Functional Analysis Of PmERF109 And PmERF109-like In Prunus Mume Under Low Temperature And Salt Stress

Prunus mume is a traditional Chinese flower species with high ornamental value and a wide range of application.P.mume is native to southern China.Low temperature in winter and soil salinization limit the introduction and cultivation of P.mume in northern China.ERF transcription factors are a class of plant-specific transcription factors,which play essential roles in plant responses to abiotic stresses.However,there have been few reports on the response of ERF transcription factors to abiotic stresses of P.mume.In this study,PmERF109 and PmERF109-like,which responded to cold stress,were screened out by the gene expression profiles of’Xue Mei’leaves at 4℃.The relative expression levels of these two genes under cold stress were analyzed,and overexpression vectors of these two genes were constructed and transformed into Arabidopsis,and the cold and salt tolerance of transgenic Arabidopsis was evaluated.The promoter of PmERF109 was cloned and its activity under abiotic stresses was analyzed.The main research results obtained are as follows:1.Cloning and protein sequence analysis of PmERF109 and PmERF109-likePmERF109 and PmERF109-like genes were cloned,which encoded 271 amino acids and 267 amino acids,respectively.The phylogenetic analysis showed that PmERF109 and PmERF109-like was most related to ERF109 of Prunus persica and ERF109-like of Prunus dulcis,respectively.PmERF109 and PmERF109-like were most related to ERF109 from group X of Arabidopsis ERF and their proteins commonly shared one conserved motif CMX-1 in the N-terminal region.2.Expression analysis of PmERF109 and PmERF109-like under cold stress.The two-year-old’Zhu Sha’potted seedlings were treated under low temperature and the leaves were sampled.The relative expression levels of PmERF109 and PmERF109-like under cold stress were analyzed by q RT-PCR.The results showed that the transcript levels of the two genes were progressively enhanced by cold stress,peaking at the 24th hour and the 6th hour,respectively,later decreased.3.Functional analysis of PmERF109 and PmERF109-like genes.The results of subcellular localization experiment showed that PmERF109 and PmERF109-like were both localized in the nucleus.The overexpression vectors of PmERF109 and PmERF109-like were constructed and transformed into Arabidopsis.We obtained 3 T₃-generation homozygous lines of each gene.The transgenic plants were treated with abiotic stresses.The results showed that the survival rate,relative electrolytic leakage,ROS accumulation of PmERF109-overexpressing Arabidopsis and PmERF109-like-overexpressing Arabidopsis were not significantly different from the wild type under cold treatment,indicating the small roles of the two genes in cold stress resistance.Overexpression of PmERF109 significantly increased the germination rate of Arabidopsis seeds under salt stress.The seedlings of PmERF109-overexpressing Arabidopsis showed a significantly lower etiolation rate than the wild type under salt stress,and the primary roots of transgenic Arabidopsis were significantly longer than the wild type.However,overexpression of PmERF109-like significantly reduced the germination rate of Arabidopsis seeds under salt and mannitol stress,which exhibited that these two genes showed opposite regulatory effects on salt stress resistance.4.Cloning and transient expression analysis of PmERF109 promoter.The promoter sequence with 1882bp of PmERF109 was obtained.It was found that there were several cis-acting elements related to hormone and stress response on the promoter.The recombinant vector of PmERF109 promoter-GUS was constructed and transiently transformed into tobacco.GUS staining results showed that the PmERF109promoter could regulate GUS expression under normal growth,Na Cl,mannitol,ABA,Me JA and 4℃treatments.