Cloning, Expression Characteristics And Functional Analysis Of Hsp90 And Wap65 Genes From Grass Carp

The grass carp (Ctenopharyngodon idellus) Hsp90 gene was cloned from mixed liver and kidney cDNA library (CiHsp90, GU258544). The full length sequence of which was 2793 bp, contained a 5'untranslated region of 104 bp and a 3'untranslated region of 292 bp. The open reading frame was 2397 bp which could code a 798 amino acids peptide. Analysis of the deduced amino acid sequence indicated that the mature peptide possessed an ATPase domain (96-255 aa). The RT-PCR analysis showed that CiHsp90 was ubiquitously expressed at lower levels in all detected tissues and up-regulated after heat shock at 34℃. To understand the function the ATPase domain of CiHsp90 involving in thermal protection, an expression vector containing its cDNA was expressed in E. coli BL21 (DE3) plysS. Upon transfer from 37℃to 42℃, these cells that accumulated PATPase (recombinant peptide of the ATPase domain) displayed greater thermoresistance than control cells. While incubated at 4℃for different periods, it could also improve viability compared with control cultures. The results showed that the ATPase domain of CiHsp90 could contribute to protecting cells against both thermal extremes. Moreover, we expressed and purified the peptide of PATPase using for the citrate synthase aggregation assays. It suggested that the ATPase domain of CiHsp90 could reduce the level of citrate synthase aggregation at the high temperature.The warm temperature acclimation related 65 kDa protein (Wap65) is a stress protein involved in thermoregulation and immune response. Wap65 plays a crucial role in heat and cold adaption in teleost. In this report, we cloned and identified a Wap65 (CiWap65, HM629798) in cDNA library (induced by Poly I:C) constructed from liver and kidney tissues of grass carp (Ctenopharyngodon idella). The full-length cDNA of Wap65 is 1510 bp with 55 bp in the 5'UTR and 327 bp in the 3'UTR. The dedued ORF is 1128 bp encoding a protein of 376 aa which contains six repeat heme binding domains at the position of 56-95 aa,102-153 aa,155-199 aa,200-243 aa,262-305 aa and 307-352 aa, respectively. The phylogenetic analysis showed that CiWap65 grouped tightly with catfish Wap65-2 in the same branch. The qPCR suggested that the CiWap65 had strong basal expression in liver, but very weak in spleen, kidney, brain and intestine. By heat shocking at 34℃for 2 hours, the expression slightly increased in spleen and was significantly up-regulated in liver but no obvious changs in other tissues tested. So CiWap65 mainly expresses in liver. These datas above support the idea that the CiWap65 isolated in this paper probably is Wap65-2. The qPCR suggested that the CiWap65 was enhanced in spleen after challenged by Poly I:C compared with the normal level but no obvious changs in other tissues tested. The qPCR suggested that the CiWap65 was down-regulated in tissues tested after challenged by LPS compared with the normal level.