| Alfalfa(Medicago sativa)is a leguminous herb,and it is the most widely cultivated forage legume and one of the most economically valuable crops in the world.Its agricultural development has been restricted by various adverse environmental conditions including water deficit,low temperature and high salinity.Compared to other crop species,alfalfa exhibited higher drought tolerance.However,under moderate and severe adverse environmental conditions,the productivity of alfalfa were severely affected.WRKY gene family is one of the largest transcription factor families in plants.Meanwhile,WRKY proteins are major regulators of plant response to abiotic stress.However,the WRKY gene family in alfalfa has not been systematically studied.Based on the previous abiotic stress transcriptional experiments,the members of MsWRKYs gene family were identified and the expression profiles of MsWRKY genes under abiotic stresses such as drought treatments were examined.The function of MsWRKY100 were identified in Arabidopsis thaliana,Medicago truncatula and M.sativa by genetics,physiology and transcriptomics.1.MsWRKYs gene family of alfalfa was studied by using bioinformatics methods.A total of 107 MsWRKYs gene family members were identified and divided into three main groups(Group ?-?),and Group ? was further divided into five subgroups.The classification,evolution,conserved motifs and expression patterns in different tissues and under different abiotic stresses of MsWRKYs members were comprehensively analyzed.A total of 27 MsWRKYs candidate genes were isolated to have changed expression levels under drought,cold,abscisic acid(ABA)and salt treatments,these candidate MsWRKYs genes might play a crucial role in the response to abiotic stresses.2.The expression of MsWRKY100 was significantly induced by drought,salt,abscisic acid and cold treatment.The GUS staining analysis showed that GUS activity was activated by mannitol,Na Cl and ABA treatments.Subcellular localization analysis of tobacco leaf epidermal cells showed that MsWRKY100 located to the nucleus.Overexpression of MsWRKY100 transgenic Arabidopsis seedlings exhibited increased primary root length and survival rate,reduced the electrolyte leakage and concentrations of malondialdehyde(MDA),and the activity of antioxidant enzymes(POD and CAT)was significantly increased than those of wildtype.Meanwhile,overexpression of MsWRKY100 also increased ABA sensitivity in Arabidopsis seedlings.MsWRKY100 transgenic alfalfa plants were obtained after Agrobacteriummediated transformation with the MsWRKY100 overexpression vector.Under control conditions,the leaf water loss rate of MsWRKY100 transgenic alfalfa was lower than that of wild type.MsWRKY100 can improve the photosynthetic efficiency of transgenic alfalfa under drought stress.These results suggest that MsWRKY100 can improve drought-stress resistance in both Arabidopsis and alfalfa.3.MtWRKY100,the homology gene of MsWRKY100 in M.truncatula.The expression level of MtWRKY100 was induced by drought and salt stresses.Two mutant lines of MtWRKY100(NF8978 and NF9753)were screened from the Tnt1 mutant library of M.truncatula.Two MtWRKY100 Tnt1 mutant lines exhibited drought sensitive phenotype.Under 300 m M and 400 m M mannitol condition,mtwrky100 exhibited decreased primary root length.Under drought stress,the survival rate were significantly lower than that of the wild type.A total of 81 lines of MtWRKY100 transgenic M.truncatula plants were obtained after Agrobacterium-mediated transformation with the MtWRKY100 overexpression vector,and the positive rates was90%.Compared with the control,MtWRKY100 overexpression lines in M.truncatula showing increased drought tolerance.During drought stress,the activity of antioxidant enzymes(SOD,POD and CAT)in overexpression of MtWRKY100 transgenic M.truncatula seedlings was significantly increased.RNA sequencing showed that overexpression of MtWRKY100 in M.truncatula activated the expression of genes involved in “antioxidative defense" and “cutin,suberine and wax biosynthesis". |
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