一种松材线虫醛脱氢酶的基因克隆及其生化性质  

Gene Cloning of an Aldehyde Dehydrogenase from Bursaphelenchus xylophilus and Biochemical Characterization

作　　者：李文硕 王林松 杜桂彩 郭群群 张廷婷 杨宏 李荣贵 LI Wen-shuo;WANG Lin-song;DU Gui-cai;GUO Qun-qun;ZHANG Ting-ting;YANG Hong;LI Rong-gui(College of Life Sciences,Qingdao University,Qingdao 266071)

机构地区：[1]青岛大学生命科学学院,青岛266071

出　　处：《生物技术通报》2022年第9期207-214,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31901313);山东省自然科学基金项目(ZR2020MC123)。

摘　　要：松材线虫(Bursaphelenchus xylophilus)是松萎蔫病的病原,可引起数十种松属树种的毁灭性破坏。通过RT-PCR 成功克隆了松材线虫体内一种醛脱氢酶基因aldh,aldh的长度为1 353 bp,编码451 个氨基酸残基,前期研究表明,杀线剂甲吡唑处理能使aldh表达水平显著上调。序列比对表明,与大多数其他醛脱氢酶中依靠半胱氨酸(Cys)残基发挥催化功能不同,aldh 编码的蛋白质保守的位置是亮氨酸(Leu)残基,但其C-端紧邻Leu残基的是组氨酸残基(His),而不是常见的Leu残基。构建了表达载体pET-15b-aldh并转化大肠杆菌BL21(DE3),IPTG诱导aldh在工程菌中实现了高表达,重组醛脱氢酶通过Ni-NTA亲和层析纯化后进行表征。以甲醛为底物的醛脱氢酶Km为27.87 mmol/L,最适pH值为7.5,最适温度为25℃,Fe^(3+)和Ni^(2+)可提高酶活性,Ca^(2+)、Mn^(2+)、Na^(+)和K^(+)可降低酶活性。重组醛脱氢酶对5种醛类化合物显示出不同的催化活性,其中香草醛是其最佳底物。该研究为深入了解醛脱氢酶在松材线虫致病过程中的作用打下了基础,也为新型工业用醛脱氢酶的研究提供了参考。Pine wood nematode(PWN),Bursaphelenchus xylophilus,is a causal pathogen of pine wilt disease(PWD)that is devastating to dozens of pine species.An gene aldh of aldehyde dehydrogenase from PWNs was successfully cloned through RT-PCR,its length was 1 353 bp and encoded a protein containing 451 amino acids residues.Prior studies demonstrated a nematicide fomepizole resulted in the up-regulation of aldh expression.Sequences alignment showed that the conserved catalytic position was Leu residue,which was different to Cys residue in most other aldehyde dehydrogenases,and its adjacent residue at C-terminal was His rather than the common nonpolar amino acid residue such as Leu.The aldh-encoding aldehyde dehydrogenase was over expressed in E.coli BL21(DE3)transformed with pET-15b-aldh via IPTG induction,and the recombinant aldehyde dehydrogenase was purified by Ni-NTA affinity chromatography.The Km of the aldehyde dehydrogenase with formaldehye as substrate was 27.87 mmol/L,the optimal pH value and temperature was 7.5 and 25℃,respectively.The enzyme activities were enhanced by Fe^(3+)and Ni^(2+),while inhibited by Ca^(2+),Mn^(2+),Na^(+)and K^(+).The recombinant aldehyde dehydrogenase showed different catalytic activities towards 5 aldehydes,and vanillin was found to be its optimal substrate in the 5 tested aldehydes.This study lays a foundation for investigating the roles of aldehyde dehydrogenase in PWD and offers a clue to explore aldehyde dehydrogenases utilized in industry.

关 键 词：松材线虫 醛脱氢酶 纯化 性质 

分 类 号：S763.7[农业科学—森林保护学]