青虾(Macrobrachium nipponense)Kazal型丝氨酸蛋白酶抑制因子基因全长cDNA的克隆及表达

THE FULL LENGTH cDNA CLONING AND EXPRESSION ANALYSIS OF KAZAL-TYPE SERINE PROTEINASE INHIBITOR GENE FROM MACROBRACHIUM NIPPONENSE

采用RACE技术,首次从青虾精巢中克隆得到KSPI基因全长cDNA。青虾KSPI基因cDNA全长890bp,包括80bp的5′UTR,546bp的3′UTR和编码87个氨基酸残基的264bp开放阅读框。预测蛋白包含1个保守的Kazal型结构域(CⅠX5CⅡX(6)VCⅢX(5)TYXNXCⅣX6CⅤX12CⅥ),结构域中P1活性位点为苏氨酸残基。应用MEGA4.1软件对青虾与已报道的虾类共15种KSPI氨基酸序列进行系统进化分析,结果表明,青虾KSPI与同样来源于精巢的罗氏沼虾KSPI进化关系最近聚为一支,其它虾类来源于肝胰腺和血细胞的KSPI聚为另外两支。采用定量PCR技术分析其组织分布,结果显示,KSPI基因在精巢、心脏和卵巢中有较高表达,其中精巢表达水平极高,并与心脏和卵巢表达差异分别达到显著(P<0.05)和极显著(P<0.01)水平,其它组织表达较弱。

A full-length KSPI cDNA sequence was cloned from the testis of Macrobrachium nipponense for the first time, using RACE technique. The full length KSPI cDNA is 890bp, containing a 80bp 5＇ UTR, a 546bp 3＇ UTR and a 246bp open reading frame encoding 87 amino acids. Amino acid sequence analysis revealed that its encoded amino acids contained a conserved Kazal-type domain. The conserved sequence was C l XsCⅡX（6）VCⅢX（5）TYXNXCX6CvX12CxⅥ, and its P1 active site was threonine. The phylogenetic tree based on 15 species KSPI proteins showed that KSPI from testis of M. nipponense shared the closetest relationship with M. rosenbergii, and KSPI from hepatopancreas and hemocytes of other species form the other two main branches. Quantitative PCR was used to detect the distribution of KSPI. According to the results, KSPi was rarely expressed in liver, brain and intestine, whereas the expression levels were relatively higher in ovary, heart and testis. Interestingly, KSPI was highly expressed in testis, while the expression of KSPI in testis was significantly different with the expression in heart （P〈0.05） and ovary （P〈0.01）.