Identification and functional characterization of transcription factors regulating the expression of the wounding-inducible sporamin gene in sweet potato (Ipomoea batatas c.v. Tainong 57)
Date Issued
2009
Date
2009
Author(s)
Lin, I
Abstract
Sporamin is a major storage protein accounting for more than 60% of the soluble proteins found in the tuberous root of sweet potato (Ipomoea batatas Lam.). The predicted protein sequence of Sporamin shares significant amino acid sequence identity with some Kunitz-type trypsin inhibitors and recombinant protein of Sporamin was shown to have strong inhibitory activity to trypsin in vitro. Northern blot analysis showed that sporamin transcripts could be systemically induced in leaf tissue of sweet potato under wounding stress. Our previous promoter assay showed that cis-element (GTAAATACGT) located at -1.1 kb~-1.02 kb of the promoter region displayed the wounding regulator. The cis-element of 10 bp--GTAAATACGT and its flanking sequences were designated as sporamin wounding responsive element, SWRE, encompassing 26 bp in length. The specific aim of this study is to further investigate the wounding mechanism of sporamin gene in sweet potato. In this work, two cDNA clones encoding SWRE-binding proteins were isolated by yeast one-hybrid screening: IbNAC1 and IbWRKY1. We demonstrated that the two SWRE-binding proteins belong to NAC and WRKY transcription family, respectively and their subcellular localizations were both in nucleus. Their transcripts were accumulated earlier than sporamin transcript once response to wounding. We analyzed the expression patterns of sporamin, IbNAC1 and IbWRKY1 genes at different tissues in sweet potato and treatment with MeJA and SA. Through EMSA, we confirmed that the two transcription factors can bind to SWRE in vitro. The binding sites of IbNAC1 and IbWRKY1 are demonstrated as A(N)3TT(N)13ACAATAT and (A)CATTT respectively. The GUS staining and GUS activity results showed that IbNAC1 had very strong transactivation ability while IbWRKY1 had less. Upon to our results, the two novel transcription factors found in sweet potato may play antagonistic function in wounding induced regulation.
Subjects
sweet potato
wounding
yeast one-hybrid
NAC family
WRKY family
transcription factors
EMSA
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