利用RNAi方法构建MS26基因雄性不育植物表达载体及农杆菌介导玉米遗传转化研究

应用RNAi技术创制了花粉彻底败育的玉米雄性不育株系,为玉米杂交制种提供雄性不育的基础材料。构建玉米MS26 RNAi植物表达载体,其中,以玉米综31未成熟的幼胚组织为受体,利用农杆菌介导法将目的基因定向转入到玉米中,以甘露糖为选择剂进行筛选获得能够稳定遗传的转基因植株。通过Taqman探针法进行分子检测,获得18株单拷贝T0代转基因植株,碘-碘化钾染色分析结果显示,12株完全不育。在田间试验中,5个转化事件的所有T1代转基因植株在散粉期都表现雄性不育,且除此之外与野生型玉米对照植株之间没有其他形态不同。实时定量PCR结果显示,MS26 RNAi转基因玉米植株中MS26基因的表达量显著下调,由此可推断,MS26 RNAiT-DNA已经整合到玉米基因组中,并能引起完全的雄性不育。

Construction of MS26 gene Male Sterility Expression Vector with RNAi and Agrobacterium-mediated Transformation in Maize

The male sterile lines were obtained through transgenic methods, creating basic materials for the application of male sterile lines in maize hybrid seed production. The plant expression vector MS26 RNAi was constructed, and immature embryonic tissue(Zong31) had been used as the receptor for the agrobacterium-mediated transformation. Transgenic plants were obtained from transformation using mannose as the selectable marker. Eighteen single copy T0 plants were identified from TaqMan assay, twelve of which showed to be male sterile in the I2- KI Dyeing analysis. In the field trials, all single copy T1 plants of the five events showed 100% male sterility at the pollen-shattering stage, while the other agronomic traits were the same as those of wild type. The target MS26 RNAi gene had integrated into maize genome. Real-time PCR analysis showed that the expression of MS26 gene was significantly decreased in MS26 RNAi transgenic maize plants. It demonstrated that the T-DNA had been fully integrated into maize genome and resulted in the complete male sterility.