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JXB Advance Access published online on February 9, 2009
Journal of Experimental Botany, doi:10.1093/jxb/erp006
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© The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RESEARCH PAPER

Pathway engineering of Brassica napus seeds using multiple key enzyme genes involved in ketocarotenoid formation

Masaki Fujisawa1, Eiji Takita2, Hisashi Harada1, Nozomu Sakurai2, Hideyuki Suzuki2, Kanji Ohyama3, Daisuke Shibata2 and Norihiko Misawa1,*

1Central Laboratories for Frontier Technology, Kirin Holdings Co. Ltd, i-BIRD, 3-570, Suematsu, Nonoichi-machi, Ishikawa 921-8836, Japan
2Kazusa DNA Research Institute, 2-6-7, Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan
3Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308, Suematsu, Nonoichi-machi, Ishikawa 921-8836, Japan

* To whom correspondence should be addressed. E-mail: n-misawa{at}ishikawa-pu.ac.jp

Brassica napus (canola) plants were genetically manipulated to increase the amount and composition of carotenoids in seeds by using seven key enzyme genes involved in ketocarotenoid formation, which originated from a soil bacterium Pantoea ananatis (formerly called Erwinia uredovora 20D3), and marine bacteria Brevundimonas sp. strain SD212 and Paracoccus sp. strain N81106 (formerly called Agrobacterium aurantiacum). The seven key gene cassettes, in which each gene was surrounded by an appropriate promoter and terminator, were connected in a tandem manner, and the resulting constructs (17 kb) were inserted into a binary vector and used for transformation of B. napus. Surprisingly, 73–85% of the regenerated plants retained all seven genes, and formed orange- or pinkish orange-coloured seeds (embryos), while untransformed controls had light yellow-coloured seeds with predominant accumulation of lutein. Three of the transgenic lines were analysed further. The total amount of carotenoids in these seeds was 412–657 µg g–1 fresh weight, which was a 19- to 30-fold increase compared with that of untransformed controls. The total amount of ketocarotenoids was 60–190 µg g–1 fresh weight. β-Carotene was the predominant carotenoid, with significant amounts of {alpha}-carotene, echinenone, phytoene, lutein, and canthaxanthin also detected in the transgenic seeds. The ratio of hydroxylated carotenoids to overall carotenoids was quite small relative to the ratio of ketocarotenoids to overall carotenoids. Interestingly, expression of many endogenous carotenogenic genes was also altered in the transgenic seeds, suggesting that their expression was affected by an increase in carotenoid biosynthesis.

Key words: Agrobacterium-mediated plant transformation, Brassica napus, canola, carotenoid, ketocarotenoid, pathway engineering, seed-specific promoter

DDBJ/EMBL/GenBank accession numbers for the nucleotide sequences of idi, CRTISO, LCYe, PDS, PSY, and ZEP in this study are AB453829 [GenBank] , AB454514 [GenBank] , AB454515 [GenBank] , AB454516 [GenBank] , AB454517 [GenBank] , and AB454518 [GenBank] , respectively.

Received 13 November 2008; Revised 29 December 2008 Accepted 7 January 2009


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