Cloning And Functional Analyses Of Fatty Acid Desaturase Genes Am FAD2s From Ammopiptanthus Mongolicus

The unsaturation degree of cellular membrane lipids is mainly determined by fatty acid desaturases(FADs),and both the unsaturation degree and FADs are closely related to the tolerance of plants to harsh environments such as low temperature and salt stress.Ammopiptanthus mongolicus is the only evergreen xerophytic broadleaf plant that is endemic to the central Asian desert and has very strong tolerance to abiotic stresses such as cold,drought and high salinity.In this study,two genes for oleic acid desaturation enzymes,namely AmFAD2-1 and AmFAD2-2,were cloned from A.mongolicus.The structural characteristics of the predicted AmFAD2-1 and AmFAD2-2 proteins and the expression changes and functions of the two genes in response and tolerance to abiotic stresses were analyzed.The main results are as follows:(1)The cDNA and gDNA coding region fragments of AmFAD2-1 and AmFAD2-2 were cloned by RT-PCR and PCR.Both the cDNA and gDNA fragments of AmFAD2-1 consist of 1149 bp,and both those of AmFAD2-2 contain 1152 bp.Therefore,the two genes have no introns in their gDNA coding regions.(2)The encoding proteins of AmFAD2-1 andAmFAD2-2 consist of 382 and 383 amino acid residues,respectively.Both the proteins contain conserved histidine clusters HXXXH,HXXHH and HXXHH that are necessary for the activity of the FAD family enzymes.The two proteins also contain the signals(YNNKL)for endoplasmic reticulum localization at their C-terminal regions.(3)RT-qPCR analysis showed that AmFAD2-1 and AmFAD2-2 were differently involved in the response of laboratory-cultured A.mongolicus seedlings to cold,heat,drought and salt stresses.During cold treatment,the transcription level of AmFAD2-1 firstly increased and then decreased,and that of AmFAD2-2 was significantly up-regulated in the late period.The transcription levels of both the genes showed a decreasing trend under heat treatment.During drought and salt treatments,the two genes showed an overall up-regulated trend in their transcription levels.In young leaves of A.mongolicus shrubs growing in the wild,the transcription levels of both AmFAD2-1 and AmFAD2-2 were overall markedly higher in the autumn and winter months compared to those in the spring and summer months.The increased changes in AmFAD2-1 transcription were obviously higher than those in AmFAD2-2 transcription during the autumn and winter.In different organs of the shrubs growing in the wild,AmFAD2-1 was highly expressed in young leaves and immature pods,while AmFAD2-2 was abundantly expressed in lateral roots and twigs.(4)Constitutive expression of AmFAD2-1 or AmFAD2-2 in Arabidopsis significantly enhanced freezing tolerance of the transgenic lines at the seedling stage and salt and drought tolerances of the transgenics at the seed germination stage.In addition,AmFAD2-2 can also improve salt and drought tolerances and water holding capacity of detached leaves of the transgenic lines at the seedling stage.