【作者】 陈晨；

【导师】 朱昌亮；

【作者基本信息】 南京医科大学 ， 病原生物学， 2012， 硕士

【摘要】 蚊媒病是全世界最重要的人类传染病之一，严重危害人类身心健康。化学防制是控制蚊媒病的一个重要手段。实践证明杀虫剂对于控制蚊媒病的传播非常有效。然而随着杀虫剂的长期、大量使用，蚊媒的抗药性也随之发生和发展。抗药性已成为当前控制蚊媒病的最大障碍。发现并研究新的抗药性靶标将有助于阐明抗药性的分子基础和寻找抗药性的治理方法。大量研究表明，蚊杀虫剂抗性性状是一种数量性状。因此，建立蚊抗性-敏感杂交系，筛选遗传标记，是研究蚊的抗性遗传机理和防制的重要基础。本研究首先采用淡色库蚊溴氰菊酯抗性（R）、敏感（S）品系进行正、反交，得到两个杂交F2代群体，并对比观察其生物学特性；以WHO成蚊接触筒生物测定法将F2个体的表型区分为10-60分钟击倒时间点和24小时恢复与未恢复8种表型，采用生存分析法分析蚊在溴氰菊酯选择压力下的生存情况；接着，采用分群分析法（BSA法)对杂交F2代溴氰菊酯抗性相关的分子标记进行筛选分析。用AFLP引物筛选所建立的抗药性2个极端水平基因池，得到与溴氰菊酯抗性性状紧密连锁的特异性DNA片段；并分别在F2抗性、敏感群体中进行验证。本研究通过淡色库蚊溴氰菊酯抗性、敏感品系进行正、反交，获得杂交F2代群体，观察到其溴氰菊酯抗性水平位于两亲本之间，符合孟德尔的遗传规律。此外，以抗性品系为母本的杂交群体的抗性水平高于以敏感品系为母本的杂交群体。以WHO成蚊接触筒生物测定法，将487只F2个体的表型区分为10-60分钟击倒时间点和24小时恢复与未恢复8种表型。并以此，编制了淡色库蚊杂交品系在杀虫剂选择压力下的生命表和生存曲线，证明R-S组生存率显著高于S-R组，而且分组因素，即亲本，为影响生存率的独立因素。以上结果均表明，抗药性相关的基因可能与性染色体连锁，母本可能提供更多的抗药性相关基因。利用BSA方法，对淡色库蚊抗性、敏感杂交F2代溴氰菊酯抗性相关的AFLP分子标记进行筛选分析。用136对AFLP引物反复筛选所建立的2个抗药性水平基因池，其中29对引物组合所扩增出的稳定可重复的108条多态性显性条带，这些条带可能与抗药性基因连锁，提示其周围可能存在抗药性基因。挑选在亲本之间扩增出稳定可重复的多态性条带的29对引物，分别在F2代溴氰菊酯抗性、敏感群体中进行验证。AFLP标记R中，L1A29.153、L1A31.135、L1A31.143标记多态率较高，在两个杂交群体中的多态率都在60%以上；AFLP标记S中，L1A25.70、L1A40.80标记多态率较高，在两个杂交群体中的多态率都在60%以上。所有标记在两群体之间的多态率均有统计学差异，p<0.05。综上，本研究首次建立了淡色库蚊溴氰菊酯抗性、敏感品系杂交F2代群体，采用BSA法，筛选到溴氰菊酯抗性性状紧密连锁的AFLP分子标记108个，并分别在F2代溴氰菊酯抗性、敏感群体中进行验证，为建立淡色库蚊溴氰菊酯抗性遗传图谱和数量性状分析（QTL）提供了作图群体和分子标记。更多还原

【Abstract】 Mosquito is an important vector which transmits life-threatening vector-bornediseases. For a long period, chemical control works as a primary strategy to managethe mosquito-borne diseases. Excessive and continuous application of insecticideshas induced rapid development of insecticide-resistance in mosquitoes, which hasbecome the major obstacle for controlling the mosquito-borne diseases now.Identification and characterization of new insecticide resistance associated genes willcontribute to clarify the mechanism of insecticide resistance and find the strategy ofinsecticide resistance management.We developed two reciprocal F2intercross segregating populations from thecrosses of the insecticide-susceptible strain（S）and deltamethrin-resistant strain（R）in Cx. pipiens pallens. We compared the biological characteristics of F2intercrossprogeny among these4groups, like spawning rate, hatching rate, growth survival rateand resistant level. We qualified the487F2hybrids into8kinds of phenotype as10min,20min,30min,40min,50min,60min and after24h recovery dead or aliveaccording to WHO adult bioassay. Their survival was analyzed in the two groups.Based on the strategy of Bulk Segregate analysis (BSA), two gene pools wasrespectively constructed according to the extreme highest and lowest resistance todeltamethrine. A total of136primer combinations was tested on the gene pools toobtain the special AFLP markers tightly linked to high or low resistance todeltamethrine. Then we identified the frequency of these AFLP markers in the highand low resistance groups of F2populations.We developed two reciprocal F2intercross segregating populations from the crosses of S-strain and R-strain. It was observed that the resistant level of the hybridswas between their parents’, and the resistant level of the hybrids fromdeltamethrin-resistant female parent is higher than that of the another.We qualified the487F2hybrids into8kinds of phenotype as10min,20min,30min,40min,50min,60min and after24h recovery dead or alive according to WHOadult bioassay. Their survivals analysis and survival curve demonstrate that thesurvival rate of the hybrids from deltamethrin-resistant female parent is higher thanthat of the another, and grouping is the only factor.These results indicate that the deltamethrine resistant genes may link to sexchromosomes, and female parents may provide more resistance related genes.Based on the strategy of Bulk Segregate analysis (BSA), two gene pools wasrespectively constructed according to the extreme highest and lowest resistance todeltamethrine. A total of136primer combinations was tested on the gene pools toobtain the special AFLP markers tightly linked to high or low resistant todeltamethrine. Among the136AFLP primer combinations,29primer combinationsproduced polymorphic fragments with clear dominance inheritance patterns andreproducibility. These AFLP markers are linked to resistance related genes, and mayexist those genes around them.We identified the frequency of these AFLP markers in the high and lowresistance groups of F2populations. Among the AFLP R markers, the highestfrequencies markers are L1A29.153、L1A31.135、L1A31.143, and among the AFLPS markers, L1A25.70、L1A40.80have the highest frequencies. The frequencies ofthese markers are above60%in both of the F2populations.To sum up, we developed two reciprocal F2intercross segregating populationsfrom the crosses of S-strain and R-strain and obtain the special AFLP markers tightlylinked to high or low resistance to deltamethrine. Our study laid the groundwork for the identification of genetic polymorphism and genetic mapping of resistance genes.更多还原