Functional Study On The Polyubiquitin-like Gene CgUbl4 Of Colletotrichum Gloeosporioides

Ubiquitin-like proteins(Ubls) are widely found in eukaryotes and play important roles in protein degradation,cell cycle regulation,cell autophagy and oxidative stress reaction.They possess essentially the same three-dimensional structure as ubiquitin,the ubiquitin or?-grasp fold.Protein covalent modification can be achieved by Ubl-conjugation pathway.Polyubiquitin-like protein is an important member of Ubls.At present,researches on ubiquitin-like proteins are mainly concentrated in animals and plants,and relatively few in filamentous fungi.In this study,the functional study of Cg Ubl4,a Ubls gene of Colletotrichum gloeosporioides,was carried out using bioinformatics analysis,gene isolation,q RT-PCR technology,yeast two-hybrid technology,and RNAi technology in order to broaden the understanding of the function of Ubls in C.gloeosporioides,as well as lay the foundation for exploring the mechanism of Ubl modification.The main research results are as follows:1.Bioinformatics analysis showed that Cg Ubls family contains four members,three of them were ubiquitin-like ribosomal fusion proteins named Cg Ubl1,Cg Ubl2,Cg Ubl3,and one was the polyubiquitin-like protein named Cg Ubl4.All the members were hydrophilic proteins without transmembrane domains and signal peptides.The secondary structure was dominated by random coils.The phylogenetic tree indicated that Cg Ubls family proteins were characterized by diversity.2.qRT-PCR was used to analyze the expression of Cg Ubls family genes during the infection process on mango leaves and fruits by C.gloeosporioides.The results showed that the relative expression of Cg Ubl4 was continuously and significantly up-regulated,indicating that Cg Ubl4 was involved in the infection process of the fungus.3.STRING version 11.0 database was used to predict protein-protein interactions,after which the yeast two-hybrid system verified that Cg Ubl4,ubiquitin-conjugating enzyme Cg UBC22 and transcription factor Cgb HLH6 could interact with each other.4.The mutant pSilent-1:Cg Ubl4 was obtained using RNAi technology,and the phenotype and pathogenicity of three mutants p Silent-1:Cg Ubl4,p Silent-1:Cg UBC22 and p Silent-1:Cgb HLH6 were comparatively analyzed.The results of phenotype observation showed that Cg Ubl4,Cg UBC22 and Cgb HLH6 were involved in the regulation of conidial morphology,conidial germination and appressorial formation in C.gloeosporioides.Further more,Cg Ubl4 and Cg UBC22 were also involved in mycelia growth,conidial production,salt stress tolerance and oxidative stress response.The virulence performances of three mutants on mangoes,apples and pears were consistent.Compared with the wild-type strain,the virulence of p Silent-1:Cg Ubl4 and p Silent-1:Cg UBC22 was significantly decreased,while the virulence of p Silent-1:Cgb HLH6 was strikingly increased,which revealed that Cg Ubl4 and Cg UBC22 could positively regulate the virulence of C.gloeosporioides,while Cgb HLH6 could negatively regulate the virulence.In this study,it was found that the polyubiquitin-like gene Cg Ubl4 of C.gloeosporioides was involved in the whole infection process of the pathogen,and it could interact with ubiquitin-conjugating enzyme Cg UBC22 and transcription factor Cgb HLH6 and regulate mycelial growth,conidial morphology,conidial production,conidial germination and appressorial formation,salt stress tolerance and oxidative stress response,as well as pathogenicity,which lay the foundation for the study of the regulatory network of polyubiquitin-like genes and the mechanism of Ubl modification,and also provide new clues to the seeking of molecule chemicals with protein inhibitor activity,together with the prevention of diseases caused by C.gloeosporioides.