【作者】 苗春波；

【导师】 孙丙耀；

【作者基本信息】 苏州大学 ， 遗传学， 2009， 硕士

【摘要】 水稻是世界上最重要的粮食作物之一,也是单子叶植物研究的模式生物。随着全基因组测序计划的完成,水稻遗传学研究已进入功能基因组时代。克隆和解析功能基因是水稻功能基因组研究的主要任务。目前水稻中已发展了多种克隆和鉴定功能基因的方法,其中最直接有效的途径是构建基因突变群体,通过对突变体的分析鉴定基因功能。采用Ac/Ds转座子标签法构建突变群体是最有效和最常用的方法之一。为了促进水稻功能基因的研究,本论文主要围绕水稻Ac/Ds标签系统做了两方面的研究:1、为促进籼稻Ac/Ds基因捕获系统的构建,以籼稻成熟胚为起始材料,进行了籼稻愈伤组织培养,希望能够优化其愈伤组织再生体系。结果如下:1)愈伤组织诱导培养基含2,4-D 0.5 mg/L时,嘉育948、盐恢559、扬籼6547、中二软占、明恢86、广恢998、遵籼3号等7个品种的愈伤组织诱导效果最佳;含2,4-D 1.0 mg/L时,镇恢084的愈伤组织诱导效果最佳;含2,4-D 1.5 mg/L时,籼小粘和扬稻6号的愈伤组织诱导效果最佳;在含不同2,4-D浓度(0.5 ~ 2.5 mg/L)的培养基中,中鉴100和湘晚籼1号都没能诱导出愈伤组织;2)在含最适2,4-D水平的诱导培养基中再添加0.2 mg/L 6-BA对愈伤组织诱导率作用不明显,但抑制愈伤组织分化成苗;3)在分化培养基中适当降低6-BA浓度既能保证愈伤组织成苗率不下降甚至上升,又可以提高再生植株的质量。2、在水稻Ds插入突变体群体中得到一株黄绿叶突变体,在对其进行研究的过程中,取得了如下结果:1)采用hiTAIL-PCR技术,建立了扩增Ds侧翼序列的稳定体系;hiTAIL-PCR比TAIL-PCR更加稳定和高效,成功率更高,且可以应用于较水稻更复杂的基因组;2)黄绿叶突变体为一单基因隐性突变体;采用hiTAIL-PCR扩增了其Ds侧翼序列,序列分析发现Ds插入到了Os03g0344200上游150 bp处;连锁分析发现该Ds插入与黄绿叶突变表型并不存在连锁关系。更多还原

【Abstract】 Rice is one of the most important food crops, and is also a model plant in monocot species. With the accomplishment of rice genome sequencing project, a new era of functional genomics has arrived in rice genetics research. The main task of rice functional genomics is functional deciphering of rice genes. Currently, several approaches have been developed in rice to clone new genes and determinate their functions, and among these strategies developing mutant population and deciphering functional genes through mutants concerned is most efficient, and mutagenesis through Ac/Ds tagging system is one of the most widely used and efficient tools for producing mutant population. To promote the development of rice functional genomics, in this paper, we will make two researches around rice Ac/Ds transposon tagging system.1. To push the development of Ac/Ds transposon tagging system in indica rice, we have made indica rice mature embryo callus culture and acquired results as follows: (1) The calli of seven varieties, including Jiayu 948, Yanghui 559, Yangxian 6547, Zhongerruanzhan, Minghui 86, Guanghui 998, and Zunxian 3, were best initiated on callus initiation media containing 2,4-D at 0.5 mg/L; The calli of Zhenhui 084 were best initiated on media containing 2,4-D at 1.0 mg/L ; When the level of 2,4-D was set at 1.5 mg/L, the calli of two varieties, including Yangdao 6 and Xianxiaonian, were best initiated; On media containing 2,4-D at 0.5 ~ 2.5 mg/L , the calli of two varieties, including Zhongjian 100 and Xiangwanxian 1, could not be initiated; (2) 0.2 mg/L 6-BA was supplemented into callus initiation media containing optimal 2,4-D level with an objective to explore the effects of 6-BA on callus initiation and plantlet regeneration, and it was found that the 6-BA treatment had no significant effects on callus initiation frequency, but could reduce plantlet regeneration frequency; (3) Not only plantlet regeneration frequency could be maintained and even increased, but also the quality of regenerated plantlets could be improved when the concentration of 6-BA in plantlet regeneration medium was properly reduced.2. We have found a yellow-green leaf mutant in Ds insertion rice lines, and during the genetic study of the mutant, we achieved the following results: (1) Using hiTAIL-PCR, we have established a stable system for identifying Ds flanking sequences; It is indicated that hiTAIL-PCR is more stable and efficient than TAIL-PCR, and can be applied to isolate Ds flanking sequences in genomes with more complexity than rice; (2) The yellow-green leaf mutant is caused by a single recessive mutant; Using hiTAIL-PCR, we isolated the Ds flanking sequence in the mutant, and sequence analysis revealed that the Ds element was inserted 150bp upstream of the rice predicted gene Os03g0344200; Analysis indicated that there is no genetic linkage between the Ds insertion and the yellow-green leaf mutant.更多还原