中国明对虾核转录因子NF-κB家族基因的克隆与表达分析

IOCAS-IR > 海洋环流与波动重点实验室

	中国明对虾核转录因子NF-κB家族基因的克隆与表达分析
其他题名	Cloning and Expression of Nuclear Transcription Factors Rel/NF-κB family genes in Chinese shrimp Fenneropenaeus chinensis
	阎慧
学位类型	博士
	2008-05-30
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	中国明对虾 Nf-κb Relish Dorsal 基因克隆表达分析鳗弧菌 Wssv
摘要	对虾流行病爆发以来，我国乃至世界的对虾产业一直受到各种虾病的困扰，对虾养殖业严重受阻。解决这一问题的关键是加强对虾免疫机制的研究，并在此基础上寻找对虾疾病防治的有效方法。Rel/NF-κB是一类核转录因子，在无脊椎动物的先天性免疫中，起着极为重要的作用。本论文根据其他无脊椎动物中NF-κB家族基因Relish和Dorsal的保守氨基酸序列分别设计简并引物，从中国明对虾血细胞cDNA中先后克隆到了Relish和Dorsal基因的部分片段，并结合SMART-RACE技术分别获得了中国明对虾Relish基因（FcRelish）和Dorsal基因（FcDorsal）的cDNA 全长。 FcRelish的cDNA 全长为2157个碱基，其中开放阅读框为1512个碱基，编码504个氨基酸；FcRelish蛋白的推导分子量为57373.5 Da，理论等电点为7.00。FcDorsal基因的cDNA 全长为1627个碱基，其中开放阅读框为1071个碱基，编码357个氨基酸；FcDorsal蛋白的推导分子量为39780.7 Da，理论等电点为8.85。分析了FcRelish基因和FcDorsal基因的在血细胞、淋巴器官、肠和肌肉等12个组织中的表达水平。组织表达结果表明FcRelish和FcDorsal在淋巴器官和血细胞中表达水平明显高于其他组织，而淋巴器官和血细胞是对虾免疫系统中最重要的两个组织，由此可以推测中国明对虾中的Relish和Dorsal可能与免疫关系密切。本论文还利用实时荧光定量PCR技术，对灭活鳗弧菌刺激，以及WSSV病毒刺激后，对虾血细胞和淋巴器官中FcRelish基因和FcDorsal基因的转录水平进行了研究。FcRelish基因在WSSV病毒刺激后，在血细胞和淋巴器官中都出现了波浪形变化，说明FcRelish对WSSV病毒刺激产生了应答。在灭活鳗弧菌刺激后，FcRelish在血细胞中变化不明显，而在淋巴器官中出现了两次明显的下调上调交替，出现这种现象的具体原因有待探究。在WSSV病毒刺激后，血细胞和淋巴器官中FcDorsal的转录表达呈波浪形变化。而在鳗弧菌刺激后，FcDorsal在血细胞和淋巴器官中的转录均在短时间内出现明显的上调表达，说明FcDorsal对鳗弧菌非常敏感。作为核转录因子的NF-κB蛋白的转录激活作用需要在细胞质中通过蛋白水解作用来激活，为了进一步阐明NF-κB对病菌感染的应答机制，需要进一步研究这两种转录因子在蛋白水平的变化，以便从分子水平阐明NF-κB在对虾天然免疫中的作用。
其他摘要	Shrimp cultivation has been suffering serious problems due to the outbreak of diseases throughout the world. The key point to solve this problem is to explore the immune mechanism of shrimp and to find effective methods to control the diseases. Rel/NF-κB family is reported as a transcription factor, which plays a central role in the innate immunity in invertebrates. In this study, degenerate primers were designed according to the sequence homologous with the same genes from other species. Then SMART RACE was used to obtain the full lengths of Relish (FcRelish) and Dorsal (FcDorsal) in Chinese shrimp Fenneropenaeus chinensis. The full length of FcRelish gene is 2157 bp and that of FcDorsal is 1627 bp respectively. The expression of above two genes was analyzed in 12 tissues of shrimp including haemocytes, lymphoid organ, intestine, muscle etc. FcRelish and FcDorsal transcripts in hemocytes and lymphoid organ are higher than those in the other tissues. Since the haemocytes and lymphoid organ play very important roles in the immune reaction of shrimp, FcRelish and FcDorsal might act as important immune factors in shrimp. Real time PCR technique was applied to quantify the transcripts of FcRelish and FcDorsal gene in haemocytes and lymphoid organ when shrimp were challenged by inactivated Vibrio and WSSV. After challenged with WSSV, the expression profile of FcRelish showed a wavy changing curve in both haemocytes and lymphoid organ, which indicate that FcRelish may play an important function when shrimp were challenged by WSSV. The expression of FcRelish in haemocytes does not change obviously after being stimulated by Vibrio, but it showed great variation in lymphoid organ instead. While the expressions of FcDorsal in hemocytes and lymphoid organ are up-regulated significantly when shrimp were stimulated by inactivated Vibrio, which might illuminate that the expression of FcDorsal is sensitive to Vibrio. As a transcription factor, only hydrolyzed NF-κB can activate the transcription of downstream genes. To study the mechanism of shrimps against bacteria and virus challenge, the variation of these two genes at translation level will be studied further.
页数	93
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/405
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	阎慧. 中国明对虾核转录因子NF-κB家族基因的克隆与表达分析[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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