CN101363059B - Molecule detection method for triazophos target resistance of rice borer - Google Patents
Molecule detection method for triazophos target resistance of rice borer Download PDFInfo
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- CN101363059B CN101363059B CN2008101561775A CN200810156177A CN101363059B CN 101363059 B CN101363059 B CN 101363059B CN 2008101561775 A CN2008101561775 A CN 2008101561775A CN 200810156177 A CN200810156177 A CN 200810156177A CN 101363059 B CN101363059 B CN 101363059B
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- rice borer
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Abstract
The invention relates to a molecular biology method for testing target enzyme resistance of chilo suppressalis which is rice insect pest for triazophos, belonging to the field of biotechnology. Auele Specific Primers which are F: 5'-GGGCAAGAAAGTAGACGCCTGGTT-3', and R: 5'- GTTCGTCAGCACTCTTCTTCCTTA-3' are used for expanding genome DNA of the chilo suppressalis, and PCR expanding products is digested by restricted enzyme MspA1 I; when the obtained segment is 534bp and 224bp, the chilo suppressalis individual is homozygote SS which is sensitive to the target enzyme of triazophos; when the obtained segment is 758bp, the chilo suppressalis individual is homozygote RR for the target enzyme resistance of triazophos; when the obtained segment is 758bp,534bp and 224bp, the chilo suppressalis individual is heterozygote RS for the target enzyme resistance of triazophos. The method has high stability, short period and high sensitivity, and is specially used for quickly testing the target enzyme resistance of the chilo suppressalis for triazophos with high sensitivity.
Description
(1) technical field
The present invention relates to striped rice borer (Chilo suppressalis Walker) and, belong to biological technical field, be exclusively used in the highly sensitive rapid detection of striped rice borer the triazophos target resistance to the molecular detecting method of triazophos (Triazophos) target resistance.
(2) background technology
Paddy rice is human important crops, and it is staple food with rice that 50% population is arranged in the world approximately, accounts for 1/3 of global cereal crop cultivated area.Paddy rice also is the staple food crop of China, and the population in the whole nation 50% is staple food with rice, and the paddy rice cultivated area accounts for 26.6% of cereal sown area, and the paddy gross output accounts for 43.6% of total grain output, and paddy production is to China's grain per unit area yield and Safety Effect maximum.Striped rice borer (Chilo suppressalis Walker) is the paddy rice primary pest, and all paddy fields all have extensive distribution in China.Since the nineties in last century, China's rice borer disaster near and top all previous records, wherein the striped rice borer disaster is particularly serious, coastal along the Yangtze valley and Jiangsu and Zhejiang Provinces is its main generating area (Sheng Chengfa etc. 2003), always mainly by chemical prevention.Before the eighties in 20th century, the medicament of control mainly contains phenyl-hexachloride, Trichlorphon, thiophos, parathion-methyl and chlordimeform etc.; The eighties, desinsection list and acephatemet etc. were by a large amount of widespread uses to the mid-90; Promote the use of triazophos the beginning of the nineties; The end of the nineties, fluorine worm nitrile and Avrmectin began to be used for the control of rice borer.Drug-fast development is the most important reason (Qu Mingjing Ph D dissertation, striped rice borer is to the resistance and the mechanism research thereof of triazophos) of long lasting effect striped rice borer great outburst.
Triazophos (Triazophos) is a kind of wide spectrum, organophosphorus desinsection miticide efficiently, 1971 Germany Hirst company (Hoechst AG) at first develop, begin to be used to prevent and treat striped rice borer the beginning of the nineties in last century.Through the more than ten years use, some regional populations are also remarkable gradually to the resistance of triazophos, and indivedual uses striped rice borer population in district have earlier produced high horizontal resistance to it.Insect insecticide produces the Mechanism of Physiological and Biochemical of resistance, mainly comprises the reduction of epidermis transmission rate, the rising of detoxification enzyme activity, target insensitivity; And the target insensitivity is topmost resistance mechanism.Triazophos target in the striped rice borer body is an acetylcholinesterase, and the transgenation of acetylcholinesterase (AChE) is the most important reason that causes resistance to produce.Mutational site among the present invention is the A314S sudden change in the conservative motif ' FGESAG ' of striped rice borer AChE I.
At present the detection of triazophos resistance is confined to biological assay, and the limitation of biological assay makes the detection method of this routine can not detect early stage resistance or low frequency resistance allele about striped rice borer.Simultaneously, the following shortcoming of biometric techniques ubiquity: (1) sensitivity is low: when the very difficult early stage resistance of detection of biological assay or low frequency resistance allele, especially resistance allele are recessive or not exclusively recessive; (2) cycle is long: bioassay results generally needed more than 24 hours, and striped rice borer give birth to survey to triazophos that testing time is 48 hours as a result; (3) require very big to examination borer population amount: measuring a typical curve needs 400 examination worms at least, and also very high to the requirement of insect raising.Survey the big or small unanimity that also will require to try worm for giving birth to of striped rice borer, strengthen, and many human factors also can influence living survey result thereby cause giving birth to the workload of surveying.Along with the extensive application of triazophos and the progressively generation of resistance, we press for a kind of quick, convenient, sensitive resistance detection method.
(3) summary of the invention
Problems such as that technical problem the objective of the invention is is low to the sensitivity of triazophos resistance detection method at striped rice borer in the prior art, cycle length, material requirements height, the molecular detecting method of striped rice borer to the triazophos target resistance is provided, reaches accuracy height, short, highly sensitive purpose of cycle.
Technical scheme
Striped rice borer is used Auele Specific Primer to the molecular detecting method of triazophos target resistance:
F：5′-GGGCAAGAAAGTAGACGCCTGGTT-3′
R：5′-GTTCGTCAGCACTCTTCTTCCTTA-3′
The genomic dna of amplification striped rice borer, pcr amplification product carries out enzyme with restriction enzyme MspAl I and cuts digestion, if when obtaining fragment and being 534bp and 224bp, illustrates that this striped rice borer individuality is to triazophos target sensitivity homozygote SS; If when obtaining fragment and being 758bp, illustrate that this striped rice borer individuality is to triazophos target resistance homozygote RR; If obtain fragment is 758bp, when 534bp and 224bp, illustrates that this striped rice borer individuality is to triazophos target resistance heterozygote RS.
The extracting method of striped rice borer genomic dna is to adopt and give birth to worker UNIQ-10 pillar animal gene group DNA extraction agent box:
1. get single head striped rice borer in four ages, in liquid nitrogen, clay into power, and shift and put in the centrifuge tube of 1.5ml;
2. the Proteinase K that adds 300 μ l ACL solution and 20 μ l then;
3. the concussion mixing is 1 minute, places 55 ℃ to place then 1-3 hours;
4. taking-up sample, light shaking mixing when waiting to reduce to room temperature, 12000rpm is centrifugal 5 minutes then;
5. draw 300 μ l supernatants to UNIQ-10 pillar, and then the AB solution of drawing 300 μ l is to UNIQ-10 pillar, the mixing 2-3 times of turning upside down, room temperature left standstill 3 minutes;
6.3000rpm centrifugal 3 minutes, take off UNIQ-10 pillar, outwell waste liquid in the collection tube;
7. UNIQ-10 pillar is put back in the collection tube, added 500 μ l elute solns, 8000rpm, centrifugal 30 seconds of room temperature;
8. repeating step (7) once;
9. take off UNIQ-10 pillar, discard the waste liquid in the collection tube, post is put back in the collection tube, 10000rpm, centrifugal 30 seconds of room temperature is to remove residual elute soln;
10. post is put into the centrifuge tube of new clean 1.5ml, added 50 μ l dissolving damping fluid in post central authorities, room temperature or 55 ℃ were placed 2 minutes.10000rpm, centrifugal 1 minute of room temperature.Liquid in the centrifuge tube is genomic dna.-20 ℃ of preservations are standby.
PCR reaction system 25 μ l:12.5 μ l2 * GC BufferII, 1.25U LA Taq DNA polymerase, 0.8 μ l single head striped rice borer genomic dna in four ages, 2 μ l2.5mM dNTPs, each 1 μ l of 10mM primer, adding distilled water is 25 μ l to reacting cumulative volume;
The PCR response procedures: 94 ℃ 2 minutes; 32 circulations then: 94 ℃ of 30S, 59 ℃ of 30S, 72 ℃ of 50S;
Restriction enzyme MspA1I digestion PCR product reaction system 10 μ l:1 μ l10 * Buffer4,0.1 μ l100 * BSA, DNA≤0.5 μ g, MspA1I0.25 μ l adds distilled water to 10 μ l;
The endonuclease reaction condition: 37 ℃ of temperature were bathed 1 hour.
Beneficial effect
The present invention compares with the prior biological determination techniques, and its advantage and positively effect show:
(1) highly sensitive: the existence of low frequency resistance homozygote or heterozygote and certain screening pressure, it is the key factor that high-level resistance produces, and the biometric detection technology is a kind of extensive detection method, can not detect early stage resistance or low frequency resistance homozygote or heterozygote, so biometric techniques is difficult to carry out the risk forecast that resistance produces.The point mutation that the present invention is relevant to the triazophos resistance according to striped rice borer utilizes sensitive strain and the resistant strain base difference in this site of allelotrope, according to the principle of RFLP-PCR, can directly judge individual genotype.The detection of the individuality by some amount can tentatively be determined resistance homozygote in certain population, heterozygote and responsive homozygous frequency, for resistance prediction, resistance management and the insect comprehensive regulation provide the most direct evidence.
(2) accuracy height: biometric techniques requires the stdn of examination worm, and the difference between the polypide is very big to result's influence, causes result's unstable, and operator's error has also strengthened this unstable.And the present invention is based on the detection technique of RFLP-PCR, because round pcr increasingly mature and promoting and the intelligent development of operation reduced this unstable to a certain extent, make detected result can be used as the ideal material that the striped rice borer control strategy is formulated.
(3) cycle is short: giving birth to survey technology needs 48 hours observation time at least.And the RFLP-PCR technology among the present invention only need just can draw ideal results less than 8 hours time.
(4) material requirements is few: measuring a typical curve in the biometric techniques needs the accurate examination of 400 leaders worm at least, the certain human and material resources of raising need cost of these examination worms.And the present invention only needs examination worm about 50 to the detection of a population.
(4) Figure of description
The special allelotrope result of Fig. 1 Different Individual digestion with restriction enzyme
Gaochun County, Nanjing, Fig. 2 Jiangsu Province (1-10) and Jiande county, Hangzhou, Zhejiang province city (11-20) striped rice borer population resistance target molecules detected result
The Maker size is: 1200bp, 900bp, 700bp, 500bp, 300bp, 100bp
(5) embodiment
1. from Gaochun County, Nanjing, Jiangsu Province and field, Jiande county, Hangzhou, Zhejiang province city generation striped rice borer population, choose 10 four-age larvas, the extraction of genomic dna (giving birth to worker UNIQ-10 pillar animal gene group DNA extraction agent box): 1) get single head striped rice borer in four ages, in liquid nitrogen, clay into power, and shift and put in the centrifuge tube of 1.5ml; (2) add the Proteinase K of 300 μ l ACL solution and 20 μ l then.(in advance Proteinase K is put 37 ℃ of insulations 1 hour, is helped the activity of activator enzyme K like this); (3) the concussion mixing is 1 minute, places 55 ℃ to place then 1-3 hours, can suitably take out mixing during this period, helps abundant cracking; (4) take out sample, light shaking mixing when waiting to reduce to room temperature, then 12, centrifugal 5 minutes of 000rpm; (5) draw 300 μ l supernatants to UNIQ-10 pillar, and then the AB solution of drawing 300 μ l is to UNIQ-10 pillar, the mixing 2-3 times of turning upside down, room temperature left standstill 3 minutes; Centrifugal 3 minutes of (6) 3,000rpm take off UNIQ-10 pillar, outwell waste liquid in the collection tube; (7) UNIQ-10 pillar is put back in the collection tube, added 500 μ l elute solns, 8,000rpm, centrifugal 30 seconds of room temperature; (8) repeating step (7) once; (9) take off UNIQ-10 pillar, discard the waste liquid in the collection tube, post is put back in the collection tube, 10,000rpm, centrifugal 30 seconds of room temperature is to remove residual elute soln; (10) post is put into the centrifuge tube of new clean 1.5ml, added 50 μ l dissolving damping fluid in post central authorities, room temperature or 55 ℃ were placed 2 minutes.10,000rpm, centrifugal 1 minute of room temperature.Liquid in the centrifuge tube is genomic dna.-20 ℃ of preservations are standby.
2. Auele Specific Primer:
F：5′-GGGCAAGAAAGTAGACGCCTGGTT-3′
R：5′-GTTCGTCAGCACTCTTCTTCCTTA-3′
3.PCR reaction system 25 μ l:
12.5 μ l2 * GC BufferII, 1.25U LA Taq DNA polymerase, 0.8 μ l single head striped rice borer genomic dna in four ages, 2 μ l dNTPs (2.5mM), each 1 μ l of primer (10mM), adding distilled water is 25 μ l to reacting cumulative volume.
4.PCR response procedures:
94 ℃ 2 minutes; 32 circulations (94 ℃ of 30S, 59 ℃ of 30S, 72 ℃ of 50S) then
Restriction enzyme MspAl I digestion PCR product reaction system 10 μ l:
1 μ l10 * Buffer4,0.1 μ l100 * BSA, DNA＜0.5 μ g, MspA1I0.25 μ l adds distilled water to 10 μ l.
5. endonuclease reaction condition: 37 ℃ of temperature were bathed 1 hour
6. enzyme is cut the electrophoresis detection of product:
10 μ l enzymes are cut product carry out electrophoresis on the sepharose of 1.5% (g/ml), current stabilization 80mA detects under ultraviolet after 40 minutes: if when fragment is 534bp and 224bp, illustrate that this individuality is responsive homozygote (SS); When if fragment is 758bp, illustrate that this individuality is resistance homozygote (RR); If fragment is 758bp, 534bp and 224bp illustrate that this individuality is heterozygote (RS).
The PCR product requires enzyme to cut according to the enzyme system of cutting, and last electrophoresis detection enzyme is cut product (Fig. 2 as a result):
Resistance homozygote individuality is (RR): 2,5,8,10,12,
The heterozygote individuality is (RS): 1,3,4,6,7,9,13,15,17
Responsive homozygote individuality is (SS): 11,14,16,18,19,20
Sequence table
＜110〉Agricultural University Of Nanjing
＜120〉striped rice borer is to the molecular detecting method of triazophos target resistance
＜130〉specification sheets
<140>00
<141>2008-08-08
<160>2
<170>PatentIn?version3.1
<210>1
<211>24
<212>DNA
＜213〉synthetic
<220>
＜221〉Auele Specific Primer F
<222>(1)..(24)
<223>
<400>1
<210>2
<211>24
<212>DNA
＜213〉synthetic
<220>
＜221〉Auele Specific Primer R
<222>(1)..(24)
<223>
<400>2
Claims (1)
1. striped rice borer comprises the molecular detecting method of triazophos target resistance, uses Auele Specific Primer:
F：5′-GGGCAAGAAAGTAGACGCCTGGTT-3′
R：5′-GTTCGTCAGCACTCTTCTTCCTTA-3′
The genomic dna of amplification striped rice borer, pcr amplification product carries out enzyme with restriction enzyme MspA1I and cuts digestion, if when obtaining fragment and being 534bp and 224bp, illustrates that this striped rice borer individuality is the homozygote SS to triazophos target sensitivity; If when obtaining fragment and being 758bp, illustrate that this striped rice borer individuality is the homozygote RR to the triazophos target resistance; If obtain fragment is 758bp, when 534bp and 224bp, illustrates that this striped rice borer individuality is the heterozygote RS to the triazophos target resistance.
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| CN103224993A (en) * | 2013-05-22 | 2013-07-31 | 江苏省农业科学院 | Detection method of target resistance mutation of Taiwan rice stem borer for organic phosphorus pesticide |
| CN111411107A (en) * | 2020-03-27 | 2020-07-14 | 武汉古奥基因科技有限公司 | Method for polyploid genome surfy |
| CN111334586A (en) * | 2020-04-02 | 2020-06-26 | 南京农业大学 | Method for rapidly detecting gene mutation of chilo suppressalis ryanodine receptor based on AS-PCR technology |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1199548A (en) * | 1998-05-18 | 1998-11-25 | 浙江省仙居农药厂 | Recompounded phentriazophos-Phoxim pesticide |
| CN101185442A (en) * | 2006-11-16 | 2008-05-28 | 郦卫弟 | Compound pesticides water emulsion agent containing fipronil and triazophos and preparation method thereof |
| WO2008104503A1 (en) * | 2007-03-01 | 2008-09-04 | Basf Se | Pesticidal active mixtures comprising aminothiazoline compounds |
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2008
- 2008-09-24 CN CN2008101561775A patent/CN101363059B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1199548A (en) * | 1998-05-18 | 1998-11-25 | 浙江省仙居农药厂 | Recompounded phentriazophos-Phoxim pesticide |
| CN101185442A (en) * | 2006-11-16 | 2008-05-28 | 郦卫弟 | Compound pesticides water emulsion agent containing fipronil and triazophos and preparation method thereof |
| WO2008104503A1 (en) * | 2007-03-01 | 2008-09-04 | Basf Se | Pesticidal active mixtures comprising aminothiazoline compounds |
Non-Patent Citations (4)
| Title |
|---|
| He YP等.《Survey of susceptibilities to monosultap, triazophos, fipronil, and abamectin in Chilo suppressalis (Lepidoptera: Crambidae)》.《J Econ Entomol》.2007,第100卷(第6期),1854-61. * |
| 曲明静等.《二化螟对三唑磷的抗性发生动态与风险评估》.《南京农业大学学报》.2005,第28卷(第3期),38-42. * |
| 曲明静等.《昆虫乙酰胆碱酯酶基因变异抗药性机制研究》.《昆虫知识》.2002,第44卷(第2期),191-194. * |
| 韩招久等.《二化螟乙酰胆碱受体α亚基的基因克隆与序列分析》.《动物学研究》.2002,第23卷(第1期),7-13. * |
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