CN102586262A - 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, reorganization 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 property, breaks out all over the world and causes disaster, and causes heavy loss example.The not only a large amount of feeding plant juice of this worm cause plant withered, and propagate various plants virus, and especially geminivirus infection often causes the viroses of plant to be very popular, and makes the serious underproduction of crop or total crop failure.Because the 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.
Owing to the abuse of traditional microbiotic and the continuous generation of Resistant strain, had a strong impact on the clinical therapeutic efficacy of infection.And that antibacterial peptide has a sterilization is strong, is difficult for causing advantages such as resistance becoming the new research focus of antibacterials.Yield of antibacterial peptides is low at present, and the separating difficulty of natural antibacterial peptide is big, thereby tends to utilize gene engineering method to obtain antibacterial peptide.
Alexin (defensin) is a cationoid antimicrobial polypeptide that extensively is formed in the bodies of aminal and plant of discovered in recent years, is made up of 29～54 amino-acid residues, contains 6～8 conservative halfcystines (normally 6), and molecular weight is less than 5KDa.The N end has 3 βZhuan Jiaos and 1 γ corner, and there is 1 amphipathic α spiral the centre, and the C end has 1 antiparallel βZhe Die, connects α spiral and βZhuan Jiao by 3～4 disulfide linkage, forms specific CS α beta structure.Alexin can synthesize in specific cell and be stored in the cytoplasmic particle; Or induced synthetic by pathogenic micro-organism; Multiple mikrobe all had stronger defence capability; Mainly act on the cytolemma of pathogenic micro-organism, make pathogenic micro-organism be difficult for it is produced resistance, alexin is the one type of candidate new antibacterials that has much magnetism at present.
Up to the present, the research report that does not still have Bemisia tabaci defensin antibacterial peptide both at home and abroad.
Summary of the invention
The technical problem that the present invention will solve is; To present Bemisia tabaci antibacterial peptide research situation and deficiency thereof; A kind of Bemisia tabaci antibacterial peptide defensin gene and its coded antimicrobial polypeptide of recombinating is provided, and said 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 shown in SEQ ID NO.1.
The present invention also provides said 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 said Bemisia tabaci defensin antibacterial peptide gene preparation to have the method for the recombinant protein of anti-microbial activity, is through recombinant gene said gene to be expressed in intestinal bacteria, obtains to have the recombinant protein of anti-microbial activity.
The present invention also provides the antibacterial peptide of said Bemisia tabaci defensin antibacterial peptide gene coding, and the aminoacid sequence of this antibacterial peptide is shown in SEQ ID NO.2.
The present invention also provides said antibacterial peptide to be used for preventing and treating the application of the medicine of Bemisia tabaci in preparation, and the Bemisia tabaci kind that this medicine is suitable for is: latent kind of Middle East-Asia Minor 1 (MEAM1), latent kind of a Mediterranean (Med), latent kind of a ZHJ1, latent kind of ZHJ2 or latent kind of a 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 among the present invention; Introduce the proteolytic enzyme Xa factor in the expression vector; Effectively enzyme is cut recombination fusion protein, and utilizes ultrafiltration mode sharp separation to obtain the defensin polypeptide according to the protein molecular size.Whole process is simple fast.
2, the reorganization Bemisia tabaci defensin antibacterial peptide among the present invention has anti-mycotic activity, can obviously suppress beauveria bassiana (Beuaveria bassiana), Metarhizium anisopliae (Metarhizium anisopliae) and the growth of botrytis cinerea insect pathogenic fungus such as (Botrytis cinerea).
Description of drawings
Fig. 1: different latent 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 synoptic diagram of Bemisia tabaci antibacterial peptide defensin mature peptide fragment fusion expression vector pET32a-Def.
Fig. 4: the SDS-PAGE gel electrophoresis figure of IPTG inductive pET32a-Def-BL21 and purified recombinant Bemisia tabaci antimicrobial peptide protein.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 behind the purifying, and kDa represents molecular weight of albumen.
Fig. 5: reorganization Bemisia tabaci antibacterial peptide defensin Tricine-SDS-PAGE gel electrophoresis figure.M is albumen maker, and 1 is the defensin protein product.
Specific embodiments
The present invention sets about from Bemisia tabaci antibacterial peptide defensin gene, but to its research of carrying out character, genetic expression and location and aspect such as recombinant expressed, has obtained the reorganization alexin defensin of obligate anti-mycotic activity.
Bemisia tabaci antibacterial peptide defensin gene according to the invention, its nucleotides sequence is 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 according to the invention, its amino acid is 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 according to the invention is that to obtain cDNA with the total RNA reverse transcription of Bemisia tabaci be template; The construction cDNA library; Make up the antibacterial peptide DB; Transcribe the local Blast screening of group with Bemisia tabaci and obtain the defensin Partial cDNA Sequence, obtain end sequence through the amplification of RACE method then, after sequence assembly obtains Bemisia tabaci antibacterial peptide defensin gene cDNA full length sequence.
The antibacterial peptide mature peptide gene order that the present invention is used to express Bemisia tabaci antibacterial peptide recombinant protein is to be template with Bemisia tabaci antibacterial peptide gene full-length gene double-stranded DNA; Obtain through the PCR method amplification, it derives from Bemisia tabaci antibacterial peptide full-length gene area relative gene fragment.
Expression vector establishment method of the present invention is by ordinary method; Will through PCR method synthetic Bemisia tabaci antibacterial peptide gene through enzyme cut with separation and purification after; Be connected to same enzyme and cut having between the corresponding restriction enzyme site (being BamH I and Xho I) behind the purifying, promptly be built into the required expression vector that contains the Bemisia tabaci antibacterial peptide gene.
The recombinant expression vector that the above-mentioned bacillus coli gene expression vector that contains the Bemisia tabaci antibacterial peptide gene of the present invention is preferentially made up by synthetic Bemisia tabaci antibacterial peptide gene of the present invention and coli expression carrier pET-32a, called after pET32a-Def.
The present invention can express the bacterial strain that the intestinal bacteria recombinant bacterial strain of Bemisia tabaci antibacterial peptide gene is preferentially obtained by the expression vector pET32a-Def transformed into escherichia coli BL21 (DE3) that contains the Bemisia tabaci antibacterial peptide, called after pET32a-Def-BL21.
The concrete preparation method of above-mentioned Bemisia tabaci antibacterial peptide gene realizes through following technical scheme: 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 cultures are got 50 μ l bacterium liquid and are joined 50ml AMP is housed
+In the aseptic triangular flask of the 150ml of LB liquid nutrient medium, 37 ℃, shaking of 150rpm cultivated 6～8h to logarithmic phase in the case, add 100mM IPTG to final concentration be 1mM, 27 ℃, the 250rpm shaking culture., the reorganization bacteria growing collects thalline after getting into plateau, through the separation and purification Bemisia tabaci antibacterial peptide product that obtains recombinating.
Above-mentioned enzyme is cut back non-fusion antibacterial peptide pure article, utilizes and adds Factor Xa sequence, uses Factor Xa enzyme to cut above-mentioned purification of Recombinant Bemisia tabaci antimicrobial peptide protein, and purifying once more, obtains about 5kDa left and right sides Bemisia tabaci antibacterial peptide defensin peptide fragment.
The present invention utilizes the above-mentioned antibacterial peptide that obtains to carry out the anti-microbial activity detection, and the result shows: the Bemisia tabaci defensin antibacterial peptide that the process enzyme is cut behind the purifying has anti-insect pathogenic fungus activity.
Bemisia tabaci antibacterial peptide gene defensin according to the invention has in the active recombinant protein of antibacterial peptide in preparation and uses.
Wherein, the method for said application is through recombinant gene said gene to be expressed in intestinal bacteria, obtains to have the recombinant protein of anti-microbial activity.
For example: the Bemisia tabaci antibacterial peptide gene defensin gene clone that obtains to pET-32a (Novagen company) expression vector, is transformed into the e. coli bl21 cell.Carry out abduction delivering, obtain to have active recombinant protein (being the antibacterial peptide of Bemisia tabaci antibacterial peptide gene defensin genes encoding).
The result who carries out bacteriostatic experiment behind the recombinant protein purification of said anti-microbial activity shows: the polypeptide of the Bemisia tabaci antibacterial peptide gene defensin genetic expression of reorganization has antimycotic active with zymic.
Further, utilize method of the present invention to produce and also can be used for the Bemisia tabaci biological control through existing gene engineering method.
Below in conjunction with laboratory concrete experimental data and combination specific embodiment, further set forth the present invention.These embodiment only are used for the present invention and are not used in restriction scope of the present invention.The experimental program of unreceipted actual conditions among the following routine embodiment; Usually according to normal condition; For example the 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.Should be pointed out that for the person of ordinary skill of the art, under the prerequisite that does not break away from the present invention's design, can also make some distortion and improvement.
Case study on implementation 1: Bemisia tabaci defensin antibacterial peptide cDNA clone
1.1, the TRIzol method extracts total RNA
(1) with a certain amount of (200 left and right sides Bemisia tabaci) freezing 1min, put into the 1.5ml centrifuge tube, add 1ml TRIzol reagent, fully grind homogenate, room temperature leaves standstill 5min.
(2) in centrifuge tube, add the 0.2ml chloroform, vibration 15s, mixed solution changes in the TIANGEN centrifuge tube, leaves standstill 2min.
(3) 4 ℃, the centrifugal 15min of 12000g gets supernatant, and it is changed in the centrifuge tube of a new 1.5ml.
(4) in centrifuge tube, add the 0.5ml Virahol, liquid mixing gently in will managing, room temperature leaves standstill 10min.
(5) 4 ℃, the centrifugal 10min of 12000g discards supernatant.
(6) in centrifuge tube, add 1ml 75% ethanol, washing precipitation gently, 4 ℃, the centrifugal 5min of 7500g discards supernatant.(add absolute ethyl alcohol this moment, can preserve) in-80 ℃ of Ultralow Temperature Freezers are medium-term and long-term
(7) dry centrifuge tube, add an amount of DEPC H
2O dissolving (65 ℃ of short dissolving), the value of spectrophotometer measurement OD260/OD280, ratio carries out next step experiment between 1.8～2.0 the time.
1.2, cDNA first chain is synthetic
(1) in the aseptic centrifuge tube of 0.5ml, add the RNA that 1 μ l extracts respectively, 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 TV reach 5 μ l.
(2) reagent in the mixing centrifuge tube and of short duration centrifugal is hatched 2min for 72 ℃.
(3) rapidly with centrifuge tube ice bath 2min, of short duration centrifugal.
(4) in centrifuge tube, add 2 μ l, 5 * Frist-strand Buffer respectively, 1 μ l 20mM WR 34678 (DTT), 1 μ l 10mM dNTP, 1 μ l Reverse Transcriptase ThermoScript II is blown and beaten mixing repeatedly, and is of short duration centrifugal.
Hatch 1h for (5) 42 ℃.
(6) centrifuge tube is placed 2～3min on ice, termination reaction.Get a part and be used 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, adds 1 μ l, the first chain synthesis reaction product respectively, 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 mixing is of short duration centrifugal.
(2) in the PCR appearance by following amplification program (95 ℃, 20s; 25～28 * (95 ℃, 5s; 68 ℃, 6min)) amplification.
(3) get 5 μ l PCR products and be used for gel detection (contained molecular weight ranges of test strip and band bright dark), get 100 times of the good 1 μ l product dilutions of detected result be used to do after the template of PCR, 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, design two upstream and downstream primers (seeing table 1) respectively according to known Defensin gene est sequence, to carry out nest-type PRC.The PCR reaction conditions is for the first time: 98 ℃, and 3min; 30 * (98 ℃, 10s; 60 ℃, 20s; 68 ℃, 1min); 68 ℃, 6min.For the second time PCR with the first time PCR product be that template increases, reaction conditions is: 95 ℃, 3min; 35 * (95 ℃, 25s; 63 ℃, 20s; 72 ℃, 1min); 72 ℃, 6min.Amplified production is connected on the pMD-19 carrier obtains recombinant plasmid, and, measure result and known array fragment and compare and sequence assembly with the universal primer M13 of pMD-19 ± carry out sequencing.The sequence assembly result carries out BlastX on NCBI, the result shows that to obtain sequence similar with maduca sexta Manduca sexta antibacterial peptide defensin gene height with greater wax moth Galleria mellonella, and similarity is respectively 75% and 74%.
Table 1.PCR amplification Bemisia tabaci antibacterial peptide defensin gene 5 ' and 3 ' terminal required primer
| The primer title | Primer sequence (5 '-3 ') |
| def-for?1 | GTTGGTGCGAAACCCGTAAATAAGAT |
| def-for?2 | GGAGTGAACTACACCTCTGACTGCTTGAA |
| def-rev?1 | TGAACTACACCTCTGACTGCTTGAAAGA |
| def-rev?2 | 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 sequence of Bemisia tabaci defensin antibacterial peptide and the cloning site of expression vector pET-32a (Novagen company), the 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 the Xa factor encoding sequence);
Def-R:CCG
CTCGAGTTATTTACGGGTTTCGCACCAAC (underscore is the XhoI site).
(2) gene amplification, clone and recombinant plasmid screening
With Bemisia tabaci dsDNA is template, carries out the PCR reaction with above-mentioned primer, and amplification condition is: 94 ℃, and the preparatory sex change of 2min; 94 ℃, 15s, 63 ℃, 30s, 72 ℃, 45s, 35 circulations; 72 ℃ are extended 6min.
Get 2 μ lPCR products, after 1% agarose gel electrophoresis detected, Clean UP test kit cleaning reclaimed the PCR product.Respectively with the DNA of PET-32a plasmid vector and above-mentioned recovery 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.Mixing is of short duration centrifugal gently, hatches 1h for 37 ℃.
(3) do agarose gel electrophoresis with the double digestion reaction product in second step, downcut sizeable DNA band and do the dna gel recovery.
(4) the PCR product after in PCR pipe, adding 2.5 μ l double digestions respectively, the pET-32a plasmid vector behind the 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 is hatched under 16 ℃ of conditions, connects more than the 1h.
(5) will connect product and be transformed in the competence, coat on the LB culture medium flat plate of penbritin 37 ℃ of overnight cultures.Choose spot culture bacteria liquid, carry out r-Tag reaction system PCR with T7ter and S-Tag universal primer and detect, get 100 μ l for every part of positive and reserve seed for planting and send survey.
(6) the comparison sequencing result is chosen the correct bacterium liquid of reserving seed for planting of result and is done next step experiment.
2.2 reorganization Bemisia tabaci defensin peptide expression and purifying
Choose order-checking positive bacteria liquid 37 ℃ of overnight shakings in the LB liquid nutrient medium that contains 50 μ g/mL penbritins and cultivate, next day, the inoculum size with 1% is connected in the liquid nutrient medium that contains 150 μ g/mL penbritins; Treat that thalli growth is to logarithmic phase; About 1.0 o'clock of OD600 adds 100mM IPTG and makes its final concentration be about 1mM, after 27 ℃/250rpm induces 5h; Survey the OD600 sampling; The centrifugal 5min of 5000rpm, PBS damping fluid (pH7.0) resuspension thalline also adds an amount of sample-loading buffer, and is used for 12%SDS-PAGE electrophoretic analysis protein expression situation; Fusion rotein Trx-Def utilizes Clontech His-tag gravity purification kit purifying to obtain Bemisia tabaci defensin antibacterial peptide recombinant protein with the form successful expression of solubility at last.The SDS-PAGE gel electrophoresis analysis of the Bemisia tabaci defensin recombinant protein warp 15% of purifying, the recombinant protein that Trx-Def merges show about 25kDa size, and be big or small consistent with expection.
2.3 recombinant protein Xa factor proteolytic cleavage
Protein concentration behind the A280 mensuration purifying, fusion rotein concentration in the 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 at first utilized the ultrafiltration pipe (Millipore of molecular weight cut-off for 10kDa; Amicon Ultra-4) removes Trx label and Xa factor etc. greater than the 10kDa protein molecular; Use molecular weight cut-off it to be carried out desalination and concentrate employing 16%Tricine-SDS-PAGE analysis then as the ultrafiltration pipe of 3kDa.
The cutting system is following:
2.5 anti-microbial activity detects
Substratum is the 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 enteron aisle Salmonellas (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).Undertaken this antibacterial tests by the liquid growth inhibition test; Concrete steps are: (bacteria culture medium is the LB substratum to strains tested at substratum; Fungi and yeast culture base are the YDP substratum) in grow into OD600 and be 0.8 after; Using the YDP substratum to be diluted to OD600 is about 0.001, and the above-mentioned enzyme that adds 20 μ L different concns in the 80 μ L yeast culture things is cut protein product.Cultivated 24 hours at 37 ℃, measure mixed culture OD600 value, confirm anti-microbial activity, each test repetition 3 times.Control group is respectively: fusion rotein Trx, Xa factor cutting 0h cutting liquid, penbritin.Through growth inhibition test, the result shows that defensin has antimycotic significantly and the zymic ability.
The anti-microbial activity result of Bemisia tabaci defensin antibacterial peptide is as shown in table 2:
Table 2
Claims (5)
1. a Bemisia tabaci defensin antibacterial peptide gene is characterized in that, the nucleotide sequence of this gene is shown in SEQ ID NO.1.
2. the said Bemisia tabaci defensin antibacterial peptide gene of claim 1 has the application in the recombinant protein of anti-microbial activity in preparation.
3. utilize the said Bemisia tabaci defensin antibacterial peptide gene preparation of claim 1 to have the method for the recombinant protein of anti-microbial activity; It is characterized in that; Be said gene to be expressed in intestinal bacteria, obtain to have the recombinant protein of anti-microbial activity through recombinant gene.
4. the antibacterial peptide of the said Bemisia tabaci defensin antibacterial peptide gene coding of claim 1 is characterized in that the aminoacid sequence of this antibacterial peptide is shown in SEQ ID NO.2.
5. the said antibacterial peptide of claim 4 is used for preventing and treating the application of the medicine of Bemisia tabaci in preparation, and the Bemisia tabaci kind that this medicine is suitable for is: latent kind of a Middle East-Asia Minor 1, latent kind of a Mediterranean, latent kind of a ZHJ1, latent kind of ZHJ2 or latent kind of a ZHJ3.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480115A (en) * | 2014-12-07 | 2015-04-01 | 浙江大学 | Bemisia tabaci defensin gene segment and dsRNA thereof based on gene silencing technology |
| CN105274007A (en) * | 2015-07-27 | 2016-01-27 | 华南农业大学 | Metarhizium anisopliae var. microsporus MaTS02 and application thereof in prevention and treatment of bemisia tabaci |
| CN106854657A (en) * | 2016-12-20 | 2017-06-16 | 广州格拉姆生物科技有限公司 | It is a kind of assistant degradation protein and the prebiotic recombinant Saccharomyces cerevisiae of antibacterial peptide to be secreted |
| CN106884007A (en) * | 2017-03-05 | 2017-06-23 | 中国农业科学院植物保护研究所 | Hidden kind of laccase LAC1 of Bemisia tabaci MED and its gene and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101220377A (en) * | 2008-01-23 | 2008-07-16 | 山东省农业科学院高新技术研究中心 | Method for horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius |
| CN101555521A (en) * | 2009-05-08 | 2009-10-14 | 中国农业科学院植物保护研究所 | Pair of Q-type bemisa babaci specificity SCAR primers, fast PCR detection method and kit |
| JP2010193755A (en) * | 2009-02-24 | 2010-09-09 | Sumitomo Chemical Co Ltd | Method for discriminating bemisia tabaci biotype |
-
2012
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101220377A (en) * | 2008-01-23 | 2008-07-16 | 山东省农业科学院高新技术研究中心 | Method for horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius |
| JP2010193755A (en) * | 2009-02-24 | 2010-09-09 | Sumitomo Chemical Co Ltd | Method for discriminating bemisia tabaci biotype |
| CN101555521A (en) * | 2009-05-08 | 2009-10-14 | 中国农业科学院植物保护研究所 | Pair of Q-type bemisa babaci specificity SCAR primers, fast PCR detection method and kit |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480115A (en) * | 2014-12-07 | 2015-04-01 | 浙江大学 | Bemisia tabaci defensin gene segment and dsRNA thereof based on gene silencing technology |
| CN105274007A (en) * | 2015-07-27 | 2016-01-27 | 华南农业大学 | Metarhizium anisopliae var. microsporus MaTS02 and application thereof in prevention and treatment of bemisia tabaci |
| CN105274007B (en) * | 2015-07-27 | 2018-11-27 | 华南农业大学 | One plant of Metarhizium anisopliae var. Anisopliae MaTS02 and its application in terms of preventing and treating Bemisia tabaci |
| CN106854657A (en) * | 2016-12-20 | 2017-06-16 | 广州格拉姆生物科技有限公司 | It is a kind of assistant degradation protein and the prebiotic recombinant Saccharomyces cerevisiae of antibacterial peptide to be secreted |
| CN106884007A (en) * | 2017-03-05 | 2017-06-23 | 中国农业科学院植物保护研究所 | Hidden kind of laccase LAC1 of Bemisia tabaci MED and its gene and application |
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