【作者】 方清；

【导师】 李冠一；

【作者基本信息】 华南热带农业大学 ， 作物遗传育种， 2002， 硕士

【摘要】 1992年以来，海南大学生物技术研究所开始用花粉管方法将红树总DNA导入普通番茄，在海滩上用海水进行耐盐性筛选，首次获得了能耐海水浇灌的耐盐番茄。本课题对获得的耐盐番茄材料进行了较系统的研究，分别从形态、细胞结构、染色体水平和分子水平等四个不同的层次考察了外源基因转入的情况及其与番茄耐性增强之间的关系，获得了与耐盐相关的一些细胞结构变异的特征，证明外源红树DNA转入了普通番茄。 本实验分两部分：第一部分、对转化番茄的形态及细胞显微结构进行观察与分析，实验表明转化番茄形态与对照相比有明显的变化，其叶面积变小，叶形收拢，这种结构可有效地减少水分的蒸发，缓解由盐胁产生的生理干旱；组织解剖结构结果显示，耐盐番茄在细胞结构上发生了明显的变异：其气孔器倾向下陷，表皮毛增多，叶脉微管组织不发达，微管束周围出现增多的贮水细胞，叶缘及其它部位表面分化出大型的多细胞分泌型结构，这些结构与转化番茄后代较强的耐盐能力有关。第二部分，通过Southern Blot和基因组原位杂交(Genomic In Situ Hybridization,GISH)，对红树转化的耐盐番茄后代分别进行了分子及细胞遗传学的鉴定，在耐盐番茄的Southern Blot中发现了特异的DNA片段，表明耐盐番茄的基因组中存在外源DNA片段；利用GISH在耐盐番茄后代中检出了外源染色体片段，同时结果还表明在不同的耐盐番茄株系中，外源DNA片段整合于番茄染色体的不同位点，其整合部位分别位于染色体的亚端部和近中部。 通过以上细胞生物学和分子生物学的研究表明，外源DNA已导入普通番茄，与其基因组整合并遗传给后代，外源红树DNA片段的渗入导致了转化植株在形态、细胞结构以及分子水平上的变异，转化番茄后代耐盐能力的增强可能与番茄基因组的这种变化有关。更多还原

【Abstract】 Since 1992,in the Institute of Biological Science and Technology of Hainan University,the pollen tube introduction method was used to breed tomato with total DNA of Rhizophora apicularta,a salt-tolerant,seashore-grown plant. The salt-tolerant transformed tomato was firstly obtained and planted on the seashore beach and watered directly with full seawater. This study is focus on the all-around analysis of the transformed tomato,which are investigated respectively on morphological,anatomical structure of the leaves,chromosome level and molecular level. From the tests,we have obtained some structural variant features related with salt resistance and demonstrated the successful introduction of Rhizophora apicularta DNA into tomato.This thesis contains two parts. In the first part,the morphological and anatomical structures of the leaves were investigated. As to the morphology,some difference between control and transformed tomato were found. The leaf dimensions of the transformed tomato with unextended leaves were smaller than that of control. These structure changes should help to decrease the evaporation of water effectively and release the physiological drought resulted from saline. The observation results on the anatomical structure of the leaves showed various changes in the transformed tomato:hollow stomata,well-developed epidermal hairs and aqueous tissue,unwell-developed vascular tissue and excretory structure consisted of multicell different from epidermic cells. These differently structural characteristics have higher relationship to their improvement to salt resistance. The second part is the investigation of molecular-cytogenetic characterization of the transformed tomato by means of Southern blot and genomic in situ hybridization (GISH). Southern blot showed the foreign DNA segments were inserted into the transformed tomato genome. Using GISH,the Rhizophora apiculota DNA sequences on tomato chromosomes were characterized. The observed cell showed signal spots and their location in different region of chromosomes in different lines.The experimental results mentioned above demonstrated that Rhizophora apiculota DNA have been introduced into tomato and integrated with tomato genome. The obvious improvement in the salt tolerance among their progenies was most probably related with the variation of their genome.更多还原