Differential Expression And Alternative Splicing Analysis Of Transcriptome Of Kandelia Obovata Under Cold Stress

Mangrove is a special ecosystem that spread in the transitional zone between coast and sea in tropical and subtropical regions.In China,Kandelia obovata is one of the most widespread and cold-resistant mangrove species.The northernmost natural distribution of K.obovata located in FuDing,Fujian Province,China.It is of great significance to study the effect of cold stress on K.obovata for the purpose of northward planting of mangrove and cold tolerance of mangrove.The current researches on the low temperature stress and cold resistance of K.obovata is most concentrated in the level of physiological and ecological.The molecular mechanism of low temperature stress on K.obovata has been rarely studied from transcriptional and molecular level.In this project,K.obovata were cultured at 25? and 5? for 4 days,respectively.The molecular mechanism of K.obovata to cope with low cold stress was analyzed by RNA-seq technology and bioinformatic methods.The main results and conclusions of my project are listed below:1.We obtained 161,275,740 clean reads by using the Illumina Hi-Seq 2500 sequencing platform.The mapping rate of six samples transcriptome data are all above 94%.We obtained 474 up-regulated genes and 892 down-regulated genes from K.obovata transcriptional data compare cold stress with normal condition.2.After comparing the different gene expression pattern between the normal condition and cold stress treated K.obovata,we found that different expression genes are mainly enriched in the circadian rhythm,plant gene signal transduction and some secondary metabolism pathways.Firstly,K.obovata used the circadian rhythm mechanism to regulate the CBF depended path way as a response to the cold stress.Treated the mangrove for a relative long time caused the disruption of the original circadian rhythm.Genes related to circadian rhythm regulated the expression of CBF genes,which broke the balance of the CBF genes expression,causing the downregulation of CBF genes.Secondly,both the RNA-seq data and qRT-PCR data reveal that ABA-dependent pathway responds to low temperature stress by promoting the transmission of ABA signaling.Third,secondary metabolism pathways are also influenced during the cold stress.3.The analyses of alternative splicing events and alternative splicing genes showed:Firstly,alternative splicing events are prevalent under cold stress in K.obovata.A total of 158,174 alternative splicing events which were distribute across 12,275 genes were identified from both of the normal and cold stress in K.obovata.Secondly,gene structure is showed to be relevant to alternative splicing.In our data sets,alternative splicing frequency of a gene was positively correlated with its exon/intron number,and negatively associated with the GC content.Third,low temperature stress caused changes in alternative splicing of K.obovata.1,707 significant differential alternative splicing genes were predicted in K.obovata under cold stress.A gene can have one or more alternative splicing types.Based on this RNA-seq data analysis results,a gene only has single type of alternative splicing is the most common and very a few genes have all five alternative splicing types.Fourth,15 K.obovata genes of SR gene family were predicted according to the RNA-seq data results;K.obovata SR gene family regulate the alternative splicing through gene expression way.4.K.obovata respond to low temperature stress in both gene expression level and alternative splicing mode.DEGs and DASGs display little overlap under low temperature treatment.No GO categories and KEGG pathway were overrepresented in both the DEGs and DASGs of K.obovata under cold stress.Transcription factor annotation and prediction were performed on DEGs and DASGs.The result shows that little overlap between the annotated gene families and the annotated transcription factors.So,alternative splicing is an additional layer of regulation in response to cold stress in K.obovata.