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GrainGenes Reference Report: PNA-96-14641

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Reference	PNA-96-14641
Title	Overexpression of thioredoxin h leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain
Journal	Proceedings of the National Academy of Sciences, USA
Year	1999
Volume	96
Pages	14641-14646
Author	Cho MJ [ Show all 6 ]
Abstract	Summary: Biochemically active wheat thioredoxin h has been overexpressed in the endosperm of transgenic barley grain. Two DNA constructs containing the wheat thioredoxin h gene (wtrxh) were used for transformation; each contained wtrxh fused to an endosperm-specific B(1)-hordein promoter either with or without a signal peptide sequence for targeting to the protein body. Twenty-two stable, independently transformed regenerable lines were obtained by selecting with the herbicide bialaphos to test for the presence of the bar herbicide resistance gene on a cotransformed plasmid; all were positive for this gene. The presence of wtrxh was confirmed in 20 lines by PCR analysis, and the identity and level of expression of wheat thioredoxin h was assessed by immunoblots. Although levels varied among the different transgenic events, wheat thioredoxin h was consistently highly expressed (up to 30-fold) in the transgenic grain. Transgenic lines transformed with the B(1)-hordein promoter with a signal peptide sequence produced a higher level of wheat thioredoxin h on average than those without a signal sequence. The overexpression of thioredoxin h in the endosperm of germinated grain effected up to a 4-fold increase in the activity of the starch debranching enzyme, pullulanase (limit dextrinase), the enzyme that specifically cleaves alpha-1,6 linkages in starch. These results raise the question of how thioredoxin h enhances the activity of pullulanase because it was found that the inhibitor had become inactive before the enzyme showed appreciable activity
Keyword	[ Hide all but 1 of 46 ] bar barley dna endosperm enhanced activity enzyme enzyme activity enzymology gene gene expression gene transfer genetic transformation glycoside hydrolases herbicide herbicide resistance hordein hordeum vulgare inhibitor limit dextrinase linkage metabolism overexpression pcr peptide plant proteins promoter protein body pullulanase recombinant dna resistance resistance gene seed germination seedling growth sequence signal signal peptide starch starch debranching enzyme sulfur proteins thioredoxin thioredoxin-h transformation transgenic transgenic barley transgenic plant triticum aestivum

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