CN111748534B - SmC4H protein and construction and expression method thereof - Google Patents

Abstract

The invention provides a SmC4H protein and a construction and expression method thereof, the SmC4H protein utilizes transcriptome sequencing of swertia mussotii Franch, obtains a SmC4H gene sequence through biological information software analysis, obtains a cDNA sequence through RT-PCR amplification, and clones; PBCV-SmC4H is firstly constructed, then an expression vector pETX-SmC4H is constructed, the expression vector is transferred into Escherichia coli Rosetta (DE3) to carry out expression of the protein, the obtained SmC4H protein is purified by a nickel column, and the purification effect is obvious.

Description

SmC4H protein and construction and expression method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a SmC4H protein and a construction and expression method thereof.

Background

Swertia mussotii, also known as Tibetan capillary artemisia, has important medicinal value, has the effects of clearing heat and removing toxicity, and clearing liver and benefiting gallbladder, is known for treating liver and gallbladder diseases, and researches show that Tibetan capillary artemisia medicinal plants have the inhibiting effect on lymphoma cells, skin cancer cells, liver cancer cells, stomach cancer and the like. The xanthone compounds mainly form the basis of antitumor drug substances of swertia mussotii.

C4H acts as the first P450 cytochrome monooxygenase upstream of the phenylpropanoid metabolic pathway and controls the biosynthesis of xanthones. At present, the C4H gene has been cloned from various plants such as Arabidopsis thaliana, Lepidium meyenii and Vitis amurensis, and a series of studies on the functions of the plants have been carried out.

Disclosure of Invention

The invention aims to provide a SmC4H protein.

The invention also aims to provide a construction and expression method of the SmC4H protein.

The technical scheme adopted by the invention is as follows:

a SmC4H protein has a sequence shown in a sequence table SEQ.1.

The cDNA sequence of the SmC4H protein has the total length of 1518bp, codes 505 amino acids, has the relative molecular mass of 51.83kDa, and is unstable hydrophilic protein.

The SmC4H protein is a key enzyme C4H gene of a phenylpropane metabolic pathway, and controls the biosynthesis of xanthone compounds.

The construction method of the SmC4H protein adopts a biological engineering means to clone, and prokaryotic expression and purification are carried out on the cloned gene to obtain an expected result.

The construction and expression method of the SmC4H protein comprises the following specific steps:

(1) extracting total RNA from swertia mussotii;

(2) reverse transcribing the RNA into cDNA, designing a specific primer based on the full-length cDNA sequence of the SmC4H gene, and performing PCR amplification;

(3) detecting and identifying the PCR product by agarose gel electrophoresis, and cutting and recovering the target fragment;

(4) connecting a cloning vector PBCV with a recovered target fragment, constructing PBCV-SmC4H, taking a connecting product, and converting the connecting product into Escherichia coli Trans5 alpha;

(5) coating the obtained product on a flat plate with kanamycin resistance, selecting positive clones, and sequencing;

(6) the recombinant vector PBCV-SmC4H and PETX are respectively subjected to enzyme digestion and connection, and an expression vector PETX-SmC4H is successfully constructed;

(7) PETX-SmC4H was transferred into Escherichia coli Rosetta (DE 3): culturing overnight at 37 deg.C and 200rpm for 12h, performing amplification culture at 25 deg.C and 200rpm for 8h, inoculating 0.3mM IPTG into the culture solution, and inducing expression at 16 deg.C and 200rpm for 12 h;

(8) and (3) crushing the thalli by using an ultrasonic crushing method, collecting supernatant, purifying target protein by using a nickel column, and detecting and analyzing the purified protein by using SDS-PAGE electrophoresis.

The invention has the beneficial effects that:

the invention provides a SmC4H protein and a construction and expression method thereof, wherein the SmC4H protein is obtained by utilizing transcriptome sequencing of swertia mussotii Franch, analyzing through biological information software to obtain a SmC4H gene sequence, obtaining a cDNA sequence through RT-PCR amplification and cloning; PBCV-SmC4H is firstly constructed, then an expression vector pETX-SmC4H is constructed, the expression vector is transferred into Escherichia coli Rosetta (DE3) to express the protein, the obtained SmC4H protein is purified by a nickel column, and the purification effect is obvious.

Drawings

FIG. 1 is a diagram showing the result of electrophoresis of a reverse transcription product of SmC4H,

M：DL2000DNA marker；

1: negative control;

2: RT-PCR amplification products of SmC 4H.

FIG. 2 is a diagram showing the result of prokaryotic expression of SmC4H,

M:Low protein marker；

BL21(DE3) strain transformed with PET-28a vector;

2: BL21(DE3) strain transformed with PET-28a-SmC4H vector;

3: purified protein of BL21(DE3) strain transformed with PET-28a-SmC4H vector.

Detailed Description

To further illustrate the present invention, reference is made to the following examples:

example 1 prokaryotic expression of the SmC4H Gene of swertia mussotii

Cloning of the gene I SmC 4H:

(1) extracting total RNA of the leaves according to the instruction of the RNA extraction kit;

(2) reverse transcription is carried out on the total RNA of the leaves according to the instruction of a Reverse transcription M-MLV (RNase H-) kit to synthesize cDNA;

(3) PCR amplification is carried out by taking the first strand of cDNA of swertia mussotii leaf as a template according to the full-length cDNA sequence of SmC4H gene obtained from swertia mussotii transcriptome.

PCR amplification detection system of PCR reaction system table

Wherein the forward primer (SmC4H-up) has a sequence shown in a sequence table SEQ.2.

Wherein, the reverse primer (SmC4H-dp) has a sequence shown in a sequence table SEQ.3.

1) The obtained PCR product was detected and identified by 1% agarose gel electrophoresis, and the results are shown in FIG. 1.

2) And cutting and recovering the target fragment according to the instructions of the rapid agarose gel DNA purification and recovery kit.

3) PBCV (having a sequence shown in SEQ.4 of the sequence Listing) and the recovered target fragment were ligated by T4 ligase.

4) The ligation products were taken, transformed into Escherichia coli Trans 5. alpha. and plated on LB (50mg/L) plates with kanamycin resistance, and inverted for 12h at 37 ℃.

5) Positive clones were selected from LB plates and sequenced.

Construction of IISmC 4H Gene expression vector

1) NdeI and XhoI restriction enzymes are selected according to the sequence of SmC4H, and the recombinant vector PBCV-SmC4H and the PETX expression vector are subjected to double enzyme digestion.

Double digestion system of digestion reaction system NdeI and XhoI

2) Detecting and identifying the enzyme digestion product by using 1% agarose gel electrophoresis, and carrying out gel cutting recovery on the enzyme digestion product by using a rapid agarose gel DNA purification recovery kit.

3) And connecting the recovered products by using T4 ligase to obtain an expression vector PETX-SmC4H which has a sequence shown in a sequence table SEQ.5.

T4 ligase ligation system

III protein expression and purification

1) PETX-SmC4H was transferred into Escherichia coli Rosetta (DE 3). After culturing overnight at 37 ℃ and 200rpm for 12 hours in a small volume, and performing scale-up culture at 25 ℃ and 200rpm for 8 hours, 0.3mM IPTG was inoculated into the culture medium, and expression was induced at 16 ℃ and 200rpm for 12 hours.

2) The ultrasonic disruption method is used for disrupting the thalli, the supernatant is collected, the target protein is purified by a nickel column, and the purified protein is detected and analyzed by SDS-PAGE electrophoresis, and the result is shown in figure 2.

The analysis result by using the LC-MS technology shows that the obtained SmC4H protein can convert cinnamic acid into p-coumaric acid. The parent ion M/z for this product was 163([ M-H ] -) and the daughter ion M/z was 119.1([ M-CO2] -). This suggests that the protein SmC4H has the biological function of cinnamate-4-hydroxylase.

The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Sequence listing

<110> Tianjin Chinese medicine university

<120> SmC4H protein and construction and expression method thereof

<141> 2020-08-02

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1518

<212> DNA

<213> SmC4H protein (SmC4H)

<220>

<221> gene

<222> (1)..(1518)

<400> 1

atggatcttc tccttttaga gaaagccctt ctgggtctct ttgccgccat cgttatagcc 60

acggtggtat ccaaactccg tggcaagcgt ttcaaactcc ctccaggccc aatcccaatt 120

cccattttcg gtaactggtt acaagtcggt gatgatttaa accaccgtaa tctcacggaa 180

tatgccaaaa aattcggtga tatactcttg ctaagaatgg gacaacgtaa tttggtggtc 240

gtctcgtcgc cggaactcgc tagagaagtt ttgcacacac aaggagttga attcggttca 300

cgaaccagaa atgtagtttt cgatattttc actgataaag gacaagatat ggtgtttacg 360

gtttacggtg aacattggag gaagatgagg aggatcatga cggtgccgtt ctttaccaac 420

aaggtggttc agcaatacag gcaaggttgg gaagatgagg ttttagaagt gattaacgat 480

gtcaagaaga atccggaggc atcaacaaat gggattgttc taaggaagag attgcaacta 540

atgatgtata ataacttgta tcggatcatg tttgatcgga gatttgagag tgaggatgat 600

cctttgttta ataagcttaa ggttttaaat ggagagagga gccgattagc acaaagcttt 660

gattacaatt atggcgattt cattcccatt ttgagacctt tcttaagagg ctacttgaaa 720

atctgccagc aagttaagga taaaagattg cagctattta aggacaattt cgtcgaagag 780

agaaagaagc tggctagcac aaaggccaca gataacaatc ggatgaaatg cgccattgat 840

tatattcttg aagcagaaca gaagggagag attaatgagg acaatgtgct tttcattgtt 900

gagaacatta atgttgctgc aattgaaacc acgttgtggt cggttgaatg gggaatcgcg 960

gagctagtta acaaccctca tatccaacag aaactccgta atgagcttga cacggtgctt 1020

ggaccaggtg tactagtgac tgaaccagac attccgaagc tgccttacct atacgcagtg 1080

gtcaaggaaa ccctgcgtct ccgaatggcc attcccttat tggtacctca catgaatctt 1140

catgatgcca agcttggtga ctatgacatt ccagcagaga gcaagatatt ggtaaacgct 1200

tggtggctgg cgaacaaccc ggcccagtgg aagaagcctg aagagtttag acccgagagg 1260

ttcttggaag aggaggcgaa agtggaagca agcggaaatg atttccggta tcttccgttt 1320

ggtgttggta ggaggagctg ccccggaatc atccttgcat tgccgattct tagtatcaca 1380

ttggggcgtt tggtgcagag ttttgagatg ttgcctcctc ctggacaatc taagattgat 1440

actactgaga aaggagggca attcagtttg cacattatga agcattcaac tattgtgttg 1500

aagccaatat ccttctaa 1518

<210> 2

<211> 34

<212> DNA

<213> Forward primer (primer)

<220>

<221> primer_bind

<222> (1)..(13)

<400> 2

ggaattccat atgatggatc ttctcctttt agag 34

<210> 3

<211> 31

<212> DNA

<213> reverse primer (primer)

<220>

<221> primer_bind

<222> (1)..(9)

<400> 3

ccgctcgagt tagaaggata ttggcttcaa c 31

<210> 4

<211> 3929

<212> DNA

<213> vector

<220>

<221> old_sequence

<222> (1)..(3929)

<400> 4

agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60

acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120

tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180

ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg 240

gtaccgagct cggatccact agtaacggcc gccagtgtgc tggaattgcc cttaagggca 300

attctgcaga tatccatcac actggcggcc gctcgagcat gcatctagag ggcccaattc 360

gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga 420

aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 480

taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 540

atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 600

accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 660

gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 720

tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 780

gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 840

agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 900

ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 960

tttaacgcga attttaacaa aattcagggc gcaagggctg ctaaaggaag cggaacacgt 1020

agaaagccag tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct 1080

ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc 1140

gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc 1200

cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga 1260

tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga 1320

ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 1380

atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 1440

aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg 1500

acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 1560

acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 1620

tcctgtcatc ccaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 1680

ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 1740

agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 1800

atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg 1860

aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 1920

gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 1980

cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 2040

tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg 2100

agttcttctg aattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 2160

tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 2220

aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 2280

cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 2340

agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 2400

ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 2460

tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 2520

tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 2580

caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 2640

accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 2700

attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 2760

ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 2820

taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 2880

taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 2940

aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 3000

agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 3060

ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 3120

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 3180

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 3240

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 3300

tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 3360

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 3420

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 3480

ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 3540

acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 3600

ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 3660

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 3720

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 3780

ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 3840

taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 3900

cagcgagtca gtgagcgagg aagcggaag 3929

<210> 5

<211> 6808

<212> DNA

<213> vector

<220>

<221> old_sequence

<222> (1)..(6808)

<400> 5

atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120

tgttagcagc cggatctcag tggtggtggt ggtggtgcaa tcttcctata accgaagttg 180

tgttatcaac ttacgaagta ttacacgttt gacttaacgg gaggaaagag tcatcatagt 240

tagaatctaa caggtcctcc tccgttgtag agttttgaga cgtggtttgc ggggttacac 300

tatgattctt agccgttacg ttcctactaa ggccccgtcg aggaggatgg ttgtggtttg 360

ccttctatgg cctttagtaa aggcgaacga aggtgaaagc ggaggagaag gttcttggag 420

agcccagatt tgagaagtcc gaagaaggtg acccggccca acaagcggtc ggtggttcgc 480

aaatggttat agaacgagag acgaccttac agtatcagtg gttcgaaccg tagtacttct 540

aagtacactc catggttatt cccttaccgg taagcctctg cgtcccaaag gaactggtga 600

cgcatatcca ttccgtcgaa gccttacaga ccaagtcagt gatcatgtgg accaggttcg 660

tggcacagtt cgagtaatgc ctcaaagaca acctatactc ccaacaattg atcgaggcgc 720

taaggggtaa gttggctggt gttgcaccaa agttaacgtc gttgtaatta caagagttgt 780

tacttttcgt gtaacaggag taattagaga gggaagacaa gacgaagttc ttatattagt 840

taccgcgtaa agtaggctaa caatagacac cggaaacacg atcggtcgaa gaaagagaga 900

agctgcttta acaggaattt atcgacgtta gaaaatagga attgaacgac cgtctaaaag 960

ttcatcggag aattctttcc agagttttac ccttacttta gcggtattaa cattagtttc 1020

gaaacacgat tagccgagga gagaggtaaa ttttggaatt cgaataattt gtttcctagt 1080

aggagtgaga gtttagaggc tagtttgtac taggctatgt tcaataatat gtagtaatca 1140

acgttagaga aggaatcttg ttagggtaaa caactacgga ggcctaagaa gaactgtagc 1200

aattagtgaa gattttggag tagaagggtt ggaacggaca taacgacttg gtggaacaac 1260

catttcttgc cgtggcagta ctaggaggag tagaaggagg ttacaagtgg catttggcat 1320

ttgtggtata gaacaggaaa tagtcacttt tatagctttt gatgtaaaga ccaagcactt 1380

ggcttaagtt gaggaacaca cacgttttga agagatcgct caaggccgct gctctgctgg 1440

tggtttaatg caacagggta agaatcgttc tcatatagtg gcttaaaaaa ccgtataagg 1500

cactctaatg ccaccaaatt tagtagtggc tgaacattgg tcaatggctt ttacccttaa 1560

ccctaacccg gacctccctc aaactttgcg aacggtgcct caaacctatg gtggcaccga 1620

tattgctacc gccgtttctc tgggtcttcc cgaaagagat tttcctcttc taggtaatat 1680

ggctgccgcg cggcaccagg ccgctgctgt gatgatgatg atgatggctg ctgcccatgg 1740

tatatctcct tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac 1800

aattccccta tagtgagtcg tattaatttc gcgggatcga gatctcgatc ctctacgccg 1860

gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg 1920

acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg 1980

tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac 2040

cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc 2100

aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt 2160

cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca 2220

gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg 2280

gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg 2340

gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg 2400

attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt 2460

aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc 2520

gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc 2580

attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt 2640

ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat 2700

gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg 2760

ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa 2820

tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg 2880

tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg 2940

gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc 3000

gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc 3060

ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc 3120

ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg 3180

gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc 3240

gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa 3300

cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag 3360

agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact 3420

tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat 3480

tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt 3540

cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg 3600

cgagaagcag gccattatcg ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct 3660

gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc 3720

gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac 3780

atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg 3840

caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac 3900

atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat 3960

ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt 4020

aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa 4080

tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg 4140

ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga 4200

acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct 4260

cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 4320

agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 4380

tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg 4440

cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatatg cggtgtgaaa 4500

taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca 4560

ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4620

taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4680

agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4740

cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4800

tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4860

tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 4920

gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 4980

acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 5040

acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 5100

cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 5160

gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 5220

gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 5280

agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 5340

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg 5400

tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct 5460

tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct 5520

cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg 5580

ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg 5640

gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt 5700

actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta 5760

ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc 5820

cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc 5880

gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag 5940

cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca 6000

ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg 6060

aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt 6120

gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa 6180

aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag 6240

tttttctaag aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca 6300

aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaaattgtaa acgttaatat 6360

tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga 6420

aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc 6480

agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac 6540

cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc 6600

gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg 6660

gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag 6720

ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc 6780

gccgctacag ggcgcgtccc attcgcca 6808

Claims (2)

1. A SmC4H protein, which is characterized in that: the nucleotide sequence is shown in a sequence table SEQ.1.

2. The method for expressing SmC4H protein as claimed in claim 1, wherein: the method comprises the following specific steps:

(1) extracting total RNA from swertia mussotii;

(2) reverse transcribing the RNA into cDNA, designing a specific primer based on the full-length cDNA sequence of the SmC4H gene, and performing PCR amplification;

(3) detecting and identifying the PCR product by agarose gel electrophoresis, and cutting and recovering the target fragment;

(4) connecting a cloning vector PBCV with a recovered target fragment, constructing PBCV-SmC4H, taking a connecting product, and converting the connecting product into Escherichia coli Trans5 alpha;

(5) coating the obtained product on a flat plate with kanamycin resistance, selecting positive clones, and sequencing;

(6) the recombinant vector PBCV-SmC4H and PETX are respectively subjected to enzyme digestion and connection, and an expression vector PETX-SmC4H is successfully constructed;

(7) the protein is transferred into Escherichia coli Rosetta (DE3) for prokaryotic expression, and the obtained SmC4H protein is purified by a nickel column.

Images