Method for improving the degradation ability of thiamethoxam by modifying tea tree csHDH1 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 CsHDH1 gene, and overexpressing the 3-hydroxyisobutyric acid dehydrogenase CsHDH1 gene of the tea tree through a genetic engineering approach; wherein the nucleotide sequence of the CsHDH1 gene is shown as SEQ ID NO. 1. The present application also provides an application of the above method for improving the degradation capacity of thiamethoxam by modifying the tea tree CsHDH1 gene in cultivating new tea tree varieties with enhanced pesticide metabolism capacity. The present application screens the CsHDH1 gene with a positive regulation effect on the degradation capacity of the commonly used insecticide thiamethoxam for tea trees, overexpresses the 3-hydroxyisobutyric acid dehydrogenase CsHDH1 gene of the 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 CsHDH1 gene of tea plants. Background Technology

[0002] The tea tree (Camellia sinensis (L.) O. Kuntze) is a plant belonging to the Camellia genus of the Theaceae family. It is one of my country's important economic crops and has great economic value.

[0003] Currently, pest control for tea trees still relies primarily on chemical methods, with thiamethoxam being widely used in tea gardens. However, in practice, improper or excessive use of pesticides has been found to potentially leave pesticide residues in tea leaves, posing a potential threat to consumers' health.

[0004] Thiamethoxam is a second-generation neonicotinoid insecticide with stomach poison, contact, and systemic properties. It selectively inhibits nicotinic acetylcholinesterase receptors (nAChR) in the central nervous system of pests, thereby blocking normal conduction and altering the electrical potential of the synaptic membrane of the pest's nerves, ultimately leading to paralysis and death.

[0005] Therefore, there is an urgent need to obtain a method that can effectively improve the ability of tea trees to degrade thiamethoxam, so as to provide a theoretical basis for the regulatory mechanism of tea trees in response to exogenous stress, and also to provide a theoretical basis for cultivating new tea varieties with enhanced pesticide metabolism. Summary of the Invention

[0006] 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 CsHDH1 gene of 3-hydroxyisobutyrate dehydrogenase in tea plants.

[0007] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0008] A method for improving the degradation capacity of thiamethoxam by modifying the CsHDH1 gene of tea plants involves overexpressing the 3-hydroxyisobutyrate dehydrogenase CsHDH1 gene of tea plants through genetic engineering; wherein the nucleotide sequence of the CsHDH1 gene is shown in SEQ ID NO.1.

[0009] As one of the preferred embodiments of the present invention, the protein sequence encoded by the CsHDH1 gene is shown in SEQ ID NO.2.

[0010] As one of the preferred embodiments of the present invention, the genetic engineering approach refers to:

[0011] The CsHDH1 gene fragment shown in SEQ ID NO.1 was ligated into the vector pCAMBIA1305.1 to obtain the tea expression vector pCAMBIA1305.1-CsHDH1; then the tea expression vector pCAMBIA1305.1-CsHDH1 was transformed into Agrobacterium tumefaciens EHA105, and Agrobacterium tumefaciens was used to mediate the transformation into the target plant.

[0012] As one of the preferred embodiments of the present invention, the function of the CsHDH1 gene was preliminarily verified using a transient transformation technique in tobacco: the tea expression vector pCAMBIA1305.1-CsHDH1 was transiently expressed in tobacco via Agrobacterium-mediated transformation. Compared with the control, transient transformation of the CsHDH1 gene in tobacco significantly reduced the content of thiamethoxam, and the residual amount of its metabolite thiamethoxam also gradually decreased over time. This indicates that transient expression of the CsHDH1 gene can enhance the degradation capacity of the target plant for thiamethoxam and its metabolite thiamethoxam.

[0013] As one of the preferred embodiments of the present invention, the CsHDH1 gene expression product is used to positively regulate the thiamethoxam degradation capacity of the target plant.

[0014] The above-mentioned method of improving the thiamethoxam degradation ability by modifying the CsHDH1 gene of tea tree is applied to the breeding of new tea varieties with enhanced pesticide metabolism.

[0015] principle:

[0016] 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 CsHDH1 gene as potentially playing a crucial role in thiamethoxam detoxification. Next, the CsHDH1 gene was cloned and validated from tea plants for the first time. Subcellular localization analysis of the CsHDH1 gene in tea plant protoplasts revealed its location in the cytoplasm. Further evidence using Agrobacterium-mediated transient tobacco transformation technology demonstrated that the CsHDH1 gene can enhance the metabolic degradation capacity of plants against the neonicotinoid insecticide thiamethoxam.

[0017] The advantages of this invention compared to the prior art are as follows: In this study, the CsHDH1 gene, which has a positive regulatory effect on the "thiamethoxam degradation ability" of tea trees, was screened. By overexpressing the CsHDH1 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

[0018] Figure 1 This is a diagram illustrating the tissue-specific expression pattern of the tea plant 3-hydroxyisobutyrate dehydrogenase gene CsHDH1 in Example 2.

[0019] Figure 2 This is the expression profile of the tea plant 3-hydroxyisobutyrate dehydrogenase gene CsHDH1 in Example 2;

[0020] Figure 3 The images show the restriction enzyme digestion diagrams of the PCR product and the pCAMBIA1305.1 vector in Example 3 (Figure A shows the CsHDH1 PCR amplification product; Figure B shows the plasmid double restriction enzyme digestion verification product after CsHDH1 is constructed and ligated into the pEASY-Blunt vector; Figure C shows the plasmid double restriction enzyme digestion verification product after CsHDH1 is constructed into the pCAMBIA1305.1 vector; DNA molecular weight standard DL2000 bp / DL10000 bp).

[0021] 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).

[0022] Figure 5 This is a graph verifying the degradation function of transiently expressed CsHDH1 in tobacco on thiamethoxam in Example 5 (Figure A shows the degradation capacity of transiently expressed CsHDH1 in tobacco on X dose of thiamethoxam, recommended dose: 0.015 kg ai / ha; Figure B shows the degradation capacity of transiently expressed CsHDH1 in tobacco on 2X dose of thiamethoxam, 2x dose: 0.03 kg ai / ha; Figure C shows the degradation capacity of transiently expressed CsHDH1 in tobacco on 3X dose of thiamethoxam, 3x dose: 0.045 kg ai / ha). Detailed Implementation

[0023] 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.

[0024] Example 1: Screening of the CsHDH1 gene:

[0025] 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 CsHDHs genes in tea plants were analyzed over time, ultimately identifying the CsHDH1 gene as potentially playing a crucial role in thiamethoxam detoxification.

[0026] The specific nucleotide sequence of the CsHDH1 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 CsHDH1 in tea plants:

[0028] The expression patterns of the CsHDH1 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 CsHDH1 was specifically highly expressed in the roots of tea plants.

[0029] in addition, Figure 2 Analysis of the transcriptome data of tea plants showed that, compared with the control group (the blank group of tea plants without thiamethoxam spraying), the expression level of CsHDH1 under thiamethoxam treatment showed a significant positive correlation with the increase of treatment time.

[0030] Example 3: Cloning and sequence structure analysis of the CsHDH1 gene:

[0031] 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 RNAprep 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 gDNA Removal 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 3-hydroxyisobutyrate dehydrogenase gene CsHDH1, and restriction enzyme sites SpeI and BamHI were added to the primers, respectively. The upstream primer was 5'-GGACTAGTATGTCTTCTCTCATCCCACAACTC-3', and the downstream primer was 5'-CGCGGATCC GACCAAAGTGGAACAAGACGAG-3'.

[0034] Using cDNA first strand as an RT-PCR template, the tea plant 3-hydroxyisobutyrate dehydrogenase gene CsHDH1 was amplified using standard PCR methods. 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 CsHDH1 gene was ligated into the pEASY-Blunt Simple Cloning Vector (TransGen Biotech, Beijing, China) to obtain the pEASY-Blunt-CsHDH1 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.

[0036] After digesting the gene sequence with the correct sequencing results using restriction endonucleases, the CsHDH1 gene was ligated to the vector pCAMBIA1305.1 to construct the expression recombinant plasmid pCAMBIA1305.1-CsHDH1.

[0037] The constructed expression vector was transformed into recombinant plasmids and then transferred into E. coli competent cells Trans1-T1 to construct recombinant bacteria.

[0038] 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).

[0039] Example 4: Subcellular localization of CsHDH1 in tea plants:

[0040] To further clarify the subcellular distribution of CsHDH1, this embodiment conducted a subcellular localization experiment on tea plant protoplasts of CsHDH1. First, plasmids were extracted from the constructed CsHDH1 subcellular localization vector pCAMBIA1305-CsHDH1. Simultaneously, protoplast cells were extracted from young leaves of tea seedlings. The empty vector pCAMBIA1305.1 and the pCAMBIA1305.1-CsHDH1 expression vector plasmids were transformed into *Shuchazao* protoplasts using the PEG transformation method. After transformation, the protoplasts were cultured in the dark for 24 hours, and GFP fluorescence was observed and photographed using a laser confocal microscope.

[0041] 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.

[0042] 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-CsHDH1 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 CsHDH1 is located in the cytoplasm.

[0043] Example 5: The ability of transiently expressed CsHDH1 gene to degrade thiamethoxam in plants:

[0044] 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 CsHDH1 gene in the plant's ability to degrade thiamethoxam.

[0045] The pCAMBIA1305.1-CsHDH1 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 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.

[0046] After confirming the transient expression of CsHDH1 in tobacco, tobacco was sprayed with different concentrations of thiamethoxam standard (Dr. Ehrenstorfer, 99.8%), namely 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.

[0047] 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).

[0048] Figure 5This is a verification of the degradation function of thiamethoxam by transient expression of CsHDH1 in tobacco. Among them, TMX-CK-X refers to "the residue of TMX when tobacco does not transiently express the CsHDH1 gene but is sprayed with X doses of thiamethoxam (TMX) pesticide"; TMX-CsHDH1-X refers to "the residue of TMX when tobacco is sprayed with X doses of thiamethoxam (TMX) pesticide after transient expression of the CsHDH1 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 CsHDH1 gene but is sprayed with X doses of TMX pesticide"; CLO-CsHDH1-X refers to "the content of CLO generated by TMX metabolism when tobacco is sprayed with X doses of TMX pesticide after transient expression of the CsHDH1 gene"; and so on.

[0049] like Figure 5 As shown, the degradation of thiamethoxam in tobacco plants transiently converted to CsHDH1 was most significant at a treatment concentration of X (recommended concentration: 0.015 kg ai / ha). At this concentration, the degradation rates of thiamethoxam in tobacco plants transiently converted to CsHDH1 were 69.1%, 64.4%, and 59.6% at 36 h, 48 h, and 72 h, respectively. Simultaneously, the residual amount of thiamethoxam also gradually decreased, suggesting that CsHDH1, after transient conversion in tobacco, may also have some degradation capacity for thiamethoxam. At a concentration of 3X (3 times the dose: 0.045 kg ai / ha), 55.2% of thiamethoxam in CsHDH1-converted tobacco was degraded at 36 h.

[0050] The above results indicate that tobacco plants transiently expressing CsHDH1 exhibit a strong degradation capacity for thiamethoxam, with the highest degradation efficiency observed at 36 hours. Based on these results, we preliminarily hypothesize that transient expression of CsHDH1 can enhance the plant's ability to degrade thiamethoxam. When applied to tea plants, the expression vector pCAMBIA1305.1-CsHDH1 can be transformed into Agrobacterium tumefaciens EHA105, and the transformation can be mediated by Agrobacterium tumefaciens into the tea plants. This invention provides a theoretical basis and functional gene resources for developing new tea varieties with enhanced pesticide metabolism capabilities using molecularly assisted breeding.

[0051] 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 tea plants or tobacco, characterized in that, Through genetic engineering, the 3-hydroxyisobutyrate dehydrogenase in tea plants was induced... CsHDH1 The gene is overexpressed in tea plants or tobacco; among them, CsHDH1 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 CsHDH1 The gene fragment was ligated into the vector pCAMBIA1305.1 to obtain the tea expression vector pCAMBIA1305.1- CsHDH1 Then, the tea plant expression vector pCAMBIA1305.1- CsHDH1 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 2, characterized in that, Using tobacco instantaneous conversion technology CsHDH1 Preliminary verification of gene function: The tea plant expression vector pCAMBIA1305.1- CsHDH1 It can be transiently expressed in tobacco through Agrobacterium-mediated expression.

4. The application of the method as described in any one of claims 1 to 3 in the breeding of new tea varieties with enhanced thiamethoxam metabolism capacity.

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