甘蓝型油菜BnCYP79B1基因的克隆与表达

摘要

Glucosinolates are secondary metabolites found mainly in Cruciferae plants.The CYP79 family,belongs to cytochromes P450,had been shown to catalyze the conversion of amino acids to their corresponding oximes in the biosynthesis of cyanogenic glucosides.In the present study,one of the glucosinolates regulator genes,named as BnCYP79B1 (GenBank JX535391.1) was cloned by homologous cloning from three stains of Brassica napus.The cDNA sequence of its open reading frame (ORF) was as long as 1625 bp,encoding a putative protein of 541 amino acid residues with a theoretical isoelectric point of 8.88.Sequence comparison with Brassica oleracea var.botrytis CYP79B1 showed that,BnCYP79B1 gene had 98.83％ similarity of nucleotide sequence and 99.26 ％ similarity of the amino acid sequence.Three different lines,w678 (high-stemed and high glucosinolates content in seed),w673 (high-stemed and low glucosinolates content in seed),and w578 (dwarf mutant with high glucosinolates content in seed),were estimated for BnCYP79B1 expression by real-time quantitative PCR analysis.The results showed that:BnCYP79B1gene mainly expressed in roots of w678 and in leaves of w578,but with the highest expression of BnCYP79B1 in w678.In conclusion,wild-type line had higher BnCYP79B1 expression level than the dwarf mutant,and also the high glucosinolates content line had rather higher BnCYP79B1 expression level than the low glucosinolates content line.%硫苷是十字花科植物的一种次生代谢产物,其合成途径受细胞色素P450的CYP79家族蛋白的调控,该实验采用同源克隆技术在甘蓝型油菜中克隆到了CYP79B1基因,命名为BnCYP79B1(GenBank登录号为JX535391.1).BnCYP79B1基因cDNA全长1 625 bp,编码一个含有541个氨基酸、理论等电点为8.88.序列对比结果显示,BnCYP79B1与花椰菜CYP79B1在DNA序列上的相似性为98.83％,推测蛋白氨基酸序列的相似性为99.26％.通过不同时期不同部位BnCYP79B1基因表达量的分析,发现BnCYP79B1基因在高秆高硫苷品系的根中表达量较高,而对矮秆高硫苷品系则是叶中表达量较高.在BnCYP79B1表达总量上,高秆品系较矮秆品系高,高硫苷品系较低硫苷品系高.