A Preliminary Study On The Function Of Ric8 Protein In

In this study, five genes (AOL_s00083g127, AOL_s00007g178, AOL_s00080g412, AOL_s00076gl8 and AOL_s00215g338) in Arthrobotrys oligospora which had significant differences in the RGS1 transcriptomic analysis were chosen for furtherly study. These five genes were deleted by homologous recombination technology, and their functions were preliminary analysed by comparing the phenotypic and the physiological and biochemical characteristics between the wild type (WT) and the mutants. Ric8 is a conserved guanine exchange factor, which exists in animals and fungi, but not in plants. Ric8 is an integral part of the G protein signaling pathway, through positive regulate Ga subunit to control the downstream signaling pathways. Ric8 plays an important role in the growth and development processes in the fungal growth and development, while the functions of Ric8 are unknown in nematode-trapping fungi. In this study, the functions of the five genes including AoRic8 were analyzed, and our results provided a basis for further understanding the trap formation and the pathogenic mechanism of the fungus A. oligospora.The main results:1. AOL_s00083g127 and AOL_s00007g178 are belonged to Gas1-like protein, and they contain a conserved domain (DUF3129, IPR021476). AOL_s00080g412 contains two carbohydrate-binding WSC (IPR002889) domains. AOL_s00076g18 contains 202 amino acids, while no conserved domain is found in Interproscan. AOL_s00215g338 has a guanine nucleotide exchange factor (Ric8, IPR019318) domain. Cluster analysis showed that Gasl-like proteins, such as AOL_s00083g127 and AOL_s00007gl78, are expanded in the nematode-trapping fungi producing adhesive traps including A. oligospora and Dactylellina haptotyla.2. Knockout vectors of the five genes were recombinant through Saccharomyces cerevisiae and In-fusion methods, and achieved positive transformations. The transformations were verified furtherly by PCR method and Southern blot, five mutants were chosen to study the functions of the five genes in the fungus A. oligospora.3. Comparison on the growth rate, spore production, trap formation and the resistance to SDS and H2O2 between the WT and mutants. Our results showed that Ric8 involved in the hyphal growth and differentiation, spore production, trap formation and the resistance to SDS and H2O2, especially, Ric8 plays a very important role in the trap formation, spore production and the resistance to SDS and H2O2. Other genes have little influence on the growth and resistance to SDS and H2O2, while the knockout of these genes have influence on the trap formation and conidia production. For example, the number of traps and conidia were decreased obviously in the mutants.Innovations in this paper:This research successfully knocked out the five genes in A. oligospora. Our results showed that five genes had different influences on the growth, conidia production, resistance, trap formation and nematocidal activity in A. oligospora.