【作者】 王贺一；

【导师】 杨毅；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 油菜（Brassica napus L．）属于十字花科芸苔属，是世界四大油料作物之一，在全球产量中处于第三位，仅次于大豆和棕榈油。近年来，真菌等病害造成了油菜产量和品质的大幅下降，而消除病害所需要的农药投入巨大，同时造成了严重的环境污染。如能通过对油菜进行转基因改良，使其自身具有良好的抗病害能力，将具有重要的经济价值和环保意义。植物防御素（Rlant defensins）是一种存在于多种植物中的小分子（45-54氨基酸）碱性多肽。其保守的含有8个Cys，而形成4个二硫键。因其与动物防御素同源而得名。植物防御素具有广谱高效的抗微生物活性，能有效的杀灭G+、G-、某些真菌、螺旋体、被膜病毒等微生物，而对植物细胞没有伤害，是植物先天性非特异性免疫的重要组成部分。2005年罗勤等从十字花科植物白芥种子中成功地克隆了编码植物防御素的基因，在原核表达载体GTK中对白芥防御素基因进行了融合表达，并通过花序浸染法转入拟南芥中进行表达，以研究防御素基因功能。本研究以甘蓝型优质高产杂交油菜新品种“蜀杂九号”恢复系材料84100-18（Brassica napus L．）为实验材料，以下胚轴为外植体，通过根癌农杆菌EHA-105的介导，将其携带的白芥防御素基因（Sad）转入油菜中，经共培养、抗生素筛选、生根获得了高抗性转化植株。对所获得的转化植株进行了PCR检测结果表明，外源的Sad基因已经整合入油菜基因组中。论文还对影响农杆菌介导法转化效率的主要因素进行了分析讨论。主要实验结果如下：1．通过对比观察外植体的诱导及分化情况，建立并优化了油菜植株的再生体系。试验表明：下胚轴的分化频率高于子叶且离生长点0.5-1cm的下胚轴切段最高；菌液稀释至OD₆₀₀0.5，浸泡时间1min，共培养2d；农杆菌再悬浮液与共培养基中加入20mg／L乙酰丁香酮可提高转化频率；AgNO₃可提高下胚轴芽的分化率；MS+2mg／L6-BA+1mg╱L 2，4-D时芽的分化率最高；卡那霉素起始选择浓度为10mg／L。2．用含有Sad基因的植物表达载体pBI121-Sad的根癌农杆菌转化油菜，对各项转化条件进行优化，确定了农杆菌EHA105转化油菜外植体的最佳条件。确定用植株再生的最佳培养基分别为：Ⅰ无菌苗培养基：MS基本培养基Ⅱ预共培养基：MS+2mg╱L6-BA+1mg╱L 2，4-D+2.5mg／LAgNO₃+19.62mg／LAsⅢ筛选培养基：MS+2mg／L 6-BA+2.5m╱LAgNO₃+500mg╱L Carb+10mg／L KanⅣ生根快繁培养基：1／2MS+0.15mg／LNAA+250mg╱LCefⅤ摇菌培养基：LB+40mg╱LRif+20mg╱LStr+50mg／LkanⅥ重悬培养基：MS+19.62mg╱L As3．转化后经卡那霉素筛选获得了油菜抗性苗，通过PCR验证，初步证明得到了含外源Sad基因的转基因植株。更多还原

【Abstract】 Rape （Brassica napus L.） is a kind of very important economic crop and oil plant at home and abroad. But everywhere the rape were threatened by the illness caused by bacteria and fungi.Plant defensins are small, basic peptides that exhibit antimicrobial activity against a broad range of bacteria and fungi. Because of their three-dimensional folding pattern, they can inhibit the growth of many fugi without damaging plant. Plant defensins can protect plant seeding and fight against pathogens which attack plant. Luo et al. （2005） had cloned the Sad gene successfully. By prokaryotic expression, the cDNA of Sad gene was inserted into the protein expression vector, GTK to induce Sad gene expression. And they had transferred to Arabidopsis by plant method to study Sad function.In the thesis, restorer line of "SHUZA No.9" which is a new sort of hybrid rape （brassica napus） is used as my experimental material, the hypocotyls were used as explants and were transformed by Sad gene via Agrobacterium tumefaciens-mediation method. Transgenic plants were obtained after co-culture, antibiotic screen and root regeneration. The transgenic plants were detected with PCR. The results suggest that foreign Sad gene has already been combined into the rape genome and were expressed. The factors that influence the frequency of agrobacterium mediated transformation were also discussed.The details are shown as followings:1. An optimized system of re-generation is formed, through comparing and observing the different situations of the induction and differentiation of the explant. Experiment shows the following results: The differentiation frequency of hypocotyl is higher than that of cotyledon and the 0.5-1cm hypocotyl which doses to the growth ponit is the best; hypocotyls were infecting for 1 min as OD₆₀₀ of Agrobacterium suspending liquid is 0.5; hypocotyls were co-cultered with Agrobacterium for 2 days. The transformation frequency of explants was promoted when 20 mg/L acetosyringone was added in Agrobacterium suspension liquid and co-cultured medium. AgNO₃ can increase the differentiation frequency of hypocotyl. the differntiation frequency of bud is the highest when MS＋3mg/L6－BA＋1mg/L 2, 4-D: it was useful for anti-Kan positive shoot to produce when initial concentration of kanmaycin was 10mg/L.2. Agrobacterium tumefaciens which contains the vector pBI121-Sad is used to transform the plants of rape （Brassica napus）. The condition of transforming is also optimized, and the best conditions of culture media are conformed as followings:Ⅰmedium of shooting: MS;Ⅱmedium of pre-culture& co-culture :MS+2mg/L6-BA+1mg/L2,4-D+2.5mg/LAgNO₃+19.62mg/LAs;Ⅲmedium of inducing callus& differentiation:MS+2mg/L6-BA+2.5mg/LAgNO₃+500mg/L Carb+10mg/L KanⅣmedium of rooting:1/2MS+0.15mg/LNAA+250mg/LcefⅤmedium of Agrobacterium activation: LB+40mg/LRif+20mg/LStr+50mg/LkanⅥmedium of Agrobacterium suspendingMS+19.62mg/LAs3. Resistant seedlings are obtained after being selected by kan, and elementarily proved to be Sad gene via PCR test.更多还原