花粉介导法获得耐草甘膦玉米植株及其耐性研究

Development of Transgenic Maize Plants Tolerant to Glyphosate via Pollen-mediated Transformation and Their Glyphosate Tolerance

为获得耐草甘膦转基因玉米植株,进而研究其除草剂耐性及外源基因的遗传特性,以质粒p BI101-aroA-M12为外源基因供体,以玉米自交系昌‘7-2’为受体,采用花粉介导法将外源基因导入玉米自交系中。PCR扩增、Southern杂交分析的结果证明获得了54个转基因植株;对其后代株系的分子检测及田间生物学鉴定,结果证明目的基因可以稳定遗传给后代植株,且赋予和提高了转基因植株的除草剂耐性。目的基因在转化体中的遗传规律研究,结果显示导入的目的基因在F2受体植株中以3∶1的比例发生遗传分离,符合孟德尔单因子显性遗传分离规律。ELISA分析和蛋白质浓度测定的结果证实目的基因在转化植株中得到表达,表达量介于40.5-112.6 ng/g叶片鲜重之间,目的基因表达量与该植株的除草剂耐性性状间呈极显著相关,r=0.942 3(P<0.01)。最终研究获得了4个高草甘膦耐性的转基因纯合株系。

In order to develop transgenic maize plants tolerant to glyphosate and further study their herbicide tolerance and the geneticcharacteristics of exogenous gene in transgenic plants,plasmid p BI101-aroA-M12 harboring aroA-M12 gene was transformed into maize inbredChang ‘7-2＇ plants by pollen-mediated transformation method. Fifty-four transgenic plants were obtained by PCR detection and Southern blotanalysis. The detection results of molecular analysis and bioassay in the field showed that target aroA-M12 gene was integrated into the genomeof transgenic plants and could stably inherit to next generation. Moreover,the aroA-M12 gene conferred the glyphosate-tolerance to transgenicplants. The result of genetic rule of target gene in transgenic plants suggested that the target gene was segregated according to the ratio of 3∶1in T2 generation transgenic plants（lines）and it was consistent with the Mendel law of single-factor heredity. The results of ELISA analysisand protein concentration determination of transgenic plants confirmed that the target gene was expressed in transgenic plants,and the proteinamount of gene expression were 40.5-112.6 ng/g leaf fresh weight,there was a significant correlation between protein amount of expression ofeach transgenic plant and corresponding glyphosate-tolerance of that plant,correlation coefficients r = 0.942 3（P0.01）. Ultimately,4 purelines of transgene with high glyphosate-tolerance were obtained from this experiment.