利用酵母单杂交文库技术筛选银杏端粒结合蛋白

Screening Telomere Binding Proteins from Ginkgo biloba L. Using Yeast One-Hybrid Library

摘要: 目的：为了获得与银杏端粒结合序列相结合的端粒结合蛋白，为木本植物端粒结合蛋白的研究提供实验依据，从而丰富对银杏端粒的研究。方法：以银杏叶片为实验材料，利用酵母单杂交文库筛选获得可能为银杏端粒结合蛋白的基因片段，并通过GFP酵母单杂交实验验证获得基因片段与端粒序列的结合特异性。结果：使用端粒特异性结合序列(TTTAGGG)₃的诱饵载体没有获得合适大小的扩增片段，而使用端粒特异性结合序列(TTTAGGG)₅的诱饵载体成功获得52个扩增片段，这说明端粒DNA序列(TTTAGGG)₃过短，不利用蛋白质的结合。比较这些扩增片段序列并去除相同序列，结果显示这52个扩增片段分别属于10个基因片段。对这10个基因片段进行进一步序列分析，并使用酵母单杂交技术验证，结果显示其中一个基因能够与银杏端粒序列特异性结合。结论：本研究通过酵母单杂交文库筛选，GFP酵母单杂交验证等实验方法建立了银杏端粒结合蛋白的筛选方法，并成功获得了一个和银杏端粒重复序列特异性结合的端粒结合蛋白。

Abstract: Objective: To provide a powerful experimental basis for the research of telomere binding proteins and telomeres in woody plants especially in Ginkgo, we investigated the telomere-binding proteins that bind with telomere sequence in ginkgo. Methods: Using Ginkgo leaves as experimental material, we obtained some gene sequences encoding telomere-binding proteins through yeast one-hybrid library screening technology, then verified their binding specificity by GFP yeast one- hybrid experiments. Results: We did not get any desired DNA sequences using bait vector contain telomere specificity sequence (TTTAGGG)₃. However, we obtained 52 DNA sequences using bait carrier contain telomere specificity sequence (TTTAGGG)₅ successfully. Our results suggested that the telomere specificity sequence (TTTAGGG)₃ might be too short to binding any proteins. After removing repeat sequences, we found that 10 genes were encoded by these 52 DNA sequences through sequences analysis. One gene among them was confirmed that could bind with Ginkgo te-lomere specificity sequence. Through GFP yeast one-hybrid technology. Conclusion: Our study es-tablished a screening method to investigate telomere binding proteins in Ginkgo through yeast one-hybrid library screening and GFP yeast one-hybrid technology, and obtained a telomere binding protein which binding with Ginkgo telomere specificity sequence specifically.