Cloning And Function Identification Of ZjERF3 In Zoysia Japonica

The adverse environment such as extreme temperature,drought and high salinity affect the growth and development of terrestrial plants,and even cause plant death.In order to adapt to poor growth conditions,plants have evolved complex and precise molecular regulatory mechanisms.ERF transcription factors are of great importance to the network.This study used “Meyer” as the test material to reveal the characteristics and biological functions of the transcription factor ZjERF3 in Zoysia japonica.The study result are as follow:1.A new member of the AP2/ERF transcription factor family,ZjERF3,was isolated from Zoysia japonica.The open reading frame of the gene is 642 bp in length,encoding 213 amino acids.Conservative domain analysis showed that ZjERF3 contains an AP2 conserved domain,which is consistent with the characteristics of ERF subfamily members.Phylogenetic analysis revealed that ZjERF3 was closely related to the ERF protein of Eragrostis pilosa.The expression of ZjERF3 gene was detected by q RT-PCR.The results showed that ZjERF3 gene expression level was varied in different tissues and different developmental stages in Zoysia japonica.Wheras,ZjERF3 was expressed most abundantly in the leaves,and especially in the mature leaves.Moreover,its expression could be induced by ET,Me JA,Na Cl or cold treatment,but suppressed by either ABA or PEG6000.2.To analyze the subcellular localization character of ZjERF3 protein,a 35S::ZjERF3::YFP vector was successfully generated.The transient expression analysis in Nicotiana benthamiana demonstrated that ZjERF3 was located in the nucleus.The p GBKT7-ZjERF3 vector was constructed and transformed into Y2 H Gold yeast cell to investigate the transcriptional activation ability.The results showed that it had strong transcription self-activation activity.3.The ZjERF3 gene was transferred into yeast strain YPH500 with pYES2 as vector,and pYES2 was used as control at the same time.Comparing the growth speed of yeast cells both recombinant plasmid and empty vector under stress condition,we found that the growth speed of yeast cell carryingpYES2-ZjERF3 was significantly faster than pYES2.The results showed that the resistance of yeast cell to three stress conditions could be improved by the transformation of ZjERF3 gene.4.The plant expression vector p3302Y3-ZjERF3 was constructed and transferred into Agrobacterium strains GV3101 to generate transgenic Arabidopsis thaliana by inflorescence infections method.There was no difference between wild type and transgenic lines under normal growth conditions.Under salt and drought stress,the germination rate and fresh weight of transgenic lines were significantly lower than wild-type lines.The relative water content,soluble sugar content,proline content of leaves were higher than wild-type lines,while the electrical conductivity(EL)and malondialdehyde(MDA)contents were lower than wild-type lines.The indicators proved that the transgenic lines is more sensitive to stress condition at germination stage,but its resistance at seedling stage is significantly better than that of wide-type lines.It is suggested that ZjERF3 gene plays important roles in the response to salt and drought stress in plants.Under extreme low temperature treatment,the survival rate of transgenic lines were significantly more than wild-type lines,indicating that ZjERF3 gene also participates in low temperature stress response.In conclusion,ZjERF3 protein had strong transcription self-activation activity and was located in the nucleus.Its overexpression improved the growth and osmotic adjustment ability of transgenic plants,Which in turn contribute to the resistance of plants under stress conditions.This study provides a theoretical basis for further elucidating the regulatory pathway of ZjERF3 in Zoysia japonica.