CN110819643A - Ginseng PgCYP309 gene and application thereof - Google Patents

Abstract

The invention discloses a ginseng PgCYP309 gene, the nucleotide sequence of which is shown in a sequence table SEQ ID NO. 1; an over-expression vector, which is inserted with a gene shown in a sequence table SEQ NO. 1; the application of the ginseng PgCYP309 gene in improving the content of ginsenoside in hairy roots of ginseng; performing PCR amplification by taking the cDNA of the fresh ginseng root as a template to obtain a PgCYP309 gene, and then constructing a pBI121-PgCYP309 overexpression vector to induce a ginseng explant to generate ginseng hairy roots; the content detection of transgenic ginseng hairy root saponin shows that the overexpression of PgCYP309 can promote the synthesis of ginsenoside.

Description

Ginseng PgCYP309 gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a ginseng PgCYP309 gene and application thereof.

Background

Ginseng (A, B)Panax ginsengC.A. Mey) is Panax of Araliaceae (Araliaceae)Panax) Perennial herb is a rare health care product, is sweet, slightly bitter and slightly warm in taste by using roots and rhizomes thereof as a medicine, enters spleen, liver, heart and kidney channels, and has the effects of greatly reinforcing primordial qi, recovering pulse, relieving depletion, tonifying liver, benefiting spleen, promoting production of body fluid, nourishing blood, calming nerves, benefiting intelligence and the like. The chemical composition of ginseng is very complex, and mainly contains various active ingredients such as saponins, polypeptides, saccharides and the like, and the main medicinal ingredient of the ginseng is ginsenoside. The ginseng has long growth period, strict requirements on the growth environment, less wild resource storage and a plurality of problems in artificial cultivation, so that the ginseng is urgently required to be developed and protected by using a bioengineering method. With Agrobacterium rhizogenes (A), (B)Agrobacterium rhizogenes) The hairy roots of ginseng generated by inducing the ginseng explant have the advantages of high growth speed, hormone autotrophy, stable hereditary property, rich secondary metabolites, high saponin content and the like, and are applied to bioreactors for producing the secondary metabolites of ginseng and analysis materials of ginseng functional genomics.

Cytochrome P450 (CYP 450) is the largest enzyme family in plant metabolism, has wide catalytic activity, and participates in the synthesis of primary and secondary metabolites of plants such as phenylpropanoids, alkaloids, terpenes and the like. CYP450 is involved in the modification of a plant triterpene skeleton, is a key gene for triterpene biosynthesis, is an important reason for causing triterpene aglycone diversity, and plays a key role in the triterpene diversity. At present, the prediction of the function of CYP450 still has certain difficulty, and thus further research on the cloning and function of CYP450 is required. The ginsenoside belongs to triterpenoids, the CYP450 gene in the ginseng is cloned and verified, the biosynthesis pathway of the triterpenoid saponin is analyzed, the foundation is laid for realizing homologous production of the ginsenoside through gene regulation, and the method has important scientific significance and application value.

Disclosure of Invention

The invention aims to provide a ginseng PgCYP309 gene and application thereof in order to solve the problems of low ginsenoside content in ginseng and homologous production of ginsenoside.

The nucleotide sequence of the ginseng PgCYP309 gene is shown in a sequence table SEQ ID NO. 1.

A super expression vector is characterized in that a gene shown in a sequence table SEQ NO.1 is inserted into a plant vector.

The application of the ginseng PgCYP309 gene in improving the content of ginsenoside in hairy roots of ginseng.

The invention provides a ginseng PgCYP309 gene, the nucleotide sequence of which is shown in a sequence table SEQ ID NO. 1; an over-expression vector, which is inserted with a gene shown in a sequence table SEQ NO. 1; the application of the ginseng PgCYP309 gene in improving the content of ginsenoside in hairy roots of ginseng; performing PCR amplification by taking the cDNA of the fresh ginseng root as a template to obtain a PgCYP309 gene, and then constructing a pBI121-PgCYP309 overexpression vector to induce a ginseng explant to generate ginseng hairy roots; the content detection of transgenic ginseng hairy root saponin shows that the overexpression of PgCYP309 can promote the synthesis of ginsenoside.

Drawings

FIG. 1 PgCYP309 gene cloning results;

FIG. 2 shows PCR results of Escherichia coli liquid;

FIG. 3 is the restriction enzyme digestion verification of the plant expression vector;

FIG. 4 shows the PCR verification result of Agrobacterium tumefaciens liquid;

FIG. 5 Ginseng hairy root induction;

FIG. 6 shows the results of PCR detection of the RolC gene and the three-stage method; 1-3, 5-7, 9-11, 13-15 are the detection results of 3 sections of the PgCYP309 gene transgenic ginseng hairy root DNA, 17-19 are the detection results of three sections of the wild type ginseng aseptic seedling DNA, 21-23 are the detection results of three sections of pBI121-PgCYP309 plasmid, 4, 8, 12 and 16 are the detection results of RolC of the PgCYP309 gene transgenic ginseng hairy root DNA, and 20 are the detection results of RolC of the wild type ginseng aseptic seedling DNA;

FIG. 7 shows the results of the total saponin content measurement of hairy roots of Panax ginseng; wild type hairy roots, positive-3, positive-63 and positive-64 hairy roots transferred with PgCYP309 gene.

Detailed Description

Example 1 cloning of Ginseng PgCYP309 Gene

1. Total RNA extraction and cDNA synthesis of ginseng root

The fresh root of the ginseng is obtained: the ginseng root of "big horse teeth" in the four-year-old haw period is collected from Jilin province Ji' an city.

Extracting total RNA of fresh roots of 4-year-old ginseng by TRIZOL Plus, and taking 1 mu g of total RNA to perform reverse transcription to obtain a cDNA template;

cloning of full-Length cDNA of PgCYP309 Gene

Primer PgCYP309-F and Primer PgCYP309-R of PgCYP309 gene added with XbaI and SmaI enzyme cutting sites are designed by using Primer5 software:

PgCYP309-F：5’-GCTCTAGAATGGAAACCCTTGATGAT-3’；

PgCYP309-R：5’-TCCCCCGGGTTAATAGATTGGACATAAC-3’；

PCR amplification is carried out by taking cDNA of fresh roots of 4-year-old ginseng as a template, thereby obtaining a full-length sequence of PgCYP309 gene (figure 1), the obtained full-length cDNA of the PgCYP309 gene is recovered by glue, the recovered product is connected with a T carrier at 16 ℃ overnight, a connecting liquid is used for transforming escherichia coli DH5 α competent cells, transformants are screened on a resistant plate containing 100 mg/L ampicillin, bacterial liquid PCR (figure 2) is carried out after single-clone bacteria shaking is selected, a positive clone is taken and sent to a sequencing company for sequencing, the length of the sequence of the PgCYP309 gene is 1563bp, and the base sequence is shown as SEQ ID NO.1 of a sequence table.

Example 2 construction of PgCYP309 Gene plant overexpression vector

Extracting plasmid DNA from the single clone with correct sequencing, carrying out double enzyme digestion by XbaI and SmaI, simultaneously carrying out double enzyme digestion on a plant expression vector pBI121 by XbaI and SmaI, respectively carrying out agarose gel electrophoresis on enzyme digestion products, recovering large fragments of the pBI121 and a PgCYP309 gene fragment after enzyme digestion, adding T4 ligase for connecting overnight at 16 ℃, transforming a connecting solution into escherichia coli DH5 α competent cells, screening transformants on a resistance plate containing 50 mg/L kanamycin, selecting single clone, carrying out bacterium liquid PCR identification and enzyme digestion identification (figure 3), wherein target bands indicate that the construction of the overexpression vector is successful, and the gene is named as pBI121-PgCYP 309.

Example 3 Agrobacterium-mediated transformation of PgCYP309 Gene

Agrobacterium rhizogenes C58C1 was transformed by freeze-thaw method, PCR was performed (FIG. 4), and glycerol was added to preserve positive transformants in a freezer at-80 ℃.

The constructed pBI121-PgCYP309 is transformed into a ginseng explant by an agrobacterium-mediated method, and the specific implementation scheme is as follows:

1) preparing an explant: carrying out sand storage lamination treatment on ginseng seeds, taking cracked seeds, removing seed shells, washing with running water to remove sandy soil on the surfaces of the seeds, disinfecting with 75% alcohol for 1 min, washing with sterile water for 3 times, disinfecting with 5% sodium hypochlorite for 10min, washing with sterile water for 3 times, inoculating to an MS culture medium, and carrying out 16-hour illumination/8-hour dark culture for 14-21d at 22 ℃;

2) induction of hairy roots of ginseng

a) Activation of strains: streaking the strain C58C1 carrying pBI121-PgCYP309 on a solid YEP plate, culturing at 28 deg.C for 2-4 days in dark, and placing in a refrigerator at 4 deg.C for use after single strain grows out;

b) pre-culturing: cutting petiole and young root of aseptic seedling of Ginseng radix into 0.5-1 cm long segments, placing in solid MS culture medium, and culturing at 22 deg.C under 16h illumination/8 h dark condition for 3 d;

c) dip dyeing and co-culturing: taking plate activated C58C1 strain carrying pBI121-PgCYP309, selecting single colony, inoculating to liquid YEP culture medium, shake culturing at 28 deg.C and 180rpm until the growth concentration of bacterial liquid reaches OD₆₀₀When =0.6, the cells were collected and resuspended to OD in 1/2 MS liquid medium supplemented with 200. mu. mol/L of As₆₀₀=0.6, placing the resuspended bacterial liquid in a shaking table at 28 ℃ and 180rpm for culturing for 15-30 min, immersing the pre-cultured petioles and radicles in the resuspended bacterial liquid, placing the solution on a shaking table at 22 ℃ and 100 rpm for infection for 15 min, sterilizing filter paper to suck dry the bacterial liquid on the surface of the material, inoculating to 1/2 MS culture medium, placing the culture medium in a dark condition at 22 ℃ for co-culturing for 3 d;

d) and (3) degerming, screening and culturing: sucking dry the bacterial liquid on the surface of the co-culture explant by using sterile filter paper, then placing the explant on a solid 1/2 MS + 200 mg/L Cef + 5 mg/L Hyg, culturing at 22 ℃ in the dark, replacing the culture medium every 14 d, subculturing for 4 times, and growing out the hairy roots (figure 5);

3) PCR molecular detection

PCR detection is carried out on the rolC gene of the Ri plasmid T-DNA region and the T-DNA region three-section method of the pBI121-PgCYP309 vector (the left arm of the vector + the 5 'end of the gene, the middle section of the gene, the 3' end of the gene + the right arm of the vector), primers are designed as follows, and the detection result is shown in figure 6, which indicates that the ginseng hairy root of the over-expression PgCYP309 gene is successfully obtained.

rolC-F：5’-ATGGCTGAAGACGACTTGTGTTC-3’;

rolC-R：5’-TTAGCCGATTGCAAACTT-3’;

pBI121- PgCYP309-LF：5’-GGAGCATCGTGGAAAAAGAAG-3’；

PgCYP309-5’R：5’-GAGAACAAAGAGTGAGAGGAGAGGG-3’；

PgCYP309-MF：5’-TAAGATGCGAAAAATGCTGCC-3’；

PgCYP309-MR：5’-TCCCCCTCACCCATAAACTTGCCAC-3’；

PgCYP309-3’F：5’-TGAGGGAGTCTACCAGGAGC-3’；

pBI121- PgCYP309-RR：5’-ACTCTAATCATAAAAACCCATCTC-3’；

Example 4 detection of transgenic Ginseng hairy root saponins content

1. Preparation of test solution

Wrapping appropriate amount of Ginseng radix hairy root powder with filter paper, soaking in 40 mL methanol overnight, and ultrasonically extracting for 30 min; placing the filter paper bag and the methanol extract in a Soxhlet extractor, adding 60 mL of methanol, extracting at 90 deg.C, collecting the extract every 12 h, adding 100 mL of methanol, and repeating for 3 times. Evaporating the extractive solution to dryness, adding 10 mL of chromatographic methanol for redissolving, and filtering with 0.45 μm microporous membrane to obtain sample solution.

2. Drawing of standard curve

Weighing a proper amount of Re standard substance to prepare a standard solution with a final concentration of 1.068 mg/mL, respectively putting 30 muL, 60 muL, 90 muL, 120 muL, 150 muL and 180 muL in a test tube with a plug and a scale of 10 mL, evaporating to dryness at 60 ℃, adding 0.2 mL of 5% vanillin glacial acetic acid solution and 0.8 mL of perchloric acid, shaking uniformly, putting the test tube in a 60 ℃ constant-temperature water bath kettle, heating for 15 min, cooling for 2 min by running water, adding 5 mL glacial acetic acid, fully shaking uniformly, taking a follow-up reagent as a blank control, measuring the absorbance value at 556 nm wavelength, and drawing a standard curve by taking the absorbance value as an X axis and the saponin content as a Y axis.

3. Determination of total saponin content in sample

Precisely sucking a test solution 0.1-10 mL into a test tube with a plug scale, measuring the absorbance value according to the method, making three samples in parallel, repeatedly measuring each sample for three times, and calculating the saponin content, wherein the result is shown in figure 7, and the total saponin content of the wild hairy roots is lower than that of the hairy roots of positive-3, positive-63 and positive-64PgCYP309 genes, thereby proving that the over-expressed PgCYP309 can promote the synthesis of ginsenoside.

Sequence listing

<110> Jilin university of agriculture

<120> ginseng PgCYP309 gene and application thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>1563

<212>DNA

<213> Ginseng radix (Panax ginseng)

<400>1

atggaaaccc ttgatgatat ttcttttctt caaaatttac aacctatcat ccccatcctc 60

ttaattgcac ccctactatt tttctttctc ttatcgcgct ttcgccgtaa accctaccca 120

ccaggcccaa gaggctggcc gctcatcggc aacatgctgc tgatggacaa cctaactcac 180

cgtgggctgg ccagactagc taataaatac ggtgggattt ttcacctccg catgggcttc 240

ctccacatgg tcaccgtgtc aacacccgac atggcccgcc aagtccttca acttcaagac 300

aacatcttct ccaaccgccc ggccaccgtc gctattagct atctcactta tgaccgagcc 360

gacatggcct tcgccaacta cggcccgttt tggcgccaaa tgcgcaaaat ctcagtcatg 420

aagctcttca gccgtaaacg ggccgagtct tgggactcgg tccgtgacga ggttgatggc 480

atgcttcgaa ttgtcgcgtc caacgctggc ttagccgtta acataggcga gctggtgttc 540

gggctgactc ggaacattat ctatcgggct gcgtttgggt ccagctcgca tgaggggaat 600

gatgatttca ttaaaatatt gcaggagttt tcgaagctgt ttggtgcttt caatatttgt 660

gattttgttc cggggttgag ctgggctgac ccacaagggt tcaccaatag gctggttaag 720

gctcgttcat cgcttgatgg gttcattgac tcgatattag acgaacacat agacaaaaag 780

aagagcaaca tgatgaatag taattgtgat gatgggaaca ctgatatggt ggacgagtta 840

ctagcatttt acagtgaaga taaggctaaa gtcaatgagt ctgatgattt acacagctcc 900

attaagctca caagagataa tattaaagcc attatcatgg acgtaatgtt tggtgggacc 960

gagacggtcg cttccgctat agagtgggcc atgtcggagc taatgagaag cccagatgac 1020

ctcaaaaagg tccaacaaga gctgaccaac gttgttgggc ttcaccgacg ggtcgaagaa 1080

agtgattttg acaagctcac ttacctcaaa tgcaccctca aagagactct ccgactccac 1140

cctcccatcc ctctcctcct ccacgaaacc gccgaggaag ccgaggttgc cggttaccgt 1200

attccggcca gatcccgtgt catcattaat tcgtgggcca tcgggcgtga caagaactcg 1260

tgggatgacc cggacacgtt caagccctct aggtttctca aacagggtgt ccccgacttc 1320

aaaggcaaca actttgagtt cttacctttt gggtcgggcc gtagatcctg tcccggcatg 1380

caacttgggc tttatgcgct tgagatggcg gtggcccacc ttactcattc ttttacttgg 1440

gaattgcctg atgggataaa acccagtgaa gtggacacaa atgatgtgtt tgggctcacc 1500

actccgaggg ctactcggct cattgcggtg ccgaacccac gactgttatg tccaatctat 1560

taa 1563

Claims (4)

1. The nucleotide sequence of the ginseng PgCYP309 gene is shown in a sequence table SEQ ID NO. 1.

2. A super expression vector is characterized in that a gene shown in a sequence table SEQ NO.1 is inserted into a plant expression vector.

3. The application of the ginseng PgCYP309 gene shown in the sequence table SEQ ID NO.1 in improving the content of ginsenoside in ginseng.

4. Use according to claim 3, characterized in that: ginseng radix is hairy root of Ginseng radix.

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