Cloning And Identification Of Deltamethrin Resistance Associated Genes Of Culex Pipiens Pallens By Combining Suppression Subtractive Hybridization And CDNA Chip*

Cloning and identification of deltamethrin resistance associated genes of Cukx p4iens pallens by combining suppression subtractive hybridization and eDNA chip*ABSTRACTInsecticides have played an important role in the control of pests since the early 20th century. Although important advances continue to be made in the development of alternative control measures, insecticides will remain a vital part in the integrated control programs of vectors for the foreseeable future. Unfortunately, the remarkable ability of vector populations to evolve resistance to every class of insecticide has been developed. Now it has been recognized that a change in the genetic composition of a population is a direct result of the selective effects of a toxicant An understanding of the insecticide-resistant mechanisms can lead to better management of pest resistance to pesticides. By strategy of functional cloning, several genes have been reported to be related with insecticide resistance, such as cytochrome P450 gene, sodium channel gene, esterase gene and so on. How many genes contribute in the mechanisms of insecticide resistance? Are there other genes acting on the resistance? In terms of the strategy of "phenotype cloning " differentially expressed genes between the deltamethrin-resistant-8-and suscePtible ed of Culexgh~ were cfoned by themethod named SUPpessha bo hybM (SSffi, andwer Med by N chiP, reverse nOrthem bfot and nOHbwt A ffe mp libop was construtriby be cloninteCboc using the deltametbrinesiStan strain of Cx twN as samPle and a full lengIh cDNA gen named twsinsen was pe by SCreenin the COnStrUctin ffe mpbol Isolation of drially mpssed pe betweedeWtw and SUSeePtile hans of Cx ptwpeuens by SSHThe deWsusCePtible bo of Cx plPbo the wasorigined from the Shangh Insect Ihatitu of Chinse Academof Science and the LC50 value was 0.0008mgh. W theMresiM H was M by seleCted wtthuefor ana the LC50 ds was 0.32mrt. In orta to setthe gens tha wer high and rpecilically exPreSsed in theresiStan strain, the - poPulation ofresistan sha was usedas M and that ofsuseqle nd as the The total RNA andM wer isolated rerpectiVely from the both for adults thathad no been fed wh blood within three-da edosion. The singlwt and Hle ed cDNAs wer Synthsed. Then theSedOUble Stran cDNAs wer digested by the An I.The teste- 9 -dscDNAs were -- added to adaPo l and adaPtO 2Kbo, the de dscDNAs were mp with the excessbe oc An bo the op pe were ~ by-- PCh The PCR Produds were cfoned into im~ vectors dri were ds bo E coli mloo. Aiwt 523 subwt clones were pe.hi de to get the pe ta were highly ana peificallyexPwt in the Hle tw the m poPulation ofHle ni was used as teSter and that ofresStan ni asdriVer The SSH was wi as above and 286 subwt cloneswere pe in the ena.The length of SSH be inserted into Pinpofor vector wasN 300600 base pairs by PCR detoCon.The results aboVe SUggested tha man gens Inay play roles onthe dehosistance orcx twpouas anu that no o*the retw W may H on insecticide-resStanCe, bot also theWle pe.2 hidendfication ofsSH clones by cDNA chiPA M chiP containin 809 SSH clones was mad. Briefly thebo in the hafor pedd vecto were amnlified by Pcnusing seqUenCes flankin the cfonin site as PriIners. The PCRwt were viW on l% Wse ge to ensure adWPCR ammpcation Prior to being robecally Printed onto nyon- l0 -meInbran. The un frOm the both ch were repernad as resistan Probe and SUSW Probe by reverseW in the M of a ?3P dCTPscanning, W and subSeqUen -- were-- It was that l55 and 42 pe were re~ 2-3 fodand >3 fold OVeWed in the resed tw l5 and 9 pewer rerpeCtively 2-3 fold and >3 fod OVeWssed in theHe the. In additio there wer 2 gens gncificallyWssed in the resiStan sha.ACcoding tD the results, we Med that vecto reSStane toinsectiCde may be rethed ed OVeWssed. lowWsed andsPecifically exPrssed gens in insecticideresiSha sha.3 M Mdri of SSH clones rewt by reversenota...