Application of Osbc1 gene in glyphosate resistance

Abstract

The invention relates to the technical field of agricultural science and plant biological genetic engineering, in particular to application of an Osbc1 gene in glyphosate resistance. According to the invention, the Osbc1-YES2-NTB plasmid is transformed into yeast, and the yeast can grow in a glyphosate-containing screening culture medium and shows glyphosate resistance. The gene regulation strategy provided by the invention provides a gene resource for cultivating herbicide-resistant crops, and has important agricultural application value.

Description

Technical Field

[0001] This invention relates to the fields of agricultural science and plant bioengineering, and in particular to the application of the Osbc1 gene in glyphosate resistance. Background Technology

[0002] Chemical herbicides have become a core means of controlling weeds in global agriculture. However, the long-term and repeated application of single-mechanism herbicides has led to the rapid evolution of herbicide resistance in weed populations, posing new threats to crop yield and quality. Breeding crop varieties tolerant to specific herbicides is considered one of the most economical and effective integrated weed management solutions. This technology involves introducing resistance genes, allowing crops to grow normally under the selective pressure of herbicides while weeds are eliminated, thus simplifying agricultural operations and improving control efficiency.

[0003] Glyphosate, with its broad spectrum of weed control, low toxicity to humans and animals, and no soil residue, is the world's leading herbicide in terms of market share. Its mechanism of action mimics phosphoenolpyruvate (PEP), competitively binding to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in chloroplasts, blocking the synthesis pathways of essential aromatic amino acids (phenylalanine, tyrosine, and tryptophan), leading to a massive accumulation of shikimic acid and ultimately plant death. Current research on glyphosate tolerance in rice mainly follows two routes: first, using gene editing technology to directionally modify endogenous OsEPSPS in rice to reduce its affinity for glyphosate; second, regulating the absorption, transport, and metabolic networks of glyphosate to reduce the accumulation of active molecules at the target site.

[0004] The continuous development of omics technologies has significantly advanced the screening of herbicide-resistant genes. Transcriptome sequencing was used to analyze the whole-genome expression of glyphosate-treated Huanghuazhan rice, and candidate genes for glyphosate resistance were screened based on changes in gene expression levels. These candidate genes were then cloned into the Huanghuazhan background, and their glyphosate resistance was further validated point-to-point using a yeast stress screening system. This provides valuable genetic resources for discovering glyphosate-resistant genes and subsequently breeding efficient and sustainable glyphosate-resistant crop varieties. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides the application of the Osbc1 gene in glyphosate resistance. The Osbc1 gene was cloned from indica rice Huanghuazhan, and yeast was transformed with the Osbc1-YES2-NTB plasmid. The yeast was able to grow in a glyphosate-containing selection medium and exhibited glyphosate resistance.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of the Osbc1 gene in glyphosate resistance.

[0008] Preferably, resistance to glyphosate is achieved by overexpressing the Osbc1 gene.

[0009] Preferably, the nucleotide sequence of the Osbc1 gene is shown in SEQ ID No. 1.

[0010] Preferably, the amino acid sequence of the protein encoded by the Osbc1 gene is shown in SEQ ID No. 2.

[0011] This invention also provides the application of the Osbc1 gene in improving yeast resistance to glyphosate.

[0012] Preferably, the application includes the following steps:

[0013] 1) The Osbc1 gene was inserted into the yeast expression vector YES2-NTB to obtain the Osbc1-YES2-NTB vector;

[0014] 2) The Osbc1-YES2-NTB vector obtained in step 1) was transformed into yeast to improve the yeast's resistance to glyphosate.

[0015] Preferably, the yeast includes W303 yeast.

[0016] Preferably, the concentration of glyphosate is 10–25 mmol / L.

[0017] This invention involves RNA extraction from Huanghuazhan rice at different time points after glyphosate application, followed by whole-transcriptome sequencing and gene expression quantification. Based on changes in gene expression levels, the Osbc1 gene, whose expression level was significantly upregulated by glyphosate, was screened as a candidate gene for glyphosate resistance. The Osbc1-YES2-NTB vector was constructed and transformed into W303 yeast competent cells. Positive clones were selected and cultured for 36 hours, then plated on selection media with different glyphosate concentration gradients. After 7 days of incubation at 30°C, yeast containing the Osbc1 gene was observed to grow in the glyphosate-containing selection medium, exhibiting glyphosate resistance. The Osbc1 gene proposed in this invention provides a gene resource for breeding herbicide-resistant rice and has significant agricultural application value.

[0018] Beneficial effects:

[0019] 1. Using a yeast stress selection system to reveal the resistance function of the Osbc1 gene.

[0020] This invention clones the Osbc1 gene from Huanghuazhan indica rice, transforms it into competent yeast cells, selects positive clone transformants, and spreads them onto selection media with different glyphosate concentrations. It was found that they can grow in selection media containing glyphosate and exhibit glyphosate resistance.

[0021] 2. Enhance the glyphosate resistance of rice and create new glyphosate-resistant rice varieties.

[0022] The glyphosate resistance gene Osbc1 obtained through screening can be applied to the molecular breeding of future new rice varieties, innovatively cultivating highly efficient glyphosate-resistant new rice varieties, effectively reducing manual weeding and herbicide use, improving the efficiency of weed control in the field, significantly saving labor and production costs, and contributing to the development of green and efficient agriculture. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0024] Figure 1 Figure A shows the glyphosate resistance results of yeast transformed with the Huang Huazhan rice Osbc1 gene.

[0025] Figure 1 B represents the change in Osbc1 expression level at different time points after glyphosate treatment. Detailed Implementation

[0026] This invention provides the application of the Osbc1 gene in glyphosate resistance.

[0027] This invention preferably utilizes overexpression of Huang Huazhan's Osbc1 gene to resist glyphosate. In this invention, the nucleotide sequence of the Osbc1 gene is shown in SEQ ID No. 1. In this invention, the amino acid sequence of the protein encoded by the Osbc1 gene is shown in SEQ ID No. 2.

[0028] SEQ ID No. 1:

[0029] ATGGGGAAGACGCCGGTGCGGATGAAGGCGGTGGTGTACGCGCTGTCGCCGTTCCAGCAGAAGGTGATGCCGGGGCTGTGGAAGGACATCACCACCAAGAATCCACCACAAGGTCTCCGAGAACTGGATCTCCGCCACGCTCCTCCTCGCCCCCATCGTCGGCACCTACGAGTACGCAATGTACTACAAGGAGCAGGAGAAGCTATCCCACAGATACTAA.

[0030] SEQ ID No. 2:

[0031] MGKTPVRMKAVVYALSPFQQKVMPGLWKDITTKIHHKVSENWISATLLLAPIVGTYEYAMYYKEQEKLSHRY*.

[0032] This invention also provides the application of the Osbc1 gene in improving yeast resistance to glyphosate.

[0033] In this invention, the application preferably includes the following steps:

[0034] 1) The Osbc1 gene was inserted into the yeast expression vector YES2-NTB to obtain the Osbc1-YES2-NTB vector;

[0035] 2) The Osbc1-YES2-NTB vector obtained in step 1) was transformed into yeast to improve the yeast's resistance to glyphosate.

[0036] In this invention, the Osbc1 gene is preferably inserted into the yeast expression vector YES2-NTB to obtain the Osbc1-YES2-NTB vector. This invention does not have specific limitations on the method of inserting the Osbc1 gene into the yeast expression vector YES2-NTB; conventional methods can be used by those skilled in the art. In this invention, the yeast preferably includes W303 yeast.

[0037] In this invention, the obtained Osbc1-YES2-NTB vector is preferably transformed into yeast to improve the yeast's resistance to glyphosate. The transformation method is not particularly limited and conventional methods are acceptable. In this invention, the concentration of glyphosate is preferably 10–25 mmol / L.

[0038] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] Cloning of the Osbc1 gene:

[0041] Based on rice genome sequence information, specific primers for amplifying the Osbc1 gene were designed.

[0042] The primer sequences are as follows: Forward primer, Osbc1-F (SEQ ID No. 3):

[0043] 5'-ATGGGGAAGACGCCGGTG-3';

[0044] Reverse primer, Osbc1-R (SEQ ID No. 4):

[0045] 5'-TTAGTATCTGTGGGATAGCTTCTCC-3'.

[0046] The complete coding sequence (CDS) of the Osbc1 gene (SEQ ID No. 1) was amplified from rice cDNA. The PCR system consisted of: 25 μl 2×buffer, 20 μl ddH2O, 1 μl dNTP, 1 μl Osbc1-F, 1 μl Osbc1-R, 1 μl cDNA, and 1 μl DNA polymerase. The PCR amplification conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles, followed by a final extension at 72℃ for 5 min. The amplified product was purified after confirmation by agarose gel electrophoresis.

[0047] Amplification of the Osbc1 gene:

[0048] The primer sequence is as follows: Forward primer, Osbc1-YES2-NTB-F (SEQ ID No. 5):

[0049] 5'-ATAAGGTACCTAAGGATCCAATGGGGAAGACGCCGGTG-3';

[0050] Reverse primer, Osbc1-YES2-NTB-R (SEQ ID No. 6):

[0051] 5'-GCTGGATATCTGCAGAATTCTTAGTATCTGTGGGATAGCTTCTCC-3'.

[0052] PCR amplification was performed using the CDS sequence of the Osbc1 gene as a template. The PCR system consisted of: 25 μl 2× buffer, 20 μl ddH2O, 1 μl dNTP, 1 μl Osbc1-YES2-NTB-F (10 μM), 1 μl Osbc1-YES2-NTB-R (10 μM), 1 μl Osbc1-CDS, and 1 μl DNA polymerase. The amplified products were purified by agarose gel electrophoresis. Based on the sequence map of the YES2-NTB vector, EcoR1 and BamH1 were selected as restriction enzyme sites. The YES2-NTB vector was digested with the following enzymes: 5 μl 10× buffer, 10 μl YES2-NTB vector, 1 μl EcoR1, 1 μl BamH1, and 33 μl ddH2O. Homologous recombination was used to ligate the cloned fragment with a linearized vector after enzyme digestion, transforming the cells into competent *E. coli* cells (DH5α) and plating them onto LB medium containing ampicillin. After overnight culture, single clones were selected for colony PCR, and positive clones were verified by double enzyme digestion. Recombinant plasmids with correct bands were sequenced to obtain the correct fusion expression vector Osbc1-YES2-NTB.

[0053] Example 2

[0054] The Osbc1 gene enhances resistance to glyphosate in yeast systems.

[0055] W303 yeast was inoculated into YPDA liquid medium and cultured at 30℃ with shaking for 18-20 h. After centrifugation, the cells were resuspended in 30 ml of sterile water, washed once, and centrifuged again, discarding the supernatant. The cells were then resuspended in 20 ml of 0.1 M LiAc, mixed, and centrifuged again, discarding the supernatant. The cells were then resuspended in 10 ml of 0.1 M LiAc, mixed, aliquoted into 1.5 ml centrifuge tubes, and centrifuged again, discarding the supernatant. The following were added sequentially to the centrifuge tubes: 240 μl of 50% PEG3350, 36 μl of 1 M LiAc, 5 μl of salmon sperm DNA (purchased from Beijing Coollab Technology Co., Ltd., which can promote plasmid entry into yeast cells and improve transformation efficiency during yeast transformation), and 5 μl of Osbc1-YES2-NTB plasmid. The mixture was then pipetted and incubated at 30℃ for 30 min. Finally, a heat shock was performed at 42℃ for 25 min. After centrifugation, the bacterial cells were resuspended in 200 μl of sterile water, spread on appropriate plates, and incubated at 30°C for 4 days. Experimental groups and empty vector control strains were then cultured in SG-U liquid medium for 1-2 days. After confirming growth, OD was adjusted. 600 =0.4, and the bacterial solution was diluted and spotted onto the plate (10 0 times, 10 -1 times, 10 -2 times, 10 -3Yeast containing Osbc1-YES2-NTB positive transformants can be grown on screening plates containing 0mM, 10mM, 20mM, and 25mM glyphosate. Figure 1 ).

[0056] These results indicate that the Osbc1 gene derived from Huang Huazhan significantly enhances yeast resistance to glyphosate. This provides a new genetic resource for the study of herbicide-resistant genes and is helpful in breeding herbicide-resistant crops.

[0057] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of Osbc1 gene in glyphosate resistance.

2. The application according to claim 1, characterized in that, Resistance to glyphosate was achieved by overexpressing the Osbc1 gene.

3. The application according to claim 1, characterized in that, The nucleotide sequence of the Osbc1 gene is shown in SEQ ID No.

1.

4. The application according to claim 1 or 3, characterized in that, The amino acid sequence of the protein encoded by the Osbc1 gene is shown in SEQ ID No.

2.

5. Application of the Osbc1 gene in improving yeast resistance to glyphosate.

6. The application according to claim 5, characterized in that, The application includes the following steps: 1) The Osbc1 gene was inserted into the yeast expression vector YES2-NTB to obtain the Osbc1-YES2-NTB vector; 2) The Osbc1-YES2-NTB vector obtained in step 1) was transformed into yeast to improve the yeast's resistance to glyphosate.

7. The application according to claim 5 or 6, characterized in that, The yeast includes W303 yeast.

8. The application according to claim 5, characterized in that, The concentration of glyphosate is 10–25 mmol / L.

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