【作者】 郑雅；

【导师】 王玉凤；

【作者基本信息】 华中师范大学 ， 动物学， 2012， 博士

【摘要】 沃尔巴克氏体(Wolbachia)是一类革兰氏阴性细菌,广泛存在于多种节肢动物的生殖细胞或体细胞中,能够通过宿主卵细胞质传递给子代。最新的研究表明,除了在丝状线虫、陆生等足动物、蜘蛛目和蜱螨目中存在外,其在昆虫中的分布更为广泛,超过65%以上的昆虫种类中都发现有Wolbachia感染,包括直翅目、膜翅目、半翅目、鞘翅目、双翅目和同翅目等。目前,Wolbachia的研究已成为国内外热点领域之一,主要原因是它能对宿主的生殖产生多种调节作用,包括雌性化、孤雌生殖、杀雄、精卵细胞质不亲和(CI)等,正是由于这些调节作用,使Wolbachia得以在宿主种群内成功传播。其中,CI是最为普遍的表型,即当Wolbachia感染的雄性宿主受精于未感染或感染了不同Wolbachia菌株的雌性宿主时,产生的后代由于发育缺陷在胚胎时期死亡。而感染相同Wolbachia菌株的雌性和雄性宿主在交配后则能产生正常发育并携带Wolbachia的后代。有关cI的分子机制直到现在仍未得到明确的解释。研究发现,在cI胚胎进行核分裂时,雄性原核出现染色质间桥,因此无法正常存活。本研究首次对我国六个不同地区黑腹果蝇Drosophila melanogaster)体内Wolbachia的感染状况进行检测,发现湖北武汉、天津、云南六库地区的黑腹果蝇均被Wolbachia感染,而其它三个地区(海南尖峰岭、河南鸡公山、西藏下察隅)无感染现象。通过克隆得到它们的wsp基因序列,提交GenBank,注册号分别为：FJ403330,FJ403331,FJ403332。并对所得序列进行系统进化分析表明,感染这三个地区果蝇的Wolbachia品系之间亲缘关系十分相近,均属于A大组的Mel亚群。Hira是一种组蛋白调节基因,其突变也能引起果蝇的雄性原核发育障碍,为了研究cI与Hira基因的关系,我们首先通过qRT-PCR的方法检测了不同感染状态下,宿主果蝇Hira基因的表达水平。结果发现,在Wolbachia感染能引起强CI表型(后代胚胎孵化率很低)的雄性果蝇中,Hira表达水平显著低于弱CI表型或未感染的雄果蝇。由于cI的强度随雄性宿主日龄的增加逐渐下降,通过分析1d和5d雄蝇中Hira的表达水平发现,5d雄蝇中Hira的表达水平显著高于1d的雄果蝇。此外,考虑到宿主果蝇幼虫发育时间与cI强度密切相关,幼虫发育期长的雄性果蝇引起的cI强度越低(后代胚胎孵化率升高),通过比较感染wMelPop但幼虫发育时间不同的雄性果蝇中Hira基因的表达,发现随着幼虫发育期的延长,其体内Hira基因表达水平逐渐升高。幼虫发育速度最快的果蝇“兄长”引起的cI强度最高,但其体内Hira基因表达水平却最低。这些结果表明,Wolbachia感染引起雄性果蝇体内Hira基因表达下调可能是导致CI的原因之一。为了探究这种现象是否只发生在果蝇这种单一宿主上,通过检测埃及伊蚊(传播登革热病毒的主要媒介昆虫)中Hira的表达,发现了与果蝇中类似的结果,即CI强度越高,引起CI的雄蚊体内Hira的表达量越低。为了进一步证明Wolbachia感染引起雄性宿主体内Hira基因表达下调是导致CI的原因,利用RNA干扰(RNAi)技术,将未感染雄蚊中的Hira基因敲降,模拟感染Wolbachia雄蚊的状况,然后将之与未感染雌蚊进行交配,结果产生了类似CI的表型,即导致子代胚胎孵化率下降,并且感染Wolbachia的雌蚊能够部分挽救这种由于雄蚊Hira基因敲降引起的缺陷。体外细胞学研究发现,一种与Hira基因具有明显互补序列的微小RNA (aae-miR-12)在感染和未感染Wolbachia的埃及伊蚊细胞中表达水平存在差异。与未感染的Aag2细胞相比,aae-miR-12在感染wMelPop-CLA的蚊子细胞中的表达水平上调,这种结果是与Hira基因表达情况截然相反,表明aae-miR-12可能与Wolbachia感染有关,并可能对Hira基因的表达起调控作用。为了进一步验证这一假说,我们将人工合成的aae-miR-12类似物和抑制物分别转染细胞,并检测细胞中Hira基因的表达情况,发现aae-miR-12对Hira基因表达具有明显抑制作用。当把aae-miR-12类似物转染到未感染Wolbachia的蚊子细胞后,细胞中Hira基因表达明显下调；然而,当把aae-miR-12抑制物转染入感染wMelPop-CLA的细胞时,Hira基因表达水平与对照相比显著上升。而利用体外构建pIZ/GFP-Hira载体,以GFP为报告基因的方法,发现aae-miR-12可以通过与Hira基因序列的互补配对来抑制其表达。因此,从以上结果推测,在埃及伊蚊中,Hira基因的表达可能受到一种微小RNA—aae-miR-12的负调控作用。Wolbachia感染可能通过上调aae-miR-12表达,抑制Hira基因表达,最终导致宿主CI的表型,这个结果将为研究Wolbachia与宿主相互关系提供一条新的思路。为了全面探究Wolbachia感染引起CI的分子机制,通过microarray技术比较了感染和未感染Wolbachia的雄性果蝇三龄幼虫的精巢中差异表达的基因,结果发现,有296个基因表达量发生至少1.5倍以上的改变(q<0.05),这些基因的功能涉及到新陈代谢、免疫、生殖等。其中,167个基因发生上调,129个基因发生下调。采用定量RT-PCR的方法进一步验证了部分基因,证明其表达改变模式与microarray的结果一致。有趣的是,Wolbachia感染导致与免疫相关的基因大部分出现上调,而与生殖相关的基因则多数下调。根据这些结果我们推测,Wolbachia可能一方面激活了宿主的免疫途径,另一方面却对其精子发生产生不利影响。具体基因功能正在进一步研究中。更多还原

【Abstract】 Wolbachia are a widespread negative bacterium of maternally inherited of arthropod, cytoplasmic transferring by host egg. Recent study have proved that Wolbachia are distributed in about 65% insect including Orthoptera, Hymenoptera, Heteroptera, Coleoptera, Diptera and Homoptera, except for ubiquitous in filar nematode, isopod, Araneae and Ticks. Though detecting Wolbachia infection of six distrincts of Drosophila melanogaster line in China, we found that populations from Wuhan in Hubei Province, Tianjin and Liuku in Yunnan Province are infected by Wolbachia, while there was no infected phenomenon in other three areas (Jianfeng mountain in Hainan Province, Jigong hill in Henan Province, Xiachayu in Tibet). The wsp gene sequences were obtained through cloning and submitted to GenBank, the accession numbers assigned are FJ403330, FJ4O3331, FJ403332. Though phylogenetic analyzing these sequences, the genetic relationship of Wolbachia strain infecting Drosophila in these three areas was very similar to each other, all belonging to Mel group in A supergroup of Wolbachia.The reason why Wolbachia has become one of investigative hotspot in domestic and abroad is that it can regulate host reproductive mode through multiple mechanism, such as feminization, parthenogenesis, male-killing and Cytoplasmic Incompatibility (CI) etc, by which way Wolbachia can be extensive disseminated. Among them, CI is the most frequent phenotype, occurs when Wolbachia infected males mate with uninfected females resulting in no or very low numbers of viable offspring. The molecular mechanisms underlying CI are currently unknown. Some study on Cytology showed that male pronucleus can not be formed normally in CI embryo due to constructing chromatin bridge in the karyokinesis process. One point mutation of Histone regulation (Hira) gene could also induce male pronucleus developmental disabilities in Drosophila. In order to study the relationship between CI and Hira gene expression level, firstly we use qRT-PCR to detect Hira gene expression level in Drosophila melanogaster infected Wolbachia with different CI strength. The results showed that in Drosophila males with strong CI strength (progeny had low egg hatch rate), the Hira expression level was significantly lower than that in males with low CI or the uninfected. Because CI strength fall-off by the increasing male age, we compared the Hira expression level between 1 day and 5 day old Drosophila males. The results demonstrated that as the increasing male age, the Hira expression level step-up gradually. In addition, it was proved that there was antagonism relationship between patrogenesis speed and CI strength, through detecting Hira expression level of infected males with different larval developmental time. The offspring embryonal hatch rate increase as the elongation of developmental time. That is, the elder brother with the most development speed had the strongest CI strength, but their Hira expression level was the lowest. All these results indicated that the down-regulation of Hira in Drosophila males caused by Wolbachia infection may be one important reason of inducing CI.In purpose of proving this phenomenon is not just happened in single species-Drosophila melanogaster, we also inspected the Hira gene expression in another host Aedes aegypti (major intermediary of Dengue Virus transmission), the analogical results were obtained. To further verify the conclusion, RNA interference technology was adopt to knock down the Hira gene in uninfected mosquito males, which mimic the infected males, then the mating between Hira-silenced males and uninfected females is performed to turn out a mimic of CI Phenotype. Besides, Wolbachia in females can’rescue’some of the damage resulting from silencing of the Hira gene in males. In vitro cytology study suggest that the expression of a micro RNA (aae-miR-12) which has obviously complementary sequences were difference between Wolbachia infected and uninfected Aedes aegypti cells. The aae-miR-12 signal was significantly stronger in Aag2.wMelPop-CLA cells compared to uninfected cells, which was opposite to the Hira expression. The result suggested that aae-miR-12 may be associated with Wolbachia infection and regulate the expression of the Hira gene. To further test this hypothesis, we adopt synthetic mimics and inhibitors of aae-miR-12 transfection experiment to validate that aae-miR-12 can repress Hira expression. Hira gene expression in Aag2 cells transfected with the aae-miR-12 mimic was significantly lower compared to mock-transfected cells or control-mimic transfected cells. Significantly higher Hira expression was found in Aag2.wMelPop-CLA cells when transfected with the aae-miR-12 inhibitor as compared to mock-transfected cells or control-inhibitor transfected cells. Additional experiments were performed in Aedes cell line using GFP as a reporter gene and constructing PIZ/GFP-Hira vector, proving that aae-miR-12 may suppress Hira expression through complementary pairing. Taken together these results suggest that Hira gene may be negatively regulated by one micro RNA aae-miR-12 in Aedes aegypti, in addition, Wolbachia infection is likely to inhibit Hira gene expression, through up-regulating aae-miR-12, finally induce CI Phenotype of host. These results will supply a firenew insight for the study on the relationship between Wolbachia and their host.Furthermore, in order to fully explore the molecular mechanism of CI caused by Wolbachia infection, we adopt Genechip to identify that a total of 296 genes expression had at least a 1.5 fold change in Wolbachia infected D. melanogaster larval testes, when comparing to the uninfected. Differential expression of genes related to metabolism, immunity, reproduction and other functions were observed. Among them,167 genes were up-regulated and 129 genes were down-regulated. Interestingly, most of the genes putatively involved in immunity were up-regulated in the presence of Wolbachia. In contrast, most of the genes putatively associated with reproduction (especially spermatogenesis) were down-regulated in the presence of Wolbachia. According to these results we infer that, Wolbachia infection may activate host immune path, however, take some adverse effect on spermatogenesis. Specific gene function is being studied.更多还原