Dexiu Zhao

Metabolic Engineering of the Phenylpropanoid Pathway Enhances the Antioxidant Capacity of Saussurea involucrata

The rare wild species of snow lotus Saussurea involucrata is a commonly used medicinal herb with great pharmacological value for human health, resulting from its uniquely high level of phenylpropanoid compound production. To gain information on the phenylpropanid biosynthetic pathway genes in this critically important medicinal plant, global transcriptome sequencing was performed. It revealed that the phenylpropanoid pathway genes were well represented in S. involucrata. In addition, we introduced two key phenylpropanoid pathway inducing transcription factors (PAP1 and Lc) into this medicinal plant. Transgenic S. involucrata co-expressing PAP1 and Lc exhibited purple pigments due to a massive accumulation of anthocyanins. The over-expression of PAP1 and Lc largely activated most of the phenylpropanoid pathway genes, and increased accumulation of several phenylpropanoid compounds significantly, including chlorogenic acid, syringin, cyanrine and rutin. Both ABTS (2,2′-azinobis-3-ethylbenzotiazo-line-6-sulfonic acid) and FRAP (ferric reducing anti-oxidant power) assays revealed that the antioxidant capacity of transgenic S. involucrata lines was greatly enhanced over controls. In addition to providing a deeper understanding of the molecular basis of phenylpropanoid metabolism, our results potentially enable an alternation of bioactive compound production in S. involucrata through metabolic engineering.

Cellular aggregate size as the critical factor for flavonoid production by suspension cultures of Saussurea medusa

Three previously established cell lines (yellow, red and white) of Saussurea medusa were investigated for jaceosidin and hispidulin production. Maximum yields of the jaceosidin and hispidulin were obtained in the red cell line at 75 ± 0.41 and 6.4 ± 0.31 mg l-1. Production of jaceosidin and hispidulin correlated with the sizes of compact callus aggregates (CCA) and cellular viability. In the red cell line, the sizes of CCA were predominantly of 2–4mm diameter and accounted for 64% biomass. This line had a sustained cell viability over 10 successive sub-cultures.

Molecular characterization and expression analysis of dihydroflavonol 4-reductase (DFR) gene in Saussurea medusa

Dihydroflavonol 4-reductase (DFR), which catalyzes the reduction of dihydroflavonols to leucoanthocyanins, is a key enzyme in the biosynthesis of anthocyanidins, proanthocyanidins, and other flavonoids of importance in plant development and human nutrition. This study isolated a full length cDNA encoding DFR, designated as SmDFR (GenBank Accession No. EF600682), by screening a cDNA library from a red callus line of Saussurea medusa, which is an endangered, traditional Chinese medicinal plant with high pharmacological value. SmDFR was functionally expressed in yeast (Saccharomyces cerevisiae) to confirm that SmDFR can readily reduce dihydroquercetin (DHQ) and dihydrokampferol (DHK), but it could not reduce dihydromyricetin (DHM). The deduced SmDFR structure shared extensive sequence similarity with previously characterized plant DFRs and phylogenetic analysis showed that it belonged to the plant DFR super-family. SmDFR also possessed flavanone 4-reductase (FNR) activity and can catalyze the conversion of eridictyol to luteoforol. Real-time PCR analysis showed that the expression level of SmDFR was higher in flowers compared with both leaves and roots. This work greatly enhances our knowledge of flavonoid biosynthesis in S. medusa and marks a major advance that could facilitate future genetic modification of S. medusa.

Methyl jasmonate stimulates jaceosidin and hispidulin production in cell cultures of Saussurea medusa

Cell cultures of Saussurea medusa produce valuable secondary metabolites, and jaceosidin and hispidulin are the major bioactive compounds. In the present study, the cultures were challenged by methyl jasmonate (MJ). The highest jaceosidin and hispidulin concentrations (65.2±3.67 mg/L and 12.3±0.47 mg/L) were achieved with 5 μM MJ added to 9-d-old subcultures, being 2.2-fold and 4.2-fold, respectively, higher than those from controls. The elicitor had little influence on cell growth, indicating that the changed biological processes did not include alterations in cell division. Furthermore, we observed that the activities of phenylalanine ammonia lyase were transiently increased after treatment with MJ, which suggests that this elicitor modifies jaceosidin and hispidulin production by regulating the phenylpropanoid pathway.

Cloning of CYP9G2 from the Diamondback Moth, Plutella xylostella (Lepidoptera: Yponomeutidae)

Cytochrome P450s constitute a superfamily of hemoproteins, important in the metabolism of endogenous and xenobiotic compounds. The full-length cDNA of a novel cytochrome P450, CYP9G2, was isolated from a cDNA library. The cDNA is 2143 bp in length and contains an open reading frame from 50 to 1615 bp, encoding a protein of 521 amino acid residues. The putative P450 protein contains a highly hydrophobic N terminus and a P450 protein signature motif, FG/S*G*R*C*G***A/G, known as the important ligand for heme binding, analysis of the NH2-terminal sequence indicated that CYP9G2 is a microsomal P450. Using polymerase chain reaction with primers specific to CYP9G2, the genomic structure of CYP9G2 was analyzed, and it was found that the gene contains seven introns and eight exons within the coding region, all the sequences of the exon-intron junctions are consistent with the AG-GT rule. Multiple alignment indicated that CYP9G2 is most similar to CYP9E2 from the Blattella germanica (42.7% identity), it is also similar to the insect P450s in family 9, including CYP9L1 from Anopheles gambiae (38.7%) and CYP9A1 from Heliothis virescens (39.5%).

Cloning and expression analysis of a hsp70 gene from Saussurea medusa

Members of the 70 kD heat-shock gene family are highly conserved across a wide range of organisms. In an effort to learn more about the evolution and possible functions of extreme environment plant Saussurea medusa Maxim hsp70, we isolated a cDNA clone encoding a putative cytosolic member (Smhsp70) of this family of proteins from a cDNA library of S. medusa cell cultures. The cDNA clone was 2224 bp in length and contained a 1941 bp open reading frame (ORF) encoding a polypeptide of 647 amino acid residues with a predicted molecular mass of 70,794 Da. The predicted protein was found to contain a C-terminal amino acid motif of “PKIEEVD” indicating that Smhsp70 was related to cytosolic members of the hsp70 family in higher plant. The secondary and three-dimensional structures of Smhsp70 were analyzed by molecular modeling. The genomic structure of Smhsp70 included one intron of 1134 bp in length. The deduced Smhsp70 protein has 93.7 and 93.2% similarity with the hsp70 of tobacco and tomato, 73.2% with the hsp70 of human, and 43.7% with DnaK of Escherichia. coli, respectively. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses indicated that the cytosolic Smhsp70 protein was constitutively expressed and markedly increased after relatively short periods of heat shock (37°C) as well as by low temperature (4°C) treatments.