Salvia miltiorrhiza gene SmWRKY71 and its application

Abstract

The present invention discloses a Salvia miltiorrhiza gene SmWRKY71 and an application thereof. The nucleotide sequence of the Salvia miltiorrhiza gene SmWRKY71 is shown in SEQ ID NO: 1, and the amino acid sequence thereof is shown in SEQ ID NO: 2. Overexpression of the Salvia miltiorrhiza gene SmWRKY71 can improve the drought resistance of Salvia miltiorrhiza, and the gene is involved in regulating the synthesis of tanshinone compounds and salvianolic acid compounds, and overexpression of the gene can improve the accumulation of tanshinone compounds and salvianolic acid compounds. The present invention provides ideas for cultivating high-quality Salvia miltiorrhiza varieties.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a salvia miltiorrhiza gene SmWRKY71 and an application thereof. Background Art

[0002] Salvia miltiorrhiza is one of the traditional major medicinal materials in my country. It is the dried root and rhizome of Salvia miltiorrhiza Bge., a plant of the genus Salvia in the family Lamiaceae. It is mostly used clinically for the treatment of cardiovascular and cerebrovascular diseases, cancer, inflammation and neurodegenerative diseases. In recent years, global climate change and extreme weather have occurred frequently, and long-term or extreme uneven distribution of water and heat has made the plant growth environment increasingly severe. Drought, the most important abiotic stress factor that limits plant growth, development and productivity, has led to a sharp drop in the production of Salvia miltiorrhiza, a major medicinal material in my country. In recent years, drought caused by uneven distribution of water and heat has plagued the production and quality of Salvia miltiorrhiza in the main production areas of Salvia miltiorrhiza, such as Shandong, Henan and Shaanxi. This has caused a shortage of medicinal materials and soaring prices, which has seriously affected the stable development of the Salvia miltiorrhiza industry. Therefore, cultivating Salvia miltiorrhiza varieties with strong stress resistance to cope with extreme climate problems is an effective way to solve this dilemma.

[0003] Studies have shown that drought stress reduces the photosynthetic rate and stomatal conductance in Salvia miltiorrhiza, affecting its growth. Water shortage can cause the expression of drought-related genes in Salvia miltiorrhiza to be upregulated, and the expression of transcription factors such as AP2 / ERF, WRKY, and bZIP that regulate terpenoid gene expression is also significantly upregulated. Under drought conditions, SmWD40 can mediate ABA and hydrogen peroxide signals to regulate stomatal movement in response to drought stress. In attempts to improve stress-resistant Salvia miltiorrhiza varieties, it was found that Salvia miltiorrhiza galactinol synthase (Gols) may be involved in the response of Salvia miltiorrhiza to temperature and water stress. Studies have shown that the introduction of Arabidopsis stress-inducible transcription factor AtDREB1B can increase the activity of enzymes such as SOD and CAT in Salvia miltiorrhiza, and reduce O 2- and H2O2 accumulation, and changes in the expression of key genes in photosynthesis, stress response, carbohydrate metabolism and cell protection, showing enhanced drought tolerance. These studies provide references for the drought resistance mechanism of Salvia miltiorrhiza and the improvement of drought-resistant varieties to a certain extent, but there is still a lack of in-depth research on the gene response regulatory network of Salvia miltiorrhiza under drought stress. This limits the in-depth understanding of the molecular mechanism of Salvia miltiorrhiza hormone network regulation under drought conditions, and also limits further breeding research on the cultivation of drought-resistant, high-yield and high-quality Salvia miltiorrhiza varieties. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a Salvia miltiorrhiza gene SmWRKY71 and its application. The present invention clones the Salvia miltiorrhiza SmWRKY71 gene from the Salvia miltiorrhiza genome data and constructs an overexpression vector, and uses Agrobacterium transformation technology to achieve overexpression of the SmWRKY71 gene in Salvia miltiorrhiza, thereby improving the drought resistance of Salvia miltiorrhiza. In addition, the expression pattern of this gene is similar to that of the key enzyme genes DXS, GGPPS, and CPS for tanshinone synthesis, and while this gene regulates the accumulation of tanshinone compounds, WRKY71, as a transcription factor, also participates in regulating the synthesis process of phenolic acid compounds. The present invention provides ideas for cultivating new varieties of Salvia miltiorrhiza.

[0005] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0006] The present invention provides a Salvia miltiorrhiza gene SmWRKY71, and the nucleotide sequence of the gene SmWRKY71 is shown as SEQ ID NO: 1.

[0007] The present invention also provides a Salvia miltiorrhiza protein SmWRKY71 encoded by the Salvia miltiorrhiza gene SmWRKY71. The amino acid sequence of the Salvia miltiorrhiza protein SmWRKY71 is shown in SEQ ID NO:2.

[0008] The present invention also provides a primer pair for obtaining the Salvia miltiorrhiza gene SmWRKY71 sequence, the primer pair is:

[0009] SmWRKY71-F: ATGTCTGATGCAGACAAT;

[0010] SmWRKY71-R:TCTTTTTCTTCAACACCCATAG.

[0011] The present invention also provides an overexpression vector pEarleyGate103-SmWRKY71, wherein the overexpression vector pEarleyGate103-SmWRKY71 is an expression vector containing the gene SmWRKY71, wherein the expression vector is pEarleyGate103.

[0012] The present invention also provides a host cell containing the overexpression vector pEarleyGate103-SmWRKY71, characterized in that: the host cell is Agrobacterium tumefaciens EHA105.

[0013] Use of any one of the following for improving the drought resistance of Salvia miltiorrhiza under drought stress, wherein:

[0014] (1) the above-mentioned gene SmWRKY71;

[0015] (2) the above-mentioned overexpression vector pEarleyGate103-SmWRKY71;

[0016] (3) The above-mentioned host cell Agrobacterium tumefaciens EHA105.

[0017] Use of any of the following for improving the accumulation of tanshinone compounds and phenolic acid compounds, wherein:

[0018] (1) the above-mentioned gene SmWRKY71;

[0019] (2) the above-mentioned overexpression vector pEarleyGate103-SmWRKY71;

[0020] (3) The above-mentioned host cell Agrobacterium tumefaciens EHA105.

[0021] The ketone compound is any one or more of dihydrotanshinone, cryptotanshinone, and tanshinone IIA;

[0022] The phenolic acid compound is any one or two of rosmarinic acid and salvianolic acid B.

[0023] Use of any of the following in breeding new varieties of drought-resistant Salvia miltiorrhiza, wherein:

[0024] (1) the above-mentioned gene SmWRKY71;

[0025] (2) the above-mentioned overexpression vector pEarleyGate103-SmWRKY71;

[0026] (3) The above-mentioned host cell Agrobacterium tumefaciens EHA105.

[0027] Beneficial effects of the present invention:

[0028] The present invention constructs a Salvia miltiorrhiza gene SmWRKY71 overexpression strain, and finds that compared with the wild-type Salvia miltiorrhiza, the photosynthetic pigment content of the SmWRKY71 overexpression strain is less affected by drought stress, the photosynthesis ability is stronger, and the SmWRKY71 overexpression strain has a stronger reactive oxygen scavenging ability and a lower reactive oxygen level, thereby showing a higher tolerance to drought stress. In addition, the present invention constructs a Salvia miltiorrhiza gene SmWRKY71 overexpression and knockout strain, and finds that the Salvia miltiorrhiza gene SmWRKY71 regulates the accumulation of tanshinone compounds, and SmWRKY71 participates in regulating the synthesis process of phenolic acid compounds as a transcription factor. The gene SmWRKY71 can enhance the drought resistance of Salvia miltiorrhiza and improve the accumulation of tanshinone compounds and phenolic acid compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the electropherogram of SmWRKY71 clone;

[0030] Figure 2 This is the evolutionary tree analysis diagram of SmWRKY71;

[0031] Figure 3 This is the SmWRKY71 coding sequence alignment;

[0032] Figure 4 This is the subcellular localization map of SmWRKY71 protein;

[0033] Figure 5 This is a diagram of the analysis of the SmWRKY71 gene expression pattern;

[0034] Figure 6 PCR verification of the strain transformed with the SmWRKY71 overexpression vector (left) and gene expression analysis of the overexpression strain (right);

[0035] Figure 7 The photosynthetic parameters of the overexpression strain and wild-type Salvia miltiorrhiza under drought stress;

[0036] In the figure, a, b, c, and d are schematic diagrams of leaf net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr), respectively;

[0037] * and # indicate significant differences, p < 0.05;

[0038] Figure 8 This is the determination of ROS and MDA content in the overexpression strain and wild-type Salvia miltiorrhiza under drought stress;

[0039] In the figure, a, b, and c are schematic diagrams of superoxide anion, H2O2, and MDA content, respectively.

[0040] * and # indicate significant differences, p < 0.05;

[0041] Fig. 9 The antioxidant enzyme system activity diagram of the overexpression strain and wild-type Salvia miltiorrhiza under drought stress;

[0042] In the figure, a, b, and c are the levels of SOD, CAT, and POD, respectively, and * and # indicate significant differences, p<0.05;

[0043] Fig.10 This is a diagram for the identification of WRKY71 knockout and overexpression strains in Salvia miltiorrhiza;

[0044] In the figure, A: amplification electrophoresis of knockout strain, B: qRT-PCR verification of overexpression strain;

[0045] Fig.11 The graph shows the content determination of dihydrotanshinone I (DT-I), cryptotanshinone (CT), tanshinone IIA (TIIA), tanshinone I (TI) and total tanshinone (TT) in the knockout and overexpression strains of WRKY71 in Salvia miltiorrhiza.

[0046] Fig.12 The graph shows the content determination of total flavonoids, total phenolic acids, total anthocyanins, rosmarinic acid (RA) and salvianolic acid B (SAB) in WRKY71 knockout and overexpression strains of Salvia miltiorrhiza.

[0047] Fig.13 This is a graph showing the expression levels of key enzyme genes in the synthesis pathway of tanshinone and tanshinone acid in transgenic plants. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0049] Example 1 Cloning and expression pattern analysis of the Salvia miltiorrhiza gene SmWRKY71

[0050] 1. Cloning of the Salvia miltiorrhiza gene SmWRKY71

[0051] Using the Unigene (c50157_g1) sequence in the transcriptome data of Salvia miltiorrhiza as a template, PCR primer pairs SmWRKY71-F and SmWRKY71-R were designed for PCR amplification. The PCR amplification program was 98°C: 3min, (98°C: 10s, 56°C: 30s, 72°C: 1min) for 30 cycles, 72°C: 10min; the PCR amplification system was 50uL containing Premix Taq: 25μL, Salvia miltiorrhiza cDNA dilution: 2μL, SmWRKY71-F (20μM): 1μL, SmWRKY71-R (20μM): 1μL, sterile water: 21μL. The PCR primers were:

[0052] SmWRKY71-F: ATGTCTGATGCAGACAAT;

[0053] SmWRKY71-R:TCTTTTTCTTCAACACCCATAG.

[0054] A gene sequence was obtained and named as gene SmWRKY71, and the nucleotide sequence is shown in SEQ ID NO:1.

[0055] 2. Analysis of the expression pattern of SmWRKY71 in Salvia miltiorrhiza

[0056] Bioinformatics software and online tools were used to predict the physicochemical properties and protein structure of the gene-encoded protein. DNAMAN and MEGA6.0 were used to perform amino acid multiple sequence alignment and phylogenetic tree construction, respectively. Real-time fluorescence quantitative PCR was used to detect the expression pattern of the gene. Results The cDNA of the SmWRKY71 gene was cloned to be 1103 bp ( Figure 1 ), encoding 244 amino acids, whose sequence is shown in SEQ ID NO: 2, and named as SmWRKY71 protein, with a relative molecular mass of 27600 and an isoelectric point of 8.19.

[0057] The protein contains the WRKY characteristic motif, does not contain a signal peptide or a transmembrane domain, and its higher-order structure is mainly composed of random coils ( Figure 3 Phylogenetic tree analysis showed that the SmWRKY71 protein was most closely related to the persimmon WRKY71 protein ( Figure 2 ).

[0058] Subcellular localization results showed that SmWRKY71 protein was localized in the nucleus, which was consistent with its transcription factor function ( Figure 4 The SmWRKY71 gene is constitutively expressed in various organs of Salvia miltiorrhiza, and its expression pattern is similar to that of the key enzyme genes DXS, GGPPS, and CPS in tanshinone synthesis, and is strongly induced by plant hormones such as methyl jasmonate, abscisic acid, and gibberellins, as well as mechanical damage ( Figure 5 ).

[0059] Example 2 Study on the drought resistance of SmWRKY71 gene of Salvia miltiorrhiza

[0060] I. Cloning of SmWRKY71 and Construction of Overexpressing Plants

[0061] The restriction sites BstI and BamHI were introduced into the CDS region of the SmWRKY71 gene of Danshen, and the CDS region of SmWRKY71 of Danshen was connected to the plant expression vector pEarleyGate103 by the method of enzyme digestion and enzyme ligation, and the competent Escherichia coli DH5α was transformed. A single clone was picked for bacterial liquid PCR verification, and the positive strain was extracted for double enzyme digestion verification. After confirmation, the pEarleyGate103-SmWRKY71 recombinant vector was obtained. The constructed recombinant vector was introduced into Agrobacteriumtumefaciens EHA105 by heat shock method, and a single clone was picked for PCR verification (the system and procedure were the same as in Example 1), and the verified strain was subjected to subsequent experiments.

[0062] The leaf disc transformation method mediated by Agrobacterium tumefaciens EHA105 was used to infect Salvia miltiorrhiza explants. After the obtained resistant buds were tested by PCR and qRT-PCR, the strain with high SmWRKY71 expression (OE-6) was selected for subsequent experiments ( Figure 6 ).

[0063] 2. Comparison of biomass between overexpression plants and wild type under drought stress

[0064] Transgenic and wild-type Salvia miltiorrhiza seedlings of 30 days old were selected and treated with 0%, 5%, 10%, and 15% PEG6000 solution to simulate drought conditions for 12 hours, with 6 individuals in each group. The biomass of seedlings in different treatment groups was measured.

[0065] The results are shown in Table 1. At the end of the treatment, the wild-type Salvia miltiorrhiza in different treatment groups showed different degrees of wilting, especially in the 10% and 15% groups; while the overexpression strain OE-6 had a good growth state when treated with different concentrations, and only some individuals in the 15% group wilted significantly. From the statistical results, when treated with 10% PEG, there was no significant difference between the overexpression strain OE-6 and the 0% PEG treatment control group, while the wild-type plants showed significant differences from the 0% PEG treatment control group, indicating that the overexpression strain OE-6 had stronger drought tolerance than the wild type when treated with 10% PEG.

[0066] Table 1 Statistics of biomass of Salvia miltiorrhiza seedlings under PEG 6000 simulated drought treatment

[0067]

[0068] 3. Comparison of photosynthesis between overexpression plants and wild type under drought stress

[0069] 1. Select the Salvia miltiorrhiza seedlings in the above-mentioned treatment group and control group, and use the colorimetric method to determine the content of chlorophyll and carotenoids in the seedling leaves.

[0070] The results are shown in Table 2. PEG treatment at different concentrations reduced the content of chlorophyll a, chlorophyll b and carotenoids in the leaves of Salvia miltiorrhiza seedlings. However, in the 5% PEG treatment group, the wild-type Salvia miltiorrhiza group showed a significant decrease in photosynthetic pigment content, while the photosynthetic pigment content of the overexpression strain OE-6 was not significantly different from that of the 0% PEG group. The results showed that the photosynthetic pigment content of the overexpression strain OE-6 was less affected by drought stress.

[0071] Table 2 Effects of drought stress on the content of photosynthetic pigments in leaves of Salvia miltiorrhiza seedlings (mg·g -1 )

[0072]

[0073] 2. The net photosynthetic rate (P n ), stomatal conductance (G s ), intercellular CO2 concentration (C i ) and transpiration rate (T r ), the photosynthetic characteristics of the overexpression line OE-6 and wild-type Salvia miltiorrhiza seedlings under drought stress were investigated.

[0074] The results show that Figure 7 ), PEG treatment led to a decrease in the net photosynthetic rate, stomatal conductance and transpiration rate in the leaves of Salvia miltiorrhiza seedlings, among which the wild-type Salvia miltiorrhiza seedlings decreased significantly; while the intercellular CO2 concentration increased with the deepening of the treatment, and the increase in the wild-type Salvia miltiorrhiza seedlings was more obvious, while the increase in the overexpression strain OE-6 was not significant. These indicators show that the overexpression strain OE-6 has a stronger photosynthetic capacity than the wild-type Salvia miltiorrhiza under drought stress.

[0075] IV. Determination of reactive oxygen levels and protective enzyme activities in overexpression plants and wild type under drought stress

[0076] 1. Detect the levels of reactive oxygen and MDA in overexpression strains and wild-type Salvia miltiorrhiza seedlings.

[0077] The results are as follows Figure 8 As shown in the figure, the levels of superoxide anion, H2O2 and MDA in the cells of wild-type seedlings increased significantly under drought treatment, and there was a significant difference when treated with 5% PEG; while the accumulation level of reactive oxygen in the cells of the overexpression strain OE-6 was not significantly different from that of the untreated group when treated with 5% and 10% PEG, and only when treated with 15% PEG did the reactive oxygen level of the overexpression strain OE-6 show a significant difference. This shows that the accumulation of reactive oxygen and MDA levels in the overexpression strain OE-6 was not significant under mild drought stress, and its intracellular reactive oxygen level was lower than that of the wild type under the same treatment conditions.

[0078] 2. Detect the activity of active oxygen system scavenging enzymes SOD, CAT, and POD in the overexpression strains and wild types.

[0079] The results are as follows Fig. 9 As shown in the figure, the SOD, CAT, and POD scavenging enzyme activities in the overexpression strain OE-6 under drought stress were stronger than those in the wild-type Salvia miltiorrhiza. This indicates that the overexpression strain OE-6 has a stronger reactive oxygen scavenging ability and a lower reactive oxygen level, thus showing a higher tolerance to drought stress.

[0080] Example 3 Study on the effect of SmWRKY71 gene on increasing the content of tanshinone and salvianolic acid in salvia miltiorrhiza

[0081] 1. Construction of SmWRKY71 gene overexpression and knockout strains in Salvia miltiorrhiza

[0082] The SmWRKY71 gene overexpression vector is the pEarleyGate103-SmWRKY71 vector plasmid in Example 2. In addition, the knockout target sequence of the SmWRKY71 gene was designed using the working principle of CRISPR-Cas9, and the SmWRKY71 knockout vector pP1C.1-WRKY71 was constructed. The constructed recombinant vector was introduced into Agrobacterium tumefaciens EHA105 by heat shock method, and a single clone was picked for PCR verification (the system and procedure were the same as in Example 1), and the verified strains were subjected to subsequent experiments. The leaf disc transformation method mediated by Agrobacterium tumefaciens EHA105 was used to infect Salvia miltiorrhiza explants. After the obtained resistant buds were tested by PCR and qRT-PCR, the OE-6 strain with high SmWRKY71 expression (same as in Example 2) and the KO-W9 strain with the largest SmWRKY71 sequence variation were selected as the SmWRKY71 gene overexpression strain (OE) and knockout strain (KO) for subsequent experiments ( Fig.10 ).

[0083] 2. Detection of secondary metabolites in overexpression and knockout strains

[0084] 1. Use HPLC to detect the content of tanshinone compounds in OE and KO strains.

[0085] The results are as follows Fig.11As shown in the figure, in the overexpression OE strain, the average contents of dihydrotanshinone, cryptotanshinone, tanshinone IIA and total tanshinone were 0.018 mg / g (4.5 times that of the control), 0.031 mg / g (1.63 times that of the control), 0.009 mg / g (2.25 times that of the control), 0.008 mg / g (8.0 times that of the control), and 0.057 mg / g (2.11 times that of the control); on the contrary, in the knockout KO strain, the contents of dihydrotanshinone, cryptotanshinone, tanshinone IIA and total tanshinone were 75%, 84.21%, 50% and 66.67% of the control, respectively. This result indicates that the expression of SmWRKY71 is positively correlated with the accumulation of tanshinone compounds and plays a key role in regulating the synthesis of tanshinone compounds.

[0086] 2. Detect the accumulation of phenolic acid compounds, including total flavonoids, total phenolic acids and total anthocyanins, in overexpression and knockout strains.

[0087] The results are as follows Fig.11 As shown in the figure, both rosmarinic acid and salvianolic acid B accumulated more in the overexpression OE strain, while the accumulation in the knockout strain was significantly lower than that in the control. This result also shows that in addition to regulating the accumulation of tanshinone compounds, SmWRKY71, as a transcription factor, is also involved in regulating the synthesis of salvianolic acid compounds.

[0088] In addition, if Fig.12 As shown in the figure, the contents of total phenolic acids, total flavonoids and total anthocyanins in the overexpression strain were significantly increased, while on the contrary, the contents of the above secondary metabolites were significantly decreased in the knockout strain.

[0089] 3. Detection of key enzyme gene expression in the metabolic pathway of tanshinone and salvianolic acid synthesis in overexpression and knockout strains

[0090] In order to further explore the regulatory effect of the SmWRKY71 gene on enzyme genes in the tanshinone and salvianolic acid synthesis pathways, this example detected the expression levels of key enzyme genes in the tanshinone and salvianolic acid synthesis pathways in transgenic plants.

[0091] The results are as follows Fig.13 As shown in the figure, the expression levels of GGPPS, CPS1, and CYP76AH1, the key enzyme genes for tanshinone synthesis, were significantly higher in the OE strain than in the control, and significantly lower in the KO strain than in the control, indicating that the expression levels of GGPPS, CPS1, and CYP76AH1 were positively correlated with the expression level of SmWRKY71. The enzyme genes PAL, C4H1, TAT1, and RAS1 in the salvianolic acid synthesis pathway had an expression pattern that was positively correlated with the expression level of SmWRKY71, indicating that SmWRKY71 has a regulatory effect on the expression of these enzyme genes.

[0092] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. An application of any one of the following in improving the drought resistance of Salvia miltiorrhiza under drought stress, characterized in that: in, (1) gene SmWRKY71, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the amino acid sequence of which is shown in SEQ ID NO: 2; (2) overexpression vector pEarleyGate103-SmWRKY71, which contains the above-mentioned gene SmWRKY71; (3) Host cell Agrobacterium tumefaciens EHA105, which contains the above-mentioned overexpression vector pEarleyGate103-SmWRKY71.

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