ISOLATION AND SEQUENCE ANALYSIS OF THE Na+/H+ ANTIPORTER cDNA FROM Atriplex halimus AND Suaeda pruinosa

Authors

  • M. I. NASR Genetic Engineering and Biotechnology Research Institute (GEBRI), Sadat City University (for-merly Menoufia University)
  • M. Z. S. AHMED Plant Genetic Resources Department, Desert Research Center (DRC) 1, Mathaf El-Matarya Street, B.O.P 11753 El-Matarya. Cairo, Egypt
  • N. A.K. RASHED Plant Genetic Resources Department, Desert Research Center (DRC) 1, Mathaf El-Matarya Street, B.O.P 11753 El-Matarya. Cairo, Egypt
  • A. A. HEMEIDA Genetic Engineering and Biotechnology Research Institute (GEBRI), Sadat City University (for-merly Menoufia University)
  • A. AGORIO Plant Genetic Resources Department, Desert Research Center (DRC) 1, Mathaf El-Matarya Street, B.O.P 11753 El-Matarya. Cairo, Egypt
  • S. FILLEUR Institut des Sciences du Vegetal (ISV), Centre National de la Recherché Scientifique (CNRS), Par-is, France
  • N. A. ABDALLAH Department of Genetics, Faculty of Agriculture, Cairo University, Giza, Egypt

Abstract

Two Egyptian native (non-domesticated) plant species, Atriplex halimus L., and Suaeda pruinosa from family Amaranthacae were collected from North West Coast of Marsa Matrouh governorate, Egypt under abiotic stress. For cDNA synthesizing and analysis, it is essential to isolate RNA in good quality and quantity to be used for RT-PCR amplification. Two protocols; TRIzol® Reagent with RNeasy Plant Mini Kit for RNA cleanup and RNeasy Plant Mini Kits plus polyethylene glycol (PEG-6000) were evaluated for their efficiency in RNA isolation. Both methods proved to be suitable for isolating RNA from plants rich in secondary metabolites and bioactive compounds as they produced pure and with reasonable amount of RNA, without contamination with DNA. The ultimate goal of this study was to identify the vacuolar antiporter NHX1 candidate genes from Egyptian Atriplex halimus and Suaeda pruinosa. Multiple sequence alignments were conducted to NHX1 genes from plants related to Atriplex and Suaeda to identify the conserved regions and to design primers for cloning those genes. According to NHX1 family conserved region, several primers were synthesis either to amplify the full genes or core regions of the genes. Phusion Taq DNA Polymerase for RT-PCR amplification for cDNAs took place. Only touchdown PCR program (TD-PCR) succeeded in amplifying the cDNAs of the Na+/H+ antiporter genes when using with Phusion Taq. This technique increased specificity, sensitivity and yield to amplify cDNA. The obtained cDNA vacuolar Na+/H+ antiporter gene AhNHX1 from both Atriplex halimus and Suaeda pruinosa were sequenced and submitted to GenBank under accession numbers KJ452334.1 and KJ452335.1, respectively. Alignment of the amino acid sequences revealed highly conserved regions representing vacuolar Na+/H+ antiporter. Computer analysis for the prediction of amino acid sequence of AhNHX1 and SpNHX1 recognized different domains including amiloride-binding domain (85LFFIYLPPI94), NHE (Na+/H+ exchange) domain, and 10 and 9 trans-membrane segments (TM). In addition, phylogenetic relationship analysis indicated that AhNHX1 and SpNHX1 were clustered into the vacuolar Na+/H+ antiporter group. The obtained results indicated that AhNHX1 and SpNHX1 genes are new members of the vacuolar Na+/H+ antiporter families.

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Published

2016-01-12

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Vol. 44 No. 1 (2015)

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