| WRKY transcription factor is a plant-specific transcription factor.Its functions in the physiological and biochemical processes,such as aging and stress response by regulating the activity of hormones and target genes.In this study,four WRKY genes in Phyllostachys edulis were studied through various methods including quantitative PCR,yeast one-hybrid and transgene,in order to figure out its specific functions initially in the process of aging,stress response and hormone signaling regulatory pathway.The main results are as follows:1.Gene cloning and sequence analysis4 WRKY genes were cloned from Phyllostachys edulis,named PheWRKY1-1,PheWRKY11-2,PheWRKY50-1 and PheWRKY76 respectively.Their c DNA sequences length were 1686 bp,1023 bp,1023 bp and 909 bp respectively.The proteins of these 4 WRKY contained no signal peptides and transmembrane domains,belonged to hydrophobic proteins and had interacting proteins.The upstream promoter region contained action elements related to stress and hormone response.Phylogenetic tree analysis and sequence alignment revealed that these 4 WRKY genes were closely related to the monocotyledonous species’ evolutionary relationship and farther from the dicotyledonous species.2.Subcellular localizationPheWRKY1-1,PheWRKY11-2 and PheWRKY50-1 were cloned into 35S-GFP respectively to construct the transient expression vector,and then transformed into the protoplasts of rice,and the expression positions were observed under the laser conclusive microscope.The results showed that these 3 WRKY genes were expressed in the nucleus.3.Transcriptional activity analysisPheWRKY1-1,PheWRKY11-2 and PheWRKY50-1 were cloned into p GBKT-7 vector and then converted into yeast respectively.The results showed that PheWRKY50-1 had transcriptional self-activation activity,while PheWRKY1-1 and PheWRKY11-2 had no transcriptional self-activation activity.4.Gene expression analysisThe expression patterns of PheWRKY1-1,PheWRKY11-2,PheWRKY50-1 and PheWRKY76 genes were analyzed by real-time quantitative PCR.The results showed that there were significant differences in the expression patterns of these three genes in different tissues and organs.PheWRKY1-1 was mainly expressed in roots,and PheWRKY50-1 was concentrated in leaves;even leaves from the same individual sample,the expression of 4 WRKY genes was significantly higher in the old leaves than in the new leaves,but with the aging of the whole plant,there was no obvious change in the gene expression;Under drought stress,PheWRKY1-1,PheWRKY11-2,PheWRKY50-1 and PheWRKY76 up-regulated in stems,PheWRKY50-1 and PheWRKY76 obviously up-regulated in leaves,PheWRKY1-1 and PheWRKY11-2 up regulated in roots;Under salt stress,the expression levels of these 4 genes up-regulated in roots and stems;Under different hormone treatments(IAA,ABA,Me JA and SA),the expression levels of PheWRKY1-1,PheWRKY11-2 and PheWRKY50-1 up or down regulated in varying degrees..5.Transformation into Arabidopsis thalianaPheWRKY11-2 and PheWRKY50-1 genes were transformed into Arabidopsis through agrobacterium tumefaciens.The transgenic plants were treated with drought and salt stress in different level,and then the phenotype of transgenic plants was observed.The results demonstrated that the transgenic lines endowed with an enhanced resistance and were better than the wild type plants comprehensively,for instance,root length,number of lateral roots and overall growth and so on. |
