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转人生长激素基因(hGH)红鲤F_4代鱼中转植基因的多态性与整合图式研究
曾志强
Subtype博士
Thesis Advisor朱作言
2000
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转基因红鲤 转植基因 分子多态性 整合图式 整合位点
Abstract鱼类转基因定向育种技术正日渐成熟,转基因鱼走向市场已为期不远。然而,人们对转植基因在受体鱼中的行为,与对其它转基因动物一样,缺乏足够的了解,尚不能实现外源基因的定点整合与受控表达。这个问题已严重制约转基因鱼纯系的建立进程。对转植基因整合机制的了解,最直接的途径是克隆转植基因及其整合位点顺序。本文通过克隆转人生长激素基因(hGH)红鲤F_4代鱼中转植基因及相关的整合位点,试图从分子水平上揭示转植基因在受体鱼中的形态,有效整合位点的顺序特征,转植基因在世代传递过程中的行为,以及外源基因何时能"内源化"等。其实际意义在于,有效整合位点顺序可用作转基因鱼不同品系的分子标记,并可为鱼类外源基因定点整合研究提供靶位点信息,在建立转基因鱼纯系的实践中有重要的应用价值。克隆转植基因及整合位点的常规方法,依外源基因构建体和转移方法的不同,有筛选基因组文库和反向PCR两种。两种方法各有其应用的局限性,是有关克隆转植基因和整合位点的报道并不多见的原因之一。本文针对显微注射法制备的转基因鱼的特点,将一种比较简单的"质粒回收法"应用于转基因鱼的转植基因克隆。主要研究内容如下: 1.建立和完善了回收转植基因的技术路线,从一尾具有快速生长 效应的F4代转基因红鲤基因组中回收了50个转植基因。2.用双酶切法对BamH I酶切基因组DNA后所回收的转植基因进行分类,并构建了它们的酶切图谱。可将回收基因分为4种类型,其中一种(占回收基因总数的18%)与构建原代转基因鱼所用的外源基因结构相同,其余3种在分子量大小、酶切种类和位置都发生了显著的变化。这意味着,外源基因在整合及四个世代的传递过程中,大多数发生了不同形式的缺失和重排事件,表现出丰富的多态性。3.对回收的2种类型转植基因侧翼顺序进行亚克隆和顺序测定,发现一种转植基因(II型)的侧翼带有宿主鲤鱼β-actin基因的5'非翻译区顺序(5'UTR),推测该拷贝转植基因的核心顺序位于宿主β-actin基因的5'UTR,单拷贝整合或位于串连体侧翼;另一种转植基因(Ⅲ型)侧翼顺序与小鼠tie2基因顺序同源,并用PCR-Southern方法对一个侧翼片段进行了验证,从普通鲤鱼基因组中检出了相应的顺序。推测该转植基因以单拷贝形式整合于宿主类tie2基因的5'侧翼区和内含子间。对整合位点与转植基因表达水平之间的关系进行了讨论,提出了一条建立转基因鱼纯系的技术路线。同时,指出了有效整合位点顺序在研究鱼类外源基因定点整合中的应用价值。 4.通过Southern杂交,结合不同酶切条件下转植基因的回收结果分析,证实了在实验鱼中,转植基因以串连体形式整合。用PCR方法确定了2尾转基因鱼个体中,转植基因串连体的形成方式为首尾相连。对转植基因拷贝间连接片段进行PCR扩增和顺序测定,发现该片段已发生了碱基的突变,其中单个碱基的插入是主要突变事件。与原始顺序的相似率下降至97.2%。在连接片段相应位置发现了转植基因末端的酶切位点,说明串连体形成时转植基因末端并没有被修饰,进一步证实了Southern杂交结果。 5.对回收转植基因的技术关键进行了讨论,并提出了提高转植基 因回收效率的技术措施。本文通过上述几个方面的研究,描述了F4代转基因红鲤中转植基因的多态性和整合图式。特别是将"质粒回收"这一简单有效的方法成功地应用于转基因鱼的转植基因克隆,并首次获得了整合位点的顺序信息,有助于我们进一步了解转植基因整合与世代传递的规律。
Other AbstractAs the great research progresses have been made in gene-transfer in fish, transgenic fishes are most likely to be put into the market on the near future. On the other hand, the behaviors of transgenes in transgenic fish are not fully understood just like those in other transgenic animals, so it is impracticable now to integrate a foreign gene into a specific site of the host genome and control its proper expression. Due to this, the process of generating homogenous transgenic fish is seriously inhibited. To find out the molecular mechanisms involved in the processes of transgene integration and inheritance, it is necessary to clone and analyze transgenes along with the sequence at integration site. In the present work, pMThGH-transgene and sequences flanking it have been cloned from a F4 hGH-transgenic red common carp, aiming at revealing evidences for transgene integration and inheritance, also the feature of sequences at functional integration site. Furthermore, the sequences of functional integration site can be used as molecular genetic markers for different transgenic fish lines, also can be severed as targeting sites for gene targeting in fish. Two kinds of traditional method, genomic DNA library screening and inverse PCR (IPCR), have been employed in cloning transgene and sequence of integration site. Apart from the present paper, there are only a few published reports about the cloning of a transgene along with flanking sequences from the DNA of transgenic animals. In some degree, this fact is due to the limitations of the application of traditional methods. Fortunately, a kind of simpler technique, plasmid rescue, has successfully employed to reisolate transfected genes from cell cultures. In the present study, the technique of plasmid rescue has been modified and used to recover transgene from transgenic fish for the first time. Results are described as follows: (1). Using a modified plasmid rescue technique, fifty transgnes were successfully recovered from the genomic DNA of a F_4 pMThGH-transgenic red common carp. (2). These recovered plasmids were classified into 4 types after double restriction with EcoRI and BamHI endonuclease. The restriction maps of these transgenes were constructed by digestion with 5 kinds of endonuclease. Only 1 type of transgene (18%) retains its original structure, whereas the other three types are very different from the original form and vary from each other on both molecular weight and physical maps. This implies that the sequences of most transgenes have been deleted and/or rearranged during integration and inheritance. Transgenes in F_4 transgenics are highly polymorphic. (3). The flank sequences in two kinds of transgene were subcloned into the pUC18 vector and sequenced. Sequencing results showed that the 5' untranslated region (5'UTR) of common carp β-actin gene flanked Type II transgene. It can be deduced that the core sequence of Type II transgene was located at 5'UTR of the host β-actin gene. This transgene was integrated into the host genome as single copy or at the end of transgene concatemers. The genomic sequence trapped by TypeIII transgene was homologous to the sequence of mouse tie2 gene. Part of flank sequence was detected in the genomic DNA of non-transgenic common carp by PCR and confirmed by Southern hybridization analysis. It is believed that TypeIII transgene was inserted as single copy into a genome site between the 5' flanking region and intron I of tie2-like gene. The relationship between transgene integration sites and levels of expression was extensively discussed. (4). Based on the results of Southern blot and transgene recovery with various endonuclease, it was confirmed that multiple copies of transgene were integrated into the fish genome as transgene concatemers. PCR result demonstrated that transgenes were arrayed in a head-to-tail manner within the concatemers. The junction fragment of transgene-transgene was amplified by PCR. Sequence aberrations, in a majority of insertion of single extraneous nucleotides, were observed at the transgene junction according to the result of sequencing analysis. The homology between the transgene junction and its corresponding region on pMThGH was lower than the expectation, with bases identity of 97.2%. The recognized site of BamHI was found at the joint of transgene-transgene. This fact strongly suggested that the ends of transgene had not been modified when transgenes were arrayed in a head-to-tail manner to form concatemers. It was also consistent with the result of Southern blot analysis. (5). Key techniques to recover transgene from genomic DNA of transgenic fish were pointed out in this paper. Further steps to improve the efficiency of transgene recovery were also proposed. Briefly, these findings reveal the molecular polymorphism and distinctive integration pattern of transgenes in F_4 transgenics. This study will substantially contribute to a better understanding of the mechanism of transgenes integration and inheritance.
Pages94
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12550
Collection学位论文
Recommended Citation
GB/T 7714
曾志强. 转人生长激素基因(hGH)红鲤F_4代鱼中转植基因的多态性与整合图式研究[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,2000.
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