Identification And Functional Analysis Of CircRNAs In Arabidopsis Thaliana Treated With MeJA

Circular RNAs(circRNAs)are a type of covalently closed circular RNAs that typically derives from a back splicing event.Although it first discovered in the 1970s,circRNAs were ignored previously.With the development of high-throughput sequencing and bioinformatics technology,circRNAs have been gradually reported in human,mice,Caenorhabditis Elegans,Arabidopsis thaliana,corn,rice,and the most in-depth research on circRNAs related to human diseases.Plant circRNAs can participate in plant growth and development,metabolic regulation,and respond to biotic or abiotic stresses,but the functions and specific mechanisms of most circRNAs remain unclear.Methyl jasmonate(MeJA)is a derivative of the plant hormone jasmonic acid(JA).Exogenous application of MeJA can activate defense responses in plants.However,it is unclear whether and how plant circRNAs are involved in MeJA-mediated defense responses.In order to explore the function of circRNAs in plant defense,this study focused on Arabidopsis thaliana seedlings.First,circRNAs sequencing was performed at 24 h,48 h,and 96 h after methyl JA(MeJA)treatment,and identified circRNAs and analysis their characteristics.The analysis of differentially expression and experimental verification were performed,and find the differentially expressed circRNAs(DEcircRNAs)under MeJA treatment.Finally,the functional analysis of DEcircRNAs was performed from the functional enrichment of the host gene of DEcircRNAs and the regulatory relationship between DEcircRNAs and miRNAs.This provided help for further explore the function and mechanism of circRNAs in Arabidopsis thaliana defense in the future.The main findings are as follows:1.A total of 8,588 circRNAs were identified using CIRI2,CIRCexplorer2,and find_circ prediction software.Characteristics analysis showed that most circRNAs were derived from exon regions,and the length of most circRNAs sequences were distributed between 200-800 bp;The splice signals are diverse,including GT/AG,CT/AG,GA/AG,CT/TC,etc;the expression level of circRNAs are low,and the number of reads that supported back splicing sites are mostly less than 20.Meanwhile,a small number of circRNAs with high expression levels,which supported reads more than 100.In addition,7,761 circRNAs identified in this study were derived from 4,928 host genes,indicated that alternative splicing patterns of circRNAs in Arabidopsis thaliana are diversity.2.Based on the number of circRNAs junction reads,circMeta software was used to identify the number of DEcircRNAs at three time points after MeJA treatment,and obtained 70,54,and 105 circRNAs respectively.There were 9 circRNAs were differentially expressed at three time points.The quantitative real-time PCR(qRT-PCR)results showed that some circRNAs expression levels were related to their host genes,which indicated that the function of DEcircRNAs may be related to their host genes.The GO and KEGG enrichment results of DEcircRNAs host genes indicated that DEcircRNAs may be involved in response to stimuli and defense responses.In addition,GSTAr software was used to construct a circRNA-miRNA-mRNA regulatory network,which contains 36 DEcircRNAs,32 miRNAs,and 1,630 mRNAs;1,630 mRNAs were analyzed by GO enrichment and found that many mRNAs in the network are related to JA metabolism and signal transduction pathways,this result suggested that some DEcircRNAs may indirectly regulate JA-mediated gene expression by affecting miRNAs,and participating in plant defense processes.In conclusion,circRNAs play important roles in the JA-mediated gene expression regulatory network.3.This study constructed a plant circRNA database,GreenCircRNA(http://greencirc.cn/).This database covers 69 species of plants and is the plant circRNAs database with the largest number of plants.GreenCircRNA provided a total of 213,494 circRNAs,of which 95,010 circRNAs belonged to exons,65,175 circRNAs belonged to intergenic regions,and 53,309 circRNAs belonged to introns.68,237 circRNAs were assembled full-length sequences by CIRI-full software,and relation information of these circRNAs were analyzed,such as chromosomal position,host genes,length of sequence,relative expression levels,and GO analysis of the host genes;Besides,circRNA-miRNA-mRNA regulatory networks were constructed for 38 plants.This database is helpful for researchers to perform functional analysis of circRNAs in the future.