Method for improving the degradation ability of thiamethoxam by modifying tea tree csHDH3 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 tea tree CsHDH3 gene, and overexpresses the 3-hydroxyisobutyric acid dehydrogenase CsHDH3 gene of tea tree through a genetic engineering approach; wherein the nucleotide sequence of the CsHDH3 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 CsHDH3 gene in cultivating new tea tree varieties with enhanced pesticide metabolism capacity. The present application screens the CsHDH3 gene with positive regulation on the degradation capacity of the commonly used insecticide thiamethoxam for tea trees, overexpresses the 3-hydroxyisobutyric acid dehydrogenase CsHDH3 gene of tea tree through a genetic engineering approach, and can obtain tea tree plants with stronger thiamethoxam degradation capacity, 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 CsHDH3 gene of tea plants. Background Technology

[0002] The tea plant (Camellia sinensis (L.) O. Kuntze) is an important woody economic crop with significant economic value. Pesticides are crucial agricultural inputs, playing a vital role in controlling pests, diseases, and weeds, ensuring agricultural product safety, and increasing agricultural yields. However, pesticide residues can also lead to a series of safety issues, causing not only environmental pollution but also potential harm to human health.

[0003] Thiamethoxam is a neonicotinoid insecticide commonly used in tea gardens. It possesses stomach poison, contact, and systemic properties, selectively inhibiting the nicotinic acetylcholinesterase receptor (nAChR) in the central nervous system of pests, thereby blocking normal conduction and altering the electrical potential of the insect's nerve synaptic membrane, ultimately leading to paralysis and death. However, as mentioned earlier, insecticide residues like thiamethoxam pose a series of safety concerns, causing not only environmental pollution but also potential harm to human health. Therefore, there is an urgent need to develop a method to effectively improve the ability of tea plants to degrade thiamethoxam, further providing a theoretical basis for the regulatory mechanisms of tea plants in response to exogenous stress, and also providing a theoretical foundation for cultivating new tea varieties with enhanced pesticide metabolism capabilities. Summary of the Invention

[0004] 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 CsHDH3 gene of tea plants.

[0005] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0006] A method for improving the degradation capacity of thiamethoxam by modifying the CsHDH3 gene of tea plants involves overexpressing the 3-hydroxyisobutyrate dehydrogenase CsHDH3 gene of tea plants through genetic engineering; wherein the nucleotide sequence of the CsHDH3 gene is shown in SEQ ID NO.1.

[0007] As one of the preferred embodiments of the present invention, the protein sequence encoded by the CsHDH3 gene is shown in SEQ ID NO.2.

[0008] As one of the preferred embodiments of the present invention, the genetic engineering approach refers to:

[0009] The CsHDH3 gene fragment shown in SEQ ID NO.1 was ligated into the vector pCAMBIA1305.1 to obtain the tea expression vector pCAMBIA1305.1-CsHDH3; then the tea expression vector pCAMBIA1305.1-CsHDH3 was transformed into Agrobacterium tumefaciens EHA105, and Agrobacterium tumefaciens was used to mediate the transformation into the target plant.

[0010] As one of the preferred embodiments of the present invention, the function of the CsHDH3 gene is initially verified by the transient transformation technology of tobacco: the tea expression vector pCAMBIA1305.1-CsHDH3 is transiently expressed in tobacco through Agrobacterium-mediated transformation.

[0011] As one of the preferred embodiments of the present invention, the CsHDH3 gene expression product is used to positively regulate the thiamethoxam degradation capacity of the target plant.

[0012] The above-mentioned method of improving the thiamethoxam degradation ability by modifying the CsHDH3 gene of tea trees is applied to the breeding of new tea varieties with enhanced pesticide metabolism.

[0013] principle:

[0014] This invention analyzes metabolomic and transcriptomic data of tea plants treated with thiamethoxam, revealing a significant positive correlation between the expression level of 3-hydroxyisobutyrate dehydrogenase (CsHDHs) and treatment time. Simultaneously, amino acid-related metabolic pathways in the metabolome were also significantly affected. Subsequently, the expression levels of multiple genes involved in 3-hydroxyisobutyrate dehydrogenase (CsHDHs) in tea plants were analyzed over treatment time, ultimately identifying the CsHDH3 gene as potentially playing a crucial role in thiamethoxam detoxification. Next, the CsHDH3 gene was cloned and validated from tea plants for the first time. Subcellular localization analysis of the CsHDH3 gene in tea plant protoplasts revealed its location in the cytoplasm. Further evidence using Agrobacterium-mediated transient tobacco transformation technology demonstrated that the CsHDH3 gene can enhance the metabolic degradation capacity of plants against the neonicotinoid insecticide thiamethoxam.

[0015] The advantages of this invention compared to the prior art are as follows: In this study, the CsHDH3 gene, which has a positive regulatory effect on the "thiamethoxam degradation ability" of tea trees, was screened. By overexpressing the CsHDH3 gene of 3-hydroxyisobutyrate dehydrogenase 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

[0016] Figure 1 This is a tissue-specific expression pattern analysis diagram of the tea plant 3-hydroxyisobutyrate dehydrogenase gene CsHDH3 in Example 2;

[0017] Figure 2 This is the expression profile of the tea plant 3-hydroxyisobutyrate dehydrogenase gene CsHDH3 in Example 2;

[0018] Figure 3 These are the enzyme digestion images of the PCR product and the vector pCAMBIA1305.1 in Example 3 (In the image, A is the PCR amplification product of CsHDH3; B is the plasmid double enzyme digestion verification product after CsHDH3 constructs and ligates the pEASY-Blunt vector; C is the plasmid double enzyme digestion verification product after CsHDH3 constructs the pCAMBIA1305.1 vector).

[0019] 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).

[0020] Figure 5 This is a verification diagram of the degradation function of tobacco transiently expressed CsHDH3 on thiamethoxam in Example 5 (Figure A shows the degradation ability of tobacco transiently expressed CsHDH3 on X dose of thiamethoxam, recommended dose: 0.015 kg ai / ha; Figure B shows the degradation ability of tobacco transiently expressed CsHDH3 on 2X dose of thiamethoxam, 2 times dose: 0.03 kg ai / ha; Figure C shows the degradation ability of tobacco transiently expressed CsHDH3 on 3X dose of thiamethoxam, 3 times dose: 0.045 kg ai / ha). Detailed Implementation

[0021] 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.

[0022] Example 1: Screening of the CsHDH3 gene:

[0023] Analysis of previous metabolomics and transcriptomics data from tea plants treated with thiamethoxam revealed a significant positive correlation between the expression level of 3-hydroxyisobutyrate dehydrogenase (CsHDHs) and treatment time, while amino acid-related metabolic pathways were also significantly affected. Subsequently, the expression levels of various genes involved in CsHDHs in tea plants were analyzed over time, ultimately identifying the CsHDH3 gene as potentially playing a crucial role in thiamethoxam detoxification.

[0024] The specific nucleotide sequence of the CsHDH3 gene is shown in SEQ ID NO.1, and the protein sequence it encodes is shown in SEQ ID NO.2.

[0025] Example 2: Tissue-specific expression pattern analysis of CsHDH3 in tea plants:

[0026] The expression patterns of the CsHDH3 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 CsHDH3 was specifically highly expressed in the roots of tea plants.

[0027] in addition, Figure 2 Transcriptome data from tea plants were analyzed, showing that under thiamethoxam treatment, the expression of CsHDH3 showed a significant positive correlation with the control group (untreated blank group) as the treatment time increased.

[0028] Example 3: Cloning and sequence structure analysis of the CsHDH3 gene:

[0029] The national-level superior variety "Shuchazao" planted in the Nongcui Garden of Anhui Agricultural University was selected, and young roots were used 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.

[0030] use The One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) kit reverse transcribes RNA into cDNA.

[0031] Primers were designed based on the coding sequence of the 3-hydroxyisobutyrate dehydrogenase gene CsHDH3, and restriction enzyme sites XbaI and BamHI were added to the primers, respectively. The upstream primer was 5'-GCTCTAGAATGGCTTCTCTTGTATCAACCC-3', and the downstream primer was 5'-CGGGATCCGTTTAAAGTGGAACAAGACGACG-3'.

[0032] Using cDNA first strand as a template, PCR was performed using standard methods to amplify the tea plant 3-hydroxyisobutyrate dehydrogenase gene CsHDH3. The 550 μL PCR reaction system 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: 98℃ for 10 sec, 98℃ for 10 sec, 57℃ for 30 sec, 72℃ for 2 min, 72℃ for 10 min, for 35 cycles.

[0033] After purification and recovery, the PCR product CsHDH3 gene was ligated into the pEASY-Blunt Simple Cloning Vector (TransGen Biotech, Beijing, China) to obtain the pEASY-Blunt-CsHDH3 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 of the sequence listing.

[0034] After digesting the gene sequence with correct sequencing results using restriction endonucleases, the CsHDH3 gene was ligated into the vector pCAMBIA1305.1 to construct the expression recombinant plasmid pCAMBIA1305.1-CsHDH3. The constructed expression vector was then transformed into E. coli competent cells Trans1-T1 to construct the recombinant bacteria.

[0035] Several positive recombinant bacteria were selected, plasmids were extracted, and restriction endonuclease digestion was performed for identification (results are shown in the figure). Figure 3 (As shown).

[0036] Example 4: Subcellular localization of CsHDH3 in tea plants:

[0037] To further clarify the subcellular distribution of CsHDH3, we conducted a subcellular localization experiment on CsHDH3 protoplasts in tea plants. First, plasmids were extracted from the constructed CsHDH3 subcellular localization vector pCAMBIA1305-CsHDH3. Simultaneously, protoplast cells were extracted from young leaves of tea seedlings. The empty vector pCAMBIA1305.1 and the pCAMBIA1305.1-CsHDH3 expression vector plasmids were transformed into tea seedling leaf cells using PEG transformation. After transformation, the cells were cultured in the dark for 24 hours, and GFP fluorescence was observed and photographed using a laser confocal microscope.

[0038] 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.

[0039] Depend on Figure 4 It can be seen that the green fluorescence signal of the empty vector pCAMBIA1305.1 fills the entire protoplast cell, but there is no specific subcellular localization region. In contrast, the green fluorescence signal of pCAMBIA1305.1-CsHDH3 is only enriched in the cytoplasm of the tea plant protoplast, and no fluorescence signal was detected in other parts. The results preliminarily confirm that CsHDH3 is located in the cytoplasm.

[0040] Example 5: The ability of transiently expressed CsHDH3 gene to degrade thiamethoxam in plants:

[0041] 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 CsHDH3 gene in the plant's ability to degrade thiamethoxam.

[0042] The pCAMBIA1305.1-CsHDH3 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 to the bacterial solution (add 1 μL AS solution per 1 mL of bacterial solution), and incubate at room temperature for 2 h; select tobacco plants with good growth, inject the bacterial solution into the back of the tobacco leaves using a disposable 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.

[0043] After confirming the transient expression of CsHDH3 in tobacco, tobacco was treated 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). Samples were collected at 36 h, 48 h, and 72 h after spraying, and only the leaves were selected for sample preparation.

[0044] The tobacco leaf sample was ground into a mortar and pestle. 0.5 g of the ground fresh tea leaf sample was placed in a 50 mL centrifuge tube. 5 mL of acetonitrile was added, and the mixture was ultrasonically extracted for 10 min. Sodium chloride (0.5 g) and anhydrous magnesium sulfate (0.5 g) were weighed into a test tube and vortexed for 2 min. The tube was then centrifuged at 5000 rpm for 5 min at room temperature. 2 mL of the supernatant was collected and transferred to a pre-filled container containing 50 mg PVPP, 5 mg PSA, 20 mg GCB, and 10 mg 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).

[0045] Figure 5This is a verification of the degradation function of thiamethoxam by transient expression of CsHDH3 in tobacco. In the figure, TMX-CK-X refers to the residual amount of TMX when tobacco does not transiently express the CsHDH3 gene but is sprayed with X doses of thiamethoxam (TMX) pesticide; TMX-CsHDH3-X refers to the residual amount of TMX when tobacco is sprayed with X doses of thiamethoxam (TMX) pesticide after transient expression of the CsHDH3 gene; ClO-CK-X refers to the content of thiamethoxam (CLO) generated by TMX metabolism when tobacco does not transiently express the CsHDH3 gene but is sprayed with X doses of TMX (CLO is a metabolite of TMX); CLO-CsHDH3-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 CsHDH3 gene; and so on.

[0046] like Figure 5 As shown, when the treatment concentration was X (recommended concentration: 0.015 kg ai / ha), tobacco plants transiently expressing CsHDH3 exhibited the best degradation effect on thiamethoxam. At this concentration, the degradation rates of thiamethoxam in tobacco plants transiently transformed with CsHDH3 were 85.3%, 81.7%, and 78.0% at 36 h, 48 h, and 72 h, respectively. Simultaneously, the residual amount of thiamethoxam gradually decreased, presumably because CsHDH3, after transient transformation in tobacco, also possesses a certain degradation capacity for thiamethoxam. At a concentration of 2X (2 times the dose: 0.03 kg ai / ha), the degradation of thiamethoxam by CsHDH3 was more significant, with degradation rates of 47.7%, 55.6%, and 48.2% at 36 h, 48 h, and 72 h, respectively. At a concentration of 3X (3 times the dose: 0.045 kg ai / ha), thiamethoxam in transiently converted tobacco CsHDH3 was found to be degraded by 52.6% after 36 h.

[0047] The above results indicate that tobacco plants transiently expressing CsHDH3 exhibit a strong degradation ability against thiamethoxam, with the highest degradation efficiency observed at 36 hours. Based on these results, we hypothesize that transient expression of the CsHDH3 gene can enhance the plant's ability to degrade thiamethoxam. When applied to tea plants, the expression vector pCAMBIA1305.1-CsHDH3 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 molecularly assisted breeding.

[0048] 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 ability of tobacco against thiamethoxam, characterized in that, Through genetic engineering, the 3-hydroxyisobutyrate dehydrogenase in tea plants was activated. CsHDH3 The gene is overexpressed in tobacco; among which, CsHDH3 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1, characterized in that, The CsHDH3 The protein sequence encoded by the gene is shown in SEQ ID NO.

2.

3. The method according to claim 1, characterized in that, The genetic engineering approach refers to: The SEQ ID NO.1 shown CsHDH3 The gene fragment was ligated into the vector pCAMBIA1305.1 to obtain the tea expression vector pCAMBIA1305.1- CsHDH3 Then, the tea plant expression vector pCAMBIA1305.1- CsHDH3 The bacteria were transferred into tobacco using Agrobacterium tumefaciens EHA105 as a medium.

4. The method according to claim 1, characterized in that, Using tobacco instantaneous conversion technology CsHDH3 Preliminary verification of gene function: The tea plant expression vector pCAMBIA1305.1- CsHDH3 It was transiently expressed in tobacco via Agrobacterium-mediated transformation.

5. The method according to any one of claims 1 to 4, characterized in that, The CsHDH3 The gene expression product is used to positively regulate the thiamethoxam degradation capacity of tobacco.

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

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