杜氏盐藻核基质附着区结合蛋白cDNA片段的克隆及序列分析

Cloning and characterization of MAR-binding protein cDNA fragment of Dunaliella salina

目的:克隆杜氏盐藻核基质附着区(MAR)结合蛋白片段。方法:根据多种生物MAR结合蛋白氨基酸的高度保守序列EWYGWHF和KYGLIYQ,设计一对简并引物,以杜氏盐藻的cDNA为模板,进行PCR扩增,PCR产物与T载体相连,转化大肠杆菌JM109后,筛选鉴定,对阳性菌落进行测序分析。将测序结果推导成氨基酸序列,与GenBank上其他物种MAR结合蛋白序列进行同源性分析。结果:在杜氏盐藻中得到的cDNA片段,核苷酸长度为474bp,编码158个氨基酸(GenBank收录号为DQ124215)。推导的氨基酸序列与稻、莲、拟南芥、豌豆、烟草、酵母、果蝇和蟾的MAR结合蛋白相比较,同源性分别为74%、73%、73%、73%、71%、70%、68%和67%。结论:所克隆的序列可能为杜氏盐藻MAR结合蛋白片段。

Aim ： To clone MAR-binding protein cDNA fragment from Dunaliella salina. Methods ：A pair of degenerate primers were designed according to the homologous amino acid conserved sequences of EWYGWHF and KYGLIYQ from known MAR-binding protein and used to amplify MAR-binding protein cDNA fragment from Dunaliella salina by PCR technique. The resulting PCR product was cloned into pMD18-T vector and then transformed into E. coll JM109. Several colonies were selected and screened to determine their sequences. Homologous analysis of the deduced amino acid sequences was performed by BLAST and compared with those of GenBank. Results： An obtained cDNA sequence was 474 bp long,which encoded 158 amino acids（ GenBank accession number：DQ124215）. The sequence shared high homology with other MAR-binding proteins, being Oryza sativa 74% , Lotus corniculatus var. japonicus 73% , Arabidopsis thaliana 73%, Pisum sativum 73%, Nicotiana tabacum 71% , Schizosaccharomyces pombe 70% , Drosophila melanogaster 68% and Xenopus laevis 67%, respectively. Conclusion： The cloned sequence is probably MAR binding protein cDNA fragment from Dunaliella salina.