【作者】 刘伟；

【导师】 贾桂霞；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2006， 硕士

【摘要】 本文以亚洲百合‘布耐罗’（‘Brunello’）为研究对象，通过对鳞片再生条件、抗生素筛选条件及农杆菌介导百合遗传转化条件的优化，建立了高效的再生及转化体系，并将IPT基因和NPR1基因导入百合，对转化再生植株进行了分子检测及生理指标分析，主要研究结果如下： 1、含NPR1基因的植物表达载体pB35SNPR1的构建。将来自拟南芥的NPR1基因插入到质粒载体pGreen0229中，构建含有卡那霉素抗性选择标记的植物表达载体pB35SNPR1（pGreen0229-35S-NPR1-GFP），并通过液氮冻融法将其导入农杆菌EHA105，用于百合的遗传转化试验。 2、运用正交试验设计，建立亚洲百合‘布耐罗’（‘Brunello’）鳞茎外植体高效再生体系。外植体以1‰（W／V）升汞溶液作为主要灭菌试剂时，内部鳞片其最适处理时间为9min，存活率为55％；中部和外部鳞片的最适处理时间均为15min，存活率分别为50％和45％。运用正交试验设计，筛选鳞茎最佳分化培养基为MS+6-BA₂+NAA_0.2mg／L，再生率达86.1％以上；筛选出小鳞茎叶的最佳分化培养基为MS+6-BA₁+NAA_0.2+2,4D_0.1mg／L，再生率达78.9％； 3、以鳞片分化的小鳞茎叶基部作为遗传转化体系的受体材料，通过卡那霉素敏感性试验，确定了小鳞茎叶诱导不定芽卡那霉素临界致死浓度为100mg／L。对影响农杆菌转化效率几个主要因素进行的研究发现：选取分化15-20d的小鳞茎叶作为受体材料，在OD值为0.5的农杆菌菌液中侵染15min，侵染后共培养2d可获得较好的转化效果，转化率在16％左右；进一步研究发现，在菌液中添加50μmol／L乙酰丁香酮，在共培养基中添加100μmol／L乙酰丁香酮浓度时，转化率较不添加乙酰丁香酮高。 4、将含质粒pB35SNPR1的农杆菌EHA105与百合小鳞茎叶共培养，通过筛选培养，获得卡那霉素抗性植株，对22株转NPR1再生植株进行GFP荧光检测和PCR检测，其中12株为阳性，经GFP荧光检测和PCR检测表明，NPR1基因已经整合到百合基因组中。对20株转IPT再生植株进行PCR检测，其中11株为阳性。 5、对PCR检测呈阳性的转基因植株进行了生理指标检测，试验结果表明：转基因植株的抗病性和抗衰老性都较阴性对照高。更多还原

【Abstract】 In this work, high frequency regeneration and transformation systems were established based on study of the factors affecting polar bulbs explants regeneration efficiency and gene transfer efficiency. IPT and NPR1 genes were introduced into Lilium through the improved protocols. The molecular detection and physiological analysis were also studied. The main research work and results were as follows:1. Construction of plant express vector pB35SNPR1 including NPR1 gene.NPR1 gene was injected into plasmid vector pGreen0229 to construct plant express vector pB35snpr1 （pGreen0229-35S-NPR1-GFP） selected noting by Kan. And then the new vector was further transferred into EHA105 by freeze-thaw method and used for transformation experiment of Lily.2. High frequence regeneration system of bulbs explants was established by orthogonal design. The results showed that the optimal time for interior bulbs was 9min, oppositely 15min for medium and exterior bulbs , and surviving rate reached 55%, 50%,45%, respectively, by 1‰ （W/V） Hg solution for explants killing agrobacteria after transformation. Meantime, the optimal regeneration medium was MS+6-BA₂+NAA_0.2mg/L for bulbs and MS+6-BA₁+NAA_0.2+2,4D_0.1mg/L for leaves, and the regeneration rate reached 86.1%, 78.9%, respectively.3. Kan sensitive test indicated that the critical Kan sensitive concentration for inducing adventitious buds was 100mg/L, based on the regenerated leaves as materials for transformation system.Several crucial factors influencing the transformation efficiency were studied. It was found that pre-culture of leaves for 15-20 days before infection with A.tumefaciens （OD-0.5） for 15min, co-culture for 2 days after infection would be favorable for the transformation, the regeneration rate was 16% around; further investigated that 50μmol/L and 100μmol/L AS was supplemented into EHA105 and co-culture medium respectively would increase transformation rate.更多还原