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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。
Jellyfish Saussurea is a traditional Chinese rare medicinal material, one of the main active ingredients luteolin with the efficacy of the prevention and treatment of cancer. The full-length cDNA of FNSII gene of Saussurea datum was obtained by RT-PCR and RACE-PCR using Smoky’s Saussurea green cell line as the test material, named SmFNSII (GenBank accession number KF170286). Sequence analysis showed that SmFNSII was 1 710 bp in length, containing 34 bp of 5? Non-coding region, 125 bp of 3? Non-coding region and 1 551 bp of a 516 amino acid open reading frame. Amino acid sequence analysis revealed that SmFNSII belongs to cytochrome P450 CYP93B subunit monooxygenase. Sequence alignment and phylogenetic analysis showed that the amino acid sequence of SmFNSII was closely related to the same plant, Salvia miltiorrhiza. The similarity was 87%. Real-time fluorescence quantitative PCR analysis showed that SmFNSII was expressed in white, green and red cell lines, the highest expression in red lines and the lowest in white lines. This result was in agreement with high performance liquid chromatography (HPLC) Hormone content of the same. The prokaryotic expression plasmid pET-SmFNSII was constructed and induced in E.coli. The induced protein was the same size as expected. By screening the high expression of FNSII gene, high yield luteolin cells of the Saussurea nivalis and plants, can cultivate a new anti-inflammatory anti-cancer activity, with high health functions Xuexian new varieties of herbs.