马铃薯α-淀粉酶基因的克隆及生物信息学分析  

Cloning and Sequence Analysis of Potato α-Amylase Gene

作　　者：王永刚[1] 马建忠[1] 马雪青[1] 周贤婧[1] 李昆鹏[1] 赵虎彪[1] 

机构地区：[1]兰州理工大学生命科学与工程学院,甘肃兰州730050

出　　处：《食品科学》2010年第19期216-220,共5页Food Science

基　　金：兰州理工大学博士启动基金项目;兰州市科技局工业科技攻关及产业化项目(07-02-44)

摘　　要：利用RT-PCR方法从马铃薯(Solanum tuberosum)茎段总RNA中扩增、克隆amyA1基因(NCBI登录号GQ406048.1)。采用半定量RT-PCR方法检测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amyA1密码子的偏好性;同时对AmyA1氨基酸的理化性质、细胞内定位、保守结构及高级结构进行预测。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。amyA1基因全长1224bp,可编码一条理论分子质量为46.40kD、407个氨基酸残基组成的、可能为亲水性的胞外酶。与NCBI已登记的马铃薯α-淀粉酶基因(登录号M79328.1)核苷酸及氨基酸序列同源性达98%。第20～348位点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349～407位点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桶状结构以及其他几个功能域结构。所构建的基因进化树表明,两个马铃薯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、玉米等单子叶植物的序列同源性较低。A cDNA for potato α-amylase was amplified by RT-PCR and then cloned. The sequence analysis showed that the cDNA had a 1224bp-ORF,which encodes an α-amylase with 407 amino acid residues （GenBank accession number：GQ406048.1）. Semi-quantitative RT-PCR assay was used to detect the expression of the α-amylase genes in potato leaves and stems,and the results showed that the expression in stems was a little stronger than that in leaves. The amino acid sequence was bioinformatically analyzed,including its codon usage bias,physical and chemical properties,subcellular localization and con-served structures. Totally 29 α-amylase genes from the 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 （M79328.1） at the amino acid level. The deducted α-amylase also contained a catalytic domain （PF00128 and SM00624） between sites 20 and 348 and a C-terminal beta-sheet domain between sites 349 and 407,which were similar to their counterparts in the amylase family 13. The postulated eight-stranded alpha/beta barrel was also found in the enzyme,which is thought as an active site of α-amylase. According to the constructed phylogenetic tree,the two genes from potato presented closer homology to those from cassava and apple,followed by kidney bean,but distant homology to those from the monocotyledon-ous plants including barley,rice and maize.

关 键 词：马铃薯 Α-淀粉酶基因 克隆 生物信息学 

分 类 号：Q785[生物学—分子生物学]