CN103898121A - Transient receptor ion channel gene MED BtTrp of bemisia tabaci MED cryptic species and application of transient receptor ion channel gene MED BtTrp - Google Patents
Transient receptor ion channel gene MED BtTrp of bemisia tabaci MED cryptic species and application of transient receptor ion channel gene MED BtTrp Download PDFInfo
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- CN103898121A CN103898121A CN201410153314.5A CN201410153314A CN103898121A CN 103898121 A CN103898121 A CN 103898121A CN 201410153314 A CN201410153314 A CN 201410153314A CN 103898121 A CN103898121 A CN 103898121A
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Abstract
The invention relates to the field of gene engineering and particularly relates to a transient receptor ion channel gene MED BtTrp of a bemisia tabaci MED cryptic species and an application of the transient receptor ion channel gene MED BtTrp. The transient receptor ion channel gene MED BtTrp has a nucleotide sequence as shown in SEQ ID No.1. According to the invention, the cDNA (Complementary Deoxyribonucleic Acid) of a transient receptor ion channel TRP (MED BtTrp) gene is cloned from the bemisia tabaci MED cryptic species, and the dsRNA (Double-Strand Ribonucleic Acid) of an MED BtTrp gene is fed, so that the survival rate of the bemisia tabaci MED cryptic species under the high temperature stress is remarkably reduced. The result provided by the invention has important significance for determining the temperature perceiving mechanism of the bemisia tabaci MED cryptic species and is expected to provide the basis for further researching a temperature adapting mechanism of bemisia tabaci and a method for controlling hazards of the bemisia tabaci through temperature adaptability in the future.
Description
Technical field
The present invention relates to genetically engineered field, be specifically related to the hidden kind of Bemisia tabaci MED instantaneous susceptor ion channel gene MED BtTrp and application thereof.
Background technology
Bemisia tabaci Bemisia tabaci (Gennadius) belongs to Hemiptera (Hemiptera) Aleyrodidae (Aleyrodidae), little Aleyrodes Bemisia, be polyphagous sucking insect, be mainly distributed in subtropical and tropical zones.The mechanism that Bemisia tabaci invasion success are expanded comprises many-side, multi-level content, and under the environment of whole world change, ecological adaptation is one of its key link.The factor that ecological adaptation relates to mainly comprises biotic factor and abiotic factor, and in abiotic factor, temperature is the important factor that affects its population dynamics and diffusion profile.There are some researches show, Bemisia tabaci invasion mechanism is closely related with Bemisia tabaci thermal adaptability.
It is one of its key factor that can be diffused as whole world important pests that the hidden kind of MED has stronger resistibility to temperature, is one of important mechanisms of its invasion success expansion.The hidden kind of MED has very strong heat-resisting ability, and it can rampantly be endangered in hot season; And in cold season, the protection ground expanding gradually provides hibernaculum for it, make it accumulate a large amount of insect population numbers so that break out.The molecular chaperones effect of the stable on heating acquisition of insect and heat shock protein (heat shock proteins, HSP) is relevant, and heat shock protein plays an important role on biological heat stress ecology adapts to and evolves.Early-stage Study shows that heat shock protein gene plays key effect in Bemisia tabaci thermotolerance, and for better understanding Bemisia tabaci thermal adaptation ability, research how perception outside temperature of Bemisia tabaci the temperature sensing mechanism in body of being passed to are particularly important.There are some researches show, instantaneous susceptor ionic channel TRP (transient receptor potential) is the critical passage of biological perception outside temperature.
Instantaneous susceptor ionic channel (transient receptor potential, TRP) is the important ionic channel of a class being positioned on cytolemma, is found in the earliest in the vision system of fruit bat.While being activated, allow the positively charged ion including calcium ion to carry out cross-film transportation.TRP, as the gate molecule in susceptor, plays the effect of outside atmosphere and neural system intermediary, thermal stimulus, chemical stimulation and mechanical stimulus etc. can be changed into inward electric current.TRP studies comparatively thorough in model animals fruit bat, mainly have photoresponse, temperature sensing, mechanical sense should with the biological functions such as biochemical response.
RNA disturbs (RNA interference, RNAi) refer to endogenous or ectogenic dsRNA(double-stranded RNA, dsRNA) there is specificity degraded in the intracellular mRNA of mediation, thereby cause target cell expression silencing, produces the phenomenon of corresponding function phenotype disappearance.RNAi, as the new technology of closing specific gene function, for the research of gene function provides quick, an easy method, has played very important effect for the research of insect.The research of RANi technology aspect gene function be one fast, the technology that can make target gene inactivation of efficient, convenient operation.In recent years, RNAi technology is more and more applied in insect research, by some gene in the reticent insect body of RNAi technology, make some ability of insect strengthen or lose, also can suppress its functional gene expression at specified time and make the growth of insect rest on certain stage, and then reach the object of utilizing or preventing and treating its harm.RNAi technology conventionally adopts and feeds, injects, immersion, electroporation, stem cell transplantation, the method such as prepare by transgenic technology.In insect research, conventionally adopt injection and feeding method to carry out the research of RNAi effect, the dsRNA of goal gene or siRNA are imported in insect body, and then realize and disturbing.Because Bemisia tabaci body volume is little, the injection restrictions such as dsRNA is not easy to operate, time-consuming, and features such as that feeding dsRNA has is easy, easy handling and being applied in Bemisia tabaci research.
The present invention is cloned into the cDNA of instantaneous susceptor ionic channel TRP (MED BtTrp) gene from the hidden kind of Bemisia tabaci MED, and the instantaneous susceptor ionic channel of the hidden kind of the Bemisia tabaci MED TRP cloning process of gene cDNA is provided, and cause the hidden kind of Bemisia tabaci MED survival rate under high temperature stress significantly to reduce by the MED BtTrp gene dsRNA result of feeding.Result of the present invention is significant for hidden kind of temperature sensing mechanism of clear and definite Bemisia tabaci MED, expects for further the thermal adaptation mechanism of research Bemisia tabaci and the method endangering by thermal adaptability control Bemisia tabaci provide foundation in the future.
Summary of the invention
The object of the invention is to be cloned into MED BtTrp gene from the hidden kind of Bemisia tabaci MED.
A further object of the present invention is to have verified MED BtTrp gene role under Bemisia tabaci high temperature stress condition, and the application of above-mentioned MED BtTrp gene is provided.
According to MED BtTrp gene of the present invention, described MED BtTrp gene cDNA full length nucleotide sequence is as shown in SEQ ID No.1:
ATGGGGAGGCCGACGGAGCGGGCCACCCGAGCCGGACAGTCTGTGCTTGCTTGTCGTTTTCGCCACGATGGAGTAATCCGTTCCATCCCAGTTTTGGGTAGTGCTCCATTTTTTGGATAACGCTTTTTCCCAGGTGCGAGGGGCAGGATGAAGCCGACGGAATCGAAGGAGGACCTCCTGGGGTCGAAGGAGAACCTAAAAAATCCCCGAGAGTCCTTGCAGTCGACCGGCGGCGAGGTTGCCCTCACCCAGATCGAGAAAAGGTTCCTCTTGCTCAGCGAAAGGGGAGATATCGCCTCGGTCAAAAGGATAATCGCTGAGCACAAAGATCACCCAGAACTATTGAACATCAACTGCGTTGATCCTCTGAACAGGTCAGCACTCATCGCAGCGATCGAAAATGAAAATATAGAGTTAATAAGAGTCCTCCTCGAACTTGGGATTGAAGTCAAGGATGCTCTTCTGCACGCAATTAAGGAAGAGTACGTTGAAGCTGTGGAGATTCTACTTGAATGGGAAGAAAAAATCCACAAACCTGGGCAGCCTTATAGTTGGGAAGCTGTTGACGGGAGCTCTTCGACATTTACACCGGACATAACACCGCTCATATTGGCTGCTCACATGAACAATTACGAAATCCTGAAAATTTTGTTGGATCGAGGCGCCACACTCCCCGCGCCACACGATGTTAGGTGTGGCTGCGACGAATGTGTAACTTCAAGTGAGCAGGATTCTTTGCGACATTCACAAGCTCGGATCAACGCGTACAGAGCTTTAACCGCACCATCCCTCATAGCCCTCTCTTCCAGGGATCCCCTCCTTACCGCCTTCGAGTTATCATGGGAGCTGCGCAGACTCAGCAAGATGGAAGCGGAATTCCGCTCTGAATATAATGAAATGAGAGGTATTTGTCAGACATTTGCAACCTCCTTGCTAGACCACGCGCGAACCTCGTAT GAACTGGAGGTGATGCTTAATCATAATCCTGATGGAGAGTCGTGGGAACCAGGAGAGAGGCAAACTCTGGAGAGACTGAAGCTAGCTATCAAATACAAACAAAAAGCGTTTGTTGCTCATCCAAATGTACAACAGCTGCTGGCAGCTATTTGGTATGATGGTCTCCCCGGATTTCGACGAAAAGGGATGATTGGTCAAGCTTTTGAGTGTGGAAAGTTAGGTGCAATGTTTCCTGTCTACGCAACCGTGTATTTGACAGCGCCTGAATCGGAGATGGGCCAGTTCATGAAAAAACCCTTTGTAAAGTTCATCTGTAATTCAACCTCCTACGCTTTCTTCCTGATGTTATTGGCCTTGGCATCACAAAGAGCGGAATTTCTTGCAATCGAATGGTTTGGACCCGACTGGATGAAAGAGATACTGAAAGAGTGGACTCGAAAGGAAAGAGGATCAATACCTGGTCTCATAGAATCCTTTATTATTTTGTATATCATAAGTTTGATTTGGGGTGAAATGAGAGCATTGTGGTCTGGTGGCTTAGAGGACTATGTTTCAGATTTGTGGAATATAGTGGATTTCATCACCAATGTATTCTATATGATATGGATTTGTCTCAGAGGAACTGCGTATATCATTGTGCAAAGAGAGCATAAAAGTGGCCTCGATCCATGGTATCCTCGTGATAAGTGGGACATGTTTGATCCTCATCTTCTATCAGAAGGTGCGTTTGCAGCCAGTATGATCTTCAGTTTCTTAAAATTGGTTCATATCTTCAGTATCAATCCTCATCTTGGTCCACTGCAAATTTCGTTGGGACGAATGATTGTCGACATCATCAAATTTTTCTTCATCTACACATTGGTCCTTTTCGCTTATGGTTGTGGTATGAATCAGTTACTTTGGTACTATGCTGATCTTGAGAGGAAAAAGTGTTATCATCTGAACGAGGATTATGCTGATTTTGATGGTCAAGATAAAGCCTGTACAACATGGAGACGTTTTGCAAATTTATTTGAAACATCTCAGTCTCTGTTCTGGGCCAGTTTCGGTTTAGTTGATCTTGTATCGTTTGATCTGACTGGTATTAAGAGTTTCACCCGGTTTTGGGCTCTTCTGCTTTTCGGATCATACTCCGTTATCAATATCATTGTCCTGCTCAACATGTTGATTGCCATGATGTCCAACAGTTACCAAATCATCTCTGAACGATCCGATACTGAATGGAAATTTGCTCGAAGTGACCTATGGATCAGTTACTTTGAGGATGGCGACACTGTTCCTCCACCATTCAACCTATCACCATCTGTTAAATCTATGAAAAGAATGATGGGAATCAAAACTGGGAAAATCAGCACTGGTTCGATGAAGAACAAAAGTCGAGCTAAAGCTATGGAGCGTCACGAAGCAGTCATGAAACTATTGGTCAGGCGATATGTTGTGGCTGAGCAAAGGAAGAGAGATGAGTTCGGCATCACAGAAGATGATGTTATGGAAATTCGACAAGATATTTCAACCCTTCGTTTTGAACTGATCGATATCCTCCGAACAAACGGAATGAAGACACCGAGCGTGGACAAAGAGGATCAAGCAGTGGGCAAGAAAGGTCGTGTGATGGAGCGTCGTTTGCTGAAGGACTTCCAGATCGGCCTGGTTGAGGGTATAATTAGTGATGTCATATCTTCTGAGAAAGAGCCAAGGGATGTCTTCAGCAAGATTGCTCGTGCAATTGGTAAGGACTCAAGTGCAAAGAAAAAGGACTGGAACGCTATTGCCGCCAAGAAAGCATCCAGCCGCGAACTTATTGGAAGCACCAGCGAGGCTGTGGAGCGTCGGCAATCACGCCAGAGTTTGCGCCGGCACATCCTCGAACATCAAGGATCAGCAATGGCAAATATGGATCCCGAGAAGCTGGTTGAGTACAATCCCAAGTTGCAAGAATATGCACCTGCTGCTCGAATCGCCTATGCCAAATTCAAGATCTCCAAGATCAAGCAAGAATATGACGATAAAGGAAATGAAGAAGAAGGCAAAGACGATGTTTTCGAGCCTGCTACAGTAACAACGAAGGACGAAACAAAACCATCCCAAAGTGGCTCCATCGCACGGCCGAGACCCAAAAGCACCAAGAAACCACCCGCACCTGGAGCCATTCCAGGTCCCGACGATGGCAAGAAAGACTTCCGGTCGCGAACTCCGATCCAAGAAGATCCAAGAGAGGCTGGCCGCACTCCAACGCCAGAACCAATCAAAATGCTCAAGACATCGATTTCCTCTCTCAAGCAAGTATCAGTCGATCAAAAAGCTGGGCCATCAGACAAAAAACCCGCCGCCCCGGGGGCTCCGGCTCCGAAAGTAGAGGTCACCGCAGCTAAATCACCTGCAAAAACAGAGGCAGCCCCGGCAAAACCTGAAGAAAAGAAGCCCACACCTGCTAAACCAGAGGAAAAGAAGCCTGCCGCTGCTGCTGCTACGCCTAAAGCAGACGAAAAGAAGGACGAGAAGAAAGAAGAAAAGAAGGACGAGAAGAAAGATGAGAAGAAAGAGGAAAAGAAAGACGATAAAAAGCCGGGCCCTGCTCCGAAAAAACCAGATGATAAGGATAAGCCTCCCTCGCCGAAGAAAACGATGATCCACACAAAACCTGGTGGTAAATCAACAGTCACCGGCGAAGTACTGACTGGATGGTTGTAAGTATTTCAAAAGAGTAACGTGTACTGGCTGCAAAAACTGATTAGTTTAATATTTCACTTCAGTGGTTTTTTTTAAACTAGCAAATTTTCTGTCTTAAAATTCTTTTTCAATTGGCAGAAACGGAGTTCTTGCATGCGTGAGGGATTTGTGATTTAAGTATTTGTTCTTACATAAAAGTTCGCGAGCAACGAGTTGCGGTGCCCCTGGTTTTCTCTGAAATCAACTCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
The present invention further provides the instantaneous susceptor ion channel gene of the hidden kind of described Bemisia tabaci MED MED BtTrp for preventing and treating the application of Bemisia tabaci
The result that the present invention obtains is that the effect of clear and definite MED BtTrp gene in hidden kind of temperature sensing of Bemisia tabaci MED lays the foundation, also for the interaction of inquiring between MED BtTrp gene and heat shock protein gene provides theoretical foundation to insect temperature sensing and adaptability.
Accompanying drawing explanation
The dsRNA of Fig. 1 MED BtTrp gene processes hidden kind of stable on heating impact of adult of Bemisia tabaci MED: hidden kind of mortality ratio of Bemisia tabaci MED after the MED BtTrp gene dsRNA that relatively feeds, the 10% sucrose solution 3h that feeds.All data are all carried out data statistic analysis with SPSS16.0 statistical software, and level of significance test is P<0.05.
Embodiment
Hidden kind of MED BtTrp full length gene cDNA sequence clone of embodiment 1: Bemisia tabaci MED
1, extract total RNA:
Get 200 of the adults of the hidden kind of Bemisia tabaci MED and put into sterilized mill, add the Trizol solution of 1mL, after grinding fully, join in the centrifuge tube of 1.5mL.Fully vibration mixes, and room temperature is placed 5min; After leaving standstill, add the chloroform of 0.2mL, fully vibration mixes 15s, and room temperature is placed 5min, uses the centrifugal 15min of high speed low temperature centrifugal machine 12000rpm; Get supernatant liquor in another centrifuge tube, add equal-volume Virahol, rock and mix, room temperature is placed 10min.The centrifugal 15min of 12000rpm at 4 ℃; Abandon supernatant liquor, add the ethanol 1mL of concentration 75%, the centrifugal 10min of 12000rpm at 4 ℃; Abandon supernatant liquor, and place 5min at Bechtop, add the DEPC water of 50 μ L, in 55-60 ℃ of water, 5min fully dissolves RNA precipitation, and-80 ℃ of Ultralow Temperature Freezers are preserved with for subsequent use.
2, hidden kind of cDNA of Bemisia tabaci MED is synthetic:
Adopt the reverse transcription test kit (Super Script First-Strand Synthesis System) of Transgen company to illustrate and carry out, specific as follows:
(1) in the PCR of 0.2mL pipe, add successively following reagent:
(2) above solution mixes gently, hatches 30min for 42 ℃, hatches 5min for 85 ℃.After finishing, reaction is placed on-20 ℃ of Refrigerator stores for subsequent use.
3, the intermediate segment of hidden kind of MED BtTrp gene of pcr amplification Bemisia tabaci MED:
(1) design primer MED BtTrp F and MED BtTrp R:
MED BtTrp-F:5’-CGCACCATCCCTCATAGCC-3’；
MED BtTrp-R:5 '-GTCGGGTCCAAACCATTCG-3 ' is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
(2) take cDNA as template, the intermediate segment of hidden kind of MED BtTrp gene of pcr amplification Bemisia tabaci MED:
Reaction system is 25 μ L:10 × PCR reaction buffer2.5 μ L, dNTP Mix (10mM) 0.5 μ L, Taq polysaccharase (2.5U) 0.5 μ L, upstream primer and the each 1.0 μ L of downstream primer (10 μ M), cDNA template 0.5 μ L, sterilizing ddH
2o19.0 μ L.
Reaction conditions is: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 35 circulations; 72 ℃ of 10min.
(3) PCR product purification: utilize the EasyPureTM Quick Gel Extraction Kit test kit of Transgen company that PCR product is cut to glue purification.Be specially: cut the sepharose that contains target DNA band, put into 1.5mL centrifuge tube and weigh; Add the GSB solution of 3 times of volumes, bathe 6-10min, interruption mixing in 55 ℃; Treat that blob of viscose melts completely, add appropriate sodium-acetate, adjust color identical with GSB color; The gelating soln melting is down to after room temperature, adds standing 1min in adsorption column, the centrifugal 1min of 12000rpm, abandoned stream fluid; Add the WB solution of 650 μ L, the centrifugal 1min of 12000rpm, abandoned stream fluid; The centrifugal 1min of 12000rpm, removes residual WB; Adsorption column is placed in to a clean centrifuge tube, uncaps and leave standstill 1min, make residual ethanol volatilization clean, add the deionized water of 20 μ L in the central authorities of post, room temperature leaves standstill 1min; The centrifugal 1min of 12000rpm, eluted dna, is put in-20 ℃ of Refrigerator stores by the DNA of wash-out.
(4) PCR product connects, transforms and identifies
Connect: in the centrifuge tube of 0.2mL, add following reagent: above-mentioned PCR purified product 4 μ L, pEASY-T1 carrier 1 μ L, mixes gently, room temperature reaction 20min, reaction finishes to be placed on ice.
Transform: be added in the Transl-T1 competent cell that volume is 50 μ L connecting product, flick and mix, ice bath 20min; 42 ℃ of heat shock 30s, are placed in 2min on ice immediately; Add 500 μ L liquid LB substratum, 200rmp, 37 ℃ of shaking tables are hatched 1h; Get 40 μ L X-gal(20mg/mL) and the IPTG(500mM of 8 μ L), after mixing, being applied to equably on LB solid plate substratum, 37 ℃ of incubators are placed 30min; Get 200 μ L bacterium liquid and be coated with flat board, 37 ℃ of overnight incubation.
Identify: the single bacterium colony of picking white is in cultivating 10h containing shaking table in the LB liquid nutrient medium of Amp, and bacterium liquid carries out bacterium colony PCR.Bacterium colony PCR reaction system is 10 μ L:2 × EasyTaq SuperMix5 μ L, M
13the each 1.0 μ L of F upstream and downstream primer (10 μ M), cDNA template 1.0 μ L, sterilizing ddH
2o2.0 μ L.
Bacterium colony PCR reaction conditions is: 94 ℃ of 10min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 30 circulations; 72 ℃, 10min.
Agarose gel electrophoresis detects bacterium colony PCR product size, selects recombinant clone, serves the order-checking of Hai Shenggong biotechnology Services Co., Ltd.
4, hidden kind of MED BtTrp gene 5 ' RACE of Bemisia tabaci MED and 3 ' RACE obtain
The intermediate segment sequence of the hidden kind of MED BtTrp gene of Bemisia tabaci MED obtaining according to step 3, design 5 ' RACE and 3 ' RACE Auele Specific Primer, 5 ' and 3 ' terminal sequence of pcr amplification MED BtTrp gene:
5’RACE Outer Primer:5’-TTGGCGGCAATAGCGTTCCAGTCC-3’
5’RACE Inner Primer:5’-CTCTTTGTCCACGCTCGGTGTCTT-3’
3’RACE Outer Primer:5’-ACACCGAGCGTGGACAAAGAGGA-3’
3’RACE Outer Primer:5’-CTATTGCCGCCAAGAAAGCATCCAG-3’
Adopt SMARTerTM RACE cDNA Amplification Kit(Clonetch) test kit, and operate in strict accordance with test kit specification sheets, amplification obtains object band, utilize DNAMAN and the splicing of GENtle software to obtain the cDNA sequence total length of TRP gene the sequence after cloning and sequencing, obtain MED BtTrp gene 3906bp sequence, the gene of gained has the sequence as shown in SEQ ID No.1.
ATGGGGAGGCCGACGGAGCGGGCCACCCGAGCCGGACAGTCTGTGCTTGCTTGTCGTTTTCGCCACGATGGAGTAATCCGTTCCATCCCAGTTTTGGGTAGTGCTCCATTTTTTGGATAACGCTTTTTCCCAGGTGCGAGGGGCAGGATGAAGCCGACGGAATCGAAGGAGGACCTCCTGGGGTCGAAGGAGAACCTAAAAAATCCCCGAGAGTCCTTGCAGTCGACCGGCGGCGAGGTTGCCCTCACCCAGATCGAGAAAAGGTTCCTCTTGCTCAGCGAAAGGGGAGATATCGCCTCGGTCAAAAGGATAATCGCTGAGCACAAAGATCACCCAGAACTATTGAACATCAACTGCGTTGATCCTCTGAACAGGTCAGCACTCATCGCAGCGATCGAAAATGAAAATATAGAGTTAATAAGAGTCCTCCTCGAACTTGGGATTGAAGTCAAGGATGCTCTTCTGCACGCAATTAAGGAAGAGTACGTTGAAGCTGTGGAGATTCTACTTGAATGGGAAGAAAAAATCCACAAACCTGGGCAGCCTTATAGTTGGGAAGCTGTTGACGGGAGCTCTTCGACATTTACACCGGACATAACACCGCTCATATTGGCTGCTCACATGAACAATTACGAAATCCTGAAAATTTTGTTGGATCGAGGCGCCACACTCCCCGCGCCACACGATGTTAGGTGTGGCTGCGACGAATGTGTAACTTCAAGTGAGCAGGATTCTTTGCGACATTCACAAGCTCGGATCAACGCGTACAGAGCTTTAACCGCACCATCCCTCATAGCCCTCTCTTCCAGGGATCCCCTCCTTACCGCCTTCGAGTTATCATGGGAGCTGCGCAGACTCAGCAAGATGGAAGCGGAATTCCGCTCTGAATATAATGAAATGAGAGGTATTTGTCAGACATTTGCAACCTCCTTGCTAGACCACGCGCGAACCTCGTATGAACTGGAGGTGATGCTTAATCATAATCCTGATGGAGAGTCGTGGGAACCAGGAGAGAGGCAAACTCTGGAGAGACTGAAGCTAGCTATCAAATACAAACAAAAAGCGTTTGTTGCTCATCCAAATGTACAACAGCTGCTGGCAGCTATTTGGTATGATGGTCTCCCCGGATTTCGACGAAAAGGGATGATTGGTCAAGCTTTTGAGTGTGGAAAGTTAGGTGCAATGTTTCCTGTCTACGCAACCGTGTATTTGACAGCGCCTGAATCGGAGATGGGCCAGTTCATGAAAAAACCCTTTGTAAAGTTCATCTGTAATTCAACCTCCTACGCTTTCTTCCTGATGTTATTGGCCTTGGCATCACAAAGAGCGGAATTTCTTGCAATCGAATGGTTTGGACCCGACTGGATGAAAGAGATACTGAAAGAGTGGACTCGAAAGGAAAGAGGATCAATACCTGGTCTCATAGAATCCTTTATTATTTTGTATATCATAAGTTTGATTTGGGGTGAAATGAGAGCATTGTGGTCTGGTGGCTTAGAGGACTATGTTTCAGATTTGTGGAATATAGTGGATTTCATCACCAATGTATTCTATATGATATGGATTTGTCTCAGAGGAACTGCGTATATCATTGTGCAAAGAGAGCATAAAAGTGGCCTCGATCCATGGTATCCTCGTGATAAGTGGGACATGTTTGATCCTCATCTTCTATCAGAAGGTGCGTTTGCAGCCAGTATGATCTTCAGTTTCTTAAAATTGGTTCATATCTTCAGTATCAATCCTCATCTTGGTCCACTGCAAATTTCGTTGGGACGAATGATTGTCGACATCATCAAATTTTTCTTCATCTACACATTGGTCCTTTTCGCTTATGGTTGTGGTATGAATCAGTTACTTTGGTACTATGCTGATCTTGAGAGGAAAAAGTGTTATCATCTGAACGAGGATTATGCT GATTTTGATGGTCAAGATAAAGCCTGTACAACATGGAGACGTTTTGCAAATTTATTTGAAACATCTCAGTCTCTGTTCTGGGCCAGTTTCGGTTTAGTTGATCTTGTATCGTTTGATCTGACTGGTATTAAGAGTTTCACCCGGTTTTGGGCTCTTCTGCTTTTCGGATCATACTCCGTTATCAATATCATTGTCCTGCTCAACATGTTGATTGCCATGATGTCCAACAGTTACCAAATCATCTCTGAACGATCCGATACTGAATGGAAATTTGCTCGAAGTGACCTATGGATCAGTTACTTTGAGGATGGCGACACTGTTCCTCCACCATTCAACCTATCACCATCTGTTAAATCTATGAAAAGAATGATGGGAATCAAAACTGGGAAAATCAGCACTGGTTCGATGAAGAACAAAAGTCGAGCTAAAGCTATGGAGCGTCACGAAGCAGTCATGAAACTATTGGTCAGGCGATATGTTGTGGCTGAGCAAAGGAAGAGAGATGAGTTCGGCATCACAGAAGATGATGTTATGGAAATTCGACAAGATATTTCAACCCTTCGTTTTGAACTGATCGATATCCTCCGAACAAACGGAATGAAGACACCGAGCGTGGACAAAGAGGATCAAGCAGTGGGCAAGAAAGGTCGTGTGATGGAGCGTCGTTTGCTGAAGGACTTCCAGATCGGCCTGGTTGAGGGTATAATTAGTGATGTCATATCTTCTGAGAAAGAGCCAAGGGATGTCTTCAGCAAGATTGCTCGTGCAATTGGTAAGGACTCAAGTGCAAAGAAAAAGGACTGGAACGCTATTGCCGCCAAGAAAGCATCCAGCCGCGAACTTATTGGAAGCACCAGCGAGGCTGTGGAGCGTCGGCAATCACGCCAGAGTTTGCGCCGGCACATCCTCGAACATCAAGGATCAGCAATGGCAAATATGGATCCCGAGAAGCTGGTTGAGTACAATCCCAAGTTGCAAGAATATGCACCTGCTGCTCGAATCGCCTATGCCAAATTCAAGATCTCCAAGATCAAGCAAGAATATGACGATAAAGGAAATGAAGAAGAAGGCAAAGACGATGTTTTCGAGCCTGCTACAGTAACAACGAAGGACGAAACAAAACCATCCCAAAGTGGCTCCATCGCACGGCCGAGACCCAAAAGCACCAAGAAACCACCCGCACCTGGAGCCATTCCAGGTCCCGACGATGGCAAGAAAGACTTCCGGTCGCGAACTCCGATCCAAGAAGATCCAAGAGAGGCTGGCCGCACTCCAACGCCAGAACCAATCAAAATGCTCAAGACATCGATTTCCTCTCTCAAGCAAGTATCAGTCGATCAAAAAGCTGGGCCATCAGACAAAAAACCCGCCGCCCCGGGGGCTCCGGCTCCGAAAGTAGAGGTCACCGCAGCTAAATCACCTGCAAAAACAGAGGCAGCCCCGGCAAAACCTGAAGAAAAGAAGCCCACACCTGCTAAACCAGAGGAAAAGAAGCCTGCCGCTGCTGCTGCTACGCCTAAAGCAGACGAAAAGAAGGACGAGAAGAAAGAAGAAAAGAAGGACGAGAAGAAAGATGAGAAGAAAGAGGAAAAGAAAGACGATAAAAAGCCGGGCCCTGCTCCGAAAAAACCAGATGATAAGGATAAGCCTCCCTCGCCGAAGAAAACGATGATCCACACAAAACCTGGTGGTAAATCAACAGTCACCGGCGAAGTACTGACTGGATGGTTGTAAGTATTTCAAAAGAGTAACGTGTACTGGCTGCAAAAACTGATTAGTTTAATATTTCACTTCAGTGGTTTTTTTTAAACTAGCAAATTTTCTGTCTTAAAATTCTTTTTCAATTGGCAGAAACGGAGTTCTTGCATGCGTGAGGGATTTGTGATTTAAGTATTTGTTCTTACATAAAAGTTCGCGAGCAACGAGTTGCGGTGCCCCTGGTTTTCTCTGAAATCAACTCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
The dsRNA of embodiment 2:MED BtTrp gene processes the stable on heating impact of the hidden kind of Bemisia tabaci MED
1, the preparation of dsRNA template:
Add the primer sequence after T7 promotor (sequence shown in underscore):
T7+ MED BtTrp-F：
TAATACGACTCACTATAGGGAGACCACGAAGACACCGAGCGTGGACAAAG；
T7+MED BtTrp-F：
TAATACGACTCACTATAGGGAGACCACGGCAATAGCGTTCCAGTCCTTTT。Synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Total RNA extracts and cDNA synthesizes: with embodiment 1.
The amplification of T7 primer PCR:
Reaction system is 50 μ L:10 × PCR buffer5.0 μ L, dNTPs1.0 μ L, cDNA template 1.0 μ L, Primer-T7F2.0 μ L, Primer-T7R2.0 μ L, TransStart Taq DNA Polymerase1 μ L, ddH
2o38.0 μ L.
Reaction conditions: 94 ℃ of 5min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ of 10min.Result detects: confirm that the band increasing is object band.
The PCR product purification of T7 primer amplification:
Use Qiaquick PCR purification kit (Qiagen, Inc., Hilden, Germany) test kit purified pcr product, operate according to test kit specification sheets.
The PCR product of purifying gained is the template of synthetic dsRNA.
2, the synthetic and purifying of dsRNA
Use
rNAi Kit test kit synthesizes and purifying dsRNA, operates according to test kit specification sheets
3, dsRNA feeds
Parafilm film uses DEPC water treatment to remove RNA enzyme in advance.The sucrose liquid that is 10% in concentration (the DEPC ddH of sterilising treatment
2o dissolving saccharose) in add dsRNA, concentration is 0.3～0.5 μ g/ μ L.According to this experiment demand, to Parafilm film folder nutritive medium method (Miles et al., 1965) done corresponding improvement: get approximately 200 of hidden kind of adults of Bemisia tabaci MED that just sprout wings and put (diameter 3cm in the penetrating Glass tubing in people two ends, high 8cm), Glass tubing upper end adds 10% sucrose liquid 200～250 μ L that contain dsRNA coated with two-layer Parafilm film and in two intermembranous spaces, ventilate with maintenance coated with gauze in Glass tubing lower end, Glass tubing around and lower end be wrapped with and be beneficial to Bemisia tabaci and assemble to top Paraflim film with black plastic paper bag, thereby better take food dsRNA mixed solution.The Glass tubing of handling well is put into growth cabinet (25 ± 0.2 ℃ of temperature, 24h illumination, relative humidity is 60-70%), feed after the dsRNA of 3 hours MED BtTrp genes, Bemisia tabaci is collected in to (long 5cm, diameter 1.2cm) in finger type pipe, puts into high temperature water bath (the Microprocessor Control MPC Huber of preheating, Germany) process 1h, treatment temp is 45 ℃.Adult after treatment recovers 1h under 26 ℃ of normal temperature, statistics survival rate.The hidden kind of Bemisia tabaci MED of 10% the sucrose solution of crossing containing dsRNA and sterilising treatment to feed not in contrast, every group of 5 repetitions.Utilize the SPSS16.0 statistical software analysis hidden kind of survival rate of Bemisia tabaci MED after different solutions of feeding, as shown in Figure 1, the survival rate of the hidden kind of Bemisia tabaci MED of the MED BtTrp gene dsRNA that feeds is significantly lower than sucrose group and the blank group survival rate (P<0.001) of feeding for result; Simultaneously at NCBI(http: //blast.ncbi.nlm.nih.gov/) BLAST shows, the target sequence sheet degree of feeding is the distinctive sequence of TRP gene, guarantee that thus interference effect is produced by the object MEDBtTrp gene of the hidden kind of Bemisia tabaci MED, therefore, the present invention illustrates that MED BtTrp gene plays keying action in hidden kind of high temperature stress of Bemisia tabaci MED and high temperature perception.
Claims (2)
1. the instantaneous susceptor ion channel gene of the hidden kind of Bemisia tabaci MED MED BtTrp, is characterized in that, its nucleotide sequence is as shown in SEQ ID No.1.
2. described in claim 1, the instantaneous susceptor ion channel gene of the hidden kind of Bemisia tabaci MED MED BtTrp is used for preventing and treating the application of Bemisia tabaci.
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| CN106884007A (en) * | 2017-03-05 | 2017-06-23 | 中国农业科学院植物保护研究所 | Hidden kind of laccase LAC1 of Bemisia tabaci MED and its gene and application |
| CN111118172A (en) * | 2020-01-06 | 2020-05-08 | 中国农业科学院植物保护研究所 | Specific probe of aleyrodids Asiai II3 cryptophyte transient receptor ion channel gene and application thereof |
| CN111893101A (en) * | 2020-08-17 | 2020-11-06 | 中国农业科学院植物保护研究所 | Bemisia tabaci MED cryptic acetyltransferase gene mof, application thereof and method for reducing low-temperature tolerance of Bemisia tabaci |
| CN111893101B (en) * | 2020-08-17 | 2022-03-25 | 中国农业科学院植物保护研究所 | Bemisia tabaci MED cryptic acetyltransferase gene mof, application thereof and method for reducing low-temperature tolerance of Bemisia tabaci |
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