Preliminary Study On Long Non - Coding RNA Involved In The Formation Of Predatory Organs In The Pathogen

Switching the lifestyles from saprophyte to parasitism is the key for fungi to carry out their pathogenicity. Therefore, understanding the molecular mechanism of the lifestyles conversion is the most important scientific question needs more studies in the future. Among fungi with both saprophytic and parasitic lifestyles, nematode-trapping fungi are a special kind of groups. Usually, these fungi live saprophytically in the soil and become predatory via formating specific traps, including constricting rings, adhesive knobs, adhesive networks, adhesive columns, and nonconstricting rings, when discovered the presence of nematodes around them. Therefore, the formation of trapping devices by nematode-trapping fungi is an important indicator of their switch from the saprophytic to the predacious lifestyles. Previous studies suggested that the formation of trap structures of Arthrobotrys oligospora maybe regulated by long noncoding RNA. In this study, the samples collected from the key time points during trap formation of A. oligospora will be high throughput sequenced based on RNA-seq. The lncRNAs that might have functions in trap formation will be predicated through bioinformatic methods. This study will help us from the perspective of long noncoding to understand the molecular mechanism of nematode-trapping fungi switching its lifestyles from saprophytic to predacious, also provides a basis for elucidating the pathogenicity mechanism of fungi and provide theoretical foundation for utilizing these fungi as high effective bio-control agents in the future. Furthermore, RNA decay mechanism is the causes of low expression levels of lncRNAs, even some lncRNAs cannot be characterized in wild type cell, which limit the function studies of lncRNA. Knock out/down of the functional proteins in RNA decay pathway would slow down the degradation of lncRNA in cell. This method will not only help us to identify more lncRNAs that cannot to be easily found in wild type cell, but also raise the expression levels of lncRNAs, providing chances to study molecular mechanisms of these functional lncRNA. In view of this, five key encoding genes in RNA decay pathway of A. oligospora, including Rrp6 (AOL_s00083g475)、Xrnl (AOL_s00007g507)、Trf4 (AOL_s00097g 578 and AOL_s00078g 371) and Dcp2 (AOL_s00004g432) were selected to knock down in this study. We constructed the knockout vectors of these five genes, and used the homologous recombination method to knockout them. This provide a bais for using RNA-seq technology to analyze the transcriptome of wild type and knock out strains in the future, and for finding more functional lncRNAs related to trap formation.The main findings of this study:1. The samples of five key points of trap formation induced by C. elegans extract. Using RNA-seq technique to sequencing lncRNAs in the 10 samples(including samples of five key stages of trap formation, each stage have 2 biological repetition). After screening single-block transcripts distant from other transcripts within 500bp, removing transcripts which are shorter than 200bp and removing transcripts which are known as non-mRNA types(similar with rRNA n tRNA、snRNA、snoRNA、pre-miRNA、 pseudogenes),3571 uncertain lncRNAs in total were identified. Then, the coding potential of these 3751 lncRNAs were analyses via the common coding potential analysis methods, such as CPC analysis, CNC1 analysis and Pfam protein domain analysis, finally,1951 candidate lncRNAs datasets were obtained.2. Five knock out vectors of the protein coding genes involved in RNA degradation mechanism were successfully constructed by electroporation of the yeast cells. Using transformation method mediated by CaCl2-PEG to transform the protoplast of A. oligospora, we got three positive knock out strains of Rrp6 gene. Compared with growth rate, spore, resistance, trap formation and nematode killing ability between WT and Δ Rrp6 strains, the results show that. Rrp6 gene has a certain impact on mycelial morphology growth, spore number and formation time and quantity of trap.The novelties of this study were described as follows:1. Although lncRNAs study is frontier science of life science, so far there has no research on lncRNAs in the nematode-trapping fungi. This study for the first time to carry out research on lncRNAs which involved in the trap formation of A. oligospora. This study will provides new ideas to reveal the molecular mechanism of life cycle changes from saprophytic to parasitic through trap formation in A. oligospora. Meanwhile, it also provides theoretical guidance for the research and development of the efficient biocontrol agents.2. Based on the idea that inactived the key proteins in RNA degradation pathway will slow down the degradation rate of ncRNAs, five knock out vectors of the protein coding genes involved in RNA degradation mechanism were successfully constructed and we got three positive knock out strains of Rrp6 gene. This study provide a bais for using RNA-seq technology to analyze the transcriptome of wild type and knock out strains in the future, and for finding more functional lncRNAs related to trap formation.