Isolation And Functional Analysis Of The Ubiquitin-Conjugating Enzyme CgUBC Family Genes Of C.gloeosporioides

Ubiquitination is an important pathway for protein processing and degradation in organisms.Ubiquitin-conjugating enzyme is one of the key enzyme groups in the process of ubiquitination.In this study,the five genes'functions of ubiquitin-conjugating enzyme familyof C.gloeosporioides(CgUBC)were studied using bioinformatics analysis,qRT-PCR,RNAi technology and TMT(Tandem Mass Tags)method.This work offers an insight into the functions of ubiquitin-conjugating enzymes in the development and pathogenicity of C.gloeosporioides.The main results are as follows:1.It was found that the ubiquitin-conjugating enzyme CgUBC gene family of C.gloeosporioides has 22 members through bioinformatics analysis.The evolutionary analysis showed that CgUBC genes and Saccharomyces cerevisiae,Fusarium graminearum,Magnaporthe oryzae UBC family genes are highly homologous,which indicated that they have similar biological functions.2.The expression of CgUBCs was quantitatively analyzed during the process of infecting tomato and under abiotic stress treatments,using qRT-PCR method.The results showed that CgUBC2,CgUBC5,CgUBC 15 and CgUBC22 were significantly up-regulated,while CgUBC10 was down-regulated significantly,indicating that CgUBCs are involved in the infection process of C.gloeosporioides and stress responses.3.The genes CgUBC2,CgUBC5,CgUBC10,CgUBC15 and CgUBC22 were separately silenced using RNA interference technique.It was found that the growth rate of CgUBC22 silenced mutants was significantly slower than that of wild-type strain,and the growth rate of CgUBC2 silenced mutants was the fastest among the mutants and wild-type strain,indicating that CgUBC2 and CgUBC22 are involved in the regulation of mycelial growth of C.gloeosporioides.The mycelial malformation of mutants of CgUBCs,such as apical expansion,different thickness and withered,indicated that these five CgUBCs were involved in the regulation of mycelial morphogenesis of C.gloeosporioides.In all the mutants,the conidial production decreased significantly,the malformation rate increased significantly,osmotic pressure tolerance weakened,appressorial formation rate decreased significantly,conidial germination process was obviously delayed,and the conidium was deformed such as hypertrophy,shortness,slenderness,intermediate depression,sharp at one end,etc.It is indicated that CgUBCs affected conidial morphogenesis,germination,and appressorial formation.All the mutants were more sensitive to the stress factors H2O2 and Congo red than wild type,but were more adaptable to NaCl stress than wild type,indicating that CgUBCs were involved in the regulation of cell wall selection pressure,oxidative stress response and high salt environment adaptability of C.gloeosporioides.The virulence of the mutants to mango,tomato,pear and apple was significantly decreased,indicating that CgUBCs were involved in regulating the pathogenicity of C.gloeosporioides.4.Proteomic quantitative analysis of mutants and wild types was conducted using TMT labeling method.It was found than the differential protein numbers of CgUBC2,CgUBC5,CgUBC10,CgUBC15 and CgUBC22 mutants were 559,151,96,182,456,respectively,and the number of up-regulated proteins were 68.7.%,84.1%,41.7%,48.9%,57.8%,respectively.The GO enrichment analysis showed that most of the differential proteins had catalytic and binding functions,and involved in biological processes such as metabolism,stress response,redox and carbohydrate metabolism.5.The pathogenicity-related polygalacturonase(PG)gene expression,enzyme activity and protein content was compared between the mutant and wild type.The results showed that the enzyme activity,gene expression level and protein content of wild-type strain were significantly higher than these of CgUBC10?CgUBC22,which was consistent with the pathogenicity results,indicating that the CgUBCs affects C.gloeosporioides virulence by regulating the activity of polygalacturonase.This study revealed that the ubiquitin-conjugating enzyme genes of C.gloeosporioides were involved in the regulation of mycelial growth,conidial germination,appressorial formation,stress tolerance,virulence and polygalacturonase activity.The work provides a base for further research on the relationship between protein ubiquitination and biological function of C.gloeosporioides.