出芽短梗霉中α-淀粉酶基因的克隆表达及生物信息学分析  

Cloning Expression and Bioinformatics Analysis ofα-amylase Gene from Aureobasidium pullulans

作　　者：刘小胖 张宁[1] 李炳学[2] LIU Xiaopang;ZHANG Ning;LI Bingxue(College of Bioscience and Biotechnoly,Shenyang Agricultural University,Shenyang,Liaoning 110161;College of Land and Environment,Shenyang Agricultural University,Shenyang,Liaoning 110161,China)

机构地区：[1]沈阳农业大学生物科学技术学院,辽宁沈阳110161 [2]沈阳农业大学土地与环境学院,辽宁沈阳110161

出　　处：《贵州农业科学》2019年第1期49-58,共10页Guizhou Agricultural Sciences

基　　金：国家自然科学基金项目(31271818);国家重点研发计划项目(2017YFD0200807-3);辽宁省自然科学基金项目(2015020763);沈阳市重点科技研发计划项目(17-150-3-00)

摘　　要：为获知出芽短梗霉(Aureobasidium pullulans)α-淀粉酶基因及其功能,为以后的体外功能验证奠定理论基础,对α-淀粉酶基因的克隆及其结构、表达、性质与功能进行了初步分析,采用反转录PCR结合普通PCR技术成功克隆出芽短梗霉α-淀粉酶基因,并对其进行生物信息学分析。结果表明:克隆的出芽短梗霉α-淀粉酶基因cDNΑ的全长1 782bp,编码区长度1 692bp,编码的蛋白质含有563个氨基酸,分子质量约为63.32kDa,理论等电点(pI)为7.24;预测该蛋白是亲水性蛋白,无跨膜结构域,无信号肽切割位点,在47～453个氨基酸存在淀粉酶结构域。α-淀粉酶的二级结构主要由无规则卷曲组成,其中无规则卷曲占47.42%,α-螺旋占27.35%,延伸链占20.06%,β-转角占4.62%。成功克隆α-淀粉酶基因并在各个时段均有表达,在108h表达量最高。出芽短梗霉普鲁兰多糖的产量随培养时间增加而增加,而粘度随着培养时间增加而降低。The cloning,structure,expression,character and function ofα-amylase gene from Aureobasidium pullulans are preliminarily analyzed and the bioinformatics of clonedα-amylase gene from A.pullulans by reverse transcription PCR and routine PCR technology was analyzed to learn the structure and function ofα-amylase gene from A.pullulans and lay a theoretical foundation for its function in vitro.Result:Total length of cDNA of clonedα-amylase gene from A.pullulans is 1 782 bp with coding region length of 1 692 bp.The encoded protein contains 563 amino acids and the molecular mass and theoretical isoelectric point are about 63.32 kDa and 7.24 separately.The forecast analysis reveals that the protein is a hydrophily protein without transmembrane domain and signal peptide cleavage sites and there exists 47~453 amino acids in the structural domain ofα-amylase.The secondary structure ofα-amylase is composed of random coil mainly and the random coil,α-helix and extended strand andβ-turn account for 47.42%,27.35%,20.06%and 4.62%respectively.The genetic expression analysis shows that the clonedα-amylase gene can express in each time period and the maximum expression is in 108 h time period.The yield and viscosity of pulullan increase and decrease with increase of culture time separately.

关 键 词：出芽短梗霉 Α-淀粉酶基因 表达 生物信息学 

分 类 号：Q93[生物学—微生物学]