Salvia miltiorrhiza SmCYP85A1 gene for improving drought resistance of plants and application of salvia miltiorrhiza SmCYP85A1 gene

Abstract

The invention relates to a salvia miltiorrhiza SmCYP85A1 gene capable of improving drought resistance of plants and application of the salvia miltiorrhiza SmCYP85A1 gene, and belongs to the field of plant growth and development gene engineering. Wherein the coding nucleotide sequence of the salvia miltiorrhiza SmCYP85A1 gene is as shown in a sequence 3; the amino acid sequence of the salvia miltiorrhiza SmCYP85A1 gene is as shown in a sequence 4. The method comprises the following steps: infecting salvia miltiorrhiza leaves with an SmCYP85A1 expression vector started by a 35S strong promoter, so as to obtain transgenic salvia miltiorrhiza SmCYP85A1 OE overexpressed by SmCYP85A1; compared with the SmCYP85A1 OE transgenic salvia miltiorrhiza, the SmCYP85A1 OE transgenic salvia miltiorrhiza can obviously enhance the drought stress tolerance of plants. Therefore, the invention provides key gene resources and technical support for genetic improvement of drought-resistant medicinal herbaceous plants.

Description

TECHNICAL FIELD

[0001] The present application relates to a Salvia miltiorrhiza SmCYP85A1 gene for improving drought resistance of plants and an application thereof, and belongs to the field of plant growth and development genetic engineering. BACKGROUND

[0002] Salvia miltiorrhiza is a perennial herb of the Labiatae family and the Salvia genus. Salvia miltiorrhiza has a long history of medicinal use and is one of the traditional Chinese medicinal materials. Traditionally, Salvia miltiorrhiza medicinal materials are mainly derived from wild resources, which have gradually been replaced by cultivated resources. However, the yield of cultivation is limited, and the demand is increasing year by year. In addition, drought stress can seriously affect the quality and yield of Salvia miltiorrhiza as a herbal medicinal plant. Therefore, it is of great significance to use genetic resources to cultivate new crop varieties with greater stress resistance.

[0003] Hormones, as important substances produced in plants, can regulate plant growth and development and stress resistance. Currently, plant hormones have become an effective method for relieving drought stress. Studies have shown that various plant hormones can improve plant stress resistance. Among them, brassinolide (BR) as a new type of plant hormone plays a core role in plant growth and development, morphological development and stress resistance regulation. The BR synthesis pathway includes early and late C-22 oxidation pathways, early and late C-6 oxidation pathways, and a synthesis shortcut between the early C-22 oxidation pathway and the late C-6 oxidation pathway. CYP85A1 is a BR oxidase gene that connects the late C-6 oxidation pathway and the early C-6 oxidation pathway, and is a very important rate-limiting enzyme gene in the BR synthesis process. Therefore, providing a SmCYP85A1 gene derived from the herbal medicinal plant Salvia miltiorrhiza to promote BR synthesis and improve plant drought resistance has become a technical problem to be solved in this field. SUMMARY

[0004] The main purpose of the present application is to provide a SmCYP85A1 gene derived from the herbal medicinal plant Salvia miltiorrhiza to promote BR synthesis and improve plant drought resistance.

[0005] The present application adopts the following technical solutions: A Salvia miltiorrhiza SmCYP85A1 gene for improving drought resistance of plants, the nucleotide sequence of which is shown in SEQ ID NO: 3, and the amino acid sequence of which is shown in SEQ ID NO: 4.

[0006] The present application also provides a cloning method of the Salvia miltiorrhiza SmCYP85A1 gene for improving drought resistance of plants, comprising the following steps: Step 1: Salvia miltiorrhiza (Salvia miltiorrhiza) sterile seedlings (provided by Shandong Yantai Ludong University of Agriculture and Forestry Engineering Research Institute) were used as test materials, and FastPure Universal Plant Total RNA Isolation Kit (Vazyme, NanJing, China, RC411-01) was used to extract RNA; the RNA was reversely transcribed into cDNA by using Superscript III first-strand synthesis system (Life Technologies, Carlsbad, USA, 18080051) reverse transcriptase; Step 2: The coding sequence of SmCYP85A1 was obtained by using Salvia miltiorrhiza genome data, and primer design was performed by using Oligo7 software; wherein the SmCYP85A1-F forward primer is shown in sequence 1; the SmCYP85A1-R reverse primer is shown in sequence 2; Step 3: The coding sequence of the target gene SmCYP85A1 was amplified The cDNA of Salvia miltiorrhiza was used as a template, and Phanta Max Master Mix high-fidelity enzyme (Vazyme, NanJing, China, P515-01) was used for PCR amplification, and finally the full-length 1392bp coding sequence of SmCYP85A1 was obtained, named as SmCYP85A1 gene (its nucleotide sequence is shown in sequence 3; its amino acid sequence is shown in sequence 4).

[0007] Preferably, in step 2, the length of the primer is 15-20bp, and the Tm value of the designed primer is suitable at 60℃, and a pK2GW7 vector linker is added to the primer.

[0008] Another object of the present application is to provide the construction of the above-mentioned Salvia miltiorrhiza SmCYP85A1 gene overexpression vector for improving plant drought resistance, and the steps are as follows: Step 1: Add fresh PCR product (coding sequence of SmCYP85A1 gene), pK2GW7 overexpression vector and Basic Mix enzyme into the PCR tube respectively, then place it in a 50℃ metal bath, react for 30min, complete the seamless cloning and assembly process; Step 2: Transform the mixed solution after the reaction in the above step into Escherichia coli DH5α and coat it on the LB solid screening medium, then pick single colonies from the screening medium for PCR detection and sequencing verification, confirm that the SmCYP85A1 overexpression vector is constructed successfully, and name it as SmCYP85A1 OE.

[0009] Preferably, in step 2, the LB solid screening medium has the following composition: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 8 g / L, spectinomycin 50 mg / L.

[0010] Another object of the present application is to provide the genetic transformation of the Salvia miltiorrhiza SmCYP85A1 gene for improving drought resistance of plants, which has the following steps: The SmCYP85A1 OE expression vector is introduced into Agrobacterium GV3101 by the Agrobacterium-mediated one-step shoot transformation method of Salvia miltiorrhiza leaves, and the 35S::SmCYP85A1 expression framework and the selection marker gene Kan are integrated into the chromosome of Salvia miltiorrhiza to obtain the SmCYP85A1 OE transgenic Salvia miltiorrhiza.

[0011] Preferably, the specific genetic transformation steps of the Salvia miltiorrhiza SmCYP85A1 gene for improving drought resistance of plants are as follows: The sterile seedlings of Salvia miltiorrhiza cultured at 24℃, 16h / 8h light, and 3000 lx light intensity are taken as materials, and the leaves are taken off for standby. The GV3101 strain containing the SmCYP85A1 OE expression vector is cultured in an LB resistant liquid medium to OD 600 =0.8, and the leaves are taken in a super-clean bench and slightly scratched with a blade. Then, the leaves are soaked in the Agrobacterium bacterial solution containing the SmCYP85A1 OE expression vector for infection. After 10 minutes, the leaves and stems are taken out and placed on filter paper to absorb the bacterial solution. Then, they are taken with tweezers and placed on a co-culture medium. After 2 days of co-culture in a dark room, the leaves after co-culture are transferred to an adventitious bud induction medium containing 50 mg / L kanamycin (Kan) and 200 mg / L timentin (Tim) for induction and selection of resistant adventitious buds. After 20 days of induction culture, the resistant adventitious buds are transferred to a resistant rooting medium containing 50 mg / L kanamycin (Kan) and 200 mg / L timentin (Tim) until adventitious roots are induced. Finally, the RNA of the transgenic plant leaves with kanamycin (Kan) resistance is extracted, and the cDNA is obtained by reverse transcription. Then, the cDNA is used as a template to analyze the expression of the SmCYP85A1 gene in different transgenic lines, and two lines OE3 and OE5 with significantly up-regulated expression are screened. Then, the SmCYP85A1 OE transgenic Salvia miltiorrhiza is subjected to DNA level identification by means of a handheld fluorescent lamp to determine that the 35S::SmCYP85A1 expression framework has been successfully inserted into the chromosome of the transgenic Salvia miltiorrhiza, and the SmCYP85A1 OE transgenic Salvia miltiorrhiza is obtained.

[0012] Preferably, the composition of the LB resistant liquid medium is as follows: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 50 mg / L of spectinomycin, 50 mg / L of gentamicin, and 20 mg / L of rifampicin.

[0013] Preferably, the composition of the co-culture medium is as follows: 2.2 g / L of MS519, 0.5 g / L of 2-morpholinoethanesulfonic acid (MES), 20 g / L of sucrose, and 7 g / L of agar.

[0014] Preferably, the composition of the adventitious bud induction resistant medium is as follows: 2.2 g / L of MS (Murashige and Skoog), 0.5 g / L of 2-morpholinoethanesulfonic acid (MES), 0.5 mg / L of 6-benzylaminopurine (6-BA), 0.05 mg / L of naphthalene acetic acid (NAA), 20 g / L of sucrose, 4 g / L of plant gel, 50 mg / L of kanamycin (Kan), and 200 mg / L of Tim.

[0015] Preferably, the composition of the resistant rooting medium is as follows: 2.2 g / L of MS (Murashige and Skoog) medium, 0.5 g / L of 2-morpholinoethanesulfonic acid (MES), 0.05 mg / L of indole-3-butyric acid (IBA), 0.02 mg / L of NAA (naphthalene acetic acid), 20 g / L of sucrose, 8 g / L of agar powder, 50 mg / L of kanamycin (Kan), and 200 mg / L of Tim.

[0016] Another object of the present application is to provide the application of the above-mentioned SmCYP85A1 gene in improving the drought resistance of Salvia miltiorrhiza.

[0017] Preferably, the application is specifically as follows: overexpression of the SmCYP85A1 gene improves the drought tolerance of Salvia miltiorrhiza.

[0018] Compared with the prior art, the present application has the following beneficial effects: By creating the transgenic Salvia miltiorrhiza SmCYP85A1 OE, it is first proved that the SmCYP85A1 gene of Salvia miltiorrhiza enhances the biological function of drought resistance of Salvia miltiorrhiza by increasing the endogenous BR content of Salvia miltiorrhiza plants, and a new germplasm of Salvia miltiorrhiza with strong drought resistance is obtained. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1The schematic structural diagram of SmCYP85A1 OE constructed by inserting the overexpression vector pK2GW7 of SmCYP85A1 in Example 1 of the present application is shown in the figure; Figure 2 The identification of the DNA level and the transcription level of the overexpressed SmCYP85A1 transgenic Salvia miltiorrhiza plant material in Example 1 of the present application and the determination of the BR content of SmCYP85A1 OE transgenic Salvia miltiorrhiza in vivo; Figure 3 The drought resistance analysis test of the WT and SmCYP85A1 OE transgenic Salvia miltiorrhiza seedlings grown in soil for one month in Example 1 of the present application; Figure 4 The determination of the physiological and biochemical indexes related to oxidative stress in the leaves of SmCYP85A1 OE transgenic Salvia miltiorrhiza under normal control and drought stress treatment in Example 1 of the present application. DETAILED DESCRIPTION

[0020] The present application will be further described below in conjunction with specific examples. It should be understood that the following description is only used to describe one or several specific embodiments of the present application and does not strictly limit the scope of protection claimed by the present application. In the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other.

[0021] Unless otherwise specified, the reagents involved in the following examples are commercially available conventional reagents, and the methods used are commonly used methods in the technical field. EMBODIMENT

[0022] Step 1: Cloning of SmCYP85A1 gene In view of the current situation that most plants are limited by drought stress and thus hinder the growth of plants, the present application provides a Salvia miltiorrhiza SmCYP85A1 gene and an application of overexpressed transgenic Salvia miltiorrhiza SmCYP85A1 OE for improving the drought resistance of plants.

[0023] Firstly, the Salvia miltiorrhiza SmCYP85A1 gene with the highest homology with CYP85A1 in Arabidopsis thaliana is selected for cloning, and the specific steps are as follows: ​(1) Using Salvia miltiorrhiza aseptic seedlings (provided by the aseptic culture room of the Ludo University of Agriculture and Forestry Engineering Research Institute in Yantai, Shandong Province) as the test material, RNA was extracted using FastPure Universal Plant Total RNA Isolation Kit (Vazyme, NanJing, China, RC411-01); the RNA was reverse transcribed into cDNA using the reverse transcriptase of the SuperScript III first-strand synthesis system (Life Technologies, Carlsbad, USA, 18080051); (2) The coding sequence of the target gene SmCYP85A1 was obtained using the Salvia miltiorrhiza genome data, and the Oligo7 software was used for primer design. The length of the primer was 15-20 bp, and at the same time, the suitable Tm value of the designed primer was between 60°C and 65°C, and the GC content was between 45% and 55%. The forward and reverse primers were added with forward and reverse adaptors for seamless cloning of the pK2GW7 vector; Among them, the forward primer SmCYP85A1-F is shown in SEQ ID NO: 1; the reverse primer SmCYP85A1-R is shown in SEQ ID NO: 2; (3) The cDNA of Salvia miltiorrhiza was used as a template, and the Phanta Max Master Mix high-fidelity enzyme (Vazyme, China, P515-01) was used for PCR amplification. The 50 μL reaction system is as shown in Table 1. Table 1 Reagent Amount Component Amount 2x Phanta Max Master Mix 25.0 μL SmCYP85A1 F forward primer (10 mM) 2.0 μL SmCYP85A1 R reverse primer (10 mM) 2.0 μL Template cDNA 1.0 μL The PCR reaction program is as follows: 95°C, 3 min, 1 cycle; 95°C, 15 s, 60°C, 15 s, 72°C, 30 s, 25 cycles; 72°C, 5 min, 1 cycle; 4°C, storage; The final coding sequence of the gene is 1392 bp long, named SmCYP85A1 gene; the nucleotide sequence is shown in SEQ ID NO: 3; the amino acid sequence of the SmCYP85A1 gene is shown in SEQ ID NO: 4; Step 2: Construction of SmCYP85A1 gene overexpression vector The construction method of the overexpression vector of SmCYP85A1 gene is as follows: (1) The cloned SmCYP85A1 coding sequence was constructed into the overexpression vector pK2GW7 using seamless cloning technology to obtain the SmCYP85A1 overexpression vector (35S::SmCYP85A1). The overexpression vector pK2GW7 is shown in sequence 5. In the reaction system shown, 100 ng of fresh PCR product (cloned SmCYP85A1 coding sequence), 20 ng of pK2GW7 overexpression vector and 2.5 μL of Basic Mix enzyme (Basic Seamless Cloning and Assembly Kit, Beijing TransGen Biotech Co., Ltd., CU201-02) were added respectively. Then, the reaction was carried out in a 50℃ metal bath for 30 min. (2) The mixture after the above reaction was transformed into Escherichia coli DH5α and plated on LB solid selection medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 8 g / L, spectinomycin 50 mg / L). Then, single clones were picked from the selection medium for PCR detection and sequencing verification to confirm that the SmCYP85A1 overexpression vector was successfully constructed and named SmCYP85A1. OE (such as) Figure 1 (as shown) Step 3: Creation of SmCYP85A1 OE transgenic Salvia miltiorrhiza The genetic transformation steps of SmCYP85A1 transgenic Salvia miltiorrhiza are as follows: The SmCYP85A1 OE expression vector was transformed into Agrobacterium GV3101 and infected with Salvia miltiorrhiza leaves using the Agrobacterium-mediated one-step bud transformation method. Finally, the 35S::SmCYP85A1 expression framework and the selection marker gene Kan were integrated into the Salvia miltiorrhiza chromosome to obtain SmCYP85A1 OE transgenic Salvia miltiorrhiza. The specific transformation steps are as follows: One-month-old sterile *Salvia miltiorrhiza* seedlings cultured at 24℃, with 16h / 8h light exposure and a light intensity of 3000 lx (day / night) were used as material. Leaves were removed for later use. The GV3101 strain containing the SmCYP85A1 OE expression vector was cultured in LB resistant liquid medium (tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, spectinomycin 50mg / L, gentamicin 50mg / L, rifampin 20mg / L) until OD500. 600 =0.8, after taking leaves in a clean bench and gently scratching the veins with a blade, soak them until they contain SmCYP85A1. Agrobacterium solution of OE expression vector was used to infect leaves for 10 minutes, then the leaves and stems were taken out, and the bacterial solution was absorbed on filter paper. The leaves were picked up with tweezers and placed on co-culture medium (MS 5192.2 g / L + MES 0.5 g / L + sucrose 20 g / L + agar 7 g / L + AS 100 mmol / L) for 2 days in dark condition at room temperature. Then, the leaves after co-culture were transferred to adventitious bud induction medium (2.2 g / L MS + 0.5 g / L MES + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 20 g / L sucrose + 4 g / L phytagel + 50 mg / L Kan + 200 mg / L Tim) containing 50 mg / L Kan and 200 mg / L Timentin (Tim) for induction and screening of resistant adventitious buds. After 20 days of induction culture, the resistant adventitious buds were transferred to resistant rooting medium (2.2 g / L MS + 0.5 g / L MES + 0.05 mg / L IBA + 0.02 mg / L NAA + 20 g / L sucrose + 8 g / L agar powder + 50 mg / L Kan + 200 mg / L Tim) containing 50 mg / L Kan and 200 mg / L Tim for induction of adventitious roots. Finally, the RNA of transgenic plants with Kan resistance was extracted, and cDNA was obtained by reverse transcription. The expression of SmCYP85A1 gene in different transgenic lines was analyzed, and two lines OE3 and OE5 with significantly up-regulated expression were screened. Then, DNA level identification of SmCYP85A1 OE transgenic Salvia miltiorrhiza was performed by means of hand-held fluorescent lamp to determine that the 35S::SmCYP85A1 expression frame had been successfully inserted into the chromosome of transgenic Salvia miltiorrhiza. The BR content in the young leaves of WT and SmCYP85A1 OE transgenic Salvia miltiorrhiza was determined, and it was found that the BR content in the leaves of OE3 and OE5 was significantly increased. As shown in Figure 1, wherein A: SmCYP85A1 OE transgenic Salvia miltiorrhiza was obtained by Agrobacterium-mediated transformation of Salvia miltiorrhiza. B: RNA extraction and cDNA synthesis were performed on seven lines of SmCYP85A1 OE transgenic Salvia miltiorrhiza and WT, and the expression change of SmCYP85A1 in them was analyzed. According to the up-regulated expression amount, two lines OE3 and OE5 were selected for further study. C: The SmCYP85A1 OE transgenic Salvia miltiorrhiza lines (OE3 and OE5) emitted blue-green fluorescence under the irradiation of hand-held fluorescent lamp, indicating that the eYFP fragment in the pK2GW7 vector had been inserted into the Salvia miltiorrhiza genome. D: The BR content in the young leaves of SmCYP85A1 OE transgenic Salvia miltiorrhiza lines (OE3 and OE5) and WT was determined. Figure 2 Step 4: Drought tolerance analysis of SmCYP85A1 OE transgenic Salvia miltiorrhiza The WT and SmCYP85A1​​​​ Using OE transgenic Salvia miltiorrhiza seedlings grown in soil as material, a drought stress treatment experiment was conducted, comparing WT and SmCYP85A1 seedlings with uniform growth. OE transgenic Salvia miltiorrhiza seedlings were randomly divided into two groups (control group and drought stress treatment group), such as Figure 3 As shown, A represents the state of WT and SmCYP85A1 OE transgenic Salvia miltiorrhiza plants in the control group before and 14 days after treatment under normal conditions; B, from top to bottom, represents the morphological changes of WT and SmCYP85A1 OE transgenic Salvia miltiorrhiza before drought stress treatment, 10 days after drought stress treatment, 14 days after drought stress treatment, and 1 day after rehydration; C represents the morphological changes of WT and SmCYP85A1 transgenic Salvia miltiorrhiza plants. The degree of stomatal opening and closing of the OE strain under normal conditions and drought stress; D represents the water loss rate of the leaves of WT and SmCYP85A1 OE plants; among them, the WT and SmCYP85A1 OE transgenic Salvia miltiorrhiza plants (OE3, OE5) in the control group showed good growth and consistent growth status before and 14 days after treatment. Figure 3 As shown in Figure A; after 10 days of drought treatment, the leaves of WT plants in the drought stress treatment group were significantly wilted and drooping; while the leaves of SmCYP85A1 overexpressing plants (OE3 and OE5), although also experiencing some water loss, showed better overall growth than WT plants and exhibited less wilting; simultaneously, after 14 days of drought treatment, all WT and SmCYP85A1OE plants in the drought stress treatment group withered; after rehydration, the leaves of SmCYP85A1OE plants in the drought stress treatment group began to unfold and regain activity, while the WT group died completely (e.g., ...). Figure 3 (As shown in Figure B). This indicates that transgenic *Salvia miltiorrhiza* with overexpression of the SmCYP85A1 gene significantly enhances its tolerance to drought stress. To elucidate the mechanism of action of SmCYP85A1 in the response of *Salvia miltiorrhiza* to drought stress, the stomatal opening of plants in the control group and the drought stress treatment group was analyzed. It was found that in the control group, the stomata were all slightly open; however, in the drought stress treatment group, the stomatal opening of SmCYP85A1 OE plants was significantly smaller than that of WT plants (e.g., ...). Figure 3 (As shown in C); WT and SmCYP85A1 were also analyzed. The water loss rate of OE transgenic Salvia miltiorrhiza detached leaves after 10 hours was found to be higher in WT leaves than in SmCYP85A1 leaves. OE transgenic Salvia miltiorrhiza leaves (such as...) Figure 3 (As shown in D). In summary, the SmCYP85A1 OE plant has a more efficient stomatal closure regulation mechanism under drought stress, which can more quickly reduce water transpiration and thus enhance drought resistance.

[0024] This invention also relates to WT and SmCYP85A1 The physiological and biochemical indicators related to oxidative stress in the leaves of OE transgenic Salvia miltiorrhiza lines were measured. Figure 4 SmCYP85A1 in Embodiment 1 of the present invention The results of the determination of physiological and biochemical indicators related to oxidative stress in the leaves of OE transgenic Salvia miltiorrhiza lines under normal control and drought stress treatment were as follows: AC represents the content of superoxide anion (A), hydrogen peroxide (B), and the content of membrane lipid peroxidation marker (malondialdehyde, MDA) (C) in the leaves of WT and SmCYP85A1 OE transgenic Salvia miltiorrhiza plants in the control and treatment groups after 10 days of drought treatment; DF represents the activities of catalase (CAT) (D), ascorbate peroxidase (APX) (E), and superoxide dismutase (SOD) (F) in the leaves of WT and SmCYP85A1 OE transgenic Salvia miltiorrhiza plants in the control and treatment groups after 10 days of drought treatment. Under drought stress, SmCYP85A1 The contents of superoxide anion and hydrogen peroxide in the leaves of OE transgenic Danshen strains were significantly lower than those in WT strains. Figure 4 As shown in Figure A), this indicates SmCYP85A1 The OE transgenic Salvia miltiorrhiza strain exhibits stronger scavenging ability for reactive oxygen species (ROS) in its leaves; meanwhile, SmCYP85A1 The MDA content in the leaves of OE transgenic Danshen strains was significantly lower than that in WT strains (e.g., Figure 4 As shown in Figure C), this illustrates SmCYP85A1. OE transgenic *Salvia miltiorrhiza* lines exhibit lower levels of membrane lipid peroxidation, resulting in less cell membrane damage and greater drought tolerance. The cellular antioxidant defense system (of which antioxidant enzymes are the most important components) can counteract ROS accumulation. Under various stress conditions, key antioxidant enzymes such as CAT, APX, and SOD help alleviate ROS accumulation. Analysis showed that under non-stress conditions, the enzyme activities of CAT, APX, and SOD did not differ significantly between the WT and SmCYP85A1 OE lines; under drought stress, the activities of these three enzymes increased in all plants; specifically, compared to WT, SmCYP85A1 showed increased activity. OE plants showed significantly higher CAT, APX, and SOD activities than WT plants (e.g., Figure 4 (As shown in DF), this illustrates SmCYP85A1 In OE plants, the accumulation of ROS is reduced by increasing the activity of antioxidant enzymes in the cellular antioxidant defense system, thereby enhancing drought resistance.

[0025] The application takes the coding genomic sequence of SmCYP85A1 in Salvia miltiorrhiza as a reference sequence to design primers, and clones to obtain the SmCYP85A1 gene of Salvia miltiorrhiza; then the coding sequence of SmCYP85A1 is constructed into a plant pK2GW7 vector through a DNA seamless cloning system; subsequently, a SmCYP85A1 overexpression transgenic Salvia miltiorrhiza strain (SmCYP85A1 OE) is obtained through a stable genetic transformation method of Salvia miltiorrhiza, and the SmCYP85A1 OE increases the BR content in vivo; finally, through comparative analysis of the drought stress tolerance of the SmCYP85A1 OE transgenic Salvia miltiorrhiza strain and WT soil culture seedlings, it is found that the drought stress tolerance of the SmCYP85A1 OE transgenic Salvia miltiorrhiza is better than that of the WT plant; finally, the function of SmCYP85A1 in improving the drought resistance of Salvia miltiorrhiza is determined, and a SmCYP85A1 OE drought-resistant Salvia miltiorrhiza new germplasm is created.

[0026] The Salvia miltiorrhiza SmCYP85A1 gene for improving the drought resistance of plants and the application thereof provided by the application provide key gene resources and technical support for the cultivation of drought-resistant herb medicinal plants.

Claims

1. A Salvia miltiorrhiza SmCYP85A1 gene for improving drought resistance of plants, whose nucleotide sequence is shown in SEQ ID NO: 3, and whose amino acid sequence is shown in SEQ ID NO:

4.

2. A cloning method of the Salvia miltiorrhiza SmCYP85A1 gene for improving drought resistance of plants according to claim 1, comprising the following steps: Step 1: extracting RNA from sterile seedlings of Salvia miltiorrhiza; and reverse-transcribing the RNA into cDNA by using a reverse transcriptase; Step 2: obtaining a coding sequence of SmCYP85A1 by using Salvia miltiorrhiza genomic data, and designing primers, wherein a forward primer SmCYP85A1-F is shown in SEQ ID NO: 1, and a reverse primer SmCYP85A1-R is shown in SEQ ID NO: 2; Step 3: amplifying a cDNA sequence of the target gene by using cDNA of Salvia miltiorrhiza as a template and performing PCR amplification with the aid of a high-fidelity enzyme, and finally obtaining a full-length coding sequence of 1392 bp of the SmCYP85A1 gene, which is named as SmCYP85A1 gene. In Step 2, the length of the primers is 15-20 bp, and the Tm value of the designed primers is 60℃. A pK2GW7 vector linker is added to the primers.

4. Construction of an overexpression vector of the Salvia miltiorrhiza SmCYP85A1 gene for improving drought resistance of plants according to claim 1, comprising the following steps: Step 1: adding the coding sequence of the fresh PCR product SmCYP85A1 gene, a pK2GW7 overexpression vector and a Basic Mix enzyme into a PCR tube, and then placing the PCR tube in a 50℃ metal bath for 30 minutes to complete the seamless cloning and assembly process; Step 2: transforming Escherichia coli with the mixed solution after the reaction in Step 1, and then coating the transformed Escherichia coli on an LB solid screening medium, and subsequently picking single colonies from the screening medium for PCR detection and sequencing verification, so as to confirm that the SmCYP85A1 overexpression vector is successfully constructed, and the SmCYP85A1 overexpression vector is named as SmCYP85A1 OE. In Step 2, the LB solid screening medium comprises the following components: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 8 g / L of agar powder and 50 mg / L of spectinomycin.

3. The cloning method for the SmCYP85A1 gene of *Salvia miltiorrhiza* to improve plant drought resistance as described in claim 2, characterized in that:

6. Genetic transformation of the Salvia miltiorrhiza SmCYP85A1 gene for improving drought resistance of plants according to claim 1, comprising the following steps: after the SmCYP85A1 OE expression vector constructed in claim 4 is transferred into Agrobacterium GV3101, the Salvia miltiorrhiza leaf is infected and transformed, so as to finally realize integration of a 35S::SmCYP85A1 expression framework and a selection marker gene kanamycin gene into the Salvia miltiorrhiza chromosome, and obtain SmCYP85A1 OE transgenic Salvia miltiorrhiza.

7. The genetic transformation of the Salvia miltiorrhiza SmCYP85A1 gene for improving drought resistance of plants according to claim 6, comprising the following specific genetic transformation steps: ​ ​ 5. The vector for overexpression of the Salvia miltiorrhiza SmCYP85A1 gene for improving drought resistance of plants according to claim 4, wherein the vector is constructed by inserting the SmCYP85A1 gene into the pCAMBIA 2300 vector. ​ ​ ​ ​ Take the annual Salvia miltiorrhiza sterile seedlings cultured at 24℃, 16h / 8h light, 3000 lx light intensity as the material, and take the leaves for standby. GV3101 strain containing SmCYP85A1 OE expression vector was cultured in LB resistant liquid medium to OD 600 =0.8, and the tender Salvia miltiorrhiza leaves were taken in the clean bench and slightly scratched with a blade, then soaked in Agrobacterium liquid containing SmCYP85A1 OE expression vector for infection, 10 minutes later, the leaves and stems were taken out and placed on filter paper to absorb the bacteria liquid, then taken with tweezers to co-culture medium, co-cultured for 2 days at room temperature in the dark, then the co-cultured leaves were transferred to the adventitious bud induction medium containing 50 mg / L kanamycin and 200 mg / L timentin for induction and selection of resistant adventitious buds; after 20 days of induction culture, the resistant adventitious buds were transferred to the resistant rooting medium containing 50 mg / L kanamycin and 200 mg / L timentin, until the adventitious roots were induced, finally, the RNA of transgenic plants with kanamycin resistance was extracted, cDNA was obtained by reverse transcription, and the expression of SmCYP85A1 gene in different transgenic lines was analyzed, and two lines OE3 and OE5 with significantly up-regulated expression were screened out; then, the SmCYP85A1 OE transgenic Salvia miltiorrhiza was DNA level identified by hand-held fluorescent lamp, and it was determined that the 35S::SmCYP85A1 expression frame was successfully inserted into the chromosome of the transgenic Salvia miltiorrhiza, and the SmCYP85A1 OE transgenic Salvia miltiorrhiza was obtained.

8. The genetic transformation of Salvia miltiorrhiza SmCYP85A1 gene for improving drought resistance of plants according to claim 7, wherein the composition of the LB resistant liquid medium is as follows: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 50 mg / L of spectinomycin, 50 mg / L of gentamicin, 20 mg / L of rifampicin; the composition of the co-culture medium is as follows: 2.2 g / L of MS519, 0.5 g / L of 2-morpholinoethanesulfonic acid, 20 g / L of sucrose, 7 g / L of agar, 100 mmol / L of AS; the composition of the adventitious bud induction medium is as follows: 2.2 g / L of MS, 0.5 g / L of MES, 0.5 mg / L of 6-BA, 0.05 mg / L of NAA, 20 g / L of sucrose, 4 g / L of plant gel, 50 mg / L of kanamycin, 200 mg / L of timentin; the composition of the resistant rooting medium is as follows: 2.2 g / L of MS, 0.5 g / L of MES, 0.05 mg / L of IBA, 0.02 mg / L of NAA, 20 g / L of sucrose, 8 g / L of agar powder, 50 mg / L of kanamycin, 200 mg / L of timentin.

9. The use of Salvia miltiorrhiza SmCYP85A1 gene for improving drought resistance of plants according to claim 1.

10. The use according to claim 9, specifically as follows: overexpression of SmCYP85A1 gene to improve the tolerance of Salvia miltiorrhiza to drought stress.

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