Molecular Cloning and Characterization of a Membrane Associated NAC Family Gene, SiNAC from Foxtail Millet [Setaria italica (L.) P. Beauv.]
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The plant-specific NAC (NAM, ATAF, and CUC) transcription factors have diverse role in development and stress regulation. A transcript encoding NAC protein, termed SiNAC was identified from a salt stress subtractive cDNA library of S. italica seedling (Puranik et al., J Plant Physiol 168:280–287, 2011). This single/low copy gene containing four exons and four introns within the genomic-sequence encoded a protein of 462 amino acids. Structural analysis revealed that highly divergent C terminus contains a transmembrane domain. The NAC domain consisted of a twisted antiparallel beta-sheet packing against N terminal alpha helix on one side and a shorter helix on the other side. The domain was predicted to homodimerize and control DNA-binding specificity. The physicochemical features of the SiNAC homodimer interface justified the dimeric form of the predicted model. A 1539 bp fragment upstream to the start codon of SiNAC gene was cloned and in silico analysis revealed several putative cis-acting regulatory elements within the promoter sequence. Transactivation analysis indicated that SiNAC activated expression of reporter gene and the activation domain lied at the C terminal. The SiNAC:GFP was detected in the nucleus and cytoplasm while SiNAC ΔC1−158:GFP was nuclear localized in onion epidermal cells. SiNAC transcripts mostly accumulated in young spikes and were strongly induced by dehydration, salinity, ethephon, and methyl jasmonate. These results suggest that SiNAC encodes a membrane associated NAC-domain protein that may function as a transcriptional activator in response to stress and developmental regulation in plants.
KeywordsFoxtail millet Development Membrane associated transcription factor NAC domain Promoter Stress
NAM ATAF1/2 CUC2
Quantitative real-time polymerase chain reaction
Basic local alignment search tool
Open reading frame
Green fluorescent protein
Cauliflower mosaic virus
Membrane transcription factors
Grateful thanks are due to the director, National Institute of Plant Genome Research, and Head, Department of Biotechnology, Jamia Hamdard, New Delhi, India for providing facilities. The authors also gratefully acknowledge the financial support from the Department of Biotechnology, Government of India (BT/PR9851/AGR/02/521/2007). Ms Swati Puranik acknowledges the award of senior research fellowship from the Council of Scientific and Industrial Research, New Delhi. Helpful discussions with Dr. Debasis Chattopadhya, Dr. Manoj Majee, and Mr. P.P. Sahu, NIPGR, are gratefully acknowledged. RPB acknowledges the start up grant from ISIRD of IIT Kharagpur. The authors would like to thank the reviewers for their constructive comments.
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