Identification And Functional Prediction Of Long Non-coding RNA Of Birch(Betula Platyphylla) In Low-temperature Stress

Betula platyphylla Suk.is a common species of Betulaceae in north China.It has the characteristics of cold-resistant and rapid growth in acidic soil,usually used in the cultivation of highly resistant excellent varieties.In recent years,the completion of its whole genome sequencing has provided convenient conditions for studying the resistance mechanism of woody plants.Long non-coding RNA(lncRNA)is a non-coding RNA with a length of more than 200 nucleotides.At present,lncRNA has deeper research in the functions of medical and animal fields,it not only regulates gene expression but also participates in the regulation of many life processes such as chromosome remodeling and transcriptional activation.In plants,studies on the specific roles and functions of lncRNA are in the initial stage,but relevant studies have proved that lncRNA is closely related to the stress resistance of plants.In order to study the potential function of lncRNA under low-temperature stress,B.platyphylla was used as experimental material in this study.Through by analysis changes of physiological indexes,and based on transcriptome sequencing to the identification of lncRNA who response to low-temperature stress and GO function analysis of target genes,to explore the mechanism of cold resistance mediated by lncRNA in B.platyphylla.The results of the study were as follows:In this study,the plants were treated at a low temperature of 8 °C for 0 h,6 h,12 h and 24 h,respectively.We found that all four physiological indexes were responsive to lowtemperature stress.With the increase of stress time,the relative electrolytic leakage and MDA content increased gradually,and the changes of SOD and POD activity were significant.This indicates that the low temperature of 8 °C has a certain effect on the growth mechanism of the birch,especially the activity of antioxidant enzymes.Transcriptome sequencing was performed on the samples,and the sequencing results were filtered and aligned to the reference genome.Integrate the data of all libraries in order to generate a unified numbered transcriptomics data set.Differentially expressed genes(DEGs)were identified at 6 h,12 h,and 24 h samples with 0 h as control,a total of 5871 DEGs were obtained,and the 24 h contained the most DEGs.GO enrichment analysis found that DEGs were mainly related to the endomembrane system,organonitrogen compound,and transcription factor activity,proving that the functions involved in DEGs were complex and extensive.According to the lncRNA identification process designed in this study,transcripts without coding ability and protein domain were screened by gffcompare,CNCI,CPC2,and Uniprot software to identify and analyze lncRNA.A total of 2622 lncRNAs were obtained,with the sequence length mainly concentrated in 200-2000 nt,and most of them were intergenic lncRNAs(linc RNA).Based on all DEGs and identification results of lncRNA,differentially expressed lncRNA(DE-lncRNA)were identified.A total of 878 lncRNA responded to low-temperature,accounting for 14.95% of the total number of DEGs(5871),and more than 1/3 of lncRNA showed differential expression,indicating that lncRNA plays a certain role in low-temperature stress.Meanwhile,co-expression analysis showed that there was a close correlation between the expression of mRNA and lncRNA.To analyze the potential functions of lncRNA,RIsearch 1.2 software was used to predict the target genes of lncRNA.878 lncRNA corresponding to 3412 lncRNA-target gene pairs,involving 1572 target genes.Combined with co-expression analysis,a total of 556 lncRNAtarget gene pairs were relevant in expression patterns.GO enrichment analysis of these target genes showed that they were related to plant immune response,transmembrane movement,and kinase activity.And these processes were proved to be closely related to cold stress,indicating that lncRNA could response to cold stress through its target genes.Since GO enrichment analysis suggested that some target genes had transcription factor activity,the lncRNA-transcription factor interaction network was constructed.Through annotations,14 co-expressed target genes belong to transcription factors,among them,8 upregulated including b HLH,ERF110,DDF1,GATA9,GRF1,LRP1,WRKY34,and WRKY9.These genes have been shown to respond positively to cold stress in other studies,so lncRNAs that related to them are equally important in low-temperature stress.In order to verify the reliability and authenticity of the results in this study,both q RT-PCR and gene clone were performed.The results show that q RT-PCR and RNA-seq are consistent,indicating that the sequencing data of this study have been trusted.At the same time,the success of 25 gene clones has also demonstrated the real presence of lncRNA identified.This study provides a certain reference for the function and the response mechanisms of lncRNA in low-temperature.