耐盐小麦中TaSC基因启动子的克隆及调控功能分析

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TaSC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(10 μmol 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 Ta S C, 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 character-istic 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.Quan-tification of GUS activity assay showed that ProTaSC was induced significantly by NaCl (200 mmol L–1) and ABA (10 μmol L–1) in the transgenic Arabidopsis seedlings,indicating ProTaSC is a functional sequence induced by NaCl or ABA treatment.