CN102586262B - Defensin gene of antimicrobial peptide of bemisia tabaci (Gennadius), antimicrobial peptide encoded by defensin gene and preparation method for defensin gene - Google Patents
Defensin gene of antimicrobial peptide of bemisia tabaci (Gennadius), antimicrobial peptide encoded by defensin gene and preparation method for defensin gene Download PDFInfo
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Abstract
The invention relates to antimicrobial peptide and application thereof, and aims to provide a defensin gene of antimicrobial peptide of bemisia tabaci (Gennadius), the antimicrobial peptide encoded by the defensin gene and a preparation method for the defensin gene. A nucleotide sequence of the gene of the antimicrobial peptide is shown as SEQ ID NO.1; and an amino acid of the antimicrobial peptide is shown as SEQ ID NO.2. A protease Xa factor is introduced into an expression vector, so that recombinant fusion protein can be effectively digested, and defensin polypeptide is obtained through rapid separation in an ultrafiltration mode according to the magnitude of protein molecules; the whole process is quick and simple; and the obtained antimicrobial peptide has antifungal activity, and can remarkably inhibit the growth of entomopathogenic fungi such as Beauveria bassiana, Metarhizium anisopliae, Botrytis cinerea and the like.
Description
Technical field
The present invention relates to a kind of Bemisia tabaci antibacterial peptide defensin gene and coded antibacterial peptide and application thereof.Relate in particular to method for preparing purified, restructuring Bemisia tabaci antibacterial peptide and the antibacterial application thereof of a kind of Bemisia tabaci antibacterial peptide defensin that recombinates.
Background technology
In recent years, Bemisia tabaci Bemisia tabaci (Gennadius) has become the disastrous insect of a kind of global invasion, all over the world, breaks out and causes disaster, and causes heavy loss example.This worm is a large amount of feeding plant juice not only, causes plant withered, and propagate various plants virus, and especially geminivirus infection, often causes the viroses of plant to be very popular, and makes crop Severe Reduction or total crop failure.Because Bemisia tabaci host range is wide, reproductivity is strong, and hazard approach is many, and the ability that sterilant is developed immunity to drugs is strong especially, therefore, when it intrudes into a new area, very easily breaks out and causes disaster.
Due to the abuse of traditional antibiotic and the continuous generation of Resistant strain, had a strong impact on the clinical therapeutic efficacy of infectious diseases.And that antibacterial peptide has sterilization is strong, be difficult for causing the advantages such as resistance, become the study hotspot that antibacterials are new.Yield of antibacterial peptides is low at present, and the separating difficulty of natural antibacterial peptide is large, thereby tends to utilize gene engineering method to obtain antibacterial peptide.
Alexin (defensin) is the cationoid antimicrobial polypeptide being extensively formed in the bodies of aminal and plant of discovered in recent years, 29～54 amino-acid residues, consists of, and contains 6～8 conservative halfcystines (normally 6), and molecular weight is less than 5KDa.N end has 3 β-bends and 1 γ corner, and there is 1 amphipathic α spiral centre, and C end has 1 antiparallel β-pleated sheet structure, by 3～4 disulfide linkage, connects α spiral and β-bend, forms specific CS α beta structure.Alexin can be synthesized and be stored in cytoplasmic particle in specific cell, or synthetic by pathogenic micro-organism induction, multiple-microorganism is all had to stronger defence capability, Main Function is in the cytolemma of pathogenic micro-organism, make pathogenic micro-organism be difficult for it to produce resistance, alexin is class candidate new antibacterials that have much at present magnetism.
Up to the present, the research report that still there is no Bemisia tabaci defensin antibacterial peptide both at home and abroad.
Summary of the invention
The technical problem to be solved in the present invention is, for current Bemisia tabaci antibacterial peptide research situation and deficiency thereof, provide a kind of Bemisia tabaci antibacterial peptide defensin gene and its coded antimicrobial polypeptide of recombinating, and described gene has the application in the recombinant protein of anti-microbial activity at gene in preparation.
Solution of the present invention is, a kind of Bemisia tabaci defensin antibacterial peptide gene is provided, and the nucleotide sequence of this gene is as shown in SEQ ID NO.1.
The present invention also provides described Bemisia tabaci defensin antibacterial peptide gene to have the application in the recombinant protein of anti-microbial activity in preparation.
The present invention also provides described Bemisia tabaci defensin antibacterial peptide gene preparation to have the method for the recombinant protein of anti-microbial activity, is by gene recombination technology, described gene to be expressed in intestinal bacteria, obtains the recombinant protein with anti-microbial activity.
The present invention also provides the antibacterial peptide of described Bemisia tabaci defensin antibacterial peptide gene coding, and the aminoacid sequence of this antibacterial peptide is as shown in SEQ ID NO.2.
The present invention also provides described antibacterial peptide in the application for the preparation of preventing and treating in the medicine of Bemisia tabaci, and the applicable Bemisia tabaci kind of this medicine is: hidden kind of Middle East-Asia Minor 1 (MEAM1), hidden kind of Mediterranean (Med), hidden kind of ZHJ1, hidden kind of ZHJ2 or hidden kind of ZHJ3.
With respect to prior art, beneficial effect of the present invention is:
1, in the Bemisia tabaci defensin antibacterial peptide gene prokaryotic expression process in the present invention, in expression vector, introduce proteolytic enzyme Xa factor, effectively enzyme is cut recombination fusion protein, and utilizes ultrafiltration mode sharp separation to obtain defensin polypeptide according to protein molecular size.Whole process is simple fast.
2, the restructuring Bemisia tabaci defensin antibacterial peptide in the present invention has anti-mycotic activity, can obviously suppress the insect pathogenic fungus growths such as beauveria bassiana (Beuaveria bassiana), Metarhizium anisopliae (Metarhizium anisopliae) and botrytis cinerea (Botrytis cinerea).
Accompanying drawing explanation
Fig. 1: different hidden kind of Bemisia tabaci antibacterial peptide defensin gene PCR amplified production electrophorograms.In Fig. 1, M is DNA maker, and 1-5 is respectively Bemisia tabaci MEAM1, Med, ZHJ1, ZHJ2 and ZHJ3 antibacterial peptide defensin gene PCR amplified production, and bp represents base pair.
Fig. 2: Bemisia tabaci antibacterial peptide gene and coli expression carrier plasmid pET-32a and defensin mature peptide gene PCR product cleavage map.In Fig. 2, M is DNA maker, 1 be plasmid pET-32a through BamH I and Xho I double digestion after product, 2 and 3 is that defensin mature peptide gene PCR product is through BamH I and Xho I double digestion after product.
Fig. 3: the structure schematic diagram of Bemisia tabaci antibacterial peptide defensin mature peptide segment composition expression vector pET32a-Def.
The SDS-PAGE gel electrophoresis figure of the pET32a-Def-BL21 of Fig. 4: IPTG induction and the restructuring Bemisia tabaci antimicrobial peptide protein of purifying.M is albumen maker, and 1-6 represents different IP TG induced concentration 0mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1mM, and 7 represent recombinant protein Trx-Def after purifying, and kDa represents molecular weight of albumen.
Fig. 5: restructuring Bemisia tabaci antibacterial peptide defensin Tricine-SDS-PAGE gel electrophoresis figure.M is albumen maker, and 1 is defensin protein product.
Specific embodiments
The present invention sets about from Bemisia tabaci antibacterial peptide defensin gene, to its carry out can character, the research of genetic expression and location and the aspect such as recombinant expressed, obtained the restructuring alexin defensin of obligate anti-mycotic activity.
Bemisia tabaci antibacterial peptide defensin gene of the present invention, its nucleotides sequence is as shown in SEQ ID NO.1.Sequence signature: length: 624 base pairs; Type: nucleic acid; Chain: two strands; Topological framework: linearity; Molecule type: cDNA.
The antibacterial peptide of Bemisia tabaci defensin antibacterial peptide gene coding of the present invention, its amino acid is as shown in SEQ ID NO.2.Sequence signature: length: 76 amino acid; Type: amino acid; Chain: strand; Topological framework: linearity; Molecule type: protein.
The cloning process of Bemisia tabaci antibacterial peptide gene cDNA of the present invention is to take the total RNA reverse transcription of Bemisia tabaci to obtain cDNA as template, construction cDNA library, build antibacterial peptide database, transcribe the local Blast screening of group with Bemisia tabaci and obtain defensin Partial cDNA Sequence, then through the amplification of RACE method, obtain end sequence, finally by sequence assembly, obtain Bemisia tabaci antibacterial peptide defensin gene cDNA full length sequence.
The present invention is that to take Bemisia tabaci antibacterial peptide gene full-length gene double-stranded DNA be template for the antibacterial peptide mature peptide gene order of expressing Bemisia tabaci antibacterial peptide recombinant protein, through PCR method amplification, obtain, it derives from the corresponding gene fragment in Bemisia tabaci antibacterial peptide full-length gene region.
Expression vector establishment method of the present invention is according to a conventional method, by by the synthetic Bemisia tabaci antibacterial peptide gene of PCR method through enzyme cut with separation and purification after, be connected to same enzyme and cut having between corresponding restriction enzyme site (being BamH I and Xho I) after purifying, be built into the required expression vector that contains Bemisia tabaci antibacterial peptide gene.
The recombinant expression vector that the Bemisia tabaci antibacterial peptide gene that the above-mentioned bacillus coli gene expression vector that contains Bemisia tabaci antibacterial peptide gene of the present invention is preferentially synthesized by the present invention and coli expression carrier pET-32a build, called after pET32a-Def.
The intestinal bacteria recombinant bacterial strain that the present invention can express Bemisia tabaci antibacterial peptide gene preferentially transforms e. coli bl21 (DE3) by the expression vector pET32a-Def that contains Bemisia tabaci antibacterial peptide and the bacterial strain that obtains, called after pET32a-Def-BL21.
The concrete preparation method of above-mentioned Bemisia tabaci antibacterial peptide gene realizes by following technical solution: choose intestinal bacteria recombinant bacterial strain pET32a-Def-BL21 and be seeded in the LB liquid nutrient medium that 10ml contains penbritin, 37 ℃ of overnight incubation, get 50 μ l bacterium liquid and join 50ml AMP is housed
+in the aseptic triangular flask of 150ml of LB liquid nutrient medium, 37 ℃, shaking in case of 150rpm cultivated 6～8h to logarithmic phase, add 100mM IPTG to final concentration be 1mM, 27 ℃, 250rpm shaking culture.After growing into plateau, collects recombinant bacterium thalline, through the separation and purification Bemisia tabaci antibacterial peptide product that obtains recombinating.
Above-mentioned enzyme is cut rear non-fusion antibacterial peptide sterling, utilize to add Factor Xa sequence, uses Factor Xa enzyme to cut above-mentioned purification of Recombinant Bemisia tabaci antimicrobial peptide protein, and purifying again, obtains about 5kDa left and right Bemisia tabaci antibacterial peptide defensin peptide fragment.
The present invention utilizes the above-mentioned antibacterial peptide that obtains to carry out anti-microbial activity detection, and result shows: it is active that the Bemisia tabaci defensin antibacterial peptide after enzyme is cut purifying has anti-insect pathogenic fungus.
Bemisia tabaci antibacterial peptide gene defensin of the present invention applies in preparation has the recombinant protein of antibacterial peptide activity.
Wherein, the method for described application is by gene recombination technology, described gene to be expressed in intestinal bacteria, obtains the recombinant protein with anti-microbial activity.
For example: the Bemisia tabaci antibacterial peptide gene defensin gene clone obtaining, to pET-32a (Novagen company) expression vector, is transformed into e. coli bl21 cell.Carry out abduction delivering, obtain the activated recombinant protein of tool (being the antibacterial peptide of Bemisia tabaci antibacterial peptide gene defensin genes encoding).
The result of carrying out bacteriostatic experiment after the recombinant protein purification of described anti-microbial activity shows: the polypeptide of the Bemisia tabaci antibacterial peptide gene defensin genetic expression of restructuring has antimycotic and activity yeast.
Further, utilize method of the present invention to produce and also can be used for Bemisia tabaci biological control by existing gene engineering method.
Below in conjunction with the concrete experimental data in laboratory and in conjunction with specific embodiments, further set forth the present invention.These embodiment are only not used in and limit the scope of the invention for the present invention.The experimental program of unreceipted actual conditions in lower routine embodiment, conventionally according to normal condition, for example Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Haebor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement.
Case study on implementation 1: Bemisia tabaci defensin antibacterial peptide cDNA clone
1.1, TRIzol method is extracted total RNA
(1) by a certain amount of (200 left and right Bemisia tabaci) freezing 1min, put into 1.5ml centrifuge tube, add 1ml TRIzol reagent, fully grind homogenate, the standing 5min of room temperature.
(2) in centrifuge tube, add 0.2ml chloroform, vibration 15s, mixed solution proceeds in TIANGEN centrifuge tube, standing 2min.
(3) 4 ℃, the centrifugal 15min of 12000g, gets supernatant liquor, is proceeded in the centrifuge tube of a new 1.5ml.
(4) in centrifuge tube, add 0.5ml Virahol, liquid in pipe is mixed gently, the standing 10min of room temperature.
(5) 4 ℃, the centrifugal 10min of 12000g, discards supernatant liquor.
(6) in centrifuge tube, add 1ml 75% ethanol, washing precipitation gently, 4 ℃, the centrifugal 5min of 7500g, discards supernatant liquor.(now add dehydrated alcohol, can in-80 ℃ of medium-term and long-term preservations of Ultralow Temperature Freezer)
(7) dry centrifuge tube, add appropriate DEPC H
2o dissolves (65 ℃ of dissolutions), the value of spectrophotometer measurement OD260/OD280, and ratio between 1.8～2.0 time, carries out next step experiment.
1.2, cDNA the first chain is synthetic
(1) toward the RNA that adds respectively in the aseptic centrifuge tube of 0.5ml 1 μ l to extract, 1 μ l SMART IV/5 ' PCR forward primer, 1 μ l CDS III/3 ' PCR reverse primer, adds 2 μ l DEPC H
2o makes cumulative volume reach 5 μ l.
(2) reagent mixing in centrifuge tube is also of short duration centrifugal, hatches 2min for 72 ℃.
(3) rapidly by centrifuge tube ice bath 2min, of short duration centrifugal.
(4) in centrifuge tube, add respectively 2 μ l 5 * Frist-strand Buffer, 1 μ l 20mM dithiothreitol (DTT) (DTT), 1 μ l 10mM dNTP, 1 μ l Reverse Transcriptase ThermoScript II, piping and druming mixes repeatedly, of short duration centrifugal.
Hatch 1h for (5) 42 ℃.
(6) centrifuge tube is placed in to 2～3min on ice, termination reaction.Get a part for further experiment, all the other are in-80 ℃ of freezing preservations of Ultralow Temperature Freezer.
1.3, dsDNA is synthetic
(1) in 0.5 centrifuge tube, add respectively 1 μ l the first chain synthesis reaction product, 5 μ l 10 * LA Buffer (Mg
2+free), 5 μ l Mg
2+, 8 μ l dNTP, 1 μ l SMART IV/5 ' PCR forward primer, 1 μ lCDS III/3 ' PCR reverse primer, 0.5 μ l LA Tag archaeal dna polymerase, 29.5 μ l ddH
2o, fully mixes, of short duration centrifugal.
(2) in PCR instrument by following amplification program (95 ℃, 20s; 25～28 * (95 ℃, 5s; 68 ℃, 6min)) amplification.
(3) get 5 μ l PCR products for gel detection (the contained molecular weight ranges of test strip and band are bright dark), get the good 100 times of templates for PCR after doing of 1 μ l product dilution of detected result, all the other are in-80 ℃ of freezing preservations of Ultralow Temperature Freezer.
1.4, pcr amplification Bemisia tabaci antibacterial peptide defensin gene 5 ' and 3 ' end
The test kit SMARTer of Clontech company is pressed in operation
tMrACE cDNA Amplification Kit specification sheets carries out.According to the requirement of test kit, according to known Defensin gene est sequence, design respectively two upstream and downstream primers (in Table 1), to carry out nest-type PRC.PCR reaction conditions is for the first time: 98 ℃, and 3min; 30 * (98 ℃, 10s; 60 ℃, 20s; 68 ℃, 1min); 68 ℃, 6min.PCR be take for the first time PCR product and is increased as template for the second time, and reaction conditions is: 95 ℃, and 3min; 35 * (95 ℃, 25s; 63 ℃, 20s; 72 ℃, 1min); 72 ℃, 6min.Amplified production is connected on pMD-19 carrier and obtains recombinant plasmid, and with the universal primer M13 of pMD-19 ± carry out sequencing, measurement result and known array fragment compare and sequence assembly.Sequence assembly result is carried out BlastX on NCBI, and it is similar with maduca sexta Manduca sexta antibacterial peptide defensin gene height to greater wax moth Galleria mellonella that result shows to obtain sequence, and similarity is respectively 75% and 74%.
Table 1.PCR amplification Bemisia tabaci antibacterial peptide defensin gene 5 ' and the required primer of 3 ' end
| Primer title | Primer sequence (5 '-3 ') |
| def-for 1 | GTTGGTGCGAAACCCGTAAATAAGAT |
| def-for 2 | GGAGTGAACTACACCTCTGACTGCTTGAA |
| def- |
TGAACTACACCTCTGACTGCTTGAAAGA |
| def- |
TAAAGGCGGACATTGTGGAAGTGC |
Embodiment 2: Bemisia tabaci antibacterial peptide recombinant expression vector structure, expression and bacteria resistance function are measured
2.1 expression vector establishment
(1) according to the cloning site of the sequence of Bemisia tabaci defensin antibacterial peptide and expression vector pET-32a (Novagen company), design primer, and 5 '-end of gene has designed identification cleavage site (IEGR) encoding sequence of Xa factor:
Def-F:CG
gGATCCaTTGAGGGTCGCGACGTCCTCATCGGAAGTT (underscore is BamH I site, and runic is Xa factor encoding sequence);
Def-R:CCG
cTCGAGtTATTTACGGGTTTCGCACCAAC (underscore is XhoI site).
(2) gene amplification, clone and recombinant plasmid screening
Take Bemisia tabaci dsDNA as template, with above-mentioned primer, carry out PCR reaction, amplification condition is: 94 ℃, and 2min denaturation; 94 ℃, 15s, 63 ℃, 30s, 72 ℃, 45s, 35 circulations; 72 ℃ are extended 6min.
Get 2 μ lPCR products, after 1% agarose gel electrophoresis detects, the clean PCR product that reclaims of Clean UP test kit.Using the DNA of PET-32a plasmid vector and above-mentioned recovery respectively as DNA profiling, prepare following reaction system: Xho I restriction enzyme 1 μ l, BamH I restriction enzyme, DNA profiling 10 μ l, ddH
2o 6 μ l, Buffer 2 μ l.Mix gently, of short duration centrifugal, hatch 1h for 37 ℃.
(3) by the double digestion reaction product of second step, do agarose gel electrophoresis, cut sizeable DNA band and do DNA gel recovery.
(4) in PCR pipe, add respectively the PCR product after 2.5 μ l double digestions, the pET-32a plasmid vector after 0.5 μ l double digestion, the T4Ligase DNA of 0.5 μ l, the T4Ligase DNA Buffer of 1 μ l, the ddH of 5 μ l
2o, hatches under 16 ℃ of conditions, more than connecting 1h.
(5) connection product is transformed in competence, coats on the LB culture medium flat plate of penbritin 37 ℃ of overnight incubation.Choose spot and cultivate bacterium liquid, with T7ter and S-Tag universal primer, carry out r-Tag reaction system PCR and detect, for every part of positive, get 100 μ l and reserve seed for planting and send survey.
(6) comparison sequencing result, chooses the bacterium liquid of reserving seed for planting that result is correct and does next step experiment.
2.2 restructuring Bemisia tabaci defensin peptide expression and purifying
Choosing order-checking positive bacteria liquid is cultivating containing 37 ℃ of overnight shakings in the LB liquid nutrient medium of 50 μ g/mL penbritins, next day, inoculum size with 1% is connected to containing in the liquid nutrient medium of 150 μ g/mL penbritins, treat that thalli growth is to logarithmic phase, OD600 approximately 1.0 o'clock, add 100mM IPTG to make its final concentration be about 1mM, after 27 ℃/250rpm induction 5h, survey OD600 sampling, the centrifugal 5min of 5000rpm, PBS damping fluid (pH7.0) resuspension thalline also adds appropriate sample-loading buffer, and for 12%SDS-PAGE electrophoretic analysis protein expression situation, fusion rotein Trx-Def is with the form successful expression of solubility, finally utilize Clontech His-tag gravity purification kit purifying to obtain Bemisia tabaci defensin antibacterial peptide recombinant protein.The Bemisia tabaci defensin recombinant protein of purifying is through 15% SDS-PAGE gel electrophoresis analysis, and the recombinant protein that Trx-Def merges shows about 25kDa size, in the same size with expection.
2.3 recombinant protein Xa factor proteolytic cleavages
Protein concentration after A280 mensuration purifying, fusion rotein concentration in 50 μ L systems (0.2 μ g/ μ L), by following system cutting, Xa factor dosage is 2U, in 23 ℃ of reaction 24h.Enzyme is cut product and is first utilized the super filter tube (Millipore that molecular weight cut-off is 10kDa, Amicon Ultra-4) remove Trx label and Xa factor etc. and be greater than 10kDa protein molecular, then the super filter tube that is 3kDa with molecular weight cut-off carries out desalination and concentrated to it, adopts 16%Tricine-SDS-PAGE to analyze.
Cutting system is as follows:
2.5 anti-microbial activities detect
Substratum is YDP substratum, and strains tested has gram-positive microorganism, Gram-negative bacteria, insect pathogenic fungus and yeast.Wherein gram-positive microorganism is streptococcus aureus (Staphylococcus aureus); Gram-negative bacteria is Salmonella enteritidis (Salmonella enteritidis) and intestinal bacteria (Escherichia coli); Insect pathogenic fungus is beauveria bassiana (Beuaveria bassiana), Metarhizium anisopliae (Metarhizium anisopliae); Plant pathogenic fungi botrytis cinerea (Botrytis cinerea) yeast is pichia spp (pichia pastoris).By liquid growth inhibition test, undertaken this antibacterial tests, concrete steps are: at substratum, (bacteria culture medium is LB substratum to strains tested, fungi and yeast culture base are YDP substratum) in grow into OD600 and be 0.8 after, with YDP substratum, being diluted to OD600 is 0.001 left and right, in 80 μ L yeast culture things, adds the above-mentioned enzyme of 20 μ L different concns to cut protein product.At 37 ℃, cultivate 24 hours, measure mixed culture OD600 value, determine anti-microbial activity, each test repeats 3 times.Control group is respectively: fusion rotein Trx, Xa factor cutting 0h cutting liquid, penbritin.By growth inhibition test, result shows that defensin has the ability of antimycotic and yeast significantly.
The anti-microbial activity result of Bemisia tabaci defensin antibacterial peptide is as shown in table 2:
Table 2
Claims (3)
1. a Bemisia tabaci defensin antibacterial peptide gene, is characterized in that, the nucleotide sequence of this gene is as shown in SEQ ID NO.1.
2. described in claim 1, Bemisia tabaci defensin antibacterial peptide gene has the application in the recombinant protein of anti-microbial activity in preparation.
3. utilize Bemisia tabaci defensin antibacterial peptide gene preparation described in claim 1 to there is the method for the recombinant protein of anti-microbial activity, it is characterized in that, be by gene recombination technology, described gene to be expressed in intestinal bacteria, obtain the recombinant protein with anti-microbial activity.
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