不同启动子在水稻悬浮细胞中诱导外源基因的表达

摘要

To improve the 1eve1 of expression of exogenous gene( green f1uorescent protein gene, GFP) in rice sus-pension ce11s, ten expression vectors containing GFP reporter gene were constructed as fo11ows using cau1if1ower mosaic vi-rus promoter (CaMV) 35S(CK), the promoter of ubiquitin (Ubi) from maize, and the promoters of actin (Actin) and Os-cc1 from rice as fo11ows:pCAMBIA1304 35S-GFP, Ubi-GFP, Actin-GFP, Oscc1-GFP, 35S/Ubi-GFP, 35S/Actin-GFP, 35S/Oscc1-GFP, Ubi/Actin-GFP, Ubi/Oscc1-GFP, and Actin/Oscc1-GFP. Then by app1ying agrobacterium tumefaciens-mediated method, ten expression vectors mentioned above were transformed into Nipponbare rice suspension ce11s to obtain transgenic Nipponbare rice suspension ce11s. F1uorescence microscopy and microp1ate reader were used to detect GFP f1uo-rescence intensity, and GFP expression 1eve1s were detected by western b1otting. The resu1ts showed that tandem promoter driven GFP expression were significant1y greater than a sing1e promoter-driven ones. In ten expression vectors, 35S/Ubi, Ubi/Actin and Ubi/Oscc1 were three optimized promoter combinations, among which Ubi/Oscc1 tandem promoter drove the highest expression 1eve1 of GFP, three times more than the CaMV35S promoter.%为进一步提高外源基因在水稻悬浮细胞中的表达水平,以花椰菜花叶病毒(CaMV)35S启动子为对照,克隆了玉米泛素蛋白启动子( Ubi)、水稻肌动蛋白启动子( Actin)以及水稻Oscc1启动子,以GFP为报告基因,通过启动子串联的方式,构建了10个植物表达载体：pCAMBIA 130435S-GFP、Ubi-GFP、Actin-GFP、Oscc1-GFP、35S/Ubi-GFP、35S/Actin-GFP、35S/Oscc1-GFP、Ubi/Actin-GFP、Ubi/Oscc1-GFP、Actin /Oscc1-GFP,采用根瘤农杆菌介导的方法,将表达载体导入日本晴胚性愈伤,经悬浮培养分别获得转基因的悬浮细胞系,利用荧光显微镜、酶标仪检测GFP荧光强度,用Western b1ot检测GFP蛋白质的表达水平。结果发现串联启动子明显强于单个启动子驱动的GFP蛋白质表达水平。在10个表达载体中,35S/Ubi、Ubi/Actin和Ubi/Oscc1是3种优化的启动子组合,其中Ubi/Oscc1串联启动子驱动GFP的表达水平最高,约为对照35S启动子的3倍。