【作者】 刘长青；

【导师】 唐学玺； 关伟军； 张洪海；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2008， 博士

【摘要】 抽取一只雄性五指山小型猪静脉血,用于制备大分子量基因组DNA,制备好的基因组DNA经HindⅢ部分酶切后,二次脉冲电泳分离所需DNA片段。电洗脱回收100～400kb范围的片段,然后与pBeoBAC11载体连接,转化DH10B感受态细胞,经过培养,挑取和保存白色单克隆。基因组文库的质量一般从文库的平均插入、基因组覆盖率、嵌合率二方面来衡量。本研究所构建中国特有五指山小型猪基因组BAC文库,含有153,600个BAC克隆(保存在40个超级池,每个超级池由10个384孔板组成)。随机挑选了270个BAC克隆的插入片段进行估计,平均为152.3kb,其中空克隆的比例为1.3%,表明文库的覆盖率7.4倍,预计从文库中筛选到单拷贝基因的机率为99.93%。文库中78%的克隆插入大于100 kb,而且有部分克隆甚至大于300kb,这样大小的插入可以满足所有试验对于片段大小的要求。从各个方面来看,本文构建的文库都适用于功能基因和物理图谱构建的研究。构建北京油鸡成纤维靶细胞系,细胞冻存个体数达45个,冻存细胞支数达170支,每支细胞含量为3～5×106细胞/ml,保证北京油鸡遗传基因的群体多样性,同时为外源基因在细胞中的表达提供了良好的体外培养靶细胞。使北京油鸡这一国家重要遗传资源在细胞水平上得以保存下来,并为相关遗传学研究提供了有效的理论依据和理想的生物材料。通过细胞形态学观察、细菌、真菌、支原体检测、生长曲线、核型分析及同工酶检测等多项细胞系质量检测,结果表明建立的北京油鸡成纤维细胞系性质稳定,生物学性能正常,各项指标均达到美国典型培养物保藏中心细胞系鉴定标准。利用6种荧光蛋白基因在所建细胞系中得到良好的表达,说明细胞具有良好的被转染能力,从基因表达的水平上进一步确定细胞系的遗传性能良好,为进行北京油鸡风味特色功能基因的表达、定位和克隆化细胞的筛选鉴定了基础。采用RT-PCR与RACE方法扩增出北京油鸡腺苷酸琥珀酸裂解酶(ADSL)基因全长cDNA序列,该基因开放阅读框长为1455个碱基,编码485个氨基酸;构建成北京油鸡ADSL基因融合表达载体pGEX-ADSL,转化大肠杆菌BL2(1DE3),IPTG诱导表达。经SDS-PAGE电泳显示重组融合蛋白在分子量约为80.5kD处有特异的蛋白条带,与预期分子量大小一致,等电点为6.79。该蛋白的表达量随诱导时间的延长而增加,5h达最高值,达到细胞总蛋白的26.9%,且主要以不可溶的包涵体形式存在,经Glutathione Sepharase 4B凝胶纯化后用Western-blotting检测表明其为北京油鸡ADSL蛋白,为其进一步的具有生物学功能研究及其应用鉴定了基础。提取北京油鸡心、肝、脾、肺、肾、脑、腿肌与胸肌等不同组织的总RNA,利用RT-PCR方法扩增检测purH基因mRNA的差异表达水平。构建带有荧光蛋白报告基因的真核重组表达载体pEGFP-N3-purH,pEYFP-N1-purH和pDsRed1-N1-purH。并利用G418药物筛选和对荧光强度高的单克隆化培养。实验结果:在转染后24、48和72h,重组融合蛋白转染率在10.3%～53.2%之间。pEGFP-N3-purH,pEYFP-N1-purH与pDsRed-N1-purH在北京油鸡成纤维细胞的细胞核与细胞质均有分布,呈弥散分布。经药物筛选和单克隆化培养,获得表达pEGFP-N3-purH,pEYFP-N1-purH与pDsRed-N1-purH融合蛋白的阳性克隆细胞株,经RT-PCR扩增与Western blot检测确认了purH融合蛋白基因已经整合到北京油鸡成纤维细胞的基因组中,获得正常表达。本研究构建了高质量的五指山小型猪基因组BAC文库,从全基因组水平上保存了五指山猪的遗传资源,为功能基因组学研究和后基因组学研究提供了重要的试验材料和实物基础。创建的北京油鸡成纤维靶细胞系从细胞水平保存了北京油鸡的基因资源,同时为风味特色基因的表达提供重要的靶细胞。由于供体细胞的质量对于转基因动物克隆非常重要,本研究利用抗性筛选和克隆化培养获得北京油鸡ADSL基因1株与purH基因3株的阳性细胞株,一方面为北京油鸡转基因动物克隆提供了重要的试验材料,为进一步培育出风味优良、肉质鲜美的转基因鸡提供优质供体细胞;另一方面对于鉴定ADSL与purH基因是否是控制北京油鸡风味特性优良的主效基因,以期对于提高我国地方鸡种种质资源创新,实现育种理论和关键技术新突破等具有一定指导作用。更多还原

【Abstract】 High-molecular-weight(HMW)DNA was prepared form blood of a male Wuzhishan pig ,with a concentration of 3.0×108 cells/ml cell suspension mixed with an equal volume of liquefied 1% low-melting-point agarose, partially digested with HindⅢand fractionated using double size selection. Digestion DNA fragments in the range of 100～400kb was recovered by elctro-elution and ligated into pBeoBAC11 vector, and then was used to transform DH10B competent cells.After incubated individual white colonies were picked and stored in -70℃.We constructed a high-redundancy bacterial artificial chromosome(BAC) library of an important chinese pig species, the Wuzhishan pig. A total153,600 clones were generated in this library and constructed in vector pBeloBAC11(ordered in 40superpools of 400×384 well plates).The average insert size of the BAC clones was estimated to be 152.3kb from 370 randomly isolated clones, and the ratio of no insert was 1.3%,indicating that the library to be approximately 7.4-fold genome coverage. 78% colonies were more than 100kb, representing a 99.99% statistical probability of obtaining at least one clone containing a unique DNA sequence in the library. We constructed the Wuzhishan pig BAC library, which will contribute to a high-resolution physical map for this species and will assist in comparative genomics studies.A fibroblast line from Chicken embryo of the 8-day-age was successfully established by direct culturing of explants. Biological analysis showed that the population doubling time (PDT) for reviving cells was approximately 48 h. Measurement of lactic dehydrogenase (LDH) and malic dehydrogenase (MDH) isoenzymes showed no cross-contamination among the cells. Tests for bacteria, fungi, viruses and mycoplasma were negative. Every index of the Luxi cattle cell line meets the quality control standards of the ATCC (American Type Culture Collection). Not only has the germline of this important chicken breed been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somacloning research. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.This research use the RT-PCR method to amplify the Adenylosuccinate lyase (ADSL)gene from Beijing fatty chicken cDNA PCR library. The results of subclone and sequences analysis shows: the open reading frame of 1455 nucleotides that encode a protein of 485 amino acid residues. The complete open reading frame of ADSL gene was inserted into expression vector pGEX-4T-1 to construct Beijing fatty chicken ADSL gene fusion expression vector pGEX-ADSL, and transformed into E. coli BL21 (DE3), screening positive cloning, expression induced by IPTG. By SDS-PAGE electrophoresis showed that there is a specific bands in the molecular weight of about 83.5kD which have to do with the expected molecular weight of the same size and isoelectric point of 6.79.The expression of the protein was increased as the increase of induced time, the maximum value of 5 h, reached 26.9% of total cells protein, and mainly in the form of insoluble inclusion bodies of existence. Through the optimization of conditions, succeeded in obtaining a soluble fusion protein, ADSL were purified by Glutathione Sepharase 4B gel-purified, and by Western blotting analysis showed that it is ADSL for Beijing chicken protein. The research is the basis for its further research with biological function of the foundation and its application identification.The specific expression of purH gene in 8 different tissues of Beijing fatty chicken was investigated by RT-PCR in this study. The full length of purH cDNA was inserted into fusion expression vector pEGFP-N3,pEYFP-N1 and pDsRed1-N1 multiple cloning sites between EcoR I and BamH I, and construct recombinant eukaryotic expression vector pEGFP-N3- purH,pEYFP-N1- purH and pDsRed1-N1- purH with GFP as reporter gene. We used lipofectin method to transfect the recombinant vectors into Beijing fatty chicken fibroblast cells. After the G418 screening and resistant colonies was picked and subcultured until use. The results showed: 24、48 and 72h after transferring, the expression efficiency of 3 kind of recombinant fusion protein genes were between10.3%～53.2%, and the fluorescence could be observed in cytoplasm and nucleus well-distributed except cryptomere vesicle; Through the G418 drug screening and monoclonal training, three cell lines of stable expression of pEGFP-N3-purH , pEYFP-N1-purH and pDsRed1-N1-purH fusion protein was cloned. RT-PCR and western blot both confirmed the pEGFP-N3-purH,pEYFP-N1-purH and pDsRed1-N1-purH have been integrated into the Beijing chicken fibroblast cell genome, and access to the normal fusion protein expression. The research is important to genetic mark, nuclear transplantation and transgenic animal clone etc.We constructed a high-redundancy bacterial artificial chromosome(BAC) library of an important chinese pig species, conserved the its whole genome resources, and supplied the research material for genome and post-genome research. Not only has the germline of this important chicken breed been preserved at the cell level, but also valuable material had been provided for gene expression. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level. These cloning cell lines of 1 ADSL and 3 purH gene would provide important material for the transgene animal cloning in order to cultivate transgene chicken and divided whether ADSL and purH are the main genes controlling the flavor characteristic or not.更多还原