甘蓝型油菜SNARE蛋白SYP122叶片瞬时表达体系的建立

Establishment of transient expression system of SNARE protein SYP122 in Brassica napus leaves

通过生物信息学鉴定了油菜中的突触融合蛋白BnSYP122,其由一个N端syntaxin结构域、一个典型的SNARE基序及一个C端跨膜结构域组成,并且N端结构域包含被称为Ha、Hb、Hc基序的3个α-螺旋束.进一步利用Gateway技术成功将目的蛋白构建于植物表达载体pEarleyGate104上,与带有绿色荧光蛋白的pEGAD分别转化农杆菌GV3101菌株,采用农杆菌介导的真空渗透法在油菜叶片中进行瞬时表达.经激光共聚焦显微镜观察和Westernblot检测表明,农杆菌介导的真空渗透法能够在油菜叶片中成功表达目的蛋白,同时验证了BnSYP122定位在细胞膜上.

A SNARE protein (BnSYP122) in Brassica napus was identified by bioinformatics. The protein composed of a N-terminal syntaxin domain with 3 α-helical bundles called Ha, Hb, and Hc motifs, a typical SNARE motif and a C-terminal transmembrane domain. Subsequently, BnSYP122 was constructed into the plant expression vector pEarleyGate 104 by Gateway technique, and transformed into GV3101 strain of Agrobacterium tumefaciens. The vector of pEGAD with green fluorescent protein as control was also transformed into GV3101 strain of A.tumefaciens. Then transient expression in B.napus leaves was performed by vacuum infiltration mediated by A.tumefaciens. Laser confocal microscopy and western blot analysis showed that vacuum infiltration method mediated by A.tumefaciens could successfully express the target protein in B.napus leaves. In addition, BnSYP122 was verified to be located on the plasma membrane.