中国明对虾重要分子伴侣蛋白基因的克隆及表达研究

IOCAS-IR > 海洋环流与波动重点实验室

	中国明对虾重要分子伴侣蛋白基因的克隆及表达研究
其他题名	Identification and characterization of chaperone protein genes in Chinese shrimp Fenneropenaeus chinensis
	栾伟
学位类型	博士
	2009-05-25
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	分子伴侣蛋白钙网蛋白葡萄糖调节蛋白78 热休克蛋白90 热休克蛋白70
摘要	对虾养殖业的可持续发展面临着种质退化、病害严重和养殖环境恶化等问题的严重挑战。养殖环境恶化造成的环境胁迫，不但影响对虾的生长性状，而且导致对虾的抵抗力下降，更容易引发病害的发生。养殖环境恶化已经严重影响了对虾养殖业的健康可持续发展。本论文针对环境恶化造成的环境胁迫对对虾影响的分子机理进行了研究。克隆了中国明对虾对环境胁迫应答的重要伴侣蛋白基因，包括钙网蛋白（FcCRT）、葡萄糖调节蛋白78 （FcGrp78）、热休克蛋白70（FcHsp70）和热休克蛋白90（FcHsp90）的全长cDNA，研究了这些基因的组织表达特征，并对这些基因在不同胁迫条件下的转录表达特征进行了分析。钙网蛋白是一种多功能的内质网钙结合蛋白，负责蛋白折叠和糖蛋白修饰。本论文首次在中国明对虾报道了钙网蛋白FcCRT基因的全长cDNA序列，编码406个氨基酸，具有保守的N-，P-和C-功能域，以及信号肽和保守的HDEL内质网回收标签。FcCRT基因与其它物种的钙网蛋白具有高度的相似性，系统进化分析表明，FcCRT在亲缘关系上更接近昆虫的钙网蛋白。Northern blot和原位杂交结果显示，FcCRT基因在中国明对虾各组织中均有表达，且在卵巢中发育早起的卵母细胞中表达量最高，说明FcCRT很可能参与了卵母细胞的成熟。FcCRT基因在不同胁迫条件下，其转录表达均呈现明显的变化。在WSSV感染实验中，中国明对虾肝胰脏和淋巴器官中FcCRT转录表达均明显上调；热休克、重金属处理均可引起FcCRT基因转录表达的变化，但不同重金属处理引起FcCRT转录表达变化的模式不同。铜离子处理6小时，会引起FcCRT基因的下调表达，但在12小时之后出现明显上调；镉离子处理12小时后引起FcCRT基因的下调表达，但在24小时又出现明显的上调表达。葡萄糖调节蛋白78（GRP78）是内质网重要的伴侣蛋白。中国明对虾的Grp78基因（FcGrp78）的cDNA全长为2325bp，编码665个氨基酸。FcGrp78具有三个Hsp70蛋白家族标签，并含有KDEL内质网回收标签。FcGrp78基因与中国明对虾已有的Hsc70和Hsp70具有高度相似性。Northern blot杂交结果显示FcGrp78基因在中国明对虾各组织中均有表达。FcGrp78基因在WSSV感染的中国明对虾肝胰脏中呈上调表达，在血细胞中下调表达，说明FcGrp78可能与对虾的免疫应答有关。热休克处理会诱导FcGrp78基因转录的上调。不同重金属离子胁迫引起的FcGrp78转录表达有所不同：铜离子处理可以诱导FcGrp78基因在处理后24小时的上调表达；镉离子的处理导致FcGrp78基因处理后12小时的下调以及处理后24小时的上调表达变化。短期低氧胁迫则抑制对虾FcGrp78基因的转录表达。本论文报道的中国明对虾FcHsp90基因 cDNA全长2552bp，编码726个氨基酸，具有保守的N端功能域、中间功能域和C端功能域，具有五个保守的Hsp90蛋白家族标签，序列上与其他物种Hsp90相似性高。Real-time RT-PCR结果显示FcHsp90基因在发育的卵巢中表达量较高，说明Hsp90可能参与了对虾卵母细胞成熟过程中的蛋白合成和卵黄蛋白原的分泌。WSSV感染引起中国明对虾肝胰脏的FcHsp90的转录表达明显上调，说明FcHsp90很可能与对虾的免疫相关。热休克处理诱导FcHsp90基因转录表达的迅速上调。铜离子处理也可诱导FcHsp90基因转录的上调表达，而镉离子处理首先引起FcHsp90基因的下调表达，24小时后开始上调。低氧胁迫也会抑制FcHsp90基因在对虾体内的转录表达。诱导型FcHsp70基因cDNA全长2511bp，编码629个氨基酸，具有三个保守的Hsp70蛋白家族标签和C末端EEVD序列。与其他物种的Hsp70蛋白具有高度的相似性。FcHsp70基因转录表达对于热休克处理和铜离子的处理非常敏感：热休克处理2小时后FcHsp70基因的转录水平是对照组的80倍；铜离子处理12小时FcHsp70基因转录表达达到对照组的15倍。而镉离子处理后没有诱导FcHsp70显著的上调表达。以上研究结果为阐明对虾对环境胁迫应答的机制奠定了重要基础，并可为抗逆对虾的培育提供依据。
其他摘要	The sustainable growth of shrimp industry in the world is challenged by diverse diseases and deteriorating environments. The deteriorative culture environments impair the immune responses of shrimp to pathogens, which easily leads to the exploration of diseases. Environmental stresses have become a huge problem to limit the development of marine aquaculture especially shrimp industry. This thesis presents cloning of calreticulin (FcCRT), glucose-regulated protein 78 (FcGrp78), heat shock protein 90 (FcHsp90) and heat shock protein 70 (FcHsp70) from Chinese shrimp Fenneropenaeus chinensis. Their transcriptional distributions in tissues of shrimp were examined. In addition, we have investigated the expression profiles of these genes in F. chinensis subject to WSSV infection, heat shock, heavy mental exposure or hypoxia exposure. As a multi-functional calcium-binding chaperone protein, calreticulin is responsible for the protein folding and glycoprotein folding in endoplasmic reticulum (ER). This is the first time to report the calreticulin gene in crustacean. Full length cDNA of FcCRT encodes 406 amino acids. FcCRT protien has conserved N-, P- and C-domains with signal peptide at N-terminal and HDEL motif at C-terminal. FcCRT shows high similarities with its homologues in other species and was more close to the homologues of insects. Northern blot or in situ hybridization analysis indicated that FcCRT expressed widely in the examined tissues of shrimp and the magnificent expression in ovary suggest its possible involvement in the maturation of oocytes. WSSV infection can induce the up-regulations of FcCRT transcripts in the hepatopancreas and lymphoid organ. Heat shock leads to the variations of FcCRT gene expression, however, copper or cadmium treatment induced distinct gene expression patterns. During Cu2+ treatment, FcCRT gene expression decrease at 6 hours after exposure and increased at 12 hour, while Cd2+ treatment caused a down-regulation of FcCRT at 12 hour post exposure, while an up-regulation at 24 hour. Glucose-regulated protein 78 (Grp78) is crucial chaperone protein in ER regulating the unfolded protein response (UPR). It is also the first report of Grp78 gene in the crustacean. Full length Grp78 cDNA is 2325bp encoding 665 amino acids. There were three Hsp70 protein family signatures found in FcGrp78 with KDEL motif at C-terminal. FcGrp78 shares high homology with Hsp70s of F. chinensis. Northern blot analysis indicated the ubiquitous existing of FcGrp78 in tissues of shrimp. During WSSV infection experiment, FcGrp78 transcriptional levels was up-regulated in the hepatopancrease and down-regulated in the hemocytes of shrimp, which implied that FcGrp78 might be involved in the process of viral infection. Other than the up-regulation following heat shock treatment, diverse heavy mental treatment leaded to different expression profiles of FcGrp78 gene。Cu2+ treatment decreased FcGrp78 gene expression at 6 hours after exposure and increased at 12 hour, while Cd2+ treatment resulted in the down-regulation of FcGrp78 at 12 hour post exposure and following up-regulation at 24 hour. It is also found that short-term hypoxia can depress FcGrp78 transcriptional level in the whole body of shrimp after 8 hour exposure. Full length cDNA of FcHsp90 is 2552 bp encoding 726 amino acids. Five Hsp90 protein family signatures were predicted in conserved N-, middle and C-domains of FcHsp90. FcHsp90 shares high similarities with reported Hsp90s in other species. Highest expression of FcHsp90 in ovary among tissues revealed by real-time RT-PCR analysis indicated that FcHsp90 may participate in the maturation of oocytes in shrimp. During WSSV infection, FcHsp90 transcriptional level increased significantly in the hepatopancrease. Heat shock or copper treatment induced significant up-regulation of FcHsp90. Cadmium treatment leaded to the down-regulation of FcHsp90 at 12 hour post exposure and subsequent up-regulation at 24 hour. Depression of FcHsp90 expression was observed after hypoxia exposure possibly due to the low metabolism level after hypoxia. In addition, a novel inducible FcHsp70 was cloned in F. chinensis. The full length cDNA is 2511 bp which encodes 629 amino acids. FcHsp70 possesses three conserved Hsp70 protein family signatures and EEVD motif at C-terminal. High similarity of FcHsp70 with other Hsp70s at amino acid level was revealed through multi-alignment and phylogeny tree analyses. FcHsp70 expression at transcriptional level was sensitive to heat shock or copper treatment. Heat shock treatment induced more than 80-fold increases of FcHsp70 gene expression in 2 hours and copper treatment leaded to a 15-fold increases in 12 hours. However, gene expression of FcHsp70 was undetectable after exposure to cadmium, which may depress FcHsp70 gene expression. Above data will be very helpful to clarify the molecular mechanism of shrimp responsive to different stresses, and provide imprehensive insights to breeding of stress resistant shrimp.
页数	153
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1171
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	栾伟. 中国明对虾重要分子伴侣蛋白基因的克隆及表达研究[D]. 海洋研究所. 中国科学院海洋研究所,2009.

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