水稻arf1基因3′-UTR片段的克隆和验证  

Cloning and validation of 3′-UTR fragments of rice arf1 gene

作　　者：苏军[1] 管其龙 陈子强[1] 陈在杰[1] SU Jun;GUAN Qilong;CHEN Ziqian;CHEN Zaijie(Fujian Provincial Key Laboratory of Genetic Engineering for Agriculture, Biotechnology Institute of Fujian Academy of Agricultural Sciences, Fuzhou 350003, China)

机构地区：[1]福建省农业科学院生物技术研究所,福建省农业遗传工程重点实验室,福州350003

出　　处：《应用与环境生物学报》2019年第2期414-419,共6页Chinese Journal of Applied and Environmental Biology

基　　金：福建省科技重大专项(2016NZ0001-2);省属公益类科研院所科研专项(2018R1019-1)资助~~

摘　　要：终止子是一段位于基因编码之后的序列,作为一种调控信号调控DNA的转录终止和RNA的释放,在基因工程技术应用中通常被构建在目的基因下游,用以调控基因的转录和表达.现有常用的终止子很少,克隆和验证新的终止子是植物基因工程技术发展的需要.通过生物信息学分析和Realtime-PCR表达验证,筛选出候选基因腺苷酸核糖基化作用因子(Similar to ADP-ribosylation factor1,arf1)基因,克隆了水稻arf1基因3′-UTR.结果显示,所克隆3′-UTR片段含有8个多聚信号元件和4个UE片段.将3′-UTR与gus基因融合后分别与玉米泛素启动子Ubiquitin和花椰菜花叶病毒(CaMV)启动子35S连接构建植物表达载体验证3′-UTR调控表达效果.烟草瞬时表达显示3′-UTR融合的gus基因在35S和Ubi启动子驱动下,在烟草叶片中能正常表达;3′-UTR调控下的gus基因在水稻根、茎、叶、花和种子中均可稳定表达,且表达量与T-Nos终止子相当.本研究表明所克隆的3′-UTR可替代T-nos终止子,可为植物基因工程技术的应用提供新的调控元件.(图5表3参29)A terminator is a sequence that is usually located at the end of a gene, and is a regulatory signal to block the transcription of DNA to RNA and release the transcript. At present, there are limited commonly used terminators;therefore, the cloning and validation of new terminators is required to develop genetically modified crops. Here, we cloned a 3′-UTR sequence of a candidate gene similar to ADP-ribosylation factor, arf1, and analyzed the control element of the sequence. The sequence of arf1 3′-UTR contained eight polyA sites and four U-rich elements. To confirm that the 3′-UTR of the arf1 gene is an important component for the regulation of transcript stability, two reporter gene constructs were made by cloning the arf1 gene 3′-UTR downstream of the bacterial b-glucuronidase gene (uidA) under the control of the CaMV 35S and Ubi promoters. An agrobacterium-mediated transient gene expression test showed that the 3′-UTR–fusion gus gene driven by the 35S or Ubi promoters can be normally expressed in tobacco leaves. The gus gene expression regulated by 3′-UTR was stable in the rice roots, stems, leaves, flowers, and seeds, and the expression level was comparable to that of T-Nos. The study showed that the clone 3′-UTR can replace the T-Nos terminator, and the results provide new regulatory elements for the application of plant genetic engineering technology.

关 键 词：水稻 3′-UTR 终止子 克隆 表达 

分 类 号：Q943.2[生物学—植物学]