番茄Lehsp23.8启动子的热诱导表达调控序列鉴定

Identification of the regulation elements in heat-inducible Lehsp23.8 promoter

摘要:

番茄线粒体小分子热激蛋白(Lehsp23.8)启动子是典型的热诱导启动子。为了研究热激条件下该启动子的调控序列, 本研究将不同长度的Lehsp23.8启动子序列与gus基因融合, 构建5￠缺失植物表达载体。然后用农杆菌介导法转化烟草, PCR及Southern blotting结果表明融合基因已经整合到烟草基因组中。GUS组织化学染色结果表明: 不同长度Lehsp23.8启动子转基因植株热激处理后, 在幼苗根、茎、叶以及花和果实中均表现出GUS活性, 只是染色强弱有差异。叶片中GUS荧光活性测定结果表明: 在热激处理条件下, 565 bp的Lehsp23.8启动子介导的GUS表达最强; 而255 bp的Lehsp23.8启动子介导的GUS表达最弱。说明Lehsp23.8启动子中255 bp的序列即能满足该启动子的热激表达, -565 bp~ -255 bp之间存在明显的增强子元件, 而-871 bp~ -565 bp之间的片段具有一定的抑制作用。

Abstract:

The promoter of mitochondria-localized small heat shock protein gene in Lycopersicon esculentum (Lehsp23.8) is characterized as strongly heat-inducible. In this study, to determine how the expression of Lehsp23.8 is regulated, we conducted five expression vectors carrying the gus gene driven by the 5￠ deletion products of the Lehsp23.8 promoter. The corresponding transgenic tobacco plants were generated via Agrobacterium tumefaciens-mediated transformation. Transgenic plants were identified by PCR and Southern blotting analysis. GUS activities under heat-shock conditions were characterized in transgenic tobacco plants. After heat shock, obvious GUS staining was detected in the leaves, shoots, roots, flowers and fruits of the transgenic tobacco plants. The result of fluorometric GUS assays in leaves showed that the heat-induced GUS activity of the 565 bp promoter was the strongest, while that of the 255 bp promoter was the lowest. Deletion analysis shows that the smallest promoter fragment (-255 to -23 bp) is sufficient for heat induction. It also indicates that the sequences between -255 bp and -565 bp serve as enhancers, while the sequences between -565 bp and -871 bp can repress the heat-induced activity of the Lehsp23.8 promoter.