【作者】 王磊；

【导师】 章晓波；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 对虾是最重要的海水养殖产品之一,但是自从1990年代以来,一直受到病害的严重威胁,尤其是对虾白斑综合症病毒(White spot syndrome virus, WSSV),已成为危害对虾养殖的最主要病原。因此,充分了解对虾的非特异性免疫系统,从而探索对虾病害防治的新途径具有重要意义。本实验室在以前的研究中,通过抑制差减杂交(SSH)发现了日本对虾中一段与细胞凋亡相关的基因序列,为了进一步了解该基因在对虾抗病毒免疫中的作用,本论文开展了该基因的功能研究,以期为对虾病毒病的防治提供依据。通过5’和3’RACE技术,从日本对虾中获得了该基因的cDNA全长,它含有一个预测的开放阅读框(ORF),长度为1524bp,编码508个氨基酸,与caspase蛋白酶家族具有同源性,因此将其命名为PjCaspase基因。相应的DNA分析发现,PjCaspase基因含有两个内含子,三个外显子。采用Genome walker技术获得了该基因的启动子,并进一步验证了其功能。将PjCaspase基因克隆至原核表达载体pGEX-4T-2中,获得了重组蛋白。研究表明,纯化的重组蛋白具有蛋白酶活性,证明PjCaspase具备Caspase的基本性质。通过RT-PCR、Northern blot和Western blot等分析发现,该基因在除心脏外的其他组织中均有转录表达。用WSSV病毒刺激后,在不同时间提取病毒对虾和抗病对虾肝胰腺总RNA,进行Real-time PCR分析,结果发现PjCaspase基因在抗病对虾中上调表达,说明该基因参与了对虾抗病毒的免疫过程。为了进一步了解PjCaspase在细胞凋亡中的作用,采用RNAi技术抑制PjCaspase基因的表达。结果表明,当PjCaspase基因的转录表达受到抑制时,对虾的细胞凋亡也受到抑制,这说明PjCaspase基因直接参与细胞凋亡。本论文初步研究发现,对虾Caspase具有基因多样性,抗病对虾中Caspase蛋白序列的变化,低于正常对虾。更多还原

【Abstract】 Shrimp is one of the most important species in aquaculture, but it has been blocked by diseases caused by white spot syndrome virus (WSSV) in the world since 1990’s. So it is important to study the innate immunity of shrimp and explore new approaches against the diseases. In our previous study, a fragment of a cDNA related to apoptosis was obtained in Peneaus japonicus by SSH. To further realize its role in shrimp antiviral immunity, in this investigation, this gene was characterized, which would be helpful for shrimp disease control.The full-length cDNA was obtained with 5’and 3’RACEs in Peneaus japonicus. It contained an open reading frame (ORF) of 1524bp encoding 508 amino acids, which was homologous with caspase protein family. This ORF was named as PjCaspase gene. Its DNA sequence revealed that PjCaspase gene cotained 2 introns and 3 exons. Its promoter was obtained using genome walker kit and further confirmed by functional analysis.After cloning of PjCaspase gene into prokaryotic vector pGEX-4T-2, the fusion protein was obtained, which presented enzyme activity of caspase. This gene was transcribed and translated in all shrimp tissues except for heart as revealed by RT-PCR, Northern blot and Western blot. Total RNAs of hepatopancreas from WSSV-infected and WSSV-resistant shrimp were extracted and subjected to real-time PCR. The results indicated that the PjCaspase gene was upregulated in WSSV-resistant shrimp, suggesting that it was involved in the shrimp antiviral immunity. To further realize the role of PjCaspase in apoptosis, RNAi was used to silence the PjCaspase gene. The results showed that the apoptosis was inhibited when the transcription of PjCaspase was inhibited, indicating that this gene played a very important role in apoptosis.In this premilitary study, it was found that the PjCaspase gene presented gene diversity. The gene diversity in WSSV-resistant shrimp was更多还原