摘要
用SMART技术构建了绒山羊 (Caprahircus)毛囊兴盛期皮肤组织的cDNA质粒文库。TrizolReagent(GIBCO/BRL)分离RNA后 ,用Oligotex (QIAGEN)提取mRNA ;以锚定引物反转录合成cDNA第一链作为模板 ,以 2个锚定引物 ,用长链PCR扩增全长cDNA双链。CHROMASPIN 4 0 0去除小片段后用SfiⅠ酶切 ,连接到SfiⅠ消化的pBluescriptⅡSK (带有SfiⅠA和B两个位点 )质粒载体中 ,转化E .coli 5α ,库容量为 1 8×10 5clones。随机挑选克隆提取质粒 ,5′测定插入片段的核苷酸序列 ,与GenBank数据库比对后发现 1个KAP6 2cDNA ,序列号为AY316 15 8。绒山羊KAP6 2由 6 75个核苷酸组成 ,编码 96个氨基酸 ,具有高甘氨酸
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 Ⅰand size fractionation,SMART cDNA was ligated into the Sfi Ⅰ digested pBluescript Ⅱ SK (with Sfi ⅠA and B site).The ligation mixture was transformed into E.coli 5α.The cDNA library contained 1 8×10 5 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.
基金
国家自然科学基金地区重点项目资助 (3 99690 0 2 )
