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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2011, Vol. 5 Issue (3) : 274-283     DOI: 10.1007/s11703-011-1101-7
RESEARCH ARTICLE |
Molecular characterization and expression analysis of phosphate transporter gene TaPT2-1 in wheat (Triticum aestivum L.)
Xirong CUI1, Yongsheng ZHANG1, Fanghua ZHAO1, Chengjin GUO1, Juntao GU2, Wenjing LU2, Xiaojuan LI2, Kai XIAO1()
1. College of Agronomy, Agricultural University of Hebei, Baoding 071001, China; 2. College of Life Sciences, Agricultural University of Hebei, Baoding 071001, China
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Abstract  

A transcript-derived fragment (TDF) showing up-regulated expression under low Pi stress and being identical to an uncharacterized phosphate transporter gene TaPT2-1 was cloned in wheat. TaPT2-1 was 2075 bp in length and encoded a 568-aa polypeptide. Transmembrane prediction analysis suggested that TaPT2-1 had 13 conserved transmembrane domains. TaPT2-1 shared much higher similarities to other four homologs from Arabidopsis thaliana, Solanum tuberosum, Capsicum frutescens, and Solanum melongena. The expression of TaPT2-1 was root specific and low Pi inducible, suggesting that it plays roles in roots and is involved in the Pi acquisition under Pi-starved condition. The promoter region of TaPT2-1 was cloned based on genome walk analysis. Several types of cis-regulatory elements, such as low Pi responding and tissue specific, were identified in TaPT2-1 promoter. The transgenic tobacco plants with the integrated TaPT2-1 promoter GUS were generated, and GUS histochemical staining analysis in the roots and leaves of the transgenic plants was performed. The results of GUS staining in roots and leaves under various Pi supply conditions were in accordance with the TaPT2-1 transcripts detected based on RT-PCR analysis. Taken together, the distinct expression of low Pi-induced and root-specific TaPT2-1 suggested that it could be used as the potential gene resource on generation of elite crop germplasms with high Pi use efficiency in the future.

Keywords wheat (Triticum aestivum L.), phosphate transporter, expression      cis-regulatory element     
Corresponding Authors: XIAO Kai,Email:xiaokai@hebau.edu.cn   
Issue Date: 05 September 2011
URL:  
http://academic.hep.com.cn/fag/EN/10.1007/s11703-011-1101-7     OR     http://academic.hep.com.cn/fag/EN/Y2011/V5/I3/274
Fig.1  Identification of a TDF responding to the starved Pi stress. A: A TDF band in a polyacrylamide gel by an arrow responds to starved Pi condition of 24 and 48 h. B: The sequence of the TDF. The restriction digestion site of I and I, two restriction endonucleases used for digestion of the cDNAs of roots of CK, 24 h, and 48 h of starved Pi treatment, are shaded.
Fig.2  The cDNA sequence and the corresponding translated polypeptide of .
Fig.3  The diagram of transmembrane domains in TaPT2-1.
Fig.4  Phylogenetic analysis of and its homologs in plant species.
Fig.5  Expression patterns of in roots and leaves under various Pi supply conditions.
Fig.6  Cloning of promoter region based on genome walking. SP1, SP2, and SP3 are the three rounds of specific PCR products of promoter.
Fig.7  The promoter sequence. The translation start codon (ATG) of was boxed. The -regulatory elements PIBS and PHO-like putatively involved in regulation of gene expression to be low Pi responding are shaded and underlined, respectively. The primers used for the promoter integration into binary vector pCAMBIA3301 are underlined and labeled concomitantly with forward arrow and reverse arrow, respectively. The three specific primers, SP1, SP2, and SP3, used for cloning of promoter in genome walking, are shown by reverse arrow.
Typecis-regulatory elementMotifOccurrence frequencyPositionPutative function
Transcription and regulationTATABOX5TTATTT2-1051; -1435Interaction of RNA polymerase
CAATBOX1CAAT8-401; -686; -1035; -1127; -1223; -1327; -1347; -1378Regulation of transcription efficiency
Low Pi respondingPIBSnnATATnC3-192; -1219; -1385Responding to low Pi
PHO-likeG(G/T/A)(C/T/A)GTGG1-1121Responding to low Pi
Tissue specificROOTMOTIFTAPOX1ATATT1-1285Root predominantly expression
GATABOXGATA3-195; -1013; -1114Tissue-specific expression, light regulated
EBOXBNNAPACANNTG4-758; -790; -834; -929Tissue-specific activation
Defense responseACGTATERD1ACGT2-320; -1180Early response to dehydration
MYCCONSENSUSATCANNTG4-758; -790; -834; -929Cold responsive
LTRECOREATCOR15CCGAC1-1531Low temperature-responsive element
WBOXNTERF3TGACY1-846Rapid and transit activation of transcription by wounding
WRKY710STGAC6-731; -847; -873; -913; -1006; -1137Defense signaling regulated via WRKY transcription factors
Auxin and salicylic acid respondingASF1MOTIFCAMVTGACG3-730; -872; -1005Transcription activation by auxin and/or salicylic acid
Signaling of light, sugar, and carbon metabolismSORLIP1ATGCCAC5-4; -87; -136; -1330; -1373Light regulated
GT1CONSENSUSGRWAAW4-559; -876; -1018; -1397Light regulated
ASF1MOTIFCAMVTGACG4-730; -872; -912; -1005Light regulated
WBOXHVISO1TGACT1-846Sugar signaling and sugar responsive
DOFCOREZMAAAG7-127; -357; -430; -604; -773; -1016; -1213Carbon metabolism
Tab.1  The putative important -regulatory elements identified in promoter
Fig.8  Molecular identification of the transgenic tobacco lines that were integrated promoter. A: The genome DNA extracted from CK and the transgenic tobacco lines. B: PCR results in which the genome DNA extracted from the CK and the transgenic tobacco lines was used as the template.
Fig.9  GUS staining analysis of the roots and leaves of control (transformed the empty binary vector) and representative transgenic plants. A: The histochemical staining of roots and leaves in which the empty binary plasmid integrated. B and C: The GUS activities of roots and leaves in which the reporter gene was driven by promoter under normal Pi supply condition (2 mM) and starved Pi condition (20 μM), respectively.
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