蚊核糖体蛋白L39基因克隆及初步功能的研究

发布时间：2018-05-26 04:45

本文选题：淡色库蚊 + 溴氰菊酯　；参考：《南京医科大学》2007年博士论文

【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，继而研究其下游蛋白质组学，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。一、淡色库蚊核糖体蛋白L39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核糖体蛋白L39基因并对其进行表达差异的鉴定，本研究采用RT-PCR技术和cDNA末端快速扩增(RACE)策略，从淡色库蚊(Culex pipiens pallens)对溴氰菊酯抗性品系4龄幼虫中克隆核糖体蛋白L39基因(RPL39)，用相应的软件进行生物信息学分析，并用荧光定量PCR和半定量RT-PCR分析敏感、抗性品系蚊的RPL39表达水平及对蚊各期RPL39进行表达差异鉴定。结果表明，成功克隆出RPL39基因。RPL39基因全长508bp，开放阅读框为156bp，编码51个氨基酸(GenBank／NCBI DQ080075)。序列分析显示，该基因在各物种中保守性很高，与已报告的冈比亚按蚊(Anopheles gambiae)核糖体蛋白L39基因(GenBank／NCBI XP 320713)有99％同源性，并具有一个经典的核糖体蛋白L39结构域，没有跨膜区域，无信号肽序列，属于胞浆蛋白。荧光定量PCR结果表明，RPL39在淡色库蚊对溴氰菊酯抗性品系中表达水平高于敏感品系，提示RPL39基因可能与对杀虫剂的抗药性相关。RPL39mRNA在淡色库蚊抗性／敏感品系各个发育阶段也有不同的表达，以雌性成蚊表达差异最大。二、淡色库蚊核糖体蛋白L39基因的初步功能研究为鉴定核糖体蛋白L39基因与淡色库蚊溴氰菊酯抗性的关系，，本研究扩增RPL39基因编码区，构建蚊细胞表达载体pIB／V5-His／RPL39，转染蚊C6／36细胞。稻瘟菌素稳定筛选和单克隆化后，细胞扩大培养，然后用RT-PCR和Western blot鉴定稳定转基因细胞的转录表达。不同浓度的溴氰菊酯处理稳定转基因细胞及对照未转基因细胞和转无关基因细胞后，用~3H-TdR测定细胞生存率法、细胞生长曲线测定及显微镜下观察细胞存活率、细胞增殖速度及细胞生长情况等。结果显示，成功构建昆虫细胞表达载体并建立稳定转染细胞系，RT-PCR和Western blot检测重组表达载体(pIB／V5-His／RPL39)在稳定转染的蚊细胞内高表达。不同浓度的溴氰菊酯处理转染细胞后，在药物压力下，转染核糖体蛋白L39目的基因细胞组的EC50显著高于转染无关基因CAT细胞组和未转染空细胞组(p＜0.05)。细胞的增值速度也快于对照细胞；转染RPL39基因细胞的形态、密度未见明显变化，而对照细胞颗粒变粗、密度降低。初步研究结果提示，RPL39基因与溴氰菊酯抗性相关，可能为溴氰菊酯抗性相关基因。三、淡色库蚊核糖体蛋白L39下游蛋白质组学的初步研究为探讨核糖体蛋白L39的抗药性作用机制及与其他蛋白质的相互作用，采用对转染核糖体蛋白L39基因细胞进行二维电泳和质谱鉴定的方法，对表达有差异的蛋白质点进行鉴定。实验结果提示，转染核糖体蛋白L39基因的细胞高表达一系列与其他物种的各种酶高度同源的蛋白，例如酚氧化酶前体、丝氨酸／苏氨酸蛋白激酶、丝氨酸蛋白酶、丝氨酸蛋白磷酸酶、精氨酸琥珀酸盐移解酶、酪氨酸磷酯酶、以及具有降解多种药物功能的多药耐药蛋白等；转染细胞还高表达一系列和其他物种转录调控相关的蛋白高度同源的蛋白，例如ATP结合转录亚家族B、跨膜GTP酶、DNA复制准许因子、转录子的稳定期调节蛋白、反转录转座子gag蛋白等，以及高表达和其他物种同源的转铁蛋白和铁反应元件结合蛋白，硒化物磷酸合成酶以及糖原合成酶等，因此推测转染核糖体蛋白L39的细胞可能是通过解毒酶合成增加、保证细胞转录和翻译蛋白的准确性以及增强细胞生存力等方面发挥抗药性作用，从而为阐明核糖体蛋白L39的抗药性作用机理提供理论基础。
[Abstract]:In the present world , there are at least 504 kinds of insects in the world , including plague , typhus , yellow fever , malaria and so on .

Molecular Cloning , Sequence Analysis and Differential Expression of the L39 Gene of the Ribosomal Protein of 1 ,

The RPL39 gene ( RPL39 ) was cloned by RT - PCR and Rapid Amplification of cDNA Ends ( RACE ) . The results showed that the RPL39 gene was successfully cloned . The total length of RPL39 gene was 508bp , the open reading frame was 156 bp , and 51 amino acids were encoded . Sequence analysis showed that the gene has high conservation in each species , and has 99 % homology with the reported L39 gene of the ribosomal protein L39 gene of the Gambia , and has a classical ribosomal protein L39 domain . There is no transmembrane region and no signal peptide sequence . The results indicate that the RPL39 gene may be related to the drug resistance of the insecticide . The RPL39 mRNA has different expression in the development stages of the resistance / sensitive strain of the anti - mosquito resistance / sensitive strain , and the expression of RPL39 mRNA is the largest in the female adult .

Preliminary Study on the L39 Gene of the Ribosomal Protein of the Second , Light - color Cutians

The expression vector pIB / V5 - His / RPL39 was amplified by RT - PCR and Western blot .
The morphology and density of transfected RPL39 gene were not changed obviously , and the density of control cells was decreased . The results suggested that the resistance of RPL39 gene to fenvalerate was related to the resistance - related genes .

A Preliminary Study on the Proteomics of the downstream Proteomics of the Ribosomal Protein L39 in the Light - color Culicidae

In order to investigate the drug resistance mechanism of ribosomal protein L39 and its interaction with other proteins , the protein spots with different expression were identified by two - dimensional electrophoresis and mass spectrometry . The results suggested that the cells transfected with ribosomal protein L39 gene were highly homologous to various enzymes of other species , such as phenol oxidase precursor , serine / threonine protein kinase , serine protease , serine protein phosphatase , arginine succinate migration enzyme , tyrosine phosphoesterase , and multidrug resistance protein with multiple drug functions .
The transfected cells also highly express a series of proteins that are highly homologous to proteins associated with transcriptional regulation of other species , such as ATP - binding transcriptional sub - family B , transmembrane GTP enzyme , DNA replication grant factors , stabilized regulatory proteins of transcripts , anti - transcription transposable gag proteins , and the like , as well as high - expression and other species - homologous transferrin and iron - reactive element binding proteins , selenides phosphate synthase , glycogen synthase , and the like , thus suggesting that the cells transfected with ribosomal protein L39 may play a drug - resistant role through detoxification enzyme synthesis , ensuring cell transcription and translation protein accuracy , and enhancing cell viability , thereby providing a theoretical basis for the mechanism of resistance to ribosomal protein L39 .
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R346

【引证文献】