【摘要】 以山新杨(Populus davidiana Dode×P.bollena Lauche)为受体材料,在筛选遗传转化条件的基础上,利用农杆菌介导叶盘转化法进行了Bt+蜘蛛杀虫肽基因转化研究。结果表明:在叶片转化时,卡那霉素的临界质量浓度为10mg/L;生根筛选培养时卡那霉素的临界质量浓度为15mg/L;利用质量浓度为600mg/L的头孢噻肟钠脱菌,对叶片生长和不定芽形成影响不大。山新杨遗传转化的工程菌菌液浓度以0.1～0.2(OD600)为最佳,其叶片分化率为73%～77%、抗性芽率为20%～23%;浸染时间以2～5min为宜,其叶片分化率和抗性芽率最高。在共培养基中加入200μmol/L的乙酰丁香酮,其叶片分化率和抗性芽率分别比对照提高1.43倍和2.61倍。对转化植株的PCR检测结果呈阳性,初步证明外源基因Bt+蜘蛛杀虫肽导入并整合进了山新杨基因组。更多还原

【Abstract】 The gene of Bt+ spider toxin was transformed using Populus davidiana Dode×P. bollena Lauche as transgenic acceptors by Agrobacterium tumefaciens mediated method. The leaf discs were immersed into 0.1~0.2 OD600 of bacteria solution for 2~5min,which was the optimal condition for infection. The threshold concentration of kanamycin was 10mg/L for differentiation and 15mg/L for rooting. 600mg/L of cefotaxime could inhibit the growth of bacteria without inhibition to the differentiation of leaf discs. As results,the disc differentiation rate was 73 to 77 percent,20 to 23 percent of which generated buds resistant to kanamycin. When 200μmol/L of acetosyringone was supplemented into the medium,the disc differentiation rate increased by 1.43 times of the control,and the rate of buds resistant to kanamycin also increased by 2.61 times of the control. PCR result shows that the exogenous gene has integrated into the genome of the poplar.更多还原