Identification Of Natural Antisense Trancripts And Circular RNA And Study On Their Potential Interactions In Ganoderma Lucidum

As a medicinal fungus in the Ganoderma of the Ganodermataceae,Ganoderma lucidum possesses pharmacological activities including anti-tumor,anti-oxidation,anti-bacterial and immunity enhancement,etc.Due to its medical and economic importance,research related to the improvement the production of its active components have been an active area of research.Non-coding RNAs have been found to be involved in the expression regulation of genes.In this study,we systematically identify and characterize the Natural Antisense Transcripts(NATs)and circular RNAs(circRNAs).Interactions between NATs and circRNAs have also been exploited.The main results are as follows:(1)A bioinformatic pipeline(NATs-finder)was developed to identify NATs from sequence data generated with high-throughput DNA sequencing technologies.NATs-finder contains three analysis models,which can identify cis-NATs,trans-NATs and non-coding cis-NATs,non-coding trans-NATs.(2)Using NATs-finder,a total of 1613 cis-NATs and 244 trans-NATs were identified in ssRNA-seq data of samples collected from three developmental stages of G.lucidum.25 pairs of NATs and Sense Transcripts(STs),which were called SATs(Sense and anti-sense transcripts pairs)in short,were found to be closely related to G.lucidum growth,development and secondary metabolism.The relative expressions of 25 pairs SATs at three developmental stages were detected by strand specific RT-qPCR(ssRT-qPCR).The Pearson correlation coefficient(r)of 60%transcripts were more than 0.9,which were consistent with the results of ssRNA-Seq detection.Secondly,expression profiles of 88%of STs and NATs had significant correlation(p ? 0.01)in three developmental stages,and 15 pairs of SATs with r>0.8%,and 4 with r<-0.8.600 STs were mapped to 409 GO terms,and 14 GO terms were significantly enriched.168 STs were mapped to 196 KEGG Pathways,and 65 Pathways were significantly enriched.There were 4 GO terms and 3 KEGG pathways showed differential enrichment among the three developmental stages(q<0.05).Combining with the results of functional enrichment analysis of STs and the correlation of expression profiles between STs and NATs at three developmental stages,it's speculated that NATs played a significant regulatory role in key enzymes such as Alocohol oxidase,Aryl Alcohol oxidase(AAO),and cellulose dehydrogenase(CDH)in lignin degradation pathway.(3)In total,250 and 2193 circRNAs were identified from strand-specific RNA-seq data generated from the polyA(-)and polyA(-)/RNase R-treated libraries,respectively.The exonic circRNAs(number of back-spliced reads>5)in the ssRNA-seq data obtained from libraries of RNase R as the main content of the subsequent analysis.Across all stages,731 exonic circRNAs(number of back-spliced reads>5)corresponding to 561 parent genes were analyzed.Ten candidate exonic circRNAs were selected validate,5 candidate exonic circRNAs can be amplified by PCR.The predicted backsplices of 3 out of 5 amplified exonic circRNAs were successfully validated.A total of 177 exonic circRNAs were differentially expressed across the three developmental stages of G.lucidum.The expression profiles of 119(16.3%)exonic circRNAs and their parent genes showed significant positive correlations(r?0.9,q<0.01),whereas 226(30.9%)exonic circRNAs and their parent genes exhibited significant negative correlations(r ?-0.9,q<0.01).380 parental genes were mapped to 335 GO terms,and 64 GO terms were significantly enriched(q<0.05),192 parental genes were mapped to 208 KEGG Pathways,73 KEGG Pathways were significant enrichment(q<0.05).There were 37 GO terms and 28 KEGG pathways showed differential enrichment among the three developmental stages(q<0.05).Combining the functional enrichment analysis of the parental genes and the correlation of expression profiles between parental genes and exonic circRNAs at three developmental stages,it's speculated that exonic circRNAs have a significant regulation effect on the glutaryl coenzyme A and GTP binding protein rhol in the MVA pathway in the synthesis of triterpenoids or polysaccharide from G.lucidum,respectively.(4)There were various interactions among parental genes,cis-NATs and exonic circRNAs:exonic circRNAs could inhibit cis-NATs,thus it could reduce the positive regulation of cis-NATs on parental genes;synergistic action existed when exonic circRNAs cooperated with cis-NATs,which would co-inhibit or co-promote the expression of genes.In addition,interactions among parental genes,cis-NATs and exonic circRNAs were closely related to the relationship of the relative position between the parental genes and cis-NATs,and relation of the number ratio among the three ones.These results not only open up new avenues for research on the regulatory role of the coding protein genes involved in the growth and development of G.lucidum,but also lay the foundation for the application of genetic engineering and synthetic biology technologies for the biosynthesis of secondary metabolites.