根癌农杆菌介导Cry1Ac基因转化‘雪青’梨获得转基因植株

Obtainment of Transgenic 'Xueqing' Pear Plants with a Synthetic Cry1Ac Gene Mediated by Agrobacterium tumefaciens

以‘雪青’梨叶片愈伤组织为外植体,经根癌农杆菌介导将CaMV35S启动子调控下的Cry1Ac基因导入‘雪青’梨。698块愈伤组织和231个Kanr芽与携带表达载体质粒pBX203的根癌农杆菌菌株LBA4404共培养3d后,转入含50mg.L-1Kan的筛选培养基培养30d,15d转接1次。结果表明,在MS+5mg.L-16-BA+0.1mg.L-1IAA筛选培养基中,Kanr芽率为34.24%,在1/2MS+2mg.L-1IBA+0.5mg.L-16-BA+500mg.L-1AC筛选培养基中,15.58%Kanr芽生根。共获得再生植株32株,经PCR和Southern-blot分析证明,其中4株的基因组已成功导入和整合Cry1Ac基因。

By using calli from leaves of Xueqing pear （Pyrus sp. ） as explants, the Cry1Ac gene regulated by CaMV35S promoter was introduced into Xueqing pear through Agrobacterium-mediated transformation. After co-culturing 698 calli and 231 Kan^r buds, respectively, with A. tumefaciens strain LBA4404 harboring a plasmid pBX203 for 3 days and transferred to the selective medium containing 50 mg·L^-1 kanamycin for 30 days at a 15-day interval, 34. 24% kanamycin-resistant （Kanr） buds （adventitious shoots） regenerated on MS ＋5 mg·L^-1 6-BA ＋0. 1·L^-1 IAA and 15.58% of Kan^r buds rooted on 1/2 MS＋2 ·L^-1 IBA ＋0.5·L^-1 6-BA ＋500 ·L^-1 AC. A total of 32 plants were obtained, but only 4 plants were confirmed by PCR and Southern-blot analysis that the Cry1Ac gene was transferred and integrated into the genome of Xueqing pear.