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  

Molecular Cloning, Characterization and Functional Analysis of a Cytochrome CYP81 Family Homologous Gene Ft P450- R4 from Fagopyrum tataricum

作　　者：李成磊[1] 赵海霞[1] 温国琴 周婧[1] 孙家宜 姚攀峰[1] 陈惠[1] 王安虎[2] 吴琦[1] 

机构地区：[1]四川农业大学生命科学学院,雅安625014 [2]西昌学院,西昌615000

出　　处：《农业生物技术学报》2015年第2期181-192,共12页Journal of Agricultural Biotechnology

基　　金：四川省教育厅青年基金(No.14ZB0008)

摘　　要：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cDNA末端快速克隆（rapid-amplification of cDNAends,RACE）技术,获得苦荞（Fagopyrum tataricum） CYP81家族同源基因FtP450-R4 （GenBank登录号：KM271986）.FtP450-R4 cDNA全长1770bp,5＇-UTR为49 bp,3＇-UTR为194bp,ORF为l 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,FtP450-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,FtP450-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,FtP450-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FtP450-R4在苦养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（Escherchia coli） BL21 （DE3）菌株对FtP450-R4蛋白进行了可溶性表达,活性鉴定表明,重组FtP450-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 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-aligument 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.

关 键 词：苦荞 细胞色素P450 克隆 分子鉴定 活性鉴定 CYTOCHROME P450 

分 类 号：S512.1[农业科学—作物学]