A rice glufosinate-resistant genetic locus and its application

Abstract

A rice herbicide resistance genetic locus and its application. This application discloses a method for cultivating glufosinate-resistant plants, comprising reducing or knocking out the expression of the SPL10 gene in plants, thereby cultivating plants with stronger glufosinate resistance than wild-type control plants.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular, the present invention relates to a plant-specific SPL transcription factor family gene OsSPL10 and its encoded protein in regulating the resistance of plants, especially rice, to the glufosinate herbicide. Background Art

[0002] Rice (Oryza sativa L.) is one of the world's most important food crops, with nearly 90% of the world's rice produced and consumed in Asia. Rice yield and quality directly impact people's living standards. Therefore, increasing rice yield and improving rice quality are crucial for ensuring livelihoods and maintaining agricultural economic stability. Furthermore, as a typical crop model plant, rice also serves as a valuable reference for research on other crops.

[0003] Crops inevitably experience various stresses throughout their lives, with weed infestation being one of the most common. Weeds can compete with crops for various beneficial resources, increase the risk of pests and diseases, and severely impact crop yield and quality. It is estimated that weed infestation reduces global crop yields by over 13% annually, equivalent to the annual food rations of one billion people worldwide. Traditional weed control methods require significant human resources, while the use of herbicides can save significant manpower and material costs. Therefore, cultivating herbicide-resistant crops is crucial for maintaining high crop yields and ensuring food quality.

[0004] Glufosinate, also known as glufosinate, is one of the most widely used herbicides, widely used for its broad-spectrum, non-selective, low toxicity, and environmentally friendly properties. To date, the development of glufosinate-resistant crops has primarily been achieved through genetic engineering. However, the application of genetic engineering in food crops remains controversial, and commercial production of genetically modified crops has yet to be established in my country. Furthermore, no crop germplasm resources with glufosinate resistance have been discovered in my country. Therefore, the development of glufosinate-resistant crops holds significant significance and potential for the development of modern agriculture in my country. Summary of the Invention

[0005] The present invention aims to identify a new gene, SPL10, involved in rice resistance to glufosinate, in addition to resistance to the glutamine synthetase (GS) target site. Functional deletion of this gene significantly enhances rice resistance to glufosinate. This invention has important reference value and potential application for research on improving plant resistance to glufosinate herbicides. Specifically, the present invention solves the technical problems of the present invention through the following technical solutions.

[0006] 1. A method for breeding glufosinate-resistant plants, comprising reducing or knocking out the expression of the SPL10 gene in a plant, thereby breeding plants that are more resistant to glufosinate than wild-type control plants.

[0007] 2. The method of item 1, wherein the nucleic acid sequence of the SPL10 transcription factor in a wild-type plant or plant cell is edited by gene editing technology to introduce or delete one or more bases, antisense nucleotide technology, or RNAi gene silencing technology to achieve reduction or knockout of the expression of the SPL10 gene in the plant.

[0008] 3. The method according to any one of items 1 to 2, wherein the plant is a monocotyledonous plant or a dicotyledonous plant.

[0009] 4. The method according to claim 3, wherein the plant is rice, corn, wheat, soybean, cotton or rapeseed.

[0010] 5. The method according to any one of items 1-2, wherein the protein encoded by the SPL10 gene comprises or consists of the following amino acid sequence:

[0011] (1) the amino acid sequence shown in SEQ ID NO: 2;

[0012] (2) amino acid sequences derived from the amino acid sequence shown in SEQ ID NO: 2 by substitution, deletion or addition of one or more amino acids;

[0013] (3) an amino acid sequence having 50% or greater, 60% or greater, 70% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.5% or greater, or 99.8% or greater sequence identity to the amino acid sequence of SEQ ID NO: 2;

[0014] Preferably, the SPL10 gene is a rice SPL10 gene and its coding region has the nucleotide sequence shown in SEQ ID NO: 1.

[0015] 6. The method according to any one of items 1-2, wherein in the plant in which the expression of the SPL10 gene is reduced or knocked out, the cytosine at position 685 in the coding region of the SPL10 gene in the nucleotide sequence corresponding to SEQ ID NO: 1 is mutated to thymine, or a single nucleotide insertion mutation occurs at position 50, resulting in a reduction or loss of the activity of the SPL10 gene expression product.

[0016] 7. Use of the SPL10 gene in screening for glufosinate-resistant plants, wherein the expression of the SPL10 gene in the glufosinate-resistant plants is reduced or knocked out compared to wild-type control plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Flowchart for screening of glufosinate-resistant rice mutants.

[0018] Figure 2 First-generation sequencing verification of the candidate mutant gene LOC_Os06g44860 (OsSPL10) in the glufosinate herbicide-resistant mutant gar6-2.

[0019] Figure 3 Phenotypes of the osSPL10 mutant in different backgrounds before and after spraying. A and B, respectively, show the phenotypes of the wild type and the gar6-2 mutant in the Longjing 31 rice background before and after spraying; C and D, respectively, show the phenotypes of the wild type and the osSPL10 mutant in the Tp309 rice background before and after spraying. The white scale represents 10 cm. DETAILED DESCRIPTION

[0020] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0021] definition

[0022] "Homology" or "identity" or "similarity" refers to the degree of sequence similarity between two peptide molecules or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be compared by alignment. When the same base or amino acid is present at a position in the compared sequences, the molecules are homologous at that position. The degree of homology between multiple sequences is a function of the number of paired or homologous sites shared by the sequences. "Unrelated" or "non-homologous" sequences have less than 40% homology to one of the sequences of the present application, but preferably less than 25% homology.

[0023] "Sequence identity" refers to a percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) to another sequence, when aligned, of the bases (or amino acids) that are identical when the two sequences are compared. Such alignments and percentage homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology, ed., Ausubel et al., 2007. Preferably, default parameters are used for alignment.

[0024] The SPL10 (squamosa protomer-binding-like 10) gene is a gene found in plants, for example, the SPL10 gene in rice (Oryza sativa L.) (also known as Os SPL10 or LOC_Os06g44860) has Gene ID No. 4341729, the SPL10 gene in Arabidopsis thaliana has Gene ID No. 839626, and the SPL10 gene in grape (Vitis vinifera) has Gene ID No. 100246757. In the context of this application, the SPL10 gene includes the rice SPL10 gene (Os SPL10) as well as SPL10 genes in other plants that are homologous to the rice SPL10 gene and have the same or similar functions in plants.

[0025] Example 1 Obtaining and gene mapping of the glufosinate-resistant rice mutant gar6-2

[0026] Wild-type seeds of the rice variety Longjing 31 (LG31) (provided by Pan Guojun, Rice Research Institute, Heilongjiang Academy of Agricultural Sciences) were induced with ethyl methanesulfonate (EMS) (CAS: 62-50-0) and self-pollinated to create an M2-generation rice mutant library. Mutants resistant to glufosinate were screened using a spray concentration of 2 g / L glufosinate (Shijiazhuang Ruikai Chemical Co., Ltd., glufosinate purity 95%-96%). The specific operation is as follows: soak the M2 generation seeds in distilled water for 2-3 days to accelerate germination, then evenly spread them in plant nutrient soil and allow them to germinate and grow normally for about two weeks, until the rice grows to the three-leaf stage; prepare a 2 g / L glufosinate herbicide and spray it evenly on the above-ground part of the seedlings with a spray bottle. After 3-5 days, the leaves of the seedlings that are not resistant to glufosinate will lose their green color and turn yellow. After 7-10 days, the seedlings whose above-ground parts are still green and can grow normally are selected as the positive seedlings for initial screening of glufosinate herbicide resistance (see Figure 1 ), and transplanted it to the field to grow normally until flowering and seeding. After the seeds matured, the M3 generation mutant seeds were harvested and screened for glufosinate resistance using the same method. The M3 generation screening further confirmed the glufosinate herbicide resistance phenotype of the above mutants and named g lufosinate a mmonium r esistance 6-2 (gar6-2).

[0027] The M3 generation seeds of the gar6-2 mutant were germinated in the field and grown to the flowering stage. The gar6-2 mutant (as the male parent) was crossed with LG31 (as the female parent) to produce BC1F1 generation hybrid seeds. The BC1F1 generation hybrid seeds were then transplanted back into the field and selfed to produce BC1F2 generation seeds, thereby constructing a BC1F2 segregating population.

[0028] The BC1F2 population was screened for glufosinate herbicide tolerance again, and the survival rate of the BC1F2 population was calculated after 7-10 days. All seedlings without glufosinate resistance died, while seedlings with glufosinate resistance survived normally. The BC1F2 segregating population of the gar6-2 mutant showed a survival:death ratio of ≈1:3 (survival:death = 79:234; x 2 = 0.001< χ 2 (0.05) =3.84), indicating that the gene that causes the gar6-2 mutant to have glufosinate resistance is a single gene recessive gene mutation inherited from the cell nucleus. Then, 30 glufosinate-resistant seedlings were selected from the BC1F2 segregating population of gar6-2, and genomic DNA was extracted from each of them. The genomic DNA from the 30 samples was mixed in equal amounts, and genome resequencing was performed using the MutMap method (Nanjing Aoweisen Biotechnology Co., Ltd.) to obtain candidate genes involved in regulating glufosinate-resistance. The candidate gene mutation sites were then further verified by first-generation sequencing, and gene association analysis was performed by first-generation sequencing. The analysis results showed that the 685th cytosine in the coding region of the LOC_Os06g44860 / OsSPL10 gene in the genome of the gar6-2 mutant (the nucleotide sequence of the coding region of the LOC_Os06g44860 / OsSPL10 gene is shown in SEQ ID NO: 1, and the amino acid sequence encoded by it is shown in SEQ ID NO: 2) was mutated to thymine (C685T), resulting in a missense mutation in the 229th amino acid of the protein encoded by the gene, from histidine to tyrosine (H311V). Figure 2 As shown (the nucleotide sequence of the OsSPL10 gene with the C685T mutation and the amino acid sequence of the polypeptide encoded thereby are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively), thereby conferring resistance to glufosinate-ammonium herbicide to the gar6-2 mutant.

[0029] Example 2 Verification of the glufosinate-resistance phenotype of rice osspl10 gene knockout mutant

[0030] To further verify that the glufosinate-resistant phenotype of the screened gar6-2 mutant was caused by a functional loss of the OsSPL10 gene, we obtained wild-type seeds from the rice Tp309 background and seeds of the OsSPL10 knockout mutant osspl10 in the Tp309 background (both provided by the research group of Professor M. Brian Traw of Nanjing University). This mutant has a thymine insertion at position 50 in the coding region of the OsSPL10 gene, resulting in premature termination of translation of the gene.

[0031] Then, the LG31, gar6-2, Tp309 and osspl10 gene-edited mutant seeds were planted in the soil for germination and growth for 2 weeks until the three-leaf stage. They were uniformly sprayed with a 2 g / L glufosinate solution. After 3-5 days, it was observed that the gar6-2 mutant under the LG31 background showed stronger glufosinate resistance than the LG31 wild type. Figure 3 As shown in AB; similarly, the osspl10 gene-edited mutant in the Tp309 background showed stronger glufosinate resistance than the Tp309 wild type, as shown in Figure 3 The experimental results again showed that the loss of OsSPL10 gene function can significantly enhance the resistance of rice to glufosinate herbicide.

[0032] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are 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. A method for cultivating glufosinate-resistant plants, comprising reducing or knocking out the expression of the SPL10 gene in a plant, thereby cultivating a plant that is more resistant to glufosinate-ammonium than a wild-type control plant, The plant is rice, and the protein encoded by the SPL10 gene consists of the amino acid sequence shown in SEQ ID NO:

2.

2. The method of claim 1, wherein the nucleic acid sequence of the SPL10 transcription factor in wild-type plants or plant cells is edited by gene editing technology to introduce or delete one or more bases, antisense nucleotide technology, or RNAi gene silencing technology to achieve reduction or knockout of the expression of the SPL10 gene in the plant.

3. The method according to any one of claims 1-2, wherein the SPL10 gene is a rice SPL10 gene and its coding region is the nucleotide sequence shown in SEQ ID NO:

1.

4. The method according to any one of claims 1 to 2, wherein in the plant in which the expression of the SPL10 gene is reduced or knocked out, the cytosine at position 685 in the nucleotide sequence of SEQ ID NO: 1 in the coding region of the SPL10 gene is mutated to thymine or a single nucleotide insertion mutation occurs at position 50, resulting in a reduction or loss of the activity of the SPL10 gene expression product.

5. Use of the SPL10 gene in screening for glufosinate-resistant plants, wherein the expression of the SPL10 gene in the glufosinate-resistant plants is reduced or knocked out compared with wild-type control plants, wherein the plants are rice, and wherein the protein encoded by the SPL10 gene consists of the amino acid sequence shown in SEQ ID NO: 2.

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