团头鲂HSP70 cDNA的克隆、序列分析以及热应激对其mRNA表达的影响

Heat shock proteins or heat stress proteins (HSPs) widely exist in the whole biosphere from prokaryote to mammal, which are highly conservative in evolution and very important in function. HSP70 family is one kind of the most conservative and important protein. In vivo and in vitro studies have shown that various stressors transiently increase production of HSP70s as protection against harmful insults. In this research, RT-PCR and RACE (Rapid amplification of cDNA ends) methods were used for the cloning of full length cDNA encoding HSP70 from liver of Megalobrama amblycephala. The length of the cDNA is 2 224 bp, containing 135 bp 5'-untranslated region , 157 bp 3'-untranslated region [excluding poly (A)] and 1 932 bp open reading frame (ORF). It encodes a peptide of 643 amino acids whose calculated molecular weight is 70.53 kD and theoretical isoelectric point is 5. 25. Intron absence in the translated region accords with the characteristic of inducible type HSP70.Sequence analysis of the protein showed that HSP70 of M. amblycephala contained three signature sequences of HSP70 family and a cytoplasm characteristic motif of EEVD. The homology analysis indicated that HSP70 of M. amblycephala shared more than 84.0% identity with the HSP70s of other vertebrates such as Danio rerio, and also shared as high as 73.4% identity with the nonvertebrate of Drosophila auraria. Bioinformatics analysis revealed that the protein encoded by HSP70 gene in M. amblycephala was hydrophilic in most region and had rich B cell antigenicity positions without signal peptide and notable transmembrane region which indicated that the protein was not secretory protein. It also contained many protein kinase C phosphorylation sites, N-myristoylation sites, casein kinase II phosphorylation sites and N-glycosylation sites, predicting that it can play an essential role in the cell signal conduction and regulation. The HSP70 had a bipartite nuclear localization signal sequences. Secondary structurs of the protein were mainly alpha helixes and random coils. Dimensional structure included ATPase domain in the amino terminus region and polypeptide-binding domain in the carboxyl terminus region. Result of real-time quantitative PCR showed that HSP70 mRNA content in liver firstly increased and then droped after high temperature (34℃)stress, which indicated that the expression of HSP70 mRNA of M. amblycephala was adjusted by heat stress. In summary, the cDNA cloned in this study belonged to inducible HSP70 gene. Cloning of this gene was very important for further investigation on anti-stress mechanism, genetic breeding and improvement of stress-tolerance in M. amblycephala.