应用双分子荧光互补技术分析米曲霉Fus3与Ste12之间的蛋白互作

Interaction between Fus3 and Ste12 in Aspergillus oryzae analysed by Bimolecular fluorescence complementation

摘要:

【背景】双分子荧光互补(Bimolecular fluorescence complementation，BiFC)在水稻恶苗病菌(Fusarium fujikuroi)等微生物蛋白互作中的应用已有报道，但在工业菌株米曲霉(Aspergillus oryzae)中还未见应用。【目的】探究米曲霉中Fus3和Ste12蛋白在生长发育中可能存在的相互作用关系，建立在米曲霉活细胞中检测蛋白互作的方法，即BiFC体系。该系统可用于特异性、可视化米曲霉目标蛋白在活细胞中的定位，并且可以更加直观地探究蛋白之间是否存在相互作用。【方法】利用Multisite Gateway复杂载体构建技术，使用切开的绿色荧光蛋白，将荧光蛋白分子的两个片段N端和C端分别与米曲霉Fus3和Ste12蛋白融合，对获得的转化株进行荧光观察。通过BiFC系统检测蛋白之间的相互作用。【结果】成功转化的米曲霉菌丝中观察到荧光，Fus3和Ste12在米曲霉中存在相互作用。【结论】通过BiFC技术证实蛋白质Fus3和Ste12在无性繁殖菌株米曲霉体内发生互作，暗示它们通过互作可能参与除了有性生殖之外的其他细胞功能，并为米曲霉蛋白互作功能研究提供一种新的检测技术和方法。

Abstract:

[Background] Bimolecular fluorescence complementation (BiFC) is applied to explore the protein-protein interactions in some organisms such as Fusarium fujikuroi, but not yet applied in Aspergillus oryzae. [Objective] We used BiFC system to detect the interactions between proteins Fus3 and Ste12 in Aspergillus oryzae during growth and development. We used this system to specifically visualize the location of the target proteins in living cells. [Methods] Complex vectors were constructer by using the Multisite Gateway cloning system. Proteins Fus3 and Ste12 were fused to C- and N-terminal green fluorescence protein fragments, respectively, and then the hyphae of the obtained transformants were observed under a fluorescence microscope. [Results] The green fluorescence protein was observed in successfully transformed cells, indicating that the interaction occurred between Fus3 and Ste12 happened in A. oryzae. [Conclusion] The method to detect protein-protein interaction in A. oryzae proves its broader application possibility.