番茄根特异表达基因LeGRP2启动子的克隆及其在拟南芥的表达分析  

Cloning of LeGRP2 Promoter from Tomato that Shows Root-Specific Expression in Arabidopsis

作　　者：李志邈[1] 杨悦俭[1] 杨飞[1,2] 叶青静[1] 王荣青[1] 阮美颖[1] 周国治[1] 姚祝平[1] Vong-Ling Ruan 

机构地区：[1]浙江省农业科学院蔬菜研究所/中澳作物改良研究中心,中国杭州310021 [2]浙江师范大学化学与生命科学学院,中国金华321004 [3]School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia

出　　处：《中国农业科学》2010年第9期1877-1882,共6页Scientia Agricultura Sinica

基　　金：浙江省自然科学基金项目(Y305280)

摘　　要：目的克隆获得番茄根特异表达启动子,为利用基因工程技术创制番茄新种质奠定基础。方法利用Clontech公司的基因组步移(genome walking)技术,扩增番茄根特异表达基因LeGRP2的上游调控序列,并构建植物表达载体,利用农杆菌介导法转化拟南芥,以GUS为报告基因研究该调控序列的组织表达特异性。结果以番茄基因组DNA为模板,经过2次基因组步移,获得了LeGRP2基因上游1959bp的调控序列(GenBank登录号:EU262719),分析发现含有9个与根特异表达相关的顺式作用元件ROOTMOTIFTAPOX1。转基因拟南芥的组织化学染色分析表明,GUS基因主要在拟南芥的根部特异表达。结论克隆获得了番茄LeGRP2基因启动子,该启动子主要在转基因拟南芥根部表达GUS基因,具有较强的根表达特异性。Objective The objective of the study was to clone a root-specific promoter from tomato for future use in generating new tomato materials through genetic engineering. Method Genomic walking technique was used to amplify the upstream regulatory sequence of LeGRP2 （glycine-rich protein）, a tomato root-specific expression gene. To investigate the tissue expression pattern of the cloned regulatory sequence, an expression vector containing this sequence fused with GUS was constructed for transformation into Arabidopsis by using agrobacterium-mediated method. Result Using tomato genomic DNA as a template, a regulatory sequence （GenBank accession number： EU262719） 1 959 bp upstream of the LeGRP2 ATG site was amplified by two consecutive genomic walking steps. Sequence analyses revealed that it contained 9 copies of ROOTMOTIFTAPOX1, a known cis-acting element related to root-specific expression. Histochemical staining of transgenic Arabidopsis showed that GUS reporter gene was predominantly expressed in root in both 7 d seedlings and 40 d adult plants. Conclusion The LeGRP2 promoter was cloned, which displayed a strong root-specific expression pattern in Arabidopsis transformed with LeGRP2 promoter fused with gene GUS.

关 键 词：番茄 根特异启动子 基因组步移 组织表达特异性 

分 类 号：S641.2[农业科学—蔬菜学]