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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。
Aim To investigate the binding protein of key recombinant protein MLH1 in spermatogenesis. Methods MLH1 was used as a bait and screened in the cDNA library of mouse testis by yeast two-hybrid system. The MLH1 interacting protein was searched and verified by GST pull-down and co-immunoprecipitation. Results By yeast two-hybrid screening, 29 proteins were screened from four testis in mouse testis, including the known MLH3 and PSM2. One of the new proteins, RHNO1 and MLH1, was verified by GST pull-down and coimmunoprecipitation. Real-time PCR detection revealed that RHNO1 was highly expressed in testis, suggesting that RHNO1 plays an important role in spermatogenesis. Conclusion Yeast two-hybrid system is a powerful means to find out the binding protein, and found a new MLH1 binding protein RHNO1, which may be involved in meiotic homologous recombination.