【作者】 林梅；

【导师】 周洁； 余文英；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2020， 硕士

【摘要】 轮枝镰刀菌(Fusarium verticillioides)是引起玉米穗腐病和甘蔗梢腐病的主要病原真菌,这些病害严重影响到作物的产量和质量。此外,轮枝镰刀菌还会在谷物上积累大量的次级代谢产物伏马毒素FB1,对人和动物的健康造成极大的危害。过氧化物酶体是真核细胞中普遍存在的由单层膜包裹的细胞器,其内含有50多种参与不同生理代谢途径的酶。子囊菌中,过氧化物酶体能够调控致病过程和次级代谢产物合成途径。过氧化物酶体基质蛋白在细胞质中合成后,通过过氧化物酶体定位信号(Peroxisome targeting signal,PTS)1型(PTS1)或2型(PTS2)分别被各自受体识别,进而转运至过氧化物酶体。在轮枝镰刀菌中,PTS2蛋白的受体是FvPex7,辅助受体是FvPex20,而二者是否参与轮枝镰刀菌致病过程以及FB1的合成途径还未见报道。本课题主要以FvPex7和FvPex20为研究对象,探索二者在轮枝镰刀菌致病力和FB1生物合成中是否发挥调控作用以及二者间的相互关系,具体研究结果如下:为了探索FvPEX7和FvPEX20基因的功能,本研究通过同源重组方法获得(35)Fvpex7、(35)Fvpex20单敲突变体及(35)Fvpex7pex20双敲突变体菌株。表型分析结果表明,与野生型Fv7600相比,(35)Fvpex7、(35)Fvpex20和(35)Fvpex7pex20突变体菌株的致病力降低,且FB1生物合成量和产孢量减少;(35)Fvpex7和(35)Fvpex7pex20突变体菌株在基本培养基上的生长速率减慢且对200μg/m L CR和CFW胁迫的敏感性降低;(35)Fvpex20对0.01%SDS胁迫的敏感性降低;在(35)Fvpex7和(35)Fvpex20突变体菌株中,PTS2信号蛋白不能定位于过氧化物酶体。随后为了探索FvPex7与FvPex20的关系,本研究通过亚细胞定位实验发现FvPex7和FvPex20主要定位于过氧化物酶体和细胞质,且二者共定位于过氧化物酶体,但在(35)Fvpex20突变体中,FvPex7不能定位于过氧化物酶体;进一步通过酵母双杂交和Co-IP实验证实FvPex7与FvPex20存在直接互作关系,且FvPex20与FvPex7的互作位点位于羧基末端。此外通过酵母双杂交实验验证到2个具有PTS2信号的蛋白与FvPex7存在互作关系。综上所述,轮枝镰刀菌PTS2蛋白受体FvPex7和辅助受体FvPex20通过影响不同的代谢途径而共同调控其致病力和FB1合成途径;FvPex7在过氧化物酶体上的定位依赖于FvPex20,且FvPex7和FvPex20共同调控PTS2蛋白的转运途径。更多还原

【Abstract】 Fusarium verticillioides is a major pathogen of maize and sugarcane,that causes ear rot and pokkah boeng disease respectively,which seriously affects their yield and quality.In addition,F.verticillioides accumulates a large amount of FB1 mycotoxins on the cereal,causing great harm to human and animal health.Peroxisome is an organelle with single membrane in eukaryotic cells and contains more than 50 enzymes involved in various physiological and metabolic pathways related to pathogenicity and secondary metabolite synthesis in the ascomycete.The peroxisomal matrix proteins containing peroxisomal targeting signal(PTS)type1(PTS1)or type 2(PTS2)are synthesized in the cytosol and then imported into peroxisomes by their respective receptors.In F.verticillioides,it is known that PTS2-containing matrix proteins are recognized by the receptor FvPex7 and co-receptor FvPex20,however their intrinsic roles in pathogenic process and FB1 biosynthesis remain elusive.Therefore,in this study we mainly explored the regulatory role of FvPex7 and FvPex20 in F.verticillioides pathogenicity and FB1 biosynthesis as well as the relationship between them.Firstly,we generated the single mutants and double mutant of FvPEX7 and FvPEX20 genes in Fv7600 wild type strain by homologous recombination method and analyzed their contribution to pathogenicity,FB1 biosynthesis and sporulation of F.verticillioides.Results obtained showed that pathogenicity,FB1 biosynthesis and sporulation were not only decreased in(35)Fvpex7 and(35)Fvpex20 single deletion mutants but also in(35)Fvpex7pex20 double deletion mutant.In addition,both the(35)Fvpex7 and(35)Fvpex7pex20 mutants grew slowly on minimum medium and showed reduced sensitivity to 200 μg/m L CR and CFW stress,while(35)Fvpex20exhibited a decreased sensitivity to 0.01% SDS stress compared to the wild type strain.Through subcellular localization,we confirmed that the localization of PTS2-containing proteins were altered in(35)Fvpex7 and(35)Fvpex20deletion mutants.Both FvPex7 and FvPex20 are localized in peroxisome and cytosol in mycelia,and they were completely colocalized in peroxisomes.But deletion of FvPEX20 resulted in a mislocalization of FvPex7 in peroxisome.Furthermore,we found that FvPex7 directly interacts with FvPex20 and the two PTS2-containing proteins interacted with FvPex7 by yeast two-hybrid.Taken together,F.verticillioides PTS2-containing proteins receptor FvPex7 and co-receptor FvPex20 contribute to pathogenicity and FB1 biosynthesis by affecting different metabolic pathways.The localization of FvPex7 on the peroxisomes is dependent on FvPex20,and FvPex7 cooperates with FvPex20 to regulate the transport pathway of PTS2-containing proteins.更多还原