甜蛋白基因MBLII 对莴苣的遗传转化

 以4 日龄莴苣(Lactua sativa L.) 无菌苗子叶为外植体, 通过根癌农杆菌介导,成功地进行了马槟榔甜蛋白基因MBLII 对莴苣的遗传转化。抗生素浓度和子叶外植体与农杆菌的共培养时间是影响转化的重要因素。附加卡那霉素( Kan) 50 mg/ L 的诱芽培养基MS I(MS+ NAA 0. 1 mg/ L+ 6􀀁BA 0. 1 mg/ L+ 羧卞青霉素500 mg/ L) 最适于侵染后子叶外植体的诱芽培养。在外植体与农杆菌共培养0~ 7 d 的范围内, 以共培养3 d 最佳( 生芽率58. 3%, 白化率29%) 。1~ 2 cm 再生芽移入诱根培养基MS II (MS+ NAA 0. 05 mg/ L+ Kan 50 mg/ L+ 羧苄青霉素300 mg/ L) 中, 诱根率可达100%。获得的抗性植株经组织化学及PCR 特异扩增鉴定和统计, 7. 6%阳性。Southern blot 结果证明MBL II 基因已整合到莴苣基因组中。

Genetic Transformation and Plant Regeneration of Lettuce with Sweet Protein Gene MBLII

The sweet protein gene MBL II was introduced into lettuce by co culturing excised cotyledon explants with Agrobacterium tumefaciens .The Agrobacterium strain LBA4404 contained the binary vector pLX10,which carried sweet protein gene MBL II,selective gene NPT II and reporter gene GUS .The best transformation efficiency was obtained when explants were co cultured with Agrobacterium for 3 days and cultured on the selective medium containing 50mg/L kanamycine(shoots regeneration rate 58.3%,albino rate 29%).Roots were induced in all adventitious shoots on MSII(MS NAA 0.05mg/L Kan 50mg/L carbencillin 300mg/L).The transgenic nature of regenerants were demonstrated by plant growth on medium containing kanamycine, GUS histochemical assay and PCR amplification.7.6% of all Kanamycine resistant shoots was GUS positive. Southern blot results confirmed that the MBL II gene had been incorporated into the genome of lettuce.