曼氏无针乌贼(Sepiella maindroni)ALSM基因的克隆及序列分析

MOLECULAR CLONING AND SEQUENCE ANALYSIS OF ALSM cDNA FROM SEPIELLA MAINDRONI

采用RT-PCR和RACE技术分离、克隆得到曼氏无针乌贼肝脏ALSM(Astacin-like squid metalloprotease)基因。序列分析表明,ALSM cDNA序列全长1418bp,其包括53bp的5’端非翻译区,1290bp阅读框以及含有poly(A)信号AATAAA的75bp3’端非翻译区,阅读框编码429个氨基酸残基。推导蛋白的相对分子质量为48916.31Da,等电点为7.978。ALSM氨基酸序列的同源性分析显示,不同进化地位动物的ALSM氨基酸序列都具有较高的同源性。系统发生树分析发现,曼氏无针乌贼ALSM首先与金乌贼、莱氏拟乌贼、长枪乌贼及太平洋褶柔鱼的ALSM-Ⅰ亚型聚类,再与其ALSM-Ⅱ及ALSM-Ⅲ亚型聚类。生物信息学分析表明,ALSM编码的蛋白为一种亲水性的分泌蛋白。这些结果对进一步研究ALSM的结构与功能以及从基因调控角度探索曼氏无针乌贼在养殖条件下生物学特性变化机理具有重要意义。

The ALSM （Astacin-like squid metalloprotease） gene of Sepiella maindroni was cloned from liver by RT-PCR and RACE technique. Sequence analysis revealed a 1418bp full-length cDNA sequence containing 53bp 5＇-untranslated region, 75bp 3＇-untranslated region and 1290bp open reading frame （ORF） and this sequence was encoded by 429 amino acids. The molecular weight of deduced protein was 48916.31Da and its pI was 7.978. The deduced amino acid sequence aligned with those of ALSM genes from different species showed high degree of sequence homology. The phylogenic analysis tree showed that ALSMs are classified into two clades： ALSM-I forms one clade, and ALSM-II and -III form the other. The ALSM protein of S. maindroni firstly clustered with ALSM-I of golden cuttlefish, bigfin reef squid, spear squid and Japanese common squid, then with ALSM-II and -III of them. The bioinformatics analysis revealed that the predicted protein was a hydrophilic secretion protein. The results are very important for further study of on structures and functions of ALSM and exploration of the mechanism of biological characteristics changes of S. maindroni in breeding conditions.