【作者】 贺骥；

【导师】 朱昌亮；

【作者基本信息】 南京医科大学 ， 病原生物学， 2009， 博士

【摘要】 蚊媒病是当今严重危害人类健康的一类常见病,蚊媒的化学防制是控制蚊媒病的一个重要手段。然而随着杀虫剂的大量使用,蚊媒的杀虫剂抗性也随之产生和发展。杀虫剂抗性已成为蚊媒病防治工作中的一大障碍。发现和研究新的抗药性相关基因将有助于阐明抗药性的分子基础和寻找新的治理抗药性的靶标。本实验室前期采用抑制性差减杂交(suppression subtractive hybridization, SSH)结合cDNA芯片及Northern blotting发现,核糖体蛋白L22基因(ribosomal protein L22, RPL22)在淡色库蚊(Culex pipiens pallens)溴氰菊酯抗性品系中高表达。在此基础上,本研究采用5’-RACE和3’-RACE法扩增淡色库蚊RPL22的全长序列,并通过实时定量PCR法研究RPL22在淡色库蚊的溴氰菊酯敏感和抗性品系及蚊敏感和抗性细胞中的差异表达;随后在细胞水平,先后通过转染后高表达、干涉后低表达和诱导后适度高表达等方法,研究RPL22对蚊细胞溴氰菊酯敏感性的影响;同时,通过实时定量PCR法检测了相应细胞色素P450 6A1基因(cytochrome P450 6A1, CYP6A1)的表达水平;此外,采用实时定量PCR法检测了淡色库蚊不同发育阶段RPL22的发育表达谱。结果显示,本研究获得的淡色库蚊RPL22全长序列为673 bp,开放阅读框为447 bp,编码148个氨基酸(GenBank登录号: EF990190)。用BlastX软件将该基因推导编码的氨基酸序列与蛋白质公共数据库Swissprot进行分析,用ClustalW软件作序列比对,以MEGA 3.1软件进行聚类分析。推导的氨基酸序列与致倦库蚊(Cx. quinquefasciatus),埃及伊蚊(Aedes aegypti)和冈比亚按蚊(Anopheles gambiae)的RPL22分别具有100%,90%和80%同源性。通过MEGA 3.1软件进行聚类分析构建进化树及序列归一化距离分析后发现,淡色库蚊的RPL22同致倦库蚊的距离最接近(0.003),其次为埃及伊蚊(0.273)和冈比亚按蚊(0.472)。实时定量PCR结果显示,RPL22在淡色库蚊抗性品系的4龄幼虫和抗药性蚊细胞中的表达水平,分别是淡色库蚊敏感品系4龄幼虫和敏感蚊细胞的2.57倍和1.71倍(p均<0.001)。将RPL22转染蚊C6/36细胞,使其在蚊细胞中高表达(23.39倍),结果发现,在溴氰菊酯浓度为100.5μg/mL,101.0μg/mL,101.5μg/mL,102.0μg/mL和102.5μg/mL时,RPL22转染组细胞的存活率分别比对照组下降8.2%,17.1%,12.1%,11.4%和6.4%(p<0.05)。实时定量PCR结果显示在高表达RPL22时,CYP6A1的表达水平降低为对照组的41.8%(p<0.001)。采用siRNA技术干涉蚊抗药性细胞的RPL22,通过实时定量PCR实验验证,干涉RPL22的效率为61.9%。结果发现,在溴氰菊酯浓度为400μg/mL和600μg/mL时,干涉组细胞的存活率分别比对照组下降7.6%和9.5%(p均<0.05)。实时定量PCR结果显示,在低表达RPL22时, CYP6A1的表达水平降低为对照组的82.5%(p<0.05)。构建RPL22诱导型表达载体,以终浓度为0.5nM CuSO4作为诱导剂,诱导48小时后RPL22的表达量为对照组的7.9倍,为接近生理水平的高表达。结果发现,在溴氰菊酯浓度为100.5μg/mL,101.0μg/mL,101.5μg/mL和102.0μg/mL时,RPL22转染组细胞的存活率分别比对照组提高8.6%,12.7%,9.6%和7.9%(p<均0.05)。实时定量PCR结果显示,淡色库蚊RPL22在不同发育阶段中,蛹期表达水平最低,比4龄第2天幼虫降低34.9%(p<0.05),而羽化后第1天的表达水平最高,比蛹期升高95.2%(p<0.001),且在羽化后的第2天RPL22的表达水平降低,接近其他各期表达水平(p>0.05)。以上结果表明,RPL22与淡色库蚊溴氰菊酯抗性相关;RPL22在适度高表达的情况下对细胞产生抗溴氰菊酯的保护作用,而过表达RPL22可能引起转录抑制或对细胞产生毒副作用,导致细胞存活率降低;RPL22在淡色库蚊的不同发育阶段存在表达差异。本研究首次报告了淡色库蚊RPL22全长序列,并发现RPL22为1个新的昆虫抗药性相关基因,为进一步阐明杀虫剂抗性的分子机制和建立抗药性检测方法提供了新的科学依据,具有重要的理论意义和潜在的实际应用价值。更多还原

【Abstract】 Many new and reemerging mosquito-borne diseases threaten the public health. Chemical control is a major method to manage mosquito-borne diseases. But under the natural selection, excessive and continuous application of insecticides has caused the development of insecticide resistance, which has become the major obstacle to controlling the mosquito-borne diseases. Identification and characterization of insecticide resistance genes will contribute to clarify the mechanism of insecticide resistance and find the target of insecticide resistance management.To investigate the insecticide resistance in mosquitoes, we had employed suppression subtractive hybridization (SSH) and cDNA microarray to identify differentially expressed genes between deltamethrin-susceptible and -resistant strains of Cx. pipiens pallens in our previous experiments, and some deltamethrin resistance related genes were isolated. One of the highly expressed genes in deltamethrin-resistant strain was a RPL22-homolog. In this study, we acquired the full length sequence of RPL22 by 5’-RACE and 3’-RACE methods, detected the differential expression by realtime PCR between deltamethrin-susceptible and -resistant strains of Cx. pipiens pallens and mosquito cells, and explored the relationship between RPL22 and deltamethrin resistance in mosquito cell line by overexpression, RNA interference and inducible higher-expression methods. At the same time, we also detected the mRNA level of CYP6A1 which is an important insecticide resistance gene, and measured the mRNA level of RPL22 in different development stages of the Culex mosquito.RPL22 (GenBank accession no. EF990190) was cloned from Cx. pipiens pallens. An open reading frame (ORF) of 447 bps was found to encode a putative 148 amino acids protein which shares 90% and 80% identity with RPL22 proteins from Aedes aegypti and Anopheles gambiae respectively.Real-time quantitative PCR analysis demonstrated that the transcription level of RPL22 in deltamethrin-resistant strain was 2.57 folds as high as that in deltamethrin-susceptible strain of Cx. pipiens pallens (p<0.001). And the transcription level of RPL22 in deltamethrin-resistant mosquito cells was 1.71 folds as high as that in deltamethrin-susceptible mosquito cells (p<0.001).The expression plasmid of RPL22 was constructed and RPL22 was overexpressed in C6/36 cells. Viability of C6/36-RPL22 cells in the presence of deltamethrin was compared with control cells to observe variance of deltamethrin resistance of these cells. The results showed that C6/36-RPL22 cells are relatively more susceptible to deltamethrin compared with the control. And the mRNA level of CYP6A1was decreased to 41.8% when RPL22 was overexpressed (p<0.001).RPL22 expression in resistant mosquito cells was knocked down by RNA interference, and the results showed that the resistant cell became more susceptible to deltamethrin compared with the control. At the same time, the mRNA level of CYP6A1 was also decreased to 82.5% when RPL22 was down-regulated.To exclude the side-effect of RPL22 overpression, we constructed the inducible expression vector of RPL22 and empty vector. Inducible expression experiment showed that transcription level of RPL22 was 7.9 folds as high as that in the control. And deltamethrin cytotoxicity assay demonstrated that RPL22 could promote the deltamethrin resistance, when the expression level of RPL22 was close to its natural higher expression levels.In this study, we cloned the full length of RPL22, and presented the first evidence that RPL22 confer the deltamethrin resistance in mosquito. And our results provided new evidence for clarifying the molecular mechanisms of insecticide resistance and establishing the new resistance testing methods. It has important theoretical value and potentially practical application .更多还原