摘要
【目的】Wolbachia是广泛存在于昆虫体内的一类通过母系传递的共生菌,能够通过多种方式影响宿主的生殖。细胞质不亲和(CI)是Wolbachia引起的最普遍的一种表型,即感染Wolbachia的雄性和未感染的雌性宿主交配后,胚胎发育停滞于早期阶段。但目前有关CI的分子机理还不清楚。本研究组前期实验表明,Wolbachia感染引起黑腹果蝇Drosophila melanogaster 3龄幼虫精巢中Mst84Db基因的表达显著下调。本研究的目的是进一步研究Mst84Db与CI的关系。【方法】我们体外合成了Mst84Db的双链RNA(dsRNA),注射雄性果蝇,将注射过的雄果蝇与野生雌果蝇交配,检测其繁殖力。基因表达采用定量RT-PCR方法进行检测。胚胎表型采用DAPI染色进行分析。【结果】注射dsRNA 24 h后,Mst84Db基因的表达水平发生显著下调。注射后72 h,基因表达下调幅度最大。与对照组相比,基因敲降后的雄蝇繁殖能力显著下降,与雌果蝇交配后胚胎孵化率显著低于对照组,这与Wolbachia诱导的CI现象类似。未孵化胚胎的表皮上没有体节出现,说明胚胎停滞于发育的早期。部分胚胎细胞核分裂不同步,且有染色质间桥出现,这也与CI胚胎中的细胞学表型一致。【结论】Wolbachia感染可能抑制果蝇精子发生过程中Mst84Db基因的表达,从而使精子失去正常功能,最终导致与雌性果蝇交配后,胚胎发育停滞,并最终死亡。Mst84Db基因在雄性果蝇中表达下调可能是产生CI的重要原因之一。
【Aim】 Wolbachia bacteria are maternallyinherited endosymbionts frequently found in insects. They can alter their hosts’ reproduction via several forms. Cytoplasmic incompatibility （CI） is the most commonly reported phenotype caused by Wolbachia infection and induces developmental arrest of early embryos resulting from mating between Wolbachia-infected males and uninfected females. The molecular mechanisms of CI, however, are not clear currently. Our previous studies showed that the Mst84Db gene exhibited significant down-regulation in the 3rd instar larval testes of Drosophila melanogaster when infected by Wolbachia. The objective of this research is to further assay whether Mst84Dbgene is associated with CI. 【Methods】 In this study, we first synthesized theMst84Db dsRNA in vitro and microinjected into male flies. Then we tested the fertility of these injected males by crossing with wild type females. Gene expression levels were examined by quantitative RT-PCR （qRT-PCR）. The embryonic phenotypes were analyzed by DAPI staining. 【Results】 The expression level of Mst84Db was significantly down-regulated since 24 h after injecting Mst84Db dsRNA. At 72 h after injection, the expression level of Mst84Db exhibited the maximum down-regulation relative to the control. The knockdown of Mst84Db in males significantly decreased the hatch rate of embryos when crossed with wild type females. This is similar to the phenotype in CI crosses. In addition, the embryonic cuticles showed almost no segmental development, indicating that some embryos stop development at early stages. Obvious CI phenotypes with asynchronized nuclear division and chromatin bridges were also observed in part of early embryos derived from Mst84Db down-regulated males. 【Conclusion】 Wolbachia infection may inhibit the expression of Mst84Db during spermatogenesis, thus reduces sperm fertility and leads to embryonic lethality. Down-regulation of Mst84Db in male Drosophila might be causally linked to CI.
出处
《昆虫学报》
CAS
CSCD
北大核心
2014年第3期293-299,共7页
Acta Entomologica Sinica
基金
国家自然科学基金项目(31172155)
