【作者】 肖达；

【导师】 高希武； 朱坤炎；

【作者基本信息】 中国农业大学 ， 农业昆虫与害虫防治， 2014， 博士

【摘要】 基因沉默是真核生物中相对保守的机制之一。赤拟谷盗具有非常敏感的基因沉默效应。但是对于dsRNA的吸收机制尚不清楚。本研究利用内吞作用抑制剂并结合基因沉默技术(RANi)来研究赤拟谷盗dsRNA的吸收机制。结果表明网格蛋白介导的内吞作用抑制剂氯丙嗪和巴佛洛霉素A可以有效阻止赤拟谷盗细胞吸收dsRNA进而影响其靶标基因沉默效率,同时赤拟谷盗中肠组织切片也显示应用以上两种内吞作用抑制剂后,中肠细胞中没有释放Cy3荧光信号,该结果更为直观的证明了氯丙嗪和巴佛洛霉素A可以阻止细胞吸收dsRNA。上述结果初步证实了网格蛋白介导的内吞作用参与dsRNA的吸收过程,因此我们从该途径中选取了4个相对重要的蛋白,即衔接蛋白2中的μ链(TcAP50)、网格蛋白重链(TcChc)、V型ATP酶H亚基(TcVhaSFD)和Rab7(TcRab7)。利用基因沉默技术将以上参与蛋白分别沉默后发现,标靶基因的沉默效率显著降低。以上研究结果表明网格蛋白介导的内吞作用参与赤拟谷盗dsRNA的吸收过程。幼虫巨大致死基因(lethal giant larvae)作为肿瘤抑制基因在上皮细胞极化过程中起着非常重要的作用。本研究中以赤拟谷盗幼虫巨大致死基因(TcLgl)为靶标基因研究dsRNA的吸收机制。我们从赤拟谷盗的基因组中鉴定并克隆了rcLgl基因。利用荧光定量PCR (RT-qPCR)检测该基因在不同发育时期及不同组织中的表达量,结果显示该基因在赤拟谷盗的不同发育时期均有表达,同时在肠组织中表达量相对较高。利用RNAi技术验证TcLgl的功能,结果显示赤拟谷盗幼虫巨大致死基因的表达量在注射其相应的dsRNA (dsTcLgl)后会显著降低。早期幼虫(第8天)在注射dsTcLgl后无法完成化蛹,到注射后第20天所有试虫全部死亡。末龄幼虫(第20天)在缺失幼虫巨大致死基因后化蛹率显著降低,化蛹率仅为50.3,36.0和18.2%。早期的蛹注射100,200和400ng的dsTcLgl后,羽化率分别为22.46,18.03和11.20%。以上结果表明幼虫巨大致死基因在昆虫生长发育过程中起着至关重要的作用。内吞作用是细胞吸收营养和大分子物质的主要途径之一。目前关于昆虫内吞作用的报道相对较少。我们从赤拟谷盗的基因组中鉴定并克隆了参与网格蛋白介导内吞作用过程中的重要蛋白,分别是衔接蛋白2中μ链(TcAP50)、网格蛋白重链(TcChc)、V型ATP酶H亚基(TcVhaSFD)和TcRab7。结果显示这4个基因位于不同的染色体上,且TcVhaSFD存在两种选择性剪切。RT-qPCR检测显示这4个基因在不同发育阶段均有表达,且蛹期表达量相对较高。组织特异性表达结果显示TcChc, TcVhaSFD和TcRab7主要在肠和脂肪体组织中表达,TcAP50主要在表皮组织中表达。利用RNAi技术研究以上基因的功能,结果表明末龄幼虫在缺失TcChc, TcAP50, TcVhaSFD和TcRab7基因后会引起赤拟谷盗的死亡。赤拟谷盗早期蛹注射dsTcChc, dsTcVhaSFD和ds TcRab7后会产生羽化畸形的成虫,其表型特征为成虫头部和胸部完全羽化而腹部还保留着蛹的形态特征。这些非正常羽化的成虫活动能力明显减弱,在羽化后的3天内全部死亡。早期蛹缺失TcAP50基因后可以正常羽化,但成虫在羽化后第6天开始出现死亡。除此之外,我们还发现注射dsTcChc和dsTcVhaSFD的雌虫卵巢发育较小且没有成熟卵的形成。这说明缺失TcChc和TcVhaSFD基因不仅影响雌虫卵巢的发育同时也影响赤拟谷盗卵的形成。更多还原

【Abstract】 RNA interference (RNAi) is a highly conserved mechanism among eukaryote organism and is used as a research tool to investigate gene function in numerous organisms. The red flour beetle (Tribolium castaneum) was considered as an emerging model organism that exhibits robust systemic RNAi response, whereas, the mechanism of dsRNA uptake in T. castaneum has not been described in detail. In this study, we proposed the possible mechanisms of dsRNA uptake in T. castaneum. With the combination of pharmacological endocytosis inhibitors and RNAi technique, we found clathrin-dependent endocytosis mediated dsRNA uptake in T.castaneum. In this study, two pharmacological endocytosis inhibitors, chlorpromazine (CPZ) and bafilomycin-Al (BafA) can disrupt exogenous dsRNA entry and decrease RNAi efficiency in T. castaneum. Midgut cell of T. castaneum was investigated to localize Cy3-labelled dsRNA and found the amount of dsRNA uptaking in cells was reduced after inhibitor application. In addition, we found that medium chain of adaptor protein2(TcAP50), clathrin heavy chain (TcChc), subunit H of vacuolar (H+)-ATPase (TcVhaSFD) and ras-related protein (TcRab7) were also involved in dsRNA uptake process in T. castaneum. This study for the first time demonstrated that dsRNA uptake is an active process involving clathrin-dependent endocytosis in T. castaneumn.Lethal giant larvae (Lgl) protein encoded by the Lgl gene plays a critical role in epithelia cell polarization. However, current knowledge on the characteristics and functions of Lgl in other insects are very limited. In this study, we sequenced and characterized TcLgl from the red flour beetle (Tribolium castaneum). Analyses of stage-and tissue-specific expression patterns revealed that TcLgl was constitutively expressed throughout all different developmental stages and was predominately expressed in the gut. In addition, the function of TcLgl was investigated using RNA interference (RNAi) in early larvae (8-d larva), late larvae (20-d larva) and early pupae (1-d pupae). The transcript level of TcLgl was dramatically suppressed after the injection of double-starnded RNA (dsRNA) of TcLgl (dsTcLgl) at all these stages. The early larvae injected with dsTcLgl failed to pupate, and led to100%cumulative mortality in two weeks after the injection. When the late larvae were injected with100,200and400ng of dsTcLgl, the pupation rates were only50.3,36.0and18.2%, respectively. However, these pupae failed to eclose and completely died at two days after eclose in normal condition. The remaining injected late larvae continue keeping larvae stage more than one week and gradually died. Similarly, when early pupae were injected with100,200,400ng of dsTcLgl, the eclosion rates were only22.46,18.03and11.20%, respectively, compared with the control pupae injected with dsGFP7d after the injection. All emerged adults which were injected with dsTcLgl during early pupal stage died within12d after eclosion. Our results indicate that TcLgl plays an essential role in insect development, especially during their metamorphosis.Endocytosis has long been thought of as simply a way for cells to internalize nutrients and membrane associated molecules. However, current knowledge on endocytosis process in insect is very limited. In this study, we have identified four major genes (TcChc,TcAP50, TcVhaSFD and TcRab7) involved in clathrin-dependent endocytosis in red flour beetle(Tribolium castaneum). Genome structure analysis show that they located on different chromosomes. TcVhaSFD have two alternative spliced transcript named TcVhaSFDa and TcVhaSFDβ. Analyses of stage-specific gene expression revealed that these four genes are constitutively expressed throughout all developmental stages and have predominately expression in pupal stage. In addition, these genes were mainly expressed in the gut and fat body tissues, except TcAP50was predominately expression in carcass. RNAi of these genes revealed that absence of TcChc, TcAP50, TcVhaSFD and TcRab7lead to100%cumulative mortality in the late larval stages. For dsRNAs injections in the early pupal stage, abnormal eclosion were observed when TcChc, TcVhaSFD and TcRab7knockdown. The obvious phenotype show that newly eclosion insects have adult’s head and thorax combined with pupal abdomen, unfortunately, these insects died within2-day post-eclosion. Early pupae of TcAP50silenced eclosion normally, however, these adults died at6-day post-eclosion and lead to100%cumulative mortality. Strikingly, we also found that TcChc and TcVhaSFD have correlation with the egg production through microscopic examine of ovaries between control and treatment. These results demonstrate for the first time that major genes involved in clathirn-dependent endocytosis, including TcChc,TcAP50, TcVhaSFD and TcRab7have crucial role in development of red flour beetle.更多还原