金龙胆草油菜素内酯合成途径C-23羟化酶基因的克隆及分析  

Cloning and Analysis of C-23 Hydroxylase Gene of Brassinosteroid Synthesis Pathway from Conyza blinii H.Lév.

作　　者：孙蓉 高静雷 刘姗[1] 刁毅[1] Sun Rong;Gao Jinglei;Liu Shan;Diao Yi(College of Biological and Chemical Engineering,Panzhihua University,Panzhihua,617000)

机构地区：[1]攀枝花学院生物与化学工程学院,攀枝花617000

出　　处：《基因组学与应用生物学》2020年第11期5214-5221,共8页Genomics and Applied Biology

基　　金：攀枝花学院博士科研启动基因项目(035200167)资助。

摘　　要：油菜素内酯具有促进植物生长、增加产量和提高植物抗逆性等作用。本研究以中草药金龙胆草(Conyza blinii)为研究对象,克隆分析其油菜素内酯合成途径C-23羟化酶(CYP90C1/ROT3)基因。根据金龙胆草叶片转录组数据,克隆获得了一条全长1512 bp的ROT3基因cDNA序列,编码504个氨基酸,命名为CbROT3,GenBank登录号为KX907782;通过生物信息学工具分析其分子特征表明CbROT3氨基酸序列与向日葵(Helianthus annuus)ROT3的相似性达87%,系统进化树分析结果也与之相吻合;该基因蛋白相对分子质量为57.51 kD,等电点为7.59,为亲水性蛋白,具有一个跨膜结构,无信号肽序列;摸索其原核表达条件,发现在22℃、0.6 mmol/L异丙基-β-D-硫代半乳糖苷(isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导条件下可获得最大量的可溶性目的蛋白。本研究结果对提高金龙胆草内源油菜素内酯含量,调控金龙胆草生长发育和增加金龙胆草抗逆性提供理论依据。Brassinosteroid(BR)plays an important role in promoting plant growth,increasing yield and enhancing plant stress resistance.In this study we cloned and characterized a C-23 hydroxylase(CYP90 C1/ROT3)gene of BR pathway from Conyza blinii.A 1512 bp length ROT3 gene was cloned according the sequence isolated from C.blinii transcript tags.It was designated CbROT3 and encoded 504 amino acids(aa).Its GenBank accession number was KX907782.The bioinformatics tools were used to analyze its molecular characteristics.The results indicated that the CbROT3 has high identities(87%)with ROT3 from Helianthus annuus,and the result of phylogenetic analysis was consistent with above.The protein had a calculated molecular mass of 57.51 kD and an isoelectric point of 7.59.It was a hydrophilic protein and contained one transmembrane domain but no signal peptide.We optimized its prokaryotic expression condition.The result showed that the temperature of 22℃and the isopropyl-beta-Dthiogalactopyranoside(IPTG)concentration of 0.6 mmol/L were the optimal condition.The results of this study provided theoretical basis to the increasing of endogenous BR content,regulating of C.blinii growth and development and improving of C.blinii stress resistance.

关 键 词：金龙胆草(Conyza blinii H.Lév) 油菜素内酯 ROT3基因 克隆 

分 类 号：S567.2[农业科学—中草药栽培]