Abstract: Based on the Southern blot analysis results, the KpnⅠ and EcoRⅠ restriction enzymes were selected to digest the genomic DNA of a cultivar of tomato ( Lycopersicon esculentum Mill . CV . zhongshu 4) . The 3 kb bands of the digested genomic DNA were inserted into the pBSⅡKS ( + ) 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 LeMTshsp 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 purified HsfA2 protein bound specifically to HSEs of the LeMTshsp promoter in vitro , and bound strongly to the proximal five HSEs than to the distal HSE . The fusion construction of LeMTshsp promoter- gus (β glucuronidase) was introduced into tomato using an Agrobacterium -mediated transformation . The resistant transgenic plants were selected on the MS medium containing 50mg􊄯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 (Cd2+ , Cu2 + , Pb2 + or Zn2 + ) 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 LeMTshsppromoter is a promoter responding to heat, cold , exogenous ABA and heavy metals.

Key words: Tomato

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, 3 kb 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2 kb 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1915 bp 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

关键词: 番茄, 线粒体小分子热激蛋白, 启动子, 逆境, 克隆FONT