Research On The LncRNAs Related To The Formation Of Predator Organs In Arthrobotrys Oligosporus Based On The Mechanism Of RNA Degradation

In recent years,to study the regulatory mechanisms of long noncoding RNAs?lnc RNAs?at epigenetic,transcriptional and post-transcriptional levels has became the hot issues.However,the studies of lnc RNAs in fungi are still in initial stage and are mainly foucued on model fungi such as yeast and Neurospora crassa.There is little knowledges about the regulatory mechanism of lnc RNAs in pathogenic fungi.Due to the“RNA decay”in cells to maintain the balance between RNA generation and degradation,the expressions of lnc RNAs in cells are usually lower,limiting the research on the production and functional mechanism of lnc RNAs.Using molecular biology method to disable the key proteins that control RNA decay will slow down the speed of lnc RNA degradation and increase the transcriptional level of lnc RNAs in cells and help us better to figure out the molecular functions of lnc RNAs.Based on this idea,Arthrobotrys oligospora,a pathogenic fungi that produce three-dimensional hyphal network to trap nematodes was used as object in this study.Five key coding genes in the RNA degradation pathway[Xrn1?AOL?s00007g507?,Trf4?AOL?s00097g578 and AOL?s00078g371?,Dcp2?AOL?s00004g432?and Dis3?AOL?s00076g625?]of this fungi have been knocked out by using the homologous recombination method.Also,RNA-seq technology was used to sequence and analyze potential lnc RNAs in the?Xrn1mutant strain.In addition,two lnc RNAs?TCONS?00026732 and TCONS?00024985?aroud the ORF regions of two functional genes[xln R?AOL?s00006g84?and pal H?AOL?s00006g291?]has been identified through bioinformatic analyses respectively.These two genes and two lnc RNAs were knocked out through homologous recombination method and their potential associations were annalysed based on the phenotypes and RT-q PCR results.Main research achievements:1.Using uracil deficient saccharomyces cerevisiae as carrier cell,the knockout vector of the five functional genes related to RNA decay were successfully constructed,and successfully obtained the knockout strains through Ca Cl₂-PEG-mediated method.Phenotypic analysis of these knockout strains and WT strains show that the growth rate of?Dcp2 were significantly reduced and?Xrn1 in TG,WA,TYGA,TG?5 m M H₂O₂?and TG?0.2 M Na Cl?mediums also shows significantly slower growth,while other knockout strains did not change significantly in compared with WT.In terms of the ability of spore production,?Dcp2 totally can not produce spores and the quantily of spore production in?Trf4?78g371?and?Trf4?97g578?were significantly decreased,while other knockout strains have no obvious change in compared with WT.In terms of trap numbers,?Dcp2 lost capacity of trap formation,and the numbers of traps in?Xrn1,?Trf4?78g371?and?Trf4?97g578?were significantly decreased.However,the abilities to kill nematodes show no obvious change in all the knockout strains except for the?Dcp2 strain.All these results suggested the the key genes in RNA decay play important roles in A.oligospora.2.Collecting the different stage samples of?Xrn1 and WT strains which induced with nematode extract.Using RNA-seq technology to sequence and identify potential lnc RNAs among these samples.Finally,a total of 1961 candidate lnc RNAs have been predicated through bioinformatics,these data provide solid support for future research on lnc RNAs during trap formation of A.oligospora.3.The encoding genes of xln R?AOL?s00006g84?and its upstream lnc RNA TCONS?00026732,and pal H?AOL?s00006g291?gene and its downstream lnc RNA TCONS?00024985 were sucessfully knocked out.Phenotypic analysis show that the growth rate,capacities to produce conidiospore and trap of?pal H?AOL?s00006g291?was significantly slower or less than the WT stain.and the number of conidiospore of?xln R?AOL?s00006g84?strain were significantly decreased.Unfortunately,the two lnc RNA knockout strains did not have significant phenotypic changes in compared with WT.However,the m RNA level of xln R?AOL?s00006g84?increased significantl in?TCONS?00026732 strain,suggeseting that xln R?AOL?s00006g84?might be regulated by lnc RNA TCONS?00026732,but not necessarily in the aspects of the production of trap and conidiospore.The innovation of this thesis:To disable the key encoding genes in the RNA decay can slow down the degradation of lnc RNAs in cells,and help us to better understand the molecular functions of lnc RNAs.Based on this idea,five key encoding genes in the RNA decay pathway in A.oligospora have been knocked out in this study,which lays the foundation for revealing the molecular mechanism from a new perspective in Arthrobotrys oligospora transformed from saprophytes to parasites by producing traps.