欧李ChCCD4基因的克隆与原核表达  

Cloning and Prokaryotic Expression of CCD1 Gene in Cerasus humilis

作　　者：张建成[1] 高利敏 王鹏飞[1] 穆霄鹏[1] 王裕添 杜灵敏 宣晓毛 杜俊杰[1] Zhang Jiancheng;Gao Limin;Wang Pengfei;Mu Xiaopeng;Wang Yutian;Du Lingmin;Xuan Xiaomao;Du Junjie(College of Horticulture,Shar,xi Agricultural University,Taigu,030801)

机构地区：[1]山西农业大学园艺学院,太谷030801

出　　处：《分子植物育种》2018年第18期5914-5919,共6页Molecular Plant Breeding

基　　金：山西省重点研发计划项目(201703D221028-4);高等学校博士学科点专项科研基金项目(20101403120006);山西省自然科学基金项目(2012011032-4);山西省科技重大专项计划项目(20121101010)共同资助

摘　　要：类胡萝卜素裂解双加氧酶(CCDs)是类胡萝卜素氧化裂解的重要酶之一,在植物中,CCDs催化裂解产物具有重要的作用。本研究以欧李品种‘农大4号’果实为试验材料,根据转录组测序数据分析,利用RT-PCR从中克隆了到了ChCCD4基因。生物信息分析发现,该序列长度1 794 bp,编码597个氨基酸的蛋白,分子量为65 717.90 Da,等电点为6.10,蛋白质二级结构主要由α-螺旋,β-折叠,随机卷须和延伸链构成,ChCCD4蛋白三级结构与CCD蛋白家族三级结构相似,具有相同的结构域。将ChCCD4基因连接到原核表达载体pet-28a,转化大肠杆菌BL21 (DE3),诱导表达,目的蛋白经过SDS-PAGE凝胶电泳检测,与空白对比,检测出了目的蛋白条带。本研究为ChCCD4基因的功能研究提供理论依据,为欧李育种方向提供了理论参考。Carotenoid cleavage dioxygenase（CCDs） is one of the important enzymes in carotenoids oxidative cleavage. In plant, CCDs catalytic pyrolysis product plays an important role. In this study, the fruit of Cerasus humilis variety ＇Nongda 4＇ was selected as experiment material, and ChCCD4 gene was cloned by RT-PCR based on the analysis of the transcriptome sequencing data. Biological information analysis found that this sequence was1 794 bp in length, encoding 597 amino acid protein. Its molecular weight was 65 717.90 Da, isoelectric point was6.10, and the protein secondary structure was mainly composed of alpha helix, beta turn, extended strand and random coil. The tertiary structure of ChCCD4 protein was similar to the tertiary structure of CCD protein family,which had the same structural domain. The ChCCD4 gene was connected to the prokaryotic expression vector pET-28 a and transformed into Escherichia coli BL21（DE3） to induce expression. After the detection of SDS-PAGE gel electrophoresis, bands were identified in target protein. This study laid the theoretical foundation for the functional study of ChCCD4 gene, which could provide reference for the breeding of Cerasus humilis.

关 键 词：欧李 ChCCD4基因克隆 生物信息分析 原核表达 

分 类 号：S662.5[农业科学—果树学]