Identification And Characterization Of Chaperone Protein Genes In Chinese Shrimp Fenneropenaeus Chinensis

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.