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

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

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。

The male sterile lines were obtained through transgenic methods, creating basic materials for the ap-plication of male sterile lines in maize hybrid seed production. The plant expression vector MS26 RNAi was con-structed, and immature embryonic tissue(Zong31) had been used as the receptor for the agrobacterium-mediatedtransformation. Transgenic plants were obtained from transformation using mannose as the selectable marker. Eigh-teen single copy T0 plants were identified from Taq Man 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 thepollen-shattering stage, while the other agronomic traits were the same as those of wild type. The target MS26 RNAigene had integrated into maize genome. Real-time PCR analysis showed that the expression of MS26 gene was sig-nificantly decreased in MS26 RNAi transgenic maize plants. It demonstrated that the T-DNA had been fully inte-grated into maize genome and resulted in the complete male sterility.