The Regulation Mechanism Of Populus Simonii×P.nigra PsnICE1 Transcription Factor In Cold Stress Response

Cold stress adversely affects plant growth and development.ICE1 could specially band to active domain of CBF3 promoter and induce transcription expression of cold-responsive genes downstream of CBF3,play important roles in cold stress response.In this study,PsnICEl gene and promoter was cloned from P.simonii×P.nigra using PCR amplification method.Multiple alignments showed that PsnCEl shares high sequence identity with other plant ICE1 proteins and contains the conserved basic helix-loop-helix(bHLH)domain and C-terminal region,but the N-terminal sequence with variation.A phylogenetic tree constructed based on complete amino acid sequences of PsnICE1 and other plant ICE Is,indicating that PsnICEl is most closely related to RcICE1 of Ricinus communis and PsICEl of Populus suaveolens.The sequence of PsnICEl promoter with a length of 1247bp was cloned from DNA of P.simonii×P.nigra using PCR amplification method.Sequence analysis showed that it consisted of a typical core promoter region of eukaryotic,besides contains the basic elements:TATA-box,CAAT-box,there are some hormone responsive elements as well as and multiple stress-induced elements,such as,ABRE,DOFCOREZM,MYBCORE,MYCCONSENSUSATHSE and W-box.It is suggested that PsnICE1 promoter may associated with adversity stress and play important roles in response to various stresses in.simonii×P.nigra.PsnICEl was fused to GFP,and introduced into onion epidermal cells by particle bombardment.The subcellular localization results showed that PsnICE1 protein was expressed in the nucleus.The expression of PsnICE1 was up-regulated by cold stress from 1.5 to 72 h,indicating that the expression of PsnICE1 is in response to cold stress.PsnICE1 is expressed in roots,stems and leaves,and the highest expression of PsnICE1 is in 72h.PsnICE1 has transcriptional activation activity,the region from 66 to 137 amino acid of PsnICE1 was found to have transactivation activity.To identify the cold tolerance function of PsnICEl gene,the overexpression vector pROK2-PsnICE1 and RNAi vector pFGC5941-PsnICE1 were constructed.The pROK2-PsnICE1,pFGC5941-PsnICE1 vectors were transformed into P.simonii×P.nigra.Futhermore,we analyzed and compared the physiological indexes such as DAB,NBT,Evas Blue staining and MDA,H2O2,proline content,relative conductivity in transgenic plants under cold stress.The results showed that overexpression of PsnICE1 in P.simonii×P.nigra had the lowest O2-,H2O2 and MDA accumulation,minimal cell death and the lowest electrolyte leakage rate among the three kinds of P.simonii×P.nigra.Conversely,the silencing expression.simonii×P.nigra displayed the opposite physiological changes.PsnICE1 can enhance the activities of SOD,POD by regulating the expression of SOD and POD,which reduced the accumulation of ROS and improved cold stress.These results indicated that overexpression of PsnICEl gene may improve the cold tolerance of P.simonii×P.nigra.Furthermore,high-throughput sequencing technology was used to analyze the gene expression profiles of overexpressed PsnICE1 P.simonii×P.nigra a and wild-type P.simonii×P.nigra.Under cold stress,1941 differentially expressed genes(DEGs)were obtained.Among them,948 were raised and 993 were lowered.The DEGs were mainly involved in the plant hormone signal transduction,cysteine and methionine metabolism,phenylpropanoid biosynthesis and phenylalanine metabolism.These results suggested that PsnICE1 gene could regulate the expression of a series of downstream target genes,especially the expression of some transcription factors such as WRKY,NAC,bHLH and so on.PsnICE1 protein was identified to bind to five motifs by TF-centered yeast one-hybrid,including E-box,DRE-motif,C-box,ABRE-motif and Unknownl.Among them,Unknownl were identified as a novel element.The yeast one-hybrid,transientiy transforme and EMSA results further demonstrated that PsnICE1 can directly bind to the five cis-acting elements.In conclusion,these results would provide a theoretical basis for the further studying the molecular mechanism response to cold stress and developing the molecular breeding in P.simonii×P.nigra.