【作者】 田海生；

【导师】 吴观陵； 朱昌亮；

【作者基本信息】 南京医科大学 ， 病原生物学， 2001， 博士

【摘要】 杀虫剂一直是人类杀灭害虫的重要武器，尤其是40年代有机合成杀虫剂问世以后，在病媒防制等方面发挥了巨大作用。然而时至今日，虫媒病无论在分布范围还是感染人数上，都呈明显的增长势头。究其主要原因，是连续、大量地使用杀虫剂导致媒介昆虫产生了抗药性（以下简称抗性）。 目前已知，昆虫抗性是可遗传的生物进化现象，是由基因决定的，在绝大多数情况下，抗性为多基因遗传。 对媒介抗性基因的研究，早期通常首先寻找杀虫剂的作用靶标（受体）或杀虫剂代谢相关酶类，然后再根据发现的特异蛋白质（酶）来克隆相应基因。如与有机磷类和氨基甲酸酯类杀虫剂抗性相关的酯酶基因，与拟除虫菊酯类杀虫剂抗性相关的钠通道基因与细胞色素P450基因等。以上经典研究途径仅能分离单一基因，难以从总体上认识抗性、阐明抗性机理。这也是抗性研究长期进展缓慢的主要原因。当前分子生物学技术的快速发展使得大通量筛选抗性相关基因成为可能。 淡色库蚊（Culex pipiens pallens）是我国重要的家栖性病媒蚊种，近年来各地主要采用拟除虫菊酯类的溴氰菊酯进行防治，已陆续有敏感性显著下降的报告。 本研究采用正向和反向抑制性差减杂交法（suppressionD 南京医科大学博士学位论文D subtractive hybridization，SSH）分离淡色库 氰菊酯 ll 抗除和敏感品系高表这或 固，并呆用 cDNA芯片、l 叹 Northern和 Northern blot g 同时，构建了淡 DD 色库诌d亢性品系cDNA支J享，从中筛 出目 困的cDNA DDde＆｝＋列。D11．用SSH分离淡色库蚊涣氰菊酯抗，队R兢因lD 糊化3天内来吸血的淡色库狮隆成蚊，以对跟菊DD 酯坑姓品系为鹏d组k ter）、对鳅菊＠郭劲感品系为g孵 DD 组Uriver），进行正向 SSH，以 性品系高表达或特异 DD 表腿因；以咖品系为tester、抗怯品系为driver，断 l 反向SSH，以获取敏感品系高表达或特异表达基固。分别忖影粑lD 2品系蚊虫的总RNA、InRNA，碾一cDNA第嘴，置换 D19：vsa．第二链，然后经Rsa酶切、接头连接、24kL减杂交 lD 和2轮叽R，获得正、反向差减杂交严物。朵交严物纯化后将D 立9。降入质陇一PinPoint“Xa－IT－－Vector，转化入大D 肠杆菌Wil 09。以趴片段两侧徽体序列PiflPoint和SP6为DD51物PCR扩增，琼脂糖凝胶电帷…X段大小。DD 结果：N、反向昭H分一瓣523和286个差献隆，Dl 构建了 2个el］含 5乃和 286个重组子的正、反向差减M。lD 枫片段大小约3m6。初步提示淡色犀刎没氰菊酯的DD 抗性可能涉及的并非个别基因，而是多基因或基因群；抗性DD 不贩抗贻因控制，而且可5腑…基因。DD．二．D 南京医科大学搏士学位论文D 2．用cDNA芯片鉴定淡色库蚊抗性和敏感品系差减支隆l 用PCR扩增棚一一的8 09个差献隆，PCR产l 物经贱、变性处理，用自动点 左制于尼龙膜戴体上，7 制成cDNA芯片。性品系和敏感品系IllRNA，反转轭经D 同赈‘？ 己为抗注探针和敏感探针。将两 与2 D 剃目同的芯片杂交，通过跟、压片、栅自显影扫和唁号扫D 描鞭弧的杂妮号。D 结果：CDNA芯片共获得差异表达克隆 2 21个，其中抗性 D 品系高表铂隆197个，卜3顾3倍以上高雄克降分另fi有D 155个和42个；髓性品系高表贩隆24个，2刁顾3倍1 以上高表达克o有15个和9个：郴卜脯2个差减氛D 怯品系中特异表达。拥究结果进一命支持本文第一ID 部分的撕，即淡色库附涣氰菊酯的抉注可能涉及的并非DDc知个别基因，6是多基因或基因群；抗性不仅受抗赈因DD 控制，而且可s腑眺基因，抗性可能航踉困8裘达D 和特异表达、敏感基因低表达综合作用的结果。 3．用叹 Northern和 Northern blot j｝L－步跟淡色犀颜D 性牙一铣感品系差减。寺。隆l 逆 Northern：＄先 PCR扩增 cDNA芯片中抗性品系 3倍］以上高一克隆16、43、30、6、38和敏感品系3倍以上l 高表达的兑隆 33、27、5L 45、4的插入基因片段，琼脂糖I 凝胶《膝梆至尼溅。然后分别 恰品B一品D 系更多还原

【Abstract】 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更多还原