藤仓镰孢菌β2-微管蛋白上与多菌灵抗药性点突变伴生的同义突变（G235G）生物学功能及其分子机制

Rice bakanae caused by Fusarium fujikuroi is one of the important diseases. Privious results of this project research group have shown that resistant molecular mechanism of F. fujikuroi against carbendazim is unique, i.e., field carbendazim-resistant strains possess not only an amino acid point mutation (Z198V or F200Y), but also a synonymous mutations (G235G) at the β2-Tub. These results are validated by both genetics method andβ2-Tub sequence analysis from nearly 200 drug-resistant strains in field. Some synonymous mutations have biological functions reported in recent years, and about 50 kinds of human diseases are involved in this kind mutation, but rarely reported in filamentous fungi. This project intends to construct non-genetically-marker and in-situ-complemented mutants of F. fujikuroi in order to reveal the biology function of the synonymous mutation G235G without the noise of genetic background. Then exclusive method is applied to scan the priority of glycine codon, gene expression, mRNA editing, mRNA　structural prediction and stability, translation, identification and functional verification for the small RNA among the mutants. The results will not only reveal the synonymous mutation is involved in the development of resistance against carbendazim and its underlying molecular mechanism, but also overturn our traditional understanding of biological function of synonymous mutations in filamentous fungi.Four SCI papers will be published and four graduate students will be brought up with the support of the grant.

本项目前期研究表明，藤仓镰孢菌对多菌灵抗药性分子机制独特----田间抗药性菌株β2-微管蛋白（β2-Tub）除氨基酸点突变（Z198V或F200Y）外，还同时发生同义突变（G235G），这得到了遗传学和对田间近200个抗药性菌株β2-Tub序列分析的验证。近年来发现，部分同义突变具有生物学功能，如与50种人类疾病有关，但在丝状真菌中鲜有报道！为揭示藤仓镰孢菌抗药性菌株β2-Tub上G235G功能及其机制，本项目拟构建无遗传标记的原位回复突变体，明确β2-Tub同义突变的生物学功能；采用排除法筛查点突变体间的氨基酸密码子使用偏好性、基因表达、mRNA剪切、结构预测和稳定性、翻译、 差异small RNA鉴定和功能验证。研究结果不仅将揭示藤仓镰孢菌β2-Tub上伴生的同义突变生物学功能及其分子机制，而且颠覆人们对丝状真菌同义突变的传统认识。发SCI论文4篇；培研究生4名。

本实验室前期研究表明：我国水稻恶苗病菌优势种藤仓镰孢菌(Fusarium fujikuroi)对多菌灵（MBC）的田间抗药性菌株存在MBCMR基因型（F200Y+ G235G）和MBCHR基因型（E198V+ G235G），为此，本研究旨在揭示与MBC抗药性氨基酸点突变伴生的同义突变G235G的生物学功能。主要研究结果如下：构建无遗传标记的原位回复突变体，明确了β2-Tub同义突变G235G在敏感菌株中单独存在，与无该同义突变的菌株没有差异显著的生物学功能，但如与F200Y或E198V相结合，能够显著增强中抗菌株的适合度，且增强其在MBC作用下微管稳定性；采用构建突变体和排除法筛查，发现与抗药性伴生的同义突变（G235G）生物学功能与编码该基因的氨基酸密码子使用偏好性无关；但链特异性的RNA-Seq和甲基化组学分析结果表明，该伴生的同义突变（G235G）功能与存在的自然反义转录本（NATs）和四个基因（FFUJ_11460、FFUJ_00061、FFUJ_05475和FFUJ_07651）的甲基化有关，前者得到了所构建的正向转录和反向转录突变体的生物学功能的验证，后者的验证工作仍在进行中；为了研究该伴生的同义突变生物学功能是否能在遗传上相近的禾谷镰孢菌中再现，分别构建了用藤仓镰孢菌的β2-Tub-GFP（带有抗药性点突变+同义突变）原位替换禾谷镰孢菌β2-Tub的替换突变体，结果表明：藤仓镰孢菌β2-Tub与抗药性伴生的同义突变生物学不能在禾谷镰孢菌中重现。该研究在国际上首次揭示NATs参与禾谷镰孢菌对多菌灵抗药性氨基酸点突变伴生同义突变（G235G）的生物学功能。