CLONING AND EXPRESSION ANALYSIS OF AN INDUC IBLE HEAT SHOCK PROTEIN 70 GENE FROM RED SWAM P CRAYFISH, PRCCAMBARUS CLARKII

Red swamp crayfish, Procambarus clarkia, with advantagesof easy cultivation and availability, can live in various tough environments, so intrigued researchers pay attention to its ecology, toxicology, physiology and immunology.We studied an inducible cDNA encoding HSP70 in P. clarkia which would contribute to extensive researches of HSP70s and environmental stresses1 An inducible heat shock protein 70 (HSP70) cDNA was cloned from red swamp crayfish by RT-PCR and RACE, which named scHSP701 The full2length cDNA of the scHSP70 was 2271bp, consisting of a partial 5′-terminal untranslated region (UTR) of 142bp, a 3′-terminal UTR of 221bp, an open reading frame of 1902bp (ORF) and a poly (A) tail and GenBank No1 DQ3015061 The gene contained only one exon according to amplification of scHSP70 from genomic DNA1 The scHSP70 cDNA encoded a polypeptide of 635 amino acids. Based on phylogenetic analysis, the gene was clustered with inducible HSP70 familymembers from other species. The evolution relationship was consisted with traditional classification1 Semi-quantitative PCR was employed to assess the temporal expression of scHSP70 mRNA levels from heat-shock treated and unstressed crayfish1 Challenge of the red swamp crayfish with 2h heat shock resulted in dramatic increases in the expression of HSP70 mRNA levels in all tissues, heart, muscle, hemocytes, digestive gland, antennal gland, testis and intestine, among which, the highest expression was found in heart. However, under normal conditions, the expressions of HSP70 mRNA levelweremuch lower in all tissues compared to treated ones, especially in haemocytes. The upregulated mRNA expression of the HSP70 in the crayfish following heat shock indicates that scHSP70 gene is inducible. These stress proteins provide invaluable information in stress response in the crayfish.