Gene Cloning, Sequence Analysis And Cold Adaptation Studies On Hsp70 And Hsp90β Genes From Chionodraco Hamatus And Trematomus Bernacchii

Channichthyidae belongs to Notothenioidei, which long live in cold, stable and oxygen-rich Southern Ocean, and have become a unique species in teleosts as the loss of Hb.Unusual characteristics in morphology and physiology, Channichthyidae provides a good opportunity for us to investigate their mechanism in cold adaption and gene evolution under extreme environment.Heat shock proteins(HSPs) are synthesised increasingly by cells under all kinds of stressors, existing in almost all organisms. HSP90 and HSP70, important members of heat shock proteins, which are also expressed under no stress in eukaryotes,is highly conserved. As protein chaperones, HSP90 and HSP70 are involved in many pathways including control of cell cycle,cell survival and hormone, and play crucial roles in cellular homeostasis. Meanwhile, they play important roles in plant development, response to stress and disease resistance.The temperature of antarctic environment keep constant all year round. Studies show that HSPs have lost the ability of temperature adaption under this prolonged cold condition. To study whether the temperature adaptive function exist under the extreme cold antarctic environment, PCR and Next-Generation sequencing technology have been used to isolate the complete opening reading frames of Hsp70 and Hsp90β genes of Chionodraco hamatus(C H) and Trematomus bernacchii(TB), both of them have a sequence of 2184 bp(Hsp90β) and 1917 bp(Hsp70). And then we used bioinformatics methods to analyse their conserved domains. The ORF of Hsp90β encodes a polypeptide of 728 amino acids, while the Hsp70 ORF encodes a polypeptide of 638 amino acids. HSP70 sequence has two signiture sequence of HSP70 family, and has a conserved GPTIEEVD motif in the C-terminal. Both HSP90β have five conserved signal areas and MEEVD motif in the C-terminal, which are characteristic of HSP90 family. Homology and phylogenetic analysis showed that the deduced Chionodraco hamatus amino acid sequence of both proteins have a very high similarity with other species, and shared 99% similarity with those of Notothenia coriiceps.To investigate the protection of Chionodraco hamatus HSP90β to cells under cold stress, recombinant p ET28-CH-Hsp90 and p ET28-ON-Hsp90 prokaryotic expression recombiant plasmids were constructed and expressed in Escherichia coli DE3(BL21) under 37 degree centigrade. Survival rates of Escherichia coli DE3 were compared after treatment of 4 degree centigrade. The rusults showed that there was no significance between C.hamatus-HSP90β group and O.niloticus-HSP90β under treatment of 4 degree centigrade for four hours; when the treatment time is extended to eight hours, the survival rate of p ET28-CH-Hsp90 group was higher than those of p ET28 vector and p ET28-ON-Hsp90 group, with a significant difference between C.hamatus- HSP90β and O.niloticus-HSP90β group(p﹤0.05); when the treatment time was twelve hours, the survival rate between them became more significant than that of eight hours. The results first indicated that both C.hamatus-HSP90β and O.niloticusHSP90β provide protection for Escherichia coli DE3. With time extension of cold treatment, the difference of cell survival rate is more significent. Compared with HSP90β in temperate fish-Oreochromis niloticus, the protection of C. hamatusHSP90β is more effective.It can be inferred that C.hamatus-HSP90β provided more effective protection for cells under cold, and C.hamatus-HSP90β showed better cold adaption. This may provide a basis for further functional study of Trematomus HSP90β in adaption of extreme environment.