The Identification And Expression Analysis Of CircRNAs In Gossypium Arboreum Under Abiotic Stress

Cotton not only plays an important role in the global economy as the most common natural raw material in the textile industry,but also has been regarded as an ideal model for studying plant cell wall formation and cellulosic biosynthesis due to its single-cell and thickened secondary wall properties.With the continuous deterioration of the global environment,high temperature,drought and soil salinization and other stress environments have seriously hindered the development of cotton industry.Therefore,it is particularly important to study the mechanism of cotton adaptation to abiotic stress and to improve the cotton stress tolerance by means of molecular biology.At present,the research on cotton stress resistance mainly focuses on the mining of important stress resistance genes,but gene expression is a complex network,which is also regulated by other factors such as microRNA.It has been reported that circular RNA(circRNA)can competitively bind miRNAs to regulate mRNA expression.Circular RNA is a kind of stable RNA without free ends,which is formed by reverse splicing,and does not contain 5’cap structure and 3’ polyA tail.However,there are few reports on circRNAs in cotton under abiotic stress.In this study,Gossypium arboreum seedlings were subjected to four abiotic stress treatments,namely cold,heat,salt and UV-B radiation,and transcriptome sequencing was performed on the seedlings after stress treatment.Then bioinformatics methods were used to analyze the characteristics of circRNAs and the expression of circRNAs and mRNA under different stress conditions.Meanwhile,the interaction between circRNAs and miRNA and the interaction between miRNA and mRNA were analyzed respectively.Finally,a coexpression and interaction network of circRNA-miRNA-mRNA was constructed.The potential regulatory roles of circRNAs in the response to abiotic stress in cotton were predicted,and the potential roles of circRNAs in the response to abiotic stress in cotton were explored,in an attempt to mine circRNAs related to abiotic stress,and provide information for further research on the mechanism of plant stress resistance in the future.The main results of this study are as follows:(1)A total of 10 circRNA sequencing libraries were constructed in this study,and a total of 209.6 G of original data were obtained after sequencing.About 98%of reads could be compared to the reference genome,with good reproducibility among samples,with Pearson’s coefficient R>0.8 and P value<0.001.(2)The circRNA was predicted and identified by Knife and CircExplorer2 software,and circRNA was identified in cold stress,heat stress,salt stress and UV radiation,respectively.By comparing the identification effect of total RNA library building sequencing and circRNA library building sequencing,it was found that circRNA library building sequencing significantly improved the detection rate of circRNA.100 circRNAs with different expression levels were randomly selected for RT-PCR and sequencing verification,and the authenticity of 33 circRNAs was verified.(3)The characteristics of circRNAs with high reliability were analyzed.It was found that circRNAs tended to produce more variable splicing genes,and multi-exon genes were more likely to produce circRNAs.Longer introns may have a certain effect on the formation of circRNAs.The circRNA producing gene types were relatively conserved,but the circRNA producing sites were not conserved.(4)The differential expression of circRNAs indicated that abiotic stress could affect the expression of circRNAs:cold stress resulted in up-regulation of 306 circRNAs and down-regulation of 15.Under heat stress,44 circRNAs were up-regulated and 14 were down-regulated.Under salt stress,33 circRNAs were up-regulated and 141 were downregulated.UV radiation resulted in the up-regulation of 290 circRNAs and downregulation of 34.However,it should be noted that the response of its parent genes to the corresponding abiotic stress was less correlated with the response of circRNAs.(5)By constructing the interaction network of circRNA,miRNA and mRNA,analysis showed that circRNA and miRNA could form a many-to-one or one-to-many interaction mode.As miR8633 targets multiple mRNAs,we speculated that CircGa01g060 and Circ-Gallg239 lead to up-regulation of heat stress response genes by inhibiting the binding of miR8633 to mRNA.