减数分裂重组关键蛋白MLH1结合蛋白的酵母双杂交筛选和鉴定

Screening and verification of proteins that interact with MLH1,the key protein in meiosis recombination,by yeast two hybrid screening

目的探寻减数分裂关键重组蛋白MLH1在精子发生中的结合蛋白。方法以MLH1为诱饵,通过酵母双杂交系统,在小鼠睾丸组织的cDNA文库中进行筛选,寻找MLH1的相互作用蛋白并通过GST pull-down和免疫共沉淀的方法进行验证。结果通过酵母双杂交的筛选,在小鼠睾丸中经四缺板筛出蛋白29个,包括己知的MLH3和PSM2等。通过GST Pull-down和免疫共沉淀验证了其中一个新的蛋白RHNO1与MLH1的结合。通过Real-time PCR检测发现RHNO1在睾丸中高表达,提示RHNO1在精子发生中具有重要功能。结论酵母双杂交系统是寻找结合蛋白的有力手段,发现了新的MLH1结合蛋白RHNO1,可能参与了减数分裂同源重组。

Objective To screen proteins that interact with MLH1, the key protein in meiosis recombination in spermatogenesis. Methods Mouse testis cDNA library was screened by the bait of MLH1 using yeast two hybrid system to find proteins interact with MLH1, and the interaction was validated by GST pull-down and co-immunoprecipitation. Results Total of 29 interaction proteins were found on the plate of SD/-Ade/-His/-Leu/-Trp by yeast two hybrid screening, including the known interaction proteins of MLH3 and PSM2. A new interaction protein was found which was named as RHNO 1, and the interaction was confirmed by GST pull-down and co-immunoprecipitation. The high expression level of RHNO1 in testis revealed by real-time PCR indicates that RHNO1 may have important function in spermatogenesis. Conclusion Yeast two hybrid system is an effective method to find new interaction proteins, and we have identified a new protein （called） RHNO 1 interacted with MLH 1, which may play roles in meiotic homologous recombination.