耐盐小麦中TaSC基因启动子的克隆及调控功能分析  

Cloning and Regulation Function Analysis of Ta SC Promoter from Salt Tolerant Wheat

作　　者：焦博 柏峰[1] 李艳艳 路佳[1] 张肖 曹艺茹 葛荣朝[1] 赵宝存[1] JIAO Bo;BAI Feng;LI Yan-Yan;LU Jia;ZHANG Xiao;CAO Yi-Ru;GE Rong-Chao;ZHAO Bao-Cun(College of Life Science,Hebei Normal University,Shijiazhuang 050024,Hebei,China)

机构地区：[1]河北师范大学生命科学学院,河北石家庄050024

出　　处：《作物学报》2018年第4期620-626,共7页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(30871471);河北省自然科学基金项目(C2011205085)资助~~

摘　　要：高盐是小麦的主要非生物胁迫因子之一,发掘小麦耐盐品种中的相关基因,分析其调控机理,有助于解析小麦耐盐性机制。本文利用TAIL-PCR和电子克隆的方法,从耐盐小麦RH8706-49中克隆了耐盐基因Ta SC的启动子序列,命名为ProTaSC。该DNA序列中存在多个顺式作用元件,包含与非生物胁迫响应有关的ABA响应元件(ABRE)和MYB蛋白结合位点(MBS)各1个。以GUS为报告基因,对克隆的启动子序列及不同长度的5′端缺失片段的表达活性分析表明,克隆的全长片段及2个5′端缺失的片段(681 bp和1096 bp)均能启动GUS表达,而小于等于343 bp的片段不具备启动功能,说明ProTaSC中从-681位到–343位核苷酸之间的区域为核心启动子区。在ProTaSC:GUS转基因拟南芥的根、叶片、花药、萼片及成熟角果的果荚壳中均检测到GUS蛋白,而在主茎、花瓣、幼果和种子中没有检测到GUS,表明ProTaSC是组织表达特异性启动子。对ProTaSC:GUS转基因拟南芥在NaCl(200 mmol L^(-1))和ABA(10μmol L^(-1))胁迫处理后的GUS定量分析表明,ProTaSC是受NaCl和ABA显著诱导表达的功能序列。High salinity is one of the major abiotic stress factors in wheat.Exploring stress related genes from salt-tolerant wheat varieties and analyzing their regulatory mechanism are helpful for elucidating the salt tolerance mechanism in wheat.In this study,the promoter sequence of a salt-tolerant related gene TaSC,designated ProTaSC,was cloned from salt-tolerant wheat mutant RH8706-49 by TAIL-PCR and silicon cloning method.A series of cis-acting elements including abscisic acid response element(ABRE),MYB protein binding site(MBS),TATA-box and CAAT-box were predicted in the promoter region.Among them ABRE and MBS are involved in abiotic stress responses.Beta-glucuronidase gene was used as reporter to study the expression characteristic of ProTaSC,showing that the full-length fragment and two 5'-progressive deletion fragments(681 bp and 1096 bp)were able to trigger GUS expression.However,GUS expression was undetectable when the fragment was less than 343 bp.These results suggest that the full-length promoter has promoting activity and the sequence between–681 to–343 nucleotides is the basic core region of ProTaSC.ProTaSC is a tissue-specific promoter because GUS gene driven by full-length ProTaSC was expressed in root,leaf,anther,sepals,and mature pods,but not in stem,petal,young fruit,and seed of Arabidopsis harboring ProTaSC:GUS.Quantification of GUS activity assay showed that ProTaSC was induced significantly by NaCl(200 mmol L–1)and ABA(10μmol L–1)in the transgenic Arabidopsis seedlings,indicating ProTaSC is a functional sequence induced by NaCl or ABA treatment.

关 键 词：小麦 耐盐突变体RH8706-49 TaSC基因启动子 TAIL-PCR 表达活性 

分 类 号：S512.1[农业科学—作物学]