HSC70 GENE AND ITS TISSUE EXPRESSION ANALYSIS IN YELLOW CATFISH

Heat shock protein 70 (HSP70s) act as a role of chaperone and play a key function in cytoprotection and cytore-pair, including protein assembly, correct folding, and membrane translocation, it also enhance the organisms' immunity and enduration to stressors. Yellow catfish (Pelteobagrus fulvidraco) is an important cultured species in China. In order to illuminate molecular mechanism of the HSP70 family members in the catfish against stressors and diseases, it is necessary to clone the gene and cDNA sequence of HSP70 family members in the first instance. Therefore, the gene and its cDNA of a HSP70 family member were cloned in yellow catfish, and mRNA expression of the gene was studied in various tissues and organs of the catfish under heat-treated or unstressed condition.A full length cDNA of 2245 bp was cloned in the gill of yellow catfish with RACE (rapid amplification of cDNA ends) technique. The cDNA contained an open reading frame (ORF) of 1938 bp, 5' untranslated region of 82 bp and 3' untranslated region of 225 bp. The deduced 645 amino acid sequence contained HSP70s' characteristic motifs (Fig. 2), and it indicated that the cDNA belonged to the family of heat shock protein 70. Carried out alignment with other organ-isms' HSC70 amino acid sequences, the deduced amino acid sequence from the eDNA showed the highest similarity (96.13%) with HSC70 amino acid sequence of Southern catfish (Silurus meridionalis). The phylogenetic tree (Fig. 3) showed that 70 kD heat shock protein of yellow catfish clustered together with other vertebrates' HSC70s but not HSP7Os. What mentioned above suggested that the sequence we cloned was a kind of heat shock cognate 70 (HSC70). Further-more, its constitutive expression in unstressed tissue cells by RT-PCR detection (Fig. 4) confirmed that the sequence we cloned was HSC70 eDNA. Subsequently, the catfish HSC70 gene was cloned by PCR amplification in the fish genome DNA. Eight introns were found in the HSCIO gene, and the longest intron (873bp) was located in 5' untranslated region and the others (length between 80 and 251 bp) in ORF region (Fig. 2). The introns with same number and similar loca-tion were found in the HSC70 genes of human (Homo sapiens), mouse (Mus musculus), rainbow trout (Oncorhynchus mykiss) and hermaphroditic teleost (Rivulus marmoratus). In order to elucidate the HSC70 mRNA expression in different tissues or organs of yellow catfish under heat-treated or unstressed condition, ten catfish were divided into two groups randomly, one (control group) was unstressed and the cat-fish were cultured in water temperature of (26+1)℃, the other (the heat shock group) was heat-treated at (36+1)℃ for 1 h, and then recovered in normal water temperature (26+1)℃ for 3h. Semi-quantitative RT-PCR method was employed to analyze the HSC70 mRNA expression in different tissues or organs, viz., blood cell, heart, liver, head kidney, spleen, gill, muscle and brain. The results were as follows: in control group, HSCIO mRNA expressed constitutively in eight tissues or organs of the unstressed catfish, and the mRNA transcripts were various in the detected tissues or organs and the highest in gill (Fig.5); in the heat shock group, HSC70 mRNA expression significantly increased in blood cells, liver, head kidney and brain (p0.05). The data above indicate that the HSC70 gene and its eDNA of yellow catfish have been cloned, and the gene ex-pressed constitutively and could be induced by heat shock in various tissues and organs.