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 microM 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.

Salicylic acid enhances Jaceosidin and Syringin production in cell cultures of Saussurea medusa.

Addition of 20 muM salicylic acid to Saussurea medusa cell cultures at day 6 resulted in jaceosidin and syringin productions up to 95 mg l(-1 )and 631 mg l(-1) which were, respectively, about 2.5- and 2.7-fold higher than in the control. The biomass was increased from 8 to 12 g l(-1). Expression of chalcone synthase gene (chs) increased sharply after 12 h treatment and was sustained up to 48 h; chalcone isomerase gene (chi) expression reached a peak at 24 h and decreased after 48 h; and phenylalanine ammonia-lyase activity increased by 7.5-fold (96 U mg(-1) protein) higher than in the control after 24 h. These results indicate that salicylic acid enhances the production of jaceosidin and syringin which is accompanied by induction of the related phenylpropanoid biosynthetic enzymes.

Overexpression of the Saussurea medusa chalcone isomerase gene in S. involucrata hairy root cultures enhances their biosynthesis of apigenin.

Saussurea involucrata is a medicinal plant well known for its flavonoids, including apigenin, which has been shown to significantly inhibit tumorigenesis. Since naturally occurring apigenin is in very low abundance, we took a transgenic approach to increase apigenin production by engineering the flavonoid pathway. A construct was made to contain the complete cDNA sequence of the Saussurea medusa chalcone isomerase (CHI) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Using an Agrobacterium rhizogenes-mediated transformation system, the chi overexpression cassette was incorporated into the genome of S. involucrata, and transgenic hairy root lines were established. CHI converts naringenin chalcone into naringenin that is the precursor of apigenin. We observed that transgenic hairy root lines grew faster and produced higher levels of apigenin and total flavonoids than wild-type hairy roots did. Over a culture period of 5 weeks, the best-performing line (C46) accumulated 32.1 mgL(-1) apigenin and 647.8 mgL(-1) total flavonoids, or 12 and 4 times, respectively, higher than wild-type hairy roots did. The enhanced productivity corresponded to elevated CHI activity, confirming the key role that CHI played for total flavonoids and apigenin synthesis and the efficiency of the current metabolic engineering strategy.

A comparison between hairy root cultures and wild plants of Saussurea involucrata in phenylpropanoids production.

Saussurea involucrata is an important medicinal plant that produces a few bioactive secondary metabolites, such as hispidulin, rutin, and syringin. Previously, we established a hairy root culture system for this species through Agrobacterium-mediated transformation. The present study addressed the issue as how hairy root cultures perform in phenylpronoid accumulation. From the ethanolic extract of a hairy root culture established for Saussurea involucrata, syringin, rutin and hispidulin, were isolated and their chemical structures were confirmed by HPLC-ESI-MS. A quantitative study of the compounds showed great levels of syringin and hispidulin (being 43.5+/-1.13 and 0.34+/-0.023 mg g-1 dry weight, respectively), about 40 and 3 times, respectively, higher than those from wild plants. But, the levels of rutin from hairy roots were much lower (0.71+/-0.043 vs. 6.59+/-0.56 mg g-1 dry weight). Compared with untransformed root cultures, syringin and hispidulin levels were also higher. An experiment on culture media showed that MS was superior to others for phenylpropanoids accumulation in hairy roots, a 28-day culture produced 405 mg l-1 syringin.

[Effects of methyl jasmonate and salicylic acid on phenylethanoid glycosides synthesis in suspension cultures of Cistanche deserticola].

The present study investigated the influence of the methyl jasmonate and salicylic acid elicitors on the formation of phenylethanoid glycosides (PeG) in the suspension cultures of Cistanche deserticola. The results showed that methyl jasmonate and salicylic acid enhanced greatly the accumulation of PeG and echinacoside (Echin), but their optimum elicitation dosage and addition time were different. The yields of PeG and Echin were significantly increased in the presence of 5 micromol/L methyl jasmonate on day 14 (up to 2.59-fold and 3.82-fold, respectively), whereas treated with 50 micromol/L salicylic acid on day 28, the maximum content of them were, respectively, 2.71 and 3.16-fold higher than the untreated cell cultures.

[Effects of physical and chemical factors on hairy root growth and flavonoids biosynthesis in the cultures of Saussurea medusa Maxim hairy root].

The effects of different physical and chemical factors on hairy root growth and flavonoids production were studied in suspension culture of Saussurea medusa hairy root in 1/2 MS medium. The results showed that the following culture conditions, nitrogen concentratiaon (involved NH4+ and NO3-), 30 mmol/L; the ratio of ammonium to nitrate, 5:25; the combination of 2% sucrose and 3% glucose; 0.5 mg/L GA3; 0.5 mg/L IBA; initial pH 5.8; light cycle, 18 h/d (3500lx); temperature, 24 degrees C; shaker revolutions per minute, 100 r/min, were favourable to hairy root growth and flavonoids production. Under the above culture conditions, up to 12.8 g/L (DW) of hairy root and 1922 mg/L of flavonoids were obtained after 21 days of culture. The content of total flavonoids in hairy root was 15%, which was about 25 times as that in the wild plantlet.

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 2-4 mm diameter and accounted for 64% biomass. This line had a sustained cell viability over 10 successive sub-cultures.

[Establishment of Saussurea involucrata hairy roots culture and plantlet regeneration].

Hairy root clones of Saussurea involucrata transformed with Agrobacterium rhizogenes strains R1601, R1000, and LBA9402 were established to investigate the flavonoid production. Opine synthesis and PCR analysis confirmed the integration of the T-DNA fragment of Ri plasmid from A. rhizogenes strain R1601 into the transformed root genome. The frequency of hairy root formation from root segments, which were pre-cultured 2 days in N6 solid medium without plant growth regulators, amounted to 100% following infection with R1601 strain of A. rhizogenes. The transformed roots were kept in hormone-free N6 liquid medium in the dark at 25 degrees C, 110r/min and routinely subcultured every 20 - 24 days. One hairy root clone, which grew vigorously with lateral branches, was periodically examined for the ability to produce flavonoid. The maximum of biomass and flavonoid yield achieved 66.7 g/L (fresh weight) and 102.3mg/g dry weight after incubation 20 days. The calli were induced from the hairy root culture in the presence of 0.5mg/L IBA and intact plantlets were regenerated from these calli. The regeneration plantlets from hairy roots, in which the flavonoid content were 53% in that of untransformed plants, weren't different in growth and morphology of the untransformed plantlets. Therefore plant regeneration from hairy roots may be also a means for producing transformed S. involucrata plants. Hairy root cultures of S. involucrata clearly showed higher flavonoid contents compared to the wild plant or the regeneration seedlings. As the wild S. involucrata grows only in special regions with peculiar climate, and cultivation of this species in a normal climate has been unsuccessful so far. The success in obtaining a method for high production of flavonoid might very well be one of the solutions for this problem in the future.

[Cloning and sequence analysis of MYB transcriptional regulator SmP gene of Saussurea medusa Maxim].

A full-length cDNA encoding a MYB-related regulatory gene was isolated from a cDNA library prepared from mRNAs of the red line callus of S. medusa by TD-PCR. The cDNA, designated SmP, is 969 nucleotides long and has an open reading frame of 771 bp with a deduced amino acid sequence of 256 residues. The putative protein of SmP has two typical conversed R2R3-Myb DNA-binding domains in N-terminal and displays a rather high degree of similarity to OsMYB from rice and LBMI from tobacco, showing 73% and 70% identity within the DNA-binding domains. However, the C-terminal domain of the SmP protein does not show obvious similarity to any other known protein sequence. It is rich in hydrophilic amino acids, especially in serine residues (18.38%), partly organized in homopolymeric stretches, a feature often found in activation domain of transcription factors.

[SSS method for screening cDNA library].

A quick and simple method subsection screening (SSS) method for screening cDNA library by PCR was established. With this method, cDNA phage plate was cut into several blocks and a couple of primers was designed according to target gene. And then the target genes were obtained by screening cDNA library. Comparing with other methods,this method has many advantages such as controlled range and clear target,and also quick and simple for obtaining the target genes. It is possible to get a target gene in one week in general by this method. Thus,the CHI gene, F3'H gene, HSP gene and one HSP partial fragment, were obtained respectively in half month in our lab. We can get twice the result with half the effort when we screen several genes in the same time. It is also suitable to screen other libraries.