• Horticultural Science & Technology
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• v.29 no.3
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• pp.267-272
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• 2011

Several reports indicated that astaxanthin and zeaxanthin have more active anticancer activity than pro-vitamin A carotenes. ${\beta}$-carotene hydroxylase is a key enzyme to synthesize zeaxanthin and astaxanthin in carotenoids biosynthesis pathway. We isolated the ChyB gene encoding ${\beta}$-carotene hydroxylase from tomato leaves. The ChyB gene (1.5Kbp) fragment was cloned into the binary vector and designated to pIG121-ChyB-tom. Agrobacterium-mediated infiltration was used for transient assay in Nicotiana benthamiana. Leaf samples were collected 0, 1, 2, 3 days after infiltration (DAI). RT-PCR result showed that the expression of ${\beta}$-carotene hydroxylase transcripts was not detected in control (0DAI), but its expression was detected after 1 DPI and increased later on. When the activity of ${\beta}$-carotene hydroxylase was measured, the 1,1-diphenyl-pricryl hydrazyl (DPPH) radical scavenging activity (27%) at 2 DAI was significantly higher than that (21%) at 0 DAI. These results indicated that anti-oxidant activity dramatically increased at 2 DAI in tobacco leaves was due to over expression of tomato ${\beta}$-carotene hydroxylase. These results can be the foundation to develop tomato cultivars with high oxy-carotenoids content using the ChyB gene transformation.

• Journal of Plant Biotechnology
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• v.31 no.3
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• pp.231-237
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• 2004

Oxycarotenoids are oxygenated carotenoids that perform critical roles in plants. $\beta$-Carotene hydroxylase adds hydroxyl groups to the $\beta$-rings of carotenes and has been cloned from several bacteria and plants including Arabidopsis. This study was carried out to investigate the effect of $\beta$-carotene hydroxylase gene (Chyb) on the oxycarotenoids biosynthesis in the transgenic Arabidopsis. Construct of pGCHYB containing Chyb was established onto Gateway vector system (pENTR3C gateway vector and pH2GW7 destination vector). Arabidopsis thaliana (cv. Columbia) was transformed with Agrobacterium tumerfacience GV3101 harboring pGCHYB construct driven by 35S promoter and hygromycin resistant gene. Seven hundred bases paired PCR products, indicating the presence of Chyb gene, were found in the transformants by PCR analysis using Chyb primers. Hygromycin resistance assay showed that transgenes were stably inherited to next generation. The overexpression of the Chyb gene resulted in the decrease carotenoid content. Especially, astaxanthin unusual oxycarotenoid in wild type Arabidopsis was detected in the transgenic plants. This means that decreased carotenoids might be converted into astaxanthin metabolism with the aid of silent gene in the host.

• Journal of Microbiology
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• v.41 no.3
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• pp.212-218
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• 2003

Citrus phytoene synthase (CitPsy) and ${\beta}$-carotene hydroxylase (CitChx), which are involved in caroteinoid biosynthesis, are distantly related to the corresponding bacterial enzymes from the point of view of amino acid sequence similarity. We investigated these enzyme activities using Pantoea ananatis carotenoid biosynthetic genes and Escherichia coli as a host cell. The genes were cloned into two vector systems controlled by the T7 promoter. SDS-polyacrylamide gel electrophoresis showed that CitPsy and CitChx proteins are normally expressed in E. coli in both soluble and insoluble forms. In vivo complementation using the Pantoea ananatis enzymes and HPLC analysis showed that ${\beta}$-carotene and zeaxanthin were produced in recombinant E. coli, which indicated that the citrus enzymes were functionally expressed in E. coli and assembled into a functional multi-enzyme complex with Pantoea ananatis enzymes. These observed activities well matched the results of other researchers on tomato phytoene synthase and Arabidopsis and pepper ${\beta}$-carotene hydroxylases. Thus, our results suggest that plant carotenoid biosynthetic enzymes can generally complement the bacterial enzymes and could be a means of carotenoid production by molecular breeding and fermentation in bacterial and plant systems.

• Plant Biotechnology Reports
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• v.3 no.4
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• pp.325-331
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• 2009

To efficiently express a gene of interest in transgenic plants, the choice of promoter is a crucial factor as it directly affects the expression of the transgene that will yield the desired phenotype. The Arabidopsis ${\beta}-carotene$ hydroxylase 1 gene (AtBch1) shows constitutive and ubiquitous expression and was thus selected as one of best candidates for constitutive promoter analysis by both in silico northern blotting and semi-quantitative RT-PCR analysis. To investigate AtBch1 promoter activity, the 1,981-bp 5'-upstream region of this gene was fused with ${\beta}-glucuronidase$ (GUS) and transformed into Arabidopsis. Through the molecular characterization of transgenic leaf tissues, the AtBch1 promoter generated strong activity that drives 1.8- and 2-fold higher GUS expression than the cauliflower mosaic virus 35S (35S) promoter at the transcriptional and translational levels, respectively. Furthermore, the GUS enzyme activity driven by the AtBch1 promoter was 2.8-fold higher than that produced by the 35S promoter. By histochemical GUS staining, the ubiquitous expression of the AtBch1 promoter was observed in all tissues of Arabidopsis. Semi-quantitative RT-PCR analysis with different tissues further showed that this promoter serves as a strong constitutive driver of transgene expression in dicot plants.

• Proceedings of the Zoological Society Korea Conference
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• 2003.08a
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• pp.187.4-187
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• 2003

No Abstract, See Full Text

• Microbiology and Biotechnology Letters
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• v.35 no.4
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• pp.292-297
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• 2007

The unicellular green alga, Haematococcus pluvialis used as a biological production system for astaxanthin. It accumulates large amounts of the red ketocarotenoid astaxanthin when exposed to various environmental stress such as active oxygen species and high light intensities. To induce astaxanthin biosynthesis of H. pluvialis, cells were incubated in either nitrate free at $25^{\circ}C$ under continuous high light intensity ($1,000\;{\mu}mol$ photons $m^{-2}s^{-1}$) for 2 days or high light stress only. Expressions of astaxanthin biosynthetic genes such as carotenoid hydroxylase, IPP isomerase and ${\beta}$-carotene ketolase were monitored under different culture conditions by using real time RT-PCR. All the subjected genes increased their expression under highlight and N-deprivation condition where a large amount of astaxanthin was accumulated.

• Plant Biotechnology Reports
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• v.4 no.2
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• pp.149-155
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• 2010

Expression of the genes for carotenoid bio-synthesis (crt) is dependent on light, but little is known about the underlying mechanism of light sensing and signalling in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter, Synechocystis). In the present study, we investigated the light-induced increase in the transcript levels of Synechocystis crt genes, including phytoene synthase (crtB), phytoene desaturase (crtP), ${\zeta}$-carotene desaturase (crtQ), and ${\beta}$-carotene hydroxylase (crtR), during a darkto-light transition period. During the dark-to-light shift, the increase in the crt transcript levels was not affected by mutations in cyanobacterial photoreceptors, such as phytochromes (cph1, cph2 and cph3) and a cryptochrome-type photoreceptor (ccry), or respiratory electron transport components NDH and Cyd/CtaI. However, treatment with photosynthetic electron transport inhibitors significantly diminished the accumulation of crt gene transcripts. Therefore, the light induction of the Synechocystis crt gene expression is most likely mediated by photosynthetic electron transport rather than by cyanobacterial photoreceptors during the dark-to-light transition.