Method for improving the degradation ability of thiamethoxam by modifying tea tree csGST1 gene and application

Abstract

The present application relates to the technical field of genetic engineering, and provides a method for improving the degradation capacity of thiamethoxam by modifying the tea tree CsGST1 gene, and overexpressing the tea tree glutathione S-transferase CsGST1 gene through a genetic engineering approach, wherein the nucleotide sequence of the CsGST1 gene is shown as SEQ ID NO. 1. The present application also provides the application of the method for improving the degradation capacity of thiamethoxam by modifying the tea tree CsGST1 gene in cultivating new tea tree varieties with enhanced pesticide metabolism capacity. In the present application, the CsGST1 gene with a positive regulation effect on the degradation capacity of the commonly used insecticide thiamethoxam for tea trees is screened, the tea tree glutathione S-transferase CsGST1 gene is overexpressed through a genetic engineering approach, and tea tree plants with stronger thiamethoxam degradation capacity can be obtained, thereby providing a theoretical basis and gene resources for cultivating new tea tree varieties with enhanced pesticide metabolism capacity.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method and application for improving the degradation capacity of thiamethoxam by modifying the CsGST1 gene in tea plants. Background Technology

[0002] The tea tree (Camellia sinensis (L.) O. Kuntze) is an evergreen woody economic plant that is frequently attacked by pests, diseases, and weeds during its growth. Currently, chemical control is still the main method used in tea gardens.

[0003] Neonicotinic pesticides are the fastest-growing and most widely used type of plant-derived pesticides after pyrethroids. Thiamethoxam, as a second-generation neonicotinic insecticide, is widely used in various economic crops such as tea, tobacco, rice, potatoes, and cotton. However, in modern agricultural production, the frequent occurrence of pests and diseases may lead to excessive or inappropriate use of pesticides, potentially resulting in pesticide residues such as thiamethoxam in tea, thus posing a potential threat to consumer health.

[0004] Therefore, we can try to develop a method to effectively improve the ability of tea trees to degrade thiamethoxam, which will provide a theoretical basis for the regulatory mechanism of tea trees in response to exogenous stress, and also provide a theoretical basis for cultivating new tea varieties with enhanced pesticide metabolism. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method and application for improving the degradation ability of thiamethoxam by modifying the CsGST1 gene of tea plants.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0007] A method for improving the degradation capacity of thiamethoxam by modifying the CsGST1 gene of tea plants involves overexpressing the glutathione S-transferase CsGST1 gene of tea plants through genetic engineering; wherein the nucleotide sequence of the CsGST1 gene is shown in SEQ ID NO.1.

[0008] As one of the preferred embodiments of the present invention, the protein sequence encoded by the CsGST1 gene is shown in SEQ ID NO.2.

[0009] As one of the preferred embodiments of the present invention, the genetic engineering approach refers to:

[0010] The CsGST1 gene fragment shown in SEQ ID NO.1 was ligated into the vector pCAMBIA1305.1 to obtain the tea expression vector pCAMBIA1305.1-CsGST1; then the tea expression vector pCAMBIA1305.1-CsGST1 was transformed into Agrobacterium tumefaciens EHA105, and Agrobacterium tumefaciens was used to mediate the transformation into the target plant.

[0011] As one of the preferred embodiments of the present invention, the function of the CsGST1 gene is initially verified by the transient transformation technology of tobacco: the tea expression vector pCAMBIA1305.1-CsGST1 is transiently expressed in tobacco through Agrobacterium-mediated transformation.

[0012] As one of the preferred embodiments of the present invention, the CsGST1 gene expression product is used to positively regulate the thiamethoxam degradation capacity of the target plant.

[0013] The above-mentioned method of improving the thiamethoxam degradation ability by modifying the CsGST1 gene of tea trees is applied to the breeding of new tea varieties with enhanced pesticide metabolism.

[0014] principle:

[0015] This invention analyzes metabolomic and transcriptomic data of tea plants treated with thiamethoxam, revealing a significant positive correlation between the expression level of glutathione S-transferase (GST) and treatment time. Simultaneously, amino acid-related metabolic pathways in the metabolome were also significantly affected. Subsequently, the changes in the expression levels of GST-related genes in tea plants over treatment time were specifically analyzed, ultimately identifying the CsGST1 gene as potentially playing a crucial role in thiamethoxam detoxification. Next, the CsGST1 gene was cloned and validated from tea plants for the first time. Subcellular localization analysis of the CsGST1 gene in tea plant protoplasts revealed its location in the cytoplasm. Further demonstration using Agrobacterium-mediated transient tobacco transformation technology showed that the CsGST1 gene can enhance the metabolic degradation capacity of plants against the neonicotinoid insecticide thiamethoxam.

[0016] The advantages of this invention compared to the prior art are as follows: In this study, the CsGST1 gene, which has a positive regulatory effect on the "thiamethoxam degradation ability" of tea trees, was screened. By overexpressing the CsGST1 gene of glutathione S-transferase in tea trees through genetic engineering, tea plants with stronger "thiamethoxam degradation ability" can be obtained, providing a theoretical basis and gene resources for cultivating new tea varieties with enhanced pesticide metabolism. Attached Figure Description

[0017] Figure 1This is a diagram illustrating the tissue-specific expression pattern of the tea plant glutathione S-transferase gene CsGST1 in Example 2.

[0018] Figure 2 This is the expression profile of the glutathione S-transferase gene CsGST1 in tea plants in Example 2;

[0019] Figure 3 The images show the enzyme digestion diagrams of the PCR product and the vector pCAMBIA1305.1 in Example 3 (Figure A shows the PCR amplification product of CsGST1; Figure B shows the plasmid double enzyme digestion verification product after CsGST1 is constructed and ligated into the pEASY-Blunt vector; Figure C shows the plasmid double enzyme digestion verification product after CsGST1 is constructed into the pCAMBIA1305.1 vector).

[0020] Figure 4 These are the results of laser confocal microscopy scanning in Example 4 (in the figures, a and e show the single channel of GFP green fluorescence; b and f show the single channel of chloroplast spontaneous red fluorescence; c and g show the bright field; d and h show the image displayed by all three channels; pCAMBIA1305.1 empty vector is the control).

[0021] Figure 5 This is a graph verifying the degradation function of transiently expressed CsGST1 in tobacco against thiamethoxam in Example 5 (Figure A shows the degradation capacity of transiently expressed CsGST1 in tobacco against X dose of thiamethoxam, recommended dose: 0.015 kg ai / ha; Figure B shows the degradation capacity of transiently expressed CsGST1 in tobacco against 2X dose of thiamethoxam, 2x dose: 0.03 kg ai / ha; Figure C shows the degradation capacity of transiently expressed CsGST1 in tobacco against 3X dose of thiamethoxam, 3x dose: 0.045 kg ai / ha). Detailed Implementation

[0022] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents and experimental methods used in the following embodiments are all conventional reagents or methods in the art and will not be described again.

[0023] Example 1

[0024] Example 1: Screening for the CsGST1 gene:

[0025] Previous analysis of metabolomics and transcriptomics data from tea plants treated with thiamethoxam revealed a significant positive correlation between glutathione S-transferase (GST) expression and treatment time, along with a significant impact on amino acid-related metabolic pathways. Subsequently, the study analyzed the changes in the expression levels of glutathione S-transferase-related genes in tea plants over treatment time, ultimately identifying the CsGST1 gene as potentially playing a crucial role in thiamethoxam detoxification.

[0026] The specific nucleotide sequence of the CsGST1 gene is shown in SEQ ID NO.1, and the protein sequence it encodes is shown in SEQ ID NO.2.

[0027] Example 2: Tissue-specific expression pattern analysis of CsGST1 in tea plants:

[0028] The expression patterns of the CsGST1 gene in eight tissues and organs of the tea plant—buds, flowers, fruits, young leaves, mature leaves, old leaves, roots, and stems—are as follows: Figure 1 As shown. Figure 1 The results showed that CsGST1 was specifically highly expressed in the roots of tea plants.

[0029] in addition, Figure 2 Transcriptome data from tea plants were analyzed, showing that under thiamethoxam treatment, the expression level of CsGST1 showed a significant positive correlation with the control group (the blank group of tea plants without thiamethoxam spraying) as the treatment time increased.

[0030] Example 3: Cloning and sequence structure analysis of the CsGST1 gene:

[0031] The young roots of the national-level superior variety 'Shuchazao', grown in the Nongcui Garden of Anhui Agricultural University, were selected for RNA extraction. Total RNA was extracted using the RNA prep Pure Plant Kit (Tiangen, Beijing, China), following the instructions. The RNA content and quality were determined using a UV spectrophotometer.

[0032] use The One-Step gDNARemoval and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) kit reverse transcribes RNA into cDNA.

[0033] Primers were designed based on the coding sequence of the glutathione S-transferase gene CsGST1, and restriction enzyme sites XbaI and BamHI were added to the primers respectively. The upstream primer was 5'-GCTCTAGA ATGGCAACGGTGAAGGTC-3', and the downstream primer was 5'-CGGGATCC GTTATTTTTCATGGCAACAACC-3'.

[0034] Using cDNA first strand as a template, PCR was performed using standard methods to amplify the tea plant glutathione S-transferase gene CsGST1. The 50 μL PCR reaction mixture consisted of: Autoclaved, distilled water (33-X) μL, 10×PCR Buffer for KOD-Plus-Neo (5 μL), 2 mM dNTPs (5 μL), 25 mM MgSO4 (3.0 μL), forward and reverse primers (1.5 μL each), KOD-Plus-Neo (1 U / μL) (1 μL), and template (1 μL). The reaction program was as follows: 98℃ for 10 sec, 98℃ for 10 sec, 57℃ for 30 sec, 72℃ for 2 min, 72℃ for 10 min, for 35 cycles.

[0035] After purification and recovery, the PCR product CsGST1 gene was ligated into the pEASY-Blunt Simple Cloning Vector (TransGen Biotech, Beijing, China) to obtain the pEASY-Blunt-CsGST1 plasmid, which was then transformed into E. coli competent cells Trans1-T1 and sent to Sangon Biotech for sequencing. The nucleotide sequence is shown in SEQ ID NO.1.

[0036] After digesting the correctly sequenced gene with restriction endonucleases, the CsGST1 gene was ligated to the vector pCAMBIA1305.1 to construct the recombinant expression plasmid pCAMBIA1305.1-CsGST1. The constructed expression vector was then transformed into *E. coli* competent cells Trans1-T1 to construct the recombinant bacteria. Several positive recombinant bacteria were selected, and the plasmid was extracted and identified by restriction endonuclease digestion (results are shown in the figure). Figure 3 (As shown).

[0037] Example 4: Subcellular localization of CsGST1 in tea plants:

[0038] To further clarify the subcellular distribution of CsGST1, this embodiment conducted a subcellular localization experiment on CsGST1 in tea plant protoplasts. First, the plasmid of the constructed CsGST1 subcellular localization vector pCAMBIA1305-CsGST1 was extracted. Simultaneously, protoplast cells from young tea seedling leaves were extracted. The empty vector pCAMBIA1305.1 and the pCAMBIA1305.1-CsGST1 expression vector plasmid were transformed into *Shuchazao* protoplasts using PEG transformation. After transformation, the protoplasts were cultured in the dark for 24 hours, and GFP fluorescence was observed and photographed using a laser confocal microscope.

[0039] Figure 4 These are images of the results observed by laser confocal microscopy. In the images, (a) and (e) show the single channel of GFP green fluorescence; (b) and (f) show the single channel of chloroplast auto-red fluorescence; (c) and (g) show the bright field; and (d) and (h) show the image displayed by all three channels.

[0040] Depend on Figure 4 It can be seen that the green fluorescent signal in the empty vector pCAMBIA1305.1 fills the entire protoplast cell, but there is no specific subcellular localization region; while the green fluorescent signal of pCAMBIA1305.1-CsGST1 is only enriched in the cytoplasm of tea plant protoplasts, and no fluorescent signal was detected in other parts; the results preliminarily confirm that CsGST1 is located in the cytoplasm.

[0041] Example 5: The ability of transiently expressed CsGST1 gene to degrade thiamethoxam in plants:

[0042] Since the transient conversion technology of tobacco is a relatively mature technology in this field, the following uses tobacco as an example to further verify the effect of the transient expression of the CsGST1 gene in the plant's ability to degrade thiamethoxam.

[0043] The pCAMBIA1305.1-CsGST1 vector was transformed into Agrobacterium tumefaciens EHA105, and positive clones were identified by conventional PCR. Single clones that were verified by PCR were picked and inoculated into 5 mL of liquid LB medium (containing 50 μg / mL RIF and 100 μg / mL Spec) and cultured until OD500. 600=0.8~1.2. Take 1 mL of Agrobacterium culture that has been cultured overnight, inoculate it into 50 mL of liquid LB medium containing (50 μg / mL RIF and 100 μg / mL Spec), and culture at 28℃ and 200 r / min for 1~2 days. Centrifuge at 5000 r / min for 5 min to collect the bacterial cells, and resuspend the bacterial cells in a resuspension solution with pH adjusted to 5.6 using 10 mM MgCl2 and 10 mM 2-(N-morpholino)ethanesulfonic acid; then resuspend the bacterial cells in MMA until the OD value of the bacterial solution is 0.6~0.8, add acetylsuccinone (1 μL AS solution per 1 mL of bacterial solution) to the bacterial solution, and incubate at room temperature for 2 h; select tobacco plants with good growth, inject the bacterial solution into the back of tobacco leaves using a disposable 1 mL syringe, and mark them; after dark treatment of the injected tobacco, observe whether the gene is transiently expressed in the tobacco using a laser confocal microscope.

[0044] After confirming the transient expression of CsGST1 in tobacco, tobacco was sprayed with different concentrations of thiamethoxam standard (Dr. Ehrenstorfer, 99.8%), including X (recommended dose: 0.015 kg ai. / ha), 2X (2 times the dose: 0.03 kg ai / ha), and 3X (3 times the dose: 0.045 kg ai / ha). Tobacco leaves were collected at 36 h, 48 h, and 72 h after spraying, and samples were prepared from only the injected leaves.

[0045] The tobacco leaf sample was ground and pulverized in a mortar. 0.5g of the pulverized fresh tea leaf sample was weighed and placed in a 50mL centrifuge tube. 5mL of acetonitrile was added, and the mixture was ultrasonically extracted for 10min. 0.5g of sodium chloride and 0.5g of anhydrous magnesium sulfate were weighed into a test tube and vortexed for 2min. The centrifuge tube was then centrifuged at 5000r / min for 5min at room temperature. 2mL of the supernatant was taken and placed in a container previously filled with 50mg PVPP, 5mg PSA, 20mg GCB, and 10mg C. 18 The extract was placed in a test tube containing 30 mg of anhydrous magnesium sulfate. The extract was vortexed for 2 min, then centrifuged at 10000 rpm for 10 min. 1 mL of the supernatant was collected and evaporated to near dryness under nitrogen. The residue was reconstituted with 1 mL of acetonitrile:water (15:85, v / v), filtered through a 0.22 μm filter membrane, and analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS).

[0046] Figure 5This is a verification of the degradation function of thiamethoxam by transient expression of CsGST1 in tobacco. In the figure, TMX-CK-X refers to the "residual amount of TMX when tobacco does not transiently express the CsGST1 gene but is sprayed with X doses of thiamethoxam (TMX) pesticide"; TMX-CsGST1-X refers to the "residual amount of TMX when tobacco is sprayed with X doses of thiamethoxam (TMX) pesticide after transient expression of the CsGST1 gene"; CLO-CK-X refers to the "content of thiamethoxam (CLO) generated by TMX metabolism (CLO is a metabolite of TMX) when tobacco does not transiently express the CsGST1 gene but is sprayed with X doses of TMX"; CLO-CsGST1-X refers to the "content of CLO generated by TMX metabolism when tobacco is sprayed with X doses of TMX after transient expression of the CsGST1 gene"; and so on.

[0047] like Figure 5 As shown, when the treatment concentration was X (recommended concentration: 0.015 kg ai / ha), the degradation of thiamethoxam in tobacco plants transiently transformed with CsGST1 was most significant. At this concentration, the degradation rates of thiamethoxam in tobacco plants transiently transformed with CsGST1 were 75.4%, 78.0%, and 69.8% at 36 h, 48 h, and 72 h, respectively. Simultaneously, the residual amount of thiamethoxam gradually decreased, suggesting that CsGST1, after transient transformation in tobacco, also has a certain degradation capacity for thiamethoxam. At a concentration of 3X (3 times the dose: 0.045 kg ai / ha), it was found that 46.45% of thiamethoxam in CsGST1 tobacco transiently transformed was degraded at 36 h. It was also observed that the degradation of thiamethoxam was inhibited with increasing treatment concentration.

[0048] The above results indicate that tobacco plants transiently expressing CsGST1 exhibit a certain ability to degrade thiamethoxam at different concentrations. Based on these results, we hypothesize that transient expression of the CsGST1 gene can enhance the plant's ability to degrade thiamethoxam. When applied to tea plants, the expression vector pCAMBIA1305.1-CsGST1 can be transformed into Agrobacterium tumefaciens EHA105, and the transformation can be mediated by Agrobacterium tumefaciens into the tea plant. This invention provides a theoretical basis and functional gene resources for developing new tea varieties with enhanced pesticide metabolism capabilities using molecular-assisted breeding.

[0049] 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, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for improving the degradation capacity of thiamethoxam in tobacco or tea, characterized in that, Through genetic engineering CsGST1 The gene is overexpressed in tobacco or tea plants; wherein, the CsGST1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1, characterized in that, The genetic engineering approach refers to: The SEQ ID NO.1 shown CsGST1 The gene fragment was ligated into the vector pCAMBIA1305.1 to obtain the expression vector pCAMBIA1305.1- CsGST1 Then, the expression vector pCAMBIA1305.1- CsGST1 Agrobacterium tumefaciens EHA105 was introduced, and the Agrobacterium tumefaciens was used to mediate the transfer into the target plant.

3. The method according to claim 1, characterized in that, Using tobacco instantaneous conversion technology CsGST1 Preliminary validation of gene function: The expression vector pCAMBIA1305.1- CsGST1 It was transiently expressed in tobacco via Agrobacterium-mediated transformation.

4. The method according to any one of claims 1 to 3, characterized in that, The CsGST1 The gene expression product is used to positively regulate the thiamethoxam degradation capacity of the target plant.

5. The application of the method as described in any one of claims 1 to 4 in the breeding of new tea varieties with enhanced thiamethoxam metabolism capacity.

Images