权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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.从蓝细菌Synechococcus sp. PCC 7942中克隆了一个新的热激基因orf7.5。该基因编码区的缺失导致了该基因的基础耐热性显著下降，表明该基因在热胁迫管理中起着重要作用。orf7.5突变体可以通过含有orf7.5的DNA片段而不是通过突变的orf7.5s（其中通过在orf7.5编码区内引入移码或点突变而产生无义突变）与野生型表型的返回互补。因此，耐热性需要适当的翻译产物，而不仅仅是转录本，温度敏感突变体中groESL或htpG转录本的积累显著减少，表明orf7.5基因可能作为正调控因子参与了这些基因的表达.为了阐明蓝藻热激基因的表达调控，采用了一种荧光定量分析系统。5 '-上游 ...更多信息将来自聚球藻属PCC 7942的groESL操纵子的调节区与无启动子的lacZ基因融合。融合基因在E.在30 ℃和42 ℃下，大肠杆菌均为组成型。然而，在蓝细菌中，相同的groESL-lacZ基因，其被引入到聚球藻属PCC 7942染色体的中性区域中，在热休克后表达。这些结果表明存在一种抑制蓝藻中groESL操纵子表达的机制。虽然编码来自硫化聚球藻（Synechococcus vulcanus）的小热休克蛋白同源物的hspA基因在大肠杆菌中的转录与groESL操纵子相似，但其在大肠杆菌中的翻译在30 ℃下被强烈抑制。结果表明蓝藻热休克蛋白基因存在转录后调控.我们克隆了一个基因编码的同源物HtpG从蓝藻聚球藻属PCC 7942。为了阐明HtpG在体内的作用，我们通过靶向突变使该基因失活。突变体细胞不能在45 ℃下生长，丧失了基础和获得性耐热性，表明HtpG在蓝藻的热胁迫管理中起着重要作用，这是原核生物的第一个这样的例子。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。我们构建了一株组成型表达HspA的蓝藻。这是首次尝试在蓝藻中组成型表达任何类型的热休克蛋白。在光照条件下从30 ℃转移至50 ℃时，与参考菌株相比，该菌株的活力有所改善。HspA的表达增加了对光系统II的热胁迫抗性，并保护捕光藻胆体免受热诱导的光漂白。