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将PCR扩增获得的水稻花药绒毡层特异表达基因Osg6B启动子(简写成6B)与来自质粒pROKⅡ上的NOS终止子相连,产生了pGEM7Z-6BNOS。然后将来自解淀粉芽孢杆菌(Bacillus amylodique faciens)中的Barnase和Barstar基因(简写成BN和BS)分别插入到pGEM7Z-6BNO上的BamH I和Nco I位点上,再用Sal I和Xho I切下2.3 kb的6BBNNOS相2.2kb的6BBSNOS片段,并分别将其插入到pDM302的Xho I位点上,经酶切分析表明6BBNNOS和6BBSNOS在pDM302上的方向相反。从而成功地构建了水稻雄性不育和其育性恢复表达载体pDM302-6BBNNOS和pDM302-6BBSNOS。用基因枪轰击水稻幼胚,获得了抗膦化麦黄酮(PPT)的水稻转基因植株,可望形成优良的水稻雄性不育和育性恢复系,以应用于生产。
The Osg6B promoter (shortened to 6B) of the anther tapetal-specific gene of rice anther obtained from PCR amplification was ligated with the NOS terminator from plasmid pROKII to generate pGEM7Z-6BNOS. The Barnase and Barstar genes (abbreviated as BN and BS) from Bacillus amylodique faciens were then inserted into the BamH I and Nco I sites on pGEM7Z-6BNO, and then digested with Sal I and Xho I The 2.3 kb 6BBNNOS-containing 6BBSNOS fragment was inserted into the site of Xho I of pDM302. The digestion analysis showed that the orientation of 6BBNNOS and 6BBSNOS was opposite to that of pDM302. Successfully constructed rice male sterility and fertility restore expression vectors pDM302-6BBNNOS and pDM302-6BBSNOS. Using gene gun to bombard rice immature embryos and obtaining transgenic rice plants resistant to phosphinothricin (PPT), it is expected to form excellent rice male sterility and fertility restorer lines for production.