Functional Characterization Of Two Cold Acclimation-related Genes MfAIR12 And MfWAK In Medicago Falcata

Medicago sativa subsp.falcata?i.e.M.falcata?is a forage legume with cold,drought and barren tolerance that can be used as a genetic pool for forage breeding.In this study,two genes related to low-temperature acclimation?MfAIR12 and MfWAK?were selected and the cold resistance mechanism were investigated.The main results are as follows:The results of MfAIR12 gene1.MfAIR12?auxin induced in root cultures?was screened from the low-temperature response profile of alfalfa,and was auxin-induced gene that affects root growth.MfAIR12codes for 231 a mino acids containing more hydrophilic a mino acids,is a close to the outer membrane of the transmembrane protein,and it is a gene cloned from Medicago alfalfa,with an ORF?Open reading frame?693 bp.MfAIR12 is a protein closed to the outer membrane of the transmembrane with more hydrophilic a mino acids.Sequence homology analysis shows that the homology of MfAIR12 and others protein is not high.MfAIR12 is not an evolutionarily conservative protein.2.The transgenic tobacco overexpressing MfAIR12 was obtained.qRT-PCR analysis showed that the expression of MfAIR12 was detected in transgenic tobacco but not in the WT.The LT50 of transgenic tobacco was significantly lower than that of WT,but the survival rate was just the opposite.Transgenic tobacco accumulates more H₂O₂,and the antioxidant enzyme activity and its gene expression were significantly increased after cold treatment.3.Higher transcript levels of NtDREB31-4 and Cor15a genes were observed in transgenic plants than in the wild type under chilling conditions by qRT-PCR.4.We also compared the cold resistance between the air12 mutant and the wild type.Under the conditions of cold acclimation or non cold-acclimation,the LT₅₀ of the air12 mutant was higher and the survival rate was lower than that of the wild type.After cold treatment,the expression levels of AtCBF1,AtCBF2,AtCBF3 and cold-responsible genes RD29A,RD29B,Cor15a,KIN1 and COR47 in air12 mutants and WT were significantly increased,but the expression was lower in air12 mutant compared with the wild type.It is suggested that Mf AIR12 plays a positive regulatory role in plant tolerance to cold through improve antioxidant enzyme activities and up-regulating expression of multiple cold responsive genes.The results of MfWAK gene1.WAK?wall associated kinase?is a cell wall-related kinase.The MfWAK gene was obtained by genetic screening from the low-temperature response profile of alfalfa.MfWAK contains an open reading frame of 2100bp encoding 699 a mino acids.The molecular weight of the protein was predicted to be 77.17 kDa and the isoelectric point was 7.1.MfWAK has an EGF domain,a transmembrane domain and a conserved intracellular kinase domain with a signal peptide sequence at the N-ter minus.Phylogenetic tree analysis showed that MfWAK was closely related to Arabidopsis thaliana AtWAKL14,and belongs to the fourth group of WAK/WAKL protein family.2.qRT-PCR results showed that MfWAK was mainly expressed petiole and roots,non expression in leaves and flower.MfWAK was induced by low temperature,and the expression of MfWAK increase prolonged treatment time.The gene was also induced by H2O2,salt and dehydration.MfWAK::GFP fusion protein is localized to the plasma membrane.3.The transgenic plants of Medicago truncatula were obtained,using Agrobacterium tumefaciens strain EHA105 harboring expression vetor of MfWAK.Under the condition of cold acclimation and non-cold acclimation,the LT50 of the Madicago truncatula plant overexpressing MfWAK was lower than the wild type.Higher transcripts level of CBF1,2,3was observed in transgenic plants than in the wild type.The expression of CAS15 and COR413 in the transgenic plants was significantly higher than that in the wild type after 2 h treatment.After 24 h cold treatment,the expression of the cold-related marker genes CAS15,COR413,LTI65 and LTI6A were significantly higher in the transgenic plants than in the wild type.The wak mutant had lower survival rate,and hinger LT₅₀ under non-stressed or cold-acclimation conditions4.Compared with wild type,the survival rate of transgenic Arabidopsis thaliana was higher,while the LT50 was lower.The expression levels of CBF gene and its downstream COR genes in transgenic Arabidopsis thaliana were higher than in the wild type.5.Overexpression of MfWAK improves the maximum photochemical efficiency?Fv/Fm?of transgenic Stylosanthes auratus,and decreased ion leakage,and improves the chilling resistance of transgenic plants.In su mMary,the expression of MfWAK gene enhances the transgenic plants'tolerance to cold and chilling.