苦荞细胞色素CYP81家族同源基因FtP450-R4的克隆、分子鉴定及其功能分析

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Abstract Cytochrome P450s are one of the largest enzyme protein families in higher plants and widely involve in secondary metabolism and stress tolerance. In this study, a CYP81 homologous gene FtP450-R4(GenBank Accession No. KM271986) was obtained from Fagopyrum tataricum. The full-length of FtP450-R4 cDNA contained a 5'-UTR (49 bp), a 3'-UTR (194 bp) and an ORF of 1 527 bp. It encoded a protein of 508 amino acid residues. Bioinformatics analysis showed that the FtP450-R4 was located in endoplasmic reticulum by the 4~24 amino acid residues in the N-terminal, and it shared the homology of 44%~46% with F3'H, I2'H, and other plants CYP450 proteins. Sequence multi-alignment showed that FtP450-R4 contained the classic motifs and conserved regions of CYP450s, but excluded the feature motifs of GGEK in F3′H. Phylogenetic tree showed that FtP450-R4, Arabidopsis thaliana CYP81 families and I2′H were gathered in a large cluster, suggesting that FtP450-R4 might involve in flavonoids hydroxylation or stress response. FtP450-R4 could be significantly induced in cotyledons by UV-B, cold and drought stress, but not in hypocotyls. The recombinant protein of FtP450-R4 expressed in a soluble form in Escherichia coli strain BL21(DE3) and activity identification showed that it could perform enzyme catalysis using reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) and kaempferol as substrates. Totally, this work can make better understand for the function of CYP450s and provides fundamental information for the flavonoids regulation mechanism in F. tataricum.

Key words： Fagopyrum tataricum Cytochrome P450 Cloning Molecular characterization Activity identification

Received: 28 August 2014 Published: 13 January 2015

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	LI Cheng-Lei
	DIAO Hai-Xia
	YUN Guo-Qin
	ZHOU Jing
	XUN Jia-Yi
	YAO Pan-Feng
	CHEN Hui
	YU An-Hu
	WU Qi

Cite this article:

LI Cheng-Lei,DIAO Hai-Xia,YUN Guo-Qin, et al. Molecular Cloning, Characterization and Functional Analysis of a Cytochrome CYP81 Family Homologous Gene FtP450-R4 from Fagopyrum tataricum[J]. , 2015, 23(2): 181-192.

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http://journal05.magtech.org.cn/Jwk_ny/EN/ OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2015/V23/I2/181

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