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 Sfi
I and size fractionation,SMART cDNA was ligated into the Sfi
I-digested pBluescript II SK (with Sfi
I A and B site).The ligation mixture was transformed into E.Coli
5α.The cDNA library contained 1.8x105
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.