【作者】 蔡剑辉；

【导师】 李冠；

【作者基本信息】 新疆大学 ， 植物学， 2005， 硕士

【摘要】 低能离子束注入生物体在理论和应用方面均取得了巨大的成功,但离子注入彩棉的研究尚未见报道。本文主要研究低能氩离子介导白棉全DNA 转化彩棉产生的生物学效应和生理生化指标的改变并应用RAPD 标记技术结合形态学鉴定从分子水平上对辐射后代个体基因组变化进行检测,分析各个处理间的遗传变异程度。选取垦绿1 号品系（绿色）进行离子注入,注入剂量为:0、6×1015Ar+/cm2、8×10¹⁵Ar⁺/cm²、10×10¹⁵Ar⁺/cm² 、20×10¹⁵Ar⁺/cm² 、40×10^<sup>15Ar⁺/cm²、60×10¹⁵Ar⁺/cm²。实验得出以下初步结果: 1)对枯萎病毒素胁迫下的各项生理生化指标（MDA、PPO、POD 等）的变化进行研究,分析它们与剂量之间的关系。实验结果显示:除了MDA 含量其它指标中处理均比CK 要高且有显著的相关性,GTP 和PPO 的活性都是叶部高于茎部,说明叶对胁迫的敏感性要高于茎。此实验间接证明了在离子束介导下外源DNA 进入了受体内和受体基因组发生整合,使其生化指标发生变化。2)对M1 代主要经济性状的变异进行检测,结果发现当剂量为10×10¹⁵Ar⁺/cm² 时彩棉的纤维长度较原种对照增加17.1%,比强度采用8×10¹⁵Ar⁺/cm² 可增长22.5%,而马克隆值却都呈现了明显的负效应。3)利用建立起来的离子束介导种子遗传转化的体系,将白棉（新海17 号）基因组DNA 导入彩棉（垦绿一号）,将处理后的种子浸种催芽播种进行抗病性筛选,对抗病转化植株进行RAPD 分析,发现S53,S176 引物可作为筛选标记进行转全DNA 植株的辅助选择。实验表明供体白棉的DNA 已部分整合到彩棉基因组中,引起其结构变化,从而反映到表观性状,并进一步说明离子束介导转基因方法的可行性。更多还原

【Abstract】 Genetic transformation is an important research domain in genetic engineering. Heavy–ion beam mediated gene transferring is a new technique that was developed recently. It has made enormous success in theory and application, But the research of this technology on colored cotton has not been reported. In this thesis, this new methodology has been used to study the transgenic technique focusing on relevant biological effects of botanic seeds after being exposed to radioactive sources. Random amplified polymorphic DNA （RAPD） was used to evaluate DNA variation of these varieties and analyzed each hereditary variation. Seeds of KL1（green） were implanted by Ar⁺, energy was 25 keV, doses were 0、6×1015Ar+/cm2、8×1015Ar+/cm2、10×10¹⁵Ar⁺/cm² 、20×10¹⁵Ar⁺/cm²、40×10¹⁵Ar⁺/cm²、60×10¹⁵Ar⁺/cm². The result showed that: 1) The physiological indexes （MDA、SS、PPO、GTP、POD、TA） were studied under Fuserium oxysporum stress condition and analyzed the relationship among the radiation doses and all the data collected. Through correlation analysis about physiological indexes, the result demonstrated: all physiological indexes except that MDA disposal content all to be higher than CK, and had the remarkable negative correlation. The activity of GTP and PPO are higher in leaves than in stems. Proved that leaf tissues were more sensitive than stems. Indirectly explained extraneous DNA entered into acceptor and the acceptor gene group caused its biochemistry index to change. 2) The main economic traits in M1 were detected, the results indicated that by using 10×10¹⁵Ar⁺/cm² the fiber length can increase 22.5%,fiber length’s improvement can be increased 17.1% by using 8×10¹⁵Ar⁺/cm². but micronaire has actually presented the obvious negative effect. 3) The genomic DNA of white cotton was delivered into colored cotton with the seeds transformation system established above. The germinated seeds were sowed in pathogenic soil and plants with resistance to wilt diseases were survived. DNA extracted from the survivals were used for RAPD analysis .The result of the RAPD analysis showed that two primers, S53, S176 can be used as molecular marker in assistance of selecting the transgenic plants after the preliminary study. This finding, for the first time, identifies DNA fragment in white cotton had integrated into the recipients’genome. And proved further the ion beam lies between the feasibility of the transgenic method.更多还原