【作者】 孙艳；

【导师】 朱昌亮；

【作者基本信息】 南京医科大学 ， 病原生物学， 2005， 硕士

【副题名】视蛋白基因（NYD-OP7）克隆、序列分析及表达载体构建

【摘要】 [目的] 在淡色库蚊溴氰菊酯抗性和敏感品系中鉴定视蛋白基因(NYD-OP7)的表达差异,获取NYD-OP7全长序列,并构建NYD-OP7原核和真核表达载体,为进一步研究奠定基础。 [方法] 用荧光定量PCR法鉴定NYD-OP7在淡色库蚊溴氰菊酯抗性和敏感品系中的表达差异。用RT-PCR法从抗性品系中扩增NYD-OP7全长序列,对NYD-OP7编码的蛋白用BlastX软件在NCBI和SwissProt数据库中进行同源性比对,用Clustalw软件进行聚类分析,用EMBL pI/Mw tool计算等电点和分子量,Signl3.0软件进行信号肽分析,ProtScale软件进行疏水性分析,PSORT Ⅱ软件进行细胞定位分析,PatSearch软件进行基序分析,SMART软件进行蛋白质结构域分析。通过基因重组方法构建NYD-OP7原核和真核表达载体。 [结果] 荧光定量PCR结果显示,NYD-OP7在淡色库蚊溴氰菊酯抗性品系中的表达量是敏感品系的9.78倍。获得淡色库蚊NYD-OP7完整开读框序列,为1116bp,经推导编码371个氨基酸(GenBank/NCBI AY749413,2004),与淡色库蚊NYD-OP1～6基因编码的视蛋白同源性最高,为91～93%。成功构建NYD-OP7原核和真核表达载体。 [结论] NYD-OP7在淡色库蚊溴氰菊酯抗性品系中高表达,提示NYD-OP7与淡色库蚊溴氰菊酯抗性相关,值得进一步研究。更多还原

【Abstract】 Objective To identify expression levels of deltamethrin-resistance associated opsin gene ( NYD-0P7 ) from the resistant strain of Culex pipiens pal lens, to clone ORF of NYD-0P7 and construct prokaryotic and eukaryotic expression plasmids of NYD-0P7.Methods Expression levels of NYD-0P7 between the resistant and the susceptible strains were identified by real time quantitative RT-PCR. The open reading frame (ORF) sequence of NYD-0P7 was cloned by RT-PCR, sequenced in an automatic DNA sequencer (ABI-100) and analyzed with bioinformatic softwares. The prokaryotic and eukaryotic expression plasmids of NYD-0P7 were constructed by gene re-construction methods.Results NYD-0P7 was found to be expressed 9. 78 fold in the resistant strain than in the susceptible strain by real time quantitative RT-PCR. The ORF sequence of 1116bp was obtained for the cDNAs, named NYD-0P7 (GenBank/NCBI AY749413,2004).The deduced protein with 371 amino acid shared 91 ~93% homology respectively, aligned with deduced proteins of NYD-0P1 ~ 6 of Cx. pipiens pallens. The expressed plasmids PGEX-6P-1/NYD-OP7 and pIEl-3/NYD-OP7 were re-constructed successsfully.Conclusion NYD-0P7 expressed higher in the resistant strain than in the susceptible strain of the Culex mosquito, indicating that NYD-OP7 may be associated with the deltamethrin resistance of Cx. pipiens pallens . The results in this work更多还原