ABSTRACT
A determination method of nosiheptide in formula feeds by HPLC-FL was developed and validated, including an inter-laboratory study. Formula feeds were extracted with acetone after adding acetic acid. Liquid chromatographic separation was performed using a ZORBAX Eclipse XDB-C18 column, with acetonitrile and water containing acetic acid as the mobile phase. Detection of NH was carried out with a fluorescence detector. Recovery tests and an inter-laboratory study were conducted using chicken and swine formula feeds fortified with nosiheptide at 0.5-27 mg-potency/kg. Mean recoveries in recovery tests ranged from 91.4 to 103%, and the repeatability in terms of relative standard deviation was within 7.8%. Mean recoveries of the inter-laboratory study ranged from 98.4 to 108%, the repeatability and reproducibility in terms of the relative standard deviations were within 8.1% and 13% respectively, and the HorRat values ranged from 0.21 to 0.75.
Subject(s)
Animal Feed/analysis , Chromatography, High Pressure Liquid/methods , Food Analysis/methods , Laboratories , Observer Variation , Reproducibility of Results , Thiazoles/analysis , Thiazoles/isolation & purificationABSTRACT
The wheat (Triticum aestivum L.) CBF gene family is assumed to play important roles in development of low-temperature and freezing tolerance through activation of the downstream Cor/Lea genes. However, no direct evidence shows association of the wheat CBF genes with stress tolerance or any interaction between wheat CBF transcription factors and Cor/Lea gene activation. Here, we introduced Wcbf2, one of the wheat CBF genes, into the tobacco (Nicotiana tabacum L.) genome. Expression of Wcbf2 significantly increased the level of freezing tolerance in the transgenic tobacco plants without phenotypic retardation, and altered the expression patterns of tobacco genes, including cold-responsive genes. A transgenic tobacco plant expressing Wcbf2 was crossed to other transgenic plants expressing a GUS reporter gene under control of the wheat Cor/Lea gene promoter. Analysis of the F(1) plants showed that the WCBF2 protein positively regulated at least the expression of Wdhn13 and Wrab17. These results strongly indicate that WCBF2 functions as a transcription factor in the development of freezing tolerance in common wheat.
Subject(s)
Freezing , Nicotiana/genetics , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Triticum/genetics , Adaptation, Physiological/genetics , Adaptation, Physiological/physiology , Gene Expression Regulation, Plant , Plant Proteins/physiology , Plants, Genetically Modified/physiology , Nicotiana/physiology , Transcriptional ActivationABSTRACT
Cold acclimation, an adaptive process for developing freezing tolerance in over-wintering plants, is associated with increased expression levels of a series of cold-responsive (Cor)/late embryogenesis abundant (Lea) genes. To investigate the function of Wcor15, a member of the wheat Cor/Lea gene family, for improvement of freezing tolerance, two types of transgenic tobacco lines expressing Wcor15-containing chimeric genes were produced and characterized. Immunoblot and gene expression analyses of a transgenic tobacco line expressing the Wcor15-GFP fusion gene under control of the CaMV35S promoter showed transport and abundant accumulation of the WCOR15 protein in the stromal compartment of the chloroplasts. The 5' upstream region of Wcor15 induced expression of the GFP reporter gene under low-temperature conditions in the transgenic tobacco. Both transgenic lines expressing the Wcor15-GFP fusion gene showed a similar and significantly improved level of freezing tolerance compared with the wild-type tobacco plants. Our results demonstrate that the induced expression of the wheat Wcor15 gene positively contributes to the development of freezing tolerance in the heterologous tobacco plants.
