The Study On DREB Type Transcription Factor Genes In Medicago

DREB transcription factors play an important role in tolerance to abiotic stress of plant.In this paper mainly we carried out researches of the DREB type transcription factors in Medicago.Three new DRE-binding protein genes MfDREB1 cDNA,MfDREB1s cDNA and MsDREB1 cDNA that encoded EREBP/AP2 type transcription factors were isolated by RACE and RT-PCR from Medicago falcata L.and Medicago sativa L.seedlings.The MfDREB1 has a open reading frame of 651bp,which coded 216 amino acid residues and the putative protein was predicted to have a molecular weight of 24.6 kDa,pl 5.95.The MfDREB1s has a open reading frame of 555bp,the putative protein is 184 amino acid long with a predicted molecular weight of 20.8 kDa,pl 9.11.The MsDREB1 has a open reading frame of 651bp, encoding 216 amino acid residues and the putative protein with a predicted molecular weight of 24.9 kDa,pl 6.11.The Protein Blast data revealed that the three proteins can be classified as typical members of the EREBP/AP2 family of DNA-binding proteins.The comparison of the MfDREB1 cDNA,MfDREB1s cDNA and MsDREB1 cDNA with their corresponding genes in genomic DNA showed that the size and nucleotide sequence of the cDNA were identical with that of the genomic DNA.This suggested that the genomic MfDREB gene,MfDREB1s gene and MsDREB1 gene have no introns.Southern blot analysis indicated that the MfDREB gene and MfDREB1s gene have three copies in Medicago falcate genome,the MsDREB1 is a double-copy gene in alfalfa.Northern blot analysis indicated that the MfDREB gene and MfDREB1s gene were induced by low temperature stress.Arabidopsis rd29A gene was induced with drought,high salt and cold treatments.There are two DRE/CRT(a DNA cis-regulatory sequence) element in the region of the rd29A promoter which was responsive to low temperature,drought and high-salt stress.The rd29A promoter has the ability to perceive abiotic stress and respond to it by activating downstream report gene.We amplified a 937bp promoter region of the rd29A gene promoter from the Arabidopsis genomic DNA by PCR.This cloned promoter region may be useful for the stress-inducible expression of foreign gene in transgenic plant.In order to establish a simple and practical transformation technique with a high transformation rate in alfalfa(Medicago sativa L.),the pollen-tube pathway transformation method has been developed.The alfalfa cultivar 'Algonguin' was used as recipient and the binary vector pPZP221 containing a GUS(β-glucuronidase) gene was used as the donor DNA of transgene.The donor DNA solution was applied to the stigma at 6,9, 12,20,24,28,32 and 48 hour after hand-pollination,respectively.PCR results showed that the transformation rates were high from 20h to 48h, with the highest rate of 16.7%at 32h after pollination.The paper established a efficient regenerative system of alfalfa tissue culture.The binary vector pPZP221(35S-MfDREB-nos) and pPZP221(RD29AP-MfDREB-nos) were transformed into alfalfa by Agrobacterium infiltration respectively.The PCR analysis suggested that the foreign gene was inserted into the recipient's genome.The over—expression of MfDREB1 activated expression of downstream genes in transgenic alfalfa, resulting in enhanced tolerance to low-temperature stress.