| bZIP(basic leucine zipper)is a very important type of transcription factor in plants,which is involved in many biological processes from plant growth and development to resistance regulation.In order to understand the characteristics of bZIP transcription factors in Medicago sativa,the biological functions of a alfalfa C type bZIP transcription factor-MsbZIP1 was analyzed,and the regulatory mechanism of MsbZIP1 gene on alfalfa resistance was explained.In this study,a full-length cDNA of MsbZIP1 gene was obtained from alfalfa using RACE technology.The full-length cDNA of this sequence is 1176 bp,encoding 361 amino acids,with a predicted molecular weight of 42.3 kD and an isoelectric point of 6.5.The analysis revealed that the protein contains the BRLZ basic domain typical of the bZIP family and a leucine zipper,and belongs to the bZIP protein family.Phylogenetic tree analysis showed that the protein belongs to the bZIP transcription factor C subfamily,and has high homology with AtbZIP63 of Arabidopsis thaliana,speculating that it may have similar functions to this protein.qRT-PCR analysis showed that the MsbZIP1 gene responded to drought,high salt,high temperature,low temperature,and abscisic acid(ABA)and auxin(Auxin)treatment to varying degrees.It is speculated that this gene may be involved in regulating alfalfa abiotic stress.In this experiment,the plant expression vector PCAMBIA3301-MsbZIP1 was constructed,and Arabidopsis was transformed by Agrobacterium-mediated inflorescence infiltration method.After screening,expansion and molecular detification of the offspring,7 positive lines of transgenic Arabidopsis were obtained.In this study,the C subfamily bZIP transcription factor was isolated from alfalfa for the first time,and the alfalfa MsbZIP1 gene was initially determined to respond to a variety of stress conditions,and positive transgenic materials were obtained.The study found that the germination of wild-type Arabidopsis seeds was earlier than that of the MsbZIP1 transgenic Arabidopsis seeds under both NaCl treatment and normal conditions.It can be inferred that the alfalfa MsbZIP1 gene delays the seed germination and may play a negative regulatory role on seed germination.After one week of salt stress treatment,relevant physiological indicators were measured,and the catalase,peroxidase,and superoxide dismutase activities of the transgenic lines were significantly higher than those of the WT.The salt resistance of transgenic lines were stronger than WT.The expression of stress-related genes was measured via qRT-PCR.The results showed that the MsbZIP1 gene regulates the expression of ABA biosynthesis genes ABA3,ABA1,AAO3,NCED3,downstream ABA response genes P5 CS and ABA signaling pathway genes ABI5,RAB18,HST,RD26,therefore enhancinge the resistance of plants to salt stress. |
