‘蜂蜜罐’枣遗传转化条件的优化及Bt基因的导入

Optimization of genetic transformation system and transformation of Bt gene for epicotyl and cotyledon from seedlings of jujube 'Fengmiguan' (Ziziphus jujuba Mill)

为建立‘蜂蜜罐’枣高效遗传转化体系,以‘蜂蜜罐’枣胚培苗上胚轴和子叶为外植体进行转化,研究了不同菌液浓度、侵染时间、共培养时间、AS(乙酰丁香酮)浓度和侵染方式对gus(β-葡糖醛酸糖苷酶)基因瞬时表达率的影响。结果表明,当菌液OD_(600)值为0.5,真空辅助侵染10 min,共培养3 d,上胚轴和子叶外植体的共培养培养基中分别添加20和10 mg·L^(-1)乙酰丁香酮时,其gus瞬时表达率均最高,为最佳遗传转化体系。利用已优化的遗传转化体系进行Bt基因转化,共获得24个PCR(聚合酶链式反应)检测呈阳性的再生芽;对PCR检测呈阳性的不定芽进行伸长培养和不定根诱导,最终获得完整转基因植株。本研究初步获得了转Bt(苏云金芽孢杆菌)基因的枣植株,为通过转基因手段进行枣抗虫新种质选育提供可能。

The establishment of a high efficient and stable genetic transformation system is the basis for genetic improvement of plant germplasm by genetic engineering.In this study,the binary vector pBI121 containing gus(β-glucuronidase)gene and the neomycin phosphotransferaseⅡ(NPTⅡ)gene was introduced into jujube‘Fengmiguan’(Ziziphus jujuba Mill)by Agrobacterium-mediated transformation using epicotyls and cotyledons as explants.The effects of different bacterial concentrations,time of infection and co-cultivation,the concentration of acetosyringone(AS)and the infection pattern on the transient expression rate of gus were investigated.Bt(Bacillus thuringiensis)gene was also introduced into jujube‘Fengmiguan’by using the optimized genetic transformation system.The results showed that the most appropriate genetic transformation system by gus staining for the epicotyls and cotyledons was when bacterial culture reached OD600=0.5 and infected for 10 min under vacuum and then co-cultivated in a medium supplemented with 20 mg·L-1,10 mg·L-1 of AS for 3 d,resepctively,the rate ofgus transient expression on epicotyls was significantly higher than cotyledons.Bt gene introduced into‘Fengmiguan’was carried out by using the optimized genetic transformation system and epicotyls and cotyledons were used as explants.After co-cultivation in a selective medium,the resistant shoots with the height of 1~1.5 cm were analyzed by PCR.Total 24 transgenic shoots were obtained and elongated from epicotyls and cotyledons.The transgenic plantlets with Bt gene were eventually obtained.Thus would provide a possibility for creating insect-resistant germplasm.