转基因水稻中HPT蛋白质的检测及表达特征研究

HPT Protein Detection and Expression Pattern in Transgenic Rice

hpt是转基因植物中最常用的筛选标记基因之一,建立了HPT蛋白质免疫印迹检测技术、了解其在转基因植物中的表达特征具有重要的理论意义和应用价值。在大肠杆菌中进行了HPT蛋白质的重组表达,利用纯化的HPT蛋白质制备了其特异性的单克隆抗体,采用免疫印迹学方法对转基因水稻4021中HPT蛋白质的表达情况进行了检测,该方法可检测出单粒稻米的10%(约1.5mg)所含的HPT蛋白质。定量分析发现水稻苗期HPT蛋白质含量约占鲜重的0.01‰。对Ca MV35S启动子驱动下的转基因水稻中HPT蛋白质的表达谱进行分析,发现其在苗期和成株期的叶片中表达量较高,在灌浆期的种子中呈现不断增加的趋势,而在茎、分蘖节、茎节、叶鞘、叶枕和根等组织中表达量较低。建立了完整的利用免疫印迹方法检测HPT蛋白质的技术,同时系统分析了HPT蛋白质在转基因水稻中的表达谱。

Hpt as one of the most commonly selective marker gene was used in transgenic plants. To establish immunoblotting method for detectting HPT protein expression profiles in transgenic plants has important theoretical significance and application value. HPT recombinant protein was expressed in E. coli,and used to prepare specific monoclonal antibody. Western blot was used to detect the expression of HPT protein in transgenic rice of 4021,the results showed that in 10% of single grain of rice（ about 1. 5mg） contained HPT protein can be detected. Quantitative analysis revealed that the content of HPT protein in rice seedling account for about 0. 01‰of fresh weight. The expression profiles of HPT protein under Ca MV 35 S promoter-driven start in transgenic rice showed that the abundance of HPT was regulated higher levels in the leaves of the seedling and adult,showing a rising trend in the seed filling period,but HPT showing low abundance in the stems,tillers,node,shears,cushion,roots and other tissues. In short,an applicable immunoblot method for detectting HPT protein was established,while the HPT protein expression profiles were analyzed in transgenic rice.