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Thesis Advisor朱作言
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline遗传学
Abstract在受精卵显微注射外源DNA,提取不同发育时期胚胎总核酸,进行DNA分子杂交,分析了五种不同构型外源DNA在泥鳅,鲫鱼,鲤鱼胚胎发育过程中的存在,复制及整合等行为。1. 注入的线型外源DNA,不论携带质粒栽体顺序与否,均可以线型单体,超环和松散不状分子,及多聚体形式存在,其中多聚体为主要存在形式,以线型二,多聚体形式注入的外源DNA则是以更大分子量的多聚体随受体总DNA一同迁移。而注入的环状分子构型未发生改变。2. 注入受体卵的不同物理构型外源DNA,在受精卵的胚胎发育中均经历不同程度的复制扩增,至原肠期达峰值后逐步或骤然降解的过程。3. pMhGH环状DNA分子在原肠期以前以松散环的形式进行了有限的扩增(3-4倍),超环分子未见明显量变;线型DNA分子则能够在卵裂早期形成大分子量,头尾相连或尾尾相连的多聚体,并主要以此形式进行有效复制。由pMhGH/BamHI形成的松散环也有少量扩增。4. 外源DNA复制量与注入外源DNA拷贝数成正相关系,与线型DNA分子片段长短亦有关联;也因不同批卵,甚至不同个体而显差异。线型外源DNA(MhGH)的最大扩增倍数可达100倍以上。借注射外源DNA的胚胎及转基因成鱼总DNA酶切后的Southern吸印转移杂交的方法,还分析了外源DNA多聚体的成因,外源DNA稳定存在的机理,整合发生的时间及整合形式等问题。
Other AbstractThe persistence, replication and integration of five different forms of MhGH gene microinjected into fertilized eggs of fish were analyzed. For all forms of novel DNA tested, the replication of the introduced sequences began appeared at the late gastrula followed by foreign DNA degradation, Injected circular molecules (FormI, II) were replicated in FormII, not FormI, without obviously increased copy numbers through blastrulation (3-4 folds). In contrast, extensive and efficient amplification of input linear DNA sequences were seen whenever the microinjected DNA was assembled into high molecular weight, head-to-tail or tail-to-tail concatemers. And the overall copy numbers significantly increased, sometimes reached more than 100 folds from the late blastrula to early neurula stages, in comparison with the copy number injected into the eggs. It must be pointed out that the fate of novel DNAs differed from batches of embryos, different individual, and the input template sizes. Although the majority of linear DNA injected was assembled into long concatemer, FormI and/or FormII DNA were slightly formed, and monomeric was remained in the host eggs. However, these 'free' DNA forms persisted during the early embyrogenetic stages without obvious replication. In addition, the input long linear concatemer was not assembled into any other forms except ligated into higher molecular weight DNA. Using the restriction enzyme digestion and Southern blotting we also analyzed aspects such as the pathways generating concatemers, the timing and the patterns of integration, and the modification or changes of some restriction endonuclease sites along the novel DNA.
Document Type学位论文
Recommended Citation
GB/T 7714
刘东. 不同构形处源DNAD在鱼类胚胎发育过程中的行为[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,1990.
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