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Int. J. Mol. Sci. 2014, 15(8), 14766-14785; doi:10.3390/ijms150814766

Molecular Cloning and Functional Characterization of the Lycopene ε-Cyclase Gene via Virus-Induced Gene Silencing and Its Expression Pattern in Nicotiana tabacum

1
Department of Chemistry, Zhengzhou University, Zhengzhou 450001, China
2
National Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute, Zhengzhou 450001, China
3
Molecular Breeding Group, Yunnan Academy of Tobacco Agricultural Sciences, Kunming 650031, China
4
College of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450001, China
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Received: 27 June 2014 / Revised: 11 August 2014 / Accepted: 12 August 2014 / Published: 22 August 2014
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Abstract

Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses. View Full-Text
Keywords: lycopene ε-cyclase; functional characterization; virus-induced gene silencing; Nicotiana tabacum; carotenoid biosynthesis lycopene ε-cyclase; functional characterization; virus-induced gene silencing; Nicotiana tabacum; carotenoid biosynthesis
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Shi, Y.; Wang, R.; Luo, Z.; Jin, L.; Liu, P.; Chen, Q.; Li, Z.; Li, F.; Wei, C.; Wu, M.; Wei, P.; Xie, H.; Qu, L.; Lin, F.; Yang, J. Molecular Cloning and Functional Characterization of the Lycopene ε-Cyclase Gene via Virus-Induced Gene Silencing and Its Expression Pattern in Nicotiana tabacum. Int. J. Mol. Sci. 2014, 15, 14766-14785.

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