CN105483131A - DsRNA (double-stranded ribonucleic acid) for silencing saliva toxic protein gene VTP of varroa destructor and application of dsRNA - Google Patents
DsRNA (double-stranded ribonucleic acid) for silencing saliva toxic protein gene VTP of varroa destructor and application of dsRNA Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 36
- 231100000331 toxic Toxicity 0.000 title claims abstract description 33
- 230000002588 toxic effect Effects 0.000 title claims abstract description 33
- 210000003296 saliva Anatomy 0.000 title claims abstract description 32
- 108091032973 (ribonucleotides)n+m Proteins 0.000 title claims description 28
- 102000040650 (ribonucleotides)n+m Human genes 0.000 title claims description 28
- 230000030279 gene silencing Effects 0.000 title abstract description 11
- 241001558516 Varroa destructor Species 0.000 title abstract description 6
- 229920002477 rna polymer Polymers 0.000 title abstract 2
- 239000002773 nucleotide Substances 0.000 claims abstract description 10
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 10
- 230000000295 complement effect Effects 0.000 claims abstract description 4
- 241000256844 Apis mellifera Species 0.000 claims description 32
- 230000004083 survival effect Effects 0.000 claims description 6
- 239000004615 ingredient Substances 0.000 claims description 4
- 201000002266 mite infestation Diseases 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000002265 prevention Effects 0.000 claims description 2
- 241000257303 Hymenoptera Species 0.000 abstract 1
- 238000000034 method Methods 0.000 description 10
- 230000008569 process Effects 0.000 description 9
- 238000010839 reverse transcription Methods 0.000 description 7
- 241000256846 Apis cerana Species 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 241000238876 Acari Species 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 238000012226 gene silencing method Methods 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 101150066002 GFP gene Proteins 0.000 description 2
- 241000382353 Pupa Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000009341 apiculture Methods 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 241001416380 Laelaps Species 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 206010067268 Post procedural infection Diseases 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 102000007365 Sialoglycoproteins Human genes 0.000 description 1
- 108010032838 Sialoglycoproteins Proteins 0.000 description 1
- 241001038073 Tropilaelaps mercedesae Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 210000000087 hemolymph Anatomy 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
- A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
- A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
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Abstract
The invention discloses dsRNA (double-stranded ribonucleic acid) for silencing a saliva toxic protein gene VTP of a varroa destructor and an application of the dsRNA. The dsRNA for silencing the saliva toxic protein gene VTP of the varroa destructor comprises a nucleotide sequence I represented as SEQ ID NO.1 and a nucleotide sequence II represented as a reverse complement sequence of the nucleotide sequence I. The dsRNA can be used for silencing the toxic protein gene of the varroa destructor, and accordingly, the harm of the varroa destructor to bees is reduced.
Description
Technical field:
The invention belongs to Biochemistry and Molecular Biology field, be specifically related to dsRNA and the application thereof of a kind of reticent Di Siwa mite saliva toxic protein gene VTP.
Background technology:
Di Siwa mite Varroadestructor (Anderson & Trueman, 2000) is the important epizoa of harm world's Apiculture, brings massive losses (Martinetal., 2004) to world's Apiculture and agricultural.Di Siwa mite can endanger honeybee capping larva, pupa and become honeybee, carry simultaneously and propagate honeybee virus, with the hot laelaps of Mei Shi (Tropilaelapsmercedesae) coinfection (Luoetal., 2011), thus cause colony productivity degradation, and even full group destroys.Di Siwa mite with the hemolymph of honeybee for food, the female mite that grows up is punched and feeds its filial generation on the epidermis of bee pupa, its sialoprotein can suppress hemagglutination, stops the wound healing of honeybee and reduces corresponding host response (Richardsetal., 2011).
From Di Siwa mite saliva, separation and purification is to one to honeybee tool virose Di Siwa mite saliva toxic protein VTP in this laboratory, and the nucleotide sequence of encoding gene-Di Siwa mite saliva toxic protein gene VTP is as shown in SEQIDNO.2.
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.
Accompanying drawing illustrates:
Fig. 1 is silencing efficiency mensuration figure, and wherein Vd-CK represents Di Siwa mite not through any process; Vd-0.9%NaCl represents Di Siwa mite and soaked massfraction 0.9%NaCl solution; Vd-dsGFP represents Di Siwa mite and soaked dsRNA-GFP; Vd-dsVTP represents that honeybee is infected by the Di Siwa mite soaking dsRNA-VTP solution.
Fig. 2 is that after silence, Di Siwa mite is to the toxicity detection figure of honeybee, and figure A is the mensuration to apis mellifera prepupa, and figure B is the toxicity test to apis cerana prepupa, and in figure, dsVTP represents that Di Siwa mite soaked dsRNA-VTP; DsGFP represents that Di Siwa mite soaked dsRNA-GFP; 0.9%NaCl represents that Di Siwa mite soaked massfraction 0.9%NaCl solution; Vd represents Di Siwa mite not through any process; NoVd: do not add the blank that watt mite is infected in honeybee prepupa.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
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 SMARTer
tM(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, ddH
2o37 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 pEASY
tMon-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 thus
tM-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 respectively
tMobtain 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%).
Claims (5)
1. a dsRNA of reticent Di Siwa mite saliva toxic protein gene VTP, 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.
2. the application of dsRNA in reticent Di Siwa mite saliva toxic protein gene VTP of reticent Di Siwa mite saliva toxic protein gene VTP according to claim 1.
3. a preparation of reticent Di Siwa mite saliva toxic protein gene VTP, is characterized in that, the dsRNA containing reticent Di Siwa mite saliva toxic protein gene VTP according to claim 1 is as effective ingredient.
4. the dsRNA of reticent Di Siwa mite saliva toxic protein gene VTP according to claim 1 improves the application in bee survival in Di Siwa mite infestationss honeybee situation.
5. prevention and control honeybee is by the preparation improving bee survival under Di Siwa mite infestationss, and it is characterized in that, the dsRNA containing reticent Di Siwa mite saliva toxic protein gene VTP according to claim 1 is as effective ingredient.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610046163.2A CN105483131A (en) | 2016-01-22 | 2016-01-22 | DsRNA (double-stranded ribonucleic acid) for silencing saliva toxic protein gene VTP of varroa destructor and application of dsRNA |
| PCT/CN2017/071717 WO2017125042A1 (en) | 2016-01-22 | 2017-01-19 | Dsrna for silencing saliva toxic protein gene vtp of varroa destructor mite and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610046163.2A CN105483131A (en) | 2016-01-22 | 2016-01-22 | DsRNA (double-stranded ribonucleic acid) for silencing saliva toxic protein gene VTP of varroa destructor and application of dsRNA |
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| WO2017125042A1 (en) * | 2016-01-22 | 2017-07-27 | 广东省生物资源应用研究所 | Dsrna for silencing saliva toxic protein gene vtp of varroa destructor mite and application thereof |
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| CN115851963B (en) * | 2022-07-18 | 2025-02-28 | 福州海关技术中心 | A primer-probe combination for detecting the honey bee thermolae mite and a detection method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101343637A (en) * | 2007-07-10 | 2009-01-14 | 中山大学 | Method for inhibiting gene expression in insects by feeding dsRNA |
| CN105085643A (en) * | 2014-05-21 | 2015-11-25 | 广东省昆虫研究所 | Varroa destructor toxic protein and coding gene thereof, and application thereof |
| CN105075987A (en) * | 2014-05-21 | 2015-11-25 | 广东省昆虫研究所 | A varroa destructor Anderson&Trueman saliva mixture and a preparation method and application thereof |
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| US20120157512A1 (en) * | 2009-08-21 | 2012-06-21 | Monsanto Technology Llc | Preventing and Curing Beneficial Insect Diseases Via Plant Transcribed Molecules |
| WO2011045796A1 (en) * | 2009-10-14 | 2011-04-21 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Compositions for controlling varroa mites in bees |
| ES3008698T3 (en) * | 2013-11-04 | 2025-03-24 | Greenlight Biosciences Inc | Compositions and methods for controlling arthropod parasite and pest infestations |
| UA119253C2 (en) * | 2013-12-10 | 2019-05-27 | Біолоджикс, Інк. | METHOD FOR VARROA TREATMENT AND VEGETABLES |
| CN105483131A (en) * | 2016-01-22 | 2016-04-13 | 广东省昆虫研究所 | DsRNA (double-stranded ribonucleic acid) for silencing saliva toxic protein gene VTP of varroa destructor and application of dsRNA |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101343637A (en) * | 2007-07-10 | 2009-01-14 | 中山大学 | Method for inhibiting gene expression in insects by feeding dsRNA |
| CN105085643A (en) * | 2014-05-21 | 2015-11-25 | 广东省昆虫研究所 | Varroa destructor toxic protein and coding gene thereof, and application thereof |
| CN105075987A (en) * | 2014-05-21 | 2015-11-25 | 广东省昆虫研究所 | A varroa destructor Anderson&Trueman saliva mixture and a preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 杨广等: "昆虫的RNA干扰", 《昆虫学报》 * |
| 郑继平: "《基因表达调控》", 31 August 2012, 中国科学技术大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017125042A1 (en) * | 2016-01-22 | 2017-07-27 | 广东省生物资源应用研究所 | Dsrna for silencing saliva toxic protein gene vtp of varroa destructor mite and application thereof |
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