthetase gene has four haplotypes, whose nucleotide sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The haplotypes all include 8 significant SNPs. Based on the rapeseed reference genome version 4.1, the 8 SNP sites are located at sites 6637711, 6637782, 6638039, 6638041, 6638119, 6639122, 6639192 and 6639500 on chromosome 2, respectively. Among them, SEQ ID NO: 6 is its excellent haplotype.

[0118] The excellent haplotype shown in SEQ ID NO: 6 can be used to screen nitrogen-efficient rapeseed varieties. The glutamine synthetase gene BnaGLN1;2a is amplified to determine its haplotype type. If it is the excellent haplotype shown in SEQ ID NO: 6, it is a nitrogen-efficient rapeseed variety.

[0119] Of course, detecting the expression level of the glutamine synthetase gene BnaGLN1;2a can also be used to screen nitrogen-efficient rapeseed varieties.

[0120] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0121] The molecular biology experiments in the following examples, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, and culture medium preparation, were primarily performed with reference to Molecular Cloning: A Laboratory Manual (3rd edition), edited by J. Sambrook et al., published by Science Press. Specific experimental conditions can be determined by simple experiments, if necessary. PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions, and can be adjusted by simple experiments, if necessary.

[0122] Plants and vectors: wild-type rapeseed Westar10, vector PMDC83-GFP;

[0123] The formula of Afudonin nutrient solution is shown in Table 1 below.

[0124] Table 1

[0125]

[0126]

[0127] Example 1 Analysis of the expression pattern of gene BnaGLN1;2a

[0128] (1) Rapeseed culture conditions at different nitrate concentrations

[0129] Select the plump and uniform wild-type rapeseed "Westar10" and soak it in tap water for 2 hours to remove the water. Then transfer it to 4℃ for purification for 4 hours and place it in a 30℃ incubator to break the shell. After the uniformly germinated seeds are grown in 0.5M CaCl2 pure water for 5 days, the uniformly growing seedlings are selected and incubated in 6mM NO3 - The seedlings were grown in the Afudao nutrient solution for 6 days, and then 0mM, 0.1mM, 0.3mM, 0.5mM, 1mM, 3mM and 6mM NO3 - The treatment lasted for 7 days, and the nutrient solution was replaced every three days. Samples were taken from both the aerial part and the root system and stored in a -80°C refrigerator for RNA extraction and expression level determination. The results are as follows: Figure 1 As shown in A.

[0130] (2) Culture conditions of rapeseed at different nitrate concentrations and time points

[0131] Select the plump and uniform wild-type rapeseed "Westar10" and soak it in tap water for 2 hours to remove the water. Then transfer it to 4℃ for purification for 4 hours and place it in a 30℃ incubator to break the shell. After the uniformly germinated seeds are grown in 0.5M CaCl2 pure water for 5 days, the uniformly growing seedlings are selected and incubated in 6mM NO3 - The seedlings were grown in the Afudao nutrient solution for 6 days, and then 6mM and 0mM NO3 - Treatment, in which 0mM NO3 - Resupply 6mM NO3 - , the nutrient solution was replaced every three days, and the nutrient solution was replaced at 0h, 6h, 12h, 18h, 24h, 3d, 5d, 7d, 9d, R1d (0mM NO3 - Resupply 6mM NO3 - 1d) and R3d(0mM NO3 - Resupply 6mM NO3 - At 3d), samples were collected from the aerial part and the root system and stored in a -80℃ refrigerator until RNA was extracted for expression level determination. The results are as follows: Figure 1 As shown in B.

[0132] (3) Subcellular localization analysis of the gene BnaGLN1;2a

[0133] The CDS sequence of the gene BnaGLN1;2a was searched in the rapeseed database (https: / / yanglab.hzau.edu.cn / ) and primers were designed to amplify the 1071 bp target fragment. The primers were F1 and R1, respectively. The target fragment was fused with the PMDC83-GFP vector to form 35S::BnaGLN1;2a-GFP (restriction site was KpnⅠ) by infusion seamless cloning technology. For the tobacco epidermal cell system, the 35S::BnaGLN1;2a-GFP plasmid was transferred into Agrobacterium GV3101 and then injected into tobacco leaves. After culturing for 24 to 48 hours, the tobacco leaf epidermal cells were torn off and their subcellular localization was observed under a stereomicroscope. For the Arabidopsis protoplast system, healthy Arabidopsis leaf protoplasts were extracted and the high-quality and high-concentration 35S-BnaGLN1;2a-GFP plasmid obtained by plasmid extraction technology was transferred into them through the PEG transformation method. After static culture for 10 hours, the subcellular localization of BnaA02.GLN1;2 was observed under a confocal microscope.

[0134] in,

[0135] The CDS sequence is as follows:

[0136]

[0137] F1: TCTGTAACCATGAGTCTTCTGAC (SEQ ID NO:7);

[0138] R1: AAGGATTCCAAAGAAGTGTAGTCTCTGCA (SEQ ID NO:8)

[0139] (4) Tissue localization analysis of the gene BnaGLN1;2a

[0140] The rapeseed database (https: / / yanglab.hzau.edu.cn / ) was searched for the 2000-bp upstream promoter sequence of the gene BnaGLN1;2a. Primers F2 and R2 were designed to amplify the target fragment. Using seamless infusion cloning, the target fragment was fused to the PBI121-GUS vector to create pBnaGLN1;2a::GUS (with HindIII and BamHI restriction sites) and transformed into Agrobacterium tumefaciens GV3101 for plant infection. Arabidopsis thaliana plants were infected with Agrobacterium carrying the pBnaGLN1;2a::GUS plasmid using the floral dip method. The resulting seeds were sown on Kana-resistant 1 / 2 MS medium and screened for positive seedlings until pure lines were obtained. Finally, the seeds of the pure and homologous lines were sown in 1 / 2MS culture medium and cultured for 10 days. After that, they were transferred to normal Afdonine nutrient solution for treatment for 7 days. Then, the plants were placed in GUS dye solution and kept warm at 37℃ for 6-12 hours. The GUS dye solution was discarded and decolorized with 75% alcohol until it was colorless. Then, the plants were observed and recorded under a microscope.

[0141] F2:GATTGGTTTGTAGGTGA(SEQ ID NO:9)

[0142] R2:GGTTACAGAGAAAATGAC(SEQ ID NO:10)

[0143] The expression pattern analysis results of gene BnaGLN1;2a are as follows Figure 1 As shown, Figure 1 A shows that the expression level of rapeseed gene BnaGLN1;2a in the root system is gradually induced with the increase of nitrate concentration, while the response in the aboveground part is not obvious and its expression level is significantly lower than that in the root system under certain nitrate concentration conditions; Figure 1 The results of B were consistent with the above results. The expression level of BnaGLN1;2a in the roots was significantly higher than that in the shoots and was significantly induced by nitrate. However, the shoots showed no response at different time points and under the condition of re-supply of nitrate. Figure 1C shows the subcellular localization of the gene BnaGLN1;2a in tobacco leaf epidermal cells and Arabidopsis protoplasts, and the results show that both can co-localize with a marker indicating cell localization (using PM999-Mcherry vector, mainly localized in the cytoplasm); Figure 1 Tissue localization of D showed that the gene was mainly expressed in all parts of the root except the root tip, and had a lower expression level in the leaves.

[0144] Example 2 Construction of BnaGLN1;2a overexpression lines in rapeseed and nitrate phenotype analysis

[0145] (1) Construction of gene BnaGLN1;2a overexpression vector

[0146] The CDS sequence of the gene was searched in the rapeseed database (https: / / yanglab.hzau.edu.cn / ) and primers were designed to amplify the 1078 bp target fragment. The primer sequences were the same as F1 and R1 in Example 1. The target fragment was fused to the PMDC83-GFP vector using infusion seamless cloning technology to form 35S::BnaGLN1;2a-GFP (with the Kpn I restriction enzyme site). After the vector was constructed, it was transformed into Agrobacterium for infection of rapeseed hypocotyls.

[0147] (2) Infection and transformation of BnaGLN1;2a overexpression vector in rapeseed

[0148] The rapeseed hypocotyls were infected with Agrobacterium carrying 35S::BnaGLN1;2a-GFP. The specific steps were as follows: 1) Sterilization: Select full-sized Weatar10 seeds and soak them in 75% alcohol for 1 minute; rinse the soaked seeds with sterile water and sterilize them with an appropriate amount of 84 disinfectant (diluted 3-4 times) for 15-20 minutes; after sterilization, rinse them 8-10 times with sterile water on a sterile workbench; 2) Sowing: Sow the sterilized seeds in the prepared M0 culture medium, about 30-50 seeds per dish; then dark-culture and incubate for 5-6 days until the hypocotyls reach a certain height, which should not be too long; 3) Shaking: Culture the seeds in a sterile Erlenmeyer flask or centrifuge tube in a shaker at 28°C and 200 rpm one day in advance according to the growth of the hypocotyls. For infection, the cultured strain (OD is generally around 0.4-0.6) is centrifuged at 6000 rpm for 10 minutes. The supernatant is discarded, and the suspension is resuspended in the same volume of DM as the bacterial solution. Centrifuge again under the same conditions, and the supernatant is discarded. The suspension is resuspended in DM and diluted for use (bacterial solution: DM = 1:10). 4) Explant Preparation and Infection: First, on a sterile workbench, use sterile forceps and a scalpel to cut the rapeseed hypocotyl into 0.8-1 cm pieces. The entire operation is performed in M1 liquid culture medium. The prepared bacterial solution is introduced into the M1 liquid culture medium and infected for 20 minutes. , shaking once every 5 minutes. After infection, the cells were transferred to M1 solid medium for culture in the dark at 24°C. After 2 days, they were transferred to M2 medium for culture in the light (24°C, 16 hours daytime and 8 hours nighttime). After two weeks, they were transferred to M3 medium until green shoots appeared. The medium was changed every 2 weeks during this period, and finally, they were transferred to M4 medium for rooting. 5) Subculture: The rooted seedlings were cultured at room temperature to obtain T0 generation seeds and the hygromycin tag and expression level were identified for breeding (quantitative primers were designed for expression level determination, F: ACCGTCGGTTGGTATCTCAG (SEQ ID NO: 11), R: CCTTCCTCCCTCATCGATTT (SEQ ID NO: 12)). After screening for positive seedlings, subculture was repeated until homozygous lines were obtained.

[0149] (3) Phenotypic analysis of BnaGLN1;2a overexpression lines in rapeseed in response to nitrate

[0150] 1) Determination of nitrate metabolism pathway-related indicators in BnaGLN1;2a overexpressing strains in rapeseed

[0151] Select the plump and consistent wild-type "Westar10" and gene BnaGLN1; 2a overexpression strain seeds, soak them in tap water for 2 hours to remove water, transfer them to 4℃ for purification for four hours, and then place them in a 30℃ incubator to break the shells. Grow the seeds that germinate consistently in 0.5M CaCl2 pure water for 5 days, then select the seedlings with consistent growth and incubate them in 6mM NO3 -The seedlings were grown in the Afudonin nutrient solution for 6 days, and then 6mM and 0.1mM NO3 - After 9-12 days of treatment, samples were taken from the aboveground parts and roots to measure relevant indicators. The biomass, total nitrogen concentration and content required dry samples, while the nitrate and free ammonium concentrations, nitrate reductase and glutamine synthetase activities required fresh samples.

[0152] Figure 2 This is the growth phenotype analysis of the gene BnaGLN1;2a after overexpression in rapeseed and treated with different nitrate concentrations. Figure 2 A, 2B and 2C show that the rapeseed gene BnaGLN1;2a overexpression line was induced by 6mM NO3 - The growth rate was significantly better than that of the wild type under 0.1mM NO3 - There was no significant difference between the two conditions. Figure 2 D and 2E, which are in the presence of 6 mM NO 3- Under these conditions, the shoot and root biomass increased significantly by 56.83% and 67.49% respectively compared with the wild type. These results indicate that overexpression of the BnaGLN1;2a gene in rapeseed can significantly promote plant growth under nitrate-sufficient conditions.

[0153] Figure 3 The nitrogen-related index analysis of the gene BnaGLN1; 2a provided in this example after overexpression of rapeseed and treatment with different nitrate concentrations, wherein 3A and 3D show that the rapeseed gene BnaGLN1; 2a overexpression line under 6mM NO3 - The nitrogen content in the aboveground part under the conditions of nitrate abundance was significantly higher than that in the wild type, and only the OEA02-3 strain reached a significant level of nitrogen concentration. 3B and 3E showed that the nitrogen concentration and content in the roots of the BnaGLN1;2a overexpression strain were significantly higher than those of the wild type under nitrate-sufficient conditions, and there was no difference under low nitrate treatment. 3C and 3F calculated the nitrogen transport coefficient and absorption efficiency of different strains, and the results showed that the nitrogen absorption efficiency of the BnaGLN1;2a overexpression strain and the wild type was significantly different only under the 6 mM condition.

[0154] Figure 4 The results of the analysis of nitrogen metabolism related indicators of the gene BnaGLN1;2a provided in this example after overexpression of rapeseed and treatment with different nitrate concentrations are shown in Figure 4A and 4D. - Under the conditions of 6 mM NO3, the glutamine synthetase activity in the shoots and roots was significantly higher than that in the wild type, while the free ammonium concentration in the rapeseed gene BnaGLN1;2a overexpression line was significantly reduced due to the accelerated assimilation rate. -The ammonium assimilation product glutamate was significantly increased under the conditions ( Figure 4 C and 4F), in 0.1 mM NO3 - There were no significant differences in the relevant indicators under different conditions;

[0155] Figure 5 The gene BnaGLN1; 2a provided in this example is overexpressed in rapeseed and treated with different nitrate concentrations. 5A and 5B show that the rapeseed gene BnaGLN1; 2a overexpression line can absorb and reduce nitrate in 6mM NO3 - The nitrate concentration in the aboveground part under these conditions was significantly higher than that in the wild type, but there was no significant difference in the root system. Figure 5 C and 5D are the nitrate reductase activity assays. Under these conditions, both the shoots and roots of the BnaGLN1;2a overexpression lines of rapeseed were significantly improved. Correspondingly, the BnaGLN1;2a overexpression lines were significantly improved in the presence of 6 mM NO3 - Under the conditions of 0.1mM NO3 - There were no significant differences in the relevant indicators under different conditions.

[0156] 2) Determination of nitrate uptake rate in BnaGLN1;2a overexpressing strains in rapeseed

[0157] Select the plump and consistent wild-type "Westar10" and gene BnaGLN1; 2a overexpression strain seeds, soak them in tap water for 2 hours to remove water, transfer them to 4℃ for purification for four hours, and then place them in a 30℃ incubator to break the shells. Grow the seeds that germinate consistently in 0.5M CaCl2 pure water for 5 days, then select the seedlings with consistent growth and incubate them in 6mM NO3 - The seedlings were grown in the Afudonin nutrient solution for 6 days and 0mM NO3 - The cells were starved for 3 days and washed in 0.1 mM CaSO4 for 1 min before being transferred to 6 mM and 0.1 mM CaSO4. 15 NO3 - Before sampling, the seedlings were washed again in 0.1 mM CaSO4 for 1 minute, dried, ground, and passed through an 80-100 mesh sieve before analysis by isotope ratio mass spectrometry. 15 The concentration of N.

[0158] Figure 5 E is the determination of nitrate absorption rate of rapeseed gene BnaGLN1;2a overexpression strain, which is in 6mM NO3 - The expression of β-actin in WT mice was significantly higher than that of wild type mice.

[0159] 3) Determination of expression levels of genes related to nitrate transport and reductase activity in BnaGLN1;2a overexpressing lines in rapeseed

[0160] Select the plump and consistent wild-type "Westar10" and gene BnaGLN1; 2a overexpression strain seeds, soak them in tap water for 2 hours to remove water, transfer them to 4℃ for purification for four hours, and then place them in a 30℃ incubator to break the shells. Grow the seeds that germinate consistently in 0.5M CaCl2 pure water for 5 days, then select the seedlings with consistent growth and incubate them in 6mM NO3 - After the seedlings were grown in the Afudonin nutrient solution for 6 days, 6mM and 0.1mM NO3 were added respectively. - After treatment under the same conditions for 7 days, the aboveground parts and roots were sampled and total RNA was extracted for reverse transcription into cDNA to determine the expression of related genes (BnaA09.NPF6;3, BnaC08.NPF6;3a, BnaA09.NPF6;3b, BnaA07.NR1a, BnaA07.NR1, BnaA07.NR1b, BnaC06.NR1, BnaC06.NR1a).

[0161] Figure 5 F is the expression level of related genes in the rapeseed gene BnaGLN1;2a overexpression line. It was found that at 6mM NO3 - conditions were significantly improved.

[0162] Example 3 Proteome Analysis of Roots of Rapeseed Varieties with Extreme Chlorate Sensitivity

[0163] Chlorate is an analog of nitrate and shares the same absorption and utilization pathway with nitrate. Therefore, varieties that absorb more chlorate have a higher absorption and utilization rate than nitrate, and are therefore nitrate-sensitive varieties.

[0164] Three chlorate-sensitive genotypes (CSG) and three chlorate-insensitive genotypes (CIG) were obtained in the early screening. Full and consistent seeds of extreme chlorate-sensitive varieties were selected and soaked in tap water for 2 hours to remove water. After that, they were transferred to 4°C for purification for four hours and then placed in a 30°C incubator to break the shells. The seeds with consistent germination were grown in 0.5M CaCl2 pure water for 5 days. After that, the seedlings with consistent growth were selected and incubated in a 6mM NO3 - The seedlings were grown in the Afudonin nutrient solution for 6 days, and then 6mM and 0.1mM NO3 - The treatment lasted for 7 days, with the nutrient solution replaced every three days. Root samples were collected from six extremely chlorate-sensitive cultivars and total protein was extracted. Root samples from three chlorate-sensitive and chlorate-insensitive genotypes were then mixed with equal amounts of protein and sequenced. The sequencing data were then subjected to PCA, differential protein count, and KEGG enrichment analysis.

[0165] Figure 6This example provides a proteomic analysis of the roots of different chlorate-sensitive extreme varieties. 6A is a PCA analysis of the proteome of chlorate-sensitive extreme varieties under normal and low nitrate conditions. The results show that there is significant differentiation between the two types of chlorate-sensitive extreme varieties under both normal and low nitrate conditions (CK and LN). Therefore, under normal conditions, a total of 554 and 400 up-regulated and down-regulated differential proteins were identified between CSG and CIG varieties, respectively ( Figure 6 B), while under low nitrate conditions, there were 526 and 344 proteins. Further KEGG enrichment analysis of the upregulated differentially expressed proteins revealed that they were enriched in multiple pathways, including ABC transport, nitrogen metabolism, etc. ( Figure 6 C), Figure 6 D Further extraction of differentially expressed proteins related to nitrate transport and utilization revealed significant differences between the two varieties, especially the expression product of the gene BnaGLN1;2a was significantly higher in CSG than in CIG.

[0166] Example 4 Genetic Variation Analysis of Gene BnaGLN1;2a

[0167] Previously, RNA samples from extremely chlorate-sensitive varieties treated with different nitrate levels were obtained and analyzed for differential expression of the gene BnaGLN1;2a. Furthermore, based on the relative fresh weight (a nitrogen efficiency indicator) of 505 resequenced rapeseed varieties after chlorate treatment, differential SNPs within the full-length gene sequence and the first 2000bp of the promoter of the BnaGLN1;2a gene were extracted and analyzed for candidate gene association analysis in conjunction with chlorate treatment indicators. Haplotypes were further divided and their indicators analyzed for differences.

[0168] Figure 7 The genetic variation analysis results of the gene BnaGLN1;2a provided in this embodiment, wherein, Figure 7AD is the gene BnaGLN1; 2a candidate gene association analysis in 505 resequenced rapeseed natural varieties detected 8 SNP sites, of which the 8 SNP sites are located at sites 6637711, 6637782, 6638039, 6638041, 6638119, 6639122, 6639192 and 6639500 on chromosome 2, respectively. Starting from the ATG position of the gene, the positions of the 8 SNP sites in the gene structure are -760, -689, -432, -430, -352, 651, 721 and 1029 bp, respectively. The first 5 SNP sites are located in the promoter, the 6th and 8th are located in the non-coding region, and the 7th site is located in the coding region. The excellent SNP sites are mainly located in the promoter region. It can be further divided into four haplotypes: Hap1, Hap2, Hap3 and Hap4, whose nucleotide sequences are SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, among which Hap4 is the optimal haplotype and can be used as an excellent haplotype for genetic breeding analysis; Figure 7 E and 7F are differential expression analyses of the gene BnaGLN1;2a between varieties with different extreme chlorate sensitivities, indicating that the expression level in the roots of CSG was significantly higher than that of CIG regardless of the nitrate level of 6 mM or 0.1 mM.

[0169] Hap1: GGTACGCA (SEQ ID NO: 3)

[0170] Hap2:GGTAGGCA (SEQ ID NO:4)

[0171] Hap3:TCGCCGCG (SEQ ID NO: 5)

[0172] Hap4:TCTAGGCG (SEQ ID NO: 6)

[0173] Example 5 Analysis of Yield and Agronomic Traits of Extreme Chlorate-Sensitive Varieties Based on Field Experiments

[0174] This experiment was conducted at the College of Resources and Environment, Huazhong Agricultural University, Wuhan, Hubei Province. Each material (CSG and CIG) was planted in six rows, with a bed width of approximately 1.8 meters, a row spacing of 0.2 meters, and a plant spacing of 0.15 meters. Three seeds were sown per hole, with 8-10 holes per row. Two treatments were set up: a control (210 kg N / ha) and a low nitrogen (70 kg N / ha) treatment. Each treatment had three replicates, using a randomized block design. Protected areas were set up in the experimental fields, with rows at least 1 meter apart. Fertilizer rates were as follows:

[0175]

[0176] Figure 8 This study analyzed the yield and agronomic traits of extremely chlorate-sensitive varieties at a fertilization level of 210 kg N / ha. Plant height, silique number, silique length, and aboveground biomass of CSG were significantly higher than those of CIG. The former also exhibited higher yield per plant and nitrogen efficiency. Figure 9 Yield and agronomic traits of extreme chlorate-sensitive varieties were analyzed at a fertilization level of 70 kg N / ha. No significant differences were observed between CSG and CIG. These data suggest that CSG, which has higher expression levels of the BnaGLN1;2a gene, performs better in the field. This suggests that interspecific variation and superior haplotypes of the BnaGLN1;2a gene can be used in nitrogen-efficient genetic breeding.

[0177] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Glutamine synthetase gene BnaGLN1;2a The application in rapeseed nitrogen efficient breeding, the glutamine synthetase gene has 4 haplotypes, namely: Hap1:GGTACGCA Hap2:GGTAGGCA Hap3:TCGCCGCG Hap4:TCTAGGCG The haplotypes all include 8 significant SNPs. Based on the rapeseed reference genome version 4.1, the 8 SNP sites are located at sites 6637711, 6637782, 6638039, 6638041, 6638119, 6639122, 6639192 and 6639500 on chromosome 2, respectively, among which Hap4 is its excellent haplotype.

2. A method for screening rapeseed nitrogen-efficient varieties, characterized in that: Amplification of the glutamine synthetase gene BnaGLN1; 2a， The haplotype type is determined. If it is the excellent haplotype shown by Hap4, it is a rapeseed nitrogen-efficient variety; the Hap4 includes 8 significant SNPs with a nucleotide sequence of TCTAGGCG. Based on the rapeseed reference genome version 4.1, the 8 SNP sites are located at sites 6637711, 6637782, 6638039, 6638041, 6638119, 6639122, 6639192 and 6639500 on chromosome 2, respectively.

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