【作者】 贾玉艳；

【导师】 陈宏； 邓继先；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2004， 硕士

【摘要】 一种新型的载体系统——细菌人工染色体(bacterial artificial chromosome，BAC)已经发展成为构建复杂基因组文库的重要方法。BAC文库在基因组研究中起着关键作用，对BAC的修饰在分析和研究基因体内和体外的功能和表达方面也有很重要的作用。利用Red系统进行的基因同源重组技术扩展了BAC文库的应用范围，并为转基因动物研究克服转基因表达的“位置效应”提供了新的思路。本研究的目的就是构建具有高产奶性能的关中奶山羊基因组DNA的BAC文库，及利用Red重组系统将天然的人组织型纤溶酶原激活剂(ht-PA)的编码序列置换掉BAC克隆中的β-酪蛋白的编码序列，用β-酪蛋白的调控序列指导ht-PA的表达，以消除转基因动物外源基因的“位置效应”, 为制备表达ht-PA的动物乳腺生物反应器提供一个有效的载体。本论文由2部分组成:1. 用脉冲电泳技术分离大片段DNA，以电透析法回收DNA后,与用BamHI酶切和CIAP（牛小肠碱性磷酸酶）脱磷的pBeloBAC11载体连接，然后电击转化感受态细胞DH10B，得到了关中奶山羊基因组的部分BAC文库，插入片段平均大小为30～40 kb。摸索了BAC载体制备、高分子量DNA制备、酶切、连接和电击转化等一系列环节对高效转化产生大片段DNA的BAC的影响，为整个文库的构建奠定了基础。2． 构建了两个携带Zeocin抗性的ht-PA的载体，一个含有牛生长激素polyA 信号 (Bovine growth hormone polyadenylation signal, BGHpA)，另一个没有BGHpA。Zeocin抗性基因含有真核和原核两个启动子，可以在真核和原核细胞中进行抗性筛选。用PCR方法获得上述两个载体的DNA片段，然后电击转化至含有BAC的E.coli DH10B细胞中，在λ Red重组系统的作用下，与BAC发生同源重组，置换了BAC中β-酪蛋白的编码区。用PCR和基因序列分析技术对BAC克隆进行了鉴定，证明获得了在β-酪蛋白基因座定位整合天然ht-PA基因的阳性克隆。更多还原

【Abstract】 A new vector system-BAC(bacterial artificial chromosome) has been developed into an important method for the construction of complex genomic library. BAC library plays a pivotal role in genome studies, and modification of BAC also has an important role for analysis and studying gene function and expression in vitro and in vivo. The homologous recombination technology with red system amplifies the application of BAC library, and provides a new path of eliminating position effect of random intrgration in transgenic animals. The goal of the research is constructing of BAC library of Guanzhong milk goat genome DNA which has high milk efficiency, and substituding encoding sequence of native ht-PA to encoding sequence of β-casein with red system, promoting the expression of native ht-PA by β-casein controling sequence, overcoming the position effect of random integration in transgenic animals, to provide an efficient vector for preparation of mammary gland bioreactors which expresses native ht-PA. This dissertation is divided into two segments:1. The former segment separated large DNA in CHEF(contour-clamped homogeneous homogeneous electric field), and extracted DNA with electroelution, the received DNA fragments were ligated to pBeloBAC11 digested by the restriction enzyme of BamHI and dephosphorylated by CIAP phosphatase, transformed competent cell DH10B by electroporation of large DNA, acquired the part BAC library of Guanzhong milk goat, whose average inserted fragments ranged from 30 kb to 40 kb. We explored many factors which influenced high efficient transformation of large DNA, such as prepartion of BAC invector and high-molecular-weight DNA, partial digestion, ligation, electroporation, ,and provided a basis of construction of the whole library. <WP=7>2. The latter segment construced two native ht-PA vectors with carrying zeocin resistance gene, native ht-PABZeo and native ht-PAZeo, one contained BGHpA, another not. Zeocin resistance gene has eukaryote and prokaryote promoters, so it is used to screen by resistance in both mammalian cell lines and bacteria. PCR fragments of above two vectors were electro-transferred into competent cell DH10B, promoted homologous recombination with the role of red recombinant system, the encoding sequence of β-casein was in vivo replaced with native ht-PA encoding sequence. BAC clones were identified by PCR and sequence analysis, and proved that native ht-PA was integrated into β-casein location as expected.更多还原