Studies On LncRNAs And Their Potential Target Genes That Regulate The Formation Of Predatory Organs In Arthrospora Oligospora

Arthrobotrys oligospora can produce special adhesive networks to transfer its lifestyle from saprophytic to parasitic through inducting of Caenorhabditis elegans.At present,people have a certain understanding about the molecular mechanism of traps,but little is known about the role of lncRNAs in the formation of traps.In this study,several lncRNAs and their potential target genes with different expression levels during traps formation were selected to study their functions.We aimed to identify the molecular machanism of traps formation based on RNA level.Firstly,TCONS₀0316346 and AOL_s00193g129 were knocked out through homologous recombination method.Compared with wild-type strain,?TCONS₀0316346 strain showed the number of traps were significantly reduced.In addition,the number of traps were significantly reduced in ?AOL_s00193g129,All these above results suggested that both TCONS₀0316346 and AOL_s00193g129 may be involved in traps formation of A.oligospora.RT-PCR results showed that the expression of AOL_s00193g129 was significantly up-regulated with the deletion of TCONS₀0316346 in fungi,implying the possiblity of interaction between them.In view of AOL_s00193g129 is a bZIP transcription factor which regulates downstream target genes,both the two mutants were also employed to transcriptome high-throughput sequence throuhg RNA-seq technique.Then,the downstream target genes that may be co-regulated by transcriptional factor AOL_s00193g129 and lncRNAs-TCONS₀0316346 were predicted via bioinformatics analysis.GO analysis suggested that these downstream target genes are mainly related to metabolic process,membrane and catalytic activity.This study laid the foundation for further study of the interaction mechanism between lncRNAs-TCONS₀0316346 and transcriptional factor AOL_s00193g129.Previous studies have been confirmed that the lncRNAs can act as competitive endogenous RNA(ceRNA)to adsorb milRNA,to regulate the expression of genes.Based on the analysis of bioinformation,lncRNA-LNC₀00315 and the 3'-UTR of functional gene AOL_s00215g45 has the same milRNA binding site,as well as the lncRNA-LNC₀00829 and the 3'-UTR of functional gene AOL_s00210g77 also possess the same milRNA binding site.RT-PCR results showed that lncRNAs and functional genes have the same expression trendency during the formation of traps,while milRNAs showed opposite expression trendency.Therefore,it is speculated that these lncRNAs may interact with milRNA to regulate the expression of functional genes.Therefore,LNC₀00315,LNC₀00829,AOL_s00215g45,AOL_s00210g77 were to furtherly selected to study their functions.Firstly,after knocking out LNC₀00315 and AOL_s00215g45,the phenotypic analysis showed that there was no significant change in growth rate and stress resistance,but the number of spores,the nematicidal activity and the number of traps were significantly reduced in ?LNC₀00315.However,There was no significant change in growth rate and stress resistance,the number of spores,the nematicidal activity and the number of traps in ?AOL_s00215g45.The dual luciferase reporter assay showed that there is not interaction between the 3'-UTR equence of AOL_s00215g45 and milRNA505,milRNA544.Same results were obtained from LNC₀00315 and milRNA505,milRNA544.In addition,after knocking out LNC₀00829,the phenotypic analysis showed that there was no significant change in growth rate and stress resistance,but the number of spores,the nematicidal activity and the number of traps were significantly reduced in ?LNC₀00829,which suggests that LNC₀00829 plays an important role in the formation of traps.Unfortunately,AOL_s00210g77 was not knocked out in this study,and the dual luciferase reporter assay also showed no interaction between the 3'-UTR sequence of AOL_s00210g77 and milRNAs.This study initially confirmed that LVC₀00315 and LNC₀00829 may play an important role during traps formation,but whether they regulate genes through ceRNA mechanism still need further studies.The innovation of this paper:This study used a series of molecular biological methods to study the regulational mechanisms of lncRNAs and their potential target genes which may have functions in the process of traps formation,understanding the molecular mechanisms of traps formation at noncdoing RNA level.