| 摘要: |
| 【目的】 研究根癌农杆菌介导的芪合酶基因转化烟草遗传体系的优化条件。【方法】 以“中烟99”叶片为受体，采用根癌农杆菌(Agrobacterium tumefaciens,GV3101)介导法，将芪合酶基因导入烟草基因组，并对其遗传体系进行了优化，对筛选获得的转基因烟草植株进行检测。【结果】 根癌农杆菌介导的芪合酶基因遗传转化的最佳条件为：将预培养2 d的烟草外植体，用稀释10倍的GV3101菌株菌液（OD600=0.6）浸染8 min后，共培养3 d，然后转入含卡那霉素（Km）50 mg/L和羧苄青霉素（Cb）500 mg/L的筛选分化培养基上诱导分化，待抗性芽长到2～3 cm后，转入含Km 50 mg/L和Cb 500 mg/L的筛选生根培养基上进行筛选，在以上最佳遗传转化条件下，初步筛选到54株转化体，经PCR和RT-PCR检测获得25株转化烟草植株。【结论】 经卡那霉素筛选、PCR、RT-PCR检测后，初步证明芪合酶基因已被整合到烟草基因组中，并可以正常转录。 |
| 关键词: 芪合酶基因 烟草 根癌农杆菌 遗传转化 |
| DOI： |
| 分类号: |
| 基金项目:陕西省“13115”科技重大专项(2008ZDKG-07) |
|
| Study on the optimization of transferring stilbene synthase gene into tobacco mediated by Agrobacterium tumefaciens |
|
|
| Abstract: |
| 【Objective】 The research studied the optimization of genetic transformation with the stilbene synthase gene by Agrobacterium mediated.【Method】 With the sterile leaves of “Zhongyan 99”,using the Agrobacterium (Agrobacterium tumefaciens,GV3101) mediation method,the stilbene synthase gene was translated into tobacco genome,and the genetic transformation system was optimized,then the transgenic plants were detected.【Result】 The best optimum conditions of the Agrobacterium mediated genetic transfornation were that leaves explants were immersed in the diluter 10 times Agrobacterium bacilliat OD600 of 0.6 for 8 minutes after being pre-cultured for 2 days,and then co-cultivated for 3 days.Then these explants were transferred to selected and rooting medium which contained 50 mg/L Kanamycin and 500 mg/L Carbenicillin.According to the transformation system, 54 transgenic plants were initially screened,then after PCR analysis and RT-PCR analysis 25 transgenic plants were obtained.【Conclusion】 The transformed plants were selected by Kanamycin,and PCR analysis and RT-PCR analysis preliminarily proved the stilbene synthase gene had been integrated into the tobacco genome and could transcript normally. |
| Key words: stilbene synthase gene tobacco Agrobacterium tumefaciens genetic transformation |
