Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.
Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.
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在红色藻类Cyanidioschyzon Merolae中的热震诱导基因表达系统的发展。
DOI：
10.1371/journal.pone.0111261
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Miyagishima SY
中科院分区：
文献类型：
--
作者：
Sumiya N;Fujiwara T;Kobayashi Y;Misumi O;Miyagishima SY
单细胞的红蓝细菌Cyanidioschyzon merolae的细胞包含单个叶绿体和叶绿体，其分裂通过光/暗循环紧密同步。在光合真核生物中，基因组内容极其简单，具有低水平的遗传冗余。此外，已经报道了通过同源重组的瞬时转化和稳定转化。然而，对于细胞生长和存活所必需的现象的分子遗传学分析，需要诱导型基因表达/抑制系统。在这里，我们报告了一个热休克诱导的基因表达系统在C。merolae。CMJ 101 C编码一种小的热休克蛋白，仅在细胞暴露于高温时才转录。使用超级折叠GFP作为报告蛋白，CMJ 101 C orf的200-bp上游区域被确定为热休克诱导的最佳启动子。诱导表达的最适温度为50°C，C. merolae细胞能够增殖。至少需要30分钟的热休克来表达感兴趣的蛋白质，并且60分钟的热休克产生最大水平的蛋白质表达。热休克后，mRNA水平迅速下降。作为该系统的一个实例，诱导了在GTdR结构域中具有突变的叶绿体分裂显性失活形式DRP 5 B蛋白的表达。显性负DRP 5 B的表达导致出现异常形状的细胞，其中两个子叶绿体和细胞仍然通过小DRP 5 B阳性管状结构连接。这一结果表明，显性负DRP 5 B抑制了叶绿体分裂位点的最终断裂，但不抑制分裂位点收缩的早期阶段。它也表明，细胞周期的进展是不逮捕的损害叶绿体分裂在最后阶段。
The cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50°C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage.
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DOI：
10.2183/pjab.86.455
发表时间：
2010
期刊：
Proceedings of the Japan Academy. Series B, Physical and biological sciences
影响因子：
--
作者：
Kuroiwa T
通讯作者：
Kuroiwa T
影响因子：
11.6
作者：
Miyagishima, S;Nishida, K;Kuroiwa, T
通讯作者：
Kuroiwa, T
影响因子：
3.5
作者：
Cormack, BP;Valdivia, RH;Falkow, S
通讯作者：
Falkow, S
影响因子：
64.8
作者：
Matsuzaki, M;Misumi, O;Kuroiwa, T
通讯作者：
Kuroiwa, T
影响因子：
4.9
作者：
Minoda, A;Sakagami, R;Tanaka, K
通讯作者：
Tanaka, K