【作者】 刘志红；

【导师】 李金泉； 李宁；

【作者基本信息】 内蒙古农业大学 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 绒山羊是我国珍贵的绒肉兼用品种,是不可多得的珍贵遗传资源,为了研究绒山羊绒毛生长发育和周期性变化的分子机制,本文构建了内蒙古绒山羊基因组的BAC文库,为绒毛生长发育的相关基因的研究、基因组物理图谱完善及BAC重叠群的构建打下了基础。随后,本文对BAC文库进行了保存。之后又建立了高效的PCR筛选系统,最后对绒山羊绒毛生长发育的相关基因进行了筛选,获得了MTNR1a、KAP8.1、BMP4、FGF5和IGFBP5等与绒山羊绒毛生长发育相关的基因的BAC克隆,为将来定位和克隆绒毛生长相关基因打下了基础,也为进一步研究绒山羊绒毛生长的分子机制打下了基础。BAC由于容量大、转化效率高、嵌合体少、易操作易回收、在宿主细胞中插入片段稳定性好等特点而成为真核细胞生物核基因组大片段插入文库制备的良好载体。BAC文库不仅可以应用于目的基因的获得,还可应用于物理图谱的构建、基因功能结构和表达的研究,定位克隆,长距离DNA测序和锚定、鉴定假定的顺式调节元件、比较基因组学研究以及转基因研究等。本试验从雄性阿尔巴斯绒山羊静脉血中提取白细胞,然后将白细胞包埋进低熔点琼脂糖凝胶中,通过HindⅢ部分酶切基因组后,通过CHEF凝胶电泳技术对大片段DNA选行两次分离,使片段大小集中。之后通过电洗脱方法回收150～400kb范围的片段。洗脱回收的大片段DNA和BAC载体按摩尔比1∶3的比例连接之后,通过点透析对连接产物进行脱盐处理,处理后的连接产物通过电转化的方法转化感受态大肠杆菌DH10B。经过复苏之后将转化菌液涂布于含有氯霉素、IPTG、X-gal的LB平板培养基上,挑选白色克隆构建文库。本研究所构建构建的阿尔巴斯绒山羊的BAC文库,结果如下:1该文库含有的克隆数为276480个,分为36个超级池,每个超级池由20块384孔板组成;2随机挑选的1132个克隆鉴定插入片段大小,估计文库的平均插入为128kb,其中空克隆比例为0.53%;文库中77%的克隆插入大于100kb,有部分克隆甚至大于300kb,文库片段大小比较集中,这样大小的插入可以满足所有试验对于片段大小的要求。3以基因组大小为3*109计,文库的基因组覆盖率为11.8倍,预计从文库中筛选到单拷贝基因的机率为99.99%。4整个文库以两步-4D的结构保存,并分别提取行池、列池、板池的DNA。5建立了高效快速的PCR筛选系统。用20个微卫星标记和5个功能基因对整个文库进行PCR筛选,所有这些标记和基因都得到了阳性克隆,克隆数从3到19不等,得到的平均阳性克隆数为11.3。说明文库具有良好的覆盖率,并且与前面鉴定结果一致。6筛选获得了绒山羊绒毛生长发育相关的基因MTNR1a、KAP8.1、BMP4、FGF5和IGFBP5等的BAC克隆,并验证其确实为要筛选的基因。本研究确定了高分子质量DNA获得的方法,优化了基因组部分酶切的条件,建立了高效的连接及转化体系,建立了一套适合于本实验室的BAC文库构建体系,并且筛选出了阳性克隆,为以后的工作奠定了基础.更多还原

【Abstract】 Cashmere goat is one of precious animal breeds in China. It is famous for supply of both mutton and cashmere for people′s lives. In order to study the molecular mechanism underlying the growth and development of cashmere and wool in cashmere goat, we constructed an Inner-Mongolian cashmere goat Bacterial Artificial Chromosome (BAC) library. Library storage and setting up of efficient PCR screening system have been done subsequently. Lastly, BAC clones related with genes affecting growth and development of cashmere goat were screened and positive clones containing MTNR1a、KAP8.1、BMP4、FGF5、IGFBP5 genes were harvested. This work will serve as the basis for mapping and cloning of functional genes as well as studying of molecular mechanism underlying the growth and development of cashmere and wool in cashmere goat.BAC library has been used widely in the research of eukaryotic organism with larger genome due to its advantages of larger capacity, stable inherited characteristic, few chimeras, easily reclaiming insertion element, convenient operation and so on. BAC library can also serve for positional cloning of target genes, constructing of physical map, researching on gene expression and function, sequencing of large DNA fragment, verification of supposed cis-regulatory, analysis of comparing genome, studying of transgene and so on.In this research, leukocytes were extracted from venous blood of male ARBAS Cashmere goat and embedded into low-melting point agarose. Embedded cells were then partially digested with enzyme HindⅢand High Molecular Weight (HMW) DNA were fractionated by twice CHEF gel electrophoresis. Digested DNA fragments in the range of 150kb~400kb were recovered by electro-elution and were ligated to BAC vector with mole ratio of 1:3. Subsequently, ligated product was desalted by dialyzing and transform to DH10B competent cells. The bacteria were revived and incubated in media with Chloromycetin, IPTG, X-gal respectively. Lastly, individual white colonies were picked and applied to library construction.Results of ARBAS Cashmere goat BAC library construction:1 This goat BAC library consists of 276,480 BAC clones in total (36 superpools, each for 20 384-well plates) 2 The average insert size of the library is 128kb, which was evaluated from analysis of 1132 randomly selected BACs. The ratio of negative clone in this library is 0.55% and clone larger than 100kb is 70%. Several clones are as large as 300kb. Generally, the size of DNA fragments in this library is unbiased and could meet the demand of further study.3 The genome coverage of the library is 11.8–fold assuming that the genome size is 3*109 bases. Then the possibility to find a single-copy gene in this library is 99.99%.4 The stored library was organized in two grades and 4-dimension structure. And DNA were extracted from row pools、column pools and plate pools , respectively.5 Establish of polymerase chain reaction (PCR) screen system with high efficient. The library was screened by using 20 microsatellite markers and 5 known functional genes with PCR. Positive BACs for all these markers and genes were identified successfully and the positive number ranges from 3 to 19 with average of 11.3. The result indicates that the coverage rate of this library is ideal and this estimation is compatible with the 11.8-fold genome redundant library.6 Positive clones containing MTNR1a、KAP8.1、BMP4、FGF5、IGFBP5et al. which are relevant to growth and development of cashmere goat were harvested and tested.In summary, we have set up a practical method to gaining HMW DNA, optimized the experimental conditions for partial enzyme digestion on genome and established a high efficient system of ligation-transformation. We believe that the establishment of this effective system for BAC library construction could serve as a basis for deep study of the molecular mechanism underlying the growth and development of cashmere and wool in the cashmere goat.更多还原