ABSTRACT
Saussurea medusa is a rare traditional Chinese medicinal herb, of which luteolin is the niain 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.
Subject(s)
5' Untranslated Regions , Amino Acid Sequence , Cell Line , Cloning, Molecular , Cytochrome P-450 Enzyme System , Genetics , Open Reading Frames , Phylogeny , Real-Time Polymerase Chain Reaction , Recombination, Genetic , Saussurea , Genetics , Sequence Alignment , Sequence Analysis, ProteinABSTRACT
Saussurea medusa is a rare traditional Chinese medicinal herb. Besides anti-inflammatory and analgesic activities, it has effects of disinhibiting cold, dispelling dampness and promoting blood circulation. Flavonoids are the main medicinal compounds in S. medusa. Contents of flavonoids and expression of flavonoids biosynthesis related genes in white and red (induced by low temperature, high sucrose and high light) callus were analyzed. The results showed that the total flavone in red line was 3.60 times higher compared to white line. The accumulation of rutin in red line (0.25% of dry weight) was 2.40 times higher compared to white line. Anthocyanins were abundant in red line, with the contents of cyanidin 3-O-glucosidechloride and cyanidin 3-O-succinyl glycoside 0.12% and 0.19% of dry weight respectively. CHS, F3'H, FNS, FLS, DFR and ANS genes were highly expressed in red line compared to white line. Expression of three transcription factors (MYB, bHLH and WD40) in red line was significantly higher than that in white line, especially the expression of MYB (19.70 times higher compared to white line). These results indicated that high expression levels of transcription factors induced high expression of structural genes in red line, thereby enhancing the flavonoids biosynthesis. The expression of bHLH and WD40 was similar, whereas it was significantly different from that of MYB, indicating that bHLH and WD40 could form a binary complex to regulate expression of structural genes and flavonoids biosynthesis.
Subject(s)
Anthocyanins , Chemistry , Cell Line , Flavonoids , Chemistry , Glucosides , Chemistry , Plant Proteins , Genetics , Metabolism , Plants, Medicinal , Chemistry , Saussurea , Chemistry , Genetics , Transcription Factors , Genetics , MetabolismABSTRACT
A fragment of chalcone synthase gene (SmCHS) was cloned from the cDNA library constructed in Saussurea medusa. The full-length cDNA sequence of SmCHS was obtained by RT-PCR. Sequence analysis showed that the full length of SmCHS was 1313 bp, containing an open reading frame (1170 bp) encoding 389 amino acids. The molecular weight of the protein was estimated to be 43 kDa. The prokaryotic expression plasmids pET28a(+)-SmCHS was constructed and transformed into Escherichia coli BL21(DE3) for expression. SDS-PAGE indicated that the fusion protein was expressed partially in soluble form after induction by IPTG. The recombinant protein was collected and purified by Ni-NTA affinity column. The enzymatic activity assay of the purified recombinant protein showed that the fusion protein had chalcone synthase activity. It could catalyze the condensation of a 4-coumaroyl-CoA with three malonyl-CoAs to produce naringenin chalcone.