Detection And Validation Of Circular RNA In Arabidopsis And Study Of Its Involvement In Interaction Between Plant And Pathogen

Circular RNA(circRNA)is a novel class of endogenous RNA formed by intramolecular reverse splicing(back splicing),forming a circular RNA structure.Because of the particular structure,circRNA was often left out by regular detection methods based on RNA terminal structure.With the rapid development of bioinformatics and high-throughput sequencing technology,circRNAs were widely identified in many eukaryotes.Arabidopsis thaliana is a model plant,in which circRNA has been reported but not deeply analyzed yet;so far,there are still no large-scale study in structure and function of circRNA in A.thaliana.In previous experiment,803 RNAs candidates were predicted in A.thaliana through bioinformatic analysis of RNA-seq database already acquired.On this basis,we validated some of these candidates in this study.The main results are as follows:(1)Four predicted circRNAs(circR4588,circR194,circR172 and circR1459)were validated by using a series of different experimental methods including a reverse PCR amplification method,RNase R enzymatic method,Poly(A)exclusive method and sequence alignment methods,etc.We were able to demonstrate that these four predicted circRNAs are indeed existed.(2)Using procedures such as specific reverse transcriptase,RNase R enzymatic method and sequence analysis,we analyzed and tested the validated circRNAs,and concluded that:a)chloroplast is rich in circRNA and may be one of the hot zones for circRNA generation;b)AG-GT splicing signals was not the necessary conditions for circRNA formeation,and typical and non-typical splicing signals both could guided the formation of circRNA;c)a single gene locus could generate a variety of circRNA variants;d)Both strands of a same gene could produce circRNA on each oritation,and this phenomenon occurred in all locations on genome.(3)By analyzing the variation of gene expression level of circRNAs after infected by Phytophthora capsici(P.35)(both 24 and 36 hours)and tomato spotted wilt virus(TSWV)(23 days),we found that circR194,circR645 and circR4235 might play a potential role in the regulation of interaction between plants and a variety of pathogenic microorganisms.