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

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。

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 Sm FNSII（Gen Bank Accession No. KF170286）, was obtained from green cell line of Saussurea medusa by RT-PCR and RACE-PCR. Sequence analysis indicated that Sm FNSII 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 Sm FNSII belonged to subfamily CYP93 B of plant cytochrome P450. Sequence alignment and phylogenetic analysis revealed that amino acid sequences of Sm FNSII shared 87% homology with the protein in Hieracium pilosella. Quantitative real-time PCR analysis indicated that Sm FNSII expression is the highest in red cell line and the lowest in white cell line, corresponding to quantitative analysis of luteolin concentration. p ET-Sm FNSII, 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.