长春花茉莉酸受体CrCOI1的生物信息学分析与原核表达  

Bioinformatics Analysis and Prokaryotic Expression of Jasmonic Acid Receptor CrCOI1 from Catharanthus roseus

作　　者：苗琪 崔馨文 周雅然 于放[1] 王燕燕[1] Miao Qi;Cui Xinwen;Zhou Yaran;Yu Fang;Wang Yanyan(School of Biological Engineering,Dalian Polytechnic University,Dalian,116034)

机构地区：[1]大连工业大学生物工程学院,大连116034

出　　处：《分子植物育种》2023年第3期747-753,共7页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31970324);辽宁省教育厅基础研究项目(J2020042)共同资助。

摘　　要：通过生物信息学方法对长春花茉莉酸受体CrCOI1氨基酸序列、结构域以及二级、三级结构进行分析,利用分子生物学方法构建重组质粒并进行原核表达。结果显示:CrCOI1编码516个氨基酸,为胞质定位,分子量为58.26 k D,等电点pI为5.45,与番薯COI1蛋白的亲缘关系比较近,CrCOI1含有F-box结构域、多个LRR富集亮氨酸重复结构域以及转运抑制响应1结构域;该蛋白二级结构由50.97%α-螺旋、31.59%无规则卷曲、12.79%延伸链以及少部分β-转角(4.65%)组成;经SWISS-MODEL预测其三级结构显示,CrCOI1蛋白由α-螺旋和无规则卷曲组成,其中F-box的3个α-螺旋从蛋白的N端伸出便于结合配体。进一步地成功构建了重组表达质粒pET28a-CrCOI1,并经IPTG诱导后在大肠杆菌BL21中实现异源表达,且发现经16℃、0.4 mmol/L IPTG诱导至12 h后蛋白表达量最高,具有时间依赖性。上述结果表明,我们成功构建了长春花茉莉酸受体CrCOI1的重组表达质粒,实现大肠杆菌中的表达,并对其进行了序列以及结构分析。这为研究CrCOI1的功能以及通过其介导茉莉酸信号调控植物生长发育以及代谢产物合成提供一定依据。The amino acid sequence,domain and secondary and tertiary structure of jasmonic acid receptor CrCOI1were analyzed using the bioinformatics method.The recombinant plasmid was constructed using the molecular biology method,and the prokaryotic expression was performed.The results demonstrated that the CrCOI1 encodes516 amino acids,molecular weight is 58.26 kD,and the isoelectric point pI is 5.45.Phylogenetic tree analysis revealed that it was closely related to COI1 from Ipomoea batatas.The conserved domain analysis indicated that CrCOI1 contained an F-box domain,a transport-inhibition response 1,and multiple LRR-enriched leucine repeat domains.The secondary structure of the protein was composed of 50.97%α-helix,31.59%random coil,12.79%elongated chain,and a small part ofβ-turn(4.65%).The results of tertiary structure prediction suggested that CrCOI1 protein was surrounded by a helix and random coil on the outside and presented a pocket-like middle.The threeα-helices of F-box stretched out at the N segment,making it convenient for binding ligand or other proteins.The recombinant expression plasmid pET28a-CrCOI1 was successfully constructed and heterologously expressed in Escherichia coli BL21 after induced by IPTG.It was discovered that the protein expression level after induction at 16℃and 0.4 mmol/L IPTG was performed for 12 h was higher,and the expression level was time-dependent.These results demonstrated that prokaryotic expression of the receptor CrCOI1 was realized and to achieve the expression in E.coli,and the sequence and structure analysis were carried out.This study provides a basis for studying the function of CrCOI1 and regulating plant growth and development and metabolite synthesis through its mediated jasmonic acid signaling.

关 键 词：长春花(Catharanthus roseus) 茉莉酸受体COI1 序列分析 结构预测 原核表达 

分 类 号：S682.19[农业科学—观赏园艺]