CN112501177B - Method for promoting development of lateral root of salvia miltiorrhiza and improving content of tanshinone in salvia miltiorrhiza root, salvia miltiorrhiza gene sequence and overexpression vector - Google Patents

Abstract

The invention provides a method for promoting the development of lateral roots of salvia miltiorrhiza and improving the content of tanshinone in salvia miltiorrhiza, which belongs to the field of genetic engineering and has the characteristics that the transgenic salvia miltiorrhiza root with better lateral root development and improved tanshinone content is obtained by over-expressing a salvia miltiorrhiza SmMYC2b gene in the salvia miltiorrhiza root, and the method comprises the following steps: step S1, constructing an overexpression vector containing the salvia miltiorrhiza SmMYC2b gene; step S2, transfecting the overexpression vector obtained in the step S1 into agrobacterium to obtain recombinant agrobacterium; step S3, transforming the salvia miltiorrhiza tissues and inducing rooting by using the recombinant agrobacterium obtained in step S2 to obtain transgenic salvia miltiorrhiza roots, wherein the sequence of the salvia miltiorrhiza SmMYC2b gene is shown as SEQ ID No: 1 is shown. The sequence of the overexpression vector is shown as SEQ ID No: 2, respectively.

Description

Method for promoting development of lateral roots of salvia miltiorrhiza and improving tanshinone content in salvia miltiorrhiza, salvia miltiorrhiza gene sequence and overexpression vector

Technical Field

The invention belongs to the field of genetic engineering, and relates to a method for promoting the development of lateral roots of salvia miltiorrhiza and improving the content of tanshinone in salvia miltiorrhiza, and a salvia miltiorrhiza gene sequence and an overexpression vector applied in the method.

Background

Salvia miltiorrhiza Bunge (Salvia miliiorrhiza Bunge) is an important Chinese medicinal material and contains various effective components. Wherein, tanshinone and phenolic acid are the main bioactive components. Modern researches show that tanshinone substances contained in salvia miltiorrhiza have various pharmacological activities of resisting tumor, resisting bacteria, diminishing inflammation, resisting oxidation, resisting atherosclerosis and the like, so that the salvia miltiorrhiza has great market demand.

However, as wild resources are increasingly reduced, and the salvia miltiorrhiza is a perennial herb, the growth cycle is longer, the content of medicinal active ingredients is low, and the problems of quality degradation of the salvia miltiorrhiza, overhigh variety breeding cost and the like exist in the traditional cultivation mode. In addition, the structure of tanshinone is complex, so the chemical synthesis process is very complicated, the cost is high and the environmental pollution is easy to cause; the cells obtained by the cell culture method have low active ingredient content and poor stability.

The rapid development of the genetic engineering provides a new idea for improving the content of tanshinone in the salvia miltiorrhiza root and solving the problem of shortage of tanshinone medicine sources.

Disclosure of Invention

In order to promote the development of lateral roots of salvia miltiorrhiza and improve the tanshinone content in salvia miltiorrhiza, in particular to the tanshinone content in salvia miltiorrhiza, the inventor provides the following technical scheme:

the invention provides a method for promoting the development of lateral roots of salvia miltiorrhiza and improving the content of tanshinone in salvia miltiorrhiza, which is characterized in that a transgenic salvia miltiorrhiza seedling with better lateral root development and improved tanshinone content is obtained by over-expressing a salvia miltiorrhiza SmMYC2b gene in salvia miltiorrhiza roots, and the method comprises the following steps:

step S1, constructing an overexpression vector containing the salvia miltiorrhiza SmMYC2b gene;

step S2, transfecting the overexpression vector obtained in the step S1 into agrobacterium to obtain recombinant agrobacterium;

step S3, transforming salvia tissue by the recombinant agrobacterium obtained in step S2 and inducing callus differentiation to obtain transgenic salvia seedlings,

wherein the sequence of the salvia miltiorrhiza SmMYC2b gene is shown as SEQ ID No: 1 is shown.

The method for promoting the development of the lateral roots of the salvia miltiorrhiza and improving the tanshinone content in the salvia miltiorrhiza provided by the invention can also have the following characteristics: wherein the tanshinone comprises dihydrotanshinone I, tanshinone I, cryptotanshinone and tanshinone IIA.

The invention also provides a salvia miltiorrhiza gene sequence for promoting the development of lateral roots of salvia miltiorrhiza and improving the tanshinone content in salvia miltiorrhiza, which is characterized in that: wherein the salvia miltiorrhiza gene sequence is a salvia miltiorrhiza SmMYC2b gene sequence, and the sequence is shown as SEQ ID No: 1 is shown.

The invention also provides an overexpression vector for promoting the development of lateral roots of salvia miltiorrhiza and improving the tanshinone content in salvia miltiorrhiza, which is characterized in that: wherein the overexpression vector contains a salvia miltiorrhiza SmMYC2b gene sequence, and the gene sequence of the overexpression vector is shown as SEQ ID No: 2, respectively.

Action and effects of the invention

According to the method for promoting the development of lateral roots of salvia miltiorrhiza and improving the tanshinone content in salvia miltiorrhiza, the salvia miltiorrhiza gene sequence and the overexpression vector, the salvia miltiorrhiza SmMYC2b gene is connected with the vector, transferred into salvia miltiorrhiza through agrobacterium and induced to differentiate, so that transgenic salvia miltiorrhiza seedlings with improved tanshinone content can be obtained, and the medicinal value of salvia miltiorrhiza is improved.

In addition, the gene SmMYC2b of the salvia miltiorrhiza can also promote the development of lateral roots of the salvia miltiorrhiza, increase the surface area of the roots and further promote the increase of the content of tanshinone.

Drawings

FIG. 1 is a schematic diagram of the construction of an overexpression vector in an example of the present invention;

FIG. 2 is a diagram showing the growth process of transgenic Salvia miltiorrhiza seedlings in an example of the present invention;

FIG. 3 is a Western-Blot analysis result of transgenic Salvia miltiorrhiza seedlings in an embodiment of the present invention;

FIG. 4 is a graph of expression levels of SmMYC2b in transgenic roots in an example of the invention;

FIG. 5 is a graph showing the content of related compounds in transgenic roots according to an embodiment of the present invention;

FIG. 6 is a root system of a root system analysis in an embodiment of the present invention;

FIG. 7 is a PCA analysis diagram of root analysis in an embodiment of the invention;

FIG. 8 is a heat map of root system analysis in an embodiment of the present invention; and

figure 9 is a volcanic image of root system analysis in an embodiment of the present invention.

Detailed Description

The following examples are given to illustrate specific embodiments of the present invention. In each of the examples described below, the reagents, enzymes and kits used were obtained from general commercial sources unless otherwise specified, and the conditions of the experiment were not specified and were either referred to the conventional conditions or followed the conditions recommended by the supplier. In addition, the sterile salvia seedlings used in the following examples were cultivated from salvia seeds, which were purchased from Shanxi Shanghai Tianshi GMP planting base as a subject group of the inventors.

Example 1: amplification of the sequence of the SmMYC2b Gene of Salvia miltiorrhiza

The purpose of this example is to obtain a gene fragment of a suspected transcription factor MYC2 from Salvia miltiorrhiza Bunge, and amplify the gene fragment to obtain an amplified related gene fragment for transformation, comprising the following steps:

step 1-1, extracting total RNA from salvia miltiorrhiza, wherein the operation method refers to TRANSGENE handbook of TransZol UP Plus RNA Kit total RNA extraction reagent, and the method comprises the following substeps:

step 1-1-1, cutting leaves of a sterile salvia miltiorrhiza seedling growing for 10 weeks in a culture room in a super clean bench, and storing in liquid nitrogen;

step 1-1-2, adding liquid nitrogen into the leaves, quickly grinding the liquid nitrogen into fine powder, immediately subpackaging the powder into eppendorf tubes added with TRNzol-UP in advance, and using 1mL of TRNzol-UP for every 50-100 mg of the leaves;

step 1-1-3, uniformly mixing by using a homogenizer, standing at room temperature for 5min, adding 0.2mL of chloroform, centrifuging at 4 ℃ for 15min by using 10,000 Xg, dividing the mixed solution into three layers, transferring the upper colorless water phase into a new centrifugal tube, adding ethanol with the same volume (about 600 mu L), and slightly and uniformly mixing;

step 1-1-4, adding the obtained solution and the precipitate into a centrifugal column, centrifuging at room temperature 1200 Xg for 30sec, and discarding the effluent (centrifuging several times if the liquid is greater than 0.8 mL);

step 1-1-5, add 0.5mLCB9 to the spin column, centrifuge at room temperature 1200 Xg for 30sec, discard the effluent and repeat the operation once;

1-1-6, adding 0.5ml of LB 9 (absolute ethanol is needed before use) into the centrifugal column, centrifuging at room temperature of 1200 Xg for 30sec, discarding the effluent, and repeating the operation once;

step 1-1-7, centrifuging at room temperature of 1200 Xg for 2min, standing at room temperature for 2min, and completely removing residual ethanol in the column;

step 1-1-8, adding 50-100 μ L of RNase-free H into the centrifugal column ₂ O, placing in an RNase-free Tube, and standing at room temperature for 1 min;

step 1-1-9, centrifuging at room temperature of 1200 Xg for 2min, eluting RNA, and storing at-80 ℃.

Step 1-2, adopting agarose gel electrophoresis to detect the concentration and purity of RNA, and specifically comprising the following substeps:

step 1-2-1, weighing 0.25g of agarose powder on an electronic balance, heating and dissolving the agarose powder in 30mL of 1 XTAE, pouring Ethidium Bromide (EB) serving as a fluorescent staining agent into a gel making groove with a well-inserted pore plate, and cooling;

step 1-2-2, mixing 2.5 mu L of RNA and 1 mu L of 10 multiplied Loading Buffer uniformly by a gun head, then Loading the mixture, setting the voltage to be 140V, and carrying out electrophoresis for 15 min;

and 1-2-3, detecting the gel after electrophoresis by using a gel imaging system, wherein three RNA bands of 28s, 18s and 5s can be clearly seen as a result. .

Step 1-2-4, directly determining the concentration of RNA by using an NANO DROP2000 ultramicro spectrophotometer: aspirate 2. mu.L of RNase-free H ₂ Performing Blank determination by taking O as a background solution, then sucking the RNA solution to be determined, and determining the total RNA concentration (mu g/mu L) according to the ratio of the absorbance at the wavelength of 260nm to the absorbance at the wavelength of 280nm (wherein, the OD260/OD280 is preferably between 1.8 and 2.0).

Step 1-3, using total RNA of salvia miltiorrhiza as a template, synthesizing cDNA according to the operation instruction of TransScript First-Strand cDNA Synthesis SuperMix kit of Beijing Omega, and the reaction system is shown in the following table 1-1:

TABLE 1-1 reaction System for cDNA Synthesis

The reaction conditions are 42 ℃ incubation for 30min, 85 ℃ heating for 5min, and the obtained cDNA is stored at-80 ℃.

Step 1-4, obtaining a suspected transcription factor MYC2 gene, comprising the following substeps:

1-4-1, sequence alignment: a gene sequence in a salvia miltiorrhiza transcriptome database is subjected to homology comparison with transcription factor MYC2 genes of multiple species (arabidopsis thaliana, wheat, poplar and the like), an Open Reading Frame (ORF) sequence of a suspected transcription factor MYC2 gene is obtained through sequence splicing and annotation, a primer sequence is designed, and the designed primer sequence is shown in tables 1-2 below.

TABLE 1-2 primer sequence Listing related to SmMYC2b Gene

And step 1-4-2, obtaining the target gene of the suspected transcription factor MYC2 through RT-PCR. Hereinafter, this suspected transcription factor MYC2 gene is referred to as SmMYC2b gene.

The procedure for RT-PCR was as follows:

a200. mu.L centrifuge tube was used to amplify using the Trans Start FastPfu Fly DNA Polymerase kit from Beijing Omegal gold Biotechnology Ltd, and the reaction system was shown in the following tables 1 to 3:

TABLE 1-3 RT-PCR reaction System

cDNA	1μL
		SmMYC2b-F(10μmol/L)	1μL
SmMYC2b-R(10μmol/L)	1μL
		dNTPs	10μL
FastPfu Fly buffer	5μL
		FastPfu Fly Polymerase	1μL
ddH 2 O	31μL
		Total volume	50μL

The PCR reaction conditions were as follows:

the target gene fragment (1824bp) is separated and recovered by agarose gel electrophoresis.

Step 1-5, connecting a target gene with a vector and transforming escherichia coli, wherein the operation method comprises the following steps:

the Blunt-Zero vector was mixed with the recovered gel product at a ratio of 1:4 and left at 25 ℃ for 25 min. Taking out the completely preserved Trans1T1 competent cells from a refrigerator at the temperature of-80 ℃, and melting by ice bath; adding the ligation product into 50 mu L of competent cells, gently mixing uniformly, and carrying out ice bath for 25 min; thermally shocking in 42 deg.C water bath for 90sec, rapidly placing into ice, and standing for 2 min; adding 500 mu L of LB culture medium without antibiotics, shaking for 1h at 37 ℃ in a shaking table at 200 rpm; sucking the bacterial liquid onto an LB culture medium plate containing 75mg/L of kanamycin, and uniformly spreading the bacterial liquid by using an aseptic spreader; and (5) inversely placing the mixture in a constant-temperature incubator at 37 ℃ for culturing for 13-15 h.

1-6, detecting the positive clone of the escherichia coli by adopting PCR, wherein the operation method comprises the following steps:

adding 1ml of LB liquid medium (containing 75mg/L of kanamycin) into 8 sterile centrifuge tubes, randomly taking 8 independent thalli, shaking the thalli in the centrifuge tubes by a constant-temperature shaking table, culturing the thalli at 37 ℃ and 200rpm for 3-4 h, and taking bacterial liquid as a next PCR template.

The reaction systems are shown in tables 1-4 below:

TABLE 1-4 PCR reaction System for detecting Escherichia coli Positive clones

The PCR reaction program was set as follows:

the PCR products were detected by agarose gel electrophoresis, and 100. mu.L of each of the bacterial solutions containing the target fragment was sequenced. Activating the correctly sequenced bacterial liquid, adding glycerol, and preserving the strain in a refrigerator at-80 ℃.

As above, a target gene SmMYC2b of a suspected transcription factor MYC2 is obtained from the salvia miltiorrhiza genome through total RNA extraction, cDNA synthesis and RT-PCR, wherein the sequence of SmMYC2b is shown as SEQ ID No: 1 is shown. The target gene has high homology with transcription factor MYC2 genes of other species, has corresponding open reading frames respectively, can encode corresponding transcription factors, and belongs to the transcription factor MYC2 genes.

Example 2: construction of overexpression vectors

The purpose of this example is to obtain a target gene from escherichia coli containing the target gene obtained in example 1, and link the target gene with a PHB-flag vector to construct an overexpression vector containing the target gene, wherein the schematic diagram of the overexpression vector construction is shown in fig. 1, and the method comprises the following steps:

and 2-1, designing a primer, and cloning the target fragment.

Wherein, the design principle of the PCR primer is as follows: selecting a monoclonal enzyme cutting site contained in PHB-flag, wherein the enzyme cutting site does not exist in the ORF region of the gene, and simultaneously removing a stop codon of the target gene. The designed primer sequences are shown in the following Table 2-1. In Table 2-1, the italic portions indicate the cleavage sites.

TABLE 2-1 primers used for cloning of fragments of interest

The PCR reaction system is shown in the following Table 2-2:

TABLE 2-2 PCR reaction System for cloning of fragments of interest

Plasmid containing target gene	1μL
		pHB-flag-SmMYC2b-F	0.5μL
pHB-flag-SmMYC2b-R	0.5μL
		Buffer	5μL
2.5mMdNTPs	2.5μL
		PCR Stimulant	5μL
FastPfu Fly Polymerase	1.5μL
		ddH 2 O	9μL
Total volume	25μL

The PCR program was set up as follows:

after the PCR is finished, gel electrophoresis is carried out, target fragments are recovered, a Bunt-Zero vector is connected, Trans1-T1 competent cells are transformed, kanamycin resistant plates are coated, and bacteria are detected, and target strips are sent to be sequenced. The plasmid was extracted and stored at-20 ℃ for correct sequencing.

Step 2-2, carrying out double enzyme digestion on the target gene plasmid and the vector, wherein the reaction system is shown in the following table 2-3:

TABLE 2-3 reaction System for double enzyme digestion

PHB-flag vector plasmid or target gene plasmid	30μL
		Enzyme
1	1.5μL
		Enzyme
2	1.5μL
		Buffer	5μL
ddH 2 O	12μL
		Total volume	50μL

And (3) carrying out gel electrophoresis on the fragments (the large fragments of the vector and the small fragments of the target gene) after enzyme digestion, and recovering.

Step 2-3, connecting the target gene plasmid and the vector with the forward target fragment and the vector enzyme-digested fragment in a molar mass ratio of 1:10, wherein the connection system is shown in the following tables 2-4:

TABLE 2-4 reaction systems for ligation of fragments of interest and vector fragments

Vector fragment	2μL
		Target fragment double enzyme digestion product	8μL
T4 connection Buffer	3μL
		T4 DNA Ligase	1μL
ddH 2 O	6μL
		Total of	20μL

Using the above reaction system, after ligation at 25 ℃ for 1h, Trans1-T1 competent cells were transferred and plated with kanamycin-resistant plates. And selecting positive strains, sequencing, extracting plasmids if the sequencing is correct, storing in a refrigerator at the temperature of-20 ℃ to be transformed into agrobacterium.

As described above, the objective gene was obtained from the E.coli plasmid obtained in example 1 by PCR, double digestion of the vector and the objective gene, and ligation to the vector to form an overexpression vector. Wherein the sequence of the overexpression vector is shown as SEQ ID No: 2, respectively.

Example 3: construction of recombinant Agrobacterium

The purpose of this example is to transfer the overexpression vector obtained in example 2 into agrobacterium to obtain recombinant agrobacterium, comprising the following steps:

step 3-1, preparing required reagents, wherein the formula of each reagent is as follows:

LB culture medium: dissolving yeast extract 5g, sodium chloride 5g and tryptone 10g in 1L deionized water, and adjusting pH to 7; adding 7.5g of plant agar powder into each L of the solid culture medium;

YEB Medium: weighing yeast extract, sucrose, beef extract, peptone, and MgSO ₄ ·7H ₂ 0.983g of O and 1000mL of deionized water were dissolved, the pH was adjusted to 7.2, and if a solid medium was prepared, 7g of agar was added. Sterilizing at 121 deg.C for 20min under high temperature and high pressure.

B5 medium: b5 powder 3.21g, MgSO ₄ ·7H ₂ 0.983g of O, 7.5g of plant agar powder, 20g of cane sugar, and sterilizing by high-pressure steam at 121 ℃ for 20 min;

kanamycin, hygromycin solution: dissolving in deionized water, filtering, sterilizing, and storing in refrigerator at-20 deg.C.

Rifampicin solution: dissolving in DMSO, filtering, sterilizing, and storing in refrigerator at-20 deg.C.

1/2MS culture medium: weighing 4.43g of MS powder and 30g of sucrose, dissolving in 1L of deionized water, and sterilizing with high pressure steam (121 ℃, 20 min). Adding 7.5g of plant agar powder into each L of the solid culture medium, and sterilizing.

And 3-2, preparing the agrobacterium tumefaciens competent cells. In this example, the agrobacterium used was EHA105 agrobacterium (purchased from shanghai virginia, inc.) and the procedure for preparing competent cells included the following substeps:

step 3-2-1, the EHA105 Agrobacterium was activated in 1mL YEB liquid medium containing 45mg/L rifampicin for 12 hours, and 20. mu.L of the activated Agrobacterium was cultured to OD in 20mL of the above medium ₆₀₀ Up to 0.5;

step 3-2-2, centrifuging for 8min at 4000rpm on a low-temperature centrifuge at 4 ℃;

step 3-2-3, 0.1M CaCl ₂ After filtration and sterilization, the mixture is placed on ice for precooling, and 10mL of resuspended thalli are taken when the temperature is reduced to about 4 ℃. Centrifuging for 8min in ice bath for half an hour, and removing the supernatant;

and 3-2-4, adding 5mL of 30% glycerol, uniformly mixing, subpackaging one tube per 100 mu L, and storing in a refrigerator at-80 ℃.

Step 3-3, transferring the overexpression vector into agrobacterium, comprising the following substeps:

step 3-3-1, taking out the prepared C58C1 competent cells from a refrigerator at the temperature of-80 ℃, placing the competent cells on ice for melting, adding 10 mu L of PHB-flag carrier plasmid containing the target gene, uniformly mixing, and carrying out ice bath for 30 min;

step 3-3-2, placing in liquid nitrogen for 5min, then placing in ice for 3min, taking out from 37 ℃ water bath for 5 min;

step 3-3-3, adding 600 mu L of non-antibiotic YEB culture medium after heat shock is finished, and activating for 5 hours at the temperature of 28 ℃ and the rpm of 200;

step 3-3-4, centrifuging at 5000rpm for 8min, removing 200 mu L of supernatant, and coating the resuspended thallus on YEB solid plate containing 75mg/L Kan and 40mg/L Rif;

and 3-3-5, performing bacteria detection after culturing for 48 hours in a constant temperature incubator at 28 ℃, wherein the bacteria detection primers are an R end primer (cgataccgtcactagtCCCGAGAGATA) on PHB-flag, an F end primer (ctctaagcttggatccATGGGGGTTGTT) on a target gene and two sections of primers of a special hygromycin resistance gene hpt on the PHB-flag. Activating the bacterial liquid with correct bacteria detection, adding glycerol, and storing the strains in a refrigerator at-80 ℃.

Example 4: culture and identification of transgenic seedlings

This example aims at transforming salvia tissue with the recombinant agrobacterium obtained in example 3 (in this example, salvia leaf disc is selected as salvia tissue to be transformed), and inducing callus to obtain salvia seedlings containing target gene SmMYC2b, comprising the following steps:

step 4-1, transforming a leaf disc, namely transforming the recombinant agrobacterium into a salvia miltiorrhiza leaf disc, comprising the following substeps:

step 4-1-1, adding 1mL of YEB culture medium containing kanamycin (75mg/L) and rifampicin (40mg/L) and 20 μ L of bacterial liquid containing the target gene vector into a 1.5mL centrifuge tube, culturing at 28 ℃ overnight at 200rpm on a constant temperature shaking bed, and using YEB culture medium containing rifampicin (40mg/L) as the empty bacteria;

step 4-1-2, adding 500. mu.L of the overnight-cultured bacterial suspension to 25mL of YEB medium containing resistance, and culturing at 28 ℃ until OD600 is equal to 0.6;

step 4-1-3, centrifuging at 8000rpm for 10min, collecting thallus, and resuspending with 1/2MS until OD600 is equal to 1.0;

step 4-1-4, cutting leaves of the salvia miltiorrhiza plant cultured in an aseptic culture tank for 60 days, cutting off the edges, and performing dark culture on an MS culture medium for two days for later use;

and 4-1-5, after the dark culture is finished, soaking the leaves in 1/2MS heavy-suspended bacterial liquid, culturing for 10min on a constant-temperature shaking table at 28 ℃, then placing the activated bacterial liquid on sterile paper by using sterile tweezers in a super clean bench, sucking off redundant bacterial liquid, and placing the bacterial liquid on a 6-BA 1.0mg/L and NAA0.1 mg/L MS antibiotic-free culture medium for dark culture for 2 days.

Step 4-2, performing induced callus and resistance reduction treatment on the transformed salvia miltiorrhiza leaf disc, comprising the following substeps:

step 4-2-1, placing the transformation leaf disc which is dark-cultured for two days on an MS solid callus induction culture medium containing 1.0mg/L of 6-BA, 0.1 mg/L of NAA, 0.5mg/L of hygromycin and 500mg/L of cefotaxime sodium, culturing under illumination at 28 ℃, subculturing once every two weeks, gradually reducing the resistance of the cefotaxime sodium to 100mg/L in the period, and continuously reducing the concentration of the cefotaxime sodium to be completely absent. The hygromycin-resistant cluster buds can be obtained after 5-6 subcultures.

And 4-2-2, cutting the cluster buds which grow vigorously, transferring the cluster buds to a rooting culture medium (1/2MS + hygromycin 0.5mg/L) for culturing until the cluster buds grow roots, and finally obtaining the hygromycin resistance regeneration salvia miltiorrhiza plants (salvia miltiorrhiza seedlings).

Step 4-3, equally selecting each group of roots obtained in the step 4-2, extracting total protein and carrying out Western-blot (WB) identification, wherein the method comprises the following substeps:

and 4-3-1, weighing the weight of the test plant tissue, and extracting the plant protein by using a plant protein extraction kit (purchased from Kangchi century).

And 4-3-2, quantifying the obtained protein by using a BCA method, adding 5 XLoading Buffer, boiling for 5-10min in boiling water, and then storing the sample at 4 ℃.

And 4-3-3, preparing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel with the concentration of 8% by using the separation gel, and performing protein separation and wet membrane transfer.

And 4-3-4, after the membrane conversion is finished, quickly putting the PVDF membrane into 5% skimmed milk powder (TBST), shaking and sealing for 1h at room temperature in a shaking table, and then adding primary antibody to incubate at 4 ℃ overnight.

And 4-3-5, rinsing the TBST for 4 times, transferring the PVDF membrane into a horseradish peroxidase-labeled secondary antibody, incubating for 2h, rinsing the TBST for 4 times, and then developing and photographing by using an ECL luminescence method.

FIG. 2 is a diagram of the growth process of transgenic Salvia miltiorrhiza seedlings. As shown in fig. 2, fig. 2(a) is a salvia miltiorrhiza leaf disc after EHA105 agrobacterium infection; FIG. 2(B) is a leaf disk of Salvia miltiorrhiza after 12 days of infestation; FIG. 2(C) shows the seedlings grown after hygromycin screening;

FIG. 2(D) is transgenic Salvia miltiorrhiza seedlings.

FIG. 3 is a Western-Blot identification result chart of transgenic Salvia miltiorrhiza seedlings. Control in FIG. 3 indicates a blank Control group (i.e., transgenic Salvia miltiorrhiza seedling transformed with a blank vector), and L14, L15, L16, L18, L19, L20 and L21 have bands corresponding to the size of MYC2b protein at 70kD, and respectively indicate different transgenic Salvia miltiorrhiza seedling strains.

As above, the lines identified as positive by Western-blot (WB) are transgenic Salvia miltiorrhiza seedlings overexpressed by SmMYC2 b.

Example 5: determination of SmMYC2b expression in roots of transgenic Salvia miltiorrhiza seedlings

The purpose of this example is to extract total RNA from the transgenic roots obtained in example 4, reverse transcribing the total RNA into cDNA, and performing real-time quantitative PCR using the cDNA as a template to detect the expression of SmMYC2b gene, comprising the steps of:

step 5-1, extracting total RNA from the root of fresh transgenic salvia miltiorrhiza seedling, wherein the operation method refers to TRANSGENE handbook of TransZol UP Plus RNA Kit total RNA extraction reagent, and the specific steps are the same as the RNA extraction method in step 1-1.

Step 5-2, adopting agarose gel electrophoresis to detect the concentration and purity of RNA, wherein the specific steps are the same as the method for detecting RNA in the step 1-2, and the calculation formula of the RNA concentration (mu g/mu L) is OD ₂₆₀ X 50X 200 (dilution factor) x 10 ^-3 。

And 5-3, synthesizing cDNA by taking total RNA of the salvia miltiorrhiza as a template according to the operational instructions of a TransScript First-Strand cDNA Synthesis SuperMix kit of Beijing all-style gold biology, Inc., wherein the specific steps are the same as the cDNA Synthesis method in the step 1-3.

Step 5-4, the detection of the expression condition of the SmMYC2b gene comprises the following substeps:

and 5-4-1, designing a primer sequence, wherein the designed primer sequence is shown in the following table 5-1.

TABLE 5-1 primer sequence Listing related to SmMYC2b Gene

And 5-4-2, judging the expression condition of the SmMYC2b gene through real-time quantitative PCR.

The real-time quantification process is as follows:

taking 200 μ L centrifuge tube, using SYBRPremix Ex Taq ^TM (AB) amplification was carried out in the following reaction scheme 5-2:

TABLE 5-2 reaction System for real-time quantitative PCR

cDNA	2μL
		qPCR-SmMYC2b-F(10μmol/L)	1μL
qPCR-SmMYC2b-R(10μmol/L)	1μL
		SYBR Premix Ex Taq TM (2×)	10μL
ddH 2 O	6μL
		Total volume	20μL

The two-step PCR reaction conditions were as follows:

the experimental data are processed by

The method is carried out. The calculation formula is as follows:

the amount of the target gene is 2 ^-ΔΔCt

ΔΔCt＝(C _{t target gene} -C _{T housekeeping gene} ) _{Experimental group} -(C _{t target gene} -C _{T housekeeping gene} ) _{Control of}

Therein, 2 ^-ΔΔCt The expression of the target gene in the experimental group was expressed in multiples compared to the control group.

FIG. 4 is a graph of expression levels of SmMYC2b in transgenic roots in an example of the invention. In the figure, CK represents a blank control group (i.e., transgenic roots into which a blank vector was transferred), and MYC2 oe represents an experimental group (transgenic roots into which SmMYC2b overexpression vector was transferred). The symbol "+" in the figure indicates that the t-test P value was less than 0.05.

As shown in FIG. 4, the expression level of SmMYC2b after overexpression is obviously changed compared with the control, and reaches 2.04 times of the control.

Example 6: determination of the content of related Compounds in the Roots of transgenic Salvia miltiorrhiza seedlings

This example aims at detecting the content of related compounds (tanshinone compounds, including dihydrotanshinone I, tanshinone I, cryptotanshinone, tanshinone IIA) in the transgenic salvia miltiorrhiza bunge obtained in example 4 to verify the effect of transferring a target gene to increase the content of tanshinone, and includes the following steps:

step 6-1, preparing a standard solution and a test solution, wherein the preparation operations of the solutions are as follows:

(1) standard substance solution

2.00mg of each of dihydrotanshinone I, tanshinone I, cryptotanshinone and tanshinone IIA is precisely weighed and respectively fixed to 2mL by methanol, and 1mg/mL of mixed standard stock solution is obtained.

When the standard substance stock solution is used, the standard substance stock solution is respectively diluted by different times by using mobile phases, and corresponding standard curves are drawn.

(2) Test solution

Weighing fresh roots of transgenic salvia miltiorrhiza seedlings, and drying in an oven at 40 ℃ until the weight is not changed any more to obtain dry roots.

Grinding the dried root into powder with tissue grinder, sieving with 100 mesh sieve, collecting 20mg, and ultrasonically extracting with 70% methanol (4mL) for 2 times, each for 30 min.

After extraction, the mixture is centrifuged at 10,000rpm for 5min in a centrifuge, and the obtained supernatant is passed through a membrane (0.22 μm) to obtain a test solution.

Step 6-2, identifying the tanshinone components by adopting a mass spectrum, wherein the mass spectrum parameters are shown in the following table 6-1:

TABLE 6-1 Mass Spectrometry parameters

The mass spectrometry conditions were set as follows: gas Temp 350 ℃; gas Flow 10L/min; nebulizer 40 psi; capillary 4000V; sheath Gas Temp 350 ℃; shear Gas Flow 10L/min.

And 6-3, measuring the content of the tanshinone component by adopting HPLC, wherein the HPLC conditions are as follows:

the instrument comprises the following steps: agilent 1200 liquid chromatogram tandem G6410A triple quadrupole mass spectrometer

A chromatographic column: waters xSELECT CSHTM C18(2.5 μm, 2.1X 50mm) PN.186006101 LN.0101313441

Mobile phase: acetonitrile- (0.1% formic acid +2mmol/L ammonium acetate) aqueous solution

Column temperature: 35 deg.C

Flow rate of mobile phase: 0.3mL/min

Sample injection amount: 10 μ L

Single needle time: 7.0 min.

FIG. 5 is a graph showing the content of related compounds in transgenic roots according to the present invention. In fig. 5, the abscissa represents different compounds, and DTI is dihydrotanshinone I; TI is tanshinone I, CT is cryptotanshinone, and TIIA is tanshinone IIA; TT is total tanshinone; the ordinate represents the relative expression amount. The symbol "-" in the figure indicates that the t-test P value was greater than 0.01 and less than 0.05.

Through the above measurement process, the tanshinone content in the transgenic root obtained in example 4 was measured, and the result is shown in fig. 4. Wherein CK represents a blank control group (i.e., transgenic hairy roots with blank vector transferred).

Example 7: analyzing the root morphology of transgenic salvia seedlings

The embodiment aims to perform root analysis and slice observation on the transgenic salvia miltiorrhiza seedlings obtained in the embodiment 4, and comprises the following steps:

and 7-1, carrying out root system analysis on the fresh transgenic salvia miltiorrhiza seedlings and the control salvia miltiorrhiza seedlings, obtaining values of Length (total root Length), SurfArea (surface area), Avgdiam (average diameter), RootVolume (total volume), Tips (root tip number), Forks (branch number) and crossovers) of different strains by using WhiRHIO software, carrying out PCA (principal component analysis) analysis and heat map drawing on the transgenic salvia miltiorrhiza groups and the control groups by using ClustVis, and carrying out volcanic map drawing by using graphpad software.

FIG. 6 is a root system of the root system analysis of the present invention; FIG. 7 is a PCA analysis diagram of the root system analysis of the present invention; FIG. 8 is a heat map of a root system analysis of the present invention; figure 9 is a volcano plot of root system analysis of the present invention.

Through the above measurement process, the root system analysis results of the transgenic roots obtained in example 4 were measured, and the results are shown in fig. 6 to 9. Wherein CK represents a blank control group (i.e., roots of transgenic seedlings transformed with a blank vector).

Effects and effects of the embodiments

As shown in FIG. 4, the expression level of SmMYC2b after MYC2b overexpression is 2.04 times of that of a control, and the content of corresponding tanshinone components is also obviously improved (FIG. 5). As shown in fig. 5, in the MYC2 bdoe of the experimental group, the contents of dihydrotanshinone I, tanshinone I, cryptotanshinone, tanshinone IIA and total tanshinone were 2.5mg/g, 13mg/g, 0.7mg/g, 1.8mg/g and 17mg/g, respectively, and the contents of dihydrotanshinone I, tanshinone I, cryptotanshinone, tanshinone IIA and total tanshinone of the control group were 0.4mg/g, 0.9mg/g, 0.1mg/g, 0.6mg/g and 1.8mg/g, respectively. The content of total tanshinone in transgenic salvia miltiorrhiza root is improved by 9.4 times.

After the SmMYC2b gene is transferred into salvia miltiorrhiza, the content of tanshinone effective ingredients in roots is obviously increased, which shows that the overexpression of SmMYC2b promotes the biosynthesis of the tanshinone effective ingredients.

As shown in FIG. 7, the PCA results show two sets of separations, each focused on two different regions, indicating that overexpression of MYC2b causes changes in root characteristics. As can be seen in the heatmap of FIG. 8, strains of CK and MYC2bOE cluster into different groups, respectively, indicating that there are differences between the two groups. In addition, three factors of Tips, Forks and Crossings are related; there is a correlation between three factors, SurfArea, RootVolume and Length. As can be seen in the volcano plot of fig. 9, the lengths, SurfArea, Tips multiples of the roots of the two groups of salvia miltiorrhiza vary significantly and differ significantly. The roots of the two groups of salvia miltiorrhiza have significant difference in difference expression fold of Forks and Crosssings, but the t-test result has no significant difference, which is caused by excessive difference in the groups. In addition, AvgDiam did not differ much between the two groups, and the direction did not coincide with the other points, and was decreasing. The above results indicate that MYC2bo oe salvia miltiorrhiza roots have a significant increase in root tip number, total root length and total surface area compared to controls, indicating that overexpression of SmMYC2b promotes development of salvia miltiorrhiza lateral roots.

As can be seen from FIGS. 6-9, the number of root tips, the total root length and the total surface area of the salvia miltiorrhiza root with the SmMYC2b overexpressed compared with the control group were significantly increased, which indicates that the salvia miltiorrhiza SmMYC2b can promote the development of lateral roots. Since the tanshinone components are mainly distributed on the periderm of the roots, the over-expression of SmMYC2b promotes the development of lateral roots of salvia miltiorrhiza and increases the surface area of the roots, thereby increasing the content of the tanshinone components.

In conclusion, after the SmMYC2b gene is transferred into the salvia miltiorrhiza, the generation of tanshinone compounds can be promoted in a metabolic way, so that the content of tanshinone in the transgenic salvia miltiorrhiza root is improved, the development of salvia miltiorrhiza lateral root can be promoted, and the content of tanshinone is further increased.

Sequence listing

<110> Shanghai Changcheng Hospital

<120> method for promoting development of lateral roots of red sage root and increasing tanshinone content in red sage root, red sage root gene sequence and overexpression vector

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1824

<212> DNA

<213> Salvia miltiorrhiza Bunge (Salvia miltiorrhiza Bunge)

<400> 1

atgggggttg ttggttggag taatgaagat aaggcgatgg cagcagcggt tttgggaact 60

aaggcttttg attacttgat gtcgagctca gtttctgctg aatgctcgtt aatggcgatg 120

gggagtgatg agaatttgca gaataagctt gcagatcttg tagagcgccc aaattcttcc 180

aatttttgtt ggaattatgc aattttctgg cagctctcta ggtccaaggc tggggacttg 240

gtgttgggat ggggggacgg gtgttgtcga gaacctcgtg atgatgagga atctgaggtt 300

actcgaattc tcaaaatgag gctcgaggat gaatctcagc agacaatgag gaaaagggtt 360

ctccaaaggt tgcatacttt gtttggagga ggcgatgagg aaaattatgc ttttggattg 420

gataaggtaa cggacactga gatgttcttc ctggcatcaa tgtacttttc cttccccagg 480

ggcgaaggag gccctgggag gtgttttgga tctggcaagt atgtctggtt gtcggattca 540

ttgaagtctt ccgttgatta ctgtgttagg tcattccttg caaagtcggc tggcatgcaa 600

actattgttt tgatcccaac tgacgttggg gtagttgaat tagggtcggt cagatgcatc 660

ccggaaagta tggagctagt caaggtggtt ggatcttcct ttttgtcgtt ttcttcactc 720

ctcaggtcga aacaagctgc agctgcggct gtggtgacag taacggacaa aaaggatacg 780

aatgccccca ttcctaacct ggctattggt aaccgaccag aagttgctcc caagattttc 840

gggcaggact taaattcaag ccatgtgcaa tttagggaaa atgtttccgt taggaaacca 900

gaagtgcaag aaaggtcttg ggacgcaagt ggaaacagga acaggctacc atttaccaac 960

aatcgcaatg gttttcatgg cgctacgtgg acacagtata gtaatgtaaa gctagggagt 1020

ccagtagagg tttacagtcc tcagactcca gccaaaaagc gtgctgaggt tgccaacggg 1080

aatagagagg agttcatgat taataatttt cagcatcaaa agccagctca aatgcacata 1140

gattttactg gagcgacctc aaggccttta acctcctcac atccacagag tctcgaatct 1200

gagctttcag atgttgaggc ttcatgcaag gaagaggctg caggcctatc agaagataag 1260

aggccgagga agcgtggtag gaagcctgcc aatggaagag acgaacccct caatcatgta 1320

gaggcagaaa gacagcgaag agagaagctg aaccagcgct tctacgctct acgagctgtt 1380

gtaccaaata tctctaagat ggataaagct tccctccttg gagatgctat tgcttacata 1440

accgaactgc agaagaagct caaggacatg gagtctgaga gagaaagact tggcagcata 1500

tcgagagaag catctgtttc agaagctaat tctaacagag aatcacaaga cctgctggct 1560

tcgagcatta acatcgaagc tggccgtgaa gaagtcactg ttaggattag ctgcccgctg 1620

gacgcccatc cagcatcaag agtcatccaa gcaatcaatg atgcacaggc aactatcatc 1680

gatgcaaaaa tggctacagg gagtgagcga gtgttccaca cattcgttgt caaatcccat 1740

ggatccgaac gactaactaa ggagaagctg atcgaagcgt tttctcgtag atccagctct 1800

ccccatcagt tatctctcgg gtaa 1824

<210> 2

<211> 12904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

catggatggc taaaatgaga atatcaccgg aattgaaaaa actgatcgaa aaataccgct 60

gcgtaaaaga tacggaagga atgtctcctg ctaaggtata taagctggtg ggagaaaatg 120

aaaacctata tttaaaaatg acggacagcc ggtataaagg gaccacctat gatgtggaac 180

gggaaaagga catgatgcta tggctggaag gaaagctgcc tgttccaaag gtcctgcact 240

ttgaacggca tgatggctgg agcaatctgc tcatgagtga ggccgatggc gtcctttgct 300

cggaagagta tgaagatgaa caaagccctg aaaagattat cgagctgtat gcggagtgca 360

tcaggctctt tcactccatc gacatatcgg attgtcccta tacgaatagc ttagacagcc 420

gcttagccga attggattac ttactgaata acgatctggc cgatgtggat tgcgaaaact 480

gggaagaaga cactccattt aaagatccgc gcgagctgta tgatttttta aagacggaaa 540

agcccgaaga ggaacttgtc ttttcccacg gcgacctggg agacagcaac atctttgtga 600

aagatggcaa agtaagtggc tttattgatc ttgggagaag cggcagggcg gacaagtggt 660

atgacattgc cttctgcgtc cggtcgatca gggaggatat cggggaagaa cagtatgtcg 720

agctattttt tgacttactg gggatcaagc ctgattggga gaaaataaaa tattatattt 780

tactggatga attgtttatg gctaaaatga gaatatcacc ggaattgaaa aaactgatcg 840

aaaaataccg ctgcgtaaaa gatacggaag gaatgtctcc tgctaaggta tataagctgg 900

tgggagaaaa tgaaaaccta tatttaaaaa tgacggacag ccggtataaa gggaccacct 960

atgatgtgga acgggaaaag gacatgatgc tatggctgga aggaaagctg cctgttccaa 1020

aggtcctgca ctttgaacgg catgatggct ggagcaatct gctcatgagt gaggccgatg 1080

gcgtcctttg ctcggaagag tatgaagatg aacaaagccc tgaaaagatt atcgagctgt 1140

atgcggagtg catcaggctc tttcactcca tcgacatatc ggattgtccc tatacgaata 1200

gcttagacag ccgcttagcc gaattggatt acttactgaa taacgatctg gccgatgtgg 1260

attgcgaaaa ctgggaagaa gacactccat ttaaagatcc gcgcgagctg tatgattttt 1320

taaagacgga aaagcccgaa gaggaacttg tcttttccca cggcgacctg ggagacagca 1380

acatctttgt gaaagatggc aaagtaagtg gctttattga tcttgggaga agcggcaggg 1440

cggacaagtg gtatgacatt gccttctgcg tccggtcgat cagggaggat atcggggaag 1500

aacagtatgt cgagctattt tttgacttac tggggatcaa gcctgattgg gagaaaataa 1560

aatattatat tttactggat gaattgtttg gtgaccagct cgaatttccc cgatcgttca 1620

aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 1680

atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 1740

tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 1800

aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 1860

gatcgggaat taaactatca gtgtttgaca ggatatattg gcgggtaaac ctaagagaaa 1920

agagcgttta ttagaataac ggatatttaa aagggcgtga aaaggtttat ccgttcgtcc 1980

atttgtatgt gcatgccaac cacagggttc ccctcgggat caaagtactt tgatccaacc 2040

cctccgctgc tatagtgcag tcggcttctg acgttcagtg cagccgtctt ctgaaaacga 2100

catgtcgcac aagtcctaag ttacgcgaca ggctgccgcc ctgccctttt cctggcgttt 2160

tcttgtcgcg tgttttagtc gcataaagta gaatacttgc gactagaacc ggagacatta 2220

cgccatgaac aagagcgccg ccgctggcct gctgggctat gcccgcgtca gcaccgacga 2280

ccaggacttg accaaccaac gggccgaact gcacgcggcc ggctgcacca agctgttttc 2340

cgagaagatc accggcacca ggcgcgaccg cccggagctg gccaggatgc ttgaccacct 2400

acgccctggc gacgttgtga cagtgaccag gctagaccgc ctggcccgca gcacccgcga 2460

cctactggac attgccgagc gcatccagga ggccggcgcg ggcctgcgta gcctggcaga 2520

gccgtgggcc gacaccacca cgccggccgg ccgcatggtg ttgaccgtgt tcgccggcat 2580

tgccgagttc gagcgttccc taatcatcga ccgcacccgg agcgggcgcg aggccgccaa 2640

ggcccgaggc gtgaagtttg gcccccgccc taccctcacc ccggcacaga tcgcgcacgc 2700

ccgcgagctg atcgaccagg aaggccgcac cgtgaaagag gcggctgcac tgcttggcgt 2760

gcatcgctcg accctgtacc gcgcacttga gcgcagcgag gaagtgacgc ccaccgaggc 2820

caggcggcgc ggtgccttcc gtgaggacgc attgaccgag gccgacgccc tggcggccgc 2880

cgagaatgaa cgccaagagg aacaagcatg aaaccgcacc aggacggcca ggacgaaccg 2940

tttttcatta ccgaagagat cgaggcggag atgatcgcgg ccgggtacgt gttcgagccg 3000

cccgcgcacg tctcaaccgt gcggctgcat gaaatcctgg ccggtttgtc tgatgccaag 3060

ctggcggcct ggccggccag cttggccgct gaagaaaccg agcgccgccg tctaaaaagg 3120

tgatgtgtat ttgagtaaaa cagcttgcgt catgcggtcg ctgcgtatat gatgcgatga 3180

gtaaataaac aaatacgcaa ggggaacgca tgaaggttat cgctgtactt aaccagaaag 3240

gcgggtcagg caagacgacc atcgcaaccc atctagcccg cgccctgcaa ctcgccgggg 3300

ccgatgttct gttagtcgat tccgatcccc agggcagtgc ccgcgattgg gcggccgtgc 3360

gggaagatca accgctaacc gttgtcggca tcgaccgccc gacgattgac cgcgacgtga 3420

aggccatcgg ccggcgcgac ttcgtagtga tcgacggagc gccccaggcg gcggacttgg 3480

ctgtgtccgc gatcaaggca gccgacttcg tgctgattcc ggtgcagcca agcccttacg 3540

acatatgggc caccgccgac ctggtggagc tggttaagca gcgcattgag gtcacggatg 3600

gaaggctaca agcggccttt gtcgtgtcgc gggcgatcaa aggcacgcgc atcggcggtg 3660

aggttgccga ggcgctggcc gggtacgagc tgcccattct tgagtcccgt atcacgcagc 3720

gcgtgagcta cccaggcact gccgccgccg gcacaaccgt tcttgaatca gaacccgagg 3780

gcgacgctgc ccgcgaggtc caggcgctgg ccgctgaaat taaatcaaaa ctcatttgag 3840

ttaatgaggt aaagagaaaa tgagcaaaag cacaaacacg ctaagtgccg gccgtccgag 3900

cgcacgcagc agcaaggctg caacgttggc cagcctggca gacacgccag ccatgaagcg 3960

ggtcaacttt cagttgccgg cggaggatca caccaagctg aagatgtacg cggtacgcca 4020

aggcaagacc attaccgagc tgctatctga atacatcgcg cagctaccag agtaaatgag 4080

caaatgaata aatgagtaga tgaattttag cggctaaagg aggcggcatg gaaaatcaag 4140

aacaaccagg caccgacgcc gtggaatgcc ccatgtgtgg aggaacgggc ggttggccag 4200

gcgtaagcgg ctgggttgtc tgccggccct gcaatggcac tggaaccccc aagcccgagg 4260

aatcggcgtg acggtcgcaa accatccggc ccggtacaaa tcggcgcggc gctgggtgat 4320

gacctggtgg agaagttgaa ggccgcgcag gccgcccagc ggcaacgcat cgaggcagaa 4380

gcacgccccg gtgaatcgtg gcaagcggcc gctgatcgaa tccgcaaaga atcccggcaa 4440

ccgccggcag ccggtgcgcc gtcgattagg aagccgccca agggcgacga gcaaccagat 4500

tttttcgttc cgatgctcta tgacgtgggc acccgcgata gtcgcagcat catggacgtg 4560

gccgttttcc gtctgtcgaa gcgtgaccga cgagctggcg aggtgatccg ctacgagctt 4620

ccagacgggc acgtagaggt ttccgcaggg ccggccggca tggccagtgt gtgggattac 4680

gacctggtac tgatggcggt ttcccatcta accgaatcca tgaaccgata ccgggaaggg 4740

aagggagaca agcccggccg cgtgttccgt ccacacgttg cggacgtact caagttctgc 4800

cggcgagccg atggcggaaa gcagaaagac gacctggtag aaacctgcat tcggttaaac 4860

accacgcacg ttgccatgca gcgtacgaag aaggccaaga acggccgcct ggtgacggta 4920

tccgagggtg aagccttgat tagccgctac aagatcgtaa agagcgaaac cgggcggccg 4980

gagtacatcg agatcgagct agctgattgg atgtaccgcg agatcacaga aggcaagaac 5040

ccggacgtgc tgacggttca ccccgattac tttttgatcg atcccggcat cggccgtttt 5100

ctctaccgcc tggcacgccg cgccgcaggc aaggcagaag ccagatggtt gttcaagacg 5160

atctacgaac gcagtggcag cgccggagag ttcaagaagt tctgtttcac cgtgcgcaag 5220

ctgatcgggt caaatgacct gccggagtac gatttgaagg aggaggcggg gcaggctggc 5280

ccgatcctag tcatgcgcta ccgcaacctg atcgagggcg aagcatccgc cggttcctaa 5340

tgtacggagc agatgctagg gcaaattgcc ctagcagggg aaaaaggtcg aaaaggtctc 5400

tttcctgtgg atagcacgta cattgggaac ccaaagccgt acattgggaa ccggaacccg 5460

tacattggga acccaaagcc gtacattggg aaccggtcac acatgtaagt gactgatata 5520

aaagagaaaa aaggcgattt ttccgcctaa aactctttaa aacttattaa aactcttaaa 5580

acccgcctgg cctgtgcata actgtctggc cagcgcacag ccgaagagct gcaaaaagcg 5640

cctacccttc ggtcgctgcg ctccctacgc cccgccgctt cgcgtcggcc tatcgcggcc 5700

gctggccgct caaaaatggc tggcctacgg ccaggcaatc taccagggcg cggacaagcc 5760

gcgccgtcgc cactcgaccg ccggcgccca catcaaggca ccctgcctcg cgcgtttcgg 5820

tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta 5880

agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg 5940

gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg 6000

gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc 6060

gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc 6120

tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 6180

acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 6240

aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 6300

cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 6360

gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 6420

tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 6480

tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 6540

cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 6600

gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 6660

ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 6720

ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 6780

ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 6840

agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 6900

aacgaaaact cacgttaagg gattttggtc atgcattcta ggtactaaaa caattcatcc 6960

agtaaaatat aatattttat tttctcccaa tcaggcttga tccccagtaa gtcaaaaaat 7020

agctcgacat actgttcttc cccgatatcc tccctgatcg accggacgca gaaggcaatg 7080

tcataccact tgtccgccct gccgcttctc ccaagatcaa taaagccact tactttgcca 7140

tctttcacaa agatgttgct gtctcccagg tcgccgtggg aaaagacaag ttcctcttcg 7200

ggcttttccg tctttaaaaa atcatacagc tcgcgcggat ctttaaatgg agtgtcttct 7260

tcccagtttt cgcaatccac atcggccaga tcgttattca gtaagtaatc caattcggct 7320

aagcggctgt ctaagctatt cgtataggga caatccgata tgtcgatgga gtgaaagagc 7380

ctgatgcact ccgcatacag ctcgataatc ttttcagggc tttgttcatc ttcatactct 7440

tccgagcaaa ggacgccatc ggcctcactc atgagcagat tgctccagcc atcatgccgt 7500

tcaaagtgca ggacctttgg aacaggcagc tttccttcca gccatagcat catgtccttt 7560

tcccgttcca catcataggt ggtcccttta taccggctgt ccgtcatttt taaatatagg 7620

ttttcatttt ctcccaccag cttatatacc ttagcaggag acattccttc cgtatctttt 7680

acgcagcggt atttttcgat cagttttttc aattccggtg atattctcat tttagccatt 7740

tattatttcc ttcctctttt ctacagtatt taaagatacc ccaagaagct aattataaca 7800

agacgaactc caattcactg ttccttgcat tctaaaacct taaataccag aaaacagctt 7860

tttcaaagtt gttttcaaag ttggcgtata acatagtatc gacggagccg attttgaaac 7920

cgcggtgatc acaggcagca acgctctgtc atcgttacaa tcaacatgct accctccgcg 7980

agatcatccg tgtttcaaac ccggcagctt agttgccgtt cttccgaata gcatcggtaa 8040

catgagcaaa gtctgccgcc ttacaacggc tctcccgctg acgccgtccc ggactgatgg 8100

gctgcctgta tcgagtggtg attttgtgcc gagctgccgg tcggggagct gttggctggc 8160

tggtggcagg atatattgtg gtgtaaacaa attgacgctt agacaactta ataacacatt 8220

gcggacgttt ttaatgtact gaattaacgc cgaattaatt cgggggatct ggattttagt 8280

actggatttt ggttttagga attagaaatt ttattgatag aagtatttta caaatacaaa 8340

tacatactaa gggtttctta tatgctcaac acatgagcga aaccctatag gaaccctaat 8400

tcccttatct gggaactact cacacattat tatggagaaa ctcgagtcaa atctcggtga 8460

cgggcaggac cggacggggc ggtaccggca ggctgaagtc cagctgccag aaacccacgt 8520

catgccagtt cccgtgcttg aagccggccg cccgcagcat gccgcggggg gcatatccga 8580

gcgcctcgtg catgcgcacg ctcgggtcgt tgggcagccc gatgacagcg accacgctct 8640

tgaagccctg tgcctccagg gacttcagca ggtgggtgta gagcgtggag cccagtcccg 8700

tccgctggtg gcggggggag acgtacacgg tcgactcggc cgtccagtcg taggcgttgc 8760

gtgccttcca ggggcccgcg taggcgatgc cggcgacctc gccgtccacc tcggcgacga 8820

gccagggata gcgctcccgc agacggacga ggtcgtccgt ccactcctgc ggttcctgcg 8880

gctcggtacg gaagttgacc gtgcttgtct cgatgtagtg gttgacgatg gtgcagaccg 8940

ccggcatgtc cgcctcggtg gcacggcgga tgtcggccgg gcgtcgttct gggctcatgg 9000

tagactcgag agagatagat ttgtagagag agactggtga tttcagcgtg tcctctccaa 9060

atgaaatgaa cttccttata tagaggaagg tcttgcgaag gatagtggga ttgtgcgtca 9120

tcccttacgt cagtggagat atcacatcaa tccacttgct ttgaagacgt ggttggaacg 9180

tcttcttttt ccacgatgct cctcgtgggt gggggtccat ctttgggacc actgtcggca 9240

gaggcatctt gaacgatagc ctttccttta tcgcaatgat ggcatttgta ggtgccacct 9300

tccttttcta ctgtcctttt gatgaagtga cagatagctg ggcaatggaa tccgaggagg 9360

tttcccgata ttaccctttg ttgaaaagtc tcaatagccc tttggtcttc tgagactgta 9420

tctttgatat tcttggagta gacgagagtg tcgtgctcca ccatgttatc acatcaatcc 9480

acttgctttg aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg 9540

ggtccatctt tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg 9600

caatgatggc atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag 9660

atagctgggc aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca 9720

atagcccttt ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg 9780

tgctccacca tgttggcaag ctgctctagc caatacgcaa accgcctctc cccgcgcgtt 9840

ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 9900

gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 9960

ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 10020

atgaccatga ttacgaattc gcccggggat ctcctttgcc ccagagatca caatggacga 10080

cttcctatat ctctacgatc tagtcaggaa gttcgacgga gaaggtgacg ataccatgtt 10140

caccactgat aatgagaaga ttagcctttt caatttcaga aagaatccta acccacagat 10200

ggttagagac gcttacgcag caggtctcat caagacgatc tacccgagca ataatctcca 10260

ggagatcaaa taccttccca agaaggttaa agatgcagtc aaaagattca ggactaactg 10320

catcaagaac acagagaaag atatatttct caagatcaga agtactattc cagtatggac 10380

gattcaaggc ttgcttcaca aaccaaggca agtaatagag attggagtct ctaaaaaggt 10440

agttcccact gaatcaaagg ccatggagtc aaagattcaa atagaggacc taacagaact 10500

cgccgtaaag actggcgaac agttcataca gagtctctta cgactcaatg acaagaagaa 10560

aatcttcgtc aacatggtgg agcacgacac gcttgtctac ctccaaaaat atcaaagata 10620

cagtctcaga agaccaaagg gaattgagac ttttcaacaa agggtaatat ccggaaacct 10680

cctcggattc cattgcccag ctatctgtca ctttattgtg aagatagtgg aaaaggaagg 10740

tggctcctac aaatgccatc attgcgataa aggaaaggcc atcgttgaag atgcctctgc 10800

cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt 10860

tccaaccacg tcttcaaagc aagtggattg atgtgataac atggtggagc acgacacgct 10920

tgtctacctc caaaaatatc aaagatacag tctcagaaga ccaaagggaa ttgagacttt 10980

tcaacaaagg gtaatatccg gaaacctcct cggattccat tgcccagcta tctgtcactt 11040

tattgtgaag atagtggaaa aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg 11100

aaaggccatc gttgaagatg cctctgccga cagtggtccc aaagatggac ccccacccac 11160

gaggagcatc gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag tggattgatg 11220

tgatatctcc actgacgtaa gggatgacgc acaatcccac tatccttcgc aagacccttc 11280

ctctatataa ggaagttcat ttcatttgga gaggacacgc tgaaatcacc agtctctctc 11340

taagcttgga tccactagtg acggtatcga taagcttatg gactacaagg atgacgacga 11400

caaggactac aaggatgacg acgacaagga ctacaaggac gacgacaaga tggatgctag 11460

cccgggccat taagctagcg agctcgaatt gatcctctag agctttcgtt cgtatcatcg 11520

gtttcgacaa cgttcgtcaa gttcaatgca tcagtttcat tgcgcacaca ccagaatcct 11580

actgagttcg agtattatgg cattgggaaa actgtttttc ttgtaccatt tgttgtgctt 11640

gtaatttact gtgtttttta ttcggttttc gctatcgaac tgtgaaatgg aaatggatgg 11700

agaagagtta atgaatgata tggtcctttt gttcattctc aaattaatat tatttgtttt 11760

ttctcttatt tgttgtgtgt tgaatttgaa attataagag atatgcaaac attttgtttt 11820

gagtaaaaat gtgtcaaatc gtggcctcta atgaccgaag ttaatatgag gagtaaaaca 11880

cttgtagttg taccattatg cttattcact aggcaacaaa tatattttca gacctagaaa 11940

agctgcaaat gttactgaat acaagtatgt cctcttgtgt tttagacatt tatgaacttt 12000

cctttatgta attttccaga atccttgtca gattctaatc attgctttat aattatagtt 12060

atactcatgg atttgtagtt gagtatgaaa atatttttta atgcatttta tgacttgcca 12120

attgattgac aacatgcatc aatcgatggc actggccgtc gttttacaac gtcgtgactg 12180

ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 12240

gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg 12300

cgaatgctag agcagcttga gcttggatca gattgtcgtt tcccgccttc agtttagctt 12360

catggagtca aagattcaaa tagaggacct aacagaactc gccgtaaaga ctggcgaaca 12420

gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca acatggtgga 12480

gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag accaaagggc 12540

aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc attgcccagc 12600

tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca aatgccatca 12660

ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc ccaaagatgg 12720

acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca 12780

agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc actatccttc 12840

gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac gggggactct 12900

tgac 12904

Claims (2)

1. A method for promoting the development of lateral roots of salvia miltiorrhiza and improving the content of tanshinone in salvia miltiorrhiza is characterized in that a transgenic salvia miltiorrhiza seedling with better lateral root development and improved tanshinone content is obtained by over-expressing a salvia miltiorrhiza SmMYC2b gene in the salvia miltiorrhiza root, and comprises the following steps:

step S1, constructing an overexpression vector containing the salvia miltiorrhiza SmMYC2b gene;

step S2, transfecting the overexpression vector obtained in the step S1 into agrobacterium to obtain recombinant agrobacterium;

step S3, transforming salvia miltiorrhiza tissues and inducing callus differentiation by using the recombinant agrobacterium obtained in step S2 to obtain transgenic salvia miltiorrhiza seedlings,

wherein the sequence of the salvia miltiorrhiza SmMYC2b gene is shown as SEQ ID No: 1 is shown.

2. The method of promoting lateral root development of red sage root and increasing tanshinone content in red sage root according to claim 1, wherein:

wherein the tanshinone comprises dihydrotanshinone I, tanshinone I, cryptotanshinone and tanshinone IIA.

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