结构域酵母三杂交诱饵载体的构建及酵母转化

Construction of yeast three hybrid system "bait" of RNA-binding domains of FXR1P

目的脆性X相关蛋白(FXR1P)是一种RNA结合蛋白,本研究构建了其N-端结构域、KH结构域的酵母三杂交诱饵载体并转化酵母菌株L40 ura3/pHyblex/Zeo-MS2,为筛选这2种结构域的相互作用RNA作基础。方法根据Genebank提供的序列设计引物,以pYESTrp3/FXR1质粒作为模板,扩增分别包含FXR1P N-端结构域、KH结构域的编码区序列,连接到用于酵母三杂交文库筛选的pYESTrp3质粒,得到诱饵质粒,导入大肠杆菌Top10扩增,筛选阳性克隆并提取其中的重组质粒,再转化酵母菌L40 ura3/pHyblex/Zeo-MS2。结果经过测序验证,该重组诱饵质粒构建成功,转化酵母菌之后对宿主菌无毒性,无自激活现象。结论成功构建了能用于酵母三杂交文库筛选的FXR1P蛋白N-端结构域、KH结构域酵母三杂交诱饵载体,为研究这两个结构域的RNA结合功能奠定了基础。

Objective Fragile X related protein(FXR1P) contains two KH domains. KH domain is a kind of RNA-binding domain, but our knowledge about what kind of RNAs FXR1P binds is extremely limited. To study the RNA-binding properties of two the three RNA-binding domains of FXR1P, namely, N-ter domain of FXR1P, KH domain, we constructed two yeast three hybrid system 'bait' plasmids that can express these two domains respectively. Methods PCR primers were designed according to FXR1 sequence we searched from genebank; then the coding region sequences of N-ter domain of FXR1P, KH domain were obtained by PCR, using pYESTrp3/ FXR1 as the template; then the PCR products and plamid pYESTrp3 were digested by EcoRI and then purified; then the two coding regions were ligated with pYESTrp3 and introduced into competent Top10; extracted the plasmid of the positive clones and screened from them the recombined plasmids through EcoRI digestion and recomformed by sequencing; the successfully recombined plasmids were introduced into yeast L40 ura3/ pHybLex/ Zeo-MS2 and tested for toxicity and self-activation at last. Results The sequencing results were consistent with the FXR1 sequence in the Genebank; the recombined plasmids did no harm to yeast host cell and there was no self-activation.Conclusion The bait plasmids that can express two of the three RNA-binding domains of FXR1P were successfully constructed and introduced into L40 ura3/ pHybLex/ Zeo-MS2; The transformed yeast strains can be used to study the RNA-binding properties of FXR1P.