秦烟遗传转化体系的优化  

Study on optimization of transformation system for the Qin tobacco

作　　者：孔娜[1] 朱锦辉[1] 陈耀锋[1] 任慧莉[1] 郭东伟[1] 

机构地区：[1]西北农林科技大学农学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2008年第9期71-75,80,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：陕西省科技攻关项目(2003K03-G1-O4)

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50mg／L卡那霉素（Kan）筛选转化外植体用800mg／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0d）的外植体用稀释150倍的农杆菌菌液（OD600=0．5）浸泡15min，转至MS1培养基上共培养48h，用无菌水冲洗后于体积分数2％NaCIO中浸泡15min，用无菌水冲洗4～5次，置800mg／LCb＋质量分数0．1％的甘露醇中浸泡约12h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。[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 kanamyein （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 0 d） in the diluted 150 times Agrobacterium bacilli （OD600 =0. 5） for 1 rain,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% NaCIO 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-me-diated genetic transformation of tobacco leaves were obtained, which effectively reduced pollution rate of the explants and increased the differentiation rate of resistance bud.

关 键 词：烟草 HAL1基因 根癌农杆菌介导法 秦烟98 

分 类 号：S572.035.3[农业科学—烟草工业]