Knockout And Functional Study Of Six LncRNAs In The Nematode-predating Fungus Arthrosporium Oligospora

Nematode-trapping fungi are a kind of fungi that can capture nematodes by producing special devices.As a model species of nematode—trapping fungi,studying the molecular mechanism of Arthrobotrys oligospora during trap formation not only lays the foundation for understanding the pathogenicity of fungi,but also provides a theory basis for developing biocontrol agent with high-efficiency.Previously,more than 1,900 lncRNAs with different expression levels during trap formation have been identified based on RNA-seq.In this study,six lncRNAs were selected to do further studies.Main research achievements:1.RT-PCR results showed that the expression levels of the four lncRNAs LNC＿00970,LNC＿001126,LNC＿000535,TCONS＿00256389 and their adjacent genes AOL＿s00188g174,AOL＿s00215g814,AOL＿s00080g55 and AOL＿s00110g71 were significant differences during trap formation.2.The knockout vectors of LNC＿000970,LNC＿001126,LNC＿000535,TCONS＿00256389 and its adjacent functional genes AOL＿s00188g174,AOL＿s00215g814,AOL＿s00080g55,AOL＿s00110g71 were successfully constructed by using homologous recombination technology and all positive mutant strains were obtained with the exception of AOL＿s00215g814.Compared with the wild-type strain,we found that the numbers of traps and nematicidal activity were significantly enhanced while the numbers of spores were significantly down-regulated in ΔLNC＿000535 strain.In ΔAOL＿s00080g55 strain,there were no significant changes of traps and conidia numbers,as well as the nematicidal activity,suggesting that lncRNA LNC＿000535 may have a certain regulational functions in the trap formation,nematicidal activity and sporulation of A.oligospora,but it has no effect on its adjacent gene AOL＿s00080g55.In addition,there were no significant differences in the numbers of traps and nematicidal activity of ΔTCONS＿00256389 strain.The conidia numbers ofΔTCONS＿00256389 were significantly down-regulated.However,the conidia numbers and nematicidal activity in the adjacent functional gene ΔAOL＿s00110g71 were significantly up-regulated,and the numbers of traps had no obvious changes.Because AOL＿s001l0g71 is the neighbouring gene of TCONS＿00256389,thus,it can speculate that TCONS＿00256389 may have functions on AOL＿s00110g71.In addition,the numbers of traps and spores were significantly reduced in ΔLNC＿001126 but the after knocking out the lncRNA LNC＿000970 and its adjacent functional gene AOL＿s00188g174,no significant changes was observed.3.More than 200 functional genes which may be involved in trap formation of A.oligospora have been identified in our laboratory before.Moreover,two lncRNAs LNC＿000189,LNC＿000775 were predicated to co-expressed with those functional genes.Therefore,both these two lncRNAs were also selected to do further studies in this thesis.Phenotypic analysis showed that the numbers of both the traps and conidia were significant increased with the deletion of LNC＿000 775 and the nematicidal activity was also significantly enhanced.However,although the traps numbers and nematicidal activity were significantly up-regulated of ΔLNC＿000189,no significant change in sporulation was observed.RT-PCR analysis showed that the expression levels of functional genes that might interact with lncRNA LNC＿000189 and LNC＿000775 and be involved in the trap formation all had changes in both the ΔLNC＿000775 andΔLNC＿000189 strains,suggesting that LNC＿000189 and LNC＿000775 may influence the trap formation through regulating the genes involved in trap formation.The innovation of this paper:Although lncRNAs have become one of the hot-points in the life sciences research field,so far,lncRNAs have not been reported to have ability to regulate the trap formations in nematode trapping fungi.In this study,functional studies of lncRNAs in A.oligospora showed that lncRNAs definitely play important functions during trap formations.These results provides foundations for studing the molecular mechanism of lncRNAs in A.oligospra in the future.