三角帆蚌精氨酸酶基因的cDNA克隆与组织表达分析

FULL-LENGTH CDNA CLONING AND TISSUE EXPRESSION ANALYSIS OF ARGINASE GENE FROM HYRIOPSIS CUMINGII

精氨酸酶(Arginase,Arg)是生物体尿素循环当中一种标志性的酶类,它不但与生物体许多疾病相关,而且是目前用于治疗肿瘤和癌症的一种重要的工具酶。根据三角帆蚌消减杂交cDNA文库获得的EST序列,运用cDNA末端快速扩增(Rapid amplification of cDNA ends,RACE)技术获得三角帆蚌精氨酸酶基因的全长cDNA序列。生物信息学方法分析表明精氨酸酶基因cDNA序列长1720 bp,开放阅读框(65—1072)1008 bp,编码335个氨基酸,5′端非编码区为64 bp,3′端非编码区为648 bp,软件推测其编码蛋白相对分子量为36.81 kD。研究采用实时荧光定量PCR(quantitative real-time PCR,qPCR)方法分析该基因在不同组织中的表达规律,结果表明,该基因在肝脏、胃、肠、鳃、心脏、外套膜、斧足共7个组织中都有表达,但主要集中在肝脏、胃和肠消化器官中表达。这可能说明低等的无脊椎动物三角帆蚌的精氨酸酶兼具有Ⅰ型和Ⅱ型精氨酸酶的特征和功能,既可以参与尿素循环,又可以在生理和病理过程中发挥重要作用,但还需进一步验证。

Arginase（Arg） is a sign enzyme among organisms of urea cycle.It is not only related to many diseases in organisms,but also used to treat tumors and cancer as an important tool enzyme.According to the Hyriopsis cumingii（H.cumingii） expressed sequence tags（EST） obtained by constructing subtractive hybridization cDNA library of H.cum-ingii liver,the gene full-length cDNA sequence of arginase from H.cumingii was cloned by RACE-PCR technique based on the designed gene-special primers.After analyzed by the software DNA Star and the bioinformatics technology,the results showed that the length of arginase gene cDNA sequence was 1720 bp,containing a complete open reading frame（65—1072） which was 1008 bp,encoding a peptide of 335 amino acid residues（aa）,flanked by a 64 bp of 5′ untrans-lated region（UTR） and a 648 bp of 3′-UTR.The deduced molecular weight of arginase was about 36.81 kD.At the transcriptional level,quantitative real-time PCR（qPCR） was used to detect the expression of the arginase gene in different tissues.The result revealed that H.cumingii arginase gene could be expressed in seven kinds of tissue con-taining liver,stomach,intestine,gill,heart,mantle,axe foot collected from H.cumingii,especially strongly expressed in digestive organs,such as liver,stomach and intestine,but weakly in heart and mantle.So it concluded that the arginase from the H.cumingii which belongs to lower invertebrate could possess the same characteristics and func-tions of arginase typeⅠandⅡfrom the higher animals.That means the arginase from the H.cumingii may not only participates in urea cycle,but also plays an important role in the processes of physiology and pathology.And the de-duction will be verified in the next experiment.