CN110305855A - Gastrodia elata GeCPR gene and its application - Google Patents

Abstract

The present invention relates to gastrodia elata GeCPR gene and application thereof, and belongs to the technical field of bioengineering, the nucleotide sequence of the gastrodia elata GeCPR gene is shown in SEQ ID NO: 1; the protein amino acid sequence encoded by the gastrodia elata GeCPR gene is as SEQ ID Shown in N0:2; Utilize GeCPR gene to construct eukaryotic expression vector pH2-35S-GeCPR, transform Armillaria armillaria, obtain positive clone transgenic Armillaria armillaria strain, can convert 4-cresol into 4-hydroxybenzyl alcohol.

Description

Gastrodia elata GeCPR gene and its application

technical field

The invention belongs to the technical field of bioengineering, and in particular relates to the Gastrodia elata GeCPR gene and its application.

Background technique

Gastrodia elata is a fungal nutritional orchid plant. It is a precious Chinese medicinal material. It can calm and hypnotize, resist syncope, improve memory, and improve immunity. Gastrodia elata contains gastrodin and p-hydroxybenzyl alcohol. Gastrodin can It has a good curative effect in the treatment of cardiovascular and cerebrovascular diseases such as myocardial hypertrophy, Alzheimer's disease, senile dementia, hyperglycemia and other diseases.

It is known that gastrodin synthesis methods include chemical synthesis and biosynthesis. The substrates required for chemical synthesis include pentaacetyl-β-D-glucose, p-cresol, p-hydroxybenzyl alcohol, etc. These substances undergo a series of biochemical reactions, and finally Synthetic gastrodin. Since the yield of gastrodin in biosynthesis is low, and most of the synthesis methods are complicated or have certain risks, increasing the yield of gastrodin has become a research hotspot. Cytochrome P450 genes can participate in catalyzing various primary and secondary metabolic reactions in plants. The main medicinal ingredients in Gastrodia elata are 4-hydroxybenzyl alcohol and gastrodin. The pharmacopoeia stipulates that the total content of these two cannot be less than 0.25%. -Hydroxybenzyl alcohol is the precursor of gastrodin, which can be synthesized by cytochrome P450. Cytochrome P450 reductase (CPR) is an important part of cytochrome P450 oxidase (CYPs), it can transfer electrons to the active center of CYP450, control the speed of redox reaction of CYP450, participate in the primary and secondary of CYPs catalysis Metabolism, current studies have shown that heterologous expression of CYP450 in Escherichia coli can achieve gastrodin biosynthesis.

Contents of the invention

In order to overcome the problems existing in the background technology, the present invention provides the GeCPR gene and its application, screening to obtain the related gene GeCPR of Gastrodia elata CPR synthesis, by constructing the GeCPR gene plant expression vector, and then transfecting Armillaria armillaria with Agrobacterium pMP90, to obtain Functional GeCPR transgenic Armillaria strains for the synthesis of 4-hydroxybenzyl alcohol.

To achieve the above object, the present invention is achieved through the following technical solutions:

Gastrodia elata GeCPR gene, its nucleotide sequence is shown in SEQ ID NO:1.

The amino acid sequence of the protein encoded by the Gastrodia elata GeCPR gene is shown in SEQ ID NO:2.

The application of the Gastrodia elata GeCPR gene in the synthesis process of 4-hydroxybenzyl alcohol, specifically, using the GeCPR gene to construct the eukaryotic expression vector pH2-35S-GeCPR, transform Armillaria armillaria, and obtain positive clones of transgenic Armillaria armillaria strains, 4-cresol can be converted into 4-hydroxybenzyl alcohol.

Beneficial effects of the present invention: the present invention screens and obtains the related gene GeCPR synthesized by Gastrodia elata CPR, constructs the GeCPR gene plant expression vector, and then transfects Armillaria mellea with Agrobacterium pMP90 to obtain the GeCPR transgene with the function of synthesizing 4-hydroxybenzyl alcohol Armillaria strains.

Description of drawings

Fig. 1 is the PCR amplification of GeCPR full-length gene;

Figure 2 shows the enzyme digestion and PCR detection of the eukaryotic expression vector pH2-35S-GeCPR. In the figure, A: enzyme digestion detection, M: DL2000DNAmaker; 1~2 pH2-35S-GeCPR plasmid double enzyme digestion; 3pENTR2B-GeCPR plasmid double enzyme digestion ; B: PCR detection;

Fig. 3 is the PCR detection of transgenic Armillaria armillaria, among the figure, M: DL5000DNAmaker, 1～4: Transgenic honey fungus, 5: Non-transgenic Armillaria armillaria, 6: Blank control;

Fig. 4 is a diagram of transformation efficiency of transgenic Armillaria armillaria.

Detailed ways

Embodiment 1: Obtaining of GeCPR full-length gene

From the transcriptome data, the P450 gene was screened out to have the gene sequence at the 5' end, and the gene sequence was amplified by nested PCR:

In the first step of nested PCR, two upstream primers and universal primers were designed. The primers were synthesized by Shanghai Qingke.

The first stage: upstream primer GeCPRF-1 (5'-AGCAACAAAGGAATATGTGC-3') and downstream primer UN36 (5'-GACTCGAGTCGACATC GATTTTTTTTTTTTTTTTT-3') for gene amplification;

The second stage: the product obtained in the first stage is diluted 10 times and used. The upstream primer GeCPRF-2 (5'-GGAGAGTCTGGTGAAGAAC-3') and the downstream primer UN36 (5'-GACTCGAGTCGACATC GATTTTTTTTTTTTTTTTT-3') will detect the target gene obtained And splicing, online software detection of the spliced P450 gene http://linuxl.soflberry.com/berry.phtml to predict the possible start codon and stop codon of P450, design primers for amplification, upstream primer: GeCPR- F (ggatccATGCAATCAGAGGCGATG) downstream primer: GeCPR-R (ctcgaaTCACCAGACATCTCTCAG), the product was recovered, TA cloned, positive clones were picked for sequencing, and the correct full-length gene was obtained.

Embodiment 2: Construction of GeCPR eukaryotic expression vector

Connect the recovered GeCPR full-length gene target fragment to the 18T vector, transform it into Escherichia coli DH5α, and obtain Escherichia coli with the PMD18T-GeCPR vector;

Activate the Escherichia coli strain with the correctly sequenced PMD18T-GeCPR carrier, pick a single colony and culture it in Amp-resistant LB liquid medium at 37°C for 12 hours, and at the same time activate PENTR ^TM -2B in the medium containing Kan antibiotics, PMD18T-GeCPR and PENTR ^TM -2B plasmids were extracted using the plasmid extraction kit purchased from Shanghai Sangon, and simultaneous double enzyme digestion was performed on the plasmid pMD18T-GeCPR and pENTR ^TM -2B vectors using BamHI and XhoI respectively. The system is as follows: 6 μL of plasmid, 1 μL of BamHI, 1 μL of XhoI, 2 μL of 10×Buffer, 10 μL of ddH ₂ O), use the gel recovery kit to recover the target gene fragments, ligate the recovered target fragments, and transform them into DH5α competent cells. Incubate on a plate containing Kan antibiotics at 37°C for 12 hours, pick a single colony and transfer it to Kan-resistant LB medium and culture for 12 hours, extract the plasmid for double enzyme digestion detection, and detect the correct entry cloning vector pENTR ^TM 2B-GeCPR and the target vector Perform LR reaction at pH2GW7.0, transform DH5α, transfer to a plate containing Spe (50 μg/mL) antibiotic and culture at 37°C for 12 hours, pick a colony formed by a single bacterium and transfer it to LB liquid medium containing the same antibiotic for cultivation, the plasmid Extraction, and simultaneous double enzyme digestion detection of the obtained plasmid, to obtain the pH2-35S-GeCPR vector.

All the constructed vectors were screened on the culture medium containing antibiotics, and the plasmids were extracted, and the correctness of the recombinant plasmids was detected by simultaneous double enzyme digestion and PCR verification of the plasmids, and the results of PCR detection showed that pH2-35S- The GeCPR vector was constructed successfully (as shown in Figure 2).

Example 3: Transformation of Armillaria armillaria

Transform the pH2-35S-GeCPR carrier into Agrobacterium competent pMP90 by electroporation method, spread it on the plate containing Spe antibiotics, incubate upside down at 37°C for 12 hours, pick a single colony for liquid culture, and use specific primers Carry out bacterial liquid PCR, after the detection is correct, the bacterial liquid is expanded and cultivated, and the method of infecting Armillaria armillaria with Agrobacterium is used to transfect Armillaria armillaria. Apply the transfected Armillaria armillaria on the PDA medium containing Hyg (100mg/L), culture at 26°C for 26 days, pick the well-grown hyphae and culture them in liquid medium for 2 weeks, and Armillaria armillaria that have not been successfully transformed Without the hygromycin resistance gene, it cannot grow normally on the medium containing Hyg (100mg/L).

Using common wild-type Armillaria armillaria and transgenic Armillaria armillaria as templates, PCR amplification was performed with the designed specific primers of GeCPR gene to verify whether the exogenous gene was inserted. The results are shown in Figure 3, the results of PCR: WT (wild type Armillaria armillaria) can not amplify the exogenous gene of 2094bp fragment, but the transgenic strain can amplify the target fragment, which proves that the GeCPR gene is successfully transferred into Armillaria armillaria .

Embodiment 4: Transformation efficiency of transgenic Armillaria armillaria 4-hydroxybenzyl alcohol

Through HPLC high-performance liquid chromatography, detect the conversion efficiency of tetrahydroxybenzyl alcohol, the precursor substance of gastrodin, an effective medicinal ingredient of Armillaria armillaria, and specifically detect the concentration of tetrahydroxybenzyl alcohol produced after 2, 4, 6, 8, 10, and 24 hours. As a result, as shown in Figure 4, the output of 4-hydroxybenzyl alcohol between the 4th and 10h was basically unchanged, and increased after 24h of catalysis.

The invention obtains the cytochrome P450 reductase (GeCPR) gene in Gastrodia elata by cloning, constructs the GeCPR gene eukaryotic expression vector, and then transfects Armillaria mellea through Agrobacterium-mediated transfection, and screens to obtain the transgenic Armillaria armillaria strain of positive clones, which can The successful conversion of 4-cresol into 4-hydroxybenzyl alcohol, the precursor of gastrodin, not only helps to increase the yield of gastrodin, but also reduces environmental pollution.

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that it can be described in terms of form and Various changes may be made in the details without departing from the invention defined in the claims.

SEQ ID No.1

2041

dna

artificial sequence

1

1 ATGCAATCAG AGGCGATGAA GTCGTCTCCG CTGGATCTTC TCTCCGCTAT CCTCACCGGC

61 AGACGAGGCG GGGAGGGCGA TTCCATTCCC GGGAATCAAG AGCTTCTTGT TCTACTTGCG

121 ACTTCAATGG CGATGCTTGT TGGCTGCGTG CTATTGATAC TATGGCGCCG TTCGTCCAAT

181 AAGAAATCTG CTTTCAAAGC CGAGCCACCG CGGCCGGCGG CCTTGAGGGT GTCCCTGGAG

241 CCGGAGATTG ACGACGGGAA GAAGAAGGTC ATTGTACTCT TCGGAACGCA GACCGGTACT

301 GCTGAAGGGT TCGCGAAGAC GTTGGCGGAG GAAGCAAAGG CACGGTACGA TAAAGCCGCT

361 TTCAAAGTTG TTGATCTGGA TGATTACGCA GCTGATGATG ATGAGTATGA AGAGAAGATG

421 AAGAAGGAGA CTCTAGCCTT GTTCTTCATG GCAACGTATG GAGATGGAGA ACCGACTGAT

481 AATGCTGCGA GGTTTCTACAA ATGGTTTTTCG GAGGGCAAAG AGAGGGGCAT TTGGCTAGAG

541 AATCTCACAT ATGCGGTATT TGGACTGGGC AACAGACAGT ATGAACACTT CAATAAGGTC

601 GCAAAGGTTG TTGATGACAT CTTAGCTGAG CAAGGCGGAA AATGTCTTGT TCCTGTTGGC

661 CTTGGGGATG ATGATCAATG CATTGAGGAT GATTTCACTG CATGGAAAGA ACAGCTCTGG

721 ACCGAGCTGG ATCAGTTGCT TCGAGATGAA GATGATGTAG CAGGCGGAAC TTATACTGTT

781 GCAGTGCCTG AATATCGTGT TGTATTCATT GATTTGCCAAG AGGCATCACA CACAGAAAAG

841 GGTTGGAATC TTGTGAATGG AAGTGCTGTT TGTGATGTTA ACCATCCTTG CAGGGCAAAT

901 GTAGCTGTAA GAAGGGAACT TCATTCTCCT GCTTCAGATC GTTCCTGCAT TCATCTGGAG

961 TTTGACATAC ATGGCACTGG TCTTATGTAT GAGGCAGGAG ATCATGTTGG TTTATATGCT

1021 GAAAATAGCT TGGAAACAGT GGAGGAAGCA GAAAAGTTAC TAGGCTATGC ACCAGATACA

1081 TTCTTTTCTA TTCATGCTGA CAAAGAAGAC GGGACACCAC TCAGCGGTGG TGCTCTTGCT

1141 TCTCCATTTC CATCTCCCTG CACATTGAGA ACTGCGTTGG CTCGATATGC TGACCTGTTA

1201 AGTTCTCCCA AAAAGGCTTC TTTAACCGCT TTGGCTGCTC ATGCGTCTGA TCCAATTGAA

1261 GCTGAACGGT TGAGATTTCT GGCTTCCCCT TCCGGAAAGG ATGAGTATTC TCAGTGGGTA

1321 ATAGCTAGCC AGAGGAGTCT TTTGGAAGTA ATGGCCGAGT TCCCTTCAGC CAAGCCACCT

1381 CTAGGTGTTT TCTTTGCAGC AATTGCTCCA AGATTACAGC ATCGCTTTTA TTCTATATCA

1441 TCTTCTTCAA GGATGTACCC AACTAGAATT CATGTGACAT GTGCTCTAGT GTATGGACCA

1501 ACACTGACCG GAAGGATTCA TAAGGGAGTA TGCTCGACTT GGATGAAGAA TGCAATTCCA

1561 TTGGAGGGAA GCGAAAATTG CAGCTGGGCT CCTATATTTG TAAGGCAATC AAATTTTAAG

1621 CTGCCTGCAG ATACCTCAGT GCCCATTATC ATGATTGGAC CTGGAACTGG CTTGGCCCCC

1681 TTCCGTGGCT TCCTACAGGA GAGGCTGGTG TTAAAAGGT CCGGTGCTGA ACTCGGCCCT

1741 GCTATGCTCT TCTTTGGATG CAGGAATCGG AAAATGGATT TCATTTATGA AGACGAGCTT

1801 AAGAATTTTG CTGAGGCTGG AGTGGTTTCT GAGCTCATTG TGACTTTCTC TCGGGAGGGA

1861 GCAACAAAGG AATATGTGCA GCATAAAATG GTCGAAAAGG CATCTGATAT TTGGGATGTT

1921 ATATCTAAAG GCGGTTACAT TTATGTATGT GGTGATGCTA AAGGCATGGC CAAAGATGTT

1981 CACCGCACTC TGCATACTAT TGTTCAGGAA CAGGGCTGTC TAGATAGCTC GAAGACGGAG

2041 AGTCTGGTGA AGAACCTGCA AATGCAAGGA AGGTATCTGA GAGATGTCTG GTGA

SEQ ID No.2

661

amino acid sequence

2

1 MQSEAMKSSP LDLLSAILTG RRGGEGDSIP GNQELLVLLA TSMAMLVGCV LLILWRRSSN

61 KKSAFKAEPP RPAALRVSLE PEIDDGKKKV IVLFGTQTGT AEGFAKTLAE EAKARYDKAA

121 FKVVDLDDYA ADDDEYEEKM KKETLALFFM ATYGDGEPTD NAARFYKWFS EGKERGIWLE

181 NLTYAVFGLG NRQYEHFNKV AKVVDDILAE QGGKCLVPVG LGDDDQCIED DFTAWKEQLW

241 TELDQLLRDE DDVAGGTYTV AVPEYRVVFI DLPEASHTEK GWNLVNGSAV CDVNHPCRAN

301 VAVRRELHSP ASDRSCIHLE FDIHGTGLMY EAGDHVGLYA ENSLETVEEA EKLLGYAPDT

361 FFSIHADKED GTPLSGGALA SPFPSPCTLR TALARYADLL SSPKKASLTA LAAHASDPIE

421 AERLRFLASP SGKDEYSQWV IASQRSLLEV MAEFPSAKPP LGVFFAAIAP RLQHRFYSIS

481 SSSRMYPTRI HVTCALVYGP TLTGRIHKGV CSTWMKNAIP LEGSENCSWA PIFVRQSNFK

541 LPADTSVPII MIGPGTGLAP FRGFLQERLV LKESGAELGP AMLFFGCRNR KMDFIYEDEL

601 KNFAEAGVVS ELIVTFSREG ATKEYVQHKM VEKASDIWDV ISKGGYIYVC GDAKGMAKDV

661 HRTLHTIVQE QGCLDSSKTE SLVKNLQMQG RYLRDVW*

Claims (4)

1. Rhizoma Gastrodiae GeCPR gene, it is characterised in that: the nucleotide sequence of the Rhizoma Gastrodiae GeCPR gene such as SEQ ID N0:1 institute Show.

2. Rhizoma Gastrodiae GeCPR gene according to claim 1, it is characterised in that: the egg of the Rhizoma Gastrodiae GeCPR gene coding White matter amino acid sequence is as shown in SEQ ID N0:2.

3. application of the Rhizoma Gastrodiae GeCPR gene according to claim 1 in 4- salicylic alcohol synthesis process.

4. application of the Rhizoma Gastrodiae GeCPR gene according to claim 3 in 4- salicylic alcohol synthesis process, feature exist In: the gene constructed carrier for expression of eukaryon pH2-35S-GeCPR of GeCPR is utilized, halimasch is converted, obtains positive colony transgenosis honey Ring bacteria strain can convert 4- cresols to 4- salicylic alcohol.