CN110305855B - Gastrodia elata GeCPR gene and application thereof - Google Patents
Gastrodia elata GeCPR gene and application thereof Download PDFInfo
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Abstract
The invention relates to a rhizoma gastrodiae GeCPR gene and application thereof, belonging to the technical field of biological engineering, wherein the nucleotide sequence of the rhizoma gastrodiae GeCPR gene is shown as SEQ ID N0: 1; the protein amino acid sequence coded by the gastrodia elata GeCPR gene is shown as SEQ ID N0: 2; the GeCPR gene is utilized to construct a eukaryotic expression vector pH2-35S-GeCPR, and to transform armillaria mellea to obtain a positive clone transgenic armillaria mellea strain which can transform 4-cresol into 4-hydroxybenzyl alcohol.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a gastrodia elata GeCPR gene and application thereof.
Background
The gastrodia elata is a fungus nutritional type orchid plant, is a rare traditional Chinese medicinal material, can calm and hypnotize, resist syncope, improve memory and improve immunity, and contains gastrodin and p-hydroxybenzyl alcohol which are important components in the gastrodia elata, wherein the gastrodin has good curative effects on treating cardiovascular and cerebrovascular diseases such as myocardial hypertrophy, Alzheimer's disease, senile dementia, hyperglycemia and the like.
The known gastrodin synthesis method comprises chemical synthesis and biosynthesis, substrates required by the chemical synthesis comprise pentaacetyl-beta-D-glucose, p-methylphenol, p-hydroxybenzyl alcohol and the like, and the substances are subjected to a series of biochemical reactions to finally synthesize the gastrodin. Because the yield of gastrodin in biosynthesis is low, and most of the synthesis methods are complex or have certain dangerousness, the improvement of the gastrodin yield becomes a research hotspot. The cytochrome P450 gene can participate in catalyzing various primary and secondary metabolic reactions in a plant body, the main medicinal components of 4-hydroxybenzyl alcohol and gastrodin in the gastrodia elata are contained in the total content which is not lower than 0.25% according to the pharmacopoeia, wherein the 4-hydroxybenzyl alcohol is a gastrodin precursor substance and can be synthesized through catalysis of the cytochrome P450. Cytochrome P450 Reductase (CPR) is an important component in cytochrome P450 oxidase Systems (CYPs), can transmit electrons to the active center of CYP450, controls the speed of the oxidation-reduction reaction of CYP450, participates in the primary and secondary metabolism catalyzed by CYPs, and researches show that the heterologous expression of CYP450 in Escherichia coli can realize the biosynthesis of gastrodin.
Disclosure of Invention
In order to overcome the problems in the background technology, the invention provides a GeCPR gene and application thereof, a related gene GeCPR synthesized by gastrodia elata CPR is obtained by screening, a GeCPR gene plant expression vector is constructed, and Armillaria mellea is transfected by agrobacterium pMP90 to obtain a GeCPR transgenic Armillaria mellea strain with the function of synthesizing 4-hydroxybenzyl alcohol.
In order to realize the purpose, the invention is realized by the following technical scheme:
the nucleotide sequence of the gastrodia elata GeCPR gene is shown as SEQ ID N0: 1.
The protein amino acid sequence coded by the gastrodia elata GeCPR gene is shown as SEQ ID N0: 2.
The application of the gastrodia elata GeCPR gene in the synthesis process of 4-hydroxybenzyl alcohol is specifically that the GeCPR gene is utilized to construct a eukaryotic expression vector pH2-35S-GeCPR, Armillariella mellea is transformed to obtain a positive clone transgenic Armillariella mellea strain, and 4-cresol can be transformed into 4-hydroxybenzyl alcohol.
The invention has the beneficial effects that: the invention obtains GeCPR related to gastrodia elata CPR synthesis through screening, constructs a GeCPR gene plant expression vector, and transfects Armillaria mellea through agrobacterium pMP90 to obtain a GeCPR transgenic Armillaria mellea strain with the function of synthesizing 4-hydroxybenzyl alcohol.
Drawings
FIG. 1 shows PCR amplification of the GeCPR full-length gene;
FIG. 2 shows the eukaryotic expression vector pH2-35S-GeCPR restriction and PCR detection, in which A: enzyme digestion detection, M: DL2000 DNAmaker; carrying out double enzyme digestion on the plasmid of 2-35S-GeCPR at the pH of 1-2; 3 pENTR2B-GeCPR plasmid double digestion; b: PCR detection;
FIG. 3 is PCR detection of transgenic Armillaria mellea, wherein M is DL5000DNAmaker, 1-4 are transgenic Armillaria mellea, 5 are non-transgenic Armillaria mellea, and 6 are blank control;
FIG. 4 is a graph of transformation efficiency of transgenic Armillaria mellea.
Detailed Description
Example 1: GeCPR full-length Gene acquisition
Screening P450 gene from transcriptome data to have 5' end forepart gene sequence, amplifying gene sequence by nested PCR:
in the first step of nested PCR, two upstream primers and a universal primer are designed, and the primers are synthesized by Shanghai engine.
The first stage is as follows: performing gene amplification by using an upstream primer GeCPRF-1 (5'-AGCAACAAAGGAATATGTGC-3') and a downstream primer UN36 (5'-GACTCGAGTCGACATC GATTTTTTTTTTTTTTTTTT-3');
and a second stage: diluting the product obtained in the first stage by 10 times and using the diluted product, detecting and splicing the obtained target gene by an upstream primer GeCPRF-2 (5'-GGAGAGTCTGGTGAAGAAC-3') and a downstream primer UN36 (5'-GACTCGAGTCGACATC GATTTTTTTTTTTTTTTTTT-3'), carrying out online software detection on the spliced P450 gene to obtain http:// linux. GeCPR-F (ggatccatGCAATCAGAGGCGATG) downstream primer: GeCPR-R (ctcgaaTCACCAGACATCTCTCAG), recovering the product, performing TA cloning, selecting positive clone and sequencing to obtain correct full-length gene.
Example 2: construction of GeCPR eukaryotic expression vector
Connecting the recovered GeCPR full-length gene target fragment with an 18T vector, and transforming the target fragment into escherichia coli DH5 alpha to obtain escherichia coli with a PMD18T-GeCPR vector;
activating Escherichia coli strain of PMD18T-GeCPR vector with correct sequencing, selecting single colony, culturing at 37 deg.C in Amp resistant LB liquid culture medium for 12h, and simultaneously, PENTRTM-2B activation in Kan antibiotic-containing media species, extraction of PMD18T-GeCPR and PENTR using plasmid extraction kit purchased from Shanghai ProducersTM-2B plasmid, plasmid pMD18T-GeCPR and pENTR, respectively, using BamHI and XhoI simultaneouslyTMCarrying out synchronous double enzyme digestion on the-2B vector by using an enzyme digestion system as follows: plasmid 6 muL, BamH I1 muL, Xho I1 muL, 10 Xbuffer 2 muL, ddH2O10 muL), recovering target gene fragments by using a glue recovery kit, connecting the recovered target fragments, transforming the target fragments into DH5 alpha competent cells, culturing the cells on a flat plate containing Kan antibiotics at 37 ℃ for 12h, selecting single colonies, transferring the single colonies into a Kan resistant LB culture medium for culturing for 12h, extracting plasmids for double enzyme digestion detection, and detecting a correct entry cloning vector pENTRTMAnd performing LR reaction on the 2B-GeCPR and a target vector pH2GW7.0, converting DH5a, transferring the product into a Spe (50 mu g/mL) antibiotic-containing flat plate, culturing for 12h at 37 ℃, selecting bacterial colonies formed by a single bacterium, transferring the bacterial colonies into an LB liquid culture medium containing the same antibiotic, culturing, extracting plasmids, and performing synchronous double enzyme digestion detection on the obtained plasmids to obtain a pH2-35S-GeCPR vector.
Screening all constructed vectors on a culture medium containing antibiotics, extracting plasmids, detecting the correctness of the recombinant plasmids by performing synchronous double enzyme digestion and PCR verification on the plasmids, and performing PCR detection on the results to show that the construction of the pH2-35S-GeCPR vector is successful (shown in figure 2).
Example 3: transformation of Armillaria mellea
Transferring a pH2-35S-GeCPR vector into an agrobacterium-infected pMP90 by adopting an electrical transformation method, coating the vector on a flat plate containing Spe antibiotic, carrying out inverted culture at 37 ℃ for 12h, selecting a single colony for liquid culture, carrying out PCR (polymerase chain reaction) on bacterial liquid by using a specific primer, carrying out amplification culture on the bacterial liquid after correct detection, and transfecting Armillaria mellea by using a method of infecting Armillaria mellea by using agrobacterium. After transfection, the armillaria mellea is smeared on a PDA culture medium containing Hyg (100 mg/L), cultured at 26 ℃ for 26d, and the grown hyphae are picked up and cultured in a liquid culture medium for 2 weeks, and the armillaria mellea which is not successfully transformed does not contain a hygromycin resistance gene and cannot normally grow on the culture medium containing Hyg (100 mg/L).
Common wild-type Armillaria mellea and transgenic Armillaria mellea are taken as templates, PCR amplification is carried out by using a designed specific primer of the GeCPR gene, and whether an exogenous gene is inserted or not is verified. Results are shown in FIG. 3, the results of PCR: WT (wild-type Armillaria mellea) can not amplify the exogenous gene of the 2094 bp fragment, and the transgenic strain can amplify the target fragment, so that the GeCPR gene is successfully transferred into the Armillaria mellea.
Example 4: transformation efficiency of transgenic Armillaria mellea 4-hydroxybenzyl alcohol
The conversion efficiency of the gastrodin precursor substance tetrahydroxybenzyl alcohol as the effective medicinal component of the armillaria mellea is detected by HPLC (high performance liquid chromatography), and the concentration of the tetrahydroxybenzyl alcohol generated after 2, 4, 6, 8, 10 and 24 hours is specifically detected, so that the yield of the 4-hydroxybenzyl alcohol is basically unchanged between 4 and 10 hours, and is improved after 24 hours of catalysis, as shown in figure 4.
The cytochrome P450 reductase (GeCPR) gene in the gastrodia elata is obtained through cloning, a GeCPR gene eukaryotic expression vector is constructed, the armillaria mellea is transfected through agrobacterium mediation, and a positively cloned transgenic armillaria mellea strain is obtained through screening, so that 4-cresol can be successfully converted into a precursor substance 4-hydroxybenzyl alcohol of the gastrodin, the yield of the gastrodin is favorably improved, and the environmental pollution is reduced.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
SEQ ID No.1
2041
DNA
Artificial sequences
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 GGTTCTACAA ATGGTTTTCG 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 GATTTGCCAG 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 TTAAAAGAGT 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 (3)
1. The gastrodia elata GeCPR gene is characterized in that: the nucleotide sequence of the gastrodia elata GeCPR gene is shown as SEQ ID N0: 1; the protein amino acid sequence coded by the gastrodia elata GeCPR gene is shown as SEQ ID N0: 2.
2. The use of the gastrodia elata GeCPR gene according to claim 1 in the synthesis process of 4-hydroxybenzyl alcohol.
3. The use of the rhizoma gastrodiae GeCPR gene of claim 2 in the synthesis process of 4-hydroxybenzyl alcohol, which is characterized in that: the Gastrodia elata GeCPR gene is used for constructing a eukaryotic expression vector pH2-35S-GeCPR, and transforming Armillariella mellea to obtain a positive clone transgenic Armillariella mellea strain which can transform 4-cresol into 4-hydroxybenzyl alcohol.
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