【作者】 李志友；

【导师】 张兴国；

【作者基本信息】 西南大学 ， 蔬菜学， 2007， 硕士

【摘要】 番茄（Lycopersicum esculentum Mill.）为茄科（Solanaceae）番茄属的一年生草本植物，因其果实营养丰富、美味可口，已成一种世界性蔬菜。低温一直是限制番茄生长与分布，影响果实品质和产量的重要环境因子。如何提高番茄对低温的耐受性一直是人们关注的焦点。快速发展的植物基因工程技术为改善番茄品种的抗寒性提供了新途径；其中，利用能够促进大量冷调节基因表达的冷应答转录因子基因并辅以强效抗寒基因的策略，是近年来尝试提高植物抗寒性的重要方式。本实验室已将拟南芥的冷应答转录因子基因AtCBF3和主效抗寒基因AtCOR15a分别受RD29A低温诱导启动子控制后导入烟草，使转基因烟草能耐受-1℃～-1.5℃的低温。本研究拟将RD29A-CBF3和RD29A-COR15a双价基因导入番茄基因组，以期得到抗寒的番茄材料，为今后进一步通过分子途径改良番茄的抗寒性奠定基础。番茄的组织培养研究开展得较早，但一直无法摆脱分化率低的问题，而分化率又受到番茄品种基因型，外植体类型和生理状态，外源激素种类和组合浓度，培养环境等诸多条件的限制和影响。因此能否综合系统地考虑各种条件因子的影响，建立高效稳定的番茄再生体系是番茄遗传转化应用的关键问题之一。同时，影响遗传转化率的因素也很多，如植物基因型、外植体类型、农杆菌菌液浓度和侵染时间、预培养和共培养时间、抗性选择抗生素浓度等。如何提高转化率，建立高效、稳定的遗传转化系统，仍是今后研究的重要课题。本实验在优化番茄高效再生体系和遗传转化体系的基础上，采用农杆菌介导的方法将RD29A-CBF₃和RD29A-COR15a双价基因导入番茄。主要实验内容和结果如下：1．以番茄高代自交系为材料，从外源激素浓度和外植体类型等方面优化了番茄再生体系。试验结果表明：浸种、暗培养对番茄种子发芽有益，能获得比较整齐的番茄幼苗；不同的激素浓度配比对番茄外植体不定芽再生有不同的影响：以MS+6-BA 1.0mg/L+IAA 0.1mg/L+3％蔗糖+0.7％琼脂（pH5.8）对子叶、MS+6-BA 0.8mg/L+IAA 0.1mg/L+3％蔗糖+0.7％琼脂（pH5.8）对下胚轴和MS+6-BA 1mg/L+IAA 0.2mg/L+3％蔗糖+0.7％琼脂（pH5.8）对真叶、茎段的作用效果比较显著；子叶再生效果优于下胚轴、真叶、茎段；生根培养基以MS+IAA0.5mg/L+3％蔗糖（pH5.8）比较合理。2．从外植体类型、农杆菌菌液浸染时间、预培养和共培养时间、抗性选择抗生素浓度五个方面对番茄遗传转化体系进行了比较系统的优化，结果证明：预培养2d、农杆菌侵染时间8min和共培养2d的转化操作的效果比较理想；子叶为60mg/L的Kan，下胚轴、真叶和茎段为70mg/L的Kan是适宜的抗性选择浓度。3．通过农杆菌介导的遗传转化将RD29A-CBF3和RD29A-COR15a双价基因导入番茄，对卡那霉素抗性植株进行PCR检测，能扩增出预期的目的带，初步证实目的基因已整合到番茄染色体基因组中。更多还原

【Abstract】 Tomato named Lycopersicum esculentum Mill in Latin, an annual herbage plant in the Solanaceae or nightshade family, becomes a world-wide vegetable because of its rich nutrition and delicious taste now. Low temperature is the key factor that limits the geographical distribution、quality and yield of tomatoes. The topic of how to improve the cold-resistance of tomatoes has always been brought into focused that people attach importance to. Recently new approaches have been achieved by speedily developing plant genetic engineering technique, among which making full use of cold response transcription activators that promote expression of cold induced genes, with the help of cold-resistant genes, is an important ways and means to try to enhance cold resistance of plant in recent year. Two genes draw up from Arabidopsis thaliana, CBF₃ and COR15a, which are controlled by cold induced promoter:RD29A, were introduced into tobacco in our lab and made it alive above -2℃. In order to obtain cold resistant material of tomato breeding to improve tomato anti-cold further by molecule path. These two genes, RD29A-CBF₃ and RD29A-COR15 a, try to be introduced to tomato in this research.These researches about tomato tissue cultivation have carried on for a long time, however, a problem that the differentiation rate of tomato tissue cultivation is low very much hasn’t always been resolved successfully. Because the differentiation rate was influenced by many factors such as: genotype, types of explants, physiological character, kinds and concentrations of exogenous hormone, culture conditions and so on, whether various factors should be taken into consideration by the numbers is one of the premises to construct a high regeneration system successfully and apply tomato genetic transformation in the round. At the same time lots of conditions impress genetic transformation rate, which is genotype, explants, the concentration and time of infection with Agrobacterium, pre-culture and co-cultivation time, selection and concentration of antibiotic resistance and so on. How to improve the genetic transformation rate of tomato and construct a genetic transformation system with high efficiency and good stability will be still one of the most important research project in the future. This experiment based on optimizing tomato regeneration system and genetic transformation system made RD29A-CBF₃ and RD29A-COR15a introduced into tomato by Agrobacteriu transformation systems. The primary experiment results and contents are as follows:1 Two key factors analyzed to optimize the regeneration system of tomato were exogenous hormone concentration and explants types and the experimental material is highly genetic. The results showed that tomato axenic seed burgeoned rapidly and tidily after seeds were soaked in water and cultivated in dark. The frequencies and amounts of shoot organogenesis were significantly affected by the hormone concentrations for a given genotype. The best culture medium for cotyledon was MS （3% sucrose） + 6-BA 1.0mg/L + IAA 0.1mg/L0.7% agar （pH5.8 ） , for hypocotyl was MS （3% sucrose） + 6-BA 0.8mg/L + IAA 0.1mg/L+0.7%agar （pH5.8） and for leaves、stems was MS（3% sucrose）+ 6-BA 1mg/L + IAA0.2mg/L +0.7% agar （pH5.8） in the experiment. For a given genotype the regeneration system of cotyledon is better than that of hypocotyls, leaves and stems. The best compatible medium for tomato taking root was MS（3% sucrose）+IAA0.5mg/L （pH5.8） in this experiment.2. Comparison among transformation systems was conducted according to five factors: the explants types, Agrobacteria infection time, pre-culture and co-cultivation time, selection and concentration of antibiotic resistance. The result indicates that the best genetic transformation system was pre-culture for 2d, Agrobacteria infection for 8 min and co-cultivation for 2d. Kan concentration is 60mg/L for cotyledon, 70mg/L for hypocotyls, leaves, stems.3. Two genes, COR15 and CBF₃, were introduced at the same time by Agrobacteriu transformation systems, the PCR checking result proved that the two genes had been integrated into the genome of tomato.更多还原