番茄多胁迫诱导型LeMTshsp启动子的分子克隆及其功能分析  

Cloning and Functional Analysis of a Multiple Stress-inducible LeMTshsp Promoter from Tomato (Lycopersicon esculentum)

作　　者：伊淑莹[1] 孙爱清[1] 赵春梅[1] 刘箭[1] 

机构地区：[1]山东师范大学生命科学学院,山东济南250014

出　　处：《云南植物研究》2007年第2期223-230,共8页Acta Botanica Yunnanica

基　　金：国家自然科学基金项目(30270132)

摘　　要：根据Southern杂交结果,选取KpnⅠ与EcoRⅠ双酶切番茄中蔬4号基因组DNA,3kb左右的酶切片段连入pBSⅡKS(+)载体,构建成含有线粒体小分子热激蛋白基因(LeMTshsp)上游2kb左右调控区的质粒文库。通过巢式PCR方法从构建的质粒文库中克隆出LeMTshsp基因上游1915bp的调控区(GenBank登录号为AB239774)。该序列含有TATA box及CAAT box等启动子基本元件,还具有6组典型的HSE元件及多个AT-rich区,另外还有许多逆境反应元件如ABRE,C-repeat—DRE,AP-1。凝胶阻滞结果表明,纯化的HsfA2蛋白与LeMTshsp启动子的HSE元件在体外具有结合活性,且与近端5组HSE的结合活性比与远端HSE的结合活性强。构建该启动子与GUS基因的融合载体,利用农杆菌介导的叶圆盘法转化番茄,GUS组织化学染色结果表明LeMTshsp启动子对热激、低温、外源ABA及重金属胁迫都有应答。Based on the Southern blot analysis results, the kpn Ⅰ and EcoR Ⅰ restriction enzymes were selected to digest the genomic DNA of a cuhivar of tomato （ Lycopersicon esculentum Mill. CV. zhongshu 4）. The 3 kb bands of the digested genomic DNA were inserted into the pBS Ⅱ KS （ ＋ ） vector. As a result, a plasmid library was generated containing about 2 kb of the 5＇-flanking sequence of the mitochondria-localized small heat shock protein gene （ LeMTshsp）. A 1915 bp of the 5＇-flanking region of l.eMTshsp was isolated from the plasmid library by nested PCR （GenBank accession number AB239774）. The 5＇-flanking region of LeMTshsp contains putative TATA box, CAAT box, a total of six HSEs and several AT-rich regions. Additionally, there are some transcription factor binding motifs related to stress response, such as ABAresponsive element [ ABRE], C-repeat_ DRE and activating protein binding sites [ AP-1 ]. Electrophoresis mobility shift assay （EMSA） showed that the puriiicd HsfA2 protein bound specifically to HSEs of the l.eMTshsp promoter in vitro, and bound strongly to the proximal five HSEs than to the distal HSE. The fusion construction of l.eMTshsp promoter-gus （β-glucuronidase） was introduced into tomato using an Agrobacterium -mediated transformation. The resistant transgenic plants were selected on the MS medium containing 50 mg/L kanamicin. PCR analysis showed that the chimeric gus gene was integrated into the tomato genome. By using the gus reporter gene system, the LeMTshsp promoter dynamics was explored under stress conditions. After heat, cold, exogenous ABA and heavy metal （Cd^2＋ , Cu^2＋ , Pb^2＋ or Zn^2＋ ） treatments, GUS staining was detected in the leaves and roots of transgenic tomato plants. The activity of the LeMTshsp promoter under heat shock conditions was comparable to that of the constitutive CaMV35S promoter. All these results show that the LeMTshsp promoter is a promoter responding to heat, cold, exogenous ABA and heavy metals.

关 键 词：番茄 线粒体小分子热激蛋白 启动子 逆境 克隆 

分 类 号：S641.2[农业科学—蔬菜学]