Molecular Cloning And Expression Profiles Of Heat Shock Protein Hsp30、hsp70 Family From Chinese Sturgeon (Acipenser Sinensis) Under High Temperature Stress And Bacterial Infection

Heat shock proteins（HSP）are a kind of stress protein commonly found in various organisms.When the organism is stimulated by various physical and chemical factors in the environment,it will produce stress feedback and synthesis the Heat shock proteins,which enhances its own resistance and reduces the damage of external stimulus to the body.Chinese sturgeon（Acipenser sinensis）,is a national Grade I aquatic protected animal and it does not have a physiological mechanism to maintain constant body temperature,so their metabolism is easily affected by the temperature of the water outside.Breeding in high temperature season,Acipenser sinensis is prone to cause bacterial diseases and bring huge economic losses to the squid farming industry.In this study,RACE and homologous sequence alignment were used to clone and identify the Chinese sturgeon HSP30 protein and HSP70 protein family genes,which play important roles in temperature stress,analyze their sequence characteristics and phylogenetic relationships,and study their tissue expression characteristics under high temperature stress and bacterial infection,which laid the foundation for studying the function of this gene under temperature stress and bacterial infection.1.Cloning HSP30 gene and analyzing its expression under high temperature stressThe full length of heat shock protein HSP30 gene was cloned from Chinese sturgeon（Acipenser sinensis）,and the molecular structure and characteristics were analyzed.Furthermore,the expression levels under high temperature stress were studied.The results showed that the full-length cDNA sequence of HSP30 gene was 1037 bp,with a 636 bp open reading frame（ORF）which encoded the protein with 211 amino acids as well as 38 bp 5’ untranslated region（5’UTR）and 363 bp 3’ untranslated region（3’UTR）.Amino acid multiple sequence alignment revealed that HSP30 has a conservedα-crystal structure.Phylogenetic analysis showed that the HSP30 amino acid sequence of Chinese sturgeon were clustered together with fish,and also showed highestsimilarity（79%）with the Acipenser ruthenus.The results of real-time PCR showed that HSP30 was extensively expressed in various tissues.The highest expression was detected in the skin,followed by the liver,and the lowest was in the intestine.After high temperature stimulation,the expression of HSP30 in the heart,spleen,kidney and skin were all significantly increased,indicating that these organs may play an important role in response to high temperature stress in Chinese sturgeon.This study aims at providing a reference for the investigation on the resistance mechanism to temperature stress in Chinese sturgeon（Acipenser sinensis）.2.Cloning HSP70 family gene and analyzing its expression under high temperature stress and bacterial infectionNine members of the HSP70 family from Acipenser sinensis（designated HSC70,HSP70-A1,HSP70-4,HSP70-4L,HSP70-12 A,HSP70-12 B,HSP70-12 Blike,HSP70-13 and HSP70-14）were cloned and identified.Domain analysis of all members of the HSP70 family found that they all have a conserved domain structure（NBD_sugar-kinase_HSP70_actin）.HSC70 and HSP70-A1 have a conserved domain structure（HSPA1-2₆-8-like_NBD）,HSP70-4 and HSP70-4Lhave a conserved domain structure（HSPA4_NBD）,HSP70-12 A,HSP70-12 B and HSP70-12B-like have a conserved domain structure（HSPA12-like_NBD）,HSP70-13 and HSP70-14 had unique domain structure（HSPA13-like_NBD and HSPA14-like_NBD）.The results of phylogenetic analysis indicated that the nine members of the Acipenser sinensis HSP70 family were distributed in five different branches in the phylogenetic tree,and members with the same domain were distributed in the same branch.Real-time PCR analysis revealed that nine members of the HSP70 family were ubiquitous and differentially expression in six tissues（liver,spleen,kindey,intestinal,brain and skin）of Acipenser sinensis.After high temperature stress and Aeromonas hydrophila infection,the expression of 9 members of HSP70 family in the spleen and kidney of the Acipenser sinensis increased significantly,which suggested that HSP70 family may play an important role in maintaining the normal physiological state of the body and coping with external temperature stress and pathogen infection.