气单胞菌低温淀粉酶基因的克隆及生物信息学分析

Gene cloning and bioinformatics analysis of the cold-adapted amylase from Aeromonas sp.

【目的】获得低温菌LA77的低温淀粉酶基因及其生物学信息。【方法】用16S r DNA序列分析对其进行了分子鉴定,并根据分类地位进行了特异引物设计和PCR扩增,通过生物学网站和软件对获得的基因及编码蛋白进行分析。【结果】低温菌LA77归属于气单胞菌属,PCR扩增获得一条编码703个氨基酸的完整开放阅读框的基因。通过生物信息学分析显示该蛋白质理论分子量约为77.9 k D,等电点为6.16,为亲水、且具有信号肽的跨膜蛋白。可能存在10个磷酸化位点,1个糖基化位点,二级结构主要由α螺旋和随机卷曲构成。基于氨基酸序列的结构域和系统进化树分析表明,该蛋白属于α-淀粉酶家族,极可能为低温淀粉酶。【结论】LA77低温淀粉酶基因的获得,将为低温淀粉酶基因的进一步改造、表达以及其酶低温催化机理的阐释奠定重要的基础。

[ Objective]To get cold- adapted amylase gene from a cold- adapted bacterium and the co- ded protein bioinformatics. [Method]The strain LA77 was identified by 16S rDNA sequence analysis, and then specific primers were designed and the gene was amplified by PCR. Furthermore, the gene and coded protein were analyzed on the website or by the software. [ Result] The strain LA77 belonged to genus Aeromonas, and the sequence of PCR products included an open reading frame which was coded for a protein with 703 amino acid residues. Bioinformatics analysis indicated that molecular weight and isoeleetrie point of the protein were 77.9 kD and 6. 16, respectively. It was a hydrophilie protein with a signal peptide, which contained eight phosphorylation sites and one N - glyeosylation site. The secondary structure was mainly constitu- ted by the α - helix and random coil. The analysis of domains and phylogenetic tree according to amino acid sequence revealed the protein belonged to the family of α- amylase, and the gene would code for cold - adap- ted amylase. [ Conclusion ] The gene would help to explain cold adaption mechanism of the amylase and further research on genetic modification and expression.