【作者】 陆芳；

【导师】 蒋明星；

【作者基本信息】 浙江大学 ， 植物保护（专业学位）， 2014， 硕士

【摘要】 共生细菌能在昆虫的生长、生殖、抵抗病原菌和有害物质等生物学过程中起重要的作用。稻水象甲(Lissorhoptrus oryzophilus Kuschel)在我国是一种重要外来入侵害虫,对水稻生产的潜在危害巨大。先期研究表明,Wolbachia是稻水象甲中的一种主要共生细菌,为宿主正常生殖所必需,但该象甲是否还存在其他共生细菌,对宿主生殖的作用如何,尚不清楚。本研究基于细菌16S rRNA序列的扩增和分析,对不同地理种群稻水象甲的肠道和卵巢中的细菌种类进行了鉴定,分析了细菌群落的多样性,并研究了主要细菌与其生殖的关系,探讨了影响宿主生殖的分子机制,具体结果如下：对5个地理种群稻水象甲(美国德克萨斯州Beaumont、阿肯色州Colt、加利福尼亚州Biggs、河北唐海和浙江乐清)体内细菌种类研究发现,其肠道细菌隶属于α-、p-、γ-变形菌纲、芽孢杆菌纲、鞘脂杆菌纲和放线菌纲,主要共生细菌有Pantoea和Wolbachia、除了Beaumont种群,其余4种群的细菌多样性均较低。在卵巢中仅发现3类细菌,即α-、Y-变形菌纲和芽孢杆菌纲,Wolbachia和Nardonella是主要的细菌。定量检测了稻水象甲经抗生素处理后体内主要共生细菌的变化情况,并结合生殖表型的变化,评价了主要细菌在该象甲中的作用。结果表明,成虫经四环素处理后(不能正常生殖)体内Wolbachia含量比水处理的显著减低,而经庆大霉素处理后(仍能正常生殖)该细菌的变化不甚明显,这与预期相符。成虫分别经四环素、庆大霉素处理后,Nardonella均保持在较高水平。成虫经庆大霉素处理后,Rickettsia的含量显著降低。表明稻水象甲生殖与Nardonella和Rickettsia含量的变化无明显关系,两者不是该象甲生殖所必需的。该结果验证了Wolbachia在该象甲生殖过程中所起的重要作用。对成虫分别进行四环素和庆大霉表处理,使其分别处于Wolbachia被去除和感染有这种细菌的状态,然后采用抑制性消减杂交技术(SSH),构建了这两类成虫的消减杂交文库。对文库中的序列进行了克隆和GO分析,对其中部分基因在Wolbachia感染与不感染状态下的表达水平进行了定量检测。发现Wolbachia可影响该象甲多种基因的表达,包括与代谢过程、细胞过程、催化活性、生殖、免疫、死亡等相关的基因,其中3个基因的表达变化显著,即核糖体RNA反义转录蛋白(transcript antisense to ribosomal RNA protein 1,TAR1)类似物、一种假设蛋白和保幼激素环氧化物水解酶类似蛋白5前体(juvenile hormone epoxide hydrolase-like protein 5 precursor)。通过本研究,明确稻水象甲体内共生细菌的主要种类和多样性,以及主要共生细菌在该象甲生殖中的作用及其部分机制。研究结果丰富了我们对象甲类昆虫与共生细菌关系的认识,为今后开展此类昆虫中共生细菌演化、生殖机制、细菌与宿主的互作机制等方面的研究提供了重要信息。更多还原

【Abstract】 Symbiotic bacteria play important roles in a number of biological processes of hosts including growth, reproduction, resistance to pathogens and harmful substances. The rice water weevil, Lissorhoptrus oryzophilus Kuschel (Coleoptera, Curculionidae), is an important invasive pest in China and can cause serious damage to rice plants. Previous studies showed that Wolbachia, one of major symbiotic bacteria in this weevil, is essential for the reproduction of its host. However, it is not clear whether there exists other symbiotic bacteria in this weevil, and whether they also have any importance for the host reproduction. In this study, based on the amplification and analysis of bacterial 16S rRNA sequences, bacterial species in guts and ovaries of L. oiyzophilus adults collected from different locations was identified and the diversity of bacterial community analyzed. Relationships between major bacteria and their reproduction as well as the underlying molecular mechanism were also explored. The results were summarized as follows.From five geographic populations of rice water weevil (Beaumont of Texas, Colt of Arkansas and Biggs of California in the USA; Tanghai of Hebei and Yueqing of Zhejiang in China), a total of 19 bacteria were identified from guts, which belong to Alpha-, Beta-, Gamma-proteobacteria, Bacilli, Sphingobacteria and Actinobacteria. The dominant symbiotic bacteria are Pantoea and Wolbachia. Except the Beaumont population, bacteria diversity in each of the investigated weevil populations is relatively low. In the ovary, only five bacteria were identified which belong to Alpha-, Gamma-proteobacteria and Bacilli, with Wolbachia and Nardonella being the dominant species.The quantitative changes of major symbiotic bacteria were detected in weevil adults treated with antibiotics, and, by combining results regarding change of reproductive phenotype in treated weevils, the relative importance of various bacteria for the reproduction of this weevil was evaluated. The results showed that, as expected, Wolbachia in the adults treated with tetracycline (leading to abnormal reproduction) was significantly lower than in the control adults (treated with water), however, the treatment using gentamicin (incapable of killing Wolbachia and not negatively impacting weevil reproduction) did not reduce the Wolbachia density significantly. In adults separately treated with tetracycline and gentamycin, Nardonella were maintained at a high level, irrespective of the status of weevil reproduction. In the adults treated with gentamicin, which can reproduce healthily, Rickettsia significantly reduced. These results indicated that changes in Nardonella and Rickettsia density has no evident relations with the reproduction status of rice water weevil, suggesting both of these two bacteria are not essential for the reproduction of this weevil. These findings strengthen our previous conclusion that Wolbachia is the essential symbiotic bacterium for the reproduction of rice water weevil.By using the adults which were separately treated with tetracycline and gentamicin and thus in two substantially different Wolbachia infection status, i.e., Wolbachia-removed and Wolbachia-infected, two cDNA libraries were constructed using the suppressive subtraction hybridization (SSH). Sequences from each library were cloned, and analyzed using by GO (Gene Ontology) annotation. Moreover,35 genes were quantitatively detected for their expression in the adults treated separately with tetracycline and gentamicin, i.e., in the adults with Wolbachia removal and those harboring Wolbachia. It was found that Wolbachia can affect the expression of multiple genes of this weevil, including the genes related to metabolic process, cellular process, catalytic activity, reproduction, immunity and death. Three genes changed their expression significantly due to the deletion of Wolbachia, namely transcript antisense to ribosomal RNA protein 1 (TAR1), a hypothetical protein, and juvenile hormone epoxide hydrolase-like protein 5 precursor.In sum, in this study the composition and diversity of bacterial community in L. oryzophilus were determined, and importance of main symbiotic bacteria for the reproduction of this weevil and some underlying molecular mechanism were investigated. The results would enrich our knowledge on the relationship between weevils and symbiotic bacteria, also provide important information as to the evolution of symbiotic bacteria, reproductive mechanism in weevils and the interaction mechanism between symbiotic bacteria and their host.更多还原