Identification And Analysis Of Grape CircRNAs And Function Research Of Vv-circATS1 On Cold Resistant

Grape is one of the most important economical fruit tree widely grown in the world,low temperature is an important limiting environmental factor that influences the growth,quality and yield of grapes.Circular RNAs（circRNAs）are a class of single-stranded non-coding RNA characterized by the presence of a covalent bond linking the 3′ and 5′ ends generated by backsplicing.Recent studies showed circRNAs play important roles in plant stress responses.Deep understanding of the characterization and function of circRNAs,especially for uncovering the possible roles in regulating the cold response in grape will be helpful to provide further understanding of circRNAs in plants and open up a new field of resistance regulation in grape.In this project,the sequencing results of mixed sample from grape tissues were analysisd via bioinformatics methods.The grape circRNA features were analysed,and flanking intronic sequences that regulate circularization in grape were tested.Moreover,based on the particularity of the circRNA structure and the complexity of its formation,efforts were made to improve circRNA expression methods in plants.In addition,expression profiles of circRNAs under different cold stress times were also recognized.Then,we screened a differentially expressed circRNA（Vv-circATS1）that is associated with grape responses to low temperatures.We ultimately functionally verified Vv-circATS1 under low-temperature adaptability through the new methods to overexpress circRNA were provided in this study.The mai1.A mixed RNA sample consisting of ’Muscat Hamburg’ tissues（root,stem,leaf,flower and berry tissues）was used for RNA sequencing（RNA-seq）after RNase R treatment.Three circRNA prediction methods,find_circ,CIRCexplorer and CIRI,were used to analyse the high-quality filtered data.8354 unique circRNAs were totally predicted in five grape tissues.Only 1432 circRNAs（17.1%）were detected by all three algorithms.The predicted number was in the order of CIRCexplorer>find_circ>CIRI.The success rates of circRNAs predicted by a single algorithm which were tested by reverse transcription PCR（RT-PCR）and Sanger sequencing was: find_circ>CIRI>CIRCexplorer.The ratio of the exonic circRNAs in grape was 91.20%;the majority of circRNAs spanned one to five exons,and the length ranged from 200 to 700 bp.We found that two alternative circularization patterns existed in grape,including alternative back-spliced circularization and alternative splicing circularization.It is worth noting that compared with circRNAs in Arabidopsis,soybean,rice,trifoliate orange,tomato and maize,grape circRNAs are not well conserved.Analysis of the relative abundance of 12 circRNAs in different tissues confirmed the tissue-specific expression of circRNAs.For all tested circRNAs,the circular transcripts were less abundant than were the linear isoforms.Our results revealed positive and weak correlations between the expression of grape circRNAs and that of their cognate linear genes in different tissues.2.The formation mechanism of grape circRNA was studied by using an Agrobacterium-mediated transient expression system in tobacco.The circRNAs were detectable after injection when cloned seven circularized exons as well as their flanking introns（circRNA₁218,circRNA₁975,circRNA₄328,circRNA₄363,circRNA₄473,circRNA₅664 and circRNA₇172）.In contrast,all seven circRNAs were not detected in tobacco leaves if the flanking introns were removed.To identify the minimal intronic sequence length for circRNA production,two circRNAs（circRNA₇172 and circRNA₁975）were chosen to test the circularization efficiency by intercepting the flanking intron sequences.We found that the length of the upstream and downstream intronic sequences can severely affect the expression level of circRNA production.A longer flanking intronic sequence appears to circularize more efficiently.Further analysis revealed that ～20-50 nt of flanking intronic sequence was sufficient for effective circularization.3.The efficiency and accuracy of back-splicing via circRNA overexpression strategy was tested using the Agrobacterium-mediated transient expression system in tobacco.We found that the "reverse complement strategy" can promote the circularization of circRNA₄328 and circRNA₄363.Nonetheless,the detected transcripts contained several circular forms.AG/GT splice sites were added to the flanking regions of circRNA₄328 and circRNA₄363 could not eliminate this handicap.Here,using flanking intron sequences supplemented by reverse complementary sequences,we improved the expression of circRNA₄328,circRNA₄363,circRNA₁975 and circRNA₇172 and ensured the accuracy of circularization.This method was the most feasible for overexpressing circRNAs on the basis of our several attempts.4.1-year-old ’Muscat Hamburg’ cuttings were treated at 4°C for 0 h（used as a control）,4 and 12 h.After high-throughput sequencing,475 differentially expressed circRNAs were found in grape leaves under cold treatment.With respect to biological processes,the GO enrichment of circRNA host genes were involved mainly in "response to cold","photosynthetic electron transport in photosystem II","MAPK signaling pathway","glycerolipid metabolism" and "phospholipase D signaling pathway" and so on.Interestingly,several cold-responsive miRNAs,including miR156,miR164,miR167,miR171,miR394,miR395,miR396,miR397,miR398,and miR408,could target the differentially expressed circRNAs.5.The size of the full-length circATS1 is 333 bp.Further,fluorescence in situ hybridization（FISH）revealed that Vv-circATS1 localizes to the cytoplasm,as well as nucleic regions.In addition,there was no significant correlation between the expression of Vv-ATS1 mRNA and that of Vv-ATS1 circRNA under cold stress.The expression of Vv-circATS1 decreased in the late stage of cold stress.Compared with control plants,transgenic plants overexpressing Vv-circATS1 were more cold resistant.Further,cold-responsive miRNAs including miR156,miR396,miR165 and miR398 exhibited specific expression patterns under cold stress between the Vv-circATS1-OE plants and the WT plants;Vv-circATS1-induced genes are primarily involved in "response to stimulus","toxin catabolic process","photosystem II assembly" and "proline transport".In summary,we revealed the widespread expression of circRNAs in grape.The basic characteristics of circRNAs in grape were investigated.In addition,we demonstrated that flanking introns are critical for the production of circRNAs.Third,new methods to overexpress circRNA in plants were provided in this study.We also found some circRNAs that may be involved in the regulation of cold stress,and it was preliminarily proved that Vv-circATS1 is involved in cold stress response.