球孢白僵菌两个FRQ节律蛋白轮班的生物钟效应解析及其调控机理

A 'classic' circadian clock has been well characterized in Neurospora crassa, in which the transcription of a single frq gene is split at the locations of different introns to encode two structurally similar Frequency (FRQ) proteins that entrain the circadian clock. Filamentous fungi may have similar or distinct circadian clocks in association with or without FRQ proteins. The applicant's previous study for doctoral degree at Zhejiang University has unveiled that the fungal insect pathogen Beauveria bassiana possesses two FRQ-domain proteins (Frq1 and Frq2) with each differing from another in both structure and molecular size. The study has further revealed that Frq1 and Frq2 of B. bassiana can be constitutively expressed at transcriptional level and shuttles periodically between cytoplasm and nucleus in different light/dark cycles and that temporal/spatial expression dynamics (time lengths and levels) of Frq1 and Frq2 within nuclei are completely complementary during 24-h exposure to light or darkness. These new findings implicate that Frq1 and Frq2 in B. bassiana may alternate their duty to entrain a novel circadian clock, which is distinct from the classic model in N. crassa. This study seeks to characterize molecular and cellular properties of the novel circadian clock by unveiling a possible interaction of Frq1 and Frq2 at both transcriptional and translational levels and an interaction of each with main blue or red light-sensing proteins through the manipulation of targeted genes and proteins. The expected results will shed light upon the Frq1/2-alternating rhythm of the novel circadian clock and its biological significance for asexual development and ecological fitness of B. bassiana, providing a new insight into the regulatory roles of Frq1 and Frq2 in the fungal adaptation to host and environment.

丝状真菌生物钟及其调控作用的科学认识主要来源于粗糙脉孢菌的经典模式，而丰富多样的丝状病原真菌中不仅存在类似该模式的生物钟，也存在可能有别于此的生物钟，它们涉及或不涉及生物钟节律蛋白FRQ。申请人前期研究发现，球孢白僵菌基因组中有两个基因编码结构和分子量均迥异的FRQ蛋白Frq1和Frq2，不仅能够相互独立地组成性表达，而且两个蛋白在细胞核内滞留时间与丰度变化呈讶异的互补状态，这不同于粗糙脉胞菌中由同一FRQ基因在不同位置内含子处剪切而形成的结构及分子量相近的两个蛋白。据此，申请人提出球孢白僵菌可能由Frq1和Frq2轮流值班运行生物钟的新假说。本项目运用基因与蛋白分子的操作技术，研究解析该新模式生物钟的分子与细胞生物学基础，揭示Frq1/2是否存在转录与翻译水平上的互作，揭示同Frq1/2互作的光敏蛋白，揭示该生物钟的运行规律及其调控机理，丰富真菌生物钟效应与生防潜能的科学认识。

本项目旨在对球孢白僵菌基因组中两个结构和分子量均迥异的生物钟蛋白Frq1和Frq2进行研究，揭示两个蛋白感应昼夜节律信号的细胞生物学基础及其生理生态效应，解析两者在细胞核内滞留时间与丰度变化呈互补状态的运行规律及其调控机理。该项目实际涉及30多个相关基因的研究。在Frq1与Frq2互作关系的研究中，发现了RNA解旋酶FRH是一个重要的互作因子，并证明FRH调控Frq1和Frq2在细胞核内的昼夜积累水平呈完全相反的动态节律，由此昼夜不间断激活中心发育通路中关键基因的持续表达而维持白僵菌不受昼夜或光周期影响的无节律产孢，并在较短时间内实现产孢水平的最大化。这一发现揭示了球孢白僵菌Frq1和Frq2呈相位差轮流值班的生物钟运行新模式以及对规模化生产作为真菌杀虫剂有效成分的高质量分生孢子粉的重要意义。在扩展研究中，还发现了一批调控白僵菌生长发育、胁迫应答、寄主侵染及毒力的重要基因，深化了白僵菌生防潜能的科学认识。项目执行三年期间，项目主持人在国际主流学术期刊以第一作者/通讯作者共发表了有标注的SCI论文19篇，参与1篇，超额完成预期指标。