CN105969808A - Application of plutella xylostella proteasome subunit alpha6 gene to control of resistance to deltamethrin - Google Patents
Application of plutella xylostella proteasome subunit alpha6 gene to control of resistance to deltamethrin Download PDFInfo
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- CN105969808A CN105969808A CN201610304811.XA CN201610304811A CN105969808A CN 105969808 A CN105969808 A CN 105969808A CN 201610304811 A CN201610304811 A CN 201610304811A CN 105969808 A CN105969808 A CN 105969808A
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- gene
- prosalpha6
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- deltamethrin
- diamondback moth
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- 241000500437 Plutella xylostella Species 0.000 title claims abstract description 34
- OWZREIFADZCYQD-NSHGMRRFSA-N deltamethrin Chemical compound CC1(C)[C@@H](C=C(Br)Br)[C@H]1C(=O)O[C@H](C#N)C1=CC=CC(OC=2C=CC=CC=2)=C1 OWZREIFADZCYQD-NSHGMRRFSA-N 0.000 title claims abstract description 31
- 239000005892 Deltamethrin Substances 0.000 title claims abstract description 17
- 229960002483 decamethrin Drugs 0.000 title claims abstract description 17
- 108050007094 Proteasome subunit alpha6 Proteins 0.000 title abstract description 3
- 101150031687 Prosalpha6 gene Proteins 0.000 claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 22
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims description 22
- 210000004027 cell Anatomy 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 10
- 239000002917 insecticide Substances 0.000 claims description 9
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 claims description 8
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 claims description 8
- 101710163270 Nuclease Proteins 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- 102000002322 Egg Proteins Human genes 0.000 claims description 2
- 108010000912 Egg Proteins Proteins 0.000 claims description 2
- 235000013305 food Nutrition 0.000 claims description 2
- 210000004681 ovum Anatomy 0.000 claims description 2
- 230000004083 survival effect Effects 0.000 abstract description 9
- 206010059866 Drug resistance Diseases 0.000 abstract description 6
- 230000002265 prevention Effects 0.000 abstract description 3
- 230000008261 resistance mechanism Effects 0.000 abstract description 3
- 241000238631 Hexapoda Species 0.000 abstract 1
- 229920002477 rna polymer Polymers 0.000 abstract 1
- 239000003814 drug Substances 0.000 description 9
- 235000013399 edible fruits Nutrition 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 6
- 230000009368 gene silencing by RNA Effects 0.000 description 6
- 108091030071 RNAI Proteins 0.000 description 5
- 229940079593 drug Drugs 0.000 description 4
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 101100298869 Drosophila melanogaster Prosalpha1 gene Proteins 0.000 description 2
- 101100465390 Drosophila melanogaster Prosalpha6 gene Proteins 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 1
- 102000001477 Deubiquitinating Enzymes Human genes 0.000 description 1
- 108010093668 Deubiquitinating Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- YBOFEONRFJMHND-UHFFFAOYSA-N [Br].N#CC#N Chemical compound [Br].N#CC#N YBOFEONRFJMHND-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000012865 response to insecticide Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
- A01K67/0337—Genetically modified Arthropods
- A01K67/0339—Genetically modified insects, e.g. Drosophila melanogaster, medfly
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
- A01K2217/054—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function
- A01K2217/058—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function due to expression of inhibitory nucleic acid, e.g. siRNA, antisense
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/70—Invertebrates
- A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
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- Life Sciences & Earth Sciences (AREA)
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- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
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- General Engineering & Computer Science (AREA)
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- Wood Science & Technology (AREA)
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- Mycology (AREA)
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Biophysics (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of insect drug resistance mechanisms and prevention and control and in particular relates to application of a plutella xylostella proteasome subunit alpha6 gene to the control of resistance to deltamethrin. After prosalpha6 gene RNAi (Ribonucleic Acid interfere) is used, the drug resistance level of larvae is remarkably reduced; after the deltamethrin with the same dosage is used for treating, the survival rate of the larvae of an interference group is reduced by 2.5 times and has remarkable difference from a control group. Individuals and cell levels prove that the gene is closely related to the resistance of the deltamethrin respectively and the gene has important meanings of further clarifying a resistance mechanism of the deltamethrin and resistance prevention and control application.
Description
Technical field
The invention belongs to pest resistance to insecticide mechanism and prevention and control field, be specifically related to diamondback moth ubiquitin protease body α 6 subunit gene
Prosalpha6 and a kind of method of deltamethrin resistance administering diamondback moth.
Background technology
Continuously, use a large amount of, single is a kind of or a class medicament is the main cause causing insecticide to develop immunity to drugs.Year after year
After a certain medicament of a large amount of use, the insecticide that this medicine drug-resistant capability is weak is eliminated after natural selection, and the individuality that drug resistance is strong
Survived and bred rapidly, thus relevant drug resistance gene is kept down also by heredity, and along with
The increase of consumption of chemical agent, these drug resistance genes are again through insecticide effect and selection, so by the selection in generation, anti-
Property of medicine accumulation is strengthened, and the medicine resistance ability of insecticide the most gradually strengthens, the insecticide of this strain that just develops immunity to drugs.But these bases relevant
Analyzing mainly at the comparison in difference of gene expression dose of cause and Drug resistance relation, does not carry out striking of related gene and subtracts and increase
The cell after strongly expressed affected resistance to insecticides level and viviperception, lack direct experimental evidence, therefore, it is difficult to enter
Row development and application.
Diamondback moth prosalpha6 gene is a Structural subunits of 20S proteasome, not yet has this gene and bromine cyanogen chrysanthemum
The report that ester resistance is relevant.
Summary of the invention
It is an object of the invention to provide diamondback moth to the resistance related gene of decis and application thereof.
The present invention, first using fruit bat Kc cell as modular system, analyzes prosalpha6 gene and deltamethrin resistance
Relation.According to fruit bat prosalpha6 gene order existing in GenBank, design primer clones fruit bat prosalpha6 base
Through experiment, the open reading frame sequence of cause, proves that fruit bat prosalpha6 gene is deltamethrin resistance related gene.
Inventor's further experiment finds that the Prosalpha6 protein gene of diamondback moth is in deltamethrin resistance strain
Expression is apparently higher than sensitive strain.
The invention discloses the application in the deltamethrin resistance administering diamondback moth of the diamondback moth prosalpha6 gene.
The dsRNA of diamondback moth proteasome Alpha 6 subunit gene prosalpha6 is preparing diamondback moth to decis sensitizer
In application.
A kind of method utilizing proteasome Alpha 6 subunit gene prosalpha6 to administer diamondback moth deltamethrin resistance, its step
Suddenly:
(1) with SEQ ID NO.1 and the SEQ ID NO.2 primer of prosalpha6, according to MEGAscriptRNAi Kit
Description operates, the dsRNA of synthesis diamondback moth proteasome Alpha 6 subunit gene prosalpha6；
(2) nuclease digestion is to remove DNA and ssRNA；
(3) purification dsRNA；
(4) dsRNA of prosalpha6 is sprayed onto on the ovum of field diamondback moth, larva and feedstuff, by permeating and taking
Food, dsRNA enters Plutella Xylostella Cell, reticent prosalpha6 gene；
Decis insecticides is executed after (5) 48 hours.
Show after deliberation: after prosalpha6 gene RNAi, the Detection of insecticide resistance of larva is decreased obviously, with same dose of
After decis processes, the survival rate of interference group larva have dropped 2.5 times, and between matched group, there were significant differences.
By individual and cellular level, we prove that this gene is closely related with the resistance of decis respectively, therefore this
Bright to being further elucidated with the resistance mechanism of decis and being respectively provided with important meaning in the control and application of resistance.
Accompanying drawing explanation
The mrna expression level (* represents p < 0.05) of prosalpha6 in Fig. 1 fruit bat Kc cell.
Variable concentrations decis is coerced the impact of lower cell survival rate by the RNAi of Fig. 2 prosalpha6.* represent p <
0.05。
Fig. 3 diamondback moth survival rate detects.* p < 0.05 is represented.
Detailed description of the invention
Embodiment one
1, experiment material
Diamondback moth is derived from the Caulis et Folium Brassicae capitatae ground in Zhong Cao township, Huaxi District, Guiyang City, Guizhou Province, surveys four-age larva after an indoor propagation generation
LD50Value, with the diamondback moth sensitive strain four-age larva LD of Wuhan City's Vegetable Research Institute50Value compares, and finds diamondback moth field population
For sensitive population.Synchronize isolation as sensitive strain to cultivate.Diamondback moth deltamethrin resistance strain is processed little by drop method
Diamond-back moth four-age larva sensitive strain is bred selection-breeding through many generations and is formed.
2, the functional study of prosalpha6
(1) the variable concentrations decis inducing action to prosalpha6
Process fruit bat Kc cell with the decis of variable concentrations, use fluorescent quantitative PCR technique, at detection variable concentrations
The expression of prosalpha6 gene in reason cell.Result shows, the expression of prosalpha6 is along with the liter of drug level
High and constantly raised, present certain linear relationship (Fig. 1)；
(2) the prosalpha6 gene RNAi impact on cell survival rate
After RNA interference 48h, process interference group (dsRNA) with the decis of variable concentrations and matched group (control) is thin
Born of the same parents 24h, trypan blue reagent detection cell survival rate, result shows: under the decis of a series of concentration is coerced, and interference group is thin
The survival rate of born of the same parents is significantly lower than the survival rate (Fig. 2) of cellular control unit.
(3) the diamondback moth prosalpha6 gene RNAi impact on deltamethrin resistance level
1. the synthesis of diamondback moth prosalpha6 gene RNAi
Conserved sequence according to prosalpha6 gene utilizes the following primer of pp5 software design, synthesizes diamondback moth
The dsRNA of prosalpha6 gene.
Forward primer:
5′-TAATACGACTCACTATAGGGATGTTTCGCAACCAGTACGA-3′(SEQ ID NO.1)
Downstream primer:
5′-TAATACGACTCACTATAGGGCTATGGACGCTGCTCGGT-3′(SEQ ID NO.2)
2. construction of expression vector
Nuclease digestion is to remove DNA and ssRNA.
Sample-adding Digestive system is as follows the most on ice
2. it is placed in 37 DEG C and hatches 1h；
Purification dsRNA.
1. reactant liquor is assembled
Piping and druming mixing assembles reactant liquor the most gently, and under the conditions of room temperature 13000rpm, centrifugal 2min, abandons filtrate；
3. drawing 500 μ L Wash Solution to join and prepare in pipe, under room temperature, 13000rpm is centrifuged 2min, abandons filter
Liquid；
4. step is repeated the most once；
Blank pipe is centrifuged 30sec to remove unnecessary liquid as far as possible the most again；
6. put into preparing pipe in a new collecting pipe, be added thereto to 50 μ L and be previously heated to more than 95 DEG C
Elution Solution, maximum (top) speed is centrifuged 2min, collects filtrate；
7. step is repeated the most once；
8. the A of spectrophotometric determination product260To detect the concentration of dsRNA by the prosalpha6 gene of amplification
DsRNA inserts pET-2P (purchased from promega company) dsrna construct expression vector pET-2P-pros.
3. double-stranded RNA expression in escherichia coli
Take 5 μ L pET-2P-pros plasmids to join in the HT115 competent cell after 50 μ L thaw, add IPTG and make end
Concentration is 0.1mM, cultivates 5h under the conditions of 37 DEG C continuously, and matched group is not added with IPTG induction, cultivates 5h under the same terms.
4. diamondback moth anti-larvae of deltamethrin feeding dsRNA
Bacterium solution and ultra-pure water are completely dissolved, by fresh Caulis et Folium Brassicae capitatae identical for size to bacterial sediment with the ratio mixing of 1:10
Blade is placed in bacterium solution immersion 10s, is put in diameter 9cm after drying, and in the glass culture dish of high 1.5cm, each culture dish is placed
15 larvas.Without IPTG induction group and only by ultra-pure water process group the most as a control group, same 15 larvas of each culture dish.Respectively
If 3 repetitions, 60 larvas of each repetition, the fresh blade of continuous feeding dsRNA dipping 3 days.
4. toxicity test
Decis is made into concentration 2500ppm using acetone as solvent, respectively to prosalpha6 gene interference group, not
Through IPTG induction group and only carry out toxicity test with the diamondback moth larvae of ultra-pure water process group.Cultivate 48 hours under suitable condition,
The dead larvae number of statistics different disposal group.Touching polypide with tweezers, inertia person is calculated as death, adds up mortality rate.Result table
Bright, disturb the survival rate of diamondback moth of prosalpha6 gene all significantly lower than matched group, and have significant difference.At water
Reason matched group is set to 1 (Fig. 3).
Claims (3)
1. proteasome Alpha 6 subunit gene prosalpha6 application in administering deltamethrin resistance.
2. utilize the method that proteasome Alpha 6 subunit gene prosalpha6 administers diamondback moth deltamethrin resistance, its step
It is:
(1) with SEQ ID NO.1 and the SEQ ID NO.2 primer of prosalpha6, according to MEGAscriptRNAi Kit explanation
Book operates, the dsRNA of synthesis diamondback moth proteasome Alpha 6 subunit gene prosalpha6；
(2) nuclease digestion is to remove DNA and ssRNA；
(3) purification dsRNA；
(4) dsRNA of prosalpha6 is sprayed onto on the ovum of field diamondback moth, larva and feedstuff, by permeating and taking food,
DsRNA enters Plutella Xylostella Cell, reticent prosalpha6 gene；
Decis insecticides is executed after (5) 48 hours.
3. the dsRNA of diamondback moth proteasome Alpha 6 subunit gene prosalpha6 is preparing diamondback moth in decis sensitizer
Application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610304811.XA CN105969808A (en) | 2016-05-10 | 2016-05-10 | Application of plutella xylostella proteasome subunit alpha6 gene to control of resistance to deltamethrin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610304811.XA CN105969808A (en) | 2016-05-10 | 2016-05-10 | Application of plutella xylostella proteasome subunit alpha6 gene to control of resistance to deltamethrin |
Publications (1)
| Publication Number | Publication Date |
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| CN105969808A true CN105969808A (en) | 2016-09-28 |
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|---|---|---|---|
| CN201610304811.XA Pending CN105969808A (en) | 2016-05-10 | 2016-05-10 | Application of plutella xylostella proteasome subunit alpha6 gene to control of resistance to deltamethrin |
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| CN (1) | CN105969808A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409430A (en) * | 2013-08-19 | 2013-11-27 | 南京师范大学 | Diamondback moth ubiquitin gene UBL40 and applications thereof in treating deltamethrin resistance of diamondback moth |
| CN105274122A (en) * | 2015-11-04 | 2016-01-27 | 重庆大学 | RNAi (ribonucleic acid interference) expression vector for TOR (target of rapamycin) genes of plutella xylostella, method for constructing RNAi expression vector and application thereof |
-
2016
- 2016-05-10 CN CN201610304811.XA patent/CN105969808A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409430A (en) * | 2013-08-19 | 2013-11-27 | 南京师范大学 | Diamondback moth ubiquitin gene UBL40 and applications thereof in treating deltamethrin resistance of diamondback moth |
| CN105274122A (en) * | 2015-11-04 | 2016-01-27 | 重庆大学 | RNAi (ribonucleic acid interference) expression vector for TOR (target of rapamycin) genes of plutella xylostella, method for constructing RNAi expression vector and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| JUNLI HU ET AL.: "Identification of Proteasome Alpha6 Subunit Associated with Deltamethrin Resistance in Drosophila melanogaster Kc cells", 《ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY》 * |
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