NPTⅡ蛋白质在转基因水稻中的表达特征研究

The Expression Properties of NPTⅡ Protein in Transgenic Rice Plant

nptⅡ编码新霉素磷酸转移酶,是转基因实验中最常用的筛选标记基因之一.本研究中,表达了重组新霉素磷酸转移酶(NPTⅡ)蛋白质,制备了单克隆抗体,建立了用免疫印迹检测转基因水稻中NPTⅡ蛋白质的方法,调查了NPTⅡ蛋白质的表达特征,包括遗传特征、表达时空特征、表达丰度和亚细胞定位等.结果表明,该方法可检测到单粒稻米的0.25%(约0.025 mg)样品中的NPTⅡ蛋白质,NPTⅡ的表达符合显性遗传规律,根据NPTⅡ的表达可对转基因T1代种子进行阴阳性及纯合株系的鉴定,对T1幼苗中NPTⅡ丰度的分析可为纯合单株的鉴定提供参考信息.定量分析表明苗期叶片中NPTⅡ蛋白质的含量约为鲜重的0.08‰.在Ca MV-35S启动子驱动下NPTⅡ蛋白质在水稻苗期、成株期的叶片和根部组织中表达量较高,而在分蘖期、孕穗期较低,NPTⅡ在不同时期的根、茎、叶、穗子、花及种子等组织中均有表达,只是在分蘖节、茎节、穗轴和花药等组织中表达量相对较低.此外,NPTⅡ蛋白质的表达主要定位在细胞质中.综上所述,本研究建立了具有应用价值的检测NPTⅡ蛋白质的免疫印记方法并系统揭示了其在水稻中的表达特征.

The npt Ⅱ gene, encoding neomycin phosphotransferase Ⅱ, is one of the most commonly used selection marker in transgenic research. In this study, recombinant NPT Ⅱ protein was expressed in E. coli and monoclonal antibodies were generated. The inheritance, temp-spatial profiling, abundance and subcellular localization properties were investigated using established Western blot（WB） analysis. The results showed that NPTⅡ protein in 0.25% of a single rice grain（about 0.025mg） was detectable, the expression of NPTⅡ protein is consistent with dominant inheritance law, positive seeds and homozygous lines can be identified in transgenic T1 generation, furthermore, the abundance of NPT Ⅱ protein can provide clues for the identification of homozygous individual in T1 seedlings. Quantitative analyses shows that NPT Ⅱ protein accounts for about 0.08‰ of total protein in the leaves at seedling stage. The expression profile of NPT Ⅱ protein in transgenic rice shows that the abundance of NPTⅡ driven by Ca MV-35 S promoter in the leaves and roots at seedling and adult stage are higher than that at tillering and booting stage. NPT Ⅱ was detectable at different stages in rice root, stem, leaf, panicle,flower and seed, however, the abundance of NPT Ⅱ in tillers, node, panicle axis and anther tissues is lower than others. In addition, the NPTⅡ protein is mainly localized in the soluble portion of cytoplasm. Taken together, an applicable immunoblot method was established and the expression properties of NPT Ⅱ protein in transgenic rice were demonstrated.