ランソウの熱ショックタンパク質(Hsp)遺伝子の発現調節機構の解明

阐明冉苏热休克蛋白（Hsp）基因的表达调控机制

基本信息

批准号：
11640641
负责人：
NAKAMOTO Hitoshi
金额：
$ 2.24万
依托单位：
Saitama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

1. A novel heat shock gene, orf7.5, was cloned from the cyanobacterium Synechococcus sp. PCC 7942. An interruption in the coding region led to a marked decrease in the basal thermotolerance at 50℃ in the transformant, indicating that the gene plays an essential role in thermal stress management. The orf7.5 mutant could be complemented with a return to the wild-type phenotype by a DNA fragment containing orf7.5, but not by mutated orf7.5s in which a nonsense mutation was generated by introducing a frameshift or a point mutation within the orf7.5 coding region. Thus, thermotolerance requires an appropriate translation product, not simply a transcript.Accumulation of the groESL or htpG transcript in the temperature-sensitive mutants was strongly reduced, suggesting that the orf7.5 gene be involved in the expression of these genes as a positive regulator.2. In order to elucidate regulation of the heat-shock gene expression in cyanobacteria, a reporter-assay system was employed. 5'-upstream … More regulatory region of the groESL operon from Synechococcus sp. PCC 7942 was fused with the promoter-less lacZ gene. The fused gene was expressed in E. coli constitutively at both 30 and 42 ℃. In cyanobacteria however the same groESL-lacZ gene, which was introduced into a neutral region of Synechococcus sp. PCC 7942 chromosome was expressed upon heat shock. These results indicate the presence of a mechanism, which represses expression of the groESL operon in cyanobacteria. Although the hspA gene encoding a small Hsp homologue from Synechococcus vulcanus was transcribed in E.coli constitutively like the groESL operon, its translation in E.coli was strongly inhibited at 30 ℃. The result indicates the presence of a post-transcriptional regulation of the cyanobacterial Hsp gene.3. We cloned a gene encoding a homologue of HtpG from the cyanobacterium Synechococcus sp. PCC 7942. In order to elucidate the role of HtpG in vivo, we inactivated the gene by targeted mutagenesis. The mutant cells were unable to grow at 45 ℃ and lost both the basal and acquired thermotolerances, indicating that HtpG plays an essential role tor the thermal stress management in cyanobacteria, the first such an example for a prokaryotic organism. Studies with the htpG mutant demonstrate that the HtpG protein contributes significantly to the ability of cyanobacteria to acclimate to low temperatures. We constructed a cyanobacterial strain, which expresses HspA constitutively. This is the first attempt to constitutively express any kind of Hsp in cyanobacteria. The transformant displayed improved viability compared with the reference strain upon transfer fro 30 to 50 ℃ in the light. Expression of HspA increased thermal stress resistance to photosystem II and protected the light-harvesting phycobilisomes from heat-induced photobleaching Less

1.从蓝藻聚球藻PCC 7942中克隆了一个新的热休克基因orf7.5。编码区的中断导致转化体在50℃下的基础耐热性显着降低，表明该基因发挥了作用。 orf7.5 突变体可以通过包含 orf7.5 的 DNA 片段恢复到野生型表型来补充，但不能通过包含 orf7.5 的 DNA 片段来补充。突变的 orf7.5s，其中通过在 orf7.5 编码区内引入移码或点突变产生无义突变，因此，耐热性需要适当的翻译产物，而不仅仅是转录产物。groESL 或 htpG 转录产物的积累。温度敏感突变体的表达量明显减少，表明orf7.5基因作为正调控因子参与了这些基因的表达。2．使用来自聚球藻 PCC 7942 的 groESL 操纵子的 5' 上游调控区与无启动子的 lacZ 基因融合。融合基因在大肠杆菌中组成型表达。然而，在蓝细菌中，相同的 groESL-lacZ 基因被引入到 30 和 42 ℃ 的中性区域。聚球藻 PCC 7942 染色体在热休克后表达，尽管编码来自聚球藻的小 Hsp 同源物的 hspA 基因在大肠杆菌中进行了组成型转录，但仍存在抑制蓝藻中 groESL 操纵子表达的机制。与groESL操纵子一样，其在大肠杆菌中的翻译在30℃下被强烈抑制。表明蓝藻Hsp基因存在转录后调控。3.我们从蓝藻聚球藻PCC 7942中克隆了编码HtpG同源物的基因。为了阐明HtpG在体内的作用，我们灭活了该基因。通过定向诱变，突变细胞无法在45℃下生长，并且失去了基础耐热性和获得性耐热性。 HtpG 在蓝藻的热应激管理中发挥着重要作用，这是原核生物的第一个例子。对 htpG 突变体的研究表明，HtpG 蛋白对蓝藻适应低温的能力有显着贡献。蓝藻菌株，其组成型表达HspA 这是在蓝藻中组成型表达任何种类的Hsp的首次尝试。与参考菌株相比，在光下从 30 ℃ 转移到 50 ℃ 时，转化体表现出更高的活力。HspA 的表达增加了对光系统 II 的热应激抵抗力，并保护光捕获藻胆体免受热诱导的光漂白。