鲈α-珠蛋白基因的克隆和序列分析

Cloning and Sequence Analysis of α-type Globin cDNA from Japanese Sea Bass(Lateolabrax japonicus)

从鲈头肾cDNA文库中随机挑取噬菌斑,作为模板,以λZAPExpress载体两端含有的T3和T7启动子序列设计引物,用PCR方法扩增插入片断,从中克隆到血红蛋白α亚基的cDNA(Genebank登录号为EU588586)。该cDNA包含435 bp的开放阅读框,编码144个氨基酸。计算其可能的分子量为15864.51,PI为8.83。序列比较分析结果表明,在近端与血红素结合的组氨酸高度保守,与大菱鲆α类珠蛋白同源性最高,达83.3%,与金鲈和大西洋鳕的同源性分别为72.7%和67.8%,与非洲爪蟾α-珠蛋白同源性最低,为2339.4%。通过构建系统进化树,对鲈α-珠蛋白的系统演化进行了分析,并对其二级和三维结构进行了预测。

Separate plaques were randomly selected from cDNA library constructed by head-kidney of Japanese sea bass（Lateolabrax japonicus） and used as templates for PCR amplification of cDNA inserted fragments with T3 and T7 primers. The expression sequence in tags （ESTs） was sequenced and cDNA of the hemoglobin α-type chain was screened from the library. It consists of an open reading frame 435 bp, encoding 144 amino acid residues. The calculated relative molecular mass of the hemoglobin α-type chain was 15864.51, with the PI of 8.83. Sequence alignment analysis revealed that the identity of amimo acid sequence in Japanese sea bass shared with other species varied much,e, g. 83.3% and 39.4% with turbot Scophthalrnus maxirnus and Xenopus laevis, 72.7% and 67.8% with yellow perch Perca flavescens and Atlantic cod Gadus morhua respectively. The author also found that the functionally important residues in the α-type globin were highly conserved, especially the proximal histidine involved in the binding heme and other subunits. In addition, the phylogenetic tree was constructed and the relationships between α- type globin and its homologues were analysized, and the secondary and 3D structure were predicted.