【作者】 沈波；

【导师】 朱昌亮；

【作者基本信息】 南京医科大学 ， 病原生物学， 2002， 硕士

【摘要】 化学防制一直是媒介防制规划中的主要方法，杀虫剂的连续、大量使用导致了抗药性的发生、发展。媒介抗性治理有赖于抗性机制的理解。目前认为抗性是由基因决定的。 本研究运用快速扩增cDNA末端法(rapid amplification of cDNA ends,RACE)从淡色库蚊抗性品系cDNA文库中扩增β-肌动蛋白基因和糜蛋白酶基因cDNA全长序列，并用荧光半定量PCR检测糜蛋白酶在抗性品系和敏感品系中的表达水平。 1．淡色库蚊β-肌动蛋白基因克隆及序列分析 采用RACE法筛选淡色库蚊溴氰菊酯抗性品系cDNA文库。根据GenBank收录的冈比亚按蚊(Anopheles gambiae)与黑腹果蝇(Drosophila melanogaster)β-肌动蛋白基因全序列设计引物，分别与文库载体序列进行PCR扩增，获得β-肌动蛋白基因的5’和3’RACE序列，然后通过T／A克隆插入pGEM-T质粒载体，经过测序、拼接获取长序列。用BLASTp软件将该基因推导的氨基酸序列与蛋白质公共数据库Swissprot比对、分析。 结果：获得淡色库蚊β-肌动蛋白基因全长序列，总长度为1290bp，开放阅读框为1132bp，编码377个氨基酸(GenBank／NCBI AY100005)。推导的氨基酸序列与公共数据库比对，发现与冈比亚按蚊β-肌动蛋白和黑腹果蝇β-肌动蛋白的同源性均为91％。蛋白质的理论分子量为41.7KD，等电点(PI)为5.4。淡色库蚊β-肌动蛋白基因全长的获取为蚊虫分子生物学的进一步研究奠定了基础。 2．淡色库蚊抗性相关糜蛋白酶基因克隆及序列分析 南京医科大学硕士学位论文 4Rib筛选额的抗。曲目关孵白酶cDNA片段，附引物，运用RACE法扩增基因的5’和丁末端，然后通过Ta克隆4趴pGEM＜质粒载体，经过测序、只帐椭…助列。用BLAS乃洲将该基因推导的氨基酸序列与蛋白质公共数据库SWISgh分析，用ClustalW yftl＝作聚类分析。并用荧光半定量PCR方法检测糜蛋白酶在抗性品系和敏感品系中的表达。 结果：踉淡色库顺蛋白鹏因全朋列，总眼为9刀hp，开放阅读框为sl3bp，编码271M 酸（GenBank／NBI AF468495）。切1剁言号鹏成熟肽的忖量为26．SKD，PI为4．5。推导的抛晰列与忖娜库比对后发现，与埃腴酬晰a娜腆蛋白颐舌虫（Gaho＊orsdan）丝氨6婚白酶的同源性 拥u为73％和50％。荧光半定量RTPCR检测，糜蛋白跄固InRNA在抗性品系比釉品系高表达。表明糜蛋白酶与淡色库蚊涣氰菊酯抗，队e关。淡色库滩蛋白跄因的获取对于媒介抗药胁测、抗药性的肝姆研究和抗药性基固资源的开发和利用等具有重要意义。更多还原

【Abstract】 Insecticides have played an important role in the control of pests since the early 2 Oth century. Insecticides will remain a vital part in the integrated control programs of vectors for the foreseeable future. Unfortunately, the vector populations in some areas have developed remarkable resistance to eveiy classes of insecticide. 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.In this paper rapid amplification of cDNA ends (RACE) was used to screen the constructed cDNA library and the full-length sequences of (3 -actin gene and chymotrypsin gene were gotten. Then LightCycler fluorescent quantification system, (3 -actin gene as control, was used to validate the different expression of chymotrypsin mRNA between deltamethrin-resistant and susceptible strains of Cx pipiens pallens.1. The acquirement and analysis of full-length (3 -actin cDNA of Cx. pipiens pallens.Two primers were designed according to the (3 -actin genes of Anopheles gambiae and Drosophila melanogaster from GenBank RACE were used to screen the constructed cDNA library and the 5’ and 3’ RACE fragments of the (3 -actin cDNA of Cx. pipiens pattern were gotten and cloned into pGEM-T vector ,then sequenced. After splicing, a sequence of 1290 base pairs including an open reading frame of 1132 basepairs was gotten (GenBank/NCBI AY 100005 ) . The deduced protein had 377 amino acids and the theoretic molecular weight and PI were respectively 41.7KD and 5.4. Aligned to Swissport, it shared 91% and 91% homology respectively with (3 -actin gene of An. gambiae and D. melanogaster. The (3 -actin gene will be used as the internal control in several experiments of Culex mosquitoes.2.The acquirement and analysis of full-length chyinotrypsin cDNA of Cx. pipiempallens.Two primers were designed according to the chymotiypsin gene fragment obtained from SSH. RACE were used to screen the constructed cDNA library and the 5’ and 3’ RACE fragments of the chymotrypsin cDNA of Cx. pipiens pallens were gotten and cloned into pGEM-T vector ,then sequenced. After splicing, a sequence of 977 base pairs including a open reading frame of 813 base pairs was gotten (GenBank/NCBI AF468495 ). The deduced protein with 271 amino acids shared 73% and 50 % homology respectively ,aligned with Aedes aegypti chymotiypsin and Glossina morsitans chymotrypsin With LightCycler fluorescent quantification system, we detected a higher expression of chymotiypsin mRNA in deltamethrin-resistant strains of Cx. pipiens pallens, indicating that the chymotrypsin gene was associated with the deltamethrin resistance of Cx. pipiens pallens. The chymotrypsin gene is very important for studying the molecular mechanisms of insect resistance to pesticides.更多还原