3种瞬时表达体系研究1个白木香倍半萜合酶的亚细胞定位

Subcellular localization of a sesquiterpene synthase from Aquilaria sinensis utilizing three transient expression systems

目的研究1个白木香倍半萜合酶(ASS)的亚细胞定位,确定其功能区域,并比较3种瞬时表达体系在亚细胞定位研究中的优劣。方法从白木香愈伤组织中提取总RNA,采用特异引物RT-PCR方法克隆倍半萜合酶ASS基因,并连接于pEZS-NL载体和pFGC5941GFP改造载体上,分别构建pFGC5941-GFP-ASS、pEZS-NL-ASS-GFP 2种植物表达载体,分别利用根癌农杆菌侵染烟草叶片、基因枪轰击洋葱表皮细胞、PEG转化白木香原生质体3种技术进行瞬时转化表达,激光共聚焦显微镜下观察表达出的绿色荧光蛋白(GFP)融合蛋白的亚细胞定位。结果在烟草叶片表皮细胞、洋葱表皮细胞及白木香原生质体中,融合蛋白绿色荧光均能被观察到。ASS基因与GFP的融合蛋白产物在烟草叶片中定位于胞质,在洋葱表皮中定位于胞质和细胞核,白木香原生质体中定位于胞质和质体。结论 ASS基因在白木香原生质体胞质及质体定位;对3种体系的结果比较表明,应用不同体系研究蛋白的亚细胞定位可能出现不同的结果,这可能与同源或异源表达的植物细胞的特性有关。

Objective To investigate the subceUular localization of a sesquitcrpene synthase from Aquilaria sinensis （ASS） and compare three transient expression systems in the subcellular localization study. Methods An ASS gene was amplified by RT-PCR using specific primers and cloned into the pEZS-NL and pFGC5941GFP to generate two plant expression vectors： pEZS-NL-ASS- GFP and p5941-GFP-ASS. Three transient expression systems, agroinflltration of tobacco leaves, particle bombardment of onion epidermal cells, and PEG transformation ofprotoplasts isolated from ASS calli were adopted and compared. The expression of the GFP fusion proteins were observed by a confocal laser scanning microscopy. Results The green fluorescence could be observed in all the three systems. However, the results were comparatively different. The cytoplasm and plastid localization of the GFP fusion protein observed in protoplasts was comparatively clear and was consistent with the studies in other plant species. Conclusion The results demonstrate that the ASS is located in the cytoplasm and plastid in the ASS protoplasts .and the different locations in three systems might be caused by the distinct characters of the heterologous or homologous cells.