CN116179593A

CN116179593A - Establishment method of Lan Maozhuang gynostemma pentaphylla genetic transformation system

Info

Publication number: CN116179593A
Application number: CN202310336045.5A
Authority: CN
Inventors: 黄鼎; 覃艳红; 明如宏; 谭勇; 姚绍嫦; 李良波; 黄荣韶
Original assignee: Guangxi University of Chinese Medicine
Current assignee: Guangxi University of Chinese Medicine
Priority date: 2023-03-31
Filing date: 2023-03-31
Publication date: 2023-05-30

Abstract

The invention discloses a method for establishing a Lan Maozhuang gynostemma pentaphylla genetic transformation system, which comprises the following steps: 1) Pre-culturing with aseptic leaves of Gynostemma pentaphyllum as explant; 2) Transforming the expression vector plasmid containing the target gene into agrobacterium rhizogenes to prepare an invasion solution and infect an explant; 3) The explant is used for sucking surface bacterial liquid by using sterile filter paper, and inoculated to a culture medium for co-culture; 4) Washing the explant producing bacterial plaque in the co-culture process of the step 3) by using sterile water containing a bacteriostat, transferring the washed explant to a sterilization culture medium, and inducing hairy roots; 5) When the hairy roots grow to about 2-3cm, cutting and placing the hairy roots in a 6,7-V solid screening culture medium containing screening antibiotics for culture, and screening the hairy roots with kanamycin resistance. The method provided by the invention has the characteristics of readily available materials, simple operation, short period and the like, and provides technical support for the gene function research of gynostemma pentaphylla and the improvement of the content of the effective active ingredients of gynostemma pentaphylla by using a biotechnology means.

Description

Establishment method of Lan Maozhuang gynostemma pentaphylla genetic transformation system

Technical Field

The invention belongs to the technical field of plant biology. More specifically, the invention relates to a method for establishing a Lan Maozhuang gynostemma pentaphylla genetic transformation system.

Background

Gynostemma pentaphylla Gynostemma pentaphyllum (thunder.) Makino is a climbing plant of Gynostemma herb of Cucurbitaceae, and is used as a whole herb, and is originally carried in the "first aid Ben Cao", also called QILANCHEN, WUYESHEN, etc., and has multiple purposes of eating, health promotion, medicine, etc. The gynostemma pentaphylla is rich in gynostemma pentaphylla saponin, the basic chemical structure of the gynostemma pentaphylla is dammarane type tetracyclic triterpene, and the gynostemma pentaphylla is the main functional component of the gynostemma pentaphylla, and has a plurality of structures which are completely the same as or similar to those of the ginsenoside. The modern pharmacological research results show that the gynostemma pentaphylla total saponin has the functions of regulating blood fat, resisting atherosclerosis, resisting oxidization, regulating immunity and the like and can be clinically used for hyperlipidemia, fatty liver and the like. Along with the deep research of key enzyme genes in the biosynthesis pathway of the gynostemma pentaphylla saponin in recent years, the biosynthesis pathway of the gynostemma pentaphylla saponin is modified by utilizing genetic engineering, metabolic engineering and synthetic biology means, so that not only can the synthesis efficiency of the gynostemma pentaphylla saponin be improved, but also the production cost can be saved.

The hairy root genetic transformation system is a commonly used gene function verification system in plants, has the advantages of short verification period, high efficiency and relatively stable genetic performance, and can ensure that the function of candidate genes is verified quickly after the candidate genes are screened. In addition, the hairy roots have the characteristic secondary metabolic pathway of the original plants, can synthesize secondary metabolites which are the same as or similar to the original plants, can be rapidly extracted from the hairy roots in large quantity, and has unique advantages in the aspects of researching the synthesis and regulation of the secondary metabolites of the plants. For example, the ginsenoside content in hairy roots of ginseng can be remarkably improved by over-expressing Farnesyl Pyrophosphate Synthase (FPS) or Squalene Synthase (SS) genes in the hairy roots of ginseng. At present, the research on patent technology or non-patent literature related to a hairy root genetic transformation system of gynostemma pentaphylla has not been reported, so that the research on the establishment method of a Lan Maozhuang genetic transformation system of gynostemma pentaphylla is necessary, and the research progress of biosynthesis and regulation related functional genes of gynostemma pentaphylla saponin is accelerated.

Disclosure of Invention

It is an object of the present invention to solve at least the above problems and to provide at least the advantages to be described later.

The invention aims to provide a method for establishing a genetic transformation system of Lan Maozhuang gynostemma pentaphylla, which aims to establish a Lan Maozhuang gynostemma pentaphylla genetic transformation system with high genetic transformation efficiency and short period, and the obtained Lan Maozhuang gynostemma pentaphylla can be applied to functional identification of genes.

To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided a method for establishing a genetic transformation system of Lan Maozhuang gynostemma pentaphylla, comprising the steps of:

1) Explant sterilization and preculture: taking gynostemma pentaphylla leaves, sterilizing to obtain sterile gynostemma pentaphylla leaves, placing the sterile gynostemma pentaphylla leaves in an MS solid culture medium containing acetosyringone after wounds are generated on the sterile gynostemma pentaphylla leaves, and performing dark culture;

2) Preparing agrobacterium rhizogenes invasion solution: transforming the expression vector plasmid containing the target gene into agrobacterium rhizogenes to prepare agrobacterium rhizogenes invasion solution;

3) Infection: the prepared agrobacterium rhizogenes invasion solution is used for dip-dyeing the pre-cultured explant in the step 1), and vibration is carried out to ensure that the explant is fully contacted with the agrobacterium rhizogenes;

4) Co-cultivation: after the dip dyeing is finished, taking out the explant, sucking the surface bacterial liquid by using sterile filter paper, and inoculating the surface bacterial liquid to a solid MS culture medium containing acetosyringone for dark culture;

5) And (3) sterilizing and culturing: washing the explant subjected to dark culture in the step 4) by using a washing liquid containing a bacteriostat, transferring the washing liquid to a degerming culture medium, and performing dark culture until the explant grows hairy roots;

6) Screening of positive hairy roots: the hairy roots are cut off and placed in a solid screening culture medium for culture, and the hairy roots with kanamycin resistance are screened.

Preferably, in step 1), acetosyringone concentration is 80-120. Mu.M, the culture temperature is 22-28 ℃, and the culture time is 2-3 d.

Preferably, the method for sterilizing the explant in step 1) comprises the following steps: sterilizing with 75% alcohol for 20-30s, sterilizing with 2% sodium hypochlorite for 10-15min, and cleaning with sterile water for 3-5 times.

Preferably, in step 1), the method of creating a wound on a sterile gynostemma pentaphylla leaf is cutting the leaf into small pieces of 0.8-1.0 cm x 0.8-1.0 cm.

Preferably, in step 2), the method for preparing the dip dyeing liquid comprises the following steps:

a) Transforming the expression vector plasmid containing the target gene into agrobacterium rhizogenes by an electrotransformation method;

b) In a sterile operation table, the agrobacterium rhizogenes transformed in the step a) is picked by an inoculating loop, streaked to an LB solid culture medium containing 40-60 mg/L spectinomycin, and inversely cultured for 48-72 hours at the temperature of 25-30 ℃;

c) Selecting single colony, inoculating the single colony into 1.0mL LB liquid culture medium containing 40-60 mg/L spectinomycin, culturing for 16-18 h at the temperature of 28-30 ℃ at the speed of 180-220 r/min;

d) Taking the bacterial liquid cultured in the step c) to be inoculated into 50mL of LB liquid culture medium containing 40-60 mg/L spectinomycin according to the inoculation amount of 0.8-1%, and carrying out shaking culture at 28-30 ℃ and 180-220 r/min until the bacterial liquid concentration reaches OD=0.8-1.0;

e) Centrifuging the bacterial liquid cultured in the step d) at 8000r/min for 5min, and pouring out the supernatant to collect bacterial cells; and (3) re-suspending the agrobacterium tumefaciens by using the MS liquid culture medium until the OD600 = 0.8-1.0, and adding acetosyringone to obtain the agrobacterium rhizogenes invasion solution with the final concentration of 100 mu M.

Preferably, the expression vector plasmid is pK7WG2D-GpbHLH4.

Preferably, the sequence of the target gene is shown as SEQ ID No. 1.

Preferably, in the step 3), the oscillating rotation speed is 80-100 r/min, the oscillating time is 10-30 min, and the temperature is 25-30 ℃.

Preferably, in step 4), the acetosyringone concentration is 100. Mu.M, the dark culture temperature is 25-28 ℃, and the dark culture time is 48 hours.

Preferably, in step 5), the washing liquid is washed 3-5 times, the dark culture time is 14 days, and the length of hairy roots is 2-3cm.

The invention at least comprises the following beneficial effects:

firstly, the invention has the advantages of easy material obtaining, simple operation and short period, can obtain enough hairy roots in 8 weeks, and the obtained hairy roots can be applied to the gene function research of gynostemma pentaphylla, including the detection of the expression quantity of the transformed genes; and detecting the expression quantity of other target genes, detecting the content of the gypenoside substances, and the like.

Secondly, the Lan Maozhuang genetic transformation system of the gynostemma pentaphylla established by the invention confirms the type of explants, agrobacterium strain, infection time, hairy root culture conditions and the like suitable for Lan Maozhuang genetic transformation of the gynostemma pentaphylla, and the establishment of the system accelerates the research progress of gynostemma pentaphylla molecular biology

Thirdly, the transcription factor GpbHLH4 gene fragment provided by the invention has the advantage that the overexpression of the transcription factor GpbHLH4 gene fragment in the Lan Maozhuang roots of gynostemma pentaphylla can obviously increase the content of gynostemma pentaphylla saponin in hairy roots.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a diagram of explant preculture; wherein a is the preculture of the leaf explant; b is the stem explant preculture;

FIG. 2 is an electrophoretogram of PCR amplified transcription factor GpbHLH 4;

FIG. 3 is a map of vector pK7WG 2D;

FIG. 4 is a PCR electrophoretogram of pK7WG2D-GpbHLH4 recombinant plasmid colonies;

FIG. 5 is a diagram of dead explants;

FIG. 6 leaf explants at different culture stages; wherein a is a rooting line induced by culturing for 10 days after infection of agrobacterium;

b is a rooting line induced by culturing for 20d after infection of agrobacterium;

FIG. 7 is a morphological feature of hairy root system after infection of gynostemma pentaphylla leaves by Agrobacterium rhizogenes K599;

FIG. 8 is a diagram showing the electrophoresis of the rolB and rolC genes of Ri plasmid in hairy root, wherein, 1-3: transgenic hairy root system; c (-). Root systems of tissue culture seedlings; c (+) agrobacterium rhizogenes K599; m is Marker;

FIG. 9 is an electrophoretogram of the gene of interest GpbHLH4 in the hairy root;

FIG. 10 shows the content of several gypenosides in Lan Maozhuang pairs of gynostemma pentaphylla over-expressed GpbHLH 4; wherein a is the average value of gypenoside A; b is the average value of ginsenoside Rb 1; c is the average value of ginsenoside Rb 3; d is the average value of ginsenoside Rd; EV is empty hairy root; OE1, OE2, OE3 represent over-expressed strains OE1, OE2, OE3, respectively; * Represents P < 0.01.

Detailed Description

The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.

It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The experimental methods described in the following embodiments are conventional methods unless otherwise indicated, and the reagents and materials are commercially available.

Screening of test one, optimal explants and hairy root Strain

1. Selection of explants

The gynostemma pentaphylla leaves and tender stem segments are used as explants.

2. Sterilization and preculture of explants

Selecting healthy gynostemma pentaphylla leaves and tender stem segments in a growth state, sterilizing the gynostemma pentaphylla leaves and tender stem segments with 70% alcohol for 20-30s, soaking the gynostemma pentaphylla leaves and tender stem segments in a 2% sodium hypochlorite solution for 10-15min, and washing the gynostemma pentaphylla leaves and tender stem segments with sterile water for 3-5 times; the sterile filter paper was blotted to dry the leaf. Cutting the leaves into small pieces of 0.8-1.0 cm multiplied by 0.8-1.0 cm by a surgical knife, gently scratching the leaves along the 45-degree direction of veins (figure 1 a), cutting the stem sections into small pieces of 1cm along the angle of 40 degrees below the tender stem sections (figure 1 b), and placing the small pieces in an MS solid culture medium containing 100 mu M acetosyringone for dark culture for 48 hours at the culture temperature of 25 ℃.

3. Selection of Agrobacterium rhizogenes

Agrobacteria MSU440 (streptomycin resistance), agrobacteria ar.qual (streptomycin and chloramphenicol resistance), agrobacteria C58C1 (streptomycin and rifampicin resistance), agrobacteria K599 (streptomycin resistance) and agrobacteria ATCC15834 were selected as the infection strains.

4. Preparation of agrobacterium rhizogenes dip-dyeing liquid

The preparation method comprises the following steps:

a) In a sterile operation table, the agrobacterium rhizogenes is picked by an inoculating loop, streaked to an LB solid culture medium containing corresponding antibiotics, and inversely cultured for 48-72 hours at the temperature of 28 ℃;

b) Picking single colony, inoculating the single colony into 1.0mL LB liquid culture medium (containing corresponding antibiotics), and culturing for 16-18 h at 220r/min and 28 ℃;

c) Inoculating the bacterial liquid cultured in the step b) into 50mL of LB liquid medium (containing corresponding antibiotics) according to the inoculum size of 0.8% -1.0%, and at the temperature of 28 ℃ and at the speed of 220r/min;

d) When the concentration OD of the bacterial liquid in the step c) is ₆₀₀ About 0.8 to 1.0, centrifuging at 8000rpm for 5min, and pouring out the supernatant;

e) Adding an equal volume of fresh MS liquid culture medium to resuspend the agrobacterium, adding 100 mu M acetosyringone, and standing for 1-2 hours at normal temperature to induce the activation of genes in the Vir region of the agrobacterium, so as to improve the infectivity and obtain the root-forming agrobacterium infection solution.

5. Infection with Agrobacterium rhizogenes

Transferring the pre-cultured explant into a prepared agrobacterium rhizogenes invasion solution, placing the solution in a shaking table, and shaking at the temperature of 28 ℃ for 10-30 min at the speed of 90r/min to ensure that the pre-cultured explant can be fully contacted with the agrobacterium rhizogenes; each treatment group infects 30 explants, respectively.

6. Co-cultivation

After infection, the bacterial liquid on the surface of the explant is sucked by sterile filter paper, and then transferred into an MS solid culture medium containing 100 mu M acetosyringone for co-culture for 2-3 d under the condition of 28 ℃ dark culture.

7. Bacteria washing

Washing the explant forming bacterial plaque in the co-culture process with sterile water (containing 300-400 mg/L carbenicillin and 150-250 mg/L timentin) for 2-3 times, and then sucking the water on the surface of the explant with sterile filter paper;

8. induction of hairy roots

The explants were transferred to 6,7-v solid medium (300 mg/L carbenicillin, 200mg/L timentin and 100. Mu.M acetosyringone) and cultured at 28℃under long sun (16 h light/8 h dark) and subcultured once every other week and gradually reduced the concentration of antibiotics until the plaques surrounding the explants had completely disappeared.

9. Root count statistics

Culturing for 20d after infection, counting the number of the root induced by different agrobacterium rhizogenes infection leaves and stem segments, and screening proper agrobacterium rhizogenes and explants for genetic transformation of gynostemma pentaphylla according to the induction rate of hairy roots. As shown in Table 1, after 20d culture, the probability of inducing rooting by using the stem segments of gynostemma pentaphylla as explants is extremely low, and all the 5 agrobacterium rhizogenes strains can induce the rooting, wherein the induction rate is 3.33% -20.00%, and the rooting rate induced by the strain K599 is the highest (20.00%). Thus, K599 was subsequently selected as an inducing strain and leaves as explants for hairy root genetic transformation of gynostemma pentaphylla.

TABLE 1 statistics of the number of root of different Agrobacterium rhizogenes infest leaves and stem segments

Genetic transformation of hairy roots

1. Selection and preculture of explants

Selecting healthy gynostemma pentaphylla leaves in a growth state, sterilizing the gynostemma pentaphylla leaves for 20-30s by using 70% alcohol, then soaking the gynostemma pentaphylla leaves in a sodium hypochlorite solution of 2% for 10-15min, and finally flushing the gynostemma pentaphylla leaves with sterile water for 3-5 times; the sterile filter paper absorbs the water of the leaf, cuts the leaf into small pieces with the size of 0.8-1.0 cm multiplied by 0.8-1.0 cm, and places the small pieces in an MS solid culture medium containing 100 mu M acetosyringone for dark culture for 48 hours at the culture temperature of 25 ℃ to obtain the precultured leaf explant. The pre-culture process can adapt the explant to in-vitro culture conditions, promote cell division, facilitate integration of exogenous DNA and improve the transformation efficiency.

2. Preparation of agrobacterium rhizogenes infection liquid

The preparation method comprises the following steps:

step one, selecting agrobacterium rhizogenes K599 as an infection strain, and converting a constructed expression vector pK7WG2D-GpbHLH4 containing a target gene into agrobacterium rhizogenes K599 by an electrotransformation method;

the construction method of the expression vector pK7WG2D-GpbHLH4 containing the target gene is as follows:

the CDS sequence of transcription factor GpbHLH4 in gynostemma pentaphylla genome is taken as reference to design specific primers SEQ.ID.NO.2 and SEQ.ID.NO.3, and the primers are synthesized by Shanghai biological company.

The nucleotide sequence of the primer is as follows:

SEQ.ID.NO.2：5’-atggctgagttggtaatatccc-3'。

SEQ.ID.NO.3：5'-ctactctaatctagaaagaagagcgatt-3'。

using gynostemma pentaphylla leaf cDNA as a template, carrying out GpbHLH4 gene fragment (SEQ. NO. 1) amplification, and a target fragment amplification system: 2X Phanta Max Buffer, 25. Mu.L; dNTP Mix (10 mmol/L), 1. Mu.L; SEQ.NO.2 (10. Mu. Mol/L), 2. Mu.L; SEQ.NO.3 (10. Mu. Mol/L), 2. Mu.L; phanta Max Super-Fidelity DNA Polymerase,1 μl; cDNA, 2. Mu.L; ddH2O,17 μL; and a total of 50. Mu.L. The amplification procedure was: 95 ℃ for 3min;94 ℃,30s,60 ℃,30s,72 ℃,1min, 32 cycles; preserving at constant temperature of 72 deg.C for 10min and 4 deg.C. After PCR amplification, the PCR products were electrophoretically detected on a 1% agarose gel, and the results are shown in FIG. 2. As can be seen from FIG. 2, the size of the target fragment obtained by PCR amplification was 1100-1200 bp, which matches the size of the GpbHLH4 gene. The target fragment was recovered using the norpraise company gel recovery kit.

(II) construction of intermediate vector pDOR 221-GpbHLH4

attB sites were added to both sides of the CDS sequence of GpbHLH4 by two rounds of PCR reactions, allowing attB sites to be placed on both ends of the fragment to form an attB-GpbHLH4 recombinant PCR product. PCR amplification system: 2X Phanta Max Buffer, 25. Mu.L; dNTP Mix (10 mmol/L), 1. Mu.L; primer (10. Mu. Mol/L) and 2. Mu.L; downstream primer (10. Mu. Mol/L), 2. Mu.L; phanta Max Super-Fidelity DNA Polymerase,1 μl; cDNA, 2. Mu.L; ddH2O,17 μL; and a total of 50. Mu.L. The PCR amplification procedure was: 95 ℃ for 3min;94 ℃ for 30s;60 ℃ for 30s;72 ℃ for 1min;32 cycles; preserving at constant temperature of 72 deg.C for 10min and 4 deg.C.

The nucleotide sequence of the first round PCR primers is shown below:

GpbHLH4-F(SEQ.ID.NO.4)：5’-aaaaagcaggctccatggctgagttggtaatatccc-3'。

GpbHLH4-R(SEQ.ID.NO.5)：5'-agaaagctgggttctactctaatctagaaagaagagcgatt-3'。

the nucleotide sequence of the second round PCR primer is as follows:

Adapter attB1(SEQ.ID.NO.6):5’-ggggacaagtttgtacaaaaaagcaggct-3'。

Adapter attB2(SEQ.ID.NO.7):5’-ggggaccactttgtacaagaaagctgggt-3'。

the PCR product was subjected to agarose gel electrophoresis and the band of interest, approximately 1200bp in size, was recovered. Preparing a BP recombination reaction system on ice: intermediate vector pDNOR221,0.6 μl; attB-GpbHLH4 recombinant PCR product, 1. Mu.L; BP clonase,0.4 μl; ddH2O, 1. Mu.L; together 3 μl. Gently sucking and beating the reaction liquid by using a liquid transfer device, and collecting the reaction liquid to the bottom of a tube by short centrifugation for reaction for 4-16h at 25 ℃; and (5) after the reaction is finished, placing the mixture on ice for cooling to obtain a recombinant product. After the connection is finished, the escherichia coli DH5 alpha transformation, colony PCR, plasmid extraction and sequencing verification are sequentially carried out. The primers used for colony PCR are as follows:

M13F(SEQ.ID.NO.8):5’-cgccagggttttcccagtcacgac-3'。

M13R(SEQ.ID.NO.9):5’-agcggataacaatttcacacagga-3'。

sequencing results show that the intermediate vector pDOR 221-GpbHLH4 is successfully constructed.

(III) overexpression vector pK7WG2D-GpbHLH4 recombination reaction

The CDS sequence of GpbHLH4 was assembled onto pK7WG2D overexpression vectors using homologous recombination by Gateway technology (vector figures are shown generally in figure 3). Preparing an LR recombination reaction system on ice: 1.0. Mu.L of intermediate vector pDNOR221-GpbHLH4 containing the fragment of interest; the overexpression vector pK7WG2D, 0.6. Mu.L; LR clonase,0.4 μl; ddH ₂ O, 1. Mu.L; together 3 μl. Gently sucking and beating the reaction liquid by using a liquid transfer device, and collecting the reaction liquid to the bottom of a tube by short centrifugation for reaction for 4-16h at 25 ℃; and (5) after the reaction is finished, placing the mixture on ice for cooling to obtain a recombinant product.

(IV) transformation of recombinant products into E.coli

E.coli DH5 alpha competent cells were thawed on ice, 3. Mu.L of the above recombinant product was added, and mixed gently and gently in an ice bath 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 the bacterial solutions were spread on LB solid medium (containing 50mg/L spectinomycin (Spe)), and after 1 hour of normal culture at 37℃the culture was inverted and cultured for 12 to 16 hours.

(fifth) identification of recombinant products

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. A20 uL system was used as follows: 2 XPCR Mix 10. Mu.L, 35S-F0.3. Mu.L, gpbHLH 4-det-R0.3. Mu.L, template 1. Mu.L, ddH2O 8.4. Mu.L.

The nucleotide sequence of the primer is as follows:

35S-F(SEQ.ID.NO.10)：5’-gacgcacaatcccactatcc-3'。

GpbHLH4-det-R(SEQ.ID.NO.11)：5'-aaagaagagcgattttgacg-3'。

the PCR procedure was: 94 ℃,10 min- & gt (94 ℃,30 s- & gt 55 ℃,30 s- & gt 72 ℃ 30 s) 32 cycles- & gt 72 ℃,5 min- & gt 4 ℃ and 10min. PCR products were electrophoretically detected in 1% agarose gel at 100V for 30min (FIG. 4). Colonies that verified positive were sent to gene company for sequencing. After sequencing is successful, preserving bacterial liquid, and extracting recombinant plasmid by using a plasmid extraction kit to obtain the expression vector plasmid pK7WG2D-GpbHLH4 containing the target gene successfully.

And secondly, picking the transformed agrobacterium rhizogenes K599 by using an inoculating loop in a sterile operation table, streaking to an LB solid medium containing 50mg/L spectinomycin, and inversely culturing at 28 ℃ for 48-72 h.

Step three, picking single colony, inoculating the single colony into 1.5mL LB liquid culture medium (containing 50mg/L spectinomycin), and culturing at 220r/min and 28 ℃ for 16-18 h.

Step four, inoculating the strain into 50mL of LB liquid medium (containing 50mg/L spectinomycin) according to the inoculum size of 0.8% -1.0%, and controlling the temperature to be 28 ℃ and 220r/min;

step five, when the concentration OD of the bacterial liquid ₆₀₀ About 0.8 to 1.0, 8000rpm, centrifuging for 5min, and pouring out the supernatant.

Step six, after re-suspending the bacteria by using an equal volume of fresh MS liquid culture medium, adding 100 mu M acetosyringone, and standing for 1-2 hours at room temperature to obtain the root-forming agrobacteria invasion solution.

3. Infection with Agrobacterium rhizogenes

Transferring the pre-cultured leaves into prepared dyeing liquid, placing the leaves in a shaking table, shaking the leaves for 10 to 30 minutes at the temperature of 28 ℃ at 90r/min, so that the pre-cultured leaves can be fully contacted with agrobacterium rhizogenes;

4. co-cultivation

After infection, the bacterial liquid on the surface of the leaf is sucked by sterile filter paper, and then transferred into an MS solid culture medium containing 100 mu M acetosyringone, and the leaf is subjected to dark culture for 2-3 d at 25 ℃. The acetosyringone is added into the co-culture medium to continuously induce the activation of the gene of the agrobacterium rhizogenes Vir region, promote the integration of exogenous genes and improve the conversion efficiency.

5. Bacteria washing

The explant forming the bacterial plaque in the co-culture process is washed for 2 to 3 times by sterile water (containing 300 to 400mg/L of carbenicillin and 150 to 250mg/L of timentin), and then the water on the surface of the stem segment is sucked by sterile filter paper.

6. Induction of hairy roots

The explants were transferred to 6,7-V solid medium (containing 300mg/L carbenicillin, 200mg/L timentin and 100. Mu.M acetosyringone), cultured at 28℃under long sun (16 h light/10 h dark) and subcultured once every other week and gradually reduced the concentration of the antibiotic until the plaques surrounding the stem were completely disappeared to prevent the proliferation of Agrobacterium resulting in death of the explants (FIG. 5). The gynostemma pentaphylla leaves are infected by agrobacterium rhizogenes K599, and are cultivated for 10 days after infection, and the wound parts of the leaves have the typical characteristics of growing out hair roots, multi-fluff, negative geotropism and the like (figure 6-a); after 20d cultivation, the induced hairy root system showed rapid elongation and thickening in the medium, with typical negative geotropism (FIG. 6-b).

7. Screening of Positive hairy roots

When the induced hairy roots grow to about 2-3cm, the hairy roots are cut off and transferred to a 6,7-V solid culture medium containing screening antibiotics (30 mg/L kanamycin), and are subjected to dark culture at 28 ℃, and the transgenic hairy root systems with kanamycin resistance, which have higher growth speed and more branches, are obtained in the culture process. As shown in FIG. 7, the root system obtained by screening has typical characteristics of multiple branches, rapid growth, negative geotropic property and the like.

8. Detection and transformation efficiency of Positive hairy root

Randomly picking 3 transgenic hairy root systems which have kanamycin resistance, high growth speed and more branches, taking the roots of tissue culture seedlings as negative control and agrobacterium rhizogenes K599 as positive control. The hairy roots were identified from the rolB and rolC genes on pRi2659 plasmid in Agrobacterium rhizogenes K599, and the target gene GpbHLH4 was detected by designing primers between the 35S promoter and the target gene GpbHLH4, and the detection primers are shown in Table 2. The PCR reaction system is as follows: 2 XTaq Master Mix 25. Mu.L, 2. Mu.L each of the upstream and downstream primers, 1. Mu.L of template DNA, ddH ₂ O20. Mu.L. The reaction procedure is: 3min at 95 ℃;95 ℃ for 30s,60 ℃ for 30s,72 ℃ for 60s,33 cycles; and at 72℃for 10min.

TABLE 2 PCR detection primers for positive hairy roots

The PCR detection result shows that: the rolB and rolC genes can be detected in the 3 induction root systems, and the efficiency is 100%; the fact that the root of the tissue culture seedling does not detect the band and the rolB and rolC genes can be detected in the rooting system shows that the rooting system is hairy root induced by agrobacterium rhizogenes K599 (figure 8). The detection result of the target gene shows that the target gene GpbHLH4 can be detected in 2 hairy root lines, which indicates that the exogenous target gene GpbHLH4 is successfully integrated into Lan Maozhuang strands through the mediation of agrobacterium rhizogenes K599, and the conversion efficiency is about 66.6 percent (figure 9).

Test three, determination of content of gypenoside component

1. Extraction of gynosaponin from Lan Maozhuang gynostemma pentaphylla

Extracting Lan Maozhuang herba Gynostemmatis with 70% ethanol as solvent under reflux for 2 times at a feed liquid ratio of 1:8 for 1.5 hr each time to obtain herba Gynostemmatis total extract. HPD400 type macroporous resin with maximum loading of 2 g.mL is selected ^-1 After the adsorption is finished, removing impurities by using 2BV of water and 3BV of 15% ethanol respectively, eluting by using 5BV of 70% ethanol, concentrating and evaporating to dryness to obtain the product. Re-dissolving with 10mL of 1:1 acetonitrile-water, filtering with a 0.45 μm microporous filter membrane, and collecting the subsequent filtrate to obtain the total saponin sample solution.

2. Detection of

The ultra-high performance liquid chromatography conditions are as follows, ACQUITY UPLC BEH C ₁₈ Chromatographic column (2.1 mm. Times.100 mm,1.7 μm), mobile phase A was acetonitrile, mobile phase B was 0.1% aqueous formic acid, gradient elution: 0-3 min, 30-35% of phase A; 3-8 min, 35-45% of phase A; 8-12 min, 45-60% of phase A; 12-15 min, 60-80% of phase A. The flow rate is 0.4mL/min; the sample injection volume is 2 mu L; the scanning range of the PDA detector is 200-400 nm.

The mass spectrum conditions are as follows, ESI ion source, negative ion mode scanning, capillary voltage of 2kV, taper hole voltage of 40V, ion source temperature of 120 ℃, desolvation gas flow rate of 600L/h, desolvation gas temperature of 400 ℃, taper hole gas flow rate of 50L/h, parent ion collision energy of 6eV, fragment ion collision energy of 20-50 eV, mass scanning range m/z of 100-2000 and scanning time of 0.2s.

3. Analysis of gypenoside content

Qualitative analysis is carried out on the control substance and the total saponin sample solution by adopting a positive ion multi-reaction monitoring (MRM) quantitative mode; and determining the sample injection peak area of the total saponin sample solution, and calculating the content according to a standard curve.

The experimental results (FIG. 10) show that the content of gypenoside A, ginsenoside Rb1, ginsenoside Rb3 and ginsenoside Rd in the Lan Maozhuang root systems of three gypenosides over-expressing GpbHLH4 is increased by 1.63-2.00 times, 2.34-2.60 times, 2.39-2.66 times and 2.82-3.31 times respectively compared with EV.

The above examples show that we clone the transcription factor gene GpbHLH4 from gynostemma pentaphylla leaves and construct the expression vector plasmid pK7WG2D-GpbHLH4 containing the target gene GpbHLH4. The transgenic hairy root system over-expressing GpbHLH4 is obtained by utilizing a genetic transformation method mediated by agrobacterium tumefaciens (K599). Experimental results show that compared with a control root system, the expression level of the key enzyme gene of the gypenoside synthesis path in the root system over-expressing GpbHLH4 is obviously improved. Meanwhile, the contents of main gypenoside components such as gypenoside A, ginsenoside Rb1, ginsenoside Rb3, ginsenoside Rd and the like in the root system of the over-expressed GpbHLH4 are also obviously improved.

Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.

Claims

1. The method for establishing the Lan Maozhuang gynostemma pentaphylla genetic transformation system is characterized by comprising the following steps of:

2. The method for constructing a genetic transformation system of gynostemma pentaphylla Lan Maozhuang according to claim 1, wherein in the step 1), the acetosyringone concentration is 80-120 μm, the culture temperature is 22-28 ℃ and the culture time is 2-3 d.

3. The method for establishing a Lan Maozhuang genetic transformation system of gynostemma pentaphylla according to claim 1, wherein the disinfection method of the explant in the step 1) is as follows: sterilizing with 75% alcohol for 20-30s, sterilizing with 2-3% sodium hypochlorite for 10-15min, and cleaning with sterile water for 3-5 times.

4. The method of constructing a genetic transformation system of gynostemma pentaphylla Lan Maozhuang according to claim 1, wherein in the step 1), the wound is formed on the leaves of the aseptic gynostemma pentaphylla by cutting the leaves into small pieces of 0.8-1.0 cm x 0.8-1.0 cm.

5. The method for establishing a Lan Maozhuang gynostemma pentaphylla genetic transformation system according to claim 1, wherein in the step 2), the method for preparing the dyeing solution comprises the following steps:

a) Transforming the expression vector plasmid containing the target gene into K599 agrobacterium rhizogenes by an electrotransformation method;

6. The method for establishing a genetic transformation system of gynostemma pentaphylla Lan Maozhuang according to claim 1, wherein the expression vector plasmid is pK7WG2D-GpbHLH4.

7. The method for establishing a Lan Maozhuang gynostemma pentaphylla genetic transformation system according to claim 1, wherein the sequence of the target gene is shown in SEQ ID No. 1.

8. The method for constructing a genetic transformation system of gynostemma pentaphylla Lan Maozhuang according to claim 1, wherein in the step 3), the oscillating speed is 80-100 r/min, the oscillating time is 10-30 min, and the temperature is 25-30 ℃.

9. The method for constructing a genetic transformation system of gynostemma pentaphylla Lan Maozhuang according to claim 1, wherein in the step 4), the concentration of acetosyringone is 100 μm, the dark culture temperature is 25-28 ℃, and the dark culture time is 48h.

10. The method for establishing a genetic transformation system of gynostemma pentaphylla Lan Maozhuang root according to claim 1, wherein in the step 5), the washing liquid is washed 3-5 times, the dark culture time is 14 days, and the length of hairy roots is 2-3cm.