Summary of the invention:
First object of the present invention is to provide a kind of dsRNA Di Siwa mite saliva toxic protein gene to extraordinary silencing efficiency.
The dsRNA of reticent Di Siwa mite saliva toxic protein gene VTP of the present invention, is characterized in that, the double-stranded RNA be made up of the nucleotide sequence shown in the nucleotide sequence shown in SEQIDNO.1 He its reverse complementary sequence.
Second object of the present invention is to provide the application of above-mentioned dsRNA in reticent Di Siwa mite saliva toxic protein gene VTP.
A preparation of reticent Di Siwa mite saliva toxic protein gene VTP, is characterized in that, containing above-mentioned dsRNA as effective ingredient.
3rd object of the present invention is to provide above-mentioned dsRNA in Di Siwa mite infestationss honeybee situation, improves application in bee survival.
Prevention and control honeybee, by the preparation improving bee survival under Di Siwa mite infestationss, is characterized in that, containing above-mentioned dsRNA as effective ingredient.
The present invention found through experiments, the dsRNA of reticent Di Siwa mite saliva toxic protein gene VTP of the present invention can reticent Di Siwa mite saliva toxic protein gene VTP, be immersed in the Di Siwa mite in the dsRNA of reticent Di Siwa mite saliva toxic protein gene VTP compared with the control, the mRNA content of its body Nei Disiwa mite saliva toxic protein gene VTP significantly reduces, and last till that honeybee prepupa sprouts wings, Di Siwa mite toxicity after reticent Di Siwa mite saliva toxic protein gene VTP significantly reduces, compared with the control, its prepupa survival rate infecting rear Apis mellifera significantly improves, and the mortality ratio of apis cerana prepupa also significantly reduces.Therefore by the toxic protein gene of reticent for dsRNA of the present invention Di Siwa mite, thus reduction Di Siwa mite can be reached to the harm of honeybee.
Embodiment 1:
1. the collection of Di Siwa mite and honeybee: the Di Siwa mite female mite that grows up is collected in APIS CERANA room, is placed in the sterile petri dish of built-in 9cm filter paper after taking-up, for subsequent use.Honeybee prepupa is then placed in 48 well culture plates being lined with aseptic filter paper, and 1 prepupa is put in every hole, for subsequent use.
2. the clone of Di Siwa mite saliva toxic protein gene VTP
Get Di Siwa mite about 60, adopt TriZolReagent (Invitrogen company, its article No. is: 15596026) extract total serum IgE, adopt purity and the amount of agarose gel electrophoresis and UV spectrophotometer measuring total serum IgE, the total serum IgE getting 1 μ g does initial reverse transcription reaction, and the Reverse Transcriptase kit of employing is SMARTertM(step of reverse transcription reaction, with reference to the operation instruction of this test kit, obtains reverse transcription product to RACEcDNAAmplificationKit for Clontech, article No.: 634923).
Take reverse transcription product as template, the Auele Specific Primer of design VTP gene:
VTPF1(5'ATGTTCAAACTTCTCGTTATCG3')
VTPR1(5'TTAGGAGGCGAGCGCCTGCTGGA3')
Employing high-fidelity Taq enzyme is carried out PCR, PCR reaction system and is: reverse transcription product 1 μ L, 10xBuffer5 μ L, dNTP (each2.5mM) 4 μ L, VTPF1 (10 μMs) 1 μ L, VTPR1 (10 μMs) 1 μ L, Taq enzyme (5U/ μ L) 1 μ L, ddH2o37 μ L.Mix after application of sample on ice.PCR reaction conditions is: 94 DEG C of 5min; 94 DEG C of 30sec, 44.5 DEG C of 30sec, 72 DEG C of 45sec, 30 circulations; 72 DEG C of 5min.Pcr amplification obtains the fragment of 405bp.After adopting agarose gel electrophoresis to reclaim this fragment, be connected to pEASYtMon-T1Simple carrier (Beijing Quan Shi gold Bioisystech Co., Ltd, its article No. is: CT111-01), concrete steps are with reference to this carrier specification sheets.This connection carrier checked order, show by analysis, this sequence contains an open reading frame, be 405 bases, its sequence is as shown in SEQIDNO.1, and called after Di Siwa mite saliva toxic protein gene VTP, obtains the pEASY being inserted with Di Siwa mite saliva toxic protein gene VTP thustM-T1Simple carrier, called after pEASY-T-VTP.
3.dsRNA synthesizes
(1) primer: T7-dsVTP-F:tAATACGACTCACTATAGGGAGAaTGTTCAAACTTCTCGTTATCG
T7-dsVTP-R:TAATACGACTCACTATAGGGAGATTAGGAGGCGAGCGCCTGCTGGA
Wherein the region of underscore mark is T7 promoter sequence;
(2) negative control primer:
T7-GFP-F：TAATACGACTCACTATAGGGCGATCAAGAAGGACCATGTGGTC；
T7-GFP-R：TAATACGACTCACTATAGGGCGATTCCATGGCCAACACTTGTCC
Wherein the region of underscore mark is T7 promoter sequence;
(3) template prepares: with pEASY-T-VTP (or the reverse transcription product in above-mentioned steps 2) and pEASY-T-GFP, (GFP gene is inserted into pEASY respectivelytMobtain in-T1Simple carrier) be template, increase respectively with above-mentioned primer, obtain corresponding PCR primer, after rubber tapping purifying, concentration reaches 0.5-1.0 μ g/ μ L.
(4) DsRNA synthesis presses test kit specification sheets (MEGAscriptKit with purifying, AM1330, Ambion), the reticent dsRNA (dsVTP) of Di Siwa mite saliva toxic protein gene VTP and the dsRNA (dsGFP) of reticent GFP gene is obtained respectively.Wherein the dsRNA of reticent Di Siwa mite saliva toxic protein gene VTP is through order-checking, and result shows, its double-stranded RNA for being made up of the nucleotide sequence shown in the nucleotide sequence shown in SEQIDNO.1 He its reverse complementary sequence.。
4. import dsRNA
Reference Campbelletal., 2010, with infusion method, dsRNA (dsVTP or dsGFP) is directed into adult female mite, concrete steps are as follows:
(1) DsRNA massfraction 0.9%NaCl is mixed with 2.5 μ g/ μ L, fills 10 μ L, put adult female mite 10 in the centrifuge tube of 500 μ L;
(2) 16 DEG C spend the night (about 15h), is placed on after taking-up on aseptic filter paper, treats that it rejuvenates.Watt mite of bringing back to life is used for subsequent experimental process.
(3) process: the female mite that grows up is soaked in Di Siwa mite
A.dsVTP；
B.dsGFP；
C.0.9％NaCl；
D. do not soak;
(4) often process 3 repetitions, often repeat 20 female mites.
5. silencing efficiency measures:
(1) in the mite alive after gene silencing, the silencing efficiency of VTP measures: the mite of above-mentioned each process, taking-up is placed on it APIS CERANA prepupa, every larva puts 2 mites with it, then ParafilmTM is used, sealed membrane pricks 10 holes, be placed in artificial culture case (34 DEG C, RH75%) to cultivate until prepupa pupates to adult.After distinguishing interval 0d, 3d, 7d and 13d during this period, get 8 mites alive and extract total serum IgE, utilize qRT-PCR to check Gene silencing efficacy.Experiment repetition twice.Concrete steps are as follows: utilize Trizol to extract the total serum IgE of watt mite, then with DNaseI process digested genomic dna, then reverse transcription obtains cDNA, with the mRNA relative content of this cDNA for the Di Siwa mite saliva toxic protein gene VTP in template detection respective handling.QPCR primer: VdVTP-qF (AACGCATTCAAGACTACATCACCAA) andVdVTP-qR (CTTTGACAACGTTCTCCTTCTGCT), reference gene is the actin gene of Di Siwa mite, and its primer is: VdActinF:CATCACCATTGGTAACGAG and VdActinR:CGATCCAGACGGAATACTT.Amplification condition: 95 DEG C, 5min; 95 DEG C, 15s, 58 DEG C, 30s, 72 DEG C, 30s, 40 circulations of increasing.
Result as shown in Figure 1, as can be seen from Figure 1, be immersed in goal gene mRNA content in its body of Di Siwa mite in dsRNA-dsVTP compared with the control significantly to reduce, when 0d, the mRNA relative content of watt mite body Nei Disiwa mite saliva toxic protein gene VTP is only 0.9% of contrast, be 1.3% after 3d, and after 7d, be 20.8%, and last till that honeybee prepupa sprouts wings, be namely 32.4% after 13d.
(2) toxicity test of the mite alive after gene silencing: the mite alive of above-mentioned each process, taking-up is placed on it Apis mellifera or middle honeybee worker bee prepupa, every larva puts 2 mites with it, then ParafilmTM is used, sealed membrane pricks 10 holes, be placed in artificial culture case (34 DEG C, RH75%) to cultivate until prepupa pupates to adult.Often process 3 repetitions, often repeat 6 prepupa.Calculate surviving rate and the vestigial wing rate of Apis mellifera respectively, the mortality ratio of middle honeybee and surviving rate.
Result as shown in Figure 2, as can be seen from Figure 2, the prepupa of the reticent postoperative infection Apis mellifera of Di Siwa mite body Nei Disiwa mite saliva toxic protein gene VTP, its toxicity reduces, the survival rate of Apis mellifera significantly improves (72.2%), but vestigial wing rate is compared in contrast does not have significant difference, and the mortality ratio of apis cerana prepupa also significantly reduces (41.1%).