The Cloning And Molecular Characterization Of Tanac Genes In Wheat

Plant-specific NAC (NAM/ATAF/CUC) comprise one of the largest plant transcription factor (TF) families. The name of NAC domain originally derived from the NAM gene from petunia and the ATAF1, ATAF2 and CUC2 genes from Arabidopsis. NAC proteins have been found to play important roles in plant development and regulators, responses to environmental stimuli and pathogens, viral infections, crop yield and seed quality. Members of this family share a highly conserved N-terminal NAC binding domain and a variable C-terminal transcriptional regulation domain.Wheat (Triticum aestivum L.) is a major important crop, which provides calories and nutrients in the diets for worldwide population. While external environmental stimuli and genetic factors seriously impact yield and seed quality. Compared to Arabidopsis and rice, investigation related to NAC TFs from wheat (T.aestivum) is rare, though extensive work has been carried out on senescence and seed quality. This paper systematically investigated molecular characterization of 15 NAC genes from wheat'Jing dong 17', our work provide potential utility in wheat genetic improvement about stress-related, development-related and multifunctional TaNAC genes.15 genes encoding TaNAC TFs were indentified by homology cloning, total RNA were extracted from treated materials (stress treatments: drought, salt, cold, ABA; and organ-specific materials: leaves, stems, and roots at four-leaf stage, senescence leaves and immature seeds at grain filling stage). In this paper, we studied molecular characterization of TaNAC genes from the following aspects:1. A phylogenetic tree was constructed combined with known functions of NAC genes from other species,we classified wheat NAC genes into three groups and predicted their functions: stress-related NAC, development-related NAM/CUC and NTL (membrane-associated transcription factor belonging to NAC), among all of the cloned genes, the TaNTLs were the first three NTL genes in wheat.2. Semi-quantitative RT-PCR analysis showed most of TaNAC were induced by abiotic stresses including drought, salinity, low temperature, and abscisic acid (ABA), and different genes showed different expression levels. TaNAC3, TaNAC4, TaNAC6, TaNAC12 were strongly induced by one kind of stress or more; TaNAC5, TaNAC7, TaNAC8 were induced by various stress treatments, but with rather low mRNA transcript levels; while TaNAC9, TaNAC10, TaNAC11, TaNTL1, TaNTL2 were significantly downregulated by drought or salinity. Thus, TaNAC genes may function in stress tolerance. All of TaNAC exhibit organ-specific expression except for TaNAC5, TaNAC7 and TaNAC10, we conclude that these genes may function in plant development and morphogenesis.3. Transient expression in onion epidermal cells indicates that TaNAC-GFP fusion proteins including TaNAC1, TaNAC2, TaNAC3, TaNAC4, TaNAC5, TaNAC6, TaNAC7, TaNAC8, and TaNAC12 were specially localized in the nucleus, and they function as transcription factors. For TaNTL1, the GFP signals of TaNTL1(full):GFP were detected in association with the plasma membranes as well as in the nucleus, predominantly association with the membranes. In contrast, TaNTL1(?TM):GFP containing residues 1-320aa (amino acid sequence) and lacking the TM motif were detected exclusively in the nucleus, so TaNTL1 is membrane-associated NAC, our results suggest that TaNTL2 and TaNTL3 also function as membrane-associated transcription factors.4. Transactivation analysis andβ-galactosidase assays showed that TaNAC2, TaNAC3, TaNAC4, TaNAC6, TaNAC8, TaNAC9, TaNTL1, TaNTL2, TaNTL3 function as transcriptional activators, and TaNAC1, TaNAC5, TaNAC7, TaNAC10, TaNAC11, TaNAC12 function as suppressors in yeast strain AH109.