Expression Profile Analysis Of Ammopiptanthus Mongloicus Under Cold Stress By RNA-Seq And AmNAC2Gene Cloning

Low temperature can lead to a series of changes in the molecular, physiological and biochemical, and morphological structure levels of plants, in which the change in gene expression level is an important genetic basis of plant response and resistance to low temperature stress. In this study, the expression profile of Ammopiptanthus mongloicus, a strong cold-resistant plant species, under cold stress was analyzed by RNA-Seq technology, and a cold-induced transcription factor gene named AmNAC2was cloned.The main results are as follows.(1) In the libraries of0h (control),2h,8h and24h after cold stress, more than8million clean reads were obtained, respectively. After the clean reads in each library were mapped to the refference unigene sequences of Ammopiptanthus mongloicus transcriptome assembled earlier in ourlaboratory,60279(70.0%),61231(71.2%),62693(72.9%) and61568(71.5%) unigenes were identified respectively.(2) Through comparing the expression levels of genes in the cold-stressed libraries with that in control library,4038,3917, and4413differentially expressed genes (DEGs) were identified in each library, among which2027,1894and1924DEGs were up-regulated and2011,2023and2489DEGs were down-regulated.(3) Eleven DEGs were selected for expression analysis by RT-qPCR method, and nine (81.8%) of them showed similar expression patterns to those detected by RNA-Seq data, indicating that the results for the identification of DEGs were considerably reliable in this study.(4) Of the DEGs identified at the three time points, genes known to respond to stresses were largely represented, and many newly identified genes were also existed. Based on GO enrichment analysis and KEGG pathway enrichment analysis, these genes were found to mainly participate in the physiological and biochemical processes of responses to stimulus and stress, secondary metabolism, lipid metabolism, plant hormone signal transduction, and starch and sucrose metabolism, the cellular components like plastid,chloroplast and membrane, as well as the molecular functions about oxidoreductase activity, antioxidant activity, hydrolase activity acting on glycosyl bonds, and transferase activity transferring glycosyl groups. (5) Through Venn diagram analysis,851commonly up-regulated DEGs and1174commonly down-regulated DEGs were identified across all three time points. Among them, the up-regulated DEGs were largely involved in the responses to stimulus and stress, defense response, secondary metabolic process, the components of plastid and chloroplast, as well as catalytic activity like oxidoreductases. Except for mainly participating in the responses to stimulus and stress and oxidoreductase activity, however, the commonly down-regulated DEGs were also closely related to the processes of lipid or fatty acid metabolism and lipid localization.(6) Using RACE and RT-PCR methods, the full-length cDNA of AmNAC2gene was obtained, whose complete coding region cDNA contains879bp and the predicted polypeptide consists of292amino acids, with a molecular weight of33.86KDa and an isoelectric point of5.86.These results laid a good foundation for further studies on the molecular mechanisms involved in cold tolerance in A. mongolicus and key cold-tolerant gene mining from this plant, as well as the subsequent functional analysis of AmNAC2.