Functional Analysis Of BHLH Transcription Factors Of Medicago Truncatula Regarding The Salt-Tolerance Regulation And Plant Shape And Development

Breeding salt-tolerant alfalfa by genetic engineering will expand its planting area to saline-alkali land,which accounts for a large portion of the China's farmland,and consequently,increase the production of the perennial forage nationwide.The bHLH transcription factor family,one of the largest transcription factor families in plants,has been reported to involve in plant growth and salt stress response.In this study,three bHLH transcription factor encoding genes were cloned from barrel clover(Medicago truncatula L.),and their functions in transgenic plants were analyzed preliminarily.The main results are as follows:Three genes encoded bHLH transcription factor family members were cloned from barrel clover and named MtbHLH147,MtbHLH148 and MtbHLH149,respectively.Bioinformatic analysis showed that all the three MtbHLHs contained highly conserved bHLH domains.As expected,the secondary structures were predominatly ?-helix and random curl,and the proteins were predicted to locate in the nucleus.Phylogenetic analysis revealed that MtbHLH147 was closely related to MtbHLH148,sharing67.4% of sequence identity.Analysis of the cis-regulatory element demonstrated that the promoters of these transcription factors contained hormone and stress response elements,suggesting their involvement in the biological processes.Analysis of the expression pattern of the three MtbHLHs in barrel clover showed that the highest levle of both MtbHLH147 and MtbHLH148 was detected in stem and MtbHLH149 was detected most abundantly in root,while the lowest level of all the three genes was detected in leaf.For treatment,all the three MtbHLHs were induced by ABA(100 ?M),and the former two genes(MtbHLH147 and MtbHLH148)were repressed by cold(4?)but up-regulated by NaCl(200 mM)within the first 8 h.For instance,under salt stress,MtbHLH147 was significantly up-regulated reaching 3.8 times of the untreated control at 4 h,and MtbHLH148 was doubled at 8 h.In contrast,MtbHLH149 was slightly up-regulated by the low temperature but showed no clear response to salt treatment.Each of the three individual MtbHLHs driven by the 35 S promoter was separately transformed into Arabidopsis and barrel clover.For Arabidopsis plants over-expressing MtbHLH147 or MtbHLH148,a significantly higher germination rate was observed in spite of their indiscernable phenotypic difference from wild type.Statistical analysis showed that the root length of the transgenic seedlings over-expressing MtbHLH148 was significantly larger than the non-transgenic plants,suggesting enhanced salt tolerance.Morphologically,MtbHLH149 over-expression Arabidopsis displayed pleiotropic defects including dwarfism and curly leaf.Under NaCl(125 mM)treatment,the germination rate of the transgenic Arabidopsis ectopically expressing MtbHLH149 was significantly lower than that of wild type.Based on these results,we infer that compared with MtbHLH147 and MtbHLH149,MtbHLH148 may play a regulatory role in plant response to salinity.