人端粒酶RNA突变体酵母三杂交诱饵质粒的构建

Construction of a bait plasmid containing human telomerase RNA mutant in yeast three-hybrid system

目的对人端粒酶RNA进行多位点的突变并构建该突变体的酵母三杂交诱饵质粒,且进行毒性检测。方法根据人端粒酶RNA(hTR)序列及突变位点设计引物,运用重叠延伸PCR法对hTR基因的第41位、第80位及第102位碱基进行T→A突变,将突变后的片段克隆入PMD18T载体,经测序正确后再亚克隆到酵母三杂交诱饵质粒PRH3'中,PCR及酶切鉴定。鉴定成功的重组诱饵质粒转化到酵母细胞L40ura3/pHyblex/ZeoMS2进行毒性检测。结果经过测序验证,该基因预期位点突变成功且其他序列未发生随机突变,重组诱饵质粒构建成功,转化酵母菌之后对宿主无毒性。结论成功构建了hTR突变体的重组质粒PRH3'-hTRm,可作为酵母三杂交系统中的"诱饵"。

Objective To construct a bait plasmid containing human telomerase RNA with multiple point mutations in a yeast three-hybrid system and evaluate the toxicity of the recombinant bait plasmid. Methods The primers were designed according to the hTR sequence and the target mutation sites for inducing T→A mutations at the 41st, the 80th and 102nd nucleotides of the hTR gene using the overlapping extension PCR （OE-PCR） method. The mutant was cloned into PMD18T vector, confirmed by sequencing, sub-cloned into the bait plasmid PRH3＇ and identified with PCR and restriction enzyme digestion. The recombinant bait plasmid was then transformed into yeast L40 ura3/pHyblex/ZeoMS2 for toxicity test. Results Sequence analysis demonstrated successful introduction of point mutations at the target sites without causing random mutation. The recombined bait plasmid constructed showed no obvious toxicity against the host yeast cells. Conclusion The recombinant plasmid containing the human telomerase RNA mutant （PRH3＇-hTRm） has been successfully constructed and can be used as the bait plasmid in yeast three-hybrid system.