Construction Of Silky BAC Library And Study Of Melanin-related Gene TYRP1 And ID

To research melanin synthesis and its accumulation mechanism, we constructed a Silky bacterial artificial chromosome (BAC) library. The library was used to study the two genes on chicken Chr. Z which involve in melanin, tyrosinase related protein 1 gene (TYRPI) and dermal melanin inhibitor gene (ID). The genomic structure of TYRP1 was determined and its correlation between melanin accumulation and TYRP1 expression was studied. Moreover, we constructed a BAC contig near ID locus which was on a region short of DNA marker in Chr.Z.I. Results of Silky BAC library construction:(1) The chicken BAC library consists of 138,240 BAC clones in total(72 superpools, each for 20 96-well plates). The stored library was organized in two grades and 3-dimension structure, and a PCR screening system with high efficiency was built.(2) Average insert size of the library is 118 kb evaluated from analysis of 452 BACs randomly picked from library. So the genome coverage of the library is 13.34 and the possibility to find a single-copy gene in the library is 99.984%. Results of screening by 40 microsatellite markers and 8 functional genes showed-the average positive clone numbers of them were 10.423 and 11.125 respectively. And maxi chimerism rate is 6% estimated from FISH results of 32 BACs. PFGE analysis of 6 EcoR] digested BACs clones which were derived from the library and contained LMBR1 sequence proved the overlap among the BACs.II. Results of study on TYRP1 and ID:(1) We first sequenced the 10,066 bp whole genomic sequences of chicken TYRP1. Analysis of the sequences indicates that: compared with human TYRP1, chicken TYRP1 is short of an intron and there is two microsatellites in introns. GeneScanning result in resource family with the microsatellite in intronl indicates that TYRP1 affects the color of skin and claw on a significant level (0.01