家蚕酪氨酸羟化酶的氨基酸序列分析和重组表达  

Amino Acid Sequence Analysis and Recombinant Expression of Bombyx mori Tyrosine Hydroxylase

作　　者：刘春[1] 周梦婷[1] 谭祥[1] 吕金凤[1] 李栋[1] 聂红毅[1] 夏庆友[1] 

机构地区：[1]家蚕基因组生物学国家重点实验室,西南大学,重庆400715

出　　处：《蚕业科学》2013年第3期467-473,共7页ACTA SERICOLOGICA SINICA

基　　金：国家重点基础研究发展计划“973”项目(No.2012CB1146-00);教育部“新世纪优秀人才支持计划”项目(No.NCET-11-0699);国家自然科学基金青年基金项目(No.31000545);中央高校基本科研业务费专项(No.XDJK200913034)

摘　　要：酪氨酸羟化酶参与昆虫体色和斑纹黑色素合成以及免疫黑化反应等重要生理过程。为了研究家蚕(Bombyx mori)酪氨酸羟化酶的生物学功能,采用生物信息学方法对家蚕与其它物种的酪氨酸羟化酶氨基酸序列进行比对分析,结果显示不同物种之间的酪氨酸羟化酶具有保守性,在昆虫物种之间尤其保守;家蚕酪氨酸羟化酶含有保守的功能位点,如铁原子结合位点His331、His336、Glu376,底物蝶呤结合位点Gly293、Leu294、Leu295、Phe300、Glu332、Tyr371和Glu376。构建重组质粒p29-Bmth,利用原核表达系统实现了家蚕酪氨酸羟化酶蛋白的重组表达,通过高效液相色谱分析证明纯化获得的重组家蚕酪氨酸羟化酶蛋白具有酪氨酸羟化活性,测定其活性为0.81 U,比活性为0.13 U/mg。研究结果为深入研究家蚕酪氨酸羟化酶的生理功能奠定了基础。Tyrosine hydroxylase plays important roles in insect physiological processes such as body coloration,skin melanin synthesis and immune melanization.In order to study biological functions of tyrosine hydroxylase in silkworm（Bombyx mori）,bioinformatic approach was employed to compare and analyze amino acid sequences of tyrosine hydroxylase from silkworm and other species.The results displayed that tyrosine hydroxylase is highly conserved in different species,especially in insect.Silkworm tyrosine hydroxylase has conserved functional sites such as iron binding sites His331,His336 and Glu376,and substrate pterin binding sites Gly293,Leu294,Leu295,Phe300,Glu332,Tyr371 and Glu376.A recombinant plasmid p29-Bmth was constructed and used for recombinant expression of silkworm tyrosine hydroxylase in prokaryotic expression system.Analysis by high-performance liquid chromatography demonstrated that the obtained recombinant silkworm tyrosine hydroxylase had tyrosine hydroxylating activity,with an activity of 0.81 U and a specific activity of 0.13 U/mg.These results established a good basis for further studies on physiological functions of silkworm tyrosine hydroxylase.

关 键 词：家蚕 酪氨酸羟化酶 氨基酸序列 功能位点 原核表达 纯化 酶活性 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]