蚊抗药性相关基因克隆与功能研究及用RNAi抑制蚊抗药性的初步研究
发布时间：2018-07-24 08:09
【摘要】：媒介昆虫严重危害人类健康,据估计传染病中2/3由媒介昆虫传播。世界史上许多危害严重的虫媒传染病如鼠疫、斑疹伤寒、黄热病、疟疾等都曾造成广泛的流行并夺去成千上万人的生命。当今世界随着人类交往的日益频繁,发现许多新的和重新出现的虫媒传染病。化学防治一直是媒介综合治理的主要方法。杀虫剂的大量、连续使用导致了媒介抗药性的发生和发展。目前,世界范围内至少已有504种昆虫产生了抗药性,包括主要的媒介昆虫,其中109种(亚种)蚊对一种或多种杀虫剂产生了抗性。杀虫剂抗性直接影响着虫媒传染病的流行和重新流行,因此,研究媒介抗药性产生的机制并进行适宜治理实乃当务之急。本研究拟从克隆抗药性相关基因入手,并对其进行功能鉴定,然后运用RNAi技术抑制抗药性基因,为探讨最终治理媒介抗药性奠定基础。为此,本文进行了以下几个方面的研究。 第一部分 淡色库蚊溴氰菊酯抗性相关基因的分子克隆与功能研究 研究一 淡色库蚊细胞色素P450 CYP6F1基因的分子克隆、序列分析及表达差异鉴定 为克隆淡色库蚊细胞色素P450(CYP6F1)基因并对其进行表达差异的鉴定,本研究采用RT-PCR技术和RACE策略,从淡色库蚊(Culex pipiens pallens)对溴氰菊酯抗性品系4龄幼虫中克隆细胞色素P450基因(CYP6F1),用相应的软件进行生物信息学分析,并用定量PCR和半定量RT-PCR分析敏感、抗性品系蚊的CYP6F1表达水平及对蚊各期CYP6F1进行表达差异鉴定。结果表明,成功克隆出CYP6F1基因及其等位基因CYP6F1v1。CYP6F1基因全长1639 bp,
[Abstract]:Vector insects are a serious hazard to human health, and it is estimated that 2 / 3 of infectious diseases are transmitted by vector insects. In the history of the world, many serious insect-borne infectious diseases such as plague, typhus, yellow fever and malaria have caused widespread epidemic and killed thousands of people. With the increasing frequency of human communication, many new and re-emerging insect-borne infectious diseases have been discovered in the world today. Chemical control has been the main method of comprehensive media management. The extensive and continuous use of insecticides has led to the development and development of vector resistance. At present, at least 504 species of insects have developed resistance to insecticides worldwide, including the major vector insects, 109 of which (subspecies) have developed resistance to one or more insecticides. Insecticide resistance has a direct impact on the prevalence and re-prevalence of insect-borne infectious diseases. Therefore, it is urgent to study the mechanism of vector resistance and to manage it properly. The purpose of this study was to clone and identify the genes related to drug resistance, and then to use RNAi technique to inhibit the resistance genes, and to lay a foundation for the final treatment of drug resistance vector. Therefore, this paper has carried on the following several aspects research. Part I Molecular cloning and functional study of Deltamethrin Resistance related Gene of Culex pipiens pallens; Molecular cloning of cytochrome P450 CYP6F1 gene from Culex pipiens pallens pallens Sequence analysis and differential expression identification were used to clone and express the cytochrome P450 (CYP6F1) gene of Culex pipiens pallens. RT-PCR technique and RACE strategy were used in this study. The cytochrome P450 (CYP6F1) gene was cloned from the (Culex pipiens pallens) of Culex pipiens pallens to the 4th instar larvae of the deltamethrin resistant strain. The bioinformatics analysis was carried out with the corresponding software, and the sensitivity was analyzed by quantitative PCR and semi-quantitative RT-PCR. The expression level of CYP6F1 and the differential expression of CYP6F1 in different stages of mosquito were identified. The results showed that the CYP6F1 gene and its allele CYP6F1v1.CYP6F1 gene were cloned successfully with a total length of 1639 BP.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R346
本文编号：2140758
[Abstract]:Vector insects are a serious hazard to human health, and it is estimated that 2 / 3 of infectious diseases are transmitted by vector insects. In the history of the world, many serious insect-borne infectious diseases such as plague, typhus, yellow fever and malaria have caused widespread epidemic and killed thousands of people. With the increasing frequency of human communication, many new and re-emerging insect-borne infectious diseases have been discovered in the world today. Chemical control has been the main method of comprehensive media management. The extensive and continuous use of insecticides has led to the development and development of vector resistance. At present, at least 504 species of insects have developed resistance to insecticides worldwide, including the major vector insects, 109 of which (subspecies) have developed resistance to one or more insecticides. Insecticide resistance has a direct impact on the prevalence and re-prevalence of insect-borne infectious diseases. Therefore, it is urgent to study the mechanism of vector resistance and to manage it properly. The purpose of this study was to clone and identify the genes related to drug resistance, and then to use RNAi technique to inhibit the resistance genes, and to lay a foundation for the final treatment of drug resistance vector. Therefore, this paper has carried on the following several aspects research. Part I Molecular cloning and functional study of Deltamethrin Resistance related Gene of Culex pipiens pallens; Molecular cloning of cytochrome P450 CYP6F1 gene from Culex pipiens pallens pallens Sequence analysis and differential expression identification were used to clone and express the cytochrome P450 (CYP6F1) gene of Culex pipiens pallens. RT-PCR technique and RACE strategy were used in this study. The cytochrome P450 (CYP6F1) gene was cloned from the (Culex pipiens pallens) of Culex pipiens pallens to the 4th instar larvae of the deltamethrin resistant strain. The bioinformatics analysis was carried out with the corresponding software, and the sensitivity was analyzed by quantitative PCR and semi-quantitative RT-PCR. The expression level of CYP6F1 and the differential expression of CYP6F1 in different stages of mosquito were identified. The results showed that the CYP6F1 gene and its allele CYP6F1v1.CYP6F1 gene were cloned successfully with a total length of 1639 BP.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R346
【引证文献】
相关博士学位论文 前1条
1 贺骥;淡色库蚊核糖体蛋白L22基因(RPL22)克隆及其与溴氰菊酯抗性关系的研究[D];南京医科大学;2009年
相关硕士学位论文 前4条
1 祝全东;桉树CAD、COMT基因的RNAi载体构建及烟草转化研究[D];中南林业科技大学;2011年
2 刘应保;淡色库蚊氯菊酯抗性相关基因XND-P450全长cDNA克隆及序列分析[D];西北农林科技大学;2010年
3 温雪梅;淡色库蚊溴氰菊酯抗性基因XN-P450的克隆、分析及CYP6F1的表达[D];西北农林科技大学;2012年
4 王晓宇;淡色库蚊氯菊酯抗性相关actin基因和P450基因全长cDNA克隆及序列分析[D];西北农林科技大学;2012年
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