Vietnamese sophora root gene StGH3.1 and application of Vietnamese sophora root gene StGH3.1 in improving stress tolerance of plants

Abstract

The invention belongs to the field of crop genetic breeding, and particularly relates to a subprostrate sophora gene StGH3.1 and application of the subprostrate sophora gene StGH3.1 in improving stress tolerance of plants. According to the application, an overexpression vector of the subprostrate sophora StGH3.1 gene is constructed and is subjected to genetic transformation to obtain a subprostrate sophora transgenic StGH3.1 tobacco plant, and it is found that the subprostrate sophora transgenic StGH3.1 tobacco plant has a remarkable improvement effect on the tolerance to heavy metal cadmium stress and drought stress; the overexpression StGH3.1 can improve the activity of SOD, POD and CAT of tobacco plant leaves under the conditions of cadmium stress and drought stress, reduce the degree of membrane lipid peroxidation and improve the tolerance of tobacco plants to cadmium stress and drought stress, so that a new idea is provided for cultivating cadmium-tolerant and drought-tolerant plants, especially subprostrate sophora.

Description

Technical Field

[0001] The invention belongs to the field of crop genetic breeding and relates to the cultivation of stress-tolerant plants. Background Art

[0002] Sophora flavescens Sophoratonkinensis ) is a local medicinal material of Guangxi Zhuang Autonomous Region, which is included in the "Ten Flavors of Guangxi" and has been included in the "Pharmacopoeia of the People's Republic of China" in all editions. It is a raw material of the "National Essential Medicines Catalogue" and the main raw material of nearly 30 Chinese patent medicines for the treatment of sore throat, hepatitis, anti-tumor, etc. According to the national production zoning of Sophora flavescens, the most suitable planting area in Guangxi Zhuang Autonomous Region accounts for about 80% of the country, mainly distributed in Hechi City, Guangxi Zhuang Autonomous Region and its counties and districts under its jurisdiction. These areas are also the main production areas of Sophora flavescens and the key planting areas of Guangxi Zhuang Autonomous Region. Hechi is the main karst landform resource distribution area in southwest my country and is also a nationally famous "hometown of non-ferrous metals". Karst areas are very prone to drought (temporary drought) and fragile environment. Drought stress has become an important environmental factor affecting the quality of Sophora flavescens during its growth. However, there are no reports on improving the adaptability of Sophora flavescens to heavy metal cadmium and drought.

[0003] The GH3 gene family is a class of early auxin response genes. A large number of studies have shown that GH3 family genes play an important role in plant stress resistance (Yuan Yuan, Enhe Bayar, Qi Yanhua. Research Progress on the Biological Functions of the Plant GH3 Gene Family [J]. Acta Botanica Sinica, 2023, 58(05): 770-782.). In Arabidopsis, it was found that Arabidopsis plants overexpressing GH3 genes had significantly enhanced resistance to drought, high salt, high temperature and other adversities, and the transcription levels of some abiotic stress-related genes were significantly increased. However, there is no report on the use of GH3 genes to simultaneously improve plant cadmium tolerance and drought tolerance. Summary of the invention

[0004] In order to solve the above problems, the present invention proposes a Sophora flavescens gene StGH3.1 And its application in improving plant stress tolerance.

[0005] The technical solution of the present invention is achieved in this way: The invention obtains the transgenic Sophora flavescens by genetically modifying technology StGH3.1 Gene-modified tobacco plants were found to have significantly improved tolerance to heavy metal cadmium stress and drought stress, which provides a new idea for breeding cadmium-resistant and drought-resistant plants, especially Sophora flavescens.

[0006] On the one hand, the present invention provides a Sophora flavescens gene StGH3.1 The nucleotide sequence has a homology of more than 90% with the sequence shown in SEQ ID No.1.

[0007] Preferably, the above-mentioned Radix Sophorae Tonkinensis gene StGH3.1 The nucleotide sequence is shown in SEQ ID No.1.

[0008] In a second aspect, a Sophora flavescens protein StGH3.1, the amino acid sequence of which has undergone one or more amino acid insertions, deletions or mutations and still has the functions of the amino acids in SEQ ID No.2.

[0009] Preferably, the amino acid sequence of the above-mentioned Sophora flavescens protein StGH3.1 is shown as SEQ ID No.2.

[0010] In the third aspect, a biological material is a biological material containing the above-mentioned Sophora flavescens gene StGH3.1 recombinant DNA, recombinant vector or recombinant bacteria.

[0011] Fourthly, the application of the above-mentioned biomaterials in improving plant stress tolerance.

[0012] Preferably, the above stress tolerance is cadmium tolerance and / or drought tolerance.

[0013] Preferably, the above application is achieved by increasing the gene of Sophora flavescens StGH3.1 , thereby improving the cadmium tolerance and / or drought tolerance of the plant.

[0014] A fifth aspect is a method for improving plant cadmium tolerance and / or drought tolerance, comprising the following steps: (1) Using Sophora flavescens cDNA as a template and a primer pair of a specific fragment as a primer, amplification was performed to recover the target fragment; (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 a plant with improved cadmium tolerance and / or drought tolerance.

[0015] Preferably, the nucleotide sequence of the upstream primer in the above primer pair is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No.4.

[0016] Preferably, the plant is tobacco or Sophora flavescens.

[0017] The present invention has the following beneficial effects: 1. This application is the first to study the StGH3.1 The gene was cloned and its subcellular localization was analyzed. StGH3.1 Gene found in tobacco plants StGH3.1 The expression of has a significant effect on improving the tolerance to heavy metal cadmium stress and drought stress, providing a new choice for screening advantageous stress resistance genes, and also providing a new idea for breeding cadmium-resistant and drought-resistant plants, especially Sophora flavescens.

[0018] 2. This application constructs Sophora flavescens StGH3.1 Gene overexpression vector was used to transform the gene and confirm StGH3.1 The gene can improve tobacco's cadmium tolerance and drought tolerance. After 30 days of Cd stress treatment, the growth of transgenic tobacco plants OE6, OE15, and OE21 was better than that of wild-type tobacco plants. After 10 days of simulated drought treatment, the growth of wild-type tobacco plants was worse than that of transgenic plants and was on the verge of wilting and death. StGH3.1 It can increase the activities of SOD, POD and CAT in tobacco leaves. Under 100 µM Cd and 200 µM Cd stress, the activities of POD, CAT and SOD increased by 35.4% and 53.9%, 11.3% and 36.6%, 41.9% and 23.8% on average; under 5% PEG and 10% PEG drought stress, the activities of POD, CAT and SOD increased by 15.9% and 31.9%, 50.9% and 57.4%, 57.9% and 83.0%, respectively; overexpression StGH3.1 It can reduce the degree of membrane lipid peroxidation in tobacco plant leaves. The MDA content under 100 µM Cd and 200 µM Cd cadmium stress conditions decreased by 12.7% and 21.2%, respectively, and the MDA content under 5% PEG and 10% PEG drought stress conditions decreased by 20.6% and 25.9%, respectively; thereby improving the tolerance of tobacco plants to cadmium stress and drought stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 for StGH3.1 Electrophoresis of gene fragment amplification products.

[0021] Figure 2 pBK-35S-GLosGFP- GH3 Schematic diagram of vector construction.

[0022] Figure 3 for StGH3.1 Nucleotide sequence and encoded amino acid sequence.

[0023] Figure 4 for StGH3.1 Subcellular localization.

[0024] Figure 5This is a growth comparison chart of wild-type tobacco and transgenic tobacco under different treatments.

[0025] Figure 6 Antioxidant enzyme activities and MDA content in wild-type tobacco seedlings and transgenic tobacco leaves treated with cadmium stress and drought stress. DETAILED DESCRIPTION

[0026] The technical solution 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 part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0028] The present application provides a Sophora flavescens gene StGH3.1 , whose nucleotide sequence is shown in any of the following: (1) The homology with the nucleotide sequence shown in SEQ ID No.1 is more than 90%; (2) a sequence that is reverse complementary to the nucleotide sequence in (1); (3) The nucleotide sequence is as shown in SEQ ID No.1.

[0029] A Sophora flavescens protein StGH3.1, whose amino acid sequence is shown in any one of the following: (1) The amino acid sequence has one or more amino acids inserted, deleted or mutated and still has the amino acid function of SEQ ID No. 2; (2) The amino acid sequence shown in SEQ ID No. 2.

[0030] A method containing the above-mentioned Radix Sophorae Tonkinensis gene StGH3.1 The biological material is a recombinant DNA, a recombinant vector, an overexpression vector or a recombinant bacterium.

[0031] Application of the above-mentioned biological material in improving the cadmium tolerance and / or drought tolerance of plants.

[0032] The above applications are achieved by increasing the gene StGH3.1 , thereby improving the cadmium tolerance and drought tolerance of plants.

[0033] A method for improving cadmium tolerance and / or drought tolerance of plants, comprising the following steps: (1) Using Sophora flavescens cDNA as a template and a primer pair of a specific fragment as a primer, amplification was performed to recover the target fragment; (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 a plant with improved cadmium tolerance and / or drought tolerance.

[0034] The invention obtains the transgenic Sophora flavescens by genetically modifying technology StGH3.1 Gene found in tobacco plants StGH3.1 The overexpression of has a significant effect on improving the tolerance to heavy metal cadmium stress and drought stress, providing a new idea for breeding cadmium-resistant and drought-resistant plants, especially Sophora flavescens.

[0035] The specific examples are further described below: Example

[0036] 1. Mountain bean root StGH3.1 Gene fragment acquisition 0.5 g of fresh leaves of Sophora flavescens were placed in a mortar and ground into powder using liquid nitrogen. Total RNA was extracted using FastPure PlantTotal RNA Isolation Kit (Vazyme), and RNA was reverse transcribed into cDNA using HiScript II 1st Strand cDNA Synthesis Kit (Vazyme). StGH3.1 gene sequence (SEQID NO:1), designed and synthesized upstream primers (SEQ ID No. 3) (cagtGGTCTCaCAACATGGCGGTTGAGACAGAGTTG) and downstream primers (SEQ ID No. 4) (cagtGGTCTCaTACATTACCCATCAATACGACGACGT), and used Sophora tonkinensis cDNA as template for PCR amplification. The reaction conditions were as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 45 s, and 35 cycles. The reaction system was as follows: 25 μL of high-fidelity enzyme 2 × Phanta Max Master Mix (Vazyme), 2 μL of upstream primers, 2 μL of downstream primers, 2 μL of cDNA, and 19 μL of dd H2O. The PCR products were detected by electrophoresis ( Figure 1) After the size of the target gene fragment matches, use FastPure Gel DNA Extraction Mini Kit (Vazyme) for gel recovery, transform into E. coli DH 5α competent cells, pick 3-5 single clones, use 2 × RapidTaq Master Mix (Vazyme) for colony PCR verification, select the target band size and bright positive clones for sequencing confirmation. Analyze the sequencing results. StGH3.1 The CD region of the gene is 1836 bp in length, encoding 612 amino acids. The encoded protein contains a GH3 auxin-responsive promoter domain (34aa-578aa) ( Figure 2 ).

[0037] 2. Construction of pBK-35S-GLosGFP expression vector The correct sequence StGH3.1 The gene fragment was subjected to restriction digestion and ligation reaction with the pBK-35S-GLosGFP expression vector. 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 relevant reagents were purchased from Boyuan Company). After the above system was mixed evenly, it was centrifuged briefly and immediately incubated at 37°C for 30-60 min; then the reaction was terminated by heating at 65°C for 20 min. 10 µL of the restriction digestion and ligation product was taken and transformed into Escherichia coli DH 5α competent cells. Ten single clones were selected for colony PCR verification, and the positive clones with correct sequencing were named pBK-35S-GLosGFP- StGH3.1 , vector construction schematic diagram as shown Figure 3 The recombinant plasmid pBK-35S-GLosGFP- StGH3.1 Transformed into Agrobacterium EHA105.

[0038] 3. Mountain bean root StGH3.1 Subcellular localization analysis Sow several tobacco seeds in a matrix containing vermiculite, and wait for 6 to 8 leaves to grow after one month of cultivation. Pick a single colony of Agrobacterium containing the pBK-35S-GLosGFP-StGH3.1 plasmid and inoculate it into 100 mL of LB liquid culture medium for expansion culture, and cultivate it at 200 rpm until the OD is 0.6~0.8. Collect the bacteria by centrifugation at 4000 rpm for 4 min, resuspend the bacteria in LB liquid suspension containing 10 mM MgCl2 and 120 µM AS, and adjust the OD to 0.6. Select tobacco leaves with good growth conditions, use a 1 mL syringe without a needle to inject from the lower epidermis of the tobacco leaves, and mark the injection area. After the injected tobacco plants are cultured in the dark for 24 hours, take the leaves at the marked area to make slides, observe and photograph them under a laser confocal microscope. The results are as follows Figure 4 As shown by Figure 4 Knowable StGH3.1 The protein expressed by the gene is localized in the nucleus and cytoplasm.

[0039] IV. Obtaining genetically modified tobacco 1. Sterilize tobacco seeds in 75% alcohol for 30 seconds, wash with sterile water for 1 minute, and then use 84 disinfectant to disinfect for 3-5 minutes, and wash with sterile water 3 times, 1 minute each time. Sow the sterilized tobacco seeds on the germination medium, and culture them at 23℃ for 16 hours of light / 8 hours of darkness for 4-5 weeks. Cut the sterilized tobacco leaves into small pieces with a scalpel and inoculate them on the pre-culture medium.

[0040] 2. Pipette 2 mL of pBK-35S-GLosGFP- StGH3.1 Agrobacterium EHA105 with the 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 and 220 rpm until the OD value was about 0.8. Centrifuged at 8000 rpm for 5 min, the supernatant was removed, and the bacteria were suspended in sterilized MS liquid medium until the OD600 was about 0.5, which was used as the engineering bacterial liquid for subsequent infection of tobacco leaves.

[0041] 3. Inoculate tobacco leaves pre-cultured for 2 to 3 days into Agrobacterium suspension for infection for 10 to 15 minutes. Dry the infected tobacco leaves on filter paper, inoculate them on co-culture medium, and culture them in the dark for 48 to 72 hours. Transfer the co-cultured leaves to the induction medium to induce callus for about 10 days. Select callus and transfer them to Basta resistance screening medium for 15 to 30 days. Transfer the selected positive callus to differentiation medium for 15-30 days. Inoculate the second screened positive callus with vigorous growth on differentiation medium. During the differentiation process, if seedlings are formed on the callus, inoculate them on seedling medium for 7-10 days. Use CTAB method to extract tobacco genomic DNA and perform PCR detection.

[0042] 4. After hardening the transgenic tobacco seedlings that are positive in the PCR test, transplant them into pots. After harvesting, sow the seeds on the resistance medium containing Basta for further screening. After germination, perform PCR testing on the seedlings and transplant them. Harvest the T1 generation seeds, and the seedlings formed after sowing the T1 generation seeds are used for subsequent analysis.

[0043] 5. Analysis of cadmium tolerance and drought tolerance Sophora tonkinensis StGH3.1 To investigate the biological functions of genes under heavy metal cadmium and drought stress, wild-type tobacco seedlings and transgenic tobacco seedlings were subjected to cadmium stress and drought stress. The experimental results showed that after 30 days of treatment with 100 µM Cd and 200 µM Cd, the growth of transgenic tobacco plants was better than that of wild-type tobacco plants ( Figure 5 ). After 10 days of simulated drought treatment with 5% PEG and 10% PEG, the growth of wild-type tobacco plants was worse than that of transgenic plants. In particular, under the 10% PEG treatment, the wild-type tobacco plants were on the verge of wilting and death ( Figure 5 ). Wild-type tobacco seedlings and transgenic tobacco leaves treated with cadmium stress and drought stress were taken to measure the antioxidant enzyme activities and MDA content. StGH3.1 Can increase the activities of SOD, POD and CAT in tobacco leaves under cadmium stress and drought stress ( Figure 6 ), under 100 µM Cd and 200 µM Cd cadmium stress, the activities of POD, CAT, and SOD increased by 35.4% and 53.9%, 11.3% and 36.6%, 41.9% and 23.8% on average; under 5% PEG and 10% PEG drought stress, the activities of POD, CAT, and SOD increased by 15.9% and 31.9%, 50.9% and 57.4%, 57.9% and 83.0%, respectively; overexpression StGH3.1It can reduce the degree of membrane lipid peroxidation in tobacco plant leaves under cadmium stress and drought stress. The MDA content under 100 µM Cd and 200 µM Cd stress conditions decreased by 12.7% and 21.2%, respectively, and the MDA content under 5% PEG and 10% PEG drought stress conditions decreased by 20.6% and 25.9%, respectively; thereby improving the tolerance of tobacco plants to cadmium stress and drought stress.

[0044] 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, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Sophora tonkinensis gene StGH3.1 The nucleotide sequence has a homology of more than 90% with the sequence shown in SEQ ID No.

1.

2. The Sophora flavescens gene according to claim 1 StGH3.1 , characterized in that: The nucleotide sequence is shown as SEQ ID No.

1.

3. A Sophora flavescens protein StGH3.1, wherein the amino acid sequence has one or more amino acids inserted, deleted or mutated and still has the function of the amino acids in SEQ ID No.

2.

4. The Sophora flavescens protein StGH3.1 according to claim 3, characterized in that: The amino acid sequence is shown in SEQ ID No.

2.

5. A biomaterial, characterized in that: The biological material contains the Sophora flavescens gene according to claim 1 or 2 StGH3.1 recombinant DNA, recombinant vector or recombinant bacteria.

6. Use of the biomaterial according to claim 5 in improving plant stress tolerance.

7. The use according to claim 6, characterized in that: The stress tolerance is cadmium tolerance and / or drought tolerance.

8. The use according to claim 7, characterized in that: The application improves the gene of Sophora flavescens StGH3.1 , thereby improving the cadmium tolerance and / or drought tolerance of the plant.

9. A method for improving plant cadmium tolerance and / or drought tolerance, characterized in that: Here are the steps: (1) Using Sophora flavescens cDNA as a template and a primer pair of a specific fragment as a primer, amplification was performed to recover the target fragment; (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 a plant with improved cadmium tolerance and / or drought tolerance.

10. The method for improving plant cadmium tolerance and / or drought tolerance according to claim 9, characterized in that: The nucleotide sequence of the upstream primer in the primer pair is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No.4; the plant is tobacco or Sophora flavescens.

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