Big Data Provides Insights Into Salt Response And Molecular Mechanism Of LncRNAs In P.trichocarpa

With the rapid development of high-throughput sequencing technology and the continuous reduction of sequencing cost,biomolecular research has entered the era of big data.In recent years,studies have found that long non coding RNA(lncRNA)plays an important role in the regulation,expression and metabolism of animal and plant genes,and can participate in various processes affecting biological molecules.Different from animals that can avoid environmental stresses,plants usually survive and thrive in fixed places,and are inevitably affected by abiotic stresses(such as salt stress).Over the years,scientists have done a lot of research on the mechanism of plant salt resistance,and made a lot of important achievements.However,previous studies on plant response to salt stress mainly focused on functional genes,and the molecular mechanism of lncRNAs in plants,especially woody plants,was rarely reported.For this reason,this study used the woody plant P.trichocarpa as the material,analyzed the whole transcriptome sequencing data of roots,stems,and leaves under short-term and long-term salt stress and combined with molecular experiments to obtain the following five aspects results:(1)Based on the characteristics of biological big data,the lncRNAs analysis platform was established and applied to the identification of lncRNAs in P.trichocarpa.Based on the data characteristics of its genome,transcriptome and lncRNAs,we set up a set of strict analysis process to analyze and identify lncRNAs.Based on this analysis process,2 988 high confidence lncRNAs were identified in P.trichocarpa.According to the relationship between lncRNAs and adjacent coding gene sites and transcription direction,lncRNAs were divided into five types:1 466 antisense,1 156 intergenic,297 divergent,47 intronic and 2 exonic.In addition,the results of characteristic analysis showed that lncRNAs were evenly distributed on 19 chromosomes and had the characteristics of tissue-specific expression.Its expression,GC content,length range and number of exons are all less than those of protein-coding genes.(2)Reveal the differential expression of lncRNAs under salt stress and their cis-regulatory effects on neighboring coding genes.Using transcriptome data,it was identified that 1 183 lncRNAs were differentially expressed under salt stress.These lncRNAs were organized into six expression clusters based on tissue specificity and salt stress.Further analysis,we found that 612 lncRNAs have a cis-regulatory effect on their neighboring coding genes.These coding genes are significantly enriched in intracellular sodium ion homeostasis,potassium and sodium ion transporter activity,redox processes,defense reactions and other abiotic stresses response pathways.This indicates that P.trichocarpa lncRNAs can regulate the expression of neighboring protein-encoding genes in cis to mediate the response to salt stress.(3)The salt responsive co regulatory network of lncRNAs was constructed,and the enrichment of salt responsive elements was found.Using WGCNA co-expression analysis method,six significant co-expression modules were identified under long-term and short-term salt stress in root,stem and leaf tissues,respectively.LncRNAs in each module had trans-regulatory effect on protein coding genes.In addition,salt responsive elements were enriched in these co-expressed lncRNAs,which were mainly involved in DNA binding,transcriptional regulation and plant hormone response.By comparing lncRNAs and TFs,we found that lncRNAs play an important role in regulating gene expression and salt tolerance as well as TFs.(4)Thirty lncRNAs and their target genes were screened,and the interaction mode of cis and trans molecules was proposed.Through the co regulatory expression analysis of(2)and(3),we screened 30 lncRNAs.The predicted cis and trans target genes are homologous genes,and are significantly enriched in signal transduction,gene expression regulation,stress response and other pathways.(5)Biochemical molecular experiments verify the bifunctional molecular action mode of Ptlinc-NAC72.Ptlinc-NAC72 is one of the intergenic lncRNAs with this regulatory potential.This gene is specifically expressed in the leaf tissue of P.trichocarpa after 7 days of salt stress.The results of color-developing in situ hybridization(CISH)indicate that it is mainly located in the cytoplasm.Furthermore,transient transformation and dual-luciferase detection experiments confirmed that Ptlinc-NAC72 has cis and trans positive regulation effects on the two target genes PtNAC72.A and PtNAC72.B,respectively.Finally,the results of co-transformation and GUS staining experiments proved that Ptlinc-NAC72 matched the 5'UTR region of the target genes through sequence complementation at the cytoplasmic post-transcriptional level,thereby stabilizing the expression of the target genes PtNAC72.A and PtNAC72.B.Thus,Ptlinc-NAC72 is one of the few identified lncRNAs that play an important regulatory role in the process of salt stress in P.trichocarpa.Focusing on the response of lncRNAs and coding genes under salt stress,a bifunctional molecular action mode for lncRNAs to regulate target gene expression in cis-and trans-manner is proposed.And the regulatory relationship between Ptlinc-NAC72 and the two target genes PtNAC72.A and PtNAC72.B has been verified.In conclusion,based on the characteristics of big data from different sources,this study first built a lncRNAs analysis platform and identified a large number of lncRNAs in P.trichocarpa.In a word,the lncRNAs analysis platform established in this study not only provides technical conditions for this study,but also provides technical support for the research of lncRNAs by our research group,collaborators and other researchers.The results obtained in this study not only increase people's understanding of the regulation mechanism of lncRNAs resistance to salt stress,but also provide a theoretical basis for molecular breeding of salt resistant trees.