绿色荧光蛋白基因作为报告基因在水稻基因转化中的应用研究

本研究中 ,构建了含有编码绿色荧光蛋白的改进型基因质粒pJPM5。用基因枪法分别把pJPM5和另一带有绿色荧光蛋白基因的质粒pSBG70 0转入水稻TNG6 7愈伤组织。用South ern杂交法证实了转基因的存在 ,而且表明多数转基因植株含有 1到 8个拷贝的转基因。取 2个月的转基因植株上的叶片用于分析绿色荧光蛋白基因表达。用SLM - 80 0 0荧光分析仪定量测定绿色荧光蛋白。多数转基因植株具有很高的绿色荧光蛋白信号。虽然水稻植株有少量自发荧光 ,但是绿色荧光蛋白基因表达出的绿色荧光蛋白信号比植株的自发荧光强得多 ,其测定不会受自发荧光的太大影响。在荧光显微镜下观察到了绿色荧光蛋白基因的表达。借助观察分析绿色荧光蛋白基因的瞬时表达 ,本研究还发现基因枪法转化中 ,如果两枪的气压为90 0psi& 135 0psi,比两枪的气压都为 90 0psi或者 135 0psi更好 ,因其能使质粒进入更多的细胞。研究结果表明 ,绿色荧光蛋白基因可以作为水稻 (甚至小麦、玉米 )转基因研究中的报告基因。研究还显示 ,MAR序列能明显增强绿色荧光蛋白基因的表达能力 (这一结果在另文讨论 ) .

The Applications of GFP as a Reporter Gene in Rice Genetic Transformation

In this study,a plasmid pJPM5 containing modified GFP gene which encode the green fluorescent protein (GFP) was constructed.Microprojectile bombardment was used to separately introduce two plasmids pJPM5 and pSBG700 into rice TNG67 calli.The transgenic plants were confirmed by Southern blot.Most of the transgenic plants contain 1 to 8 copies of transgene.GFP gene expression was measured in the extracts from rice leaf of two month old transgenic plants.And the quantification of gfp expression is carried by the SLM-8000 Fluorescence Analyzer. Most of the transgenic plants have showed significant GFP signal. Even though the rice plant has auto fluorescence, the expression of GFP is high enough to give out much higher fluorescence signal compared with auto fluorescence of rice plants.The GFP was also observed in transformed calli under microscope.The transient expression of GFP helped to demonstrate that the bombardment shots of 900psi & 1350 psi make the particles carrying plasmids reach more cells than the bombardment shots of 2X 900psi or 2X 1350psi. Our results showed that this modified GFP gene can be used as a reporter gene for rice genetic transformation and even for other cereal crops such as wheat and corn etc.The result also professes that the MAR sequence can enhance the expression of GFP gene significantly(data was shown in another paper).