A rice glufosinate herbicide-resistant gene and its application

Abstract

The present invention discloses a glufosinate herbicide-resistant gene GLR6 of rice and its applications, relating to the field of biotechnology. The nucleotide sequence of the gene GLR6 is as follows: (1) as shown in SEQ ID No.1 or SEQ ID No.2; or (2) the nucleotide sequence of a mutant, allele or derivative generated by adding, substituting, inserting or deleting one or more nucleotides. The present invention also provides the encoded protein of the gene GLR6, a recombinant construct, a recombinant host cell, and a method and application for conferring glufosinate herbicide resistance to rice by using the gene GLR6. The beneficial effects of the present invention are as follows: in the allelic variation of GLR6 and the loss-of-function mutants obtained by gene editing, they have glufosinate herbicide resistance at the seedling stage. By using these mutant rice plants in direct seeding cultivation, glufosinate herbicide can be used to control weeds in the field, thereby quickly achieving the removal of weeds in the field.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically relates to a glufosinate herbicide-resistant gene of rice and its application. Background Art

[0002] Rice is one of the important food crops, and more than half of the global population takes rice as the staple food. In China, its total area, total output, and yield per unit area rank first among various food crops. During the production process of rice, the harm of weeds in the field seriously affects the yield of rice, and the situation of controlling paddy weeds is severe. With the popularization of the direct-seeded rice cultivation technology, the problem of weed control becomes more prominent. The harm of weeds in direct-seeded paddy fields is more serious than that in transplanted paddy fields, and it is more difficult to control.

[0003] Glufosinate belongs to phosphonic acid herbicides, which can inhibit glutamine synthetase in the plant nitrogen metabolism pathway, thereby interfering with the metabolism of plants and causing plant death. Glufosinate has the characteristics of a broad herbicide spectrum, low toxicity, high activity, and good environmental compatibility, and has broad application prospects. However, ordinary rice does not have resistance to glufosinate herbicides, resulting in that glufosinate cannot be applied to the weed control in paddy fields.

[0004] In order to reduce weeds in paddy fields and ensure the growth and yield of rice, it is of great significance to obtain rice with resistance to glufosinate herbicides. Obtaining genes resistant to glufosinate herbicides plays a very important role in quickly cultivating new varieties of glufosinate herbicide-resistant rice.

[0005] Chinese Patent with the publication number of CN1335886A discloses a glufosinate-tolerant rice, in which the bar gene is under the control of the CaMV35S promoter at a specific locus in the rice genome. However, due to the complex application procedures for the commercialization of transgenic varieties and people's concerns about the safety of transgenic rice, it is very difficult to be popularized and applied in production at the present stage, and the popularization and application of glufosinate herbicide-resistant transgenic rice varieties in China are also restricted by foreign patents. Therefore, this patent needs to be further improved. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to solve the dependence of the application of glufosinate resistance genes in the prior art on transgenic technology, and provide a non-transgenic glufosinate-resistant mutant gene GLR6. By using the site-directed modification of the GLR6 gene, new varieties of glufosinate herbicide-resistant rice can be cultivated.

[0007] The present invention realizes the solution of the above technical problems through the following technical means:

[0008] The mutation site of a glufosinate herbicide-resistant rice mutant glr6, the mutation site is a single-base substitution and a single-base deletion, and the mutation site is located on the LOC_Os06g47150 gene.

[0009] The LOC_Os06g47150 gene is the glufosinate herbicide resistance gene GLR6.

[0010] A gene GLR6 that controls the glufosinate herbicide-resistant rice mutant glr6, the nucleotide sequence of the gene GLR6, (1) is shown in SEQ ID No.1 or SEQ ID No.2; or (2) the nucleotide sequence of a mutant, allele or derivative generated by adding, substituting, inserting or deleting one or more nucleotides; (3) or a nucleotide sequence that can hybridize under stringent conditions with the nucleotide sequence shown in SEQ ID No.2 and simultaneously encodes a nucleotide sequence with the ability to control glufosinate herbicide resistance in rice.

[0011] The stringent conditions refer to placing the hybridization membrane in pre-hybridization solution (0.25 mol / L sodium phosphate buffer, pH 7.2, 7% SDS), pre-hybridizing at 65 °C for 30 minutes; discarding the pre-hybridization solution, adding hybridization solution (0.25 mol / L sodium phosphate buffer, pH 7.2, 7% SDS, isotope-labeled nucleotide fragment), hybridizing at 65 °C for 16 hours; discarding the hybridization solution, adding washing solution I (20 mmol / L sodium phosphate buffer, pH 7.2, 0.1% SDS), washing the membrane at 65 °C twice, 10 - 15 minutes each time; adding washing solution II (10 mmol / L sodium phosphate buffer, pH 7.2, 0.1% SDS), washing the membrane at 65 °C for 10 - 15 minutes.

[0012] The nucleotide sequence of SEQ ID No.1 relates to the promoter, coding region and regulatory region of the gene GLR6.

[0013] The present invention uses heavy ion irradiation on the Chinese japonica rice variety Jinjing 818 to obtain a rice mutant glr6 that does not affect yield-related agronomic traits such as plant height, tiller number, spikelet number per panicle, 1000-grain weight, etc., and has glufosinate herbicide resistance.

[0014] The present invention constructs a genetic analysis population for the glr6 mutant and conducts genetic behavior analysis on it, and finds that the glufosinate herbicide resistance of the glr6 mutant is recessively controlled by the mutated single gene GLR6.

[0015] Beneficial effects: The present invention isolates and identifies a glufosinate herbicide resistance gene GLR6 by map-based cloning. Through phenotypic analysis and genetic complementation experiments on the materials, it is confirmed that the GLR6 gene has a certain function in regulating glufosinate herbicide resistance in rice.

[0016] The gene GLR6 provides a theoretical basis, materials and gene support for clarifying the regulatory genetic basis of rice's glufosinate herbicide resistance at the molecular level in the future, as well as for breeding new environmentally friendly rice varieties based on molecular design.

[0017] A protein encoded by the control gene GLR6 for rice glufosinate herbicide resistance, the amino acid sequence of the protein encoded by the gene GLR6, (1) as shown in SEQ ID No. 3; or (2) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID No. 3 by substitution, deletion, and / or insertion of one or more (such as 1-25, 1-20, 1-15, 1-10, 1-5, 1-3) amino acid residues; or (3) an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, especially at least 95% or 98% or 99% identity with the amino acid sequence shown in SEQ ID No. 3; or an active fragment of the amino acid sequence of (1), (2), or (3).

[0018] A recombinant construct containing the nucleotide sequence of the control gene GLR6 for rice glufosinate herbicide resistance, and the vector used for the recombinant construct is a cloning vector or an expression vector for expressing the nucleotide.

[0019] A recombinant host cell, including a host cell containing the above recombinant construct, or a polynucleotide sequence of the control gene GLR6 for rice glufosinate herbicide resistance of the present invention integrated into its genome. The host cell can be selected from plant cells or microbial cells, such as Escherichia coli cells or Agrobacterium cells, preferably plant cells, and most preferably rice cells. The cells can be isolated, in vitro, cultured, or a part of a plant.

[0020] The present invention discloses a rice variety resistant to glufosinate herbicide by using glr6 mutants and obtaining it through glr6 mutation sites, as well as a rice variety resistant to glufosinate herbicide obtained by making the GLR6 gene function-deficient mutation by various means, including the above physical mutagenesis, chemical mutagenesis, biological mutagenesis, and gene editing technology.

[0021] Preferably, the host cell is a plant cell or a microbial cell.

[0022] Preferably, the plant cell is a rice cell; the microbial cell is an Escherichia coli cell or an Agrobacterium cell.

[0023] A method for cultivating rice resistant to glufosinate herbicide, comprising the following steps: using mutagenesis means, the mutagenesis means including physical mutagenesis, chemical mutagenesis, and biological mutagenesis, or making the control gene GLR6 of the above rice glufosinate herbicide mutant glr6 function-deficient by gene editing technology, wherein the obtained rice plants exhibit a glufosinate herbicide-resistant phenotype.

[0024] A method for cultivating glufosinate - resistant rice, comprising the following steps: hybridizing the glufosinate - resistant rice mutant glr6 with the above - mentioned mutation site with other rice varieties, and obtaining glufosinate - resistant rice plants through progeny segregation.

[0025] Use of the rice plants or varieties obtained by the above method in glufosinate resistance.

[0026] The advantages of the present invention are as follows:

[0027] In the allelic variation of GLR6 and the loss - of - function mutants by gene editing of the present invention, they have glufosinate resistance at the seedling stage. Using this mutant rice in direct seeding cultivation, glufosinate can be used to treat field weeds, thus quickly achieving the removal of field weeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a comparison diagram before and after glufosinate treatment of wild - type WT and mutant glr6 in Example 1 of the present invention;

[0029] Figure 2 It is a genetic linkage analysis diagram of wild - type WT and mutant glr6 in Example 2 of the present invention;

[0030] Figure 3 It is a gene alignment diagram of wild - type WT and mutant glr6 in Example 2 of the present invention;

[0031] Figure 4 It is a comparison diagram of glufosinate resistance between positive transgenic seedlings and negative seedlings in the T1 generation in Example 2 of the present invention;

[0032] Figure 5 It is a sequencing peak diagram of the mutation sites of several homozygous lines crispr - glr6 with GLR6 gene knockout and wild - type WT in Example 3 of the present invention;

[0033] Figure 6 It is a comparison diagram of glufosinate resistance between several homozygous lines crispr - glr6 with GLR6 gene knockout and wild - type WT in Example 3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0035] The test materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0036] For those not specifying the specific technology or conditions in the examples, they can all be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications.

[0037] Example 1:

[0038] Phenotypic analysis of the glufosinate-resistant rice mutant glr6

[0039] Using heavy ions 12 C 6+ mutagenesis (energy 80 MeV, dose 120 Gy) of the japonica rice variety Jinjing 818 to obtain the glr6 mutant. The phenotypic characteristics of this mutant are: under the spraying of glufosinate herbicide, glr6 has obvious resistance, as Figure 1 shown.

[0040] Using the glr6 mutant to cross with the wild-type Jinjing 818 to construct a backcross population. In the segregating population of 480 F2 generations, after spraying with glufosinate herbicide and after recovery growth, among them, 112 plants survived and 368 plants died. The segregation ratio conforms to 1:3 (χ2[1:3] = 0.18 < χ20.05 = 3.84; P > 0.05). Therefore, it can be inferred that the glufosinate herbicide resistance of the glr6 mutant is controlled by a single pair of recessive genes.

[0041] Example 2:

[0042] Gene mapping of the glufosinate-resistant gene GLR6

[0043] (1) Construction of the mapping population

[0044] Using the glr6 mutant to cross with indica rice varieties such as 93-11, Minghui 63, and Nanjing 11, respectively obtaining seeds of the segregating population by self-crossing the F1 generations of different hybridization combinations. These seeds were planted in the field. At the three-leaf stage, they were sprayed with glufosinate herbicide, and 50 surviving individual plants were selected as the mutant pool. Another 50 individual plants that died were used as the wild-type pool.

[0045] (2) Second-generation resequencing and MutMap analysis

[0046] DNA was extracted from the above-mentioned mutant pool and wild-type pool, and then second-generation resequencing and gene linkage analysis were carried out. It was found that there was obvious linkage on chromosome 6 (as Figure 2 shown). Further analysis and sequencing alignment of the genes in this interval were confirmed, and it was found that there was a base substitution and a base deletion in the LOC_Os06g47150 gene (as Figure 3As shown in the figure, it results in the premature termination of the encoded amino acid. Therefore, the LOC_Os06g47150 gene was used as a candidate gene for GLR6.

[0047] (3) Functional complementation verification of the GLR6 gene

[0048] To verify that the LOC_Os06g47150 gene is the GLR6 gene, wild-type seedling leaves were used as materials to extract RNA and reverse-transcribe and amplify to obtain cDNA. A LOC_Os06g47150 expression vector pGLR6::GLR6 driven by the LOC_Os06g47150 self-promoter (a 2500bp fragment upstream of ATG) was constructed; the pGLR6::GLR6 expression vector was transferred into the glr6 mutant. When sufficient seeds were harvested, the positive transgenic seedlings in the next generation (T1 generation) were sprayed with glufosinate herbicide, and it was found that none of them had glufosinate herbicide resistance, while the isolated negative seedlings still had glufosinate herbicide resistance ( Figure 4 as shown in the figure).

[0049] Example 3:

[0050] Method for cultivating rice varieties resistant to glufosinate herbicide

[0051] (1) Cultivating rice varieties resistant to glufosinate herbicide using the glr6 mutation site

[0052] The glr6 mutant was crossed, backcrossed, and self-crossed with normal rice varieties, and during this process, the glr6 mutation site was verified by sequencing and the genetic background was selected. Finally, new glufosinate herbicide-resistant varieties with homozygous glr6 mutant genes under different genetic backgrounds were obtained. The specific implementation steps are as follows:

[0053] 1. Using receptor parents such as 93-11, Minghui 63, Wushan Simiao, etc. as male parents to cross with the glr6 mutant to obtain F1;

[0054] 2. Using F1 as the female parent to backcross with receptor parents such as 93-11, Minghui 63, Wushan Simiao, etc. to obtain BC1F1;

[0055] 3. Plant BC1F1, and use sequencing analysis to select the glr6 heterozygous genotype;

[0056] 4. Use molecular markers evenly distributed on 12 pairs of rice chromosomes (including but not limited to SSR, SNP, InDel, EST, RFLP, AFLP, RAPD, SCAR type markers) and polymorphic between the glr6 mutant and the recurrent parent to identify the genetic background of the single plants selected in step 3, and select plants with a high similarity (such as greater than 75%) to the recurrent parent genotype;

[0057] 5. Use the plants selected in step 4 to backcross with recipient parents, such as 93-11, Minghui 63, Wushan Si Miao, etc. to obtain BC2F1;

[0058] 6. Plant BC2F1, repeat steps 3 and 4, select plants with heterozygous glr6 genotype and high genetic background recovery rate (such as greater than 95%), and collect them from the hybrid BC2F2.

[0059] 7. Plant BC2F2, repeat steps 3 and 4, select the plants with the highest genetic background homozygosity rate and glr6 genotype heterozygosity, and collect BC2F3. The glr6 homozygous plants isolated from the offspring of BC2F3 are sprayed with glufosinate herbicide and show the phenotype of glufosinate herbicide resistance.

[0060] (2) Cultivating new rice varieties resistant to glufosinate using gene editing technology

[0061] The GLR6 gene was edited using CRISPR / Cas9 technology to create a mutant with loss of GLR6 gene function. Several independent homozygous strains with GLR6 gene knockout showed resistance to glufosinate herbicide.

[0062] The construction and transformation methods of CRISPR / Cas9 vectors are as follows:

[0063] According to the gDNA sequence of the GLR6 gene, the target primers were designed as follows:

[0064] GLR6-CRISPR-U3: 5'-TTTCGTGGATTCCGCGGCGAAGG-3' (SEQ ID No. 4)

[0065] For the specific construction method, please refer to the article published by Professor Liu Yaoguang of South China Agricultural University (A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. (2015) Molecular Plant, 8 (8): 1274-1284). The vector was introduced into Jinjing 818 by Agrobacterium-mediated transformation (rice transformation was completed by the applicant's laboratory). Comparative phenotypic analysis of wild-type and transgenic rice revealed that several homozygous strains of GLR6 knockout gene CRISPR-glr6 (sequencing peaks of mutation sites are shown in Figure 2) were Figure 5 ) showed obvious resistance to glufosinate-ammonium herbicide at the seedling stage ( Figure 6 ).

[0066] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for cultivating glufosinate - resistant rice, characterized in that, A rice plant with a loss-of-function of the control gene GLR6 of the glufosinate herbicide mutant glr6 obtained by gene editing technology, wherein the obtained rice plant exhibits a phenotype of glufosinate herbicide resistance, and the nucleotide sequence of the gene GLR6 is as shown in SEQ ID No.1 or SEQ ID No.2.

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