Ishinishi Ryo, Matsuura Hideyuki, Tanaka Satoshi, Nozawa Saaya, Tanada Keisuke, Kawashita Norihito, Fujiyama Kazuhito, Miyasaka Hitoshi, Hirata Kazumasa
Applied Environmental Biology Laboratory, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Applied Environmental Biology Laboratory, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Gene. 2018 Jan 15;640:14-20. doi: 10.1016/j.gene.2017.10.012. Epub 2017 Oct 7.
The genetic basis of stress resistance in extremophilic microalgae is not well studied. In this study, a gene of unknown function, the cluster58 or CL58 gene, was identified from the halotolerant green alga Chlamydomonas W80 and characterized. The CL58 gene encodes a protein containing a domain of unknown function, the CHRD domain, and a putative secretory signaling sequence at its N-terminus. The levels of CL58 mRNA increased in response to high copper levels and low temperatures. When the CL58 gene was heterologously expressed as a fusion gene with the NanoLuc luciferase gene in Chlamydomonas reinhardtii, a majority of the NanoLuc activity was detected in the culture medium compared with that in the intracellular fraction. A mutagenic analysis revealed that the putative secretory signaling sequence was sufficient for the secretion of the CL58-NanoLuc fusion protein. In addition, we expressed the protein encoded by the CL58 gene in Escherichia coli; the recombinant, soluble protein was then purified. In summary, we identified a novel gene from C. W80 that appears to encode a stress-responsive, CHRD domain-containing secreted protein.
极端嗜盐微藻抗逆性的遗传基础尚未得到充分研究。在本研究中，从耐盐绿藻莱茵衣藻W80中鉴定并表征了一个功能未知的基因cluster58或CL58基因。CL58基因编码一种蛋白质，该蛋白质包含一个功能未知的结构域CHRD结构域，并且在其N端含有一个假定的分泌信号序列。CL58 mRNA的水平在高铜水平和低温条件下会升高。当CL58基因与纳米荧光素酶基因作为融合基因在莱茵衣藻中异源表达时，与细胞内部分相比，大部分纳米荧光素酶活性在培养基中被检测到。诱变分析表明，假定的分泌信号序列足以分泌CL58-纳米荧光素酶融合蛋白。此外，我们在大肠杆菌中表达了CL58基因编码的蛋白质；然后纯化了重组可溶性蛋白。总之，我们从莱茵衣藻W80中鉴定出一个新基因，该基因似乎编码一种应激反应性、含CHRD结构域的分泌蛋白。
