Cloning And Characterization Of MfNAC48,MfWRKY3 And MfWRKY22 Genes In Medicago Falcata

Medicago falcata is used as germplasm resource and gene pool for alfalfa breeding because of its excellent characteristics such as cold resistance,drought resistance,salt and alkali resistance.NAC and WRKY,which respectively play an important role in plant growth,development and resistance to biotic and abiotic stresses,are plant-specific transcription factors.In order to explore the characteristic and function of NAC and WRKY transcription factor in Medicago falcata responding to abiotic stresses,the c DNA sequences of MfNAC48,MfWRKY3 and MfWRKY22 genes were cloned in this study.It was fonud that the MfNAC48,MfWRKY3 and MfWRKY22 were expressed in roots,stems and leaves of Medicago falcata by real-time PCR analysis,and their expression level in leaves was significantly higher than in roots and stems.Meanwhile,the expression of them were induced by salt stress,drought stress and low temperature,and regulated by ABA treatment.Then,the expression vector encoding full-length and partial fragments of MfNAC48,MfWRKY3 and MfWRKY22 were constructed with p GBKT7 vector as skeleton,and the transcriptional activation function of MfNAC48,MfWRKY3 and MfWRKY22 was verified.The results showed that MfNAC48,MfWRKY3 and MfWRKY22 had the transcriptional activation function and the transcriptional activation domains were located at their C-terminal region.The transient expression vectors of p CAMBIA1300(35S-MfNAC48-s GFP),p CAMBIA1300(35S-MfWRKY3-s GFP)and p CAMBIA1300(35S-MfWRKY22-s GFP)were constructed and transformed into Agrobacterium tumefaciens respectively,and then injected into the lower epidermal cell of tobacco.It was found that MfNAC48,MfWRKY3 and MfWRKY22 were all localized in the nucleus by detection using laser confocal microscope.The upstream1888 bp promoter sequence of MfWRKY22 was obtained by Genome Walker kit,which contains stress response element,light response element and WRKY transcription factor specific W-box element.Finally,the p PZP221(35S-MfNAC48-NOS),p PZP221(35S-MfWRKY3-NOS)and p PZP221(35S-MfWRKY22-NOS)were constructed,and transgenic Arabidopsis thaliana plants were obtained.Under salt,drought,low temperature stresses and ABA treatment,the stresses resistance of transgenic Arabidopsis thaliana overexpressing of MfWRKY22 were enhanced,and the role of MfWRKY22 playing in plant growth was regulated by ABA.