叶用莴苣高温胁迫响应因子LsHsp70的功能研究

The cultivation area of lettuce is small in our country a decade ago, but it is developing fast in recent years. Lettuce adapted to cool climate and the high temperature in summer seriously reduces its quality, even a large area destroyed. The study of the genes function which closely related to the high temperature stress response have great significance to reveal the mechanism of its adaptive growth after response to high temperature. In previous researches, we obtained one HSP70 protein related to high temperature resistance in lettuce. We named this gene as LsHsp70, which encode the HSP70 protein. It was isolated by two-dimensional electrophoresis and mass spectrometer. The result showed that this gene is closely related with the lettuce resistance which analyzed by western blotting and real time RT-PCR. In order to clear the function of the gene, this project is designed as follows. We will transformed LsHsp70-eGFP fusion protein vector into protoplast and observed the subcellular localization of genes by the laser confocal microscopy; at the same time we will over-express and silence LsHsp70 in lettuce, that enable us to understand the function of Hsp70 in lettuce; at the last we will screen the cDNA libraries to isolated target interaction protein using yeast two hybrid. We hope to explore the molecular regulation mechanism of LsHsp70 under high temperature, thus we can guide the production and develop the high temperature –resisted varieties. The ultimate goal is make the lettuce can stability and safe production in whole year.

叶用莴苣目前是我国发展较快的绿叶蔬菜，其性喜冷凉气候，常因夏季高温严重影响品质，甚至导致大面积绝收。研究其重要的高温胁迫响应基因的功能，对破解叶用莴苣响应高温胁迫后进行适应性生长的分子机制具有重要意义。在前期研究中，我们利用双向电泳技术和质谱分析得到了与叶用莴苣耐热性相关的一个HSP70蛋白，将编码这条蛋白的基因命名为LsHsp70，western blotting分析和实时定量RT-PCR分析表明此条基因确实与叶用莴苣耐热性密切有关。为了进一步明确该基因的功能，本项目拟构建LsHsp70与eGFP的融合表达载体，PEG介导法转化原生质体后利用激光共聚焦显微技术观察此基因的亚细胞定位；同时构建LsHsp70基因的过表达和干涉载体, 研究此基因的功能；采用酵母双杂交系统筛选与该基因互作的靶蛋白，分析该条基因响应高温胁迫的分子机制，进而指导生产培育出耐热品种，保障叶用莴苣稳定、安全、周年生产。

叶用莴苣目前是我国发展较快的绿叶蔬菜，其性喜冷凉气候，常因夏季高温严重影响品质，甚至导致大面积绝收，是困扰生产的一大难题。研究其重要的高温胁迫响应基因的功能，对破解叶用莴苣响应高温胁迫后进行适应性生长的分子机制具有重要意义。在前期研究中，我们利用双向电泳技术和质谱分析得到了与叶用莴苣耐热性密切相关的HSP70蛋白，在本项目中，我们利用实时定量RT-PCR方法研究了两条Hsp70基因在高温胁迫抽薹前后的表达差异；通过转基因研究，明确了它们的功能，同时利用酵母双杂交技术，找到了13个与Hsp70蛋白互作的蛋白，初步阐明了叶用莴苣高温抽薹的分子机理，即“高温促进LsHsp70基因和钙调蛋白的互作，促进了GA积累，导致叶用莴苣抽薹”，为指导生产培育耐热叶用莴苣新品种，保障叶用莴苣稳定、安全、周年生产奠定了坚实的理论基础。