Expression of Exogenous Genes Under the Control of Endogenous HSP70 and CAB Promoters in the Closterium peracerosum-strigosum-littorale complex
Expression of Exogenous Genes Under the Control of Endogenous HSP70 and CAB Promoters in the Closterium peracerosum-strigosum-littorale complex
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DOI：
10.1093/pcp/pcn039
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发表时间：
2008-04-01
影响因子：
4.9
通讯作者：
Sekimoto, Hiroyuki
中科院分区：
文献类型：
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作者：
Abe, Jun;Hiwatashi, Yuji;Sekimoto, Hiroyuki
为了获得有关植物有性繁殖的基本信息，对单细胞轮藻类新月藻（Closterium peracerosumstrigosumlittorale 复合体）（C. psl. 复合体）进行了研究。使用腐草霉素抗性 (ble) 和衣藻绿色荧光蛋白 (cgfp) 基因作为选择标记，开发了内源基因的基因导入和瞬时表达系统。这些基因的密码子使用与 C. psl 中的基因相似。复杂的。强烈驱动这些基因进入 C. psl。复杂细胞，C. psl 的两个天然启动子。复杂的基因组CpHSP70和CpCAB1与ble::cgfp融合基因连接并通过粒子轰击引入细胞中。孵育 2 天后，我们发现 500 个在 CpHSP70 启动子控制下表达 GFP 的细胞（在 42℃ 热激处理后进行鉴定）和 100 个在 CpCAB1 启动子控制下表达 GFP 的细胞（在光照条件下观察）。相反，当引入花椰菜花叶病毒 35S 启动子控制下的 ble::cgfp 时，仅在两个细胞中检测到 GFP 信号。在细胞核中检测到 ble::cgfp 融合蛋白，而在细胞质中检测到单一 cgfp 蛋白。我们的结果表明，新分离的 CpHSP70 和 CpCAB1 天然启动子分别是在热激和光照条件下诱导外源基因表达的有用工具。此外，该策略还可用于瞬时测定，例如 C. psl 中未知基因产物的细胞内定位。复杂的。
A unicellular charophyte alga, Closterium peracerosumstrigosumlittorale complex (C. psl. complex), has been studied in order to obtain basic information regarding sexual reproduction in plants. Systems for gene introduction and transient expression were developed for endogenous genes using phleomycin resistance (ble) and Chlamydomonas green fluorescent protein (cgfp) genes as selection markers. These genes have codon usage similar to that of genes in the C. psl. complex. To drive these genes strongly into C. psl. complex cells, two native promoters of the C. psl. complex genomeCpHSP70 and CpCAB1were linked to a ble::cgfp fusion gene and introduced into the cells by particle bombardment. Following 2 d of incubation, we found 500 cells expressing GFP under the control of the CpHSP70 promoter, which were identified following heat shock treatment at 42C, and 100 cells expressing GFP under the control of the CpCAB1 promoter, which were observed in lit conditions. In contrast, the GFP signal was only detected in two cells when ble::cgfp under control of the cauliflower mosaic virus 35S promoter was introduced. The ble::cgfp fusion protein was detected in the nucleus, whereas the single cgfp protein was detected in the cytoplasm. Our results indicate that the newly isolated native promoters of CpHSP70 and CpCAB1 are useful tools for inducing exogenous gene expression under heat shock and lit conditions, respectively. In addition, this strategy can be used for transient assays, such as the intracellular localization of unknown gene products in the C. psl. complex.