水母雪莲黄酮合成酶FNSII基因克隆及其在3种细胞系中的表达

Cloning of flavone synthase (FNSII) gene and expression in three cell lines of Saussurea medusa

摘要:

水母雪莲是中国传统珍稀药材，其主要活性成分之一木犀草素具有预防和治疗癌症的功效。以水母雪莲绿色细胞系为试材，采用RT-PCR和RACE-PCR方法获得1个水母雪莲FNSII基因cDNA全长，命名为SmFNSII (GenBank登录号为KF170286)。序列分析结果表明：SmFNSII全长1 710 bp，包含34 bp的5?非编码区、125 bp的3?非编码区和1个长度为1 551 bp编码516个氨基酸的开放阅读框。氨基酸序列分析表明SmFNSII属于细胞色素P450 CYP93B亚家族单氧化酶。序列比对和系统进化分析表明，SmFNSII氨基酸序列与同科植物毛山柳菊的亲缘关系最近，相似性达87%以上。实时荧光定量PCR分析表明，SmFNSII在白色、绿色和红色细胞系均有表达，红色系中的表达量最高，白色系中表达量最低，此结果与高效液相色谱分析3种颜色细胞系中木犀草素含量结果一致。构建pET-SmFNSII原核表达重组质粒，并在大肠杆菌中诱导表达，诱导蛋白大小与预期一致。通过筛选FNSII基因表达水平高、高产木犀草素的水母雪莲细胞系和植株，可培育抗炎抗癌活性较高、具有高保健功能的雪莲生药新品种。

Abstract:

Saussurea medusa is a rare traditional Chinese medicinal herb, of which luteolin is the main active medicinal compound for cancer prevention and treatment. A full-length FNSII gene, namely SmFNSII (GenBank Accession No. KF170286), was obtained from green cell line of Saussurea medusa by RT-PCR and RACE-PCR. Sequence analysis indicated that SmFNSII is 1 710 bp in full length, containing a 34 bp 5?-untranslated region (5?-UTR), a 125 bp 3?- UTR, and a 1 551 bp open reading frame (ORF) encoding 516 amino acid residues. Amino acid sequence analysis indicated that SmFNSII belonged to subfamily CYP93B of plant cytochrome P450. Sequence alignment and phylogenetic analysis revealed that amino acid sequences of SmFNSII shared 87% homology with the protein in Hieracium pilosella. Quantitative real-time PCR analysis indicated that SmFNSII expression is the highest in red cell line and the lowest in white cell line, corresponding to quantitative analysis of luteolin concentration. pET-SmFNSII, a prokaryotic expression recombinant plasmid, was constructed and transferred into Escherichia coli, and the expressed protein band was the same size with predicted protein. Saussurea medusa cultivars with high anti-inflammatory, anti-cancer activities and health care function would be cultivated through filtering cell lines and plants with high expression level of FNSII gene and luteolin accumulation.