黄颡鱼HSC70基因及其组织表达分析

HSC70 GENE AND ITS TISSUE EXPRESSION ANALYSIS IN YELLOW CATFISH

热休克蛋白70(HSP70)与生物体的抗胁迫能力密切相关。本文采用RACE(Rapid amplification of cDNA ends)技术,从黄颡鱼Pelteobagrus fulvidraco克隆到一种组成型热休克蛋白(HSC70)基因及其cDNA。该cDNA全长2245bp,包括5′非编码区82bp,3′非编码区225bp,开放阅读框(ORF)1938bp,编码645个氨基酸组成的蛋白质。黄颡鱼HSC70基因含有8个内含子,与人、鼠、虹鳟和花斑溪鳉的HSC70基因内含子数目相同,位置相似。其中,最长内含子(873bp)位于5′端非编码区,其余内含子(长度在80—251bp之间不等)均在编码区以内。黄颡鱼HSC70基因编码的氨基酸序列与南方鲶的相似度最高,达96.13%,与欧洲银鲫和团头鲂的相似度分别为94.45%和94.14%。RT-PCR检测显示,正常情况下黄颡鱼HSC70在血细胞、心脏、肝、头肾、脾、鳃、肌肉和脑中均有表达,但表达量在鳃中最高,肌肉中最低;统计结果显示,热激后HSC70在血细胞、肝、头肾和脑中的表达量显著上升(p<0.05),而在其余组织中热激前后的表达差异不显著(p>0.05)。

Heat shock protein 70 （HSP70s） act as a role of chaperone and play a key function in cytoprotection and cytorepair, 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 eDNA sequence of HSP70 family members in the first instance. Therefore, the gene and its eDNA 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 eDNA of 2245 bp was cloned in the gill of yellow catfish with RACE （rapid amplification of eDNA ends） technique. The eDNA 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 eDNA belonged to the family of heat shock protein 70. Carried out alignment with other organisms＇ 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 HSP70s. What mentioned above suggested that the sequence we cloned was a kind of heat shock cognate 70 （HSC70）. Furthermore, 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 HSC70 gene, and the longest intron （873bp） was located in 5＇ untranslated region and the others （length between 80 and 251bp） in ORF region （Fig. 2）. The introns with same number and similar location 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 catfish were cultured in water temperature of （26 ±1 ） ℃ , the other （ the heat shock group） was heat-treated at （36 ± 1 ） ℃ for lh, 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, HSC70 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 （p 〈 0.05） , but did not significantly in the other four detected tissues or organs （heart, spleen, gill and muscle） （p〉0.05）. The data above indicate that the HSC70 gene and its eDNA of yellow catfish have been cloned, and the gene expressed constitutively and could be induced by heat shock in various tissues and organs.