CN113637680B

CN113637680B - Application of salvia miltiorrhiza transcription factor SmbHLH124 in improving hairy root yield traits of salvia miltiorrhiza

Info

Publication number: CN113637680B
Application number: CN202110877918.4A
Authority: CN
Inventors: 张勇; 邓科君; 郑雪莲; 仲昭辉; 刘晓佩; 方庆; 李梦珂
Original assignee: University of Electronic Science and Technology of China
Current assignee: University of Electronic Science and Technology of China
Priority date: 2021-07-30
Filing date: 2021-07-30
Publication date: 2023-08-11
Anticipated expiration: 2041-07-30
Also published as: CN113637680A

Abstract

The invention belongs to the technical field of plant biology, and particularly relates to a novel application of a salvia miltiorrhiza transcription factor SmbHLH 124. The invention aims to solve the technical problem of improving the yield characteristics of hairy roots of red sage roots. The technical scheme for solving the technical problem is to provide the application of the salvia miltiorrhiza transcription factor SmbHLH124 or the encoding DNA molecule thereof in improving the hairy root yield trait of the salvia miltiorrhiza and the method for improving the hairy root yield trait of the salvia miltiorrhiza. The technical scheme of the invention can obtain hairy roots with obviously increased biomass and tanshinone content, and effectively improve the yield characteristics of the hairy roots of the red sage root. The invention can provide ideal materials for tanshinone medicine source production and has good application prospect in solving the problem of insufficient medicine sources of tanshinone compounds.

Description

Application of salvia miltiorrhiza transcription factor SmbHLH124 in improving hairy root yield traits of salvia miltiorrhiza

Technical Field

The invention belongs to the technical field of plant biology, and particularly relates to application of a salvia miltiorrhiza transcription factor SmbHLH124 in improving hairy root yield traits.

Background

The red sage root (Salvia miltiorrhiza Bunge) is a medicinal plant with long use history, is recorded in Chinese pharmacopoeia for a long time, is used as a medicament by roots and rhizomes, and is widely applied clinically. Tanshinone compounds are fat-soluble active ingredients in salvia miltiorrhiza, and representative ingredients are Cryptotanshinone (CTS), dihydrotanshinone (DTS), tanshinone I (TSI) and tanshinone II A (TSA), and have definite medicinal effects in various aspects such as cardiovascular diseases, anti-inflammatory and immune, anti-tumor, liver protection, neuroprotection and the like (Liu Huiying, 2016).

The hairy root of red sage root is the same as other plant hairy roots, has the characteristics of fast growth rate, high bifurcation, autotrophy of hormone and the like, and is one of ideal medicine source raw materials for producing active ingredients in red sage root. During hairy root culture, changing culture conditions or utilizing related biotechnology means in an effort to enhance accumulation of secondary metabolites in hairy roots has become a research hotspot. Common genetic engineering means for improving the yield of secondary metabolites using biotechnology methods are: constructing over-expression and antisense inhibition carrier technology, and genetically transforming the red sage root to obtain hairy root. But currently there is more room for lifting.

Basic helix-loop-helix (bHLH) transcription factors are one of the largest families of transcription factors in plants, and play an important role in regulating plant growth and development, metabolism, coping with physiological processes such as biotic and abiotic stress and the like. At present, the members of the bHLH transcription factor family in the red sage root, such as SmbHLH1, smbHLH 3,SmbHLH 53,SmbHLH 10 and the like, have a certain regulation and control effect on the biosynthesis of secondary metabolites, but have no report on the improvement of the growth of hairy roots of the red sage root.

Disclosure of Invention

The invention aims to solve the technical problem of improving the yield characteristics of hairy roots of red sage roots.

The technical scheme for solving the technical problem is to provide the application of the salvia miltiorrhiza transcription factor SmbHLH124 or the encoding DNA molecule thereof in improving the hairy root yield character of the salvia miltiorrhiza.

Wherein the amino acid sequence of the salvia miltiorrhiza transcription factor SmbHLH124 in the application is shown as SEQ ID No. 10.

Wherein the nucleotide sequence of the DNA molecule encoded by the salvia miltiorrhiza transcription factor SmbHLH124 in the application is shown as SEQ ID No. 1.

Wherein the hairy root yield trait in the above application is the hairy root biomass and/or the hairy root tanshinone content.

Wherein the tanshinone in the application is at least one of tanshinone I, cryptotanshinone, dihydrotanshinone I or tanshinone IIA.

The invention also provides a method for improving the yield traits of hairy roots of the red sage root. The method comprises the following steps:

a. constructing an over-expression vector for expressing a coding gene of the salvia miltiorrhiza transcription factor SmbHLH 124;

b. c, transforming the red sage root by using the over-expression vector obtained in the step a to obtain genetically transformed hairy root of the red sage root;

c. c, screening and identifying the transformed hairy roots obtained in the step c, and screening hairy roots of the coding genes of the overexpression salvia miltiorrhiza transcription factor SmbHLH 124;

d. liquid culture is carried out on hairy roots of the coding gene of the overexpression red sage root transcription factor SmbHLH124, and hairy roots with improved yield traits are obtained.

Wherein the amino acid sequence of the salvia miltiorrhiza transcription factor SmbHLH124 in the method is shown as SEQ ID No. 10.

Furthermore, the nucleotide sequence of the coding gene of the salvia miltiorrhiza transcription factor SmbHLH124 in the method is shown as SEQ ID No. 1.

Further, the genetic transformation method in the above method may be at least one of electroporation, gene gun method, agrobacterium-mediated method, liposome-mediated method, PEG-mediated method and virus-mediated method.

Wherein, the hairy root yield trait in the method is the biomass of hairy roots and/or the tanshinone content of hairy roots.

Further, the tanshinone in the above method is at least one of tanshinone I, cryptotanshinone, dihydrotanshinone I or tanshinone IIA.

The invention has the beneficial effects that: the invention creatively discovers that the SmbHLH124 gene overexpression can obtain hairy roots with increased growth quantity and tanshinone content. Furthermore, the invention constructs an over-expression SmbHLH124 gene vector aiming at the hairy root system of the red sage root. Experiments show that the method can improve the expression of SmbHLH124 genes in the root of the red sage root, so that the biomass and the tanshinone content of the hairy root are obviously increased, and the yield character of the hairy root of the red sage root is effectively improved. The invention provides ideal materials for tanshinone medicine source production and has good application prospect in solving the problem of insufficient medicine sources of tanshinone compounds.

Drawings

FIG. 1, smbHLH124cDNA clone.

FIG. 2, smbHLH124 overexpression vector construction. a, a pLXP003 vector schematic diagram; b, constructing colony PCR electrophoresis patterns by using the pLXP003 vector.

FIG. 3, hairy root genetic transformation flow. a, inducing root culture; b, inducing root to culture for 15 days; c, subculturing for 0 days; d, subculturing for 20 days.

FIG. 4, identification of root systems over-expressing SmbHLH 124. a, root system rolC gene identification; b, positive identification of the SmbHLH124 root line. M, markers; PC, positive control; NC, negative control, numbers 6, 29, 39, 41, 43, etc. represent root systems of overexpressed SmbHlH124 that survived the screening culture, respectively.

FIG. 5, control root system and over-expressed SmbHLH124 root system growth were compared. a, performing liquid culture on a control root system and an over-expressed SmbHLH124 root system for 45 days, and performing phenotype graph; and b, c, carrying out statistical analysis on the growth data. C58C1-7, and controlling root systems; pLXP003-6, -39, -41 is the over-expressed root system.

FIG. 6, overexpression of SmbHLH124 levels of several tanshinones in hairy roots of Salvia Miltiorrhiza. a, average value of dihydrotanshinone I (mg/g); b, tanshinone average (mg/g); c, tanshinone I average value (mg/g); d, tanshinone IIA average (mg/g).

Detailed Description

In order to obtain the hairy roots of the red sage root with improved growth and tanshinone and other yield traits, the invention carries out identification analysis on the red sage root bHLH transcription factor family members in the whole genome range reported previously. It is predicted that SmbHLH124 is probably involved in tanshinone biosynthesis regulation.

On the basis, the invention constructs an expression vector capable of over-expressing the transcription factor SmbHLH124 gene, and transfers the expression vector into agrobacterium, and the hairy root material for over-expressing the SmbHLH124 is obtained by an agrobacterium-mediated method. As a result, it was found that the obtained overexpressed SmbHLH124 had increased both in the growth amount of hairy roots and in the tanshinone content. The new application of the salvia miltiorrhiza transcription factor SmbHLH124 and the encoding gene thereof in improving the yield traits of hairy roots of the salvia miltiorrhiza is obtained.

The above-mentioned overexpression vector can be constructed according to a common expression vector. In one example of the present invention, the vector pGSD-AV3 was used.

The transformation of the root of red-rooted salvia using the above-described overexpression vector to obtain genetically transformed hairy root of red-rooted salvia may be performed using various transformation methods in the art.

For example, agrobacterium-mediated genetic transformation methods may be used. The agrobacterium may use a variety commonly used in the art, such as C58C1 or LBA4404, etc.

Specifically, the method for improving the hairy root yield traits of the red sage root in the technical scheme of the invention can be specifically carried out by referring to the following methods:

(1) Obtaining of SmbHLH124 Gene fragment

Specific primers SmbHLH124-F and SmbHLH124-R are designed according to the published sequence information of SmbHLH124 genes in the genome and transcriptome data of the red sage root, the sequences of the specific primers are shown as SEQ ID No.2 and SEQ ID No.3, and the SmbHLH124 gene fragment amplification is carried out by taking cDNA (20 ng/. Mu.L) of the annual red sage root underground root as a template.

(2) Construction of SmbHLH124 overexpression vector

The obtained SmbHLH124 gene fragment is assembled on a vector pGSD-AV3 in a Golden Gate mode, the constructed recombinant vector is used for transforming escherichia coli DH5 alpha competent cells, monoclonal is selected, PCR detection is carried out by using primers TC013-F (with a primer sequence as SEQ ID No. 4) and TC013-R (with a primer sequence as SEQ ID No. 5), sanger sequencing verification is carried out on the plasmid, an over-expression vector pLXP003 of the salvia miltiorrhiza SmbHLH124 is obtained, the pLXP003 is transformed into agrobacterium C58C1, and the agrobacterium strain used for genetic transformation of the salvia miltiorrhiza hairy root and containing the over-expression vector of the SmbHLH124 gene plant is obtained.

(3) Genetic transformation of hairy roots of red sage root

The plant over-expression vector pLXP003 is obtained through genetic transformation, screening and transformant of hairy root of red sage root mediated by agrobacterium C58C 1.

Explant preparation: selecting dark green healthy leaves in the middle leaves of the strong red sage root aseptic seedling, and cutting the leaves into leaf discs with the length of about 0.5cm multiplied by 0.5cm for standby.

Infection with agrobacterium: centrifuging the cultured agrobacterium to collect thalli, and re-suspending the thalli by using an equal volume of MS+200 mu M acetosyringone liquid culture medium; taking out the prepared leaf disc, and soaking the leaf disc in the re-suspension bacteria liquid for infection.

Co-cultivation: the bacterial solution was decanted and the leaf discs were inoculated with a layer of filter paper on the surface facing upward onto SmCCM (MS+200 uM AS) co-culture medium and incubated at 25℃for about 60h.

Induction culture: the leaf disk is inoculated into an induction culture medium SmHRM (1/2MS+500 mg/L Cef 50mg/L Kan) with a photoperiod of 25 ℃ and 16hr light/8 hr darkness, and is continuously cultured for 20d. White hairy roots start to grow about 5-10 days, the sequence of the growing roots is marked, the hairy roots growing vigorously are cut off from the edges of leaves at 20 days, and the roots are put into a screening culture medium for screening, wherein 5-7 roots are planted in each dish.

Screening culture (20-30 d, screening 2-3 times): root systems with the length of 3-5cm are cut from leaf discs, placed in order and transferred into a screening culture medium SmSSM (B5+500 mg/L Cef+50mg/L Kan), marked with root tip initial sites and placed in a dark condition at 25 ℃ for continuous culture. And (3) carrying out subculture for 1 time every 10-15d, carrying out subculture for 1-2 times, and culturing for 20-30d. In the process, some root systems obviously grow; some root systems stop growing and the root tips are browned. Taking the root system with growth to carry out the next experiment.

(4) Molecular characterization of transformed hairy root material

Extracting genomic DNA of hairy roots of red sage root by CTAB method, and carrying out positive detection of transgenosis. Specific primers rolC-F (primer sequence shown as SEQ ID No. 6) and rolC-R1 (primer sequence shown as SEQ ID No. 7) contained on a hairy root plasmid vector and specific primers contained on pLXP003 were used: the BHlH124-F2 (primer sequence is shown as SEQ ID No. 8) and nptII-R2 (primer sequence is shown as SEQ ID No. 9) are amplified to obtain target fragments, the target fragments detect positive transgenes, and the obtained root system is determined to be hairy root of the SmbHLH124 over-expression vector.

Further, the overexpressed SmbHLH124 obtained by the present invention can be assayed for yield traits using the following method. Overexpression hairy root biomass analysis: after harvesting hairy roots of 45 days of liquid culture, weighing fresh weight on a balance with precision of ten-thousandth, recording, transferring into an oven, drying to constant weight at 40 ℃, and weighing to obtain dry weight. And (3) measuring the tanshinone content: the content of tanshinone in hairy roots of the salvia miltiorrhiza Bunge transferred into the SmbHLH124 gene overexpression vector is determined by high performance liquid chromatography, so that the screening of hairy root lines of the salvia miltiorrhiza Bunge gene with obviously improved tanshinone is facilitated.

The present invention will be described in further detail by the following examples.

EXAMPLE 1 construction of the Salvia Miltiorrhiza SmbHLH124 overexpression vector

(1) SmbHLH124cDNA fragment acquisition

Specific primers SmbHLH124-F and SmbHLH124-R are designed according to the sequence information of SmbHLH124 genes in published genome and transcriptome data of the red sage root, the sequences of which are shown as SEQ ID No.2 and SEQ ID No.3, and the primers are synthesized by Shanghai Hairyholder company. The cDNA of the underground root of annual red sage root (20 ng/. Mu.L) is used as a templateSmbHLH124 gene fragment amplification, target fragment amplification system: 10 XKOD Plus Buffer 5. Mu.L, dNTP (2.5 mM) 5. Mu.L, mgSO4 (25 mM) 3. Mu.L, KOD Plus Neo (1U/. Mu.L) 1. Mu.L, smbHLH124-F (10. Mu.M) 1. Mu.L, smbHLH124-R (10. Mu.M) 1. Mu.L, cDNA 1. Mu.L, ddH ₂ O33. Mu.L. The PCR amplification procedure was as follows: 95 ℃/5 min- > (95 ℃/30 s- > (56 ℃/30 s- > (68 ℃/30 s). Times.40 cycles- > 68 ℃/5 min- > 12 ℃/10 min). And (3) carrying out agarose electrophoresis on the PCR product, recovering a target fragment of about 300bp by using an AxyPrep DNA gel recovery kit, wherein an electrophoresis diagram is shown in figure 1, and sending the gel recovery product to Shanghai Marine engineering company for Sanger sequencing, wherein the sequencing result shows that the obtained fragment is SmbHLH124, and the sequence is shown as SEQ ID No. 1.

(2) Construction of intermediate vector pGE01-bHLH124

Amplifying the SmbHLH124cDNA fragment by using a specific primer to enable specific enzyme cutting sites to be carried on two ends of the fragment, wherein the primer-F is SmbHLH124-F (with a sequence shown as SEQ ID No. 2), the primer-R is pFQ-R (with a sequence shown as SEQ ID No. 9), carrying out agarose gel electrophoresis on a PCR product, recovering a target strip with a size of about 300bp, and carrying out double enzyme cutting on the recovered SmbHLH124cDNA fragment with the specific enzyme cutting sites and a skeleton vector pGE01 respectively, wherein an enzyme cutting system is as follows: 10xGreen Buffer 5. Mu.L, kpnI (1U/. Mu.L) 1. Mu.L, pstI (1U/. Mu.L) 1. Mu.L, fragment/backbone vector (300. Mu.g) 10. Mu.L, ddH2O 33. Mu.L. And enzyme cutting at 37 ℃ for 1.5 hours. The recovered SmbHLH124cDNA fragment after cleavage was T4 ligated with the backbone vector pGE 01. T4 connection system: 10×T4Ligase Buffer 2. Mu.L, T4 Ligase 1. Mu.L, smbHLH124cDNA fragment 2. Mu.L, pGE01 (digested) 3. Mu.L, ddH2O 12. Mu.L. T4 ligation reaction conditions: 25℃and 12h of connection. After the connection is finished, the escherichia coli DH5 alpha transformation, colony PCR, plasmid extraction and sequencing verification are sequentially carried out. primer-F used in colony PCR: smbHLH124-F (SEQ ID No. 2), primer-R: smbHLH124-R (SEQ ID No. 3). Sequencing results show that the intermediate vector pGE01-bHLH124 is successfully constructed.

(3) Overexpression vector pLXP003 assembly

The cDNA of SmbHLH124 was assembled onto pGSD-AV3 backbone vector by means of Golden Gate. pGSD-AV3 backbone vector is a T-DNA vector, full length 7050bp, wherein 265-289 is the left border repeat of the transfer DNA; 340-350, 976-986 are BsaI recognition and cleavage sites; 351-975 is a virulence gene of E.coli, used to screen clones; 987-1022 are multiple cloning sites; 1037-1061 are right border repeats of the transfer DNA. The Golden gate reaction system is: t4 DNA ligase buffer (10X) 2. Mu.L, T4 DNA ligase 1. Mu.L, restriction enzyme BsaI 1. Mu.L, pGSD-AV3 (65 ng/. Mu.L), pPE-CaMV35S (140 ng/. Mu.L) 1. Mu.L, pTE-35ST (35 ng/. Mu.L) 1. Mu.L, pGE01-bHLH124 (100 ng. Mu.L) 1. Mu.L, SE01-NptII (200 ng. Mu.L) 1. Mu.L, ddH2O 10.5. Mu.L. The Golden gate reaction procedure is: (5 min at 37 ℃ C., 10min at 16 ℃) for 15 cycles, 10min at 37 ℃ C., 10min at 65 ℃ C., 4 ℃ C./10 min. After the reaction is finished, the reaction product of escherichia coli DH5 alpha conversion, colony PCR, plasmid extraction and sequencing verification are sequentially carried out. Wherein the primer-F used in colony PCR is TC013-F (primer sequence is shown as SEQ ID No. 4), and the primer-R is TC013-R (primer sequence is shown as SEQ ID No. 5).

(4) Plasmid transformed E.coli competent cells

Melting competent cells of Escherichia coli DH5 alpha on ice, adding the above 20ul of the ligation product, flicking, mixing, and ice-bathing for 30min. Heat shock at 42deg.C for 1min, and ice-bathing on ice for 2min. 500. Mu.L of LB liquid medium was added thereto, and incubated at 37℃for 45min with shaking at 200 rpm. After 12000rpm centrifugation for 2min, 400. Mu.L of the supernatant was discarded, and the resuspended cells were gently beaten with a pipetting gun. All bacterial solutions are coated on LB solid medium (containing 50mg/L Kan), and are cultivated for 1h at 37 ℃ in a normal way and then are cultivated for 12-16h in an inverted way.

(4) Colony PCR

The monoclonal on LB plate was picked up with sterile toothpick and placed in 50. Mu.L ddH ₂ In O water, 1uL of the bacterial liquid is taken as a template for PCR amplification. A25 uL system was used as follows: 10 XPCR Buffer 2.5. Mu.L, dNTP 0.5. Mu.L, primer-F0.5. Mu.L, primer-R0.5. Mu.L, taq DNA enzyme 0.2. Mu.L, template 1. Mu.L, ddH ₂ O19.8. Mu.L. The PCR procedure was: 94 ℃,2 min- & gt (94 ℃,30 s- & gt 55 ℃,30 s- & gt 72 ℃ for 30 s) 35 cycles- & gt 72 ℃,5 min- & gt 4 ℃ for 10min (Taq DNA enzyme, dNTP and the like are purchased from Tiangen biological company). PCR products were electrophoretically detected in a 1% agarose gel at 130V for 30min, see FIG. 2b.

(5) Plasmid extraction and sequencing verification

And adding 50 mu L of bacterial liquid into 4-5ml of culture medium to perform amplification culture for 12-16h, and extracting plasmids. The extraction of plasmid DNA was performed according to the AXYGEN AxyPrepTM Plasmid Miniprep Kit instructions. Sequencing verification is carried out on the extracted plasmid by Shanghai worker company, and sequencing results show that the over-expression vector pLXP003 is successfully constructed, and the structural schematic diagram of the constructed pLXP003 vector is shown in figure 2a.

EXAMPLE 2 Agrobacterium-mediated genetic transformation of hairy roots of Salvia Miltiorrhiza

Methods disclosed in Agrobacterium-mediated genetic transformation of hairy roots of Salvia Miltiorrhiza (Toki et al, early infection of scutellum tissue with Agrobacterium allows high-speed transformation of price. The Plant Journal,2006,47 (6): 969-976.). The genetic transformation steps of the red sage root are specifically as follows, and the flow chart is shown in figure 3:

(1) Agrobacterium preparation

Picking agrobacterium tumefaciens C58C1 (Shanghai Taitan technology Co., ltd.) carrying a target vector pLXP003 stored at-80 ℃ by an inoculating loop, streaking on LB+50mg/L Kan+50mg/L Rif plates, and inversely placing in a 28 ℃ incubator for culturing for 48 hours; picking single colony on the LB plate by using a toothpick, inoculating the single colony into a test tube (or 10mL EP tube) containing 5mL of LB+50mg/L Kan+50mg/L Rif liquid medium, and performing activation culture at 28 ℃ and 180rpm for 48 hours; on the day of infection, the bacterial liquid is sucked into a 100mL or 250mL triangular flask containing 50mL of LB liquid culture medium according to the proportion of 1:20 to 1:50, and the bacterial liquid is subjected to expansion culture at 28 ℃ and 180rpm for about 4-6 hours until OD600 = 0.5.

(2) Explant preparation

Selecting a strong red sage root aseptic seedling middle leaf (middle dark green healthy leaf, over tender or over old leaf can affect conversion efficiency), removing main leaf vein and leaf edge on an ultra clean workbench by using a sharp blade (a new blade is suitable), cutting into leaf discs of about 0.5cm multiplied by 0.5cm, and putting into sterile water or MS liquid culture medium to keep moist for standby.

(3) Agrobacterium infection

Transferring the cultured agrobacterium into a sterilizing centrifuge tube, centrifuging at 4200rpm for 10min at normal temperature, collecting thallus, and re-suspending thallus with equal volume of MS+200 mu M acetosyringone liquid medium; taking out the prepared leaf disc, soaking in the re-suspension bacteria liquid, infecting for 30min, and properly and gently oscillating to ensure that the leaf disc is completely immersed in the bacteria liquid.

(4) Co-culture (2 d):

pouring out the bacterial liquid, flushing the leaves for 2-3 times by using sterile water, thoroughly sucking the bacterial liquid on the surface of the leaf disc by using sterile water absorbing paper (sucking as dry as possible), and carrying out dark culture for 60h at 25 ℃ on SmCCM with the front surface of the leaf disc facing upwards and a layer of filter paper paved on the surface.

(5) Induction culture (20 d):

the leaf disk is inoculated into an induction culture medium SmHRM (1/2MS+500 mg/L Cef+50mg/L Kan) with a photoperiod of 25 ℃ and 16hr light/8 hr darkness for 20d continuously. White hairy roots start to grow about 5-10 days, the sequence of the growing roots is marked, the hairy roots growing vigorously are cut off from the edges of leaves at 20 days, and the roots are put into a screening culture medium for screening, wherein 5-7 roots are planted in each dish.

(6) Screening culture (20-30 d, screening 2-3 times):

root systems with the length of 3-5cm are cut from leaf discs, placed in order and transferred into a screening culture medium SmSSM (B5+500 mg/L Cef+50mg/L Kan), marked with root tip initial sites and placed in a dark condition at 25 ℃ for continuous culture. And (3) carrying out subculture for 1 time every 10-15d, carrying out subculture for 1-2 times, and culturing for 20-30d. In the process, some root systems obviously grow; some root systems stop growing and the root tips are browned. Taking the root system with growth to carry out the next experiment.

EXAMPLE 3 molecular characterization of Salvia Miltiorrhiza over-expressed SmbHLH hairy root

(1) Extraction of genomic DNA from hairy root of red sage root

The extraction of the hairy root DNA of the red sage root adopts a CTAB method, and the specific operation steps are as follows:

the CTAB extract was preheated in a 65℃water bath. Taking a single part 1-2cm away from the root tip, placing the single part into a 2mL centrifuge tube with steel balls, placing liquid nitrogen for quick freezing, and vibrating the single part into powder. 500. Mu.L of preheated CTAB extract is added, and the mixture is fully and uniformly mixed in a water bath at 65 ℃ for 30-50 min. 500 μl chloroform was added: isoamyl alcohol (24:1), mixing completely, and centrifuging at 12000rpm at room temperature for 10min. Taking supernatant, adding equal volumeIsopropanol precipitation of (C) at-20℃for 1h. Centrifuging at 12000rpm for 10min at room temperature, and collecting precipitate. The supernatant was removed, rinsed with 75% ethanol, and centrifuged at 12000rpm for 2min. The supernatant was removed and the DNA was dried at room temperature. Add 30. Mu.L ddH ₂ O dissolves DNA and is preserved in a refrigerator at-20 ℃ for standby.

(2) Positive detection of root of red sage transgenic

Specific primers rolC-F (primer sequence shown as SEQ ID No. 6) CTCCTGACATCAAACTCGTC and rolC-R1 (primer sequence shown as SEQ ID No. 7) GCTGCTGTACCTCTACGTCGA contained on the hairy root plasmid vector and specific primers contained on pLXP003 were used: the BHlH124-F2 (primer sequence is shown as SEQ ID No. 8) GGCAATCTGGATCTTCAAGAATC and nptII-R2 (primer sequence is shown as SEQ ID No. 9) TGTGCCCAGTCATAGCCG amplified target fragments detect transgene positivity, the amplified fragments are about 400bp in size, and the PCR amplification system and the reaction program are the same as those of the previous colony PCR. The results showed that the regenerated plants detected were positive plants (see FIG. 4).

EXAMPLE 4 biological analysis of root systems in hairy form of Saviae Miltiorrhizae radix overexpressing SmbHLH

(1) Liquid culture of hairy root of red sage root

In the identified materials of the overexpressing SmbHLH124 and the C58C1, 10 roots of hairy roots of red sage roots (with similar growth state) of about 3-5cm are selected from each root system, inoculated in a 250mL conical flask containing 100mL of liquid 1/2B5 culture medium for liquid culture (three flasks are repeatedly cultured for each root system), the conical flask is wrapped by black shading cloth, the black shading cloth is cultivated in a constant temperature shaking table set at 25 ℃ and 150rpm, the growth condition of the hairy roots is recorded every 15 days, and the liquid culture is ended until the culture is completed until 45 days. (see FIG. 5 a)

(2) Analysis of root biomass of hairy roots of red sage root

Hairy roots were harvested after 45 days of liquid multiplication culture, surface moisture was absorbed as much as possible using a piece of absorbent paper, and their fresh weights were weighed on a one-ten thousandth scale, and as a result, it was found that the fresh weights of overexpressed hairy roots were significantly improved compared to control roots (see fig. 5 b).

After the harvested hairy roots were oven dried to constant weight at 40 ℃, their dry weight was obtained by weighing them with a ten-thousandth balance, and as a result, it was found that the dry weight of the overexpressed hairy roots was significantly improved compared to the control roots (see fig. 5 c).

EXAMPLE 5 detection of tanshinone content in root system with hairy root system overexpressing SmbHLH from Salvia Miltiorrhiza

(1) Extraction of tanshinone from hairy root of red sage root

Hairy roots of Salvia Miltiorrhiza Bunge cultured in 100mL liquid culture medium for 45 days are dried at 40deg.C to constant weight. Grinding into powder in a mortar, precisely weighing 0.050g of fine powder of hairy roots of radix salviae miltiorrhizae, adding into a 10mL volumetric flask, adding 8mL of 80% methanol (80% methanol, 20% water) into the volumetric flask, performing ultrasonic extraction at 25 ℃ for 60min, using 80% methanol to fix the volume to 10mL, mixing uniformly, filtering by using quantitative filter paper, and collecting filtrate; filtering with 0.34 μm filter membrane, and loading into automatic sampler bottle.

(2) HPLC (high Performance liquid chromatography) detection of tanshinone content in hairy roots of salvia miltiorrhiza

The tanshinone content in the hairy root samples of Salvia Miltiorrhiza was determined by high performance liquid chromatography (High Performance Liquid Chromatography, HPLC). The detection conditions were as follows:

liquid chromatography: waters e2695 HPLC

A detector: waters 2489UV/Vis Detector

Chromatographic column: venusil XBP C18 (L) (4.6. Times.250 mm;5 μm; 150A)

Mobile phase: 0.02% phosphoric acid aqueous solution as mobile phase A (aqueous phase) and 100% acetonitrile as mobile phase B (organic phase)

Flow rate: 1m L/min

Column temperature: 35 DEG C

Detection wavelength: 270nm

Sample injection amount: 20 mu L

Gradient elution conditions were as follows: 0-5min:5% acetonitrile-10% acetonitrile; 5-15min:10% acetonitrile-20% acetonitrile; 15-50min:20% acetonitrile-60% acetonitrile; 50-65min:60% acetonitrile

Preparing a reference substance solution: precisely weighing four tanshinone: the control substances of tanshinone I (T-I), cryptotanshinone (CT), dihydrotanshinone I (DT I) and tanshinone IIA (T-IIA) are respectively 5mg in 6 10mL volumetric flasks, dissolved in 80% methanol respectively, and fixed to volume to 10mL, so as to obtain 500 mug/mL of control mother liquor. The mother solution is used for preparing a control working solution with proper concentration according to the requirement.

Drawing a standard curve: and (3) measuring the working solutions of the 4 reference substances under the chromatographic condition, namely preparing a series of reference substance working solutions with gradient concentration, respectively measuring the peak areas of the working solutions, carrying out linear regression by taking the sample injection quantity X (mug or ng) of each reference substance as an abscissa and the peak area Y (mug.s) as an ordinate, and drawing a tanshinone standard curve.

And (3) component content measurement: the peak area of the sample was determined according to the above chromatographic conditions, and the content of various tanshinones in the control root system C58C1 and the overexpressed SmbHLH124 root system was calculated using the external standard method.

The experimental results (FIG. 6) show that the content of dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone IIA in the root system of three hairy roots of red sage root which overexpress SmbHLH124 is increased by 3.05-3.69 times, 1.93-2.70 times, 1.68-2.13 times and 1.07-1.82 times respectively compared with C58C 1.

The above examples show that we clone the transcription factor gene SmbHLH124 from root of red sage root and construct the vector for over-expressing SmbHLH 124. The transgenic hairy root system of the SmbHLH124 is obtained by utilizing a genetic transformation method mediated by agrobacterium tumefaciens (C58C 1). Experimental results found that the fresh and dry weights of roots overexpressing SmbHLH124 were significantly increased relative to control roots. Meanwhile, the contents of main tanshinone components such as dihydrotanshinone I, cryptotanshinone, tanshinone I, tanshinone IIA and the like in root systems of the over-expressed SmbHLH124 are also obviously improved.

Sequence listing

<110> university of electronic science and technology

<120> use of the transcription factor SmbHLH124 of Salvia Miltiorrhiza in improving hairy root yield traits of Salvia Miltiorrhiza

<160> 10

<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

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atgtcgagcc gaagatcaag atcaaggcaa tctggatctt caagaatcac agatgatcaa 60

atcaacgaac tcgtttccaa gctgcaacag cttcttcccg agatgcataa taggcgctca 120

gacaagaagt cagcaaccaa agtgttgcag gagacatgca actacattag aagcttgcac 180

agagaagttg atgacttgag tgagaggctg tctgaattgc ttgaaaacgc cgacactact 240

caagctgctc ttattagaag cttacttatg cagtag 276

<210> 2

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atggtaccgg tctccgaaaa tgtcgagccg aagatcaaga t 41

<210> 3

<211> 49

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atctgcaggg tctctcgttc tgcataagta atgatctaat aagagcagc 49

<210> 4

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

agatttgcct tttcaatttc agaa 24

<210> 5

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

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agtcccccgt gttctctcca 20

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<213> Artificial sequence (Artificial Sequence)

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ctcctgacat caaactcgtc 20

<210> 7

<211> 21

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<213> Artificial sequence (Artificial Sequence)

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gctgctgtac ctctacgtcg a 21

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

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ggcaatctgg atcttcaaga atc 23

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

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tgtgcccagt catagccg 18

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<212> PRT

<213> Artificial sequence (Artificial Sequence)

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Met Ser Ser Arg Arg Ser Arg Ser Arg Gln Ser Gly Ser Ser Arg Ile

1 5 10 15

Thr Asp Asp Gln Ile Asn Glu Leu Val Ser Lys Leu Gln Gln Leu Leu

20 25 30

Pro Glu Met His Asn Arg Arg Ser Asp Lys Lys Ser Ala Thr Lys Val

35 40 45

Leu Gln Glu Thr Cys Asn Tyr Ile Arg Ser Leu His Arg Glu Val Asp

50 55 60

Asp Leu Ser Glu Arg Leu Ser Glu Leu Leu Glu Asn Ala Asp Thr Thr

65 70 75 80

Gln Ala Ala Leu Ile Arg Ser Leu Leu Met Gln

85 90

Claims

1. Use of the transcription factor SmbHLH124 or its coding DNA molecule in improving hairy root yield of Saviae Miltiorrhizae radix, wherein the hairy root yield is the biomass of hairy root and tanshinone content of hairy root.

2. The use according to claim 1, wherein the amino acid sequence of the transcription factor SmbHLH124 of Salvia Miltiorrhiza is shown in SEQ ID No. 10.

3. The use according to claim 2, wherein the nucleotide sequence of the DNA molecule encoded by the transcription factor SmbHLH124 of salvia miltiorrhiza is shown in SEQ ID No. 1.

4. The use according to claim 3, wherein the tanshinone is at least one of tanshinone i, cryptotanshinone, dihydrotanshinone i or tanshinone iia.

5. The method for improving the hairy root yield traits of the red sage root is characterized by comprising the following steps:

a. constructing an over-expression vector for expressing the SmbHLH124 coding gene;

c. b, screening and identifying the transformed hairy roots obtained in the step b, and screening hairy roots which over-express SmbHLH124 genes;

d. performing liquid culture on hairy roots over-expressing SmbHLH124 genes to obtain hairy roots with improved yield traits;

the hairy root yield traits are the biomass of hairy roots and the tanshinone content of hairy roots.

6. The method according to claim 5, wherein the amino acid sequence of the transcription factor SmbHLH124 of Salvia Miltiorrhiza is shown in SEQ ID No. 10.

7. The method according to claim 5, wherein the SmbHLH124 encoding gene has the nucleotide sequence shown in SEQ ID No. 1.

8. The method according to any one of claims 5 to 7, wherein the tanshinone is at least one of tanshinone i, cryptotanshinone, dihydrotanshinone i, or tanshinone iia.

9. The method according to claim 8, wherein: the method for transforming the red sage root by the over-expression vector in the step b can be at least one of electroporation method, gene gun method, agrobacterium-mediated method, liposome-mediated method, PEG-mediated method or virus-mediated method.