摘要
为克隆得到毛白杨CPR基因、成功在体外表达可溶性蛋白并通过构建GFP偶联载体观察其亚细胞定位,本研究通过RT-PCR的方法克隆得到了毛白杨CPR基因,登录号为MK575137。进一步通过生物信息学分析,发现毛白杨CPR与毛果杨CPR同源性最高,均有着典型的细胞色素C还原酶结构域。随后在酵母表达体系中表达该蛋白,验证了该蛋白不能被分泌表达,表明CPR蛋白为膜蛋白。进一步的亚细胞定位实验表明,CPR蛋白定位于内质网。CPR蛋白是P450单加氧化酶催化木质素合成的相关过程中必不可少的辅酶,该研究可为今后体外验证毛白杨P450单加氧化酶在木质素合成的相关过程中的酶活活性提供基础。
In order to study the expression and subcellular localization of PtoCPR,we cloned the CPR gene from Populus tomentosa by RT-PCR,and the accession number was MK575137.Further bioinformatics analysis showed that the PtoCPR had the highest homology with Populus trichocarpa CPR,and had a typical cytochrome C reductase domain.Successfully expressed the protein in yeast expression system,which verified that the protein could not be secreted and expressed,indicating that the protein was a membrane protein.Subcellular localization experiments showed that CPR protein was localized in endoplasmic reticulum.CPR protein is a necessary coenzyme in the process of lignin synthesis catalyzed by P450 monooxygenase.This study provides the necessary basis for further in vitro verification of P450 monooxygenase activity related to lignin synthesis in Populus tomentosa.
作者
曾琪琦
蒋湘宁
盖颖
Zeng Qiqi;Jiang Xiangning;Gai Ying(The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of Chinese Forestry Administration,College of Biological Sciences and Biotechnology,Beijing Forestry University,Beijing,100083)
出处
《分子植物育种》
CAS
CSCD
北大核心
2020年第8期2423-2430,共8页
Molecular Plant Breeding
基金
国家自然科学基金(31300498)资助。
