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ABSTRACT: The Arabidopsis thaliana At1g68290 gene encoding an endonuclease was isolated and designated ENDO2, which was cloned into a binary vector to overexpress ENDO2 with a C-terminal 6 × His-tag in A. thaliana. Our Arabidopsis transgenic lines harboring 35SP::ENDO2 produced stable active enzyme with high yield. The protein was affinity purified from transgenic plants, and its identity was confirmed by liquid chromatography-mass spectrometry and automatic Edman degradation. ENDO2 enzyme digests RNA, ssDNA, and dsDNA, with a substrate preference for ssDNA and RNA. The activity toward ssDNA (361.7 U/mg) is greater than its dsDNase activity (14.1 U/mg) at neutral pH. ENDO2 effectively cleaves mismatch regions in heteroduplex DNA containing single base pair mismatches or insertion/deletion bases and can be applied to high-throughput detection of single base mutation. Our data also validated that the removal of sugar groups from ENDO2 strongly affects its enzymatic stability and activity.
Journal of Agricultural and Food Chemistry 04/2012; 60(20):5169-79. · 2.82 Impact Factor
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ABSTRACT: Three novel ethylene response factor (ERF) genes, BkERF1, BkERF2.1 and BkERF2.2, were isolated from a medicinal plant, Bupleurum kaoi. The deduced BkERFs contain a canonical nuclear localization signal and an ERF/AP2 DNA binding domain. RNA gel blot analysis revealed that BkERF1 and BkERF2.1 were ubiquitously expressed at low levels in all parts of mature plants, and that BkERF2.2 was expressed at moderate levels in vegetative tissues. Exogenous application of methyl jasmonate induced BkERF1/2.1/2.2 transcripts. BkERF2.2 transcript levels were slightly increased by addition of ethephon and salicylic acid. BkERFs were localized in the plant nucleus and functioned as transcriptional activators. In B. kaoi cells overexpressing BKERFs, inoculation with Botrytis cinerea increased expression of some defense genes which are associated with enhanced disease resistance. Similarly, overexpression of BkERFs in transgenic Arabidopsis thaliana resulted in elevated mRNA levels of the defense gene PDF1.2, and in enhanced resistance to B. cinerea. Collectively, these results provide evidence that BkERFs mediate the expression of defense-related genes in plants.
Journal of plant physiology 03/2011; 168(4):375-81. · 2.50 Impact Factor
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ABSTRACT: The conversion of flavonoid aglycones to their glycosides by plant glycosyltransferases may affect a wide range of outcomes, including stability, solubility and bioavailability. Scutellaria barbata, rich in flavonoid glycosides, is widely used as a traditional Chinese herbal medicine. In this study, a flavonoid glycosyltransferase cDNA (SbUGT) and its promoter from S. barbata were cloned and characterized as a flavonoid glycosyltransferase using whole-cell biotransformation. Fragments of different lengths of the 5'-flanking region of the SbUGT gene were fused to the beta-glucuronidase (GUS) gene and analyzed with transgenic Arabidopsis plants using histochemical and fluorometric assays. GUS activity in transgenic plants carrying the SbP-850U construct (-850 to +86 relative to the transcription start site) displayed the highest level and was enhanced by salt and methyl jasmonate, similar to the expression patterns of the endogenous SbUGT. GUS activity disappeared when the promoter was deleted to -98, and deletion analyses indicated the existence of positive and negative regulatory element(s). Unexpectedly, plants carrying the construct SbP-102U (-102 to +86) exhibited strong GUS activity exclusively in the roots. Our experiments revealed that the specific expression is mediated by different promoter regions and the unique region driving root-preferred expression can be used as a root-specific promoter.
Planta 09/2010; 232(4):963-74. · 3.00 Impact Factor
