赤霉菌致病相关基因CR1的鉴定和功能分析

Fusarium graminearum is the predominant species causing head blight disease of wheat and other cereal crops. In addition to substantial yield losses, the mycotoxins produced by F. graminearum are harmful to human and animals. In order to identify crucial genes involved in the pathogenicity and toxingenicity of this fungus, we have constructed a mutant library with a representative strain isolated from Wuchang by restriction enzyme mediated transformation. By screening the library, we have isolated several important mutants including M594, from which we cloned the cytokinesis related gene CR1 by TAIL-PCR. M594 produces significant less deoxynivalenol and has reduced virulence on wheat heads. It also shows abnormal conidiospore as well as the collapsed septum. Analysis of the flanking seqeunces of the insert region indicated that the inserted region contains a cytokinesis-related gene, referred as CR1. To further reveal the function and role of the CR1 gene in pathogenicicity and mycotoxin biosynthesis, we will generate knockout mutant cr1 and complementary strain CCR1. Foor those strains, pathogenicity assay and mycotoxin production will be studied. Green fluorescent protein will be fused to the C terminus of CR1 protein in wild type strain 5035and the Nikon eclipse 90i epifluorescent microscopy will be used to observe and document CR1-GFP in specific phrase.The time lapse observation will reveal its cellular localization and dynamic changes in the developmental process. We will also observe the fixed and sectioned mutant hypha/conidiaspore by transmission electron microscopy to investigate the fine structure of mutant cells. Meanwhile, protein localization will be revealed by immunoelectron microscopy with polyclonal antibody against CR1 protein. This will provide insights into the proper location of this protein in the cell. In addition, we will combine gene expression profile and NMR-based metabonomic analysis, to reveal differentially expressed genes and significantly changed pathways between the mutant and wild type. After completion of this study, we will identify the function and cellular localization of the CR1 protein and reveal its roles in infection process and toxin production of F. graminearum. With the informations provided by microarray assay and NMR-based metabonomic analysis, the function and regulatory networks of CR1 gene will be illustrated comprehensively.

麦类赤霉病不仅降低作物产量，还产生对人畜有害的生物毒素。为了鉴定赤霉病菌致病产毒关键基因用于发展病菌控制新靶点，我们选择代表性菌株武昌菌株，创建随机突变体库，筛选获得了多个致病力显著降低的突变体，其中一个突变体 M594不仅致病力低，分生孢子、隔膜、产毒量均发生变异；分析其插入突变边界序列表明，该突变体涉及一个细胞分裂相关基因CR1。本项目研究将在此基础上，创建CR1缺失突变体及其回复突变体，分析比较CR1基因缺失体﹑回复突变体及野生型菌株的产毒、致病发育性状，解析CR1基因在赤霉病菌致病和产毒过程中的作用和功能；构建CR1-GFP融合蛋白，研究CR1在细胞周期中的动态过程及作用位点；以抗CR1蛋白抗体，免疫定位CR1在细胞超微结构中的结合特征；利用基因芯片和核磁共振技术，分析CR1基因缺失对基因组转录和代谢的影响及方式；本项目的完成，将为全面揭示CR1基因的功能及其调控网络提供依据。

麦类赤霉病不仅降低作物产量，还产生对人畜有害的生物毒素。为了鉴定赤霉病菌致病产毒关键基因用于发展病菌控制新靶点，我们选择代表性菌株武昌菌株，创建随机突变体库，筛选获得了多个致病力显著降低的突变体，其中突变体 M594 不仅致病力低，分生孢子、隔膜、产毒量均发生变异。通过TAIL-PCR和序列分析比对发现，FG07919基因发生了突变，该基因与酵母和动物中二型肌球蛋白同源，将其命名为Myo2基因。构建同源敲除载体将禾谷镰刀菌野生型5035中Myo2基因进行敲除，Myo2基因缺失后，禾谷镰刀菌胞质分裂过程发生严重异常，隔膜形成过程受阻，进而菌丝生长速率极大降低，产孢能力极大下降，分生孢子畸形、串生。此外，其有性生长受到完全抑制，无法形成子囊壳和子囊孢子。更为重要的是，Myo2基因缺失后，禾谷镰刀菌完全丧失其侵染能力，毒素水平也极显著降低。由此可见，Myo2蛋白在禾谷镰刀菌整个生长发育、致病、产毒和有性繁殖等过程中都发挥着举足轻重的作用。透射电镜观察发现，Δmyo2隔膜异常，数量下降且常不完整，为了进一步观察，将绿色荧光蛋白与Myo2蛋白进行融合，在荧光显微镜下观察到了Myo2蛋白在细胞中的位置和作用过程，发现Myo2蛋白在隔膜形成之前便聚集到分裂处，并形成环状结构，随后Myo2蛋白环收缩，隔膜由外自内开始形成，当Myo2蛋白环收缩成一个亮点时，隔膜形成过程结束，随后Myo2蛋白逐渐消失。在分生孢子隔膜形成中的作用过程中相似。分析Δmyo2突变体基因表达谱发现，Myo2基因缺失后，禾谷镰刀菌次生代谢发生明显异常，参与黄色镰刀菌素和脱氧雪腐镰刀菌烯醇合成的基因表达受到抑制，这也解释了Δmyo2突变体中毒素水平下降的原因。此外，还发现一个参与真菌与植物特异性系统应答反应过程的基因FGSG_00051，在Δmyo2突变体中几乎无法检测到，下调水平达4万倍。该基因表达量的急剧下降也解释了Δmyo2突变体致病力的缺失。对该基因具体功能的研究将有助于我们进一步了解禾谷镰刀菌致病机理。真菌细胞胞质分裂过程非常保守且完全不同于植物细胞，本研究中的Myo2基因并不存在于植物细胞中，可以为新型杀菌剂的研究提供新的作用靶标。同时，由于Myo2蛋白及其作用在真菌中的保守性，基于其所开发的新型杀菌剂将具有很好的广谱性。