拟南芥atfes1a突变抑制因子的功能分析

The atfes1a line, defective in thermotolerance and having a decreased level of cytosolic Hsp70, was subjected to mutation. A suppressor line, with significant increases in both its thermotolerance compared with atfes1a and the level of cytosolic HSP70, was picked out from the library of the mutants, and hereafter was named as Su(fes1a). The locus of the suppressor was located, by means of both gene mapping and Tail-PCR methods, at at2g45100, and the accuracy of Su(fes1a) location was further confirmed by characterization of the two independent salk T-DNA lines, each of them having a T-DNA insert in Su(fes1a) gene. .Both of two independent salk T-DNA lines showed the up-regulation of heat response and the improvement in resistance to higher night temperature. Su(fes1a) is similar to Brf protein, a regulative subunit of RNA polymerase III in yeast, and is clustered together with Brf1 transcription regulators in plant. .Because there is no any experimental date available about plant Brf1 yet, we shall, in this project, carry out a few studies as follows. (1) To reveal the subcell location of Su(Fes1A) as well as its possible movement between nucleus and chloroplast; (2) To evaluate the effect Su(Fes1A) on "molecular chaperion machine" in vivo；(3）To sort out the genes regulated by Su(Fes1A); (4) To elucidate the key responding elements within the promoter that interacts with Su(Fes1A); (5) To search for the proteins probably connecting with Su(Fes1A) in nucleus and chloroplast. In the end of the project, we could provide the knowledge in detail for well understanding how Su(Fes1A), in the dark, regulates the thermotolarance and heat response in plant.

以热敏感拟南芥atfes1a突变株为原始材料，进行二次突变,从突变体库中，我们筛选到耐热性明显恢复,且细胞质HSP70表达上调的一株突变体，简称Su(fes1a)。我们定位了Su(fes1a)基因，Su(Fes1A)类似于酵母RNA polymearse III 复合体中的一个调节亚基，归属植物转录调控因子Brf1家族。无光条件下，两个Su(fes1a)的独立T-DNA突变体的热激应答明显上调，并呈现耐夜间高温的表型，Su(fes1a)是新发现的严格受光控制的热激应答调控因子。我们将就Su(Fes1a)的亚细胞定位、Su(fes1a)可能在细胞器间的移动、Su(fes1a)对细胞分子伴侣机器效率的影响、Su(fes1a)所调控基因的种类、Su(fes1a)靶标序列之核心元件特征、Su(fes1a)互作蛋白等问题展开研究，阐明无光条件下Su(Fes1A)调控热激应答的分子机理。

全球变暖的趋势已成事实, 植物高温应答的分子机理是植物学理论研究的重要内容, 本课题立项中提出, atfes1a的高温敏感抑制子是一全新的发现, 研究此抑制子可以深入理解植物应对高温胁迫的分子机理。本课题的核心任务是解析atfes1a抑制子的工作机理，通过4年的研究，我们发现：.①.突变atfes1a导致植物热敏感的主要原因。缺失atfes1a导致了高温剧烈胁迫后蛋白合成缓慢，AtFes1a是HSP70的结合蛋白，HSP70是新生蛋白合成的必须分子伴侣，突变atfes1a导致HSP70丢失NEF，特别是高温胁迫后，以致呈现热伤害后无法修复，植物呈现高温敏感性。.② 本课题研究的抑制子（基因）AtBrf1是RNA聚合酶Ⅲ的 TFBⅢ复合体中的核心亚基，由于AtBrf1的突变导致了RNA聚合酶Ⅲ和RNA聚合酶Ⅱ之间的平衡偏离，即在高温胁迫后，由于AtBrf1突变或表达抑制，导致RNA聚合酶Ⅱ途径得到强化，抗热相关基因，如热激蛋白，表达强化，导致抑制子呈现耐热性提高。.③ TBP介入了抑制子（基因）AtBrf1的调控途径。本研究发现,TBP作为RNA polymase Ⅲ 和 RNA polymaseⅡ 共用因子，在调控arfes1a耐热性方面起重要作用，atbrf1调控atfes1a的信号途径是经过了TBP。RNA polymase Ⅲ 和 RNA polymaseⅡ途径为平行途径，突变atbrf1导致RNA polymase Ⅲ对TBP的需求下降，这也导致RNA polymaseⅡ途径强化，导致atfes1a的抗热性提高。.④ Atfes1a 是剧烈高温胁迫后，蛋白组装的必须因子。突变Atfes1a并不影响植物发育，也不影响温和高温胁迫下的植物生长。致死性的高温胁迫导致了TBP蛋白严重突变，TBP的蛋白合成组装进入RNA polymase复合体过程需要HSP70等分子伴侣，Atfes1a是HSP70分子伴侣行使其功能的必须NEF，缺失Atfes1a（NEF）导致TBP组装低效，在atfes1a突变体中，强高温胁迫后，TBP组装进入RNA polymaseⅡ成为恢复期蛋白质合成的瓶颈。.本研究发现Atfes1a的核心功能是介入了强高温后的蛋白质合成。TBP 组装低效是导致Atfes1a突变体热敏感的原因之一。