淡色库蚊EU073017基因的克隆和初步功能鉴定

发布时间：2018-02-09 09:24

本文关键词： 淡色库蚊溴氰菊酯抗药性 EU073017基因克隆定量PCR 稳定转染细胞存活率　出处：《南京医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 【目的】克隆淡色库蚊EU073017基因,研究其与蚊抗药性关系。【方法】根据本试验室抑制性差减杂交(suppression subtractivehybridization,SSH)结合cDNA芯片筛选获得的EST片段(GenBankno.BE 247823),设计引物,从淡色库蚊溴氰菊酯抗性品系cDNA文库中分别扩增出目的基因的5'和3'末端,T/A克隆于pGT载体,转化、测序、对位拼接获取基因序列。生物信息学软件分析EU073017基因编码的氨基酸序列。实时荧光定量PCR验证EU073017基因在淡色库蚊溴氰菊酯抗性品系和敏感品系(4龄幼虫期)的表达差异。应用基因重组方法构建EU073017基因的昆虫表达载体,并转染入白纹伊蚊C6/36细胞,经药物筛选获得稳定转染单克隆细胞系;RT-PCR鉴定目的基因在转染细胞的转录水平。Cell Counting Kit-8(CCK-8)试剂盒检测转染细胞系对溴氰菊酯的敏感性变化。【结果】获得淡色库蚊EU073017基因,长度为398bP,编码序列(coading sequence,CDS)长度为270 bp,推导编码89个氨基酸(GenBank no.ABU49655,2007)。该推导氨基酸序列与致死按蚊(Anopheles funestus)、冈比亚按蚊(An.gambiae)和埃及伊蚊(Aedesaegypti)同源基因推导氨基酸序列同源性分别为86%、84%和80%。实时荧光定量PCR结果显示,EU073017基因在溴氰菊酯抗性品系(4龄幼虫期)中的表达是敏感品系(4龄幼虫期)中表达的1.65倍。成功构建了EU07301基因的昆虫表达载体,并构建了稳定转染EU073017基因的单克隆细胞系。稳定转染细胞对溴氰菊酯敏感性测定结果显示:在40、60、80、100和120μM浓度的溴氰菊酯处理下,转染EU073017基因的蚊细胞存活率要高于对照组细胞系的存活率(p＜0.05)。【结论】本研究克隆了淡色库蚊EU073017基因。该基因在淡色库蚊溴氰菊酯抗性品系中高表达。转染EU073017基因可提高蚊细胞对溴氰菊酯的抵抗性。
[Abstract]:[objective] to clone the EU073017 gene of Culex pipiens pallens and study its relationship with the drug resistance of Culex pipiens pallens. [methods] according to the suppression subtractive subtractive hybridization (SSH) and cDNA chip screening of EST fragment GenBankno.be 247823 in this laboratory, primers were designed. From the cDNA library of Culex pipiens pallens deltamethrin resistance strain, the 5 'and 3'terminal TPA of the target gene were cloned into pGT vector, transformed and sequenced. Acquisition of gene sequences by pairwise splicing. Analysis of amino acid sequences encoded by EU073017 gene by bioinformatics software. Real-time fluorescence quantitative PCR verification of EU073017 gene in 4th instar larvae of Culex pipiens pallens deltamethrin resistant and sensitive strains). The insect expression vector of EU073017 gene was constructed by gene recombination method. After transfection into C6 / 36 cell line of Aedes albopictus, stable transfection monoclonal cell line was obtained by RT-PCR. The sensitivity of the transfected cell line to deltamethrin was detected by using the cell Counting Kit-8 CCK-8 kit. [results] the EU073017 gene of Culex pipiens pallens was obtained. The length of the deduced amino acid sequence was 398bPand the length of the coding sequence was 270bp.The deduced amino acid sequence was GenBank no. ABU49655Anopheles funestusus, Anopheles gambiae (Anopheles gambiae) and Aedes aedesaegypti (Aedes aegypti). The deduced amino acid sequence was homologous to Anopheles funestusus, Anopheles gambiaeus and Aedes aedesaegyptii, respectively. The deduced amino acid sequence was homologous to Anopheles funestusus. The results of real-time fluorescence quantitative PCR showed that the expression of EU073017 gene in deltamethrin resistant strain was 1.65 times higher than that in sensitive strain of 4th instar larval stage. The insect expression vector of EU07301 gene was successfully constructed. A monoclonal cell line stably transfected with EU073017 gene was constructed. The sensitivity of stable transfection cells to deltamethrin was determined. The survival rate of mosquito cells transfected with EU073017 gene was higher than that of control cell lines (p < 0.05). [conclusion] in this study, the EU073017 gene of Culex pipiens pallens was cloned and overexpressed in the deltamethrin resistant line of Culex pipiens pallens. Transfection of EU073017 gene can enhance the resistance of mosquito cells to deltamethrin.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R383

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