溴氰菊酯抗性白纹伊蚊抑制消减cDNA文库的构建

Construction of Suppression Subtracted cDNA Library of Deltamethrin-resistant Aedes albopictus

目的构建溴氰菊酯抗性白纹伊蚊抑制消减cDNA文库。方法分别提取白纹伊蚊溴氰菊酯抗性株(R-lab株)和敏感株(S-lab株)总RNA,纯化后获得mRNA,并反转录为双链cDNA。以R-lab株mRNA作为实验方(tester),S-lab株mRNA作为驱动方(driver),以及R-lab株mRNA为driver方,S-lab株mRNA为tester方,进行正、反双向抑制性消减。富集的差异表达cDNA克隆到pMD18-T载体,构建白纹伊蚊对溴氰菊酯抗性和敏感双向消减文库。结果分别从正向文库和反向文库中获得580和477个阳性克隆,从所构建文库随机挑选150个克隆进行PCR鉴定,阳性克隆率为93%,cDNA片段主要分布于150～750bp。结论构建了抗溴氰菊酯白纹伊蚊抑制消减cDNA文库。

Objective To construct the suppression subtracted cDNA library of deltamethrin-resistant Aedes albopictus. Methods Total RNA was extracted from the dehamethrin-resistant （R-lab） and -sensitive （S-lab） isolates, mRNA was obtained after purification. Double stranded eDNAs were synthesized after reverse transcription. Two subtractions were performed by suppression subtraetive hybridization with S-lab as tester and R-lab as driver or S-lab as driver and R-lab as tester. Enriched different expressed eDNA was cloned into pMD18-T vector to construct subtractive libraries. Results The subtracted eDNA libraries contained 580 and 477 positive clones respectively. The PCR results of 150 clones picked randomly from each library showed that the positive ratio of constructed eDNA libraries was 93%, with a length of eDNA fragments ranged from 150 bp to 750 bp. Conclusion The suppression subtracted eDNA library of dehamethrin-resistant Ae. albopictus is constructed.