CN105085643B - A kind of Di Siwa mites toxic protein and its encoding gene and application - Google Patents
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Abstract
The invention discloses a kind of Di Siwa mites toxic protein and its encoding gene and application.The present invention is cloned into new Di Siwa mite toxic proteins VMP encoding gene VMP from the genome of Di Siwa mites, its expression product Di Siwa mite toxic protein VMP have toxic action to honeybee, therefore can be applied to the Di Siwa mite toxic protein VMP of the present invention and its encoding gene and prepare in honeybee lethal drug.The present invention is also that the mechanism that honeybee is endangered in basic scientific research Di Siwa mites creates condition.
Description
Technical field：
The invention belongs to Biochemistry and Molecular Biology field, and in particular to a kind of Di Siwa mites toxic protein and its volume
Code gene and application.
Background technology：
Di Siwa mites are always one of most important harm of world's apiculture, and only Australia is not sent out in global range at present
Existing (Zhou Ting, 2005；Rosenkranz et al.,2010).Di Siwa mites suck honeybee into the body fluid of honeybee or larva, cause honey
Honeybee constitution declines, while reduces the cognitive ability of honeybee and sense of direction (Kralj et al., 2007), and reduction honeybee of turning out for work returns nest
Rate, shorten the life-span (Dainat et al., 2012) of honeybee, bee colony group's gesture is weak.Most importantly Di Siwa mites also propagate honey
Honeybee bacterium and virus disease (Tentcheva et al., 2004；Yan et al.,2009；Prisco et al.,2011,Ai
Et al., 2012), and by suppress host immune suppress reacting activation these latent virus (Yang＆Cox-foster, 2005；
Santillán-Galicia et al.,2008).Made in the honeybee bee colony collapse imbalance sick (CCD) that the states such as the U.S., Greece occur
It is panic into the world, cause the great attention of scientist, through studying its pathogenic factor (Cox-Foster relevant with the harm of honeybee mite
et al.,2007).Annual China's apiculture only caused by honeybee mite direct losses just reach several hundred million RMB, and because preventing and treating honeybee
Mite using medicine cause bee product pollute (Wang Qiang etc., 2006；Wu Jie etc., 2007；Week is graceful etc., and 2007) and the Di Siwa mite resistances to the action of a drug
And influence of the medicament residue to environment be even more can not estimate (Hu Fuliang etc., 2004；Huang Wencheng, 2005；Satta et al.,
2005；Zeng Zhi generals etc., 2007；Adamczyk et al.,2010；Damiani et al.,2011；González-Gómez et
al.,2012)。
With the diffusion of Di Siwa mites, and being mutually adapted between host, some apis mellifera Linnaeuses and Africanized Apis mellifera
(Africanized Apis mellifera) also show to the tolerances of Di Siwa mites (Aumeier et al., 2000；
Navajas et al.2008；Le Conte et al.,2011).Therefore, sight is gathered in honeybee pair by researcher again
In terms of the tolerance studies of Di Siwa mites, and think that effective cleaning behavior is most important tolerance factor (Moretto et
al.,2006；Calderón et al.,2009；Le Conte et al.,2011).But how cleaning behavior controls
It is smellBecause honeybee may make it that in the saliva that Di Siwa mites secrete when thorn sucks thing containing certain material
Perceive the presence of mite.And in fact middle honeybee only Di Siwa mites have just enter into honeycomb or it is mobile when strong reaction, and Di
(Rath, 1999) that honeybee can not perceive when this watt of mite transfixion, it is clear to compare Apis mellifera by molecular biology method
The transcript profile of clean honeybee does not also support that cleaning honeybee has stronger olfactory sensibility (Le Conte et al., 2011), and pointed out can
Can be honeybee herd immunity cause bee colony be resistant to Di Siwa mites.Et al. (2012) then think the cleaning of Apis mellifera
Behavior is based on the larva (Di Siwa mites and the common caused vestigial wing of DWV viruses) being damaged, and belongs to the immune response of honeybee.Make
Tolerance is produced during long-term be mutually adapted for the apis cerana and Di Siwa mites of original host, is received significant attention.
And extensive apis cerana (A.cerana) is raised in China seldom by the catastrophic collapse of Di Siwa mites.Greatly
Amount document shows that the apis cerana as original host produces tolerance with Di Siwa mites during long-term be mutually adapted,
Apis cerana by Di Siwa mites Population Control within 800/case, then can maintain bee colony stabilization (Penget al.,
1987a,b；Delfinado-Baker&Peng,1995；Rath,1999).Research shows, influences middle bee colony Nei Disiwa mite kinds
The factor of group's size mainly has three aspects：Di Siwa mites reproductive capacity (Reproduction), the anti-mite behavior of apis cerana with
And the natural death of Di Siwa mites.The factor for influenceing Di Siwa mite reproductive capacity has：1) fertility of female mite；2) capping of honeybee pupa
Go through the phase；3) attraction of bee larva；4) lair size.The anti-mite behavior of apis cerana has grooming (grooming
Behaviour) and cleaning behavior (Hygienic behaviour) (Rath, 1999).Early literatures (Rothenbuhler,
1964) it is narrower to the definition for cleaning behavior, honeybee " open room lid and remove infected disease pest " in referring to merely.But
It is this to uncap and remove behavior and be not specific to watt mite, but common cleaning behavior, for all foreign substances or
Larva of the person by harms of microbe.Later, Rath (1999) was supplemented this, and cleaning behavior is defined as " uncapping, removing
And bury larva " behavior.Honeybee can remove the dead larva in worker cell quickly and the dead larva in drone cell is difficult then quilt
Remove, the worker bee of middle honeybee allow the drone larvae of death to stay in the room in without going to uncap, (drone cell has thick cocoon to be also not easy dozen
Open), so as to bury by Di Siwa mite infestationss or ill lethal drone larvae.
The content of the invention：
It is an object of the invention to provide a kind of Di Siwa mite toxic protein VMP and its coding to honeybee with toxic action
Gene VMP.
The Di Siwa mite toxic protein VMP of the present invention, its amino acid sequence is as shown in SEQ ID NO.2.
The Di Siwa mite toxic proteins VMP of present invention encoding gene VMP, its nucleotide sequence such as SEQ ID NO.1 institutes
Show.It should be understood that, it is contemplated that the degeneracy of codon, on the premise of amino acid sequence is not changed, to above-mentioned encoding gene
Nucleotide sequence is modified, and is fallen within protection scope of the present invention.
The present invention is cloned into new Di Siwa mite toxic proteins VMP encoding gene from the genome of Di Siwa mites
VMP, its expression product Di Siwa mite toxic protein VMP have toxic action to honeybee, therefore can be by the Di Siwa of the present invention
Mite toxic protein VMP and its encoding gene, which are applied to, to be prepared in honeybee lethal drug.The present invention is also in basic scientific research
The mechanism that Di Siwa mites endanger honeybee creates condition.
Brief description of the drawings：
Fig. 1 is VMP gene cloning PCR electrophoretograms, and M represents Wide range DNA Marker, is respectively from top to bottom
6kb, 4kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp, 100bp, expand to obtain contains VMP genes
The DNA fragmentation of reading frame is 405bp (swimming lane 1)；
Fig. 2 is the SDS-PAGE of expressing protein VMP purifying, and M represents pre-dyed albumen marker, distinguished from top to bottom
Purifying is represented for 170kDa, 130kDa, 95kDa, 72kDa, 55kDa, 43kDa, 34kDa, 26kDa, 17kDa, 10kDa, Line1
Expressing protein VMP, elution albumen when Line2 is purifying VMP.
Fig. 3 is toxicity test analysis charts of the purifying expression Di Siwa mite toxic protein VMP to honeybee, and the VMP of purifying dilutes
For following concentration：10.08ng/ μ L, 2ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L inject apis cerana respectively, Italy
Honeybee worker bee prepupa and 5 age greater wax moths.About 0.2 μ L are injected per cephalont.It is control (1) with following processing：In the saliva of separation
Toxic protein (purified saliva protein), background expressing protein (background expression product) (Flow of (2) desalination
Through), (3) PBS, (4) do not inject the larva (CK) of any material.Often processing sets 3 repetitions, often repeatedly 8 cephalont.34
DEG C, 80%RH cultures.Postscript calculates the death rate within 11 days.Data processing：SPSS16.0, ANOVA, one-way analysis of variance
(Duncan).Fig. 4 is toxicity test aspect graphs of the purifying expression Di Siwa mite toxic protein VMP to honeybee, and the VMP of purifying dilutes
For following concentration：10.08ng/ μ L, 2ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L inject apis cerana respectively, Italy
Honeybee worker bee prepupa and 5 age greater wax moths.About 0.2 μ L are injected per cephalont.It is control with following processing：(1) in the saliva of separation
Toxic protein (purified saliva protein), (2) VMP expression bacterial strain background expressing protein (background expression production
Thing) (Flow through), (3) PBS, (4) do not inject the larva (CK) of any material.After 11 days, apis cerana and Italy
The representative configuration figure of honeybee different disposal.
Embodiment：
Following examples are to further explanation of the invention, rather than limitation of the present invention.In the following example not
It is usually conventional meanses well-known to those skilled in the art to indicate specific experiment condition and method, used technological means.
Embodiment 1：
First, the clone of toxin gene
Di Siwa mites about 60 are taken, (Invitrogen companies, its article No. are using TriZolReagent：15596026)
Total serum IgE is extracted, using the purity and amount of agarose gel electrophoresis and UV spectrophotometer measuring total serum IgE, takes 1 μ g total serum IgE
Starting reverse transcription reaction is done, the Reverse Transcriptase kit used is SMARTerTM RACE cDNA Amplification Kit
(Clontech, article No.：634923), the step of reverse transcription reaction obtains reverse transcription product with reference to the operation instruction of the kit.
Using reverse transcription product as template, the specific primer of VMP genes is designed：
VMPF1(5'ATGTTCAAACTTCTCGTTATCG3')
VMPR1(5'TTAGGAGGCGAGCGCCTGCTGGA3')
Performing PCR is entered using high-fidelity Taq enzyme, PCR reaction systems are：Reverse transcription product 1 μ L, 10xBuffer5 μ L, dNTP
(each2.5mM) 4 μ L, VMPF1 (10 μM) 1 μ L, VMPR1 (10 μM) 1 μ L, Taq enzyme (5U/ μ L) 1 μ L, ddH2O37μL.On ice
Mixed after sample-adding.PCR reaction conditions are：94℃5min；94 DEG C of 30sec, 44.5 DEG C of 30sec, 72 DEG C of 45sec, 30 circulations；72
℃5min.PCR amplifications obtain 405bp fragment.Electrophoresis result is as shown in Figure 1.The fragment is reclaimed using agarose gel electrophoresis
Afterwards, it is connected to pEASYTM(Beijing Quan Shi gold Bioisystech Co., Ltd, its article No. are-T1Simple carriers：CT111-01 on),
Specific steps are with reference to the carrier specification.The connection carrier is sequenced again, through analysis shows, the sequence contains an open reading
Frame, it is 405 bases, its sequence compares analysis as shown in SEQ ID NO.1, through BLAST and do not find similar sequences, by this gene
It is named as Di Siwa mite virulent genes VMP.Its encoding proteins has 134 amino acid residues, its sequence such as SEQ ID NO.2 institutes
Show, analyzed through BLASTx, homology is up to 44% (XM_003740358.1), and the albumen is named as into Di Siwa mite toxicity eggs
White VMP.
2nd, embodiment 2：Transetta is converted, expresses VMP
With the pEASY containing Di Siwa mite virulent gene VMP gene cDNAs in embodiment 1TM- T1Simple recombinant plasmids
For template, primers F 1 (5 ' is designedGAATTCATGTTCAAACTTCTCGTTATCG3 ') (underscore represents EcoRI restriction enzyme sites)
With R1 (5 'AAGCTTTTAGGAGGCGAGCGCCTGCTGGA3 ') (underscore represents Hind III digestions site).Reaction system
It is：PEASY containing Di Siwa mite virulent gene VMP gene cDNAsTM- T1Simple recombinant plasmids (50ng/ μ L) 1 μ L,
10xBuffer5 μ L, dNTP (each2.5mM) 4 μ L, F1 (10 μM) 1 μ L, R1 (10 μM) 1 μ L, Taq enzyme (5U/ μ L) 1 μ L,
ddH2O37μL.Mixed after being loaded on ice.Reaction condition is：94℃5min；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 45sec,
30 circulations；72℃5min.405bp DNA fragmentation is obtained after PCR reactions, by the fragment purification, forward direction insertion expression vector
At pET-32a (+) EcoRI restriction enzyme sites.
PCR fragment restructuring is as follows into the flow of pET-32a (+) EcoR I restriction enzyme sites：1. take 5 μ g pET-32a
(+) plasmid, linearization for enzyme restriction processing is carried out using EcoR I and Hind III restriction enzymes, and the plasmid of linearisation is pure
Change, be dissolved in ddH2In O, make its final concentration of 50ng/ μ L；2. PCR fragment is dissolved in ddH after purification2In O, it is final concentration of to adjust its
50ng/μL；3. with reference to T4DNA Ligase (TAKARA, article No. D2011A) specification, restructuring coupled reaction is carried out.Specific step
Suddenly it is：Following coupled reaction system (cumulative volume is 20 μ L), PCR fragment 8 μ L, EcoRI and Hind III are prepared in centrifuge tube
μ L, 10 × T4DNALigase Buffer2 μ L, T4DNA the Ligase1 μ L of pET-32a (+) carrier 1 of linearization process,
ddH2O7μL.Mix, 16 DEG C of connections are overnight.4. the μ l of connection product 10 are taken to be added to the expressive host Escherichia coli impression of 50 μ l defrostings
In state cell Transetta DE3, coating is cultivated on the resistance LB plates containing carbenicillin (Car, 100 μ g/mL), positive bacteria
Fall and identify that primer is still F1 and R1 using regular colony PCR method.Positive colony send company's sequencing to reaffirm, Di Siwa mites
Virulent gene VMP is inserted in expression vector pET-32a (+), and is converted and entered expressive host competent escherichia coli cell
In Transetta DE3.
Thus obtain turning the expression bacterial strain for having the expression vector pET-32a (+) containing Di Siwa mite virulent gene VMP genes
Transetta DE3-pET-32a(+)-VMP。
3rd, toxic protein VMP expression and purifications
Picking turns the expression bacterial strain Transetta DE3-pET- for having the expression vector pET-32a (+) containing VMP genes
32a (+)-VMP is inoculated in 50mL LB (the g/ μ of μ containing Car100 L) fluid nutrient medium, and 37 DEG C of concussion and cultivates are stayed overnight.Switching 9mL kinds
Sub- liquid is in the 500mL triangular flasks of fresh 300mL (the g/ μ of μ containing Car100 L) LB fluid nutrient mediums, 37 DEG C of 180rpm cultures
It is 0.4-0.6 to OD values, adds a certain amount of IPTG (final concentration 0.2mM) and induce 6h at 16 DEG C.4 DEG C, 8000g, centrifuge 10min
Collect thalline；PBS washing precipitations in preweighted centrifuge tube, weigh after removing supernatant after centrifugation, every gram of thalline weight in wet base
Add 10mL protein purification solution, ice-bath ultrasonic ripple cracking thalline：300W, 4s, interval 6s are acted on, repeat 100 times and followed for one
Ring, totally two circulations.4 DEG C, 12000 × g, 10min is centrifuged, collects supernatant.Supernatant cross 0.22 μm of filter membrane go the removal of impurity with
Bubble, cross Ni- post (IMAC, BIO-RAD, article No.：7324612), purification step by specification is carried out, expression Di Si after purification
Through carrying out SDS-PAGE electrophoretic analysis (Fig. 2), concentration mensuration is Bradford methods, is using kit by watt mite toxic protein VMP
Bradford Protein Assay Kit (give birth to work, article No. in Shanghai：SK3041).Gained purifying expression Di Siwa mite toxic proteins
VMP concentration is 10.08ng/ μ L.
4th, toxicity tests of the VMP to honeybee
Above-mentioned purifying expression Di Siwa mite toxic proteins VMP is diluted to following concentration respectively：10.08ng/ μ L, 2ng/ μ
L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L, inject apis cerana, apis mellifera worker bee prepupa and 5 age greater wax moths respectively.
About 0.2 μ L are injected per cephalont.It is control with following processing：(1) toxic protein (the purified saliva in the saliva of separation
Protein), the background expressing protein (background expression product) (Flow through) of (2) desalination, (3) PBS, (4), which are not injected, appoints
The larva (CK) of what material.Often processing sets 3 repetitions, often repeatedly 8 cephalont.34 DEG C, 80%RH cultures.12 days postscripts are calculated dead
Rate.Data processing：SPSS16.0, ANOVA, one-way analysis of variance (Duncan), as a result find purifying expression Di Siwa mite poison
Property albumen VMP concentration>There is obvious lethal toxicity (Fig. 3,4) during 1.01ng/ μ L to apis cerana.
Claims (3)
1. a kind of Di Siwa mites toxic protein VMP, it is characterised in that its amino acid sequence is as shown in SEQ ID NO.2.
A kind of 2. encoding gene of the Di Siwa mite toxic proteins VMP described in claim 1, it is characterised in that its nucleotides sequence
Row are as shown in SEQ ID NO.1.
3. applications of the Di Siwa mite toxic protein VMP in apis cerana lethal drug is prepared described in claim 1.
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| CN101613684A (en) * | 2009-05-20 | 2009-12-30 | 广东省昆虫研究所 | Have the active chitinase of Di Siwa mite, serratia marcescens and the application thereof of killing |
| CN101613683A (en) * | 2009-05-20 | 2009-12-30 | 广东省昆虫研究所 | Have the active chitinase ChiB of Di Siwa mite and the application thereof of killing |
| CN101613682A (en) * | 2009-05-20 | 2009-12-30 | 广东省昆虫研究所 | Have the active chitinase ChiC1 of Di Siwa mite and the application thereof of killing |
| CN101613685A (en) * | 2009-05-20 | 2009-12-30 | 广东省昆虫研究所 | Have the active combined chitinase of Di Siwa mite and the application thereof of killing |
| EP2356901A1 (en) * | 2010-02-12 | 2011-08-17 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Induction of gene expression in arthropods |
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| US8962584B2 (en) * | 2009-10-14 | 2015-02-24 | Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. | Compositions for controlling Varroa mites in bees |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613684A (en) * | 2009-05-20 | 2009-12-30 | 广东省昆虫研究所 | Have the active chitinase of Di Siwa mite, serratia marcescens and the application thereof of killing |
| CN101613683A (en) * | 2009-05-20 | 2009-12-30 | 广东省昆虫研究所 | Have the active chitinase ChiB of Di Siwa mite and the application thereof of killing |
| CN101613682A (en) * | 2009-05-20 | 2009-12-30 | 广东省昆虫研究所 | Have the active chitinase ChiC1 of Di Siwa mite and the application thereof of killing |
| CN101613685A (en) * | 2009-05-20 | 2009-12-30 | 广东省昆虫研究所 | Have the active combined chitinase of Di Siwa mite and the application thereof of killing |
| EP2356901A1 (en) * | 2010-02-12 | 2011-08-17 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Induction of gene expression in arthropods |
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| 狄斯瓦螨生物学及生物防治研究进展;苏晓玲等;《蜜蜂杂志(月刊)》;20111231(第5期);第5-8页 * |
| 狄斯瓦螨综合防治方法最新研究进展;赵红霞等;《中国农学通报》;20111231;第27卷(第12期);第271-276页 * |
| 蜜蜂寄生螨—狄斯瓦螨的研究进展;张祎等;《环境昆虫学报》;20120331;第34卷(第3期);第345-353页 * |
| 蜜蜂抗狄斯瓦螨机制的研究进展;潘娇等;《应用昆虫学报》;20121231;第49卷(第5期);第1360-1365页 * |
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