真盐生植物盐穗木HcWD-40蛋白的亚细胞定位

摘要

[目的]WD-repeat蛋白是一类结构高度保守的蛋白质家族,常分布于细胞质或细胞核内,具信号转导、染色体组装,转录调控等多种功能.实验对极端耐盐的藜科真盐生植物盐穗木的WD-40蛋白进行亚细胞定位,初步探讨该基因参与盐穗木耐盐抗旱的机制.采用绿色荧光蛋白(GFP)基因为报告基因,对真盐生植物盐穗木HcWD-40基因的表达产物进行亚细胞水平定位.[方法]PCR扩增获得序列正确的HcWD-40,构建pCAMBIA 1301-1-HcWD-40-mGFP融合基因表达载体,采用伯乐(Bio-RAD)基因枪介导的方法转化到洋葱表皮细胞中,28℃暗培养16～24 h后,以仅表达GFP的洋葱细胞作为对照,利用激光共聚焦显微镜观察GFP在洋葱表皮细胞中的分布.[结果]重组载体pCAMBIA 1301-1-HcWD-40-mGFP可成功转入洋葱表皮细胞且HcWD-40蛋白正确表达.激光共聚焦显微镜检测结果表明该基因表达产物分布于细胞质与细胞核中.[结论]目的蛋白HcWD-40定位于细胞质和细胞核内,可能间接或直接参与多种细胞过程的调控.%[Objective]WD-repeat protein belongs to a family with a highly conserved structure.It exists in cytoplasm or nucleus and have essential roles in signal transduction,transcriptional regulation and chromosome assembly.Halostachys caspica is a kind of extremely salt-tolerant euhalophyte of Chenopodiaceae.To explore the mechanism of the WD-repeat protein from H.caspica (HcWD-40) is involved in salt-and-drought tolerance; The subcellular localization of HcWD-40 was examined.[Method] The open reading frame of HcWD-40 gene without the stop codon was obtained by PCR amplification.Plant expression plasmid pCAMBIA 1301-1-HcWD-40-mGFP was constructed for transforming into onion epidermal cells by particle gun bombardment using Helios Gene-Gun system.The transformed onion epidermal cells were then incubated for 16-24 h at 28℃ in dark condition.After the incubation,the onion epidermal cells were observed for the distribution of GFP through laser confocal microscope.[Result] The recombinant plant expression vector pCAMBIA 1301-1-HcWD-40-mGFP was transformed into onion epidermal cells and expressed successfully.The confocal observation results showed that HcWD-40-GFP fusion protein was transiently expressed in onion epidermal cells and localized both in the cytosol and nucleus.[Conclusion] Our results indicate that HcWD-40 is located both in cytosol and nucleus,which suggested that it might be directly or indirectly involved in a variety of cellular processes.