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转人生长激素基因鲤鱼F_4代中转植基因特性研究
Alternative TitleCharacters of transgenes in F_4 hGH-transgenic common
吴波
Subtype博士
Thesis Advisor朱作言
2002
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
Keyword转基因鲤鱼 转植基因 整合位点 分子特性
Abstract该文以两尾转人生长激素基因红鲤F<,4>仔鱼为材料,采用经改进的质粒回收法对上述问题进行了研究,主要内容如下:1.从两尾转人生长激素基因红鲤F<,4>仔鱼个体(A和B)基因组中分别回收了上千个转植基因克隆,各随机取200个转植基因进行BamHⅠ和EcoRⅠ双酶切分析.2.构建每类转植基因的六种限制性内切酶BamHⅠ、HindⅢ、EcoRⅠ、PstⅠ、KpnⅠ和BglⅡ的酶切图谱.3.通过Southern杂交对转植基因在回收质粒上的位置进行了定位分析,并对三种回收到的变异型转植基因旁侧顺序进行了亚克隆和顺序测定.4.对转植基因回收方法进行了完善,对转植基因的世代传递和遗传稳定性进行了讨论,对转植基因所表现的多态性成因进行了分析.通过上述几个方面的研究结果和分析,首次较全面和细致地刻画了F<,4>转基因红鲤中转植基因的整合图景,发现了外源基因整合和世代传递过程中发生的变异现象并分析了成因,获得了转植基因整合位点旁侧宿主基因组顺序的资料,为下一步克隆全长新基因打下了基础.其结果有力地证实了建立稳定转基因动物品系的可能性,并有助于深入了解外源基因整合机制和世代传递规律.
Other AbstractGene-transfer in fish has been studied deeply and great progresses have been made. However, some essential problems still remain unsolved such as the molecular mechanisms of integration and generation-transmitted of foreign gene. As the development made in transgenic research and breakthrough of gene-targeting made in fish, one emphasis of current transgenic research is to answer these questions. To find out the key, cloning and analyzing the transgenes and their integration sites from host genome is the most direct and persuaded solution. "A model of transgenic fish", /zG#-transgenic common carp, which was constructed in our lab, has been raised into F6 fish from generation to generation. All individuals were found transgenic positive in F4 group. While many doubts on transgenes in F4 genome have attracted researchers great attentions. For example, after four generations being transmitted, researchers want to know which forms the transgenes exist in; whether the transgenes are stable and whether there are aberrances happened in them. Study on these problems is of great importance for the foundation of stable transgenic animal strains and the sequence data of integration sites can also be accumulated to facilitate the wide appliance of gene-targeting in fish. In this research, two /zG-ff-transgenic common carp F4 fish were picked out and studied with the method "plasmid rescue". Main results are described as following: More than one thousand transgenes clones were recovered from two /iGff-transgenic red common carp F4 individuals' (A and B) genomes, respectively. Two hundred, clones were randomly taken out from each individual and double-digested with BamB. I and EcoR I one by one. According to that, the two hundred clones in individual A were characterized into 6 classes (A-l to A-6) and those in individual B 4 classes (B-l to B-4). Restriction map of each class was constructed with six enzymes: Bamli I, Hind III, EcoR I, Pst I, Kpn I and Bgl II. Through comparisons of restriction maps, transgenes of individual B: B-l, B-2, B-3 and B-4 were found to be the same as those of individual A: A-l, A-2, A-4 and A-5, respectively. A-l and B-l, 83% and 84.5% in proportion respectively, maintained their original forms while the remaining transgenes classes (less han 17% of total recovered transgene numbers) were different from the original composition in molecular weight, restriction enzyme class and position, implying that a minor proportion of the transgene was polymorphic and the aberrances like deletion and arrangement had happened. PCR analysis of 10 classes of transgenes also revealed the presence of spontaneous mutation. 3. Locations of transgenes in the recovered plasmids were determined by southern blotting. And then adjacent sequences of transgenes integration sites in three recovered aberrant classes were subcloned and analyzed. Transgene of A-2 was inserted into common carp DNA sequences homologous to the mouse phosphoglycerate kinase-1 gene and those of A-3 and A-6 were inserted into common carp DNA sequences homologous to the human epidermal keratin 14 gene and sequence of common carp J3 -actin gene, respectively. Moreover, An 188bp tract in A-3 (nt. 313-501) was found 94% homologous to the "MIR" repeat family. The sequence traits of transgenes integration sites were also discussed. 4. The method of transgenes recovering was improved. The mechanism of generation transmitted, the inheritance stability of transgenes and the reason of polymorphism were discussed. Foundation of stable transgenic animal strains was thought to be possible in light of our result and the possible approach was presented. Through our present research, the integration patterns of transgenes in transgenic common carp F4 were meticulously and comprehensively depicted for the first time. The aberrances happened during the courses of foreign gene integration and the generation transmitted was found and the causes of which were discussed. Some data of the sequences of host genome adjacent to the transgenes were accumulated and would conduce to clone the full-length new gene of common carp in future. Our results are helpful to realize the mechanism of foreign genes integration and inheritance and prove strongly that it is of great possibility to construct a stable transgenic animal strain.
Pages91
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12650
Collection学位论文
Recommended Citation
GB/T 7714
吴波. 转人生长激素基因鲤鱼F_4代中转植基因特性研究[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,2002.
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