【作者】 田敏；

【导师】 朱睦元；

【作者基本信息】 浙江大学 ， 遗传学， 2003， 博士

【摘要】 植物组织培养技术是植物体细胞遗传学的基础，对理论基础研究和植物基因工程以及农作物品种改良等都有重大的意义。离体诱导和培养愈伤组织具有多种用途。一方面可研究植物生长发育及分化的机制、遗传变异规律；另一方面可作为植物基因工程的受体材料，生产有价值的药用蛋白质。本研究建立了一套完整的草莓愈伤组织再生体系，探讨其分化的生理生化基础，以及小鼠EGF基因克隆及其植物转基因研究，主要结果如下。 一、草莓体外再生体系的建立 采用不同的植物生长调节物质(生长素和细胞分裂素)的浓度及其组合以及不同的光照条件对草莓的多种外植体，如叶片、叶柄、根段、芽尖等，进行愈伤组织的诱导，产生了3类在外形、颜色、质地、生长速率、分化能力等方面具有明显差异的愈伤组织，即松软、湿润、灰白色的Ⅰ型，颗粒状、紧实呈浅黄色的Ⅱ型和绿色致密的Ⅲ型。Ⅰ型愈伤组织生长快，但分化率低，并能在一定条件下转变为Ⅲ型。Ⅲ型具有较高的分化能力。并且通过对它们分化过程的观察发现，其形态建成的方式是直接的器官发生，而不是通过体细胞胚的发生再生植株。 同时，研究了激素、外植体、光照条件等因素对草莓体外培养的影响，发现产生的愈伤组织的形态主要与培养基中的激素种类和光照条件有关，而与外植体来源无明显相关性。 二、愈伤组织分化的生理生化基础分析 主要研究了不同类型愈伤组织及其分化过程中和继代培养中的糖类、蛋白质及活性氧和抗氧化酶活性的变化。 (1)Ⅰ型愈伤组织含有较高的可溶性糖，而Ⅱ型和Ⅲ型则具有相对较高的蔗糖含量。愈伤组织器官发生过程中，可溶性糖和蔗糖含量均呈现下降趋势。继代过程中可溶性总糖含量无明显变化，蔗糖含量在培养15周后迅速下降。三类愈伤组织蛋白质含量差异显著，Ⅱ型和Ⅲ型中的含量约是Ⅰ型中的6倍。分化过程中蛋白质含量逐渐上升，SDS-PAGE表明芽分化和根分化的愈伤组织中蛋白质合成量显著增强；继代培养中Ⅱ和Ⅲ型愈伤中蛋白质含量有平缓下降趋势，Ⅰ型愈伤组织中则下降较快。同时，在继代培养过程中，愈伤组织的形态发生能力下降，Ⅰ型愈伤组织逐渐失去再生能力。这些实验结果表明，愈伤组织的再生能力与可溶性糖的含量无关，而与蔗糖、蛋白质的含量有明显相关性。浙大学博士学位论文 (2)三类愈伤组织在活性氧代谢方面表现出极大的差别。n和川型初生愈伤组织H刃2的含量分别是I型的5倍和9倍，02甲的释放速率是3倍和4倍，并且具有较高的抗氧化酶 (soD，cAT、PoD)活性，特别是500的活性，在初生n型愈伤组织是高达990 u n-lg一1Fw，而在I型中只有36 uh一19一1 Fw。在愈伤组织分化过程中，HZoZ的水平先升高后下降，O:甲释放速率一直下降，SOD活性的变化趋势同HZq一致，CAT活性一直下降而PoD先降后升。培养基中添加外源HZO:能在一定程度上提高愈伤组织的再生率，而SOD的抑制剂DDC则使再生率下降。 另外，在18周的继代培养中，也观察到所有类型愈伤组织中HZO:含量缓慢上升，q一释放速率较快增长，尤其是在I型中，培养18周后其速率已明显高于n型和川型.同步检测抗氧化酶活性，发现其活性呈现降低趋势，特别是I型愈伤组织在培养12周时已测不到SOD的活性。这说明内源HZOZ在愈伤组织的分化中起着重要作用，并且一定水平的外源H202能促进植物细胞的再分化。并推测，继代培养中活性氧与抗氧化酶之间的不平衡所造成的氧化胁迫对细胞正常的代谢、生长和发育产生破坏性的影响，与其分化能力的降低存在着直接的相关性。 综上所述，对于草毒愈伤组织的再生，活性氧可能起着双重作用。一方面，一定浓度的活性氧对愈伤组织的再生起促进作用，甚至是启动再生过程所必须的:另一方面，培养中高水平的活性氧可能又会对细胞造成氧化胁迫，从而抑制愈伤组织的再生潜能。三、小鼠EGF基因克隆及其植物转基因研究 从小鼠肾组织中提取总RNA，用君G尸基因的下游引物将其反转录为cDNA第一链，利用RT一PcR的方法克隆到长约1 7obP的编码成熟EGF蛋白质的基因序列，并将其构建到克隆载体pUC18中，序列分析表明所得到的片段与文献报道的小鼠百G尸基因序列完全一致。构建了百G尸基因的植物表达载体:pCAMBIA一EGF和pBI 121一EGF。通过农杆菌转化法得到了转基因烟草再生植株，转基因草毒的研究仍在进行中。更多还原

【Abstract】 Plant tissue culture is very important for the somatic genetics, and it can be used not only to study on the mechanism of growth, development and differentiation of plant cell, but also to product valuable protein as bioreactor and to improve crop in breeding. In this study, plant regeneration through organogenesis of shoot bud from callus cultures of strawberry was established and the metabolism of sugar, protein and reactive oxygen species (ROS) was investigated in order to understand the mechanism of differentiation of plant tissue and cell in vitro. In addition, the cloning of EGF gene and its transformation to the tobacco and strawberry callus were carried out. The main main results are as follows:1.The establishment of plant regeneration system through organogenesis from callus cultures of strawberryThree types of callus cell lines were established due to the growth regulator added to the MS medium and the culture condition, and they showed different potential for morphogeny after being transferred to the same regeneration medium. The type I callus was induced quickly, and was soft and grayish white, with a smooth, wet-looking surface. The type II callus was light yellow, compact and granular, and grew much more slowly. The type III callus was green, compact and tubercular. The type I callus had lower regeneration capacity and the type III had higher. The callus expressed their totipotency through the direct organogenesis rather than the somatic embryogenesis.The formation of different types of callus were mainly controlled by the growth regulator and light, and there is little relationship between the explants and the type of callus.2. The possible relationship between the metabolism of sugar, protein and ROS(1) Three types of callus had different sugar and protein levels. The type I callus possessed higher soluble sugar and decreased sucrose, in the contrast with II and IIIcallus. The II and III calli showed 6-fold as much as the content of water soluble protein, compared with the I callus. SDS-PAGE suggested that the protein was strongly expressed in the callus that differentiated to form shoot cluster and root. During the period of subculture, the sucrose in calli sharply decrease in 15 week, and lover of the soluble sugar keep constant. The protein level in type II and III callus decreased slowly, while what in the type I callus quickly. The ability of expression of totipotency in calli decreased, especially the callus I lost the ability after 12 week subculture. It seemed that there existed some relationship between the regeneration capacity and level of sucrose and protein.(2) During the differentiation and development of callus, H2O2 productioncoincided with emergence of meristemoid and formation of bud primordium in morphogenic calli, SOD activity increased in the early regeneration culture and decreased thereafter, and catalase activity constantly declined while peroxidase decreased during the 5-d culture and gradually increased thereafter. The inhibition of H2O2 production by DDC decreased the percentage of calli generating shoot bud, and exogenous addition of H2O2 slightly promoted the potential for regeneration of shootbud.In the freshly induced calli, the II and III types of callus had 5-fold and 9-fold higher contents of intracellular H2O2, and 3 -fold and 4-fold higher level of O2-, compared with the I type of callus, respectively. Also, the calli II and III had much higher activities of antioxidant enzymes than the callus I. During the subculture, H2O2 level increased gradually and O2’- rapidly, while the activities of antioxidant enzymes gradually declined. The results indicated that the ROS might play a dual role in the regeneration of strawberry calli. On the one hand, a certain level of ROS may have a positive effect on the regeneration, and on the other hand, high level of ROS is inhibitory for the expression of totipotency in calli.3. The cloning of EGF gene and its transformed to the tobacco and strawberry callusTotal RNA was isolated from m更多还原