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Molecular Biotechnology

, Volume 49, Issue 2, pp 138–150 | Cite as

Molecular Cloning and Characterization of a Membrane Associated NAC Family Gene, SiNAC from Foxtail Millet [Setaria italica (L.) P. Beauv.]

  • Swati Puranik
  • Ranjit Prasad Bahadur
  • Prem S. Srivastava
  • Manoj PrasadEmail author
Research

Abstract

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.

Keywords

Foxtail millet Development Membrane associated transcription factor NAC domain Promoter Stress 

Abbreviations

NAC

NAM ATAF1/2 CUC2

qRT-PCR

Quantitative real-time polymerase chain reaction

BLAST

Basic local alignment search tool

ORF

Open reading frame

GFP

Green fluorescent protein

X-GAL

β-d-Galactopyranoside

CaMV

Cauliflower mosaic virus

TM

Transmembrane

MTFs

Membrane transcription factors

Notes

Acknowledgments

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.

Supplementary material

12033_2011_9385_MOESM1_ESM.doc (112 kb)
Supplemental Table S1. Accession numbers for 82 NACs used for phylogenetic analysis.Supplemental Table S2. Summary of putative cis-acting regulatory elements identified in SiNAC promoter region.(DOC 112 kb)
12033_2011_9385_MOESM2_ESM.doc (52 kb)
Supplemental Figure S1. Nucleotide sequence of the 5′ upstream region of SiNAC. The start codon ATG is defined as +1. Numbers on the left indicate nucleotide position relative to the ATG translational start codon. The transcription start site and TATA-box are shown in bold and italics, respectively. Nucleotide sequences representing putative cis-regulatory elements are underlined.Supplemental Figure S2. Phylogenetic tree of NAC proteins from various organisms. The numbers beside the branches represent bootstrap values based on 100 replications. The scale bar indicates the relative amount of change along branches. SiNAC is marked in box. The NAC subgroup names are shown at the right of brackets.(DOC 52 kb)

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Swati Puranik
    • 1
    • 2
  • Ranjit Prasad Bahadur
    • 3
  • Prem S. Srivastava
    • 2
  • Manoj Prasad
    • 1
    Email author
  1. 1.National Institute of Plant Genome Research (NIPGR)New DelhiIndia
  2. 2.Department of BiotechnologyFaculty of ScienceNew DelhiIndia
  3. 3.Department of BiotechnologyIndian Institute of Technology KharagpurKharagpurIndia

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