Radix sophorae tonkinensis gene stgh3.1 and application thereof in improving plant stress resistance

Abstract

This invention belongs to the field of crop genetics and breeding, and specifically relates to a gene from Sophora tonkinensis. StGH3.1 And its application in improving plant stress tolerance. This application describes the construction of Sophora tonkinensis... StGH3.1 Gene overexpression vectors were used to genetically transform the gene, resulting in the transgenic Sophora tonkinensis gene. StGH3.1 Tobacco plants were found to have a significant effect on improving their tolerance to heavy metal cadmium stress and drought stress; overexpression StGH3.1 It can increase the activity of SOD, POD, and CAT in tobacco plant leaves under cadmium and drought stress, reduce the degree of membrane lipid peroxidation, and improve the tolerance of tobacco plants to cadmium and drought stress. This provides a new idea for cultivating cadmium-tolerant and drought-tolerant plants, especially Sophora tonkinensis.

Description

Technical Field

[0001] This invention belongs to the field of crop genetics and breeding, and relates to the cultivation of stress-resistant plants. Background Technology

[0002] Sophora tonkinensis ( Sophora tonkinensis Sophora tonkinensis is one of the "Ten Delicacies of Guangxi" traditional Chinese medicinal herbs from Guangxi Zhuang Autonomous Region. It is included in all editions of the *Pharmacopoeia of the People's Republic of China* and is a key ingredient in nearly 30 kinds of traditional Chinese medicine products, including those listed in the *National Essential Medicines List*, for treating sore throat, hepatitis, and anti-tumor purposes. According to the national production zoning of Sophora tonkinensis, Guangxi Zhuang Autonomous Region accounts for approximately 80% of the most suitable planting area nationwide, mainly distributed in Hechi City and its subordinate counties. These areas are also the main producing areas of Sophora tonkinensis and the key areas for its development in Guangxi Zhuang Autonomous Region. Hechi is a major karst landform resource distribution area in Southwest my country and a nationally renowned "hometown of non-ferrous metals." Karst areas are highly susceptible to drought (temporary drought), making drought stress a significant environmental factor affecting the quality of Sophora tonkinensis. However, there are currently no research reports on improving the adaptability of Sophora tonkinensis to heavy metal cadmium and drought.

[0003] The GH3 gene family is a class of early auxin response genes, and numerous studies have shown that GH3 family genes play an important role in plant stress response (Yuan Yuan, Enhe Bayar, Qi Yanhua. Research progress on the biological functions of plant GH3 gene family [J]. Acta Botanica Sinica, 2023, 58(05):770-782.). In Arabidopsis thaliana, it was found that Arabidopsis plants overexpressing the GH3 gene showed significantly enhanced resistance to drought, high salinity, and high temperature, and the transcription levels of some abiotic stress-related genes were significantly increased. However, there are currently no reports on using the GH3 gene to simultaneously improve plant cadmium tolerance and drought tolerance. Summary of the Invention

[0004] To address the above problems, this invention proposes a Sophora tonkinensis gene. StGH3.1 And its application in improving plant stress resistance.

[0005] The technical solution of this invention is implemented as follows:

[0006] This invention obtains *Sophora tonkinensis* through transgenic technology. StGH3.1 The study found that the genetically modified tobacco plants significantly improved their tolerance to heavy metal cadmium stress and drought stress, providing new insights for breeding cadmium- and drought-tolerant plants, especially Sophora tonkinensis.

[0007] On the one hand, this invention proposes a Sophora tonkinensis gene. StGH3.1 Its nucleotide sequence is more than 90% homologous to the sequence shown in SEQ ID No. 1.

[0008] Preferably, the S. tonkinensis gene StGH3.1 The nucleotide sequence of the S. tonkinensis gene is shown in SEQ ID No. 1.

[0009] In a second aspect, a S. tonkinensis protein StGH3.1 is provided, which has an amino acid sequence with one or more insertions, deletions or mutations of amino acids and still has the function of the amino acids in SEQ ID No. 2.

[0010] Preferably, the amino acid sequence of the S. tonkinensis protein StGH3.1 is shown in SEQ ID No. 2.

[0011] In a third aspect, a biological material is provided, which is a recombinant DNA, a recombinant vector or a recombinant bacteria containing the S. tonkinensis gene StGH3.1 Preferably, the S. tonkinensis gene is StGH3.1.

[0012] In a fourth aspect, the biological material is used for improving the stress tolerance of a plant.

[0013] Preferably, the stress tolerance is cadmium tolerance and / or drought tolerance.

[0014] Preferably, the use is to improve the cadmium tolerance and / or drought tolerance of a plant by increasing the expression of the S. tonkinensis gene StGH3.1 Preferably, the S. tonkinensis gene is StGH3.1.

[0015] In a fifth aspect, a method for improving the cadmium tolerance and / or drought tolerance of a plant is provided, which comprises the following steps:

[0016] (1) using S. tonkinensis cDNA as a template and a primer pair specific to a fragment as primers to amplify the target fragment;

[0017] (2) performing enzyme digestion and ligation of the target fragment and an intermediate vector to obtain a recombinant vector, and transforming the recombinant vector into a plant to be improved to obtain a plant with improved cadmium tolerance and / or drought tolerance.

[0018] Preferably, the nucleotide sequence of the upstream primer in the primer pair is shown in SEQ ID No. 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 4.

[0019] Preferably, the plant is tobacco or S. tonkinensis.

[0020] The present application has the following advantages:

[0021] 1. The present application first clones and analyzes the subcellular localization of the S. tonkinensis genes in the nucleus and cytoplasm, and obtains transgenic tobacco plants with the S. tonkinensis genes through transgenic technology, and finds that StGH3.1 the S. tonkinensis genes can improve the cadmium tolerance and / or drought tolerance of the plants. StGH3.1 StGH3.1 ​The expression of this gene significantly enhances tolerance to heavy metal cadmium stress and drought stress, providing a new option for screening dominant stress-resistant genes and offering new ideas for cultivating cadmium-tolerant and drought-tolerant plants, especially Sophora tonkinensis.

[0022] 2. This application constructs a *Sophora tonkinensis* plant. StGH3.1 Gene overexpression vectors were used for genetic transformation to confirm... StGH3.1 The gene can improve the cadmium and drought tolerance of tobacco. After 30 days of Cd stress treatment, the transgenic tobacco plants OE6, OE15, and OE21 all showed better growth than wild-type tobacco plants. After 10 days of simulated drought treatment, the wild-type tobacco plants showed worse growth than the transgenic plants and were on the verge of wilting and dying. Overexpression StGH3.1 It can enhance the activities of SOD, POD, and CAT in tobacco plant leaves. Under cadmium stress of 100 µM Cd and 200 µM Cd, the average activities of POD, CAT, and SOD increased by 35.4% and 53.9%, 11.3% and 36.6%, and 41.9% and 23.8%, respectively. Under drought stress of 5% PEG and 10% PEG, the activities of POD, CAT, and SOD increased by 15.9% and 31.9%, 50.9% and 57.4%, and 57.9% and 83.0%, respectively. Overexpression StGH3.1 It can reduce the degree of membrane lipid peroxidation in tobacco plant leaves. Under cadmium stress conditions of 100 µM Cd and 200 µM Cd, the MDA content decreased by 12.7% and 21.2%, respectively, and under drought stress conditions of 5% PEG and 10% PEG, the MDA content decreased by 20.6% and 25.9%, respectively; thereby improving the tolerance of tobacco plants to cadmium stress and drought stress. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 for StGH3.1 Electrophoresis diagram of gene fragment amplification products.

[0025] Figure 2 pBK-35S-GLosGFP- StGH3 Schematic diagram of carrier construction.

[0026] Figure 3 for StGH3.1 Nucleotide sequence and encoding amino acid sequence.

[0027] Figure 4 For StGH3.1 Subcellular localization.

[0028] Figure 5 Figure is a growth comparison chart of wild type tobacco and transgenic tobacco under different treatments.

[0029] Figure 6 Figure is the antioxidant enzyme activity and MDA content of wild type tobacco seedlings and transgenic tobacco leaves under cadmium stress and drought stress treatments. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0031] The experimental methods used in the following experimental examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials available through commercial channels unless otherwise specified.

[0032] The present application provides a sophora flavescens gene StGH3.1 The nucleotide sequence thereof is as shown in any one of the following:

[0033] (1) The homology of the nucleotide sequence shown in SEQ ID No. 1 is more than 90%;

[0034] (2) The sequence reverse complementary to the nucleotide sequence in (1);

[0035] (3) The nucleotide sequence shown in SEQ ID No. 1.

[0036] A sophora flavescens protein StGH3.1, the amino acid sequence of which is as shown in any one of the following:

[0037] (1) The amino acid sequence has one or more insertions, deletions or mutations of amino acids and still has the function of the amino acid sequence in SEQ ID No. 2;

[0038] (2) The amino acid sequence shown in SEQ ID No. 2.

[0039] A biological material containing the above-mentioned sophora flavescens gene StGH3.1 The above-mentioned biological material is recombinant DNA, recombinant vector, overexpression vector or recombinant bacteria.

[0040] The above-mentioned biological material is used for improving the cadmium tolerance and / or drought tolerance of plants.

[0041] The above applications enhance the gene expression of Sophora tonkinensis. StGH3.1 The expression level of [certain substances] can be increased, thereby improving the cadmium tolerance and drought tolerance of plants.

[0042] A method for improving cadmium tolerance and / or drought tolerance in plants, comprising the following steps:

[0043] (1) Using Sophora tonkinensis cDNA as a template, amplification was performed using primer pairs for the specific fragment, and the target fragment was recovered;

[0044] (2) The target fragment is ligated with the intermediate vector by enzyme digestion to obtain a recombinant vector, which is then transferred into the plant to be improved to obtain plants with improved cadmium tolerance and / or drought tolerance.

[0045] This invention obtains *Sophora tonkinensis* through transgenic technology. StGH3.1 Tobacco plants with genetically modified organisms were discovered. StGH3.1 Overexpression of this compound significantly enhances tolerance to heavy metal cadmium stress and drought stress, providing new insights for cultivating cadmium- and drought-tolerant plants, especially Sophora tonkinensis.

[0046] The following embodiments will provide further details: Example

[0047] 1. Mountain bean root StGH3.1 Gene Fragment Acquisition

[0048] 0.5 g of fresh Sophora tonkinensis leaves were placed in a mortar and ground into powder using liquid nitrogen. Total RNA was extracted using the FastPure PlantTotal RNA Isolation Kit (Vazyme), and the RNA was reverse transcribed into cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme). Based on the description of Sophora tonkinensis... StGH3.1 The gene sequence (SEQ ID NO:1) was used to design and synthesize the upstream primer (SEQ ID No. 3) (cagtGGTCTCaCAACATGGCGGTTGAGACAGAGTTG) and the downstream primer (SEQ ID No. 4) (cagtGGTCTCaTACATTACCCATCAATACGACGACGT). PCR amplification was performed using *Sophora tonkinensis* cDNA as a template. The reaction conditions were: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 58℃ annealing for 15 s, and 72℃ extension for 45 s, for 35 cycles. The reaction system consisted of: 25 μL of high-fidelity enzyme 2 × Phanta Max Master Mix (Vazyme), 2 μL of upstream primer, 2 μL of downstream primer, 2 μL of cDNA, and 19 μL of dd H2O. The PCR products were detected by electrophoresis.Figure 1 )After the target gene fragment size is consistent, use FastPure Gel DNA Extraction Mini Kit (Vazyme) for gel recovery, and transform into E. coli DH 5α competent cells. Pick 3-5 single colonies, and use 2 × RapidTaq Master Mix (Vazyme) for colony PCR verification. Select the positive clones with correct target band size and bright color for sequencing confirmation. Analyze the sequencing results, StGH3.1 The full-length CD region is 1836 bp, encoding 612 amino acids, and the encoded protein contains a GH3 auxin-responsive promoter domain (34aa-578aa) Figure 2 ).

[0049] II. Construction of pBK-35S-GLosGFP expression vector

[0050] The correct sequencing StGH3.1 The gene fragment and pBK-35S-GLosGFP expression vector were subjected to enzyme digestion and ligation reaction, and the reaction conditions were as follows: pBK-35S-GLosGFP 1 µL, PCR product 1 µL, BioRun Eco31I 1 µL, T4 DNA Ligase 1 µL, 10 × Reaction Buffer 2 µL, ddH2O 14 µL (all reagents were purchased from Boyuan). After mixing the above system uniformly, centrifuging briefly, and immediately incubating at 37℃ for 30-60 min; then heating at 65℃ for 20 min to terminate the reaction. Take 10 µL of enzyme digestion and ligation product, transform into E. coli DH 5α competent cells, pick 10 single colonies for colony PCR verification, and the positive clone with correct sequencing is named pBK-35S-GLosGFP- StGH3.1 , and the schematic diagram of vector construction is shown in Figure 3 The recombinant plasmid pBK-35S-GLosGFP- StGH3.1 was transformed into Agrobacterium EHA105 by heat shock method.

[0051] III. Radix Sophorae Tonkinensis StGH3.1 Subcellular localization analysis

[0052] A few tobacco seeds were sown in the substrate containing vermiculite, and after one month of cultivation, 6-8 leaves were grown. The Agrobacterium containing the pBK-35S-GLosGFP-StGH3.1 plasmid was inoculated into 100 mL of LB liquid medium for expansion culture, and cultured at 200 rpm until the OD was 0.6-0.8. The bacteria were collected by centrifugation at 4000 rpm for 4 min, and the bacteria were resuspended in LB liquid containing 10 mM MgCl2 and 120 µM AS, and the OD was adjusted to 0.6. The tobacco leaves in good growth condition were selected, and the lower epidermis of the tobacco leaves was injected with a 1 mL needle-free syringe, and the injection area was marked. After 24 h of dark culture of the injected tobacco plants, the leaves at the marked position were made into slides, and observed and photographed under a laser confocal microscope. The results are shown in Figure 4 , and it can be seen that Figure 4 the protein expressed by the gene is located in the nucleus and cytoplasm. StGH3.1

[0053] Four, transgenic tobacco acquisition

[0054] 1. The tobacco seeds were sterilized with 75% alcohol for 30 s, washed with sterile water for 1 min, and then sterilized with 84 disinfectant for 3-5 min, and washed with sterile water for 3 times, 1 min each time. The sterilized tobacco seeds were sown on the germination medium, and cultured at 23°C with 16 h light / 8 h dark for 4-5 weeks. The sterile tobacco leaves were cut into small pieces with a scalpel and inoculated on the pre-culture medium.

[0055] 2. Agrobacterium EHA105 containing the pBK-35S-GLosGFP- StGH3.1 recombinant plasmid was inoculated into 100 mL of LB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and cultured at 28°C, 220 rpm until the OD value was about 0.8. The supernatant was removed by centrifugation at 8000 rpm for 5 min, and the bacteria were resuspended in sterilized MS liquid medium to an OD600 of about 0.5, which was used as the engineering bacteria liquid for subsequent infection of tobacco leaves.

[0056] ​3. The tobacco leaves pre-cultured for 2-3 days are inoculated in the Agrobacterium suspension for 10-15 min, the infected tobacco leaves are dried on filter paper and inoculated on co-cultivation medium for dark culture for 48-72 h. The co-cultured leaves are transferred to induction medium for induction of callus for about 10 d. The callus is selected and transferred to Basta-resistant screening medium for culture for 15-30 d, and the positive callus screened is transferred to differentiation medium for culture for 15-30 d; the positive callus with vigorous growth is inoculated on differentiation medium, and if the callus has seedling formation, the callus is inoculated on a strong seedling medium for growth for 7-10 d, tobacco genomic DNA is extracted by CTAB method, and PCR detection is performed.

[0057] 4. The transgenic tobacco seedlings with positive PCR detection are acclimatized and transplanted to flower pots, and the seeds are sowed on Basta-containing resistant medium for continuous screening, and the seedlings are transplanted after PCR detection after germination, and T1 generation seeds are harvested, and the seedlings formed after sowing of the T1 generation seeds are used for subsequent analysis.

[0058] Five, cadmium tolerance and drought tolerance analysis

[0059] To study the biological function of Sophora tonkinensis StGH3.1 Girard under heavy metal cadmium and drought stress, wild type tobacco seedlings and transgenic tobacco seedlings are subjected to cadmium stress and drought stress treatment. The experimental results show that after 100 µM Cd and 200 µM Cd stress treatment for 30 d, the growth of the transgenic tobacco plants is better than that of the wild type tobacco plants (P<0.05) Figure 5 ). After 5% PEG and 10% PEG simulated drought treatment for 10 d, the growth of the wild type tobacco plants is worse than that of the transgenic plants, especially under 10% PEG treatment, the wild type tobacco plants are close to wilting and death (P<0.05) Figure 5 ). The wild type tobacco seedlings and the transgenic tobacco leaves subjected to cadmium stress and drought stress treatment are subjected to determination of antioxidant enzyme activity and MDA content. The experimental results show that overexpression of Sophora tonkinensis StGH3.1 can improve the SOD, POD and CAT activities of the tobacco plant leaves under cadmium stress and drought stress conditions (P<0.05) Figure 6 ). Under 100 µM Cd and 200 µM Cd cadmium stress, the POD, CAT and SOD activities are increased by 35.4% and 53.9%, 11.3% and 36.6%, and 41.9% and 23.8% on average; under 5% PEG and 10% PEG drought stress, the POD, CAT and SOD activities are increased by 15.9% and 31.9%, 50.9% and 57.4%, and 57.9% and 83.0% respectively; overexpression of Sophora tonkinensis StGH3.1The tobacco plant can reduce the degree of membrane lipid peroxidation under cadmium stress and drought stress conditions, and the MDA content is reduced by 12.7% and 21.2% under 100 µM Cd and 200 µM Cd cadmium stress conditions, and the MDA content is reduced by 20.6% and 25.9% under 5% PEG and 10% PEG drought stress conditions, thereby improving the tolerance of the tobacco plant to cadmium stress and drought stress.

[0060] The above merely describes preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A gene from Sophora tonkinensis. StGH3.1 Its nucleotide sequence is shown in SEQ ID No.

1.

2. The Sophora tonkinensis gene as described in claim 1 StGH3.1 The expressed protein has the amino acid sequence shown in SEQ ID No.

2.

3. A biomaterial, characterized in that: The biological material is a root of Sophora tonkinensis containing the gene described in claim 1. StGH3.1 Recombinant DNA, recombinant vectors, or recombinant bacteria.

4. The application of the biomaterial according to claim 3 in improving plant cadmium tolerance and / or drought tolerance, characterized in that: The plant in question is either tobacco or Sophora tonkinensis.

5. The application according to claim 4, characterized in that: The application improves the gene expression of Sophora tonkinensis. StGH3.1 The expression level of [the substance] can be increased, thereby improving the cadmium tolerance and / or drought tolerance of tobacco or Sophora tonkinensis.

6. A method for improving cadmium tolerance and / or drought tolerance in plants, characterized in that, The steps are as follows: (1) Using Sophora tonkinensis cDNA as a template, amplification was performed using primer pairs for the specific fragment, and the target fragment was recovered; (2) The target fragment is ligated with the intermediate vector by enzyme digestion to obtain a recombinant vector, which is then transferred into the plant to be improved to obtain plants with improved cadmium tolerance and / or drought tolerance. The nucleotide sequence of the target fragment is shown in SEQ ID No. 1; the plant is tobacco or Sophora tonkinensis.

7. The method for improving cadmium tolerance and / or drought tolerance in plants according to claim 6, characterized in that: The nucleotide sequence of the upstream primer in the primer pair is shown in SEQ ID No. 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 4.

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