Application of Salvia miltiorrhiza guide protein SmDIR18 and its encoding gene

Abstract

The present invention discloses a Salvia miltiorrhiza guide protein SmDIR18 and an application of its encoding gene. The present invention clones a guide protein SmDIR18 from the medicinal plant Salvia miltiorrhiza for the first time, which can significantly affect plant growth and development, increase the content of tanshinone in Salvia miltiorrhiza, and further enhance the environmental adaptability of Salvia miltiorrhiza. The research results show that the Salvia miltiorrhiza guide protein SmDIR18 is induced to express by salt after the plant is treated with salt for 4 hours, and the gene expression amount increases significantly. Overexpression of the Salvia miltiorrhiza guide protein SmDIR18 gene can increase the content of tanshinone substances in Salvia miltiorrhiza. The Salvia miltiorrhiza guide protein SmDIR18 gene knockdown plant grows slowly and the content of tanshinone is reduced. The regulation of the gene can be applied to the cultivation of stress-resistant Salvia miltiorrhiza species, and the content of tanshinone, an effective active ingredient in Salvia miltiorrhiza, can be increased by stress resistance, which has high economic value and broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of a Salvia miltiorrhiza guiding protein SmDIR18 and a coding gene thereof. The coding gene of the Salvia miltiorrhiza guiding protein SmDIR18 described in the present invention can regulate the growth and development of Salvia miltiorrhiza plants, increase the tanshinone content in Salvia miltiorrhiza, and thereby enhance the plant's adaptability to the environment and its stress resistance under harsh natural conditions. Background Art

[0002] Salvia miltiorrhiza is a perennial herbaceous plant of the Lamiaceae family and the genus Salvia. The main uses are the dried roots and rhizomes of Salvia miltiorrhiza. As a commonly used clinical medicinal material, Salvia miltiorrhiza contains the effective active ingredient tanshinone, which can be used to treat heart diseases such as coronary heart disease and angina pectoris. As the raw material of many Chinese patent medicines, the demand for Salvia miltiorrhiza has increased accordingly, but the wild Salvia miltiorrhiza resources are scarce, resulting in a shortage of supply. Guidance proteins are directly involved in the synthesis of plant lignin, regulating plant root growth and plant growth and development, and can directly participate in regulating the entry and exit of water, oxygen and ions into and out of the vascular bundle through the endodermal Casparian strip. However, there are currently no reports on the function of guidance proteins in Salvia miltiorrhiza. In-depth exploration of growth and development-related genes in the Salvia miltiorrhiza guiding protein family, increasing the content of tanshinone, the effective active ingredient of the plant, and cultivating other stress-resistant germplasm resources of Salvia miltiorrhiza will greatly promote the further improvement of the economic value of Salvia miltiorrhiza. It will also provide a reference for improving and extracting compounds through stress resistance for some low-content but high-bioactive compounds such as paclitaxel, whose biological pathways are not fully elucidated and cannot be produced through biosynthetic pathways. This is of great significance to the development of the traditional Chinese medicine industry and the scientific use of land. Summary of the invention

[0003] The purpose of the present invention is to provide a Salvia miltiorrhiza guide protein SmDIR18 and an application of its encoding gene in view of the technical problem that the demand for Salvia miltiorrhiza is increasing, but the quality of cultivated Salvia miltiorrhiza is decreasing. The present invention screens and obtains a guide protein SmDIR18 from Salvia miltiorrhiza plants. Studies have found that the guide protein SmDIR18 can regulate the growth and development of Salvia miltiorrhiza, increase the content of tanshinone, an effective active ingredient in Salvia miltiorrhiza, and thus enhance the adaptability of Salvia miltiorrhiza to the environment; therefore, it has high practical application value and economic value.

[0004] The purpose of the present invention can be achieved by the following technical solutions:

[0005] In the first aspect, the present invention seeks to protect the use of the Salvia miltiorrhiza guide protein SmDIR18 with an amino acid sequence as shown in SEQ ID NO.2 or a biological material related to the Salvia miltiorrhiza guide protein SmDIR18 in at least one of the following (a1)-(a9):

[0006] (a1) Application in regulating the growth and development of Salvia miltiorrhiza plants;

[0007] (a2) Use in the preparation of products for regulating the growth and development of Salvia miltiorrhiza plants;

[0008] (a3) application in regulating the content of tanshinone in Salvia miltiorrhiza;

[0009] (a4) Use in the preparation of products for regulating the content of tanshinone in salvia miltiorrhiza;

[0010] (a5) Application in regulating stress resistance of Salvia miltiorrhiza;

[0011] (a6) Use in the preparation of products for regulating stress resistance of Salvia miltiorrhiza;

[0012] (a7) Application in breeding new Salvia miltiorrhiza germplasm with improved growth and development ability;

[0013] (a8) Application in breeding new Salvia miltiorrhiza germplasm with increased tanshinone content;

[0014] (a9) Application in breeding new Salvia miltiorrhiza germplasm with improved stress resistance.

[0015] Furthermore, in the above application, the biological material related to the Salvia miltiorrhiza guide protein SmDIR18 is at least one of the following (b1)-(b11):

[0016] (b1) a gene encoding the Salvia miltiorrhiza guidance protein SmDIR18;

[0017] (b2) a primer pair for cloning the gene described in (b1);

[0018] (b3) an expression cassette containing the gene described in (b1);

[0019] (b4) a recombinant vector containing the gene described in (b1), or a recombinant vector containing the expression cassette described in (b3);

[0020] (b5) a recombinant microorganism containing the gene described in (b1), or a recombinant microorganism containing the expression cassette described in (b3), or a recombinant microorganism containing the recombinant vector described in (b4);

[0021] (b6) a transgenic Salvia miltiorrhiza plant cell line containing the gene described in (b1), or a transgenic Salvia miltiorrhiza plant cell line containing the expression cassette described in (b3), or a transgenic Salvia miltiorrhiza plant cell line containing the recombinant vector described in (b4);

[0022] (b7) transgenic Salvia miltiorrhiza plant tissue containing the gene described in (b1), or transgenic Salvia miltiorrhiza plant tissue containing the expression cassette described in (b3), or transgenic Salvia miltiorrhiza plant tissue containing the recombinant vector described in (b4);

[0023] (b8) transgenic Salvia miltiorrhiza plant organs containing the gene described in (b1), or transgenic Salvia miltiorrhiza plant organs containing the expression cassette described in (b3), or transgenic Salvia miltiorrhiza plant organs containing the recombinant vector described in (b4);

[0024] (b9) a transgenic Salvia miltiorrhiza plant containing the gene described in (b1), or a transgenic Salvia miltiorrhiza plant containing the expression cassette described in (b3), or a transgenic Salvia miltiorrhiza plant containing the recombinant vector described in (b4);

[0025] (b10) a tissue culture produced from regenerable cells of the transgenic Salvia miltiorrhiza plant described in (b9);

[0026] (b11) Protoplasts produced from the tissue culture described in (b10).

[0027] Furthermore, the nucleotide sequence of the gene encoding the Salvia miltiorrhiza guide protein SmDIR18 in (b1) is shown as SEQ ID NO.1.

[0028] Furthermore, the nucleotide sequences of the primer pair in (b2) are shown as SEQ ID NO.3 and SEQ ID NO.4.

[0029] Furthermore, the above-mentioned application is to increase the activity and / or content of the Salvia miltiorrhiza guiding protein SmDIR18 in the target Salvia miltiorrhiza plant, or overexpress the gene encoding the Salvia miltiorrhiza guiding protein SmDIR18, which can promote the growth and development of Salvia miltiorrhiza plants or / and increase the content of tanshinone in Salvia miltiorrhiza or / and improve the stress resistance of Salvia miltiorrhiza.

[0030] In a second aspect, the present invention claims a method, which is at least one of the following (c1) to (c3):

[0031] (c1) a method for promoting the growth and development of Salvia miltiorrhiza;

[0032] (c2) a method for increasing the content of tanshinone in salvia miltiorrhiza;

[0033] (c3) Methods for improving stress resistance of Salvia miltiorrhiza;

[0034] The method is to increase the activity and / or content of the Salvia miltiorrhiza guiding protein SmDIR18 in the target Salvia miltiorrhiza plant, or to overexpress the gene encoding the Salvia miltiorrhiza guiding protein SmDIR18.

[0035] In a third aspect, the present invention claims a method for cultivating transgenic Salvia miltiorrhiza, which comprises overexpressing a gene encoding the Salvia miltiorrhiza guide protein SmDIR18 in a target Salvia miltiorrhiza plant to obtain at least one of the transgenic Salvia miltiorrhiza plants described in (d1) to (d3):

[0036] (d1) Transgenic plants of Salvia miltiorrhiza with improved growth and development ability;

[0037] (d2) transgenic plants with increased tanshinone content;

[0038] (d3) Transgenic plants with improved stress resistance.

[0039] In a specific embodiment of the present invention, the vector containing the gene encoding the Salvia miltiorrhiza guide protein SmDIR18 is transferred into the leaves of sterile Salvia miltiorrhiza seedlings through Agrobacterium infection to obtain the corresponding transgenic Salvia miltiorrhiza plants.

[0040] The gene encoding the Salvia miltiorrhiza guidance protein SmDIR18 is as described above.

[0041] The stress resistance described in the present invention is salt tolerance.

[0042] Beneficial effects of the present invention:

[0043] The invention screens a guide protein SmDIR18 from salvia miltiorrhiza, and obtains the full-length sequence of the gene by reverse transcription of RNA extracted from salvia miltiorrhiza plants into cDNA through PCR technology. An overexpression vector and a knockdown vector are constructed and transferred into sterile salvia miltiorrhiza seedling leaves. Transgenic plants show that the guide protein SmDIR18 can affect the growth and development of salvia miltiorrhiza plants, increase the content of tanshinone in salvia miltiorrhiza, and further enhance the adaptability of salvia miltiorrhiza to the environment, and have high economic value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Salvia miltiorrhiza plants treated with different salt concentrations for different periods of time.

[0045] Figure 2 The content of effective active ingredients after salt treatment;

[0046] Among them, A: the content of tanshinone IIA after salt treatment; B: the content of tanshinone I after salt treatment; C: the content of dihydrotanshinone I after salt treatment; D: the content of cryptotanshinone after salt treatment.

[0047] Figure 3 This is the evolutionary tree of the Salvia miltiorrhiza DPs family genes and six Arabidopsis thaliana DPs genes.

[0048] Figure 4 Screening of guide protein genes in Salvia miltiorrhiza;

[0049] Among them, A: the expression level of 5 DPs genes of Salvia miltiorrhiza after salt treatment; B: the gene expression diagram of tanshinone synthesis pathway of Salvia miltiorrhiza after salt treatment; C: the expression level of SmDIR18 gene in the root of Salvia miltiorrhiza after salt treatment.

[0050] Figure 5 It is a positive verification of the transgenic plants of Salvia miltiorrhiza;

[0051] Among them, A: the first lane is a marker, and the second lane is the amplified SmDIR18 gene CDS sequence fragment; B, C: positive verification of transgenic plants, in which the first lane is a marker, the second to fourth lanes are all wild-type groups, and the fifth to seventh lanes are positive verifications of the SmDIR18 gene knockdown group and the SmDIR18 gene overexpression group, respectively.

[0052] Figure 6 is the expression level of SmDIR18 and the content of tanshinones in transgenic plants;

[0053] Among them, A: photos of transgenic plants; B: expression level of SmDIR18 gene in transgenic plants; C: content of tanshinone I in transgenic plants; D: content of tanshinone IIA in transgenic plants; E: content of cryptotanshinone in transgenic plants. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is described below by specific examples, but the present invention is not limited thereto, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0055] The present invention screened and obtained a guide protein SmDIR18 from the plant Salvia miltiorrhiza, and its amino acid sequence is shown in SEQ ID NO. 2. The protein can be artificially synthesized, or its encoding gene can be synthesized first and then biologically expressed. The nucleotide sequence of the gene encoding the Salvia miltiorrhiza guide protein SmDIR18 is shown in SEQ ID NO. 1.

[0056] Example 1: Danshen salt treatment experiment

[0057] 1.1 Salvia miltiorrhiza seed breeding and growth

[0058] The seeds of Salvia miltiorrhiza were placed in an appropriate container, soaked in water accounting for about 80% of the entire container for half a day, and the mucus on the surface of the seeds was gently removed with gauze, and sown in a nutrient soil with a vermiculite and soil mass ratio of 1:3 for breeding and germination, the temperature was 25°C, and the light-dark time ratio was 16 h / 8 h. After the Salvia miltiorrhiza seeds germinated and grew seedlings, they were transplanted into pots and continued to grow for 80 days for this experiment. The Salvia miltiorrhiza seeds in this experiment were purchased in Shandong.

[0059] A total of 45 Salvia miltiorrhiza plants with similar growth status were selected, and each group of 3 plants was divided into 15 groups on average. Five time points (0 h, 4 h, 8 h, 12 h, and 24 h) and three gradient salt concentrations (0 mol / L, 0.1 mol / L, and 0.3 mol / L) were selected to treat the Salvia miltiorrhiza plants. After the treatment, the Salvia miltiorrhiza plants were washed (such as Figure 1 The samples were stored at -80°C after being photographed, and each group of samples had three replicates.

[0060] 1.2 Determination of Tanshinone Content

[0061] Weigh an appropriate amount of the root of the Salvia miltiorrhiza sample stored in a -80℃ refrigerator and treated with salt stress, wash thoroughly with ultrapure water, place in a 65℃ oven, and dry until the weight of the sample does not change after three consecutive weighings. Put the dried Salvia miltiorrhiza sample into a clean and sterilized mortar, pour in liquid nitrogen and grind it with a pestle, and pass through a 100-mesh sieve. Weigh 0.65 g of the ground powder, add 1.5 ml of pure methanol for extraction, shake at 1800 rpm for 20 min at room temperature, ultrasonicate for 30 min, centrifuge at 4℃, 13000 rpm for 20 min, take the supernatant and pass through a 0.22 μm microporous filter to remove tiny particles, take 100 μL and put it into an Agilent injection bottle, and store the remaining processed samples at -40℃.

[0062] The samples in this experiment were determined by LC / MS of Agilent 1290 / 6545 Q-TOF. Chromatographic conditions: Agilent 1290 high performance liquid chromatography system was used, the column model was ZORBAX SB-C18 (5 μm, 3.0×150 mm; Agilent, USA), the mobile phase A was 0.1% formic acid (1 mL / L) aqueous solution, the mobile phase B was pure acetonitrile, the column temperature was set to 35°C, the injection chamber temperature was 4°C, the sample injection volume was 5 μL, the flow rate was 0.4 mL / min, the post-run time was 3 min, and the mobile phase composition in the positive ion mode was shown in Table 1.

[0063] Table 1 shows the mobile phase composition in positive ion mode

[0064]

[0065] Mass spectrometry conditions: The mass spectrometer was performed using an Agilent 6545A Q-TOF mass spectrometer with the following operating parameters: drying gas was N2, the gas flow rate was 8 L / min, the positive ion mode was used, the spray needle pressure was 35 psig, the capillary voltage was 3500 V, the secondary mass spectrometry collision voltage was 10 V, 20 V, and 40 V, and the scanning frequency was 1.5 images / s.

[0066] The samples were detected by mass spectrometry, and the retention time, molecular weight and secondary fragment ions of tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone contained therein were consistent with those of the standard. By injecting standards of different concentrations and extracting the peak area to establish a standard curve of concentration and peak area, the peak area of ​​the sample was extracted and the corresponding concentration was calculated through the standard curve, and finally the concentration of tanshinones in the sample was obtained. The results of the changes in the content of tanshinone IIA, tanshinone I, dihydrotanshinone I and cryptotanshinone showed that in the plants of Salvia miltiorrhiza treated with salt, the contents of tanshinone IIA, tanshinone I, dihydrotanshinone I and cryptotanshinone all changed. At 0-12 h, the contents of the four tanshinones increased to a certain extent, and the contents began to decrease in 12-24 h. The content of tanshinones increased more under the treatment of 0.1 mol / L salt concentration than that under the treatment of 0.3 mol / L concentration (such as Figure 2 The study found that when plants were treated with salt solutions of different concentrations, the concentration of tanshinone first increased with the increase of salt treatment time, and then slowly decreased.

[0067] Example 2 Screening of Salvia miltiorrhiza leader protein gene

[0068] 2.1 Screening of candidate genes for Salvia miltiorrhiza leader proteins

[0069] Tanshinones are mainly found in the roots of Salvia miltiorrhiza. Salvia miltiorrhiza guides the expression of root genes, which affects the development of plant roots and is closely related to the adaptability of plants to abiotic stress conditions such as salt stress and osmotic stress. This experiment first found the DPs family in Salvia miltiorrhiza, which is mainly divided into four subfamilies. Among them, six DPs (AT4G13580, AT3G24020, AT1G07730, AT2G28670, AT3G55230, AT2G39430) family genes have been found in the Arabidopsis thaliana family, which directly participate in the polymerization of lignin in plant roots and regulate the development of the endothelium of plant roots. The protein sequences of DPs genes were extracted by TBtools software. The protein sequences of Salvia miltiorrhiza DPs and six Arabidopsis thaliana DPs were compared by MEGA 11 software for phylogenetic tree analysis. It was found that five DPs in Salvia miltiorrhiza DPs had high homology with Arabidopsis thaliana DPs, namely GWHPAOSJ011677, SmDIR18 (GWHPAOSJ012058), GWHPAOSJ009894, GWHPAOSJ016925, and GWHPAOSJ009237 (e.g. Figure 3 as shown).

[0070] 2.2 Expression of candidate guide protein genes in Salvia miltiorrhiza

[0071] The Salvia miltiorrhiza plants treated with 0.1 mol / L salt solution at three time points, 0 h, 4 h, and 12 h, were selected, and RNA was extracted according to the instructions of the total RNA extraction kit provided by Nanjing Novozyme Biotechnology Co., Ltd., and the RNA concentration and purity were measured by the instrument Nano. After passing the test, the RNA was stored in a -80°C refrigerator. According to the reverse transcription reagent provided by Yisheng Biotechnology Co., Ltd., the residual DNA in the RNA was first removed according to the reaction system shown in Table 2.

[0072] Table 2 shows the reverse transcription reaction system

[0073]

[0074] Directly add 5 μL of 4×Hifair III SuperMix plus to the reaction solution in the previous step, mix well, and then place the sample in a PCR instrument for reverse transcription according to the program in Table 3.

[0075] Table 3 shows the reverse transcription reaction procedure

[0076]

[0077] After reverse transcription, the samples were stored at -20°C.

[0078] Primers for genes in the tanshinone biosynthesis pathway and five DPs genes were designed using the software Primer Premier 6. qPCR experiments were performed using the cDNA obtained by reverse transcription as a template. The qPCR amplification primers are as follows:

[0079] SmDIR18-F: ACCATGTCGAGGCTGTCCATC (SEQ ID NO.5)

[0080] SmDIR18-R:AAGGTCTGAACGCCGTCCAA (SEQ ID NO.6)

[0081] GWHPAOSJ009894-F: AGCTCCGAGGATGGCAACAG (SEQ ID NO.7)

[0082] GWHPAOSJ009894-R: ATGACGGCGAGATGCGACTC (SEQ ID NO.8)

[0083] GWHPAOSJ011677-F: CGTCCAACACCGTCGTCCAA (SEQ ID NO.9)

[0084] <h2 style=";text-align:left;direction:ltr">GWHPAOSJ011677-R:CTCGTTGTCAACCACCGTCAC(SEQ ID NO.10)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0085] <h2 style=";text-align:left;direction:ltr"> GWHPAOSJ016925-F:TGGCAATACAGTCCAGTTAGGC(SEQ ID NO.11)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0086] <h2 style=";text-align:left;direction:ltr"> GWHPAOSJ016925-R:CGCAGCAGTCAGGTAGTCATC(SEQ ID NO.12)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0087] <h2 style=";text-align:left;direction:ltr"> GWHPAOSJ009237-F:GGCGGCGTTCTCCTTCTTCA(SEQ ID NO.13)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0088] <h2 style=";text-align:left;direction:ltr"> GWHPAOSJ009237-R:AGATCGGGTTGCTCGGCTTC(SEQ ID NO.14)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0089] <h2 style=";text-align:left;direction:ltr"> SmCYP71D373-F:CAGGAACGAGAGTGATAGTGAAC(SEQ ID NO.15)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0090] <h2 style=";text-align:left;direction:ltr"> SmCYP71D373-R:GATAGAGAAGCATAGATAGCGGAAG(SEQ ID NO.16)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0091] <h2 style=";text-align:left;direction:ltr"> SmCYP71D375-F:CGGTGGTGCTGGATATGTTC(SEQ ID NO.17)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0092] <h2 style=";text-align:left;direction:ltr"> SmCYP71D375-R:TCTTCTTGTGCCTTGTTGAGTT(SEQ ID NO.18)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0093] <h2 style=";text-align:left;direction:ltr"> SmCYP71D411-F:AGTGCTCGTGGATGTGTT(SEQ ID NO.19)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0094] <h2 style=";text-align:left;direction:ltr"> SmCYP71D411-R:GCCTTGGTGAGTGTGCTA(SEQ ID NO.20)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0095] <h2 style=";text-align:left;direction:ltr"> SmCYP76AK1-F:CTACTCCACCCCGACAA(SEQ ID NO.21)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0096] <h2 style=";text-align:left;direction:ltr"> SmCYP76AK1-R:CGGATTCCTCCACGAT(SEQ ID NO.22)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0097] SmCPS1-F: ACTACCGTTCATCAAGGCCA (SEQ ID NO.23)

[0098] SmCPS1-R: CCTCGAGTTGATTCTGCACG (SEQ ID NO. 24)

[0099] SmCYP76AH1-F:TCGTCGGAAACATGCTGCAA (SEQ ID NO.25)

[0100] SmCYP76AH1-R: GCGGGCCGTATCTCTTTGAG (SEQ ID NO.26)

[0101] SmDXR-F:TGAGCCCGGTCGTATGACTT (SEQ ID NO.27)

[0102] SmDXR-R: TGAACCAGCAGCAAGTGCAA (SEQ ID NO. 28)

[0103] SmKSL1-F: AAGGTTGGGAGTCGACCGAT (SEQ ID NO.29)

[0104] SmKSL1-R: AGCATACGGAGCCAGTTCCT (SEQ ID NO.30)

[0105] SmActin-F: AGGAACCACCGATCCAGACA (SEQ ID NO.31)

[0106] SmActin-R: GGTGCCCTGAGGTCCTGTT (SEQ ID NO. 32).

[0107] The qPCR reaction solution was prepared according to the reaction system shown in Table 4.

[0108] Table 4 shows the qPCR reaction system

[0109]

[0110] Data processing is based on 2 -△△Ct The expression of related genes in the samples was calculated by this method, and SmActin was used as the internal reference gene for data normalization.

[0111] After salt treatment, the expression levels of the five DPs genes in the roots of Salvia miltiorrhiza first increased, and the expression of the SmDIR18 gene was strongly induced at 4 h of salt treatment, and then the gene expression level decreased at 12 h. Compared with the other four DPs genes, the expression level of the SmDIR18 (GWHPAOSJ012058) gene changed most significantly (e.g. Figure 4 The expression of tanshinone pathway genes in the roots showed a trend of first increasing and then decreasing. The expression of tanshinone pathway genes was higher at 4 h, which was consistent with the expression trend of DPs genes in the roots of Salvia miltiorrhiza (e.g. Figure 4 The results showed that the expression of GWHPAOSJ011677 and GWHPAOSJ009237 genes was different from the expression pattern of tanshinone pathway genes, while the expression pattern of SmDIR18, GWHPAOSJ009894, and GWHPAOSJ016925 genes was consistent with the expression trend of tanshinone pathway genes, among which the expression of SmDIR18 gene changed significantly, which may be significantly induced by salt stress.

[0112] Example 3: Construction of gene vector and acquisition of transgenic plants

[0113] 3.1 Gene vector construction

[0114] The cDNA extracted from the RNA in Example 2 was used as a template to amplify the ORF of the target gene using primers SmDIR18-F (underlined is the restriction endonuclease BamH I recognition sequence) and SmDIR18-R (underlined is the restriction endonuclease Sac I recognition sequence). The amplification primers are as follows:

[0115] SmDIR18-F: CTCTCTCTCAAGCTT GGATCC ATGTCCAAGAAATCAGCAATAGTGTT (SEQ IDNO.3)

[0116] SmDIR18-R:GCCCTTGCTCACCAT GAGCTC TCAGTAAGTAAGATGCACAGTGATCCT (SEQ ID NO. 4).

[0117] The amplification procedure is shown in Table 5.

[0118] Table 5 PCR amplification program

[0119]

[0120] The amplification system is shown in Table 6.

[0121] Table 6 PCR amplification system

[0122]

[0123] After PCR amplification, the product was verified by 1.5% agarose gel electrophoresis. After development, the target band of the product was consistent with the length position. The target band was recovered using a full-gold column recovery kit. At the same time, the pHB overexpression vector (kindly donated by Professor Tang Kexuan of Shanghai Jiaotong University) was double-digested with BamH I and Sac I. The enzyme digestion system is shown in Table 7.

[0124] Table 7 Enzyme Digestion System

[0125]

[0126] The digested plasmid was recovered using a full-gold column recovery kit, and homologous recombination was performed according to the system shown in Table 8.

[0127] Table 8 Homologous recombination system

[0128]

[0129] The homologous recombination product was stored at 4°C, and the product was transformed into the competent Escherichia coli DH5ɑ, added to LB liquid culture medium at 37°C and 120 rpm for 1 h, added to LB solid culture medium containing kanamycin, 100 μL of Escherichia coli was added and evenly spread on the solid culture medium, placed in a 37°C incubator for 16 h, picked a single colony and placed it in LB liquid culture medium containing kanamycin, placed in a 37°C shaker at 180 rpm for 6 h, and after the culture medium became turbid, the bacterial liquid PCR positive verification was performed according to the system shown in Table 9.

[0130] Table 9 Bacterial liquid PCR positive verification system

[0131]

[0132] The amplification procedure is the same as the above gene cloning procedure. After the PCR is completed, the product of the bacterial solution PCR is detected by agarose gel electrophoresis. After development, the positive bacterial solution containing the target band length is sent to Anshengda Company for sequencing. According to the sequencing results, the ORF sequence consistent with the PCR amplification is selected. The constructed vector is transformed into Agrobacterium by the plasmid heat shock method, and the screening and identification methods are the same as above.

[0133] At the same time, an RNA interference vector plasmid was constructed. The PCR product was cloned into the pDONR vector (purchased from Thermo Fisher Scientific) by BP recombination reaction to form the pENTR-SmDIR18 entry cloning vector plasmid. The pK7GWIWG2D-SmDIR18 (pK7GWIWG2D plasmid was purchased from Thermo Fisher Scientific) expression vector was constructed by LR reaction. The amplification primer sequences are as follows:

[0134] RNAi-SmDIR18-F:CACCGCACGATCACGGTGATCGACGAC (SEQ ID NO.33)

[0135] RNAi-SmDIR18-R: CGAAGCCGCAGGCGTTCTTGAAC (SEQ ID NO. 34).

[0136] The BP recombination reaction was carried out according to the system shown in Table 10.

[0137] Table 10 BP recombination reaction system for constructing pENTR-SmDIR18 vector

[0138]

[0139] After the system was mixed, the metal bath was reacted at 22°C for 1 h. The transformation, screening and identification of E. coli were the same as above. After the positive strains were screened, the LR reaction was carried out according to the system shown in Table 11.

[0140] Table 11 LR reaction system for constructing pK7GWIWG2D-SmDIR18 expression vector

[0141]

[0142] After mixing, the reaction was incubated at 25°C overnight, and 1 μL of proteinase K was added. After mixing, the reaction was terminated by reacting in a metal bath at 37°C for 15 min. The transformation, screening and identification methods of E. coli were the same as above. After screening the positive strains, they were transferred to Agrobacterium, and the screening and identification methods were the same as above.

[0143] 3.2 Obtaining transgenic plants

[0144] Select sterile Salvia miltiorrhiza seedlings with good growth conditions, cut their leaves into small pieces of 1cm×1cm, and culture them in the dark for 2 days. Add the positive Agrobacterium bacterial solution to the LB liquid medium containing kanamycin and rifampicin, and culture it in a 28℃ incubator until the OD value is about 0.8. Centrifuge the bacterial solution at 4℃ and 5000 rpm for 5 minutes, resuspend it with 1 / 2 MS liquid medium, and culture it at 28℃ for 1 hour. Put the leaves after dark culture into the bacterial solution, shake it at 28℃ and 100 rpm for 15 minutes, take out the leaves and wipe off the bacterial solution on the surface, and culture them in 1 / 2 MS solid medium containing acetosyringone for 15 days. After the hairy roots and plants grow, transfer the plants to 1 / 2 MS solid medium for further culture.

[0145] Example 4, positive plant identification and index determination

[0146] 4.1 Identification of positive plants

[0147] Select transgenic plants, extract the DNA of transgenic plants using the plant tissue DNA extraction kit provided by Tiangen Biochemical Technology Co., Ltd., and use the extracted DNA as a template to amplify the T-DNA fragment on the overexpression vector plasmid and the rolC on the Ri plasmid on Agrobacterium. The amplification primers are as follows:

[0148] SmDIR18-OE-F: GGTAATATCCGGAAACCTCCTCGG (SEQ ID NO.35)

[0149] SmDIR18-OE-R: CCGTCCTCCTTGAAGTCGATGC (SEQ ID NO.36)

[0150] rolC-F: CTCCTGACATCAAACTCGTC (SEQ ID NO.37)

[0151] rolC-R: TGCTTCGAGTTATGGGTACA (SEQ ID NO. 38).

[0152] The amplification procedure and system were consistent with the method for obtaining the ORF of SmDIR18. After the amplification, the reaction products were detected by agarose gel electrophoresis, and it was found that the target band fragments were contained in the corresponding positions, indicating that the plants were transgenic plants.

[0153] The full length of the CDS sequence of the SmDIR18 gene is 747 bp. After the primers were designed, it was amplified by PCR and verified by preparing a 1.5% agarose gel. The first lane on the left was a marker. The results showed that the amplified band of the SmDIR18 gene was at the corresponding position (such as Figure 5A). A 1.5% agarose gel was prepared for agarose gel electrophoresis, where the first lane was a marker, the second to fourth lanes were all wild-type groups, and the fifth to seventh lanes were positive verifications of the SmDIR18 gene knockdown group and the SmDIR18 gene overexpression group, respectively. The agarose gel electrophoresis results of the SmDIR18 transgenic plants showed that the amplified bands were all at the corresponding positions (such as Figure 5 B and C in ).

[0154] 4.2 Determination of transgenic plant indicators

[0155] Three plants were selected from each of the three groups of SmDIR18 gene overexpression group, knockdown group and wild type as three biological replicates. The results showed that among the transgenic plants of Salvia miltiorrhiza, the SmDIR18 knockdown Salvia miltiorrhiza plants were shorter than the wild type plants, and their growth and development were affected (e.g. Figure 6 In the SmDIR18 overexpressing transgenic plants, the gene expression level was higher than that of the wild type, while the expression level of the SmDIR18 gene in the knockdown group was lower than that of the wild type plants (e.g. Figure 6 The contents of tanshinone I, tanshinone IIA and cryptotanshinone in the overexpression group were higher than those in the wild-type group and the knockdown group (e.g. Figure 6 C, D and E in the figure). The study found that the plants in the overexpression group of the SmDIR18 gene grew better than the wild-type plants, and the expression level of SmDIR18 and the content of tanshinone were higher than those of the wild-type plants. The knockdown group plants grew shorter. At the same time, the expression level of SmDIR18 and the content of tanshinone were lower than those of the wild-type plants. Salvia miltiorrhiza can adapt to salt stress by increasing the content of tanshinone under salt stress conditions. SmDIR18 can affect the growth of Salvia miltiorrhiza plants and the content of tanshinone in the body, and has a certain positive effect on Salvia miltiorrhiza's resistance to adverse stress.

[0156] SEQ ID NO.1

[0157] Salvia miltiorrhiza

[0158] >GWHTAOSJ012058

[0159] ATGTCCAAGAAATCAGCAATAGTGTTCCTATTCACACTCTTCGTCGCGATCACCTCGAGCGGCGCGGTGGTCCCGGCCGGCGGCGGCGAAGTCCCCGTTCTGGAGCTATACATGCACGACATCCTGGGCGGCAGCAACCCGACGGCGCGGCCGGTGACGGGGCTGCTGGGCAACATATACAGCGGCCAAGTCCCCTTCGCCCGCCCCCTCGGCTTCCAACCGCCCAAGAACGGCGTGGCCATCCCCAACGCCAACGGCGCCATCCCGACCATCAACGCCAACGGCATCCCGCTGGGCACGGGCCTGGCCGGCACGGCCTTCGCGGGAAACCCTAACAACAACAACAATAACAACAACATAAACACGGCCCAGCTGGGGCCGGACGGGCTGGGGCTGGGGTTCGGCACGATCACGGTGATCGACGACTACCTGACGGCGGCGCCGGAGCTGGGATCGCAGCAGGTGGGGAAGGCGCAGGGGGTGTACGTGGCGAGCTCGGCGGACGGGTCGACGCAGATGATGGCGTTCACGGCGATGATGGAGGGAGGGGAGTTCGGAGACAGCATCAACTTCTTTGGGGTGTACAGGATCGGGAGCACCATGTCGAGGCTGTCCATCACCGGCGGCACCGGCAAGTTCAAGAACGCCTGCGGCTTCGCCGAGCTCAGGTCGCTCATTCCGGCCGGCCAGCATGTTTTGGACGGCGTTCAGACCTTGCTCAGGATCACTGTGCATCTTACTTACTGA

[0160] SEQ ID NO.2

[0161] Salvia miltiorrhiza

[0162] >GWHPAOSJ012058

[0163] MSKKSAIVFLFTLFVAITSSGAVVPAGGGEVPVLELYMHDILGGSNPTARPVTGLLGNIY

[0164] SGQVPFARPLGFQPPKNGVAIPNANGAIPTINANGIPLGTGLAGTAFAGNPNNNNNNNNI

[0165] NTAQLGPDGLGLGFGTITVIDDYLTAAPELGSQQVGKAQGVYVASSADGSTQMMAFTAMMEGGEFGDSINFFGVYRIGSTMSRLSITGGTGKFKNACGFAELRSLIPAGQHVLDGVQTLLRITVHLTY

[0166] SEQ ID NO.3

[0167] SmDIR18-F:CTCTCTCTCAAGCTT GGATCC ATGTCCAAGAAATCAGCAATAGTGTT

[0168] SEQ ID NO.4

[0169] SmDIR18-R:GCCCTTGCTCACCAT GAGCTC TCAGTAAGTAAGATGCACAGTGATCCT。

Claims

1. Application of the Salvia miltiorrhiza directing protein SmDIR18 with an amino acid sequence as shown in SEQ ID NO.2 or a biological material related to the Salvia miltiorrhiza directing protein SmDIR18 in at least one of the following (a1) to (a9): (a1) Application in regulating the growth and development of Salvia miltiorrhiza plants; (a2) Use in the preparation of products for regulating the growth and development of Salvia miltiorrhiza plants; (a3) application in regulating the content of tanshinone in Salvia miltiorrhiza; (a4) Use in the preparation of products for regulating the content of tanshinone in salvia miltiorrhiza; (a5) Application in regulating salt tolerance of Salvia miltiorrhiza; (a6) Use in the preparation of products for regulating the salt tolerance of Salvia miltiorrhiza; (a7) Application in breeding new Salvia miltiorrhiza germplasm with improved growth and development ability; (a8) Application in breeding new Salvia miltiorrhiza germplasm with increased tanshinone content; (a9) Application in breeding new Salvia miltiorrhiza germplasm with improved salt tolerance; The biological material related to the Salvia miltiorrhiza guide protein SmDIR18 is at least one of the following (b1)-(b11): (b1) a gene encoding the Salvia miltiorrhiza guidance protein SmDIR18; (b2) a primer pair for cloning the gene described in (b1); (b3) an expression cassette containing the gene described in (b1); (b4) a recombinant vector containing the gene described in (b1), or a recombinant vector containing the expression cassette described in (b3); (b5) a recombinant microorganism containing the gene described in (b1), or a recombinant microorganism containing the expression cassette described in (b3), or a recombinant microorganism containing the recombinant vector described in (b4); (b6) a transgenic Salvia miltiorrhiza plant cell line containing the gene described in (b1), or a transgenic Salvia miltiorrhiza plant cell line containing the expression cassette described in (b3), or a transgenic Salvia miltiorrhiza plant cell line containing the recombinant vector described in (b4); (b7) transgenic Salvia miltiorrhiza plant tissue containing the gene described in (b1), or transgenic Salvia miltiorrhiza plant tissue containing the expression cassette described in (b3), or transgenic Salvia miltiorrhiza plant tissue containing the recombinant vector described in (b4); (b8) transgenic Salvia miltiorrhiza plant organs containing the gene described in (b1), or transgenic Salvia miltiorrhiza plant organs containing the expression cassette described in (b3), or transgenic Salvia miltiorrhiza plant organs containing the recombinant vector described in (b4); (b9) a transgenic Salvia miltiorrhiza plant containing the gene described in (b1), or a transgenic Salvia miltiorrhiza plant containing the expression cassette described in (b3), or a transgenic Salvia miltiorrhiza plant containing the recombinant vector described in (b4); (b10) a tissue culture produced from regenerable cells of the transgenic Salvia miltiorrhiza plant described in (b9); (b11) Protoplasts produced from the tissue culture described in (b10).

2. The use according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the Salvia miltiorrhiza guide protein SmDIR18 in (b1) is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The nucleotide sequences of the primer pair described in (b2) are shown in SEQ ID NO.3 and SEQ ID NO.

4.

4. The use according to claim 1, characterized in that: Increasing the activity and / or content of the Salvia miltiorrhiza guiding protein SmDIR18 in the target Salvia miltiorrhiza plant, or overexpressing the gene encoding the Salvia miltiorrhiza guiding protein SmDIR18, can promote the growth and development of the Salvia miltiorrhiza plant or / and increase the content of tanshinone in Salvia miltiorrhiza or / and improve the salt tolerance of Salvia miltiorrhiza.

5. A method, characterized in that The method is at least one of the following (c1) to (c3): (c1) a method for promoting the growth and development of Salvia miltiorrhiza; (c2) a method for increasing the content of tanshinone in salvia miltiorrhiza; (c3) Methods for improving the salt tolerance of Salvia miltiorrhiza; The method is to increase the activity and / or content of the Salvia miltiorrhiza guiding protein SmDIR18 in claim 1 in the target Salvia miltiorrhiza plant, or to overexpress the gene encoding the Salvia miltiorrhiza guiding protein SmDIR18.

6. A method for cultivating transgenic Salvia miltiorrhiza, characterized in that: Overexpressing the gene encoding the Salvia miltiorrhiza guide protein SmDIR18 described in claim 1 in the target Salvia miltiorrhiza plant to obtain at least one of the following transgenic Salvia miltiorrhiza plants (d1) to (d3): (d1) Transgenic plants of Salvia miltiorrhiza with improved growth and development ability; (d2) transgenic plants with increased tanshinone content; (d3) Transgenic plants with improved salt tolerance.

7. The method according to claim 5 or 6, characterized in that: The nucleotide sequence of the gene encoding the Salvia miltiorrhiza guide protein SmDIR18 is shown in SEQ ID NO.1.

Images