Functional Analysis Of The Long Non-coding RNA Gene AsBRM In Arabidopsis Thaliana

LncRNAs are a class of RNAs greater than 200 nt in length,with low or no protein-coding capacity.They are abundant and widely distributed in eukaryotes,and can regulate gene expression or directly affect the function of some proteins at epigenetic,transcriptional and post-transcriptional levels.Several studies have shown that plant LncRNAs play an important role in plant growth and development as well as stress response.However,the current understanding of plant LncRNAs and their functions is very limited.In this study,we identified a novel Arabidopsis LncRNA—asBRM,and its function and regulatory mechanism in root hair development were investigated using the approaches of promoter-driven GUS reporter system,overexpression and CRISPR knockout of AsBRM combined withRNA-seq and plant Ch IRP(Chromatin Isolation byRNA Purification)technology.The main results are as follows:1.Bioinformatics analysis revealed that asBRM is a natural antisense transcript of theBRAHMA(BRM)gene,a component of the chromosome remodeling complex,and it fails to have the capacity to encode protein.GUS staining showed that asBRM has a different distribution pattern fromBRM in the roots,and q RT-PCR analysis indicated that asBRM has a strong inhibitory effect on the expression ofBRM in the roots.2.The full-length sequence of asBRM and its partial fragments were cloned separately,and their overexpression lines were obtained.It was found that up-regulation of asBRM expression promoted root hair elongation in Arabidopsis,and the degree of promotion was positively correlated with the integrity of asBRM sequence.3.The asBRM promoter region was knocked out by CRISPR technology,and a loss-of-function Arabidopsis mutant was obtained.After phenotypic verification,the root hair length of the deletion mutant at 3 d was extremely shorter than that of the wild type,further demonstrating that asBRM has a promotional effect on root hair elongation in Arabidopsis.4.qRT-PCR analsysis showed that overexpression of asBRM transcriptionally repressed theBRM gene,while the CRISPR-based mutaion of asBRM enhanced the expression ofBRM.Moreover,two transgenetic lines overexpressing BRM driven by 35 S promoter and its native promoter were generated,respectively.Subsequently,their root hairs were analyzed,and the length of root hairs was significantly reduced in both BRM overexpression mutants,supporting that BRM plays an opposite function to asBRM in root hair elongation.These results indicated that AsBRM might affect root hair development through negatively regulating the expression ofBRM.5.The transcriptome analysis showed that asBRM overexpression resulted in the up-regulation of 161 genes and down-regulation of 163 genes in 14 d seedlings,and the suppressive effect of asBRM onBRM expression was verified using qRT-PCR.Meanwhile,the combination of our RNASeq data with the previous transcriptome data of BRM deletion mutants showed that the common up-regulated genes were concentrated in the JA pathway(JAZ3,JAZ10,OPR3,LOX3,JAO2,LOX4)and the common down-regulated genes were concentrated in the ABA pathway(SNRK3.6,CYP707A2),suggesting that asBRM may affect hormone-associated genes through regulating the expression ofBRM.6.Combination analysis of our transcriptome data with previous Ch IP-Seq data onBRM protein,showed that many differentially expressed genes(JA-related,ARFs,root hair developmentrelated)harbored the binding sites of BRM protein.Furthermore,several genes were transcriptionally validated using qRT-PCR,suggesting that asBRM regulates downstream genes through BRM.7.To further verify the regulatory effect of asBRM on the expression ofBRM,35S:S1streptavdin-tagged asBRM transgenic lines were generated to conduct Ch IRP-q PCR analysis.Consequently,a significant enrichment of genomic DNA of BRM was detected,indicating that asBRM was able to occupy the genomic DNA of BRM.These findings suggest that asBRM promotes root hair elongation,whileBRM located in the opposite strand inhibits root hair elongation.asBRM regulates root hair development through occupying theBRM genomic DNA and thus inhibiting BRM expression,which expands the understanding of lncRNA function and root hair regulatory network.