SAD和RRP3在禾谷镰刀菌有性生殖中的作用及其分子机制研究

Fusarium graminearum is an important plant pathogenic fungus, and wheat head blight caused by this fungus seriously endangers China's grain production. Controlling the release of ascospores during sexual reproduction is one of the important measures to achieve effective control of wheat head blight. A number of studies have shown that the mechanism of RNA silencing in the asexual and sexual reproduction of Fusarium graminearum is quite different, and RNA silencing in sexual reproduction regulates the development of ascus. In addition, the A-I RNA editing is also involved in the sexual reproduction of Fusarium graminearum. In previous study, the applicant found that two RdRP genes (Fgrrp3 and Fgsad) of Fusarium graminearum were significantly expressed during sexual reproduction and the release of ascospores was reduced in Fgsad deletion mutant. Based on the role of RdRP in the RNA silencing pathway of Neurospora crassa, this project intends to further study the role of Fgsad and Fgrrp3 in the RNA silencing pathway during sexual reproduction and its association with RNA editing, and to elucidate the role of these two genes in the sexual reproduction of Fusarium graminearum and its molecular mechanism. The results of this project are conducive to improving the RNA silencing mechanism of Fusarium graminearum and help to understand the regulation mechanism of sexual reproduction of Fusarium graminearum.

禾谷镰刀菌是一种重要的植物病原真菌，由该菌引起的小麦赤霉病严重危害我国的粮食生产。减少有性生殖时期子囊孢子的释放是实现有效防控小麦赤霉病的重要措施之一。多项研究表明，禾谷镰刀菌无性生殖与有性生殖时期的RNA沉默机制存在较大差异性，并且有性生殖中的RNA沉默调控着子囊发育。此外，A-I型RNA编辑同样参与了禾谷镰刀菌的有性发育过程。申请人在前期研究中发现，禾谷镰刀菌的两个RdRP基因（Fgsad和Fgrrp3）在子囊壳中高量表达，并在Fgsad缺失后显著降低子囊孢子的释放。基于RdRP在粗糙脉孢霉RNA沉默途径中的作用，本项目拟进一步研究Fgsad和Fgrrp3在禾谷镰刀菌有性生殖特有的RNA沉默途径中的作用及其与RNA编辑现象的关联性，阐明两基因在有性生殖中的作用及其分子机制。本项目的研究结果有利于完善禾谷镰刀菌的RNA沉默机制，并有助于深入了解禾谷镰刀菌有性生殖过程的调控机理。

禾谷镰刀菌的子囊孢子是小麦赤霉病的主要初侵染源，与其相关的有性生殖期的调控机制非常复杂。本项目前期研究发现，禾谷镰刀菌RdRP家族基因SAD的缺失将导致子囊孢子释放量减少，然而其作用机制以及另一高诱导表达RRP3的功能尚未明确。因此，本项目围绕RdRP展开生物学功能和分子机制研究，主要结果如下：. 1. 证实RdRP1、SAD和RdRP5参与子囊孢子释放，而RRP3不参与。. 2. 明确RdRP1、SAD和RdRP5均参与来源于外显子负链sRNA（ex-siRNA）的累积或形成，其中以RdRP1和SAD为主要参与因子，此外△Fgrdrp1和△Fgsad的ex-siRNA的表达水平分别与△Fgdcl2和△Fgdcl1呈正相关。. 3. ex-siRNA可负调控来源基因，并且部分来源基因参与子囊发育和子囊孢子释放。. 4. 发现RNA编辑并不直接影响ex-siRNA对来源mRNA的降解，预测sRNA临近序列的二级结构可能作为支架影响RNA编辑相关蛋白结合。. 5. 通过分子实验证实一天然负链转录本GzmetE-AS通过产生sRNA负调控GzmetE进而影响子囊孢子释放。. 综合以上结果，本研究阐明了RdRP通过参与ex-siRNA介导的RNA沉默途径调控有性生殖的分子机制，并证明天然负链转录本可通过该途径调控子囊孢子释放。本研究结果深入解析了禾谷镰刀菌有性生殖阶段的RNA沉默机制，并进一步完善了参与禾谷镰刀菌有性生殖的调控因子。..