SSH法构建奶牛乳房炎抗性相关基因的cDNA文库

Construction of mastitis resistant cDNA library in dairy cows using suppression subtractive hybridization

以经产荷斯坦奶牛的乳腺组织为材料,抽提总的RNA,分离提纯Poly(A)+RNA,反转录并合成单链和双链的cDNA,经酶切形成200～800bp的片段。将患乳房炎奶牛的cDNA分成2组,分别与不同的接头连接,再与正常奶牛的cDNA进行2次消减杂交和2次抑制性的PCR扩增;将第2次PCR产物与pGEM-T载体连接,转化大肠杆菌TOP10感受态细胞进行文库扩增,构建了具有高消减效率的奶牛乳房炎抗性相关cDNA文库。文库扩增后得到361个白色阳性克隆,随机挑选克隆进行PCR鉴定,插入片段主要分布在200～800bp。文库的成功构建为进一步筛选、克隆与奶牛乳房炎抗性相关基因奠定了基础,对研究奶牛乳房炎抗性的分子机制以及乳房炎的综合防制具有重要意义。

Poly （A）＋ RNA were purified using Oligotex mRNA Kits （Qiagen） from mammary tissue of 21 Hol- stein cows with mastitis and 7 health Holstein cows in lactation as control. The single-and double-stranded cDNA were synthesized from the poly （A） ＋ RNA using PCR-SelectTM cDNA Subtraction Kit （Clontech） and further diges- ted using Rsa I into cDNA fragments sized frorri 400 to 600 bp （dscDNA）. Then the dscDNAs from the mastitis cows （as tester） were div＇ded into two portions which were ligated separately with a different cDNA adaptor. The ligated cDNA were hybridized twice with the dscDNA of the healthy cows at 68℃ for 8 h each. The products after double hybridizations were diluted by 200 times and then used for suppression PCR twice;the secondary PCR products was inserted into pGEM-T vector and transformed into E. coli TOP10 competent cells;361 positive clones were obtained;identification of the inserted cDNA fragments in subtractive library was done using PCR. The results showed that there were inserted fragments sized by 200-800 bp in the 16 randomly selected positive clones, which would provide useful baseline for screening and cloning specific mastitis resistant genes and under standing the mo- lecular mechanism of mastitis resistance in dairy cow.