岩原鲤HSC70基因的克隆、序列分析及其组织表达研究
Cloning, Sequence Analysis and Gene Expression of cDNA for the HSC70 Gene in Procypris rabaudi
章海滨1，张修月1，宋昭彬1, 2*
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作者单位：(1. 四川大学生命科学学院, 四川省濒危野生动物保护生物学重点实验室, 成都610065; 2. 四川大学生命科学学院，生物资源与生态环境教育部重点实验室, 成都610065)
中文关键字：HSC70；克隆；序列分析；基因表达；岩原鲤
英文关键字：heat shock cognate 70; cloning; sequence analysis; gene expression; Procypris rabaudi
中文摘要：采用RT-PCR (reverse transcription-polymerase chain reaction) 技术从岩原鲤 (Procypris rabaudi) 脑组织中克隆出长1043bp的组成型热休克蛋白HSC70 (heat shock cognate 70) 基因的cDNA (complementary deoxyribonucleic acid) 部分片段，其编码的347个氨基酸包含HSP70家族签名序列(IVLVGGSTRIPKIQK)，核定位信号标签(KRKHKKDISDNKRAVRR)及2个糖基化位点 (NKSI/NVSA)。采用相同引物从岩原鲤基因组DNA中扩增出长1418 bp的HSC70基因片段，比对分析显示其含有3个分别长200 bp、98 bp和77 bp的内含子。氨基酸序列同源性分析显示，HSC70基因氨基酸序列具有高度保守性，岩原鲤HSC70基因氨基酸序列与草鱼Ctenopharyngodon idellus的同源性达到了100%，与其他脊椎动物的同源性为95.68%~99.71%。FQ-PCR (Real-time fluorescent quantitative PCR) 方法检测显示，岩原鲤HSC70基因存在明显的组织表达差异，在脑中相对表达量最高，其次为卵巢、心、肌肉和肾，在肝中相对表达量最少。
英文摘要：The HSC70 (heat shock cognate 70) cDNA (complementary DNA) was obtained from brain of rock carp (Procypris rabaudi) using RT-PCR. The length of HSC70 cDNA sequence was 1043 base pairs (bp) encoding a polypeptide of 347 amino acids. Analysis of this amino acid sequence indicated that HSC70 contained a signature sequence (IVLVGGSTRIPKIQK) of HSP70 family, a putative biparitite nuclear localization signal (NLS) (KRKHKKDISDNKRAVRR) and two glycosylation sites (NKSI/NVSA). To investigate whether there were introns existed in the coding regions of HSC70 gene, the full length of HSC70 gene was amplified by the same primers in genomic DNA. Then the sequence structure analysis revealed that HSC70 gene contained three introns, which were 200 bp, 98 bp and 77 bp in length, respectively. Homology analysis suggested that the amino acid sequence of HSC70 gene was highly conserved. The highest sequence similarity (100%) was observed between rock and grass carp (Ctenopharyngodon idellus), and the sequence of rock carp shared 95.68%~99.71% identity with other vertebrates. The results of Real-time fluorescent quantitative PCR (FQ-PCR) indicated that HSC70 ubiquitously expressed in all six tissues examined, and the highest expression level was detected in brain, followed by in ovary, heart, muscle, kidney, and liver.