甘蓝短散布元件对转基因表达效果的研究

Effect of Short Interspersed Nuclear Element(SINE) of Cabbage on Transgene Expression In Transgenic Tobacco

为检验甘蓝短散布元件(SINE)对植物转基因表达的影响,采用PCR方法,从甘蓝基因组中克隆了一段短散布元件序列(Short Interspersed Nuclear Element,SINE),该SINE具有核基质结合区(matrix attachmentregion,MAR)的结构特征,将其构建到β-葡糖醛酸酶(β-glucuronidase,GUS)基因(uidA)的两侧翼,形成SINE调控的植物表达载体,采用农杆菌介导法,将含SINE序列和不含SINE序列的植物表达载体导入烟草中。对转基因植株进行GUS活性定量测定,结果表明,SINE表现出类似MAR的功能,可以提高外源uidA基因的表达水平,与不含SINE的转化植株相比,外源基因的平均表达水平提高了2倍,但转基因植株个体间表达水平存在较大的差异。

In this study, a SINE fragment was cloned from cabbage （Brassica oleracea var. capitata L, ） by PCR method in order to test its effect on transgene expression in plant. This SINE fragment has the structure and feature of matrix attachment region （MAR）. The plant expression vector was constructed by SINE sequence used to flank the β-glucuronidase （GUS） report gene within the T-DNA of the binary vector. Vectors containing or lacking SINEs were then used to transform tobacco plants by Agrobacteriurn turnfaciens. The biologic activity of GUS was quantitatively determined; the result showed that the expression level of uidA gene could be improved by regulation of SINE fragment, and the function of SINE was similar to MAR. Furthermore, transgenic plants containing the SINE and flanking the GUS gene exhibited higher levels of transgene expression, compared with the transgenic plants that lacked the SINE. On average, plants transformed with SINE-containing vector expressed GUS at level 2-fold higher than the controls. However, the expression differences among individual transformants were still obvious.