Salvia miltiorrhiza gene smcyp85a1 for improving drought resistance of plants and application thereof

Abstract

This invention relates to a *Salvia miltiorrhiza* SmCYP85A1 gene for improving plant drought resistance and its application, belonging to the field of plant growth and development genetic engineering. The encoding nucleotide sequence of the *Salvia miltiorrhiza* SmCYP85A1 gene is shown in Sequence 3; the amino acid sequence of the *Salvia miltiorrhiza* SmCYP85A1 gene is shown in Sequence 4. This invention obtains transgenic *Salvia miltiorrhiza* SmCYP85A1 OE overexpressing *Salvia miltiorrhiza* by infecting *Salvia miltiorrhiza* leaves with a 35S strong promoter-driven SmCYP85A1 expression vector. Compared with non-transgenic *Salvia miltiorrhiza*, the SmCYP85A1 OE transgenic *Salvia miltiorrhiza* significantly enhances the plant's tolerance to drought stress. Therefore, this invention provides key gene resources and technical support for the genetic improvement of drought-resistant medicinal herbs.

Description

A SmCYP85A1 gene for enhancing drought resistance in tanshinone plants and its application Technical Field

[0001] This invention relates to a Danshen SmCYP85A1 gene for improving plant drought resistance and its application, belonging to the field of plant growth and development genetic engineering. Background Technology

[0002] Salvia miltiorrhiza, a perennial herb belonging to the Lamiaceae family and the Salvia genus, has a long history of medicinal use and is one of my country's traditional major medicinal materials. Traditionally, Salvia miltiorrhiza was mainly sourced from wild resources, but it has gradually transitioned to cultivation. However, cultivated yields are limited, while demand is increasing year by year. Furthermore, as a medicinal herb, drought stress severely affects the quality and yield of Salvia miltiorrhiza. Therefore, utilizing genetic resources to cultivate new crop varieties with greater stress resistance is of great significance.

[0003] Hormones, as important substances produced within plants, regulate plant growth, development, and stress resistance. Currently, plant hormones have become an effective method for alleviating drought stress. Studies have shown that various plant hormones can enhance plant stress resistance, among which brassinolide (BR), a novel plant hormone, plays a central role in plant growth, development, morphogenesis, 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 synthetic shortcut between the early C-22 and late C-6 oxidation pathways. CYP85A1 is the BR oxidase gene, connecting the late C-6 oxidation pathway and the early C-6 oxidation pathway, and is a crucial rate-limiting enzyme gene in BR synthesis. Therefore, providing a SmCYP85A1 gene derived from the herbaceous medicinal plant *Salvia miltiorrhiza* to promote BR synthesis and thus improve plant drought resistance has become a pressing technical challenge in this field. Summary of the Invention

[0004] The main objective of this invention is to provide a SmCYP85A1 gene derived from the herbaceous medicinal plant Salvia miltiorrhiza, which can be used to promote BR synthesis and thereby improve the plant's drought resistance.

[0005] The present invention adopts the following technical solution:

[0006] A Danshen SmCYP85A1 gene that enhances plant drought resistance has the nucleotide sequence shown in Sequence 3 and the amino acid sequence shown in Sequence 4.

[0007] This invention also provides a method for cloning the SmCYP85A1 gene of *Salvia miltiorrhiza* that enhances plant drought resistance, comprising the following steps:

[0008] Step 1: Using sterile Salvia miltiorrhiza seedlings (provided by the Institute of Agricultural and Forestry Engineering, Ludong University, Yantai City, Shandong Province) as experimental material, RNA was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China, RC411-01); the RNA was reverse transcribed into cDNA using the SuperScript III first-strand synthesis system (Life Technologies, Carlsbad, USA, 18080051).

[0009] Step 2: Using the tanshinone genome data, obtain the coding sequence of SmCYP85A1, and design primers using Oligo7 software; the SmCYP85A1-F forward primer is shown in Sequence 1; the SmCYP85A1-R reverse primer is shown in Sequence 2.

[0010] Step 3: Amplification of the target gene SmCYP85A1 coding sequence

[0011] Using cDNA from Salvia miltiorrhiza as a template, and with the aid of Phanta Max Master Mix high-fidelity enzyme (Vazyme, NanJing, China, P515-01), PCR amplification was performed to obtain the full-length 1392bp coding sequence of SmCYP85A1, which was named the SmCYP85A1 gene (its nucleotide sequence is shown in Sequence 3; its amino acid sequence is shown in Sequence 4).

[0012] Preferably, in step 2, the primer length is 15-20 bp, and the Tm value of the designed primer is suitable at 60℃. The pK2GW7 vector adapter is added to the primer.

[0013] Another objective of this invention is to provide the construction of the above-mentioned overexpression vector for the SmCYP85A1 gene in *Salvia miltiorrhiza* to enhance plant drought resistance, the steps of which are as follows:

[0014] Step 1: Add fresh PCR product (coding sequence of SmCYP85A1 gene), pK2GW7 overexpression vector and Basic Mix enzyme to PCR tubes respectively, then place them in a 50℃ metal bath and react for 30 min to complete the seamless cloning and assembly process.

[0015] Step 2: Transform Escherichia coli DH5α into the mixture after the above steps and plate it on LB solid selection medium. 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 SmCYP85A1OE.

[0016] Preferably, in step 2, the LB solid screening medium has the following composition: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 8 g / L agar powder, and 50 mg / L spectinomycin.

[0017] Another object of the present invention is to provide the genetic transformation of the SmCYP85A1 gene in *Salvia miltiorrhiza* that improves plant drought resistance, the steps of which are as follows:

[0018] 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.

[0019] Preferably, the specific genetic transformation steps of the Tanshinone SmCYP85A1 gene for improving plant drought resistance are as follows:

[0020] One-month-old sterile *Salvia miltiorrhiza* seedlings cultured at 24℃, with a light intensity of 3000 lx for 16h / 8h, 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 until OD200. 600=0.8. Leaves were taken in a clean bench and the veins were gently incised with a blade. They were then immersed in an Agrobacterium tumefaciens solution containing the SmCYP85A1OE expression vector for infection. After 10 minutes, the leaves and stem segments were removed and blotted dry on filter paper. They were then transferred to a co-culture medium with forceps and co-cultured at room temperature in the dark for 2 days. Subsequently, the co-cultured leaves were transferred to a medium containing 50 mg / L kanamycin (Kan) and 200 mg / L... Resistant adventitious shoots were induced and screened on a termethin (Tim) adventitious shoot induction medium. After 20 days of induction culture, the resistant adventitious shoots were transferred to a resistant rooting medium containing 50 mg / L kanamycin (Kan) and 200 mg / L termethin (Tim) until adventitious roots were induced. Finally, RNA was extracted from the leaves of transgenic plants that had acquired kanamycin (Kan) resistance. After obtaining cDNA through reverse transcription, cDNA was used as a template to analyze the expression of the SmCYP85A1 gene in different transgenic lines, and two lines, OE3 and OE5, with significantly upregulated expression levels were screened. Subsequently, the DNA level of SmCYP85A1 OE transgenic Salvia miltiorrhiza was identified using a handheld fluorescent lamp, confirming that the 35S::SmCYP85A1 expression framework had been successfully inserted into the chromosome of the transgenic Salvia miltiorrhiza, thus obtaining SmCYP85A1 OE transgenic Salvia miltiorrhiza.

[0021] Preferably, the composition of the LB-resistant liquid culture medium is as follows: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 mg / L spectinomycin, 50 mg / L gentamicin, and 20 mg / L rifampin.

[0022] Preferably, the co-culture medium consists of: MS519 2.2 g / L, 2-morpholinoethanesulfonic acid (MES) 0.5 g / L, sucrose 20 g / L, agar 7 g / L, and acetosyringone (AS) 100 mmol / L.

[0023] Preferably, the adventitious bud resistance-inducing medium comprises the following components: 2.2 g / L MS (Murashige and Skoog), 0.5 g / L 2-morpholinoethanesulfonic acid (MES), 0.5 mg / L 6-benzylaminopurine (6-BA), 0.05 mg / L naphthaleneacetic acid (NAA), 20 g / L sucrose, 4 g / L plant gel, 50 mg / L kanamycin (Kan), and 200 mg / L termetine (Tim).

[0024] Preferably, the composition of the resistance rooting medium is: 2.2 g / L MS (Murashige and Skoog) medium, 0.5 g / L 2-morpholinoethanesulfonic acid (MES), 0.05 mg / L indole-3-butyric acid (IBA), 0.02 mg / L NAA (naphthaleneacetic acid), 20 g / L sucrose, 8 g / L agar powder, 50 mg / L kanamycin (Kan), and 200 mg / L termetine (Tim).

[0025] Another object of the present invention is to provide the application of the above-mentioned SmCYP85A1 gene in improving the drought resistance of Salvia miltiorrhiza.

[0026] Preferably, the application is as follows: overexpressing the SmCYP85A1 gene to improve the tolerance of Salvia miltiorrhiza to drought stress.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] By creating a transgenic Danshen SmCYP85A1OE that overexpresses the SmCYP85A1 gene in Danshen, we have for the first time demonstrated the biological function of the SmCYP85A1 gene in Danshen to enhance the drought resistance of Danshen by increasing the endogenous BR content in Danshen plants. At the same time, we obtained a new Danshen germplasm with strong drought resistance. Attached Figure Description

[0029] Figure 1 is a schematic diagram of the structure of SmCYP85A1 OE constructed by inserting SmCYP85A1 into the overexpression vector pK2GW7 in Embodiment 1 of the present invention;

[0030] Figure 2 shows the identification of SmCYP85A1 transgenic Salvia miltiorrhiza plants overexpressing SmCYP85A1 at the DNA and transcriptional levels, and the determination of BR content in SmCYP85A1 OE transgenic Salvia miltiorrhiza.

[0031] Figure 3 shows the drought resistance analysis test of WT and SmCYP85A1 OE transgenic Salvia miltiorrhiza seedlings grown in soil for one month in Example 1 of the present invention;

[0032] Figure 4 shows the determination of physiological and biochemical indicators related to oxidative stress in the leaves of the SmCYP85A1 OE transgenic Salvia miltiorrhiza line in Example 1 of this invention under normal control and drought stress treatment. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments. It should be understood that the following text is only used to describe one or more specific implementations of the present application and does not strictly limit the scope of protection specifically claimed in the present application. In the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other.

[0034] Unless otherwise specified, all reagents used in the following examples are commercially available conventional reagents, and all methods used are methods commonly used in this technical field. Examples

[0035] Step 1: Cloning of the SmCYP85A1 gene

[0036] In response to the current situation where the growth of most plants is hindered due to drought stress, this invention provides a method to improve the drought resistance of plants by using the SmCYP85A1 gene of Danshen and the application of transgenic Danshen with SmCYP85A1OE overexpression.

[0037] First, the SmCYP85A1 gene from *Arabidopsis thaliana*, which has the highest homology with CYP85A1 in *Arabidopsis thaliana*, was selected for cloning. The specific steps are as follows:

[0038] (1) Using sterile seedlings of Salvia miltiorrhiza (provided by the sterile culture laboratory of the Institute of Agricultural and Forestry Engineering, Ludong University, Yantai City, Shandong Province) as experimental materials, RNA was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, NanJing, China, RC411-01); RNA was reverse transcribed into cDNA using SuperScript III first-strand synthesis system (Life Technologies, Carlsbad, USA, 18080051) reverse transcriptase.

[0039] (2) The coding sequence of the target gene SmCYP85A1 was obtained using the tanshinone genome data. Primers were designed using Oligo7 software. The primer length was 15-20 bp. The suitable Tm value of the designed primers was between 60℃ and 65℃, and the GC content was between 45% and 55%. The forward and reverse adapters of the pK2GW7 vector seamless clone were added to the forward and reverse primers respectively.

[0040] The forward primer SmCYP85A1-F is shown in sequence 1; the reverse primer SmCYP85A1-R is shown in sequence 2.

[0041] (3) Using cDNA of Salvia miltiorrhiza as a template, PCR amplification was performed using Phanta Max Master Mix high-fidelity enzyme (Vazyme, China, P515-01). The 50 μL reaction system is shown in Table 1 below.

[0042] Table 1

[0043] Reagent dosage: Component Amount 2×Phanta Max Master Mix 25.0 μL, L mCYP85A1-F forward primer (10 mM) 2.0 μL, L mCYP85A1-R reverse primer (10 mM) 2.0 μL, template cDNA 1.0 μL surface

[0044] The PCR reaction program is as follows: 95℃, 3 min, 1 cycle; 95℃, 15 s, 60℃, 15 s, 72℃, 30 s, 25 cycles; 72℃, 5 min, 1 cycle; 4℃, store.

[0045] The final coding sequence of the gene, 1392 bp in length, was obtained and named SmCYP85A1; its nucleotide sequence is shown in Sequence 3; the amino acid sequence of the SmCYP85A1 gene is shown in Sequence 4.

[0046] Step 2: Construction of the SmCYP85A1 gene overexpression vector

[0047] 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.

[0048] (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 SmCYP85A1OE (as shown in Figure 1).

[0049] Step 3: Creation of SmCYP85A1 OE transgenic Salvia miltiorrhiza

[0050] The genetic transformation steps of SmCYP85A1 transgenic Salvia miltiorrhiza are as follows:

[0051] 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.

[0052] 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. Leaves were taken in a clean bench and the veins were gently scratched with a blade. They were then immersed in Agrobacterium tumefaciens containing the SmCYP85A1OE expression vector for 10 minutes. The leaves and stem segments were then removed, blotted dry on filter paper, and transferred to a co-culture medium (MS 519 2.2 g / L + MES 0.5 g / L + sucrose 20 g / L + agar 7 g / L + AS 100 mmol / L). The medium was co-cultured for 2 days in the dark at room temperature. Subsequently, the co-cultured leaves were transferred to an adventitious shoot induction medium containing 50 mg / L Kan and 200 mg / L Timentin (Tim) (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 plant gel + 50 mg / L Kan + 200 mg / L...). Resistant adventitious shoots were induced and screened on Tim. After 20 days of induction culture, the resistant adventitious shoots were transferred to a resistant rooting medium containing 50 mg / L Kan and 200 mg / L Tim (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) until adventitious roots were induced. Finally, RNA was extracted from the leaves of transgenic plants that had acquired Kan resistance. After reverse transcription, cDNA was obtained and used as a template to analyze the expression of the SmCYP85A1 gene in different transgenic lines. Two lines, OE3 and OE5, with significantly upregulated expression levels were screened. Subsequently, SmCYP85A1 was subjected to fluorescence under a handheld fluorescent lamp. DNA-level identification of OE transgenic Salvia miltiorrhiza confirmed that the 35S::SmCYP85A1 expression framework had been successfully inserted into the chromosome of the transgenic Salvia miltiorrhiza. The BR content in the young leaves of WT and SmCYP85A1 OE transgenic Salvia miltiorrhiza was measured, revealing a significant increase in BR content in the leaves of OE3 and OE5. As shown in Figure 2, A: RNA extraction and cDNA synthesis were performed on the seven obtained SmCYP85A1OE transgenic Salvia miltiorrhiza lines and WT, and the expression changes of SmCYP85A1 were analyzed. Based on the upregulated expression level, OE3 and OE5 lines were selected for further investigation. B: The SmCYP85A1OE transgenic Salvia miltiorrhiza lines (OE3 and OE5) emitted blue-green fluorescence under handheld fluorescent light, indicating that the eYFP fragment in the pK2GW7 vector had been inserted into the Salvia miltiorrhiza genome. C: The BR content in the young leaves of the SmCYP85A1OE transgenic Salvia miltiorrhiza lines (OE3 and OE5) and WT was measured.

[0053] Step 4: Drought tolerance analysis of SmCYP85A1 OE transgenic Salvia miltiorrhiza

[0054] Using WT and SmCYP85A1OE transgenic Salvia miltiorrhiza seedlings grown in soil as materials, a drought stress treatment experiment was conducted. The uniformly growing WT and SmCYP85A1OE transgenic Salvia miltiorrhiza seedlings were randomly divided into two groups (control group and drought stress treatment group), as shown in Figure 3. A shows the state of the WT and SmCYP85A1OE transgenic Salvia miltiorrhiza plants in the control group before and 14 days after treatment under normal conditions; B, from top to bottom, shows the morphological changes of WT and SmCYP85A1OE 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 shows the stomatal opening and closing degree of WT and SmCYP85A1OE lines under normal conditions and drought stress; D shows the water loss rate of the leaves of WT and SmCYP85A1OE plants; The control group showed the WT and SmCYP85A1OE plants before and 14 days after treatment. The OE transgenic Salvia miltiorrhiza plants (OE3 and OE5) showed good growth and consistent growth status, as shown in Figure 3A. In the drought stress treatment group, the WT plants exhibited significant leaf wilting and drooping after 10 days of drought treatment. While the leaves of the SmCYP85A1 overexpressing plants OE3 and OE5 also experienced some water loss, their overall growth was better than that of the WT plants, with less wilting. Meanwhile, after 14 days of drought treatment, all WT and SmCYP85A1OE plants in the drought stress treatment group withered. After rehydration, the SmCYP85A1 plants in the drought stress treatment group... The leaves of the OE plants began to unfold and regain activity, while the WT group died completely (as shown in Figure 3B). 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 *Salvia miltiorrhiza*'s response to drought stress, the stomatal opening of the control and drought-stressed groups was analyzed. It was found that in the control group, the stomata were slightly open; however, in the drought-stressed groups, the stomatal opening of the SmCYP85A1 OE plants was significantly smaller than that of the WT plants (as shown in Figure 3C). Simultaneously, the water loss rate of detached leaves of WT and SmCYP85A1OE transgenic *Salvia miltiorrhiza* after 10 hours was analyzed, revealing that the water loss rate of WT leaves was consistently higher than that of SmCYP85A1OE transgenic *Salvia miltiorrhiza* leaves (as shown in Figure 3D). In conclusion, SmCYP85A1... OE plants have a more efficient stomatal closure regulation mechanism under drought stress, which can more quickly reduce water transpiration and thus enhance drought resistance.

[0055] This invention also measured the physiological and biochemical indicators related to oxidative stress in the leaves of WT and SmCYP85A1OE transgenic Salvia miltiorrhiza lines. Figure 4 shows the results of the measurement of physiological and biochemical indicators related to oxidative stress in the leaves of SmCYP85A1OE transgenic Salvia miltiorrhiza lines under normal control and drought stress treatment in Example 1 of this invention. Among them, AC represents the superoxide anion content (A), hydrogen peroxide content (B), and the content of membrane lipid peroxidation marker (Malondialdehyde, MDA) (C) in the leaves of WT and SmCYP85A1 OE transgenic Salvia miltiorrhiza plants after 10 days of drought treatment in the control group and treatment group; DF represents the catalase (CAT) activity (D), ascorbate peroxidase (APX) activity (E), and superoxide dismutase (SOD) activity (F) in the leaves of WT and SmCYP85A1 OE transgenic Salvia miltiorrhiza plants after 10 days of drought treatment in the control group and treatment group. Under drought stress, the contents of superoxide anion and hydrogen peroxide in the leaves of the SmCYP85A1OE transgenic Salvia miltiorrhiza line were significantly lower than those in the WT line (Figure 4A), indicating that the SmCYP85A1OE transgenic Salvia miltiorrhiza line had a stronger ability to scavenge reactive oxygen species (ROS). At the same time, the MDA content in the leaves of the SmCYP85A1OE transgenic Salvia miltiorrhiza line was significantly lower than that in the WT line (Figure 4C), indicating that the SmCYP85A1OE transgenic Salvia miltiorrhiza line had a lower degree of membrane lipid peroxidation, resulting in less cell membrane damage and making it more drought resistant. The cellular antioxidant defense system (of which antioxidant enzymes are the most important components) can counteract the accumulation of ROS. Under various stress conditions, key antioxidant enzymes such as CAT, APX, and SOD help to alleviate the accumulation of ROS. Analysis showed that under non-stress conditions, there were no significant differences in the enzyme activities of CAT, APX, and SOD between the WT and SmCYP85A1 OE lines; under drought stress, the activities of the above three enzymes increased in all plants; specifically, compared with WT, the activities of CAT, APX, and SOD in SmCYP85A1OE plants were significantly higher than those in WT (as shown in DF in Figure 4), indicating that SmCYP85A1OE plants reduced ROS accumulation by increasing the enzyme activity of antioxidant enzymes in the cellular antioxidant defense system, thereby enhancing drought resistance.

[0056] This invention uses the coding genome sequence of SmCYP85A1 in Salvia miltiorrhiza as a reference sequence to design primers and clone the SmCYP85A1 gene. Then, using a seamless DNA cloning system, the coding sequence of SmCYP85A1 is constructed into the plant pK2GW7 vector. Subsequently, a stable genetic transformation method is used to obtain a transgenic Salvia miltiorrhiza line overexpressing SmCYP85A1 (SmCYP85A1OE), with increased BR content. Finally, by comparing the drought tolerance of the SmCYP85A1OE transgenic Salvia miltiorrhiza line with that of WT soil-cultured seedlings, this invention found that the SmCYP85A1OE transgenic Salvia miltiorrhiza has a better drought tolerance than the WT plants. Ultimately, this invention clarifies the function of SmCYP85A1 in improving the drought resistance of Salvia miltiorrhiza and creates a new drought-resistant Salvia miltiorrhiza germplasm, SmCYP85A1OE.

[0057] The SmCYP85A1 gene for improving drought resistance in tanshinone plants and its application provided by this invention offer key gene resources and technical support for the cultivation of drought-resistant herbaceous medicinal plants.

Claims

1. An application of the SmCYP85A1 gene in improving the drought resistance of *Salvia miltiorrhiza*, characterized in that: The nucleotide sequence of SmCYP85A1 is shown in Sequence 3; the amino acid sequence is shown in Sequence 4; the application is to overexpress the SmCYP85A1 gene of Salvia miltiorrhiza to improve the tolerance of Salvia miltiorrhiza to drought stress.

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