【作者】 李娟；

【导师】 夏玉先；

【作者基本信息】 重庆大学 ， 生物学， 2019， 硕士

【摘要】 蝗绿僵菌(Metarhizium acridum)是一类十分重要的模式真菌。在真菌中,肽酶在影响真菌适应性方面发挥着重要的作用,而二肽酶(dipeptidase)的功能研究仍然少见。二肽酶能够将二肽水解形成两个游离的氨基酸,对生物体糖代谢及氨基酸代谢具有十分重要的意义。目前,C69家族二肽酶dipeptidase(PepD)在真菌中功能尚未见报道。因此,本文以昆虫病原真菌蝗绿僵菌为研究对象,分析了其酶学特性,阐明了该二肽酶基因(MaPepD)在蝗绿僵菌中的功能,并对该基因参与调控相关表型的机制进行了探索。主要获得了以下研究结果:(1)大肠杆菌(Escherichia coli)表达获得MaPepD蛋白,该蛋白分子量为56-kDa,酶活性为240 U/mg,K_m为1640 mM。该二肽酶最适pH 6.0最适温度是55°C。(2)缺失MaPepD基因后,分生孢子在1/4 SDAY培养基上萌发率提高,对紫外、湿热、细胞壁破坏剂抗性增加,但菌株毒力不受影响。(3)收集1/4 SDAY培养基上培养3 d的孢子进行RNA-Seq分析,结果表明:MaPepD可能介导了细胞壁合成、氧化应激、DNA修复相关基因的转录以影响正常产孢孢子的抗逆性。(4)二肽酶基因MaPepD缺失后,菌株在SYA培养基上的产孢模式发生转变:由微循环产孢转变为正常产孢。(5)氨基酸代谢组学分析表明:MaPepD突变体(ΔMaPepD)中有19种氨基酸(包括其衍生物)的含量水平发生了显著变化;当外源添加4 mMβ-Ala水溶液于SYA培养基中,敲除菌株由正常产孢回复为微循环产孢。(6)收集SYA培养基培养12 h的菌丝进行RNA-Seq分析,结果表明:MaPepD可能介导了孢子萌发、菌丝生长、产孢、氨基酸代谢等相关基因的转录以调控微循环产孢转换。实验结果表明:MaPepD具有较高二肽酶活性,MaPepD缺失后,在SYA培养基上产孢模式发生转换,一些氨基酸的含量水平发生变化。在1/4 SDAY培养基上,蝗绿僵菌分生孢子的萌发率提高、酶活性降低、抗紫外和抗湿热能力极显著增加。更多还原

【Abstract】 Metarhizium acridum is one of the most important entomopathogenic fungi,and widely studied as a model fungi.In fungi,peptidases play crucial roles in regulating fungal adaptability.Dipeptidase(PepD),an important class of peptidases,can cleave dipeptides into two amino acids,playing important roles for sugar and amino acid metabolism.The function of PepD is largely unknown in fungi.In this study,we analyzed the dipeptidase,enzymatic characteristices,and the relevant mechanism in M.acridum.The main results are as follows:1.The SDS-PAGE showed that MaPepD,a molecular mass of 56-kDa,displayed a high activity at specified conditions.The purified dipeptidase has an optimal activity at pH 6.0 and 55°C.2.Disruption of MaPepD increased conidial germination on 1/4SDAY and significantly improved the resistance to UV-B and heat-stock.Exceptionally,virulence was unaffected in ΔMaPepD.3.Digital gene expression data revealed that ΔMaPepD displayed increased UV-B and heat shock tolerances compared to wild type by mediating transcription of sets of genes involved in conidiation,polarized growth,cell cycle,DNA replication and repair,and some important signaling pathways,such as DNA repair and stress response.4.The MaPepD was upregulated in sucrose yeast extract agar(SYA)medium.Deletion of MaPepD resulted in a shift of conidiation pattern: from microcycle conidiation to normal conidiation.5.Amino acid metabolism analysis demonstrated that the levels of 19 amino acids and derivatives significantly changed in mutant ΔMaPepD.6.RNA-Seq analysis revealed that MaPepD mediates transcription of sets of genes involved in conidiation,polarized growth,cell cycle,cell proliferation,stress tolerance,and some important cell growth signaling pathways.更多还原