Transcriptome And CircRNA Analysis Of Medicago Lupulina Treated With High Calcium Stress

Medicago lupulina is a large perennial legume forage,which plays an important role in grassland animal husbandry.In karst areas,soil high calcium stress is one of the important factors affecting the growth and development of Medicago lupulina,which affects the germination of Medicago lupulina seeds and the growth of seedlings.Therefore,studying the effect of high calcium stress on Medicago lupulina is of great significance for exploring effective calcium regulation measures.Since there is currently no reference genome of Medicago lupulina,the analysis of Medicago lupulina transcriptome data with reference to the alfalfa reference genome is not reliable.Therefore,in order to fully reveal the changes and regulation of gene expression in Medicago lupulina in response to calcium stress,this study processed 9 transcriptome data of Medicago lupulina seedlings with different calcium concentrations of0.1mmol/L,10.0mmol/L,and 25.0mmol/L.Carrying out the transcript integrated library calculation workflow,splicing,assembling,annotating,and analyzing the Medicago lupulina transcriptome data under different calcium concentration treatments.Finally,a Medicago lupulina transcriptome data containing 240,280 transcripts(Supertranscript)and155,971 Unigene were obtained.Based on the transcriptome data,we screened out 1,473 differentially expressed genes under different calcium treatments through differential gene analysis.Through GO enrichment analysis,we found that the differential genes were significantly enriched in the biological processes of "reproductive shoot system development","response to metal ion","flower development","vesicle-mediated transport","response to salt stress",etc.Through KEGG enrichment analysis,we found that the differential genes were significantly enriched in the metabolic pathways of "Spliceosome","Lysine degradation","beta-Alanine metabolism" and "Endocytosis".Further,the weighted gene co-expression network analysis(WGCNA)analysis was performed on the differentially expressed genes.A network module that is significantly related to calcium stress was obtained,and the four key regulatory genes VGLYAH,FRL4 A,GGPPSI and fus A2 genes of the network module were screened out.It is speculated that they have important regulatory effects on the calcium-regulated gene network module.Circular RNA(circRNA)is an endogenous non-coding RNA(nc RNA)that often exists in eukaryotes.It does not have a 3’poly(A)tail and a 5’ cap.It has a characteristic covalent The closed ring form exists and is difficult to degrade.The latest research shows that certain circRNAs act as miRNA sponges by isolating and preventing miRNAs from binding to the corresponding target genes,and play an important role in regulating gene expression after transcription.In plants,the exploration of circRNA has just begun,and the circRNA information possessed by most plants is still unknown.Therefore,this study uses a specific algorithm to predict the circRNA information of Medicago lupulina from the transcriptome data,and analyzes the effect of high calcium treatment on the expression of Medicago lupulina circRNA,which is of great significance to advance the research of plant circRNA.In this study,110 circRNAs were predicted using Medicago lupulina transcriptome data,and ath-miR854,ath-miR5652,and ath-miR398 that could interact with circRNA were predicted by Reg RNA 2.0 software.It was found that these miRNAs can interact with plants under drought,flood,and heat Different expressions are shown under stress and water shortage.Using PASmiR,Plant circ Net and other data,22 target genes that can interact with circRNA and predicted miRNA were retrieved,and two possible circRNA-miRNA-gene networks were predicted.It is speculated that these two regulatory networks may also exist.In Medicago lupulina,it is also related to the resistance of Medicago lupulina.Through Pathway enrichment analysis of 22 related target genes of circRNA,it is found that 7 target genes are involved in "Peroxisome","Oxidative phosphorylation" and "Ribosome","PPAR signaling pathway" and "Ubiquitin mediated proteolysis" and other 23 signaling pathways.