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

1.从蓝藻聚球藻中克隆到一个新的热休克基因orf7.5。PCC 7942。编码区的突变导致转化子在50℃时的基础耐热性显著降低，表明该基因在热胁迫管理中起重要作用。Orf7.5突变体可以通过含有orf7.5的DNA片段恢复到野生型表型，但不能通过在orf7.5编码区引入移码或点突变而产生无义突变的orf7.5突变来补充。因此，耐热性需要一个合适的翻译产物，而不是简单的转录。在对温度敏感的突变体中，groESL或htpG转录本的累积明显减少，这表明orf7.5基因作为正调控基因参与了这些基因的表达。为了阐明蓝藻中热休克基因的表达调控，采用了报告-分析系统。5‘-上游…聚球藻groESL操纵子的更多调控区域。将PCC7942与无启动子的lacZ基因融合。融合基因在大肠杆菌中分别在30和42℃处进行了组成性表达。而在蓝藻中，同样的groESL-lacZ基因被引入聚球藻的中性区域。PCC7942染色体在热休克时有表达。这些结果表明，蓝藻中存在一种抑制groESL操纵子表达的机制。虽然编码来自聚球藻的一个小的热休克蛋白同源物的hspA基因与groESL操纵子一样在大肠杆菌中转录，但它在大肠杆菌中的翻译在30℃时受到强烈的抑制。这一结果表明蓝藻HSP基因存在转录后调控。我们从蓝藻聚球藻中克隆了编码HtpG同源物的基因。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。突变细胞在45℃时不能生长，失去了基础耐热性和获得性耐热性，这表明htpG在蓝藻的热应激管理中发挥了重要作用，这在原核生物中是第一个这样的例子。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著贡献。我们构建了一株结构性表达HSPA的蓝藻菌株。这是首次尝试在蓝藻中组成性表达任何一种热休克蛋白。在30~50℃的光照条件下，转化子的活力明显高于参比菌株。HSPA的表达增加了对光系统II的热胁迫抗性，并减少了对光诱导漂白的捕光藻胆体的保护作用

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