中国水仙DFR基因启动子的克隆及功能  

Cloning and functional analysis of DFR gene promoter in Narcissus tazetta var. chinensis

作　　者：姚红 周平 范雨昕 孙瑞琦 Muhammad Anwar 曾黎辉[1] YAO Hong;ZHOU Ping;FAN yuxin;SUN ruiqi;Muhammad Anwar;ZENG Lihui(College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002)

机构地区：[1]福建农林大学园艺学院

出　　处：《应用与环境生物学报》2019年第4期993-998,共6页Chinese Journal of Applied and Environmental Biology

基　　金：福建农林大学科技创新基金(KFA17352A,KFA17602A);福建农林大学国际科技合作与交流项目(Kxb16013A)资助~~

摘　　要：为了解中国水仙花青素合成途径关键基因DFR(NtDFR)的表达调控以及中国水仙不能合成花青素的分子机制,采用染色体步移法从中国水仙中克隆NtDFR基因起始密码子上游962 bp的启动子序列.生物信息学分析结果表明启动子序列除包含TATA-box、CAAT-box等基本启动子元件外,还包含光调控元件、植物激素响应元件、胁迫响应元件等多个顺式作用元件.此外,该启动子序列还含有MYB转录因子结合位点.为验证启动子的表达特性,将NtDFR启动子取代植物表达载体pBI121上35S启动子,构建pBI121-pNtDFR::GUS载体,利用农杆菌转化烟草叶片瞬时表达,通过GUS组织染色法确定了克隆的启动子的活性.将中国水仙R2R3-MYB转录因子NtMYB2、NtMYB5分别和pBI121-pNtDFR::GUS共同注射烟草,定量PCR和GUS组织化学染色结果表明NtMYB2和NtMYB5都使NtDFR启动子诱导的烟草叶片GUS颜色变浅以及GUS基因表达量下降,表明NtMYB2和NtMYB5是NtDFR的抑制因子.本研究结果有助于了解中国水仙花青素合成途径的分子调控机制.To understand how dihydroflavonol 4-reductase gene (NtDFR) expression is regulated, the key gene in the pathway of anthocyanin biosynthesis in Chinese narcissus (Narcissus tazetta var. chinensis) and the molecular mechanism of no anthocyanin accumulation in Chinese narcissus. The 962 bp promoter fragment of the NtDFR gene was cloned from genomic DNA by the genome walking method. The plant expression vector pBI121 was used to construct the pBI121-pNtDFR::GUS vector. Promoter activity was determined by GUS histochemical staining with agro-infiltrated tobacco leaves. The results of promoter sequence analysis showed that the promoter contains a number of TATA-box, CAAT-box, and other promoter elements, such as light responsive elements, hormone response elements, and stress-response elements. Moreover, the promoter also contains the MYB transcription factor combination elements. In addition, NtMYB2 and NtMYB5, R2R3-MYB genes of Chinese narcissus, were transiently expressed with pBI121-pNtDFR::GUS in tobacco leaves separately. The results of real-time quantitative PCR and GUS histochemical staining analysis indicated that NtMYB2 and NtMYB5 repress the activity of the NtDFR promoter. Thus, NtMYB2 and NtMYB5 are the repressors of NtDFR.

关 键 词：中国水仙 二氢黄酮醇4-还原酶基因 启动子 功能鉴定 

分 类 号：S682.21[农业科学—观赏园艺]