Cloning And Functional Analyses Of NAC Transcription Factor Genes AmNTL1 And AmNAC6 From Ammopiptanthus Mongolicus

NAC(NAM,ATAF,and CUC)transcription factor family is a class of plant-specific transcriptional regulatory proteins that play important roles in the organ formation,hormone regulation,and resistance to biotic and abiotic stresses of plants.Ammopiptanthus mongolicus is the only evergreen broadleaf plant distributed in the central Asian desert.It has a very high tolerance to harsh environments such as severe cold and drought.In this study,two NAC genes,AmNTL1 and AmNAC6,were cloned from this species and their functions in abiotic stress resistance were analyzed.The main results are as follows:(1)The encoding protein of AmNTL1 consists of 533 amino acid residues and contains a conserved NAC domain at its N-terminus.The C-terminal region of AmNTL1 is a transcriptional activation domain that contains a transmembrane motif near its C-terminus.(2)Subcellular localization analysis showed that the AmNTL1 protein may be localized in plasma membrane.In the yeast self-activation system,the AmNTL1 protein showed transcriptional activation activity.(3)In laboratory-cultured A.mongolicus seedlings,the transcription level of AmNTL1 was up-regulated by cold and salt stresses but tended to decrease under heat stress.In the early stage of the drought treatment,the transcription level was higher than that of the control,and then it dropped back to the control level with the treatment.In young leaves of the A.mongolicus shrubs naturally growing in the wild,the transcription level of AmNTL1 was higher in January to April,lower in May and June,and middle in other months of the year of sampling.In different organs of the shrubs,the transcription levels in lateral roots,young leaves and flower buds were higher than those in twigs and immature pods.(4)Constitutive expression of AmNTL1 in Arcibidopsis significantly enhanced the resistance of transgenic lines to freezing,chilling and high salinity as well as their sensitivity to exogenous ABA.The tolerance of transgenic lines to osmotic stress also increased.In addition,the transgenic lines showed higher stability in cellular membranes and chlorophyll and stronger oxidative stress tolerance compared to the control wild type.(5)The encoding protein of AmNAC6 contains 304 amino acid residues and consists of an N-terminal NAC domain(containing two nuclear localization signals)and a C-terminal transcriptional activation region.Subcellular localization experiment showed that the AmNAC6 protein was located in the nucleus.(6)In laboratory-cultured A.mongolicus seedlings,the transcription level of AmNAC6 was up-regulated by drought stress and changed in an alternating pattern ot up-and down-regulation under cold,heat or salt stress.In young leaves of A.mongolicus shrubs growing in the wild,no obvious changes in the transcription level of AmNAC6 were observed among lost months of the year of sampling.In different organs of the shrubs,the transcription level was relatively low in young leaves.