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细胞色素P450(cytochrome P450,CYP450)是高等植物中最大的酶蛋白家族之一,广泛参与植物的次生代谢和抗逆生理。本研究采用同源克隆和c DNA末端快速克隆(rapid-amplification of c DNA ends,RACE)技术,获得苦荞(Fagopyrum tataricum)CYP81家族同源基因Ft P450-R4(Gen Bank登录号:KM271986)。Ft P450-R4 c DNA全长1 770 bp,5’-UTR为49 bp,3’-UTR为194 bp,ORF为1 527 bp,其ORF序列可编码508个氨基酸残基的蛋白。生物信息学分析表明,Ft P450-R4蛋白通过N-端4~24位氨基酸残基定位于内质网,与F3’H(flavonoid 3’-hydroxylase)、I2’H(isoflavone 2’-hydroxylase)和其他CYP450的同源性在44%~46%之间;多重序列比对显示,Ft P450-R4具有CYP450中经典的基序和保守序列,但不含F3’H的特征基序(GGEK);系统进化树分析显示,Ft P450-R4与拟南芥(Arabidopsis thaliana)CYP81家族和I2’H聚为一大簇,说明其可能参与苦荞黄酮类化合物的羟化或对逆境胁迫的应答。UV-B、寒冷和干旱均能引起Ft P450-R4在苦荞子叶中表达量的显著变化,而在胚轴中其表达量变化不显著。利用大肠杆菌(Escherichia coli)BL21(DE3)菌株对Ft P450-R4蛋白进行了可溶性表达,活性鉴定表明,重组Ft P450-R4蛋白能以还原型烟酰胺腺嘌呤二核苷酸磷酸(reduced form of nicotinamide-adenine dinucleotide phosphate,NADPH)和山奈酚为底物进行酶促反应,具有生物学活性。本研究可为了解CYP450在黄酮合成代谢中的功能,明确苦荞黄酮代谢调控的分子机制提供了基础资料。
Cytochrome P450 (CYP450) is one of the largest enzyme protein families in higher plants. It is widely involved in plant secondary metabolism and anti-stress physiology. In this study, homologous cloning and rapid amplification of cDNA ends (RACE) were used to obtain Ft P450-R4 (GenBank accession number KM271986), a homolog of CYP81 family of Fagopyrum tataricum. Ft P450-R4 cDNA has a total length of 1 770 bp, a 49 bp 5’-UTR, a 194 bp 3’-UTR, and an ORF of 1 527 bp. The ORF sequence encodes a protein of 508 amino acid residues. Bioinformatics analysis showed that the Ft P450-R4 protein was located in the endoplasmic reticulum through amino acid residues 4 to 24 of N-terminus, and was highly homologous to flavonoid 3’-hydroxylase, I2’H (isoflavone 2’-hydroxylase) Homology analysis revealed that Ft P450-R4 possesses the classical motif and conserved sequence of CYP450, but does not contain the characteristic motif of F3’H (GGEK) Phylogenetic tree analysis showed that Ft P450-R4 was clustered with the CYP81 family and I2’H in Arabidopsis thaliana, suggesting that Ft P450-R4 may be involved in hydroxylation of Tartary buckwheat flavonoids or in response to stress. UV-B, both cold and drought induced significant changes in the expression level of Ft P450-R4 in tartary buckwheat cotyledons, while its expression did not change significantly in the hypocotyls. The Ft P450-R4 protein was expressed in Escherichia coli BL21 (DE3) strain. The activity of the recombinant Ft P450-R4 protein showed that the recombinant Ft P450-R4 protein expressed in reduced form of reduced nicotinamide adenine dinucleotide phosphate nicotinamide-adenine dinucleotide phosphate (NADPH) and kaempferol as substrates for enzymatic reaction, with biological activity. This study may provide basic information for understanding the function of CYP450 in the flavonoid anabolism and clarifying the molecular mechanism of the metabolism regulation of buckwheat flavonoids.