Functional Analysis Of Alfalfa MsABF2 In Abiotic Stress Tolerance

Abiotic stress seriously affects plant growth and destabilizes crop yield.In plants,Abscisic acid（ABA）plays a key role in responding to abiotic stress.Under salt,drought,low temperature and other abiotic stresses,the synthesis of ABA is significantly induced followed by activating a series of the signal pathways to stimulate the plant resistance mechanisms.Remarkably,ABF（ABA-responsive element binding factors）transcription factors play a crucial role in the ABA signaling pathway.In this study,the alfalfa MsABF2 gene was transformed into tobacco to analyze the function of this gene under abiotic stress.The main results are showed as follows:1.Tissue-specific expression and expression patterns under abiotic stresses of MsABF2 in alfalfaThe results of q RT-PCR analysis showed that alfalfa MsABF2 gene was expressed in roots,stems,leaves,flowers and pods of alfalfa,most notably,the highest expression was found in leaves.The expression of MsABF2 gene could be induced by low temperature,salt,saline-alkali and ABA treatment,moreover,the expression level is influenced by the increasing of treatment time.In roots and leaves,when under temperature treatment,the expression of MsABF2 gene reached the peak at 24 h;while under ABA treatment,the expression levels reached their peaks at 1 h and 12 h,respectively.When under salt and saline-alkali treatment,the expression of MsABF2gene reached the peak at 1 h in leaves.2.Genetic transformation of alfalfa MsABF2 gene to tobaccoThe MsABF2 gene was transformed into tobacco by Agrobacterium-mediated method,then 25 strains of MsABF2 transgenic tobacco were obtained via hygromycin（Hyg）resistance screening and PCR identification.Transgenic lines with the same growth and growth status were selected for q RT-PCR detection.Three high expression transgenic lines,OE8,OE37 and OE40 were used in further experimental studies.3.Stress tolerance analysis of MsABF2 transgenic tobaccoThe perforated phenotype and whole plant phenotype experiment were carried out on the transgenic tobacco with MsABF2 gene under 300 mmol/L Na Cl and 30 mmol/L Na HCO₃.The results showed a stronger salt and alkali resistance ability of MsABF2transgenic tobacco as its degree of yellowing and leaf wilting were both lower than in wild type.The germination experiment under ABA stress and vertical plate root length experiment were carried out on generation tobacco seeds with MsABF2 gene.Results showed that the germination rate,root length and fresh weight of MsABF2 transgenic tobacco were all lower than those of the wild type.This indicates that overexpression of MsABF2 gene increased the sensitivity of transgenic tobacco to ABA.Under salt and saline-alkali stress,the contents of malondialdehyde,superoxide anion and hydrogen peroxide in MsABF2 transgenic tobacco were lower than those of wild-type tobacco.However,the contents of proline,soluble sugar,soluble protein and antioxidant enzyme activity are all higher than in the wild type.Besides,the DAB and NBT staining experiments for tobacco treated under salt and saline-alkali stress showed that the coloring degree of MsABF2 transgenic tobacco was much lower than that of wild-type,indicating less reactive oxygen species accumulation compared with WT.These results showed that the MsABF2 gene can enhance the resistance of plants to salt and saline-alkali stress.No matter under normal or saline-alkali stress,the ABA contents in MsABF2 are all significantly higher than that in wild type,demonstrating that the MsABF2 gene can promote the biosynthesis of ABA in plants,therefore play an important role in the ABA signaling pathway thus improving the salt and alkali resistance of plants.Compared with wild type,ABA synthesis-related gene ABA1 and NCED2 were up-regulated,while ABI1 and ABI2 were down-regulated under saline-alkali stress,revealing that MsABF2 is a positive regulator of the ABA signalling pathway,but it could also inhibit the negative regulation mechanism of the ABA pathway,hence to enhance the salt-alkali tolerance of plants.Meanwhile,the expression of SOS1,SOS2gene were induced under other stresses,indicating MsABF2 gene can also response to salt-alkali stress through other ways.