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。一种新型的热休克基因ORF7.5是从蓝细菌Synechococcus sp中克隆的。 PCC 7942。编码区域的中断导致转化剂中50℃的基本热耐耐含量显着降低，表明该基因在热应力管理中起着至关重要的作用。 ORF7.5可以通过包含ORF7.5的DNA片段返回到野生型表型，但不能通过突变的ORF7.5s来完成，在该突变的ORF7.5s中，通过引入ORF7.5编码区域内引入框架突变或点突变而产生了废话。这是，热耐含量需要适当的翻译产物，而不仅仅是转录物。在温度敏感的突变体中GroESL或HTPG转录本的蓄能被大大降低，这表明ORF7.5基因作为阳性调节剂的表达与这些基因的表达有关。2。为了阐明蓝细菌中热震基基因表达的调节，采用了记者测定系统。 5'上游…来自Synechococcus sp。 PCC 7942与无启动子的LACZ基因融合在一起。融合基因在30和42℃的组成型大肠杆菌中均表达。然而，在蓝细菌中，同一个Groesl-lacz基因被引入了Syechococcus sp的中性区域。 PCC 7942染色体在热休克后表达。这些结果表明存在一种机制，该机制反映了蓝细菌中Groesl算子的表达。尽管编码来自Synechococcus vulcanus的小HSP同源物的HSPA基因像Groesl oberator一样在大肠杆菌中转录，但其在E.Coli中的翻译被强烈抑制在30℃时。结果表明存在蓝细菌HSP基因的转录后调节3。我们克隆了一个基因，该基因编码了cyanobacterium synechococcus sp。 PCC 7942。为了阐明HTPG在体内的作用，我们通过靶向诱变使该基因失活。突变细胞无法在45°C下生长，并且失去了碱性和获得的热液，这表明HTPG在蓝细菌中的热应力管理起着至关重要的作用，这是核生物体的第一个这样的例子。对HTPG突变体的研究表明，HTPG蛋白有显着促进蓝细菌适应低温的能力。我们构建了一种蓝细菌菌株，该菌株构成了HSPA。这是首次在蓝细菌中表达任何类型的HSP的尝试。与在光线30到50℃转移时的参考应力相比，转化剂的生存力提高了。 HSPA的表达增加了对光系统II的热应力耐药性，并保护了轻度捕获的植物质体免受热诱导的光漂白较少