玉米FAD2基因RNA干涉表达载体的构建及转化

根据已克隆的ZmFAD2基因(GenBank登陆号:DQ496227)设计引物,通过RT-PCR扩增得到120 bp的特异性基因片段作为hpRNA干涉片段。利用pUCCRNA i与pUb i.cas为中间载体,成功构建了含Ub iqu itin启动子和hpRNA i片段的干涉表达载体p1 300 UFIFN。以自主选育的高油玉米自交系幼胚为材料,诱导、继代出胚性愈伤组织,利用携带p1 300 UFIFN质粒的根癌农杆菌LBA4404对其进行遗传转化。对抗性植株进行PCR检测,共获得6株转基因阳性株。

Construction of Maize FAD2-hpRNAi Expression Vector and Its Transformation

According to the sequence of the published delta-12 desaturase genes of maize(GeneBank accession DQ496227),a specific cDNA fragment was amplified by RT-PCR from the total RNA of maize immature embryos.The intron-spliced hairpin RNA plant expression vector p1 300 UFIFN was constructed succesessfully,incorporating Ubiquitin promoter and the 120 bp specific fragment.The embryogenic calli were initiated from hybrid lines of high oil maize breeded by ourselves,and transformed by Agrobacterium tumefaciens strain LBA4404 carrying p1 300 UFIFN vector.Six positive transgenic plants were obtained by the PCR analysis using hpt-specific primers.