| 摘要: |
| [目的]优化烟草的高效遗传转化体系.[方法]以陕西地区广泛种植的烟草品种秦烟98为试材,采用根癌农杆菌(Agrobacterium tumefaciens,LBA4404)介导法,将从酿酒酵母细胞中克隆到的具有较强耐盐表型的HAL1基因进行遗传转化,对农杆菌介导的遗传转化条件及其影响因素进行研究.[结果]用50 mg/L卡那霉素(Kan)筛选转化外植体用800 mg/L羧苄青霉素(Cb)除菌可以获得理想的筛选和除菌效果.以优化的农杆菌介导烟草叶片的遗传转化条件为:将未经过预培养(预培养0 d)的外植体用稀释150倍的农杆菌菌液(0D600=0.5)浸泡1min,转至MS1培养基上共培养48 h,用无菌水冲洗后于体积分数2%NaClO中浸泡15 min,用无菌水冲洗4～5次.置800 mg/L Cb 质量分数0.1%的甘露醇中浸泡约12 h除菌.[结论]获得了农杆菌介导烟草叶片遗传转化的最佳优化条件,有效地减少了外植体的污染率,提高了抗性芽分化率. |
| 关键词: 烟草 HAL1基因 根癌农杆菌介导法 秦烟98 |
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| 基金项目:陕西省科技攻关项目（2003K03-G1-O4） |
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| Study on optimization of transformation system for the Qin tobacco |
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| Abstract: |
| 【Objective】 The study was to optimize the efficiency of tobacco genetic transformation system.【Method】 With the Qinyan 98 extensively planted in the region of Shaanxi as test matericals,using the traditional Agrobacterium (Agrobacterium tumefaciens,LBA4404)-mediated,the Agrobacterium mediated genetic transformation conditions and factors with the gene from yeast cells were studied to clone the table with strong salt-tolerant gene of the HAL1.【Result】 Ideal result was obtained after scanning the explants with 50 mg/L kanamycin (Kan) and degerming with 800 mg/L carboxymethyl benzylpenicillin (Cb).The optimization conditions of Agrobacterium-mediated genetic transformation were:dipping the explants which were not been pre-trained (pre-culture 0d) in the diluted 150 times Agrobacterium bacilli (OD600=0.5) for 1 min,then transferred the explants to the medium MS1.After culturing for 48 h,the explants were washed with sterilized distilled water and then dipped into volume scores 2% NaClO for 15 min.The explants,rinsed with sterilized distilled water four to five times,were dipped into 800 mg/L Cb+quality scores of 0.1% mannitol about 12 h.【Conclusion】 The best optimum conditions of the Agrobacterium-mediated genetic transformation of tobacco leaves were obtained,which effectively reduced pollution rate of the explants and increased the differentiation rate of resistance bud. |
| Key words: tobacco HAL1 gene Agrobacterium tumefaciens mediated Qinyan 98 |
