毛果杨漆酶基因LAC-1的克隆与原核表达研究

Cloning and Prokaryotic Expression of LAC-1 Gene from Populus trichocarpa Torr. & Gray

目的:克隆毛果杨漆酶基因LAC-1,对其进行生物信息学分析,并构建原核表达载体在大肠杆菌中进行融合蛋白的诱导表达并纯化目的蛋白,为植物漆酶基因蛋白体外结构与功能的研究提供一定的理论基础。方法:根据同源克隆的原理,利用已经研究公布的拟南芥中的漆酶基因序列对毛果杨基因组数据库进行同源检索,利用PCR技术克隆毛果杨漆酶基因的序列,构建重组表达载体,转化大肠杆菌BL21并表达重组蛋白,通过Ni-NTA亲和层析进行纯化。结果:克隆获得了毛果杨漆酶基因的序列(命名LAC-1,基因登录号XP_002310245),序列分析表明LAC-1具有漆酶基因的保守结构域,进化树分析表明该序列与拟南芥的漆酶基因AtLAC4有较高的同源性,利用所构建的原核表达载体,经IPTG诱导和SDS-PAGE电泳检测结果表明表达蛋白与预期蛋白大小一致。结论:LAC-1属于毛果杨基因家族的一员,该漆酶基因可以在体外成功进行原核表达,为毛果杨漆酶基因家族成员的鉴定及后续活性功能的分析提供了一定的理论基础。

Objective: Cloning, sequence analysis of the LAC-1 gene from Populus trichocarpa Torr. &Gray, then induced expression and purify of the fusion protein in E. coli after construct prokaryotic expression vector to provide a foundation for studying the function of the target protein. Method: According to the principle of homologous cloning, laccase gene from Arabidopsis thaliana was used to blast the database JGI of Populus trichocarpa. The laccase gene was isolated by PCR and transformed into E. coli by the individual expression construct pET-30a and expression the recombination protein, then purified by Ni-NTA affinity chromatography. Result: Populus trichocarpa laccase gene was isolated (renamed LAC-1, Genebank: XP_002310245). Seqence analysis revealed that LAC-1 had the key residues of laccase, phylogenetic analysis showed LAC-1 had high homologous with AtLAC4. The results of SDS-PAGE demonstrated that the expressed proteins were consistent with the size of expected protein in the prokaryotic expression system. Conclusion: The LAC-1 genes belonged to the family of laccase and successfully expressed in E. coli. This study will supply theoretical foundation in identifying and functional analysis of members from laccase family.