| 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. |
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