Camellia sinensis csGSTU8 gene and application thereof in relieving and degrading toxic effect of glyphosate

Abstract

This invention discloses the CsGSTU8 gene of tea plant and its application in alleviating and degrading glyphosate toxicity, relating to the field of genetic engineering technology. The nucleotide sequence of the CsGSTU8 gene is shown in SEQ ID NO.1 of the sequence listing. The amino acid sequence of the protein encoded by the CsGSTU8 gene is shown in SEQ ID NO.2 of the sequence listing. CsGSTU8 is upregulated in both roots and leaves in response to glyphosate stress; CsGSTU8 is specifically highly expressed in tea plant roots. The pCAMBIA1305 plasmid (containing a GFP tag) constructed from this gene, after transformation with Agrobacterium and infection of tobacco leaves, was observed to be localized in the nucleus and cytoplasm. The CsGSTU8 protein is expressed in prokaryotes, and its enzyme activity is strongest at pH 8.0. Transforming the pYES2 plasmid constructed from this gene into the glyphosate-sensitive strain BY4741 restored the growth ability of the yeast mutant on high-concentration glyphosate medium. The cloning of this gene elucidates its role in tea plant resistance to glyphosate stress, providing data support for research on the molecular mechanisms of tea plant resistance to glyphosate stress.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the CsGSTU8 gene in tea trees and its application in mitigating and degrading glyphosate toxicity. Background Technology

[0002] Tea (Camellia sinensis) is an important leaf crop with significant economic, cultural, and health value. Weeds are one of the most serious problems affecting tea plantation production, and glyphosate is currently the most common herbicide used in agriculture. The widespread use of glyphosate and other herbicides severely impacts the growth and development of tea plants and the quality and safety of tea. Therefore, identifying key genes involved in the detoxification of tea plants by glyphosate is crucial for understanding the molecular mechanisms of tea plant responses to glyphosate stress. This research provides target genes and theoretical support for solving the problem of glyphosate damage in tea plants, and is of significant research importance.

[0003] Chinese patent application CN113512550A discloses the application of the tea plant CsHAC1 gene and protein in altering plant stress resistance. This patent constructs a recombinant expression vector in *Saccharomyces cerevisiae* containing the tea plant CsHAC1 gene. Transforming this vector into *Saccharomyces cerevisiae* and inducing overexpression of the gene with galactose enhances the yeast's tolerance to H2O2 and NaCl stress, demonstrating that the gene can improve the yeast's tolerance to hydrogen peroxide and / or sodium chloride stress. The tea plant CsHAC1 gene and its encoded protein provide a theoretical basis for subsequent genetic engineering techniques to cultivate stress-resistant tea plants, and have significant application value for developing superior stress-resistant tea varieties. However, this patent does not disclose the tea plant CsGSTU8 gene of this invention or its application in the degradation of glyphosate. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to find the key gene for detoxification of glyphosate in tea trees and apply it.

[0005] The present invention solves the above-mentioned technical problems through the following technical means:

[0006] The first aspect of the present invention provides a tea plant CsGSTU8 gene, wherein the tea plant CsGSTU8 gene is a glutathione S-transferase gene, and the nucleotide sequence of the tea plant CsGSTU8 gene is shown in SEQ ID NO.1.

[0007] Beneficial effects: The CsGSTU8 gene in tea trees and its application demonstrate its response to glyphosate stress in tea trees and its detoxification effect on glyphosate in yeast. This provides a target gene for addressing the problem of glyphosate phytotoxicity in tea trees and offers theoretical support for breeding new herbicide-resistant tea varieties.

[0008] A second aspect of the present invention provides a protein encoded by the CsGSTU8 gene of the tea plant, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] A third aspect of the present invention provides a tea tree expression vector containing the aforementioned tea tree CsGSTU8 gene.

[0010] Preferably, it is obtained by digesting the fragment shown in SEQ ID NO.1 into vector pYES2 or vector pMALC5X.

[0011] The fourth aspect of this invention proposes the application of the above-mentioned tea tree CsGSTU8 gene in mitigating and degrading the toxic effects of glyphosate on yeast strains.

[0012] The fifth aspect of this invention proposes the application of the protein encoded by the CsGSTU8 gene of the tea plant in mitigating and degrading the toxic effects of glyphosate on yeast strains.

[0013] The sixth aspect of this invention provides a method for constructing a yeast strain with the ability to alleviate and degrade glyphosate toxicity, comprising the following steps:

[0014] (1) Construction of pYES2-CsGSTU8 vector:

[0015] Using pEASY-T1::CsGSTU8 plasmid as a template, PCR amplification was performed with upstream primer 5'-TTGGTACCGAGCTCGGATCCATGGCAGAACAAGTGAAGTTG-3' (SEQ ID NO.10) and downstream primer 5'-GATGGATATCTGCAGAATTCCTACTCATATTTAGGACCGC-3' (SEQ ID NO.11) to obtain the target gene. The pYES2 vector was purified by enzyme digestion, and the target gene fragment was ligated into the pYES2 vector using homologous recombination to obtain the recombinant plasmid pYES2-CsGSTU8 vector.

[0016] (2) The recombinant plasmid pYES2-CsGSTU8 vector was transformed into a yeast mutant, and the mutant was screened and cultured to obtain a yeast strain that could alleviate and degrade glyphosate toxicity.

[0017] Preferably, the yeast mutant is a glyphosate-sensitive strain.

[0018] Preferably, the glyphosate-sensitive strain includes yeast BY4741.

[0019] The seventh aspect of the present invention proposes a yeast strain that has the effect of mitigating and degrading glyphosate toxicity, obtained by the above-described construction method.

[0020] The advantages of this invention are:

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] (1) In this invention, a glutathione S-transferase located in the nucleus and cytoplasm of tea plants was cloned, and its detoxification function against glyphosate was verified in yeast. This gene was upregulated in both tea leaves and roots in response to glyphosate stress. This invention also provides a recombinant plasmid containing the CsGSTU8 gene and a transgenic engineered bacterium. This invention enriches the research on glyphosate resistance in tea plants, helps to elucidate the molecular mechanism of tea plant response to glyphosate stress, and can provide theoretical support for solving the problem of glyphosate damage in tea plants. The CsGSTU8 protein in tea plants exhibits the strongest prokaryotic expression and protease activity at pH 8.0.

[0023] (2) CsGSTU8 in tea tree leaves and roots were upregulated in response to glyphosate stress. CsGSTU8 was highly expressed in tea tree roots. The pCAMBIA-1305 plasmid (containing GFP tag) constructed from this gene was transformed into Agrobacterium and then infected into tobacco leaves. It was observed that the gene was located in the nucleus and cytoplasm. The pYES2 plasmid constructed from this gene was transformed into glyphosate-sensitive strain BY4741 and was able to restore the growth ability of yeast mutants on high concentration glyphosate medium. Attached Figure Description

[0024] Figure 1 The expression pattern of CsGSTU8 in leaves and roots after glyphosate treatment;

[0025] Figure 2 The expression level of CsGSTU8 in different tissues and organs of tea plant;

[0026] Figure 3 Subcellular localization analysis of CsGSTU8 in tobacco;

[0027] Figure 4 For prokaryotic expression of CsGSTU8 protein ( Figure 4 A) and its enzyme activity under different pH conditions ( Figure 4 B);

[0028] Figure 5 The detoxification effect of CsGSTU8 on different concentrations of glyphosate in yeast;

[0029] Figure 6 OD of different yeast strains under different concentrations of glyphosate pressure 600 Value change. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0032] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0033] Example 1:

[0034] 1. Cloning and sequence structure analysis of the CsGSTU8 gene

[0035] The CsGSTU8 gene in tea plants is a glutathione S-transferase gene. Its cloning and sequence structure analysis are as follows:

[0036] The material used was mature tea leaves of the national-level superior variety 'Shuchazao' from the Tea Germplasm Resource Garden of Anhui Agricultural University. After thoroughly grinding the tea samples in liquid nitrogen, RNA was extracted from the tissue samples using an RNA extraction kit (Vazyme, Nanjing, China) according to the manufacturer's instructions. RNA quality was assessed using agarose gel electrophoresis. RNA concentration was then measured using a nucleic acid analyzer, and the RNA was reverse transcribed into cDNA using both a standard reverse transcription kit (Vazyme, Nanjing, China) and a quantitative reverse transcription kit (Vazyme, Nanjing, China). The cDNA obtained from the standard reverse transcription was used for RT-PCR gene cloning, while the quantitative reverse transcription sample was used for subsequent quantitative real-time PCR experiments. The CsGSTU8 gene was amplified by RT-PCR using the reverse-transcribed cDNA as a template. The upstream primer was 5'-GAGGTCTAAGTAACAAGTCTTG-3' (SEQ ID NO. 3).

[0037] Downstream primer: 5'-ACTAGTCTGCAGTTTGACAATG-3' (SEQ ID NO.4). The 25 μl PCR reaction system consisted of: 12.5 μL LA-taq (Takara, Beijing, China), 9.5 μL sterile water, 1 μL each of upstream and downstream primers and cDNA template for conventional reverse transcription.

[0038] The reaction program was as follows: 95℃ for 10 sec, 98℃ for 10 sec, 58℃ for 15 sec, 72℃ for 2 min, 72℃ for 10 min, for a total of 32 cycles. The PCR amplified product, CsGSTU8 gene, was separated by agarose gel electrophoresis. The product was then ligated into the pEASY-T1 (TransGen, Beijing, China) vector and transformed into Trans-T1 (TransGen, Beijing, China) competent cells. After 24 h of growth on LB medium supplemented with kanamycin, single clones were picked for sequencing. The obtained CsGSTU8 gene nucleotide sequence is shown in SEQ ID NO.1, as follows:

[0039] ATGGCAGAACAAGTGAAGTTGTTTGGTGCTTGGGGAAGCCCTTGTGTTCGCAGAGTTGAGATTGCCCTGAAAATGAAAGGCGTCGAATACGAATTCATTGAAGAAGATCTAACTAACAAGAGTCCTCTTCTTCTTAAATACAACCCTGTTCACAAGAAGATTCCAGTG CTGTTTGCACAATGGAAAGCCGATTTGTGAGTCGGTTGTGATTATTGAGTACATTGATGAGACCTGGAAAGGCACTCCCATCTTGCCTGCAGATCCTTATGACAGAGCCATGGCACGTTTTTTGGGCTAAGTTCATCGATGAGAAGTGCTTGCCTGCACTATGGAAGGCTT GTTGGAGCAAAGATGAGGAACAAGAGAAGGCCATGGAAGAAGCATGTGAGCTCCTGAAAACACTAGAAGCCCAACTCAAGGGTAACAAGTTCTTTGGAGGAGATAACATTGGACTTGTAGACATTGTTGCTAACTTCATCGGATTCTGGGTTGAAGTCCTTCTAGAAGC CGCGGGATCAACAGATTTGTTGACAGACGAGAAATTTCCCATCTTATGCAAATGGAAGCATGCCTATGTGAACTGCCCAGTTATCAAAGAAAATCTGCCTCCCAAAGAGAAACTTTTGGCCTTCTTCAGAGCTCGCCAACAACCCGCTAGCGGTCCTAAATATGAGTAG

[0040] The protein encoded by the CsGSTU8 gene has the following amino acid sequence as shown in SEQ ID NO.2:

[0041] MAEQVKLFGAWGSPCVRRVEIALKMKGVEYEFIEEDLTNKSPLLLKYNPVHKKIPVLLHNGKPICESVVIIEYIDETWKGTPILPADPYDRAMARFWAKFIDEKCLPALWKA CWSKDEEQEKAMEEACELLKTLEAQLKGNKFFGGDNIGLVDIVANFIGFWVEVLLEAAGSTDLLTDEKFPILCKWKHAYVNCPVIKENLPPKEKLLAFFRARQQPASGPKYE

[0042] 2. Expression pattern of CsGSTU8 gene in tea leaves and roots after glyphosate treatment

[0043] Material 1 used in this study was a mature 'Shuchazao' tea tree, a national-level superior variety, from the Tea Germplasm Resource Garden of Anhui Agricultural University. Glyphosate at a concentration of 6.25 g / L was sprayed on both sides of the leaves until droplets fell. The second leaf of the tender shoots was collected on day 1 and day 5, respectively. Material 2 used one-year-old 'Shuchazao' clonal plug seedlings. The roots were treated with the same concentration of glyphosate, and tender root tissue was collected on day 1 and day 5, respectively. Both materials were treated with water using the same method as controls. Three biological replicates were performed for each of the experimental and control groups. Samples were rapidly frozen in liquid nitrogen for RNA extraction. RNA extraction and reverse transcription methods were the same as above.

[0044] The qRT-PCR reaction system consisted of 10 μL: 5 μL of TBGreen Premix (Takara, Beijing, China), 3.2 μL of sterile water, 0.3 μL each of forward and reverse primers, and 1.2 μL of cDNA template for quantitative reverse transcription. qRT-PCR analysis was performed on a CFX96™ real-time PCR system (Bio-Rad). The analysis procedures were as follows: ① 95℃ for 5 min; ② 39 cycles of 95℃ for 10 sec, 58℃ for 30 sec, and 72℃ for 30 sec; ③ Melting curves were plotted from 65℃ to 95℃ at a rate of 0.1℃ / sec. Upstream primer: 5'-GAGGTCTAAGTAACAAGTCTTG-3' (SEQ ID NO.3), downstream primer: 5'-AATGAATTCGTATTCGACGCC-3' (SEQ ID NO.5). Using the tea plant CsGADPH gene as an internal control, upstream primer: 5'-TTGGCATCGTTGAGGGTCT-3' (SEQ ID NO.6), downstream primer: 5'-CAGTGGGAACACGGAAAGC-3' (SEQ ID NO.7). The results were analyzed using 2... -△Ct The relative expression level of CsGSTU8 was calculated using this method.

[0045] like Figure 1 The results showed that CsGSTU8 expression was significantly upregulated in tea tree leaves after 1 and 5 days of glyphosate treatment, with increases of 4.15 and 3.22 times compared to the control (CK), respectively. In the root tissues of tea trees treated with glyphosate, CsGSTU8 expression was not significantly different from the control (CK) at 1 day, but was significantly upregulated (2.19 times) at 5 days. These results indicate that the CsGSTU8 gene in tea trees responds to glyphosate stress.

[0046] 3. Analysis of CsGSTU8 gene expression patterns in different tissues of tea plants

[0047] Different tissues and organs of mature 'Shuchazao' tea trees were collected from the tea germplasm resource nursery of Anhui Agricultural University, including buds, first leaves, second leaves, third leaves, mature leaves, stems, roots, and fruits. These samples were immediately flash-frozen in liquid nitrogen and stored at -80°C. RNA was then extracted from these samples and reverse-transcribed into cDNA. The expression pattern of the target gene was analyzed by qRT-PCR using the cDNA as a template, following the same method.

[0048] Depend on Figure 2The results of qRT-PCR showed that CsGSTU8 was expressed at the highest level in roots, followed by mature leaves, while its expression level in other organs was much lower than that in roots.

[0049] 4. Subcellular localization of CsGSTU8 in tea plants

[0050] (1) Construction of pCAMBIA1305-CsGSTU8 vector

[0051] Using pEASY-T1::CsGSTU8 plasmid as a template, PCR amplification was performed with the upstream primer 5'-GACAGCCCAGATCACTAGTATGGCAGAACAAGTGAAGTTG-3' (SEQ ID NO.8) and the downstream primer 5'-CTTGC TCACCATGGATCCCTAGCGGTCCTAAATATGAG-3' (SEQ ID NO.9). The PCR product was recovered by 1.5% agarose gel electrophoresis. The pCAM BIA1305 (containing a GFP tag) vector was purified by double digestion at specific restriction enzyme sites using restriction endonucleases. The target gene fragment was ligated into the pCAMBIA1305 vector using homologous recombination. The recombinant plasmid was transformed into Trans-T1 E. coli competent cells, and single clones were selected for sequencing.

[0052] (2) Agrobacterium infection of tobacco

[0053] Plasmids were extracted from correctly sequenced monoclonal strains and transformed into EHA105 Agrobacterium competent cells. Screening was performed on LB medium supplemented with rifampicin and kanamycin. After 48 hours, monoclonal strains were selected for PCR verification. Positive monoclonal strains were cultured at 28°C with shaking until the OD reached 0.8–1.0. After centrifugation, the cells were resuspended until the OD reached 0.6. The resuspended bacterial solution was injected into healthy Tobacco Benzovia plants (the injected bacterial solution completely submerged the tobacco leaf). After culturing in the dark for 48 hours, infected leaves were injected with DAPI (4',6-diamidinyl-2-phenylindole) as a nuclear localization marker. The injected sites were then excised and prepared for observation using a laser confocal microscope (Leica, Germany). Figure 3 This is a subcellular localization map of CsGSTU8 in tobacco. (Example) Figure 3 As shown, GFP represents pCAMBIA1305-CsGSTU8 green fluorescent protein; DAPI represents the nuclear localization marker; Bright represents the bright-field image of pCAMBIA1305-CsGSTU8; and Merged represents the fusion image of GFP, DAPI, and Bright.

[0054] Depend on Figure 3It can be seen that the GFP signal of the empty pCAMBIA1305 vector can be observed throughout the cell, and the CsGSTU8-GFP fluorescence signal can be detected in both the nucleus and cytoplasm, and it overlaps with the nuclear region stained by DAPI, indicating that CsGSTU8 is located in the nucleus and cytoplasm.

[0055] 5. Prokaryotic expression and in vitro enzyme activity verification of CsGSTU8 protein

[0056] (1) Prokaryotic expression and purification of CsGSTU8 protein

[0057] The positive monoclonal strain was placed in LB medium supplemented with ampicillin and cultured at 37°C with shaking until OD. 600 =0.5, add 0.1M IPTG and transfer to a 16℃ shaker for 12h to induce the expression of the target protein. After that, centrifuge the bacterial culture at 3500rpm for 10min, discard the supernatant and resuspend in PBS buffer. Place the resuspended bacterial culture in a cell sonicator to disrupt the cells until the bacterial culture is clear. Then, use the principle of interaction between starch sugar resin (NEB, USA) and MBP tag to purify the protein. The protein purity is detected by SDS-Page gel electrophoresis.

[0058] (2) CsGSTU8 enzyme activity assay

[0059] The concentration of purified CsGSTU8 protein was detected using the Coomassie brilliant blue assay, and the required amount of protein solution to be added to the enzyme activity system was calculated based on the measured protein concentration. Enzyme activity was measured using CDNB and GSH as substrates. The substrates were added to prepared phosphate and Tris-HCl buffers at different pH values, and the target protein was added last. The reaction was carried out at room temperature for 2 min, and the activity of the target protease was measured using a microplate reader (SpectraMax M2).

[0060] like Figure 4 As shown in the figure, SDS-PAGE electrophoresis reveals that the extracted CsGSTU8 protein has good purity, and the molecular weight of the CsGSTU8 protein linked to the MBP tag is approximately 70 kDa. Figure 4 (A) Excluding the MBP-tagged protein molecular weight, the CsGSTU8 protein has a molecular weight of approximately 25 kDa. Its enzyme activity at different pH concentrations was verified using phosphate-buffered saline (pH gradient set at 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0) and Tris-HCl buffer (pH gradient set at 7.5, 8.0, 8.5, and 9.0). The results showed that the optimal pH for CsGSTU8 protease activity was 8.0. Figure 4 B).

[0061] 6. Functional verification of the CsGSTU8 gene in yeast

[0062] (1) Construction of pYES2-CsGSTU8 vector

[0063] Using pEASY-T1::CsGSTU8 plasmid as a template, PCR amplification was performed with the upstream primer 5'-TTGGTACCGAGCTCGGATCCATGGCAGAACAAGTGAAGTTG-3' (SEQ ID NO.10) and the downstream primer 5'-GATGGATATCTGCAGAATTCCTACTCATATTTAGGACCGC-3' (SEQ ID NO.11). The PCR product was recovered by 1.5% agarose gel electrophoresis. After purification by double digestion of specific restriction enzyme sites of the pYES2 vector with restriction endonucleases, the target gene fragment was ligated into the pYES2 vector using homologous recombination. The recombinant plasmid was transformed into Trans-T1 E. coli competent cells, and single clones were selected for sequencing.

[0064] (2) CsGSTU8 transformation of yeast mutant

[0065] Yeast BY4741 is a glyphosate-sensitive strain that exhibits slow or even no growth in the presence of glyphosate, and can be used to verify the glyphosate stress resistance of candidate genes.

[0066] Transform the recombinant plasmid into BY4741 competent yeast cells (Weidi, Shanghai, China): (1) Insert the carrier DNA into a 95℃ metal bath for 5 min, then quickly insert it into ice after heating. (2) Take 100 μl of BY4741 competent cells and place them on ice to thaw. Add 1 μg of pre-chilled target plasmid, 10 μl of pretreated carrier DNA, and 500 μL of PEG / LiAc, and mix well. (3) Incubate at 30℃ for 30 min, turning and mixing every 15 min. (4) Transfer the competent cells to a 42℃ water bath for 15 min, turning and mixing every 7.5 min. (5) Centrifuge the competent cells at 5000 rpm for 40 s and discard the supernatant. Resuspend in sterile water and centrifuge at 5000 rpm for 40 s, then discard the supernatant. (6) Resuspend in 50 μL of sterile water and plate on yeast culture dishes lacking uracil. Select and culture for 72 h. (7) Pick a single clone and place it in sterile water. Boil it at 100℃ for 10 min and then centrifuge it. Take the supernatant as a template for PCR to verify the positive single clone strain.

[0067] (3) Verification of the yeast dosing function of tea tree CsGSTU8

[0068] Positive monoclonal strains were placed in YPDA and cultured on a shaker at 28°C for 24 hours. After centrifugation at 3500 rpm, the supernatant was discarded. Yeast strains transfected with the pYES2 empty vector plasmid served as a negative control. Yeast cultures containing both the empty vector plasmid and the CsGSTU8 recombinant plasmid were resuspended in sterile water to OD200. 600 The value was 0.6 (inter-sample error less than 0.02). Each bacterial culture was diluted sequentially to four gradients (1, 10, 100, and 1000 times dilution) and then spotted onto the plate.

[0069] All carbon sources (glucose, lactose, raffinose) and glyphosate isopropylamine saline solutions in the yeast culture media were sterilized by filtration through a 0.22 μm membrane before use. When needed, these solutions were added to uracil-free yeast solid culture medium at approximately 60°C, and then poured into petri dishes. A grid was drawn at the bottom of SD-Ura solid culture media containing different concentrations of glyphosate. 10 μl of bacterial solution was taken and dropped onto the intersections of the grid, with each horizontal row representing different concentrations of the same bacterial solution, and each vertical column representing different concentrations of the same bacterial solution. The media were completely air-dried in a clean bench before being sealed and incubated upside down in a 28°C incubator. Yeast growth was observed after 5 days.

[0070] like Figure 5 As shown, when the empty yeast vectors pYES2 and pYES2-CsGSTU8 were transformed into glyphosate-sensitive yeast BY4741, the growth of BY4741 transformed with the empty pYES2 vector was significantly inhibited under different concentrations of glyphosate (0.05%, 0.1%, and 0.2%), while the growth of BY4741 transformed with CsGSTU8 was normal. This indicates that CsGSTU8 is a functional gene protein that can detoxify glyphosate toxicity.

[0071] (4) Growth status of yeast strains under different concentrations of glyphosate stress

[0072] Positive monoclonal strains were revived and cultured in YPDA, then centrifuged at 3500 rpm and the supernatant was discarded. Yeast strains transformed with the pYES2 empty vector plasmid were used as negative controls. Yeast cultures containing both the empty vector plasmid and the CsGSTU8 recombinant plasmid were resuspended in sterile water at OD. 600 The value was 0.6 (error less than 0.02). 500 μL each of the positive monoclonal strain and the empty vector control were added to 25 mL of SD-Ura liquid medium containing different concentrations of glyphosate (0.05% and 0.1%). Three biological replicates were set up for each of the yeast strains transformed with the empty vector and the recombinant vector. The OD of the bacterial suspension was measured every 12 h using a UV spectrophotometer. 600The values ​​were measured eight times over a period of 96 hours. Note that the carbon sources (glucose, lactose, raffinose) and glyphosate isopropylamine saline in the yeast culture medium were sterilized by passing them through a 0.22 μm filter before use. They were then added to the uracil-deficient yeast liquid culture medium when needed.

[0073] like Figure 6 As shown, the empty yeast vectors pYES2 and pYES2-CsGSTU8 were transformed into glyphosate-sensitive yeast BY4741. The BY4743 bacterial culture containing the empty pYES2 vector was then transferred into liquid media containing different concentrations of glyphosate (0.05% and 0.1%). 600 This indicates that its growth was significantly inhibited within 96 hours, and the OD of the BY4743 yeast culture transformed with CsGSTU8 was [data missing]. 600 At 60 h, a significant increase began to appear compared to the control, and at 96 h, the OD in liquid growth medium with glyphosate concentrations of 0.5 g / L and 1 g / L was [value missing]. 600 The concentrations were 0.767 and 0.72, respectively, which were 9.13 and 5 times that of the control. These results indicate that CsGSTU8 has a detoxifying effect against glyphosate stress.

[0074] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Tea tree CsGSTU8 The application of genes in mitigating and degrading the toxic effects of glyphosate on yeast strains is characterized by, The tea tree CsGSTU8 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A method for constructing a yeast strain with the ability to alleviate and degrade glyphosate toxicity, characterized in that, Includes the following steps: (1) Construction of pYES2-CsGSTU8 vector: Using pEASY-T1::CsGSTU8 plasmid as a template, with upstream primer: 5' - TTGGTACCGAGCTCGGATCCATGGCAGAACAAGTGAAGTTG - 3' and downstream primer: 5' - GATGGATATCTGCAGAATTCCTACTCATATTTAGGACCGC - 3', PCR amplification was performed to obtain the target gene. The pYES2 vector was purified by enzyme digestion, and the target gene fragment was ligated into the pYES2 vector by homologous recombination to obtain the recombinant plasmid pYES2-CsGSTU8 vector. (2) The recombinant plasmid pYES2-CsGSTU8 vector was transformed into a yeast mutant, and the mutant was screened and cultured to obtain a yeast strain that could alleviate and degrade glyphosate toxicity.

3. The construction method according to claim 2, characterized in that, The yeast mutant is a glyphosate-sensitive strain.

4. The construction method according to claim 2, characterized in that, The glyphosate-sensitive strains include yeast BY4741.

5. The construction method according to claim 2, characterized in that, The screening culture specifically involves resuspending the sample in sterile water and then spreading it onto a yeast culture dish lacking uracil for screening culture.

6. The construction method according to claim 5, characterized in that, Screening and culturing for 72 hours.

7. The construction method according to claim 2, characterized in that, In (1), the pYES2 vector was purified by enzyme digestion, and the enzyme was a restriction endonuclease.

8. A yeast strain with the ability to alleviate and degrade glyphosate toxicity obtained by the construction method according to any one of claims 2-7.

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