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为了研究花粉致死基因ZmAA1的功能,进而创制转ZmAA1基因玉米不育新材料。在Gen Bank中找到花粉致死基因ZmAA1及启动子Pg47,并在目的片段的上下游设计増加酶切位点,基因合成构建到克隆载体puc57上,采用传统酶切连接的方法构建了植物表达载体pCAMBIA3300-Pg47-ZmAA1-35S-bar,并利用农杆菌介导法转化优良玉米自交系郑58萌动胚。成功构建植物表达载体pCAMBIA3300-Pg47-ZmAA1-35S-bar;共获得94株除草剂抗性植株,其中47株目的片段PCR呈阳性反应,对温室中表现为花粉败育的PCR阳性植株进行目的片段及筛选标记bar基因RT-PCR与蛋白免疫学试纸条检测,结果表明,花粉致死基因ZmAA1及启动子Pg47已经整合到玉米基因组并表达。
In order to study the function of the pollen lethal gene ZmAA1, a new maize sterility transgenic ZmAA1 gene was created. The pollen-lethal gene ZmAA1 and promoter Pg47 were found in GenBank. The restriction sites were designed upstream and downstream of the target fragment. The gene was synthesized and cloned into the vector pUC57. The plant expression vector pCAMBIA3300 -Pg47-ZmAA1-35S-bar, and the transformed maize inbred line Zheng 58 germinated by Agrobacterium-mediated transformation. A total of 94 herbicide-resistant plants were obtained, of which 47 were positive for PCR. The PCR-positive plants displaying pollen abortion in the greenhouse The results showed that the pollen lethal gene ZmAA1 and promoter Pg47 had been integrated into maize genome and expressed.