Functional Study Of LncRNAs And Their Potential Target Genes In Arthropoda Oligospora

Nematode-trapping fungus Arthrobotrys oligospora growth in saprophytic lifestyle as usual,it can produce special devices named adhensive networks to kill nematode when it senses the exist of nematodes.These trap devices have been considered as the key point of this fungus transform from saprophytic to parasitic lifestyles.It is very important to study the molecular mechanism of trap formation.Previous studies of our research group used the RNA-seq technology to sequnce the fungus samples during seveal key stages of trap formation,and successfully obtained 1,951 candidate lncRNAs.Also,54 bioinformatic predicted lncRNAs with different expressions were identified through RT-qPCR and 30 of them were finally confirmed Because lncRNAs can regulate their neighboring genes through cis action,the 2,000 bp upstream and downstream of these confirmed lncRNAs were analysed and five lncRNAs(TCONS?00193747,TCONS?00349760,TCONS?00182722,TCONS?00188549,TCONS?00316812)and its functional genes(AOL?s00080g63,AOL?s00215g323utp215,AOL?s00080g393,AOL?s000193g139)were selected to study their functions in this study.Main research achievements in this study:Five knockout vectors of lncRNAs and their adjacent functional genes were constructed and their corresponding knockout strains were successfully obtained(AOL?s00215g323 and AOL?s000193g139 genes were knocked out by others in the laboratory).1.Phenotypic analysis of?AOL?s00080g393 and its two downstream lncRNAs knockout strains,?TCONS?00182722 and?TCONS?00188549,revealed that the spore production of?AOL?s00080g393 was lower than that of wild-type strains,and the ability to produce traps were increased.However,both the capacities to produce spores and traps in?TCONS?00182722 and?TCONS?00188549 were reduced.The traps of mutant strains and wild-type strains were induced on the plate with addition of(NH₄)₂SO₄and Na NO_3,and the strain?TCONS?00182722 can produce more traps in compared with other stains.RT-qPCR results showed that the transcription level of gene AOL?s0080g393 was significantly up-regulated in the lncRNAs knockout strains?TCONS?00182722 and?TCONS?00188549,suggesting that these two lncRNAs may affect trap formation by negatively regulating AOL?s00080g393.2.The number of spores and traps were both reduced in the knockout strain?TCONS?00349760,and RT-qPCR and Northern blot results showed that the expression level of AOL?s00215g323 in the?TCONS?00349760 knockout strain was significantly upregulated,Because the number of spores and traps were also reduced in the?AOL?s00215g323 mutant strain It can speculate that TCONS?00349760 might affect the formation of conidia andtraps of A.oligospora by regulating AOL?s00215g323.3.The ability to prodece traps was greatly attenuated in the lncRNA knockout strain?TCONS?00316812,as well as in the?AOL?s00193g139 mutant strain,indicating that lncRNA TCONS?00316812 may have the ability to regulate AOL?s00193g139.In additon,the?AOL?s00080g63 mutant strain loss the ability to produce conidia and the number of traps was reduced,the number of spores were significantly reduced in the?TCONS?00193747 strain but there no obvious changes about the trap formaton,suggesting that lncRNA TCONS?00193747 may regulate AOL?s00080g63 to influence the ability of sporulation.The innovation of this study:In this study,we performed functional studies on lncRNAs and their adjacent functional genes that may be involved in the trap formations of A.oligospora.Our results showed that the capcities to produce traps or spores of this fungus were influenced more or less with the the knockout of lncRNAs and their adjacent coding genes,and the expression levels of genes were also affected after the knockout of lncRNAs.All these suggested that lncRNAs can regulte their adjacent coding genes.This study lays the foundation for the further studies on the mechanisms of lncRNAs to regulate trap formation in nematode-trapping fungi.