Construction of a Skin cDNA Library of Cashmere Goat and Cloning of KAP6-2 Fulllength cDNA

In order to understand the molecular mechanism of the characteristic of cashmere,we constructed a cDNA library using the SMART cDNA library construction kit (Clontech).Total RNA was isolated from the goat (Capra hircus) skin tissue with hair follicle angen.Oligotex (QIAGEN) was used to isolate mRNA from total RNA.The "anchor firststrand cDNA" synthesized by reverse transcription with the SMART technique.The LD-PCR was performed using a modified oligo (dT) primer and an anchor primer as the primer set,and anchor first-strand cDNA as the template to enrich the cDNA population for full-length sequences.After digestion with SfiⅠand size fractionation,SMART cDNA was ligated into the SfiⅠ -digested pBluescript Ⅱ SK (with SfiⅠA and B site).The ligation mixture was transformed into E.Coli 5α.The cDNA library contained 1.8×105 independent clones.Randomly select cDNA clones and sequence with the 5 prime end,a full-length KAP6-2 cDNA was found by compared with those in the NCBI database (nr) using the Blast-N programs,it showed 75.5% identity in amino acids with mouse KAP6-2 and the accession number in GenBank is AY316158.