The Expression And Function Of Circular RNA In Etiolation And De-etiolation Process Of Arabidopsis Thaliana

Circular RNAs（circRNAs）are endogenous non-coding RNAs and characterized by the presence of a covalent bond linking the 3’ and 5’ ends,which is different from traditional linear RNA.Due to the covalently closed circular structure of circRNAs can escape degradation by RNase R,so it is not only abundantly and stably expressed in organic organism,but also can regulated the gene expression.The broadness,conservation and tissue specificity of circRNAs may become a novel biomarker in the future.Although it has been demonstrated that circRNAs play important roles in a range of biological and developmental processes in animals,the knowledge of circRNAs in plants is limited.Light is one of the most crucial environmental factor for plants,which a wide of range of reactions during the growth and development process,such as photomorphogenesis,phototropism,cholorplast development and construction.Light signal triggers de-etiolation process of Arabidopsis thaliana by regulating expression of related genes.Development of the chloroplasts and construction of functional chloroplasts are based on the expression of both plastid and nuclear genes with transcriptional and post-transcriptional regulation mechanisms.In this study,the model plant A.thaliana was selected as the experimental material.Using high-throughput sequencing,bioinformatics approaches and molecular biology,circRNAs detected in etiolated and de-etiolation seedlings.The main results are as follows:（1）A total of 499 circRNAs were identified by RNase digestion,high-throughput sequencing and bioinformatics.The identified circRNAs were generated from all of the chromosomes,as well as from the mitochondrial and chloroplast genomes.Among the identified circRNAs,173 were generated from exons of a protein-coding gene,that is,they were exonic circRNAs.Only 27 of the circRNAs were generated by introns.Furthermore,a total of 308 circRNAs were derived from intergenic regions.In total,485 DEGs were found between etiolated and de-etiolation,there were 369 significantly up-regulated and 116 significantly down-regulated circRNAs in the de-etiolation samples.（2）Functional annotation analysis was performed to evaluate the potential functions of the parent genes of circRNAs.GO categories were assigned to the parent genes of circRNAs,found that important functions such as cellular metabolic,response to stimulus,plastid/chloroplast components,catalytic activity and protein binding.The biological interpretations of the circRNA parent genes were further analyzed using the Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway database.The most highly represented pathways included photosynthesis and carbon metabolism.Therefore,many parent genes of circRNAs are involved in the light-regulated A.thaliana de-etiolation process.In addition,33 circRNAs had putative varies miRNA-binding sites,suggesting that some circRNAs are potential miRNA targets.Therefore,plant circRNAs may play a role in the regulation of gene expression by interacting with miRNAs.（3）Here 10 sequences of chloroplast genome（cpDNA）encoded circRNAs（cp-circRNA）from via next-generation sequencing.Their parental sequences were referred to exonic,intronic,and intergenic gene sequences.Moreover,6 protein-coding genes of cp-circRNAs were located in the long single copy（LSC）and the short single copy（SSC）region,among them two（ath circ 477 and ath circ₄81）had the canonical GT/AG splicing.（4）Using digital polymerase chain reaction（dPCR）to confirm the expression of cp-circRNAs and quantify their absolute values,four of these circRNAs were confirmed:ath_circ₄76,ath_circ₄77,ath circ₄78,and ath_circ₄80.With similar the RNA-Seq,the light regulation of ath_circ₄77,ath circ 478 and ath circ 480 resulted in higher expression levels in de-etiolated seedling and ath circ₄76 was highly expressed in etiolated seedlings.The differentially expressed parent genes shared a common expression trend with the corresponding circRNAs and were expressed more in etiolated than in de-etiolated seedlings,suggesting co-regulation of circRNAs and their parent genes.（5）Among 499 of circRNA sequences,parental genes of 5 circRNAs were annotated to the three genes encoding the small subunits of Rubisco holoenzyme,namely RBCS-1B,RBCS-2B and RBCS-3B.The divergent primers and convergent primers were designed to valid the sequences and then sanger sequencing,the stable circRBCS was verified and contains only the sequences transcripted from the antisense strands of the exons 4 and 5 from the RBCS-2B and exon 3 from the RBCS-3B.The expression of circRBCS is similar to parent genes RBCS in leaves,stems,pods,and flowers,these so-called photosynthetic organs,but both did not the expression of both in roots.In the 3-day-old dark-grown seedlings,the expression of circRBCS and RBCS continued to increase and then decreased with the illumination times increased.It was speculated that circRBCS can co-expression with the parental gene and regulate at the transcriptional level.（6）Using direct-differentiation system,overerpressin of circRBCS was transformed intoA.thaliana,and 4 positive lines were produced.It was found that the increased expression of circRBCS did not lead to RBCS expression significant change.As well as,no significant change in phenotype was observed.This possible reason is that the construction of the overexpression vector is not suitable for plants.In summary,by using A.thaliana from Columbia 0 ecotype as material,researched expression of circRNAs on the process of seedlings development by high-throughput sequencing,bioinformatics analysis and molecular biology.CircRNAs stable expressed in chloroplasts and photosynthesis process,may be involved in the regulation of expression of the parental genes.These results lay the foundation for the further investigation circRNAs in plants.