Method for Establishing Genetic Transformation System of Hairy Roots of Gynostemma pentaphyllum

Abstract

The invention discloses a method for establishing a genetic transformation system of hairy roots of Gynostemma pentaphyllum, which comprises the following steps: 1) pre-cultivating with sterile leaves of Gynostemma pentaphyllum as explants; 2) transforming the expression vector plasmid containing the target gene into Agrobacterium rhizogenes, preparing an infection solution and infecting the explants; 3) the explants are blotted with sterile filter paper to dry the surface bacterial solution, inoculated on a culture medium for co-cultivation; 4) the explants producing bacterial plaques in step 3) during co-cultivation are washed with sterile water containing an antibacterial agent, transferred to a sterilization culture medium, and hairy roots are induced; 5) when the hairy roots grow to about 2 to 3 cm, they are cut off and placed in a 6,7-V solid screening culture medium containing screening antibiotics for cultivation, and hairy roots with kanamycin resistance are screened out. The method established by the present invention has the characteristics of easy material collection, simple operation, short cycle, etc., and provides technical support for the research on the gene function of Gynostemma pentaphyllum and the use of biotechnology to improve the content of the active ingredients of Gynostemma pentaphyllum.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology. More specifically, the invention relates to a method for establishing a genetic transformation system of gynostemma pentaphyllum hairy roots. Background Art

[0002] Gynostemma pentaphyllum (Thunb.) Makino is a herbaceous climbing plant of the genus Gynostemma in the Cucurbitaceae family. The whole plant is used as medicine. It was first recorded in the "Jiuhuang Bencao". It is also known as Qiyedan ​​and Wuyeshen. It has many uses such as food, health care, and medicine. Gynostemma pentaphyllum is rich in gypenosides. Its basic chemical structure is a dammarane-type tetracyclic triterpene. Many of them are exactly the same or similar to ginsenosides. They are the main active ingredients of Gynostemma pentaphyllum. Modern pharmacological research results show that total gypenosides have the effects of regulating blood lipids, anti-atherosclerosis, anti-oxidation, and immunomodulation. They can be used clinically for hyperlipidemia and fatty liver. With the in-depth study of the key enzyme genes in the biosynthetic pathway of gypenosides in recent years, the biosynthetic pathway of gypenosides can be modified by genetic engineering, metabolic engineering, and synthetic biology, which can not only improve the synthesis efficiency of gypenosides, but also save production costs.

[0003] The hairy root genetic transformation system is a commonly used gene function verification system in plants. It has the advantages of short verification cycle, high efficiency and relatively stable genetic performance. It can ensure that after the candidate gene is screened, the candidate gene function can be verified quickly. In addition, the hairy root has the characteristic secondary metabolic pathway of the original plant, can synthesize the same or similar secondary metabolites as the original plant, and the plant secondary metabolites can be quickly and massively extracted from the hairy root, which has unique advantages in studying the synthesis and regulation of plant secondary metabolites. For example, by overexpressing the farnesyl pyrophosphate synthase (FPS) or squalene synthase (SS) genes in ginseng hairy roots, the ginsenoside content in the hairy roots can be significantly increased. At present, there is no report on the patent technology or non-patent literature related to the hairy root genetic transformation system of Gynostemma pentaphyllum. Therefore, it is necessary to study the establishment method of the hairy root genetic transformation system of Gynostemma pentaphyllum to accelerate the research process of the functional genes related to the biosynthesis and regulation of Gynostemma pentaphyllum saponins. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] An object of the present invention is to provide a method for establishing a genetic transformation system of Gynostemma pentaphyllum hairy roots, which aims to establish a genetic transformation system of Gynostemma pentaphyllum hairy roots with high genetic transformation efficiency and short cycle, and the obtained Gynostemma pentaphyllum hairy roots can be used for gene function identification.

[0006] In order to achieve these objects and other advantages of the present invention, a method for establishing a genetic transformation system of gynostemma pentaphyllum hairy roots is provided, which comprises the following steps:

[0007] 1) Explant disinfection and pre-culture: Gynostemma pentaphyllum leaves were taken and sterilized to obtain sterile Gynostemma pentaphyllum leaves. After wounds were made on the sterile Gynostemma pentaphyllum leaves, the leaves were placed in MS solid culture medium containing acetosyringone for dark culture.

[0008] 2) Preparation of Agrobacterium rhizogenes infection fluid: transforming the expression vector plasmid containing the target gene into Agrobacterium rhizogenes to prepare Agrobacterium rhizogenes infection fluid;

[0009] 3) Infection: The prepared Agrobacterium rhizogenes infection solution is infected with the explants pre-cultured in step 1), and shaken to ensure full contact between the explants and the Agrobacterium rhizogenes;

[0010] 4) Co-culture: After the infection, the explants were taken out and the bacterial liquid on the surface was dried with sterile filter paper, and then inoculated onto solid MS medium containing acetosyringone for dark culture;

[0011] 5) sterilization culture: the explants cultured in the dark in step 4) are washed with a cleaning solution containing an antibacterial agent, transferred to a sterilization culture medium, and cultured in the dark until the explants grow hairy roots;

[0012] 6) Screening of positive hairy roots: Cut the hairy roots and culture them on solid screening medium to screen out hairy roots with kanamycin resistance.

[0013] Preferably, in step 1), the concentration of acetosyringone is 80-120 μM, the culture temperature is 22-28° C., and the culture time is 2-3 days.

[0014] Preferably, the explants are disinfected in step 1) by using 75% alcohol for 20-30 seconds, 2% sodium hypochlorite for 10-15 minutes, and then washed with sterile water for 3-5 times.

[0015] Preferably, in step 1), the method for producing wounds on the sterile Gynostemma pentaphyllum leaves is to cut the leaves into small pieces of 0.8-1.0 cm×0.8-1.0 cm.

[0016] Preferably, in step 2), the method for preparing the infection solution comprises the following steps:

[0017] a) transforming the expression vector plasmid containing the target gene into Agrobacterium rhizogenes by electroporation;

[0018] b) In a sterile operating table, use an inoculation loop to pick up the Agrobacterium rhizogenes transformed in step a), streak it onto LB solid medium containing 40-60 mg / L spectinomycin, and invert and culture it at 25-30° C. for 48-72 hours;

[0019] c) Pick a single colony and inoculate it into 1.0 mL of LB liquid medium containing 40-60 mg / L spectinomycin, and culture it at 180-220 r / min and 28-30°C for 16-18 h;

[0020] d) taking the bacterial solution cultured in step c) and inoculating it into 50 mL of LB liquid culture medium containing 40-60 mg / L spectinomycin at an inoculum size of 0.8%-1%, and shaking and culturing the culture at 28-30° C. and 180-220 r / min until the bacterial solution concentration reaches OD=0.8-1.0;

[0021] e) The cultured bacterial solution of step d) was centrifuged at 8000 r / min for 5 min, and the supernatant was discarded to collect the bacterial cells; Agrobacterium was resuspended in MS liquid medium to OD600=0.8-1.0, and acetosyringone was added to a final concentration of 100 μM, which was the infection solution of Agrobacterium rhizogenes.

[0022] Preferably, the expression vector plasmid is pK7WG2D-GpbHLH4.

[0023] Preferably, the sequence of the target gene is as shown in SEQ.ID.NO.1.

[0024] Preferably, in step 3), the oscillation speed is 80-100 r / min, the oscillation time is 10-30 min, and the temperature is 25-30°C.

[0025] Preferably, in step 4), the concentration of acetosyringone is 100 μM, the dark culture temperature is 25-28° C., and the dark culture time is 48 h.

[0026] Preferably, in step 5), the cleaning solution is used for 3-5 times, the dark culture time is 14 days, and the length of the hairy roots is 2-3 cm.

[0027] The present invention has at least the following beneficial effects:

[0028] First, the present invention is easy to obtain materials, simple to operate, and has a short cycle. A sufficient amount of hairy roots can be obtained in 8 weeks. The obtained hairy roots can be used for the study of the gene function of Gynostemma pentaphyllum, including the detection of the expression amount of the transformed gene; the detection of the expression amount of other target genes and the detection of the content of Gynostemma pentaphyllum saponin substances.

[0029] Second, the genetic transformation system of Gynostemma pentaphyllum hairy roots established by the present invention clarifies the explant type, Agrobacterium strain, infection time and hairy root culture conditions suitable for genetic transformation of Gynostemma pentaphyllum hairy roots. The establishment of this system has accelerated the research progress of Gynostemma pentaphyllum molecular biology.

[0030] Third, the transcription factor GpbHLH4 gene fragment provided by the present invention can significantly increase the content of gypenoside in the hairy roots by overexpression in the hairy roots.

[0031] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Figure 1 is a diagram of explant pre-culture; wherein a is a leaf explant pre-culture; b is a stem segment explant pre-culture;

[0033] Figure 2 This is the electropherogram of PCR amplified transcription factor GpbHLH4;

[0034] Figure 3 is a map of the vector pK7WG2D;

[0035] Figure 4 This is the PCR electrophoresis diagram of the recombinant plasmid colony of pK7WG2D-GpbHLH4;

[0036] Figure 5 Figure 1 shows a dead explant.

[0037] Figure 6 Leaf explants at different culture stages; a is the root system induced after 10 days of culture after Agrobacterium infection;

[0038] b shows the root system induced by Agrobacterium infection after 20 days of culture;

[0039] Figure 7 The morphological characteristics of the hairy root system of Gynostemma pentaphyllum leaves infected with Agrobacterium rhizogenes K599;

[0040] Figure 8 To verify the electrophoresis of rolB and rolC genes of Ri plasmid in hairy roots, 1-3: transgenic hairy root root system; C(-): tissue culture seedling root system; C(+) Agrobacterium rhizogenes K599; M: Marker;

[0041] Fig. 9 To verify the electrophoresis pattern of the target gene GpbHLH4 in hairy roots;

[0042] Fig.10Figure 3 The effect of overexpression of GpbHLH4 on the contents of several gypenosides in Gynostemma pentaphyllum hairy roots; a is the average value of gypenoside A; b is the average value of ginsenoside Rb1; c is the average value of ginsenoside Rb3; d is the average value of ginsenoside Rd; EV is the empty type hairy root; OE1, OE2, OE3 represent the overexpression lines OE1, OE2, OE3, respectively; ** represents P < 0.01. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0044] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0045] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0046] Experiment 1: Screening of the most suitable explants and root-producing strains

[0047] 1. Selection of explants

[0048] The leaves and young stem segments of Gynostemma pentaphyllum were used as explants.

[0049] 2. Disinfection and pre-cultivation of explants

[0050] Select healthy leaves and young stems of Gynostemma pentaphyllum, disinfect them with 70% alcohol for 20-30 seconds, soak them in 2% sodium hypochlorite solution for 10-15 minutes, and finally rinse them with sterile water for 3-5 times; use sterile filter paper to absorb the moisture of the leaves. Use a scalpel to cut the leaves into small pieces of 0.8-1.0cm×0.8-1.0cm, and gently scratch the leaves at a 45° angle along the veins ( Figure 1 a), cut the young stem segment into 1 cm segments at a 40 degree angle ( Figure 1 b), and cultured in MS solid medium containing 100 μM acetosyringone for 48 h in the dark at 25°C.

[0051] 3. Selection of Agrobacterium rhizogenes

[0052] Agrobacterium rhizogenes MSU440 (streptomycin resistance), Agrobacterium rhizogenes Ar.Qual (streptomycin and chloramphenicol resistance), Agrobacterium rhizogenes C58C1 (streptomycin and rifampicin resistance), Agrobacterium rhizogenes K599 (streptomycin resistance) and Agrobacterium rhizogenes ATCC15834 were selected as the infecting strains.

[0053] 4. Preparation of Agrobacterium rhizogenes infection solution

[0054] The preparation method comprises the following steps:

[0055] a) In a sterile operating table, pick up the above-mentioned Agrobacterium rhizogenes with an inoculation loop, streak it onto LB solid medium containing the corresponding antibiotics, and incubate it inverted at 28°C for 48 to 72 hours;

[0056] b) Pick a single colony and inoculate it into 1.0 mL of LB liquid medium (containing the corresponding antibiotics), and culture it at 220 r / min and 28°C for 16 to 18 hours;

[0057] c) inoculating the bacterial solution cultured in step b) into 50 mL of LB liquid culture medium (containing corresponding antibiotics) at an inoculation rate of 0.8%-1.0%, 28° C., 220 r / min;

[0058] d) When the bacterial solution concentration in step c) is OD 600 When the value is about 0.8-1.0, centrifuge at 8000 rpm for 5 min and discard the supernatant;

[0059] e) adding an equal volume of fresh MS liquid culture medium to resuspend the Agrobacterium, and adding 100 μM acetosyringone, and leaving at room temperature for 1 to 2 hours to induce activation of the Vir region gene of Agrobacterium and improve the infectivity, thereby obtaining the root-inducing Agrobacterium infection solution.

[0060] 5. Infection of Agrobacterium rhizogenes

[0061] The pre-cultured explants were transferred to the prepared Agrobacterium rhizogenes infection solution, placed in a shaker, and shaken at 90 r / min and 28°C for 10 to 30 min to ensure sufficient contact between the pre-cultured explants and Agrobacterium rhizogenes; 30 explants were infected in each treatment group.

[0062] VI. Co-cultivation

[0063] After infection, the bacterial liquid on the surface of the explants was dried with sterile filter paper, and then transferred to MS solid culture medium containing 100 μM acetosyringone and co-cultured at 28°C in the dark for 2 to 3 days.

[0064] 7. Washing bacteria

[0065] The explants that formed plaques during the co-culture process were washed 2-3 times with sterile water (containing 300-400 mg / L carbenicillin and 150-250 mg / L timentin), and then the water on the surface of the explants was dried with sterile filter paper;

[0066] 8. Induction of Hairy Roots

[0067] The explants were transferred to 6,7-v solid medium (containing 300 mg / L carbenicillin, 200 mg / L timentin and 100 μM acetosyringone), cultured at 28°C under long-day conditions (16 h light / 8 h dark), subcultured every other week, and gradually reducing the concentration of antibiotics until the plaque around the explants completely disappeared.

[0068] 9. Hair root count

[0069] After infection, the number of roots induced by different rhizogenes infecting leaves and stem segments was counted after 20 days of culture. According to the induction rate of hairy roots, suitable rhizogenes and explants were selected for genetic transformation of Gynostemma pentaphyllum. As shown in Table 1, after 20 days of culture, the probability of inducing roots using stem segments of Gynostemma pentaphyllum as explants was extremely low. All five rhizogenes strains were able to induce roots, with an induction rate of 3.33% to 20.00%, among which strain K599 had the highest rooting rate (20.00%). Therefore, K599 was subsequently selected as the inducing strain, and leaves were used as explants for genetic transformation of hairy roots of Gynostemma pentaphyllum.

[0070] Table 1. Statistics of the number of roots in leaves and stems infected with different Agrobacterium rhizogenes

[0071]

[0072] Experiment 2: Genetic transformation of hairy roots

[0073] 1. Selection and pre-culture of explants

[0074] Select healthy Gynostemma pentaphyllum leaves, first disinfect them with 70% alcohol for 20-30 seconds, then soak them in 2% sodium hypochlorite solution for 10-15 minutes, and finally rinse them with sterile water for 3-5 times; use sterile filter paper to absorb the moisture of the leaves, cut the leaves into 0.8-1.0 cm × 0.8-1.0 cm small pieces, place them in MS solid culture medium containing 100 μM acetosyringone, and culture them in the dark for 48 hours at a culture temperature of 25°C to obtain pre-cultured leaf explants. The pre-culture process can adapt the explants to in vitro culture conditions, promote cell division, facilitate the integration of exogenous DNA, and improve the efficiency of transformation.

[0075] 2. Preparation of Agrobacterium rhizogenes infection solution

[0076] The preparation method comprises the following steps:

[0077] Step 1: Select Agrobacterium rhizogenes K599 as the infection strain, and transform the constructed expression vector pK7WG2D-GpbHLH4 containing the target gene into Agrobacterium rhizogenes K599 by electroporation;

[0078] Among them, the construction method of the expression vector pK7WG2D-GpbHLH4 containing the target gene is as follows:

[0079] (I) Specific primers SEQ.ID.NO.2 and SEQ.ID.NO.3 were designed based on the CDS sequence of transcription factor GpbHLH4 in the Gynostemma pentaphyllum genome and were synthesized by Shanghai Bioengineering Co., Ltd.

[0080] The nucleotide sequences of the primers are as follows:

[0081] SEQ.ID.NO.2: 5'-atggctgagttggtaatatccc-3'.

[0082] SEQ.ID.NO.3: 5'-ctactctaatctagaaagaagagcgatt-3'.

[0083] Using cDNA from Gynostemma pentaphyllum leaves as a template, the GpbHLH4 gene fragment (SEQ.NO.1) was amplified. The target fragment amplification system was as follows: 2×Phanta Max Buffer, 25μL; dNTP Mix (10mmol / L), 1μL; SEQ.NO.2 (10μmol / L), 2μL; SEQ.NO.3 (10μmol / L), 2μL; Phanta Max Super-Fidelity DNA Polymerase, 1μL; cDNA, 2μL; ddH2O, 17μL; 50μL in total. The amplification procedure was as follows: 95℃, 3min; 94℃, 30s, 60℃, 30s, 72℃, 1min, 32 cycles; 72℃, 10min, and constant temperature storage at 4℃. After PCR amplification, the PCR product was detected by electrophoresis on 1% agarose gel. If the result Figure 2 As shown. Figure 2 It can be seen that the size of the target fragment obtained by PCR amplification is between 1100 and 1200 bp, which is consistent with the size of the GpbHLH4 gene. The target fragment was recovered using the gel recovery kit of Novozymes.

[0084] (II) Construction of the intermediate vector pDNOR221-GpbHLH4

[0085] The attB sites were added to both sides of the CDS sequence of GpbHLH4 through two rounds of PCR reaction, so that the attB sites were added to both ends of the fragment to form attB-GpbHLH4 recombinant PCR products. PCR amplification system: 2×Phanta Max Buffer, 25μL; dNTP Mix (10mmol / L), 1μL; upper primer (10μmol / L), 2μL; downstream primer (10μmol / L), 2μL; PhantaMax Super-Fidelity DNA Polymerase, 1μL; cDNA, 2μL; ddH2O, 17μL; a total of 50μL. The PCR amplification program was: 95℃, 3min; 94℃, 30s; 60℃, 30s; 72℃, 1min; 32 cycles; 72℃, 10min, and constant temperature storage at 4℃.

[0086] The nucleotide sequences of the first round PCR primers are as follows:

[0087] GpbHLH4-F (SEQ. ID. NO. 4): 5'-aaaaagcaggctccatggctgagttggtaatatccc-3'.

[0088] GpbHLH4-R (SEQ. ID. NO. 5): 5'-agaaagctgggttctactctaatctagaaagaagagcgatt-3'.

[0089] The nucleotide sequences of the second round PCR primers are as follows:

[0090] Adapter attB1(SEQ.ID.NO.6):5'-ggggacaagtttgtacaaaaaagcaggct-3'.

[0091] Adapter attB2(SEQ.ID.NO.7):5'-ggggaccactttgtacaagaaagctgggt-3'.

[0092] The PCR product was subjected to agarose gel electrophoresis and the target band of about 1200bp was recovered. Prepare the BP recombination reaction system on ice: intermediate vector pDNOR221, 0.6μL; attB-GpbHLH4 recombinant PCR product, 1μL; BP clonase, 0.4μL; ddH2O, 1μL; a total of 3μL. Use a pipette to gently mix the reaction solution, centrifuge briefly to collect the reaction solution to the bottom of the tube, react at 25℃ for 4-16h; cool on ice after the reaction is completed to obtain the recombinant product. After the connection is completed, Escherichia coli DH5α transformation, colony PCR, plasmid extraction and sequencing verification are carried out in sequence. The primers used for colony PCR are as follows:

[0093] M13F(SEQ.ID.NO.8):5'-cgccagggttttcccagtcacgac-3'.

[0094] M13R (SEQ. ID. NO. 9): 5'-agcggataacaatttcacacagga-3'.

[0095] Sequencing results showed that the intermediate vector pDNOR221-GpbHLH4 was successfully constructed.

[0096] (III) Recombination reaction of overexpression vector pK7WG2D-GpbHLH4

[0097] The CDS sequence of GpbHLH4 was assembled into the pK7WG2D overexpression vector using homologous recombination using Gateway technology (see vector diagram). Figure 3 LR recombination reaction system was prepared on ice: intermediate vector pDNOR221-GpbHLH4 containing the target fragment, 1.0 μL; overexpression vector pK7WG2D, 0.6 μL; LR clonase, 0.4 μL; ddH2O, 1 μL; 3 μL in total. Use a pipette to gently mix the reaction solution, centrifuge briefly to collect the reaction solution to the bottom of the tube, react at 25°C for 4-16 hours; after the reaction is completed, cool it on ice to obtain the recombinant product.

[0098] (IV) Transformation of recombinant products into E. coli

[0099] Thaw the competent E. coli DH5α cells on ice, add 3 μL of the above recombinant product, flick to mix, and place on ice for 30 minutes. Heat shock at 42°C for 1 minute, place on ice for 2 minutes. Add 500 μL of LB liquid culture medium, incubate at 37°C, 200 rpm and shake for 45 minutes. Centrifuge at 12000 rpm for 2 minutes, discard 400 μL of supernatant, and gently blow and resuspend the bacteria with a pipette. Spread all the bacterial liquid on LB solid culture medium (containing 50 mg / L spectinomycin (Spe)), culture upright at 37°C for 1 hour, and then culture inverted for 12 to 16 hours.

[0100] (V) Identification of recombinant products

[0101] Use a sterile toothpick to pick a single clone on the LB plate, place it in 50μL ddH2O water, and take 1uL of this bacterial solution as a template for PCR amplification. Use a 20uL system, the system is as follows: 2×PCR Mix 10μL, 35S-F 0.3μL, GpbHLH4-det-R0.3μL, Template 1μL, ddH2O 8.4μL.

[0102] The nucleotide sequences of the primers are as follows:

[0103] 35S-F (SEQ. ID. NO. 10): 5'-gacgcacaatcccactatcc-3'.

[0104] GpbHLH4-det-R (SEQ. ID. NO. 11): 5'-aaagaagagcgattttgacg-3'.

[0105] The PCR program was: 94°C, 10 min → (94°C, 30 s → 55°C, 30 s → 72°C, 30 s) 32 cycles → 72°C, 5 min → 4°C, 10 min. The PCR products were detected by electrophoresis in 1% agarose gel at 100 V for 30 min ( Figure 4 ). The colonies that were verified as positive were sent to a gene company for sequencing. After successful sequencing, the bacterial solution was preserved, and the recombinant plasmid was extracted using a plasmid extraction kit to successfully obtain the expression vector plasmid pK7WG2D-GpbHLH4 containing the target gene.

[0106] Step 2: In a sterile operating table, pick up the transformed Agrobacterium rhizogenes K599 with an inoculation loop, streak onto LB solid medium containing 50 mg / L spectinomycin, and culture inverted at 28°C for 48 to 72 hours.

[0107] Step 3: Pick a single colony and inoculate it into 1.5 mL LB liquid culture medium (containing 50 mg / L spectinomycin), and culture at 220 r / min and 28°C for 16 to 18 h.

[0108] Step 4: Inoculate the culture medium into 50 mL of LB liquid culture medium (containing 50 mg / L spectinomycin) at an inoculum size of 0.8%-1.0%, 28°C, 220 r / min;

[0109] Step 5: When the bacterial solution concentration OD 600 When the concentration is about 0.8-1.0, centrifuge at 8000 rpm for 5 min and discard the supernatant.

[0110] Step 6. Resuspend the bacteria in an equal volume of fresh MS liquid culture medium, add 100 μM acetosyringone, and leave at room temperature for 1 to 2 hours to obtain the Agrobacterium rhizogenes infection solution.

[0111] 3. Infection of Agrobacterium rhizogenes

[0112] Transfer the pre-cultured leaves to the prepared infection solution, place them on a shaker, and shake at 90r / min and 28°C for 10-30min to ensure sufficient contact between the pre-cultured leaves and Agrobacterium rhizogenes;

[0113] 4. Co-cultivation

[0114] After infection, the bacterial liquid on the surface of the leaves was blotted dry with sterile filter paper, and then transferred to MS solid medium containing 100 μM acetosyringone and cultured in the dark at 25°C for 2 to 3 days. Adding acetosyringone to the co-culture medium can continuously induce the activation of the Vir region genes of Agrobacterium rhizogenes, promote the integration of exogenous genes, and improve the transformation efficiency.

[0115] 5. Washing bacteria

[0116] The explants that formed plaques during the co-cultivation process were washed 2-3 times with sterile water (containing 300-400 mg / L carbenicillin and 150-250 mg / L timentin), and then the moisture on the surface of the stem segment was absorbed with sterile filter paper.

[0117] 6. Induction of Hairy Roots

[0118] The explants were transferred to 6,7-V solid medium (containing 300 mg / L carbenicillin, 200 mg / L timentin and 100 μM acetosyringone) and cultured at 28°C under long-day conditions (16 h light / 10 h dark). The culture was subcultured every other week, and the concentration of sterilizing antibiotics was gradually reduced until the bacterial plaque around the stem segment completely disappeared to prevent the reproduction of Agrobacterium and cause the death of the explants ( Figure 5 ). The leaves of Gynostemma pentaphyllum were infected with Agrobacterium rhizogenes K599 and cultured for 10 days after infection. Hairy roots grew out from the wounds of the leaves, showing typical characteristics such as multiple hairs and negative geotropism ( Figure 6-a); After 20 days of culture, the induced hairy roots rapidly elongated and thickened on the culture medium, showing typical negative geotropism ( Figure 6 -b).

[0119] VII. Screening of positive hairy roots

[0120] When the induced hairy roots grow to about 2 to 3 cm, they are cut off and transferred to a 6,7-V solid medium containing a selection antibiotic (30 mg / L kanamycin) and cultured in the dark at 28°C. During the culture process, transgenic hairy roots with kanamycin resistance, fast growth rate, and more branches are selected. Figure 7 As shown, the screened root system exhibits typical characteristics such as multiple branches, rapid growth, and negative geotropism.

[0121] 8. Detection of positive hairy roots and transformation efficiency

[0122] Three transgenic hairy root systems with kanamycin resistance, fast growth rate and more branches were randomly selected as the detection objects, the roots of tissue culture seedlings were used as negative controls, and Agrobacterium rhizogenes K599 was used as positive controls. Hairy roots were identified based on the rolB and rolC genes on the pRi2659 plasmid in Agrobacterium rhizogenes K599. Primers were designed between the 35S promoter and the target gene GpbHLH4 to detect the target gene GpbHLH4. The detection primers are shown in Table 2. The PCR reaction system was: 2×Taq Master Mix 25μL, 2μL of upstream and downstream primers, 1μL of template DNA, and 20μL of ddH2O. The reaction program was: 95℃3min; 95℃30s, 60℃30s, 72℃60s, 33 cycles; 72℃10min.

[0123] Table 2. Primers for positive hairy root PCR detection

[0124]

[0125] The results of PCR detection showed that rolB and rolC genes could be detected in all three induced root systems, with an efficiency of 100%. No bands were detected in the roots of tissue culture seedlings, but rolB and rolC genes could be detected in the root system, indicating that the root system was hairy roots induced by Agrobacterium rhizogenes K599 ( Figure 8 ). The target gene detection results showed that the target gene GpbHLH4 could be detected in two hairy root strains, indicating that the exogenous target gene GpbHLH4 was successfully integrated into the hairy roots of Gynostemma pentaphyllum through the mediation of Agrobacterium rhizogenes K599, and the transformation efficiency was about 66.6% ( Fig. 9 ).

[0126] Test 3: Determination of Gynostemma Saponin Content

[0127] 1. Extraction of Gynostemma pentaphyllum saponins from Gynostemma pentaphyllum hairy roots

[0128] Take the hairy roots of Gynostemma pentaphyllum, use 70% ethanol as solvent, reflux extract twice at a solid-liquid ratio of 1:8, each time for 1.5 hours, to obtain the total extract of Gynostemma pentaphyllum. The maximum loading capacity of HPD400 macroporous resin is 2g·mL -1 After adsorption, remove impurities with 2BV water and 3BV 15% ethanol, then elute with 5BV 70% ethanol, concentrate and evaporate to dryness. Redissolve with 10mL 1:1 acetonitrile-water, filter through 0.45μm microporous membrane, and take the filtrate, which is the total saponin sample solution.

[0129] 2. Detection

[0130] The ultra-high performance liquid chromatography conditions are as follows: ACQUITY UPLC BEH C 18 Chromatographic column (2.1 mm × 100 mm, 1.7 μm), mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid aqueous solution, gradient elution: 0-3 min, 30%-35% phase A; 3-8 min, 35%-45% phase A; 8-12 min, 45%-60% phase A; 12-15 min, 60%-80% phase A. Flow rate is 0.4 mL / min; injection volume is 2 μL; PDA detector scanning range is 200-400 nm.

[0131] The mass spectrometry conditions were as follows: ESI ion source, negative ion mode scanning, capillary voltage of 2 kV, cone voltage of 40 V, ion source temperature of 120 °C, desolvation gas flow rate of 600 L / h, desolvation gas temperature of 400 °C, cone gas flow rate of 50 L / h, parent ion collision energy of 6 eV, fragment ion collision energy of 20-50 eV, mass scanning range of m / z 100-2000, and scanning time of 0.2 s.

[0132] 3. Analysis of Gynostemma Saponin Content

[0133] The reference substance and the total saponin sample solution were qualitatively analyzed using the positive ion multiple reaction monitoring (MRM) quantitative mode; the injection peak area of ​​the total saponin sample solution was measured, and its content was calculated based on the standard curve.

[0134] Experimental results ( Fig.10 ) showed that compared with EV, the contents of gypenoside A, ginsenoside Rb1, ginsenoside Rb3 and ginsenoside Rd in the three GpbHLH4-overexpressing Gynostemma hairy root systems increased by 1.63-2.00 times, 2.34-2.60 times, 2.39-2.66 times and 2.82-3.31 times, respectively.

[0135] The above examples show that we cloned the transcription factor gene GpbHLH4 from the leaves of Gynostemma pentaphyllum, and constructed the expression vector plasmid pK7WG2D-GpbHLH4 containing the target gene GpbHLH4. The transgenic hairy root system overexpressing GpbHLH4 was obtained by using the genetic transformation method mediated by Agrobacterium tumefaciens (K599). The experimental results showed that compared with the control root system, the expression level of the key enzyme gene of the Gynostemma pentaphyllum saponin synthesis pathway in the root system overexpressing GpbHLH4 was significantly increased. At the same time, the content of the main Gynostemma pentaphyllum saponin components such as Gynostemma pentaphyllum saponin A, Ginsenoside Rb1, Ginsenoside Rb3, Ginsenoside Rd in the root system overexpressing GpbHLH4 was also significantly increased.

[0136] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for establishing a genetic transformation system of Gynostemma pentaphyllum hairy roots, characterized in that: The following steps are involved: 1) Explant disinfection and pre-culture: Take Gynostemma pentaphyllum leaves, disinfect them to obtain sterile Gynostemma pentaphyllum leaves, and place them in MS solid culture medium containing acetosyringone after wounds are produced on the sterile Gynostemma pentaphyllum leaves for dark culture; 2) Preparation of Agrobacterium rhizogenes infection fluid: transforming the expression vector plasmid containing the target gene into Agrobacterium rhizogenes to prepare Agrobacterium rhizogenes infection fluid; the sequence of the target gene is shown in SEQ ID NO: 1; 3) Infection: The prepared Agrobacterium rhizogenes infection solution is infected with the explants pre-cultured in step 1), and the explants are shaken to ensure full contact between the explants and the Agrobacterium rhizogenes; 4) Co-culture: After the infection, remove the explants, dry the surface bacterial liquid with sterile filter paper, and inoculate them on solid MS medium containing acetosyringone for dark culture; 5) Sterilization culture: The explants cultured in the dark in step 4) are washed with a cleaning solution containing an antibacterial agent, transferred to a sterilization culture medium, and cultured in the dark until the explants grow hairy roots; 6) Screening of positive root hairs: Cut off the root hairs and place them on solid screening medium for culture to screen out the root hairs with kanamycin resistance.

2. The method for establishing the Gynostemma pentaphyllum hairy root genetic transformation system according to claim 1, wherein In step 1), the concentration of acetosyringone is 80-120 μM, the culture temperature is 22-28° C., and the culture time is 2-3 d.

3. The method for establishing the Gynostemma pentaphyllum hairy root genetic transformation system according to claim 1, wherein In step 1), the explants are disinfected using 75% alcohol for 20-30 seconds, 2-3% sodium hypochlorite for 10-15 minutes, and then washed with sterile water for 3-5 times.

4. The method for establishing the Gynostemma pentaphyllum hairy root genetic transformation system according to claim 1, characterized in that: In step 1), the method for creating wounds on sterile Gynostemma pentaphyllum leaves is to cut the leaves into small pieces of 0.8-1.0 cm×0.8-1.0 cm.

5. The method for establishing the Gynostemma pentaphyllum hairy root genetic transformation system according to claim 1, characterized in that: In step 2), the method for preparing the infection solution comprises the following steps: a) Transforming the expression vector plasmid containing the target gene into K599 Agrobacterium rhizogenes by electroporation; b) In a sterile operating table, use an inoculation loop to pick up the Agrobacterium rhizogenes transformed in step a), streak it onto LB solid medium containing 40-60 mg / L spectinomycin, and incubate it upside down at 25-30°C for 48-72 hours; c) Pick a single colony and inoculate it into 1.0 mL of LB liquid medium containing 40-60 mg / L spectinomycin, and culture at 180-220 rpm and 28-30°C for 16-18 h; d) Inoculate the cultured bacteria in step c) into 50 mL of LB liquid medium containing 40-60 mg / L spectinomycin at an inoculum size of 0.8%-1%, and culture at 28-30°C and 180-220 r / min with shaking until the bacterial concentration reaches OD = 0.8-1.0; e) The cultured bacterial solution of step d) was centrifuged at 8000 r / min for 5 min, and the supernatant was discarded to collect the bacterial cells; the Agrobacterium was resuspended in MS liquid medium to OD600 = 0.8-1.0, and acetosyringone was added to a final concentration of 100 μM, which was the infection solution of Agrobacterium rhizogenes.

6. The method for establishing the Gynostemma pentaphyllum hairy root genetic transformation system according to claim 1, characterized in that: In step 3), the oscillation speed is 80-100 r / min, the oscillation time is 10-30 min, and the temperature is 25-30°C.

7. The method for establishing the Gynostemma pentaphyllum hairy root genetic transformation system according to claim 1, characterized in that: In step 4), the concentration of acetosyringone is 100 μM, the dark culture temperature is 25-28°C, and the dark culture time is 48 h.

8. The method for establishing the Gynostemma pentaphyllum hairy root genetic transformation system according to claim 1, characterized in that: In step 5), the washing solution is used for 3-5 times, the dark culture time is 14 days, and the length of the hairy roots is 2-3 cm.

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