Functional Analysis Of MfNAC87 And MfNAC63 Genes In Medicago Falcata

Medicago falcata,which has excellent characteristics such as drought resistance,cold resistance,and salt-alkali tolerance,is an important germplasm resource for resistance breeding.NAC protein is one of transcription factors in plant and not only involves in plant growth,fruit ripening,leaf senescence and other physiological processes,but also plays an important role in the process of stresses response.In this study,the 1290 bp ORF of MfNAC87,which encodes a protein with a predicted molecular mass of 48.32 KDa and an isoelectric point of 5.83,was cloned from Medicago falcata.Then the expression vector p CAMBIA1300（35S-MfNAC87-s GFP）was constructed and expressed in tobacco（Nicotiana benthamiana）leaf epidermal cell,the results showed that the MfNAC87 was localized in the nucleus.It was confirmed that the MfNAC87 played a role in transcriptional activation in yeast cell and the transcriptional activation domain was located at the C-terminal of MfNAC87 protein.The T₃ lines of overexpresssion the MfNAC87 and MfNAC63 in Arabidopsis thaliana respectively were obtained.It were found that over-expresssion of MfNAC87 could inhibit the growth of transgenic Arabidopsis thaliana seedlings under the drought,salt stresses and ABA treament,but over-expresssion of MfNAC63could enhance the growth of transgenic Arabidopsis thaliana seedlings.But,under the low temperature treatment,the growth was enhanced in MfNAC87 over-expresssing transgenic Arabidopsis thaliana seedlings and inhibited in MfNAC63 over-expresssing transgenic Arabidopsis thaliana seedlings.Finally,the 1404 bp and 1439 bp fragments upstream of MfNAC87 and MfNAC63 were obtained respectively by Genome Walker technology.Meanwhile,the cis-acting elements regulated by light,low temperature,gibberellin,methyl jasmonate were found in promoter sequence of MfNAC87 gene,and the cis-acting elements regulated by light,gibberellin,abscisic acid,auxin were found in promoter sequence of MfNAC63 gene.The promoter sequence of MfNAC63 were obtained by PCR and the p ORER1-pro._MfNAC63::GUS expression vector was construced,then the transgenic Arabidopsis thaliana were obtained and GUS histochemical staining showed that the expression of gene drived by the MfNAC63 promoter was regulated by ABA,IAA and GA₃.