马铃薯α-淀粉酶基因的克隆及生物信息学分析(英文)

Cloning and Bioinformatic Analysis of Potato α-Amylase Gene amyA1

本研究利用RT-PCR从马铃薯(Solanum tuberosum)茎段总RNA中扩增、克隆了一cDNA分子。该cDNA分子含有一长为1224bp的开放读框,可编码一含407个氨基酸残基的多肽、理论分子量为46.40kD、可能为亲水性的胞外酶。因其氨基酸序列同源于α-淀粉酶,故将该基因命名为amyA1(NCBI收录号:GQ406048.1)。采用半定量RT-PCR方法检测了amyA1基因在马铃薯茎、叶等不同组织中的表达强度,表明在茎组织中的表达丰度略高。利用生物信息学软件分析了amyA1密码子的偏好性,以期为选择适宜的表达系统提供依据;同时对amyA1的理化性质、细胞内定位、保守结构及高级结构进行了预测。基于NCBI数据库中有物种代表性的29种α-淀粉酶基因序列构建了基因进化树。与NCBI收录的马铃薯α-淀粉酶基因(NCBI收录号:M79328.1)的核苷酸及氨基酸序列同源性达98%。第20至第348范围内的氨基酸残基含有与淀粉酶13家族及亚家族相似的催化活性域(PF00128、SM00624),第349至第407范围内的氨基酸残基含有α-淀粉酶C-末端β折叠区域(PF07821)。蛋白质结构预测表明氨基酸残基序列有维持淀粉酶活性的(β/α)8桶状结构以及其它几个功能域结构。所构建的基因进化树表明,2个马铃薯α-淀粉酶基因与木薯、苹果的序列同源性较高,与菜豆的次之,与水稻、大麦和玉米等单子叶植物的序列同源性较低。

In this research, we amplified a cDNA for potato α-amylase by RT-PCR and cloned it. Sequence analysis showed that the cDNA had a 1 224 bp open reading frame and was referred to as amyA1, which encodes for an α-amylase with 407 amino acid residues （GenBank accession number： GQ406048.1） with the MW 46.40 kD. After that we used semi-quantitative RT-PCR assay to detective the expression of the amyA1 gene in potato leaves and stems. The result showed that the expression in stems is a little stronger than in leaves. Then we analyzed the amino acid sequence bioinformatically, including its codon usage bias, physical and chemical properties, subcellular localization, and conserved structures. 29 α-amylase genes from same or different species were taken from the GenBank for constructing a phylogenetic tree. The bioinformatical analyses showed that the putative protein shared 98% identity with a published potato α-amylase （GenBank accession number： M79328.1） at the amino acid level. The deducted α-amylase also contains a catalytic domain （PF00128、SM00624） between 20 to 348 and a C-terminal beta-sheet domain （PF07821）between 349~407, which are similar to ones of the amylase family 13. The postulated eight-stranded alpha/beta barrel was also found in the enzyme, which was thought as an active site of α-amylase. According to the phylogenetic tree, the two genes from potato presents more close homology to those from cassava and apple than from barley, rice and maize.