钩藤UrIO基因及其启动子的克隆与分析  

Cloning and Analysis of the UrIO Gene and Its Promoter in Uncaria rhynchophylla

作　　者：郑浩杰 王晓红[1] 李永权 李雪[1] 上官黎阳 田宇航 张明生[1] ZHENG Hao-Jie;WANG Xiao-Hong;LI Yong-Quan;LI Xue;SHANGGUAN Li-Yang;TIAN Yu-Hang;ZHANG Ming-Sheng(College of Life Sciences/Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education),Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学生命科学学院/山地植物资源保护与种质创新教育部重点实验室,贵阳550025

出　　处：《农业生物技术学报》2023年第4期741-753,共13页Journal of Agricultural Biotechnology

基　　金：贵州省生物学一流学科建设项目(GNYL[2017]009FX1KT02);贵州省科技计划重大专项(黔科合平台人才[2017]5411-06);国家喀斯特石漠化防治工程技术研究中心建设项目(2012FU125X13);贵州省中药材现代产业技术体系建设项目(GZCYTX-02)。

摘　　要：环烯醚萜氧化酶(iridodial oxidase,IO)属于CYP76家族A26亚家族,是萜类吲哚生物碱上游合成途径中环烯醚萜途径的第4步限速酶,能催化环烯醚萜生成7脱氧马钱子酸。本研究基于转录组数据注释,克隆得到钩藤(Uncaria rhynchophylla)UrIO基因(GenBank No.ON227515),编码516个氨基酸;系统进化关系显示,钩藤UrIO与金鸡纳树(Cinchona calisaya)CcIO亲缘关系最近,其次是长春花(Catharanthus roseus)CrIO;多序列比对分析表明,钩藤UrIO具有细胞色素P450(cytochrome P450 proteins,CYP)家族的3个典型结构域。组织表达谱显示,UrIO在钩藤的茎中表达水平最高,其次是果实中;并且在外源茉莉酸甲酯(methyl jasmonate,MeJA)处理下显著上升(P<0.05),不受外源脱落酸(abscisic acid,ABA)诱导;利用融合引物与巢式整合PCR(fusion primer and nested integrated PCR,FPNI-PCR)技术克隆UrIO的启动子;植物顺式调节元件(plant cis-acting regulatory elements,PlantCARE)分析显示,该启动子包含胁迫响应和激素响应等元件,瞬时烟草(Nicotiana tabacum)转化实验表明,该启动子能驱动下游GUS基因表达,进一步的酵母单杂交(yeast one hybrid,Y1H)证实了UrMYC2和UrIO启动子互作。本研究为进一步解析钩藤萜类吲哚生物碱合成途径提供参考。Iridoid oxidase(IO)belonging to the CYP76 family A26 subfamily and is the step 4 rate-limiting enzyme of the iridodial pathway in the upstream synthetic pathway of terpenoid indole alkaloids,catalyzing the synthesis of 7-deoxyloganetic acid from iridoid.In this study,UrIO(GenBank No.ON227515)was cloned based from Uncaria rhynchophylla transcriptome data,which encoded 516 amino acids.Phylogenetic relationship showed that the relationship between UrIO of U.rhynchophylla and CcIO of Cinchona calisaya was the closest,followed by CrIO of Catharanthus roseus.Multiple sequence alignment analysis showed that UrIO had 3 typical domains of cytochrome P450 proteins(CYP)family.The expression level of UrIO in stem was the highest,followed by fruit,and the expression of UrIO was significantly increased under methyl jasmonate(MeJA)treatment.However,it was not induced by abscisic acid(ABA).The promoter of UrIO was cloned by fusion primer and nested integrated PCR(FPNI-PCR)method as well,which contained stress response and hormone response elements by plant cis-acting regulatory elements(PlantCARE)analysis.Transient transformation experiments in tobacco(Nicotiana tabacum)showed that the promoter could drive GUS reporter gene expression.Furthermore,yeast one hybrid(Y1H)confirmed the interaction between UrMYC2 and UrIO promoter.This study provides a reference for further analysis of the synthetic pathway of indole alkaloids from U.rhynchophylla.

关 键 词：钩藤 环烯醚萜氧化酶 启动子 酵母单杂交 

分 类 号：S2[农业科学—农业工程] Q78[生物学—分子生物学]