Constrcution Of Xinjiang Military Reclamation Of Wastel And Type Fine-Fleece Sheep Genome BAC Library And Study On Loci Were Related To MHC Gene Of Chromosome 20 Near Region

Xinjiang was an important livestock production base, fleece and fine-breeding origin of China. Because of its natural and social condition also folk custom's affect, xinjiang had been a breed pattern which giving first place to plant-eating livestock, especially to the sheep breeding industry. In xinjiang the distribution of sheep was broad and had lots of breeds. With the deep developing of the west, how to estimate the system position of xinjiang sheep, application prospect and livestock industry's reasonable arrangement would be the key to our region and whole livestock industry. Construction of xinjiang military reclamation of wasteland type fine-fleece sheep genome bacterial artificial chromosome library (BAC) and study of loci were related to MHC gene of chromosome 20 near region was a basic research project whose kernel was gene recombination technology.Construction high-resolution physical mapping of a species could represent a country's research level and actual strength in bio-technology region on some extent in which human genome project was a successful case and prove. Sheep genome mapping gained from genome sequencing project had big gap, which needed other BAC library being no used to genome sequencing. For this reason, constructing xinjiang military reclamation of wasteland type fine-fleece sheep genome BAC library and researching loci were related to MHC gene of chromosome 20 near region were not only offering technology to construct xinjiang military reclamation of wasteland type fine-fleece sheep's high-resolution physical mapping but also filling the blank in sheep research field. This work would push on sheep's total genome research and application process of our country, offer research base for constructing total genome high-resolution physical mapping and locate functional gene of sheep on the world, which would produce deep influence to all kinds of regions in animal life science researching.To research loci were related to MHC gene of chromosome 20 near region in xinjiang military reclamation of wasteland type fine-fleece sheep, we constructed its genome BAC library, which offered of the utmost useful resource for the following up works including the study on MHC functional gene and construction the high-resolution physical map. We used PCR screening system of 2 times genome library to screen the positive clones of selectedmolecular markers, sequence the PCR production and contrast the affinity of the related sequences. We used complete molecular biology technology to construct the BAC library, which, showed that the library accorded to the international library constructed standard and had higher quality after examining which establishing the foundation for the further research work. The results were as follows:(1) la this present study, we used our special regional fostering breed xinjiang military reclamation of wasteland type fine-fleece sheep's genome to construct its genome library, including 190,464 clones which more than the others sheep BAC library. The average insert fragment of the library was 133kb which longer than other constructed sheep BAC library's insert fragment. At the same time 92.5% clones' insert fragment of the library larger than 100kb and some larger than 300kb. The insert fragment will content to all experiment design that demanding the fragment's size.(2) If we supposed sheep's genome contenting 3x106 kilobase, on the basis of the average insert fragment was 133kb the library covered 8 times genome of xinjiang military reclamation of wasteland type fine-fleece sheep. The probability of screening the tagged fragment from the library was 98.208% that could fully contented the need of construction the physical map and screening the functional gene.(3) Four molecular markers: DMB_EX2> MCMA36^ CP73 and BM1258 located to MHC gene of chromosome 20 near region in xinjiang fine-fleece sheep had been screened positive clones from the constructed 2 times genome library PCR screening system and the average positive clones was 1.5. Considering with the screening result the constructed genome library was fairly closed to the 8 times genome coverage and had no erroneous tendency. This showed that the new construction BAC genome library had fairly rate of coverage which made the library being of the utmost useful resource of studying functional gene, position cloning and improving the genome physical map of sheep.(4) To further confirm the validity of positive clones' sequences screened from the 2 times genome library PCR screening system, we were contrasting affinity of the PCR production from the 4 different molecular markers DMBJEX2> MCMA36^ CP73 and BM1258. The result showed that all of the four sequences had higher affinity that the related species come from: DMB_EX2 locus had 89% affinity with cattle;MCMA36 locus had 97% affinity with goat;CP73 locus had 96% affinity with sheep;BM1258 locus had 97% affinity with cattle.(5) Because of the foreign fragment being inserted to the library came from the xinjiangfrom: DMB_EX2 locus had 89% affinity with cattle;MCMA36 locus had 97% affinity with goat;CP73 locus had 96% affinity with sheep;BM1258 locus had 97% affinity with cattle.(5) Because of the foreign fragment being inserted to the library came from the xinjiang fine-fleece sheep' genome, so we could directly determine the genome sequences. The genome sequences affinity among sheep, cattle and goat were higher than other species so we could use cattle and goat's genome research doing compare genome research to clarify the sheep's genome function, which would be benefited to study different between the xinjiang fine-fleece sheep and other sheep breeds or species of the special characters.Although technology of construction a large fragment genome library was fairly skilled, there still had some problem on technique relative to common library. In this present, we took following improved steps: (1) Constructing the library, we used agarose incubated method extracting genomic DNA to ensure the quality of insert DNA fragment;(2) Taking two-round recovery by three-step pulsed field gel electrophoresis to depart the small fragment and large fragment by digested. This would reduce the proportion of low weight DNA in high weight DNA and important to improve the size of insert fragment;(3) Using electroporation to recover large DNA fragment to decrease mostly mechanism loss and ensure the purity of recovery DNA also improve the efficiency of ligation and transformation;(4) Determining the most appropriate condition of ligation and transformation to improve the ligation and transformation efficiency of large insert fragment and vector : insert fragment was 10:1, 12°C had higher ligation efficiency. To lower 40kb's plasmid using 200Q resistance, 25^iF capacitance and 18, OOOv/cm voltage could gain the highest transformation efficiency.We had successfully constructed the xinjiang military reclamation of wasteland type fine-fleece sheep genome BAC library, but the study on special functional gene of the particular regional breed of China, construction its genome physical map and improvement its gene map need further supplement, improve and deep-going.