Effects of exogenous IBA and fungal elicitor on growth of in vitro roots culture of Dysosma versipellis and production of podophyllotoxin / 中国中药杂志

Using the White as basic medium, the effects of the exogenous IBA and endophytic fungal elicitor on the growth of in vitro roots cultures of Dysosma versipellis and production of podophyllotoxin were investigated in this study. The results showed that the IBA and the endophytic fungus Zasmidium syzygii elicitor could increase the content of podophyllotoxin of in vitro roots of D. versipellis after 3 weeks. The White medium added with 3 mg·L~(-1) IBA induced the highest increase of podophyllotoxin(1 830.86 μg·g~(-1)), which was 2.07 folds greater than the control, and followed by 1.5 mg·L~(-1) IBA, fungal elicitor, 1 mg·L~(-1) IBA, 0.5 mg·L~(-1) IBA and 4.5 mg·L~(-1) IBA, which was 1.82, 1.71, 1.63, 1.43 and 1.1 folds greater than the control, respectively. The results also showed that the growth of roots was certain positively correlated with the change of IBA concentration. Therefore, 3 mg·L~(-1) IBA was the most suitable for the production of podophyllotoxin in the in vitro roots of D. versipellis, and the stimulating effect of Z. syzygii fungal elicitor was between 1.5 mg·L~(-1) and 1 mg·L~(-1) IBA, which was a potential natural elicitor to induce the accumulation of podophyllotoxin in future production.

Real-time fluorescent quantitative PCR detection of key enzyme genes expression of alkaloid biosynthesis promoted by endophytic fungal elicitor in Sophora alopecuroides / 中草药

Objective To establish a method of detecting the expression of Lysine decarboxylase (LDC) -a key enzyme for the synthesis of alkaloid in the host promoted by the endophytic fungal elicitor of Sophora alopecuroides by using real-time fluorescence quantitative PCR (qRT-PCR). Methods Target gene primers QLDC-F/QLDC-R and reference gene primers Lectin-F/Lectin-R were designed according to LDC and Lectin gene sequences of S. alopecuroids; Five-fold gradient dilution of cDNA was used as the standard sample for the construction of the standard curve of target gene and the reference gene. Reaction system and reaction conditions of qRT-PCR were optimized, and the sensitivity of semi-quantitative PCR and qRT-PCR were analyzed and compared. Under different eliciting time of endophytic fungal elicitors NDZKDF13 of S. alopecuroides, the content of oxymatrine in the host was determined by HPLC, the expression of LDC gene was detected by qRT-PCR, and the relationship between LDC gene expression and the accumulation of OMA was analyzed. Results The results of qRT-PCR were better when the cDNA content in the system was 200 ng/μL and the annealing temperature was 61 ℃. The standard curve of the target gene and the reference gene was constructed, in which the cycle threshold and template concentration showed a good linear relationship, the amplification efficiency was above 99%, and the sensitivity was 25 times that of semi-quantitative PCR. Under the induction effect of endophytic fungal elicitor NDZKDF13, expression of host LDC gene reached the peak on the 6th day, which was 25.58 times that of the control. The increase of OMA content lagged the change of the LDC gene expression and reached the highest amount on the 9th day after the induction. Conclusion The qRT-PCR technique was successfully applied to the functional gene research of S. alopecuroides. Through the optimization of various conditions, a platform for accurate and simple detection of functional gene expression in S. alopecuroides was established.

Signal transduction of atractylodin biosynthesis in Atractylodes lancea cell induced by endophytic fungal elicitor mediated with nitric oxide followed by salicylic acid / 中草药

Objective: To investigate the signal molecules and signal transduction involved in endophytic fungal elicitor-induced atractylodin biosynthesis and the effect of an endophytic fungal elicitor on the key enzyme activity in Atractylodes lancea. Methods: Content changes of nitric oxide (NO), salicylic acid (SA), and atractylodin were detected under the endophytic fungal elicitor treatment by plant cell suspension culture technology. Results: The endophytic fungal elicitor remarkably promoted NO burst and the biosynthesis of SA and atractyodin by activating nitric oxide synthase (NOS), phenylalanine ammonia lyase (PAL), and acetyl coenzyme A carboxylase (ACC), respectively. NOS inhibitor PBITU could inhibit the NO and SA accumulation and the atractyodin biosynthesis induced by the elicitor. And atractyodin biosynthesis could also be triggered by exogenous NO or SA. The results indicated that NO and SA were the necessary signal molecules and NO burst was mediated by NOS induced by endophytic fungal elicitor. NO quencher cPITO could effectively remove NO burst in A. lancea cell induced by endophytic fungal elicitor and notably inhibit the biosynthesis promotion of SA and atractyodin in A. lancea cell induced by endophytic fungal elicitor. Exogenous SNP could reverse the cPITO inhibition on the activity of PAL and ACC and the synthesis of SA and atractylodin. This suggested that NO was an upstream signal molecule mediated endophytic fungal elicitor to accelerate the biosynthesis of SA and atractyodin. Conclusion: Endophytic fungal elicitor mediated through NO followed by SA could promote atractyodin biosynthesis by activating ACC in A. lancea.

Polyamine-mediated triterpenoid synthesis in suspension cells of Betula platyphylla induced by fungal elicitor / 中草药

Objective: To clarify whether polyamine-mediated triterpenoid synthesis in birch (Betula platyphylla) suspension cells induced by fungal elicitor. Methods: Fungal elicitor (40 μg/mL), putrescine (Put, 1 mmol/L), and D-arginine (D-Arg, 2 mmol/L) were added to the eight-day-old suspension cell culture, the content changes of triterpenoid and free polyamines were analyzed by chemical colorimetry and HPLC. The effect of polyamine on triterpenoid synthesis in B. platyphylla suspension cells induced by fungal elicitor was analyzed by pharmacology and restoration experiment. Results: After the treatment of fungal elicitor or Put, the contents and yields of free polyamines and triterpenoid were increased. Among of them, the triterpenoid content was the highest after 24 h treatment, the increasing rates were 68.54% and 30.34%, respectively. The triterpenoid content was increased by the cotreatment of fungal elicitor and Put, but the increasing degree of yield was lower than that by the treatment of fungal elicitor alone. Compared with the fungal elicitor alone, the cotreatment of fungal elicitor and D-Arg decreased the triterpenoid content by 40.57% in the highest degree of decreasing after 24 h treatment. In restoration experiment, with the treatment time prolonging, the effect of fungal elicitor, Put, or cotreatment of fungal elicitor and D-Arg on triterpenoid synthesis was decreasing to the control level finally. Conclusion: Polyamine could mediate the triterpenoid synthesis in cells of B. platyphylla induced by fungal elicitor.

Interaction between putrescine and hydrogen peroxide on regulating triterpenoid production in Betula platyphylla / 中草药

Objective: To analyze the interaction between putrescine (Put) and hydrogen peroxide (H2O2) on the regulation of triterpenoid production in Betula platyphylla suspension cell culture. Methods: Put (1 mmol/L), H2O2 (0.1 mmol/L), and fungal elicitors (40 μg/mL) were added into the eight-day-old B. platyphylla suspension cell culture, and the changes of triterpenoid content, polyamine content, and H2O2 fluorescence intensity were analyzed by chemical colorimetry, HPLC, and fluorescence microscopy, respectively. Results: Put (1 mmol/L) increased the H2O2 fluorescence intensity and triterpenoid content in B. platyphylla suspension cell culture. H2O2 (0.1 mmol/L) promoted the triterpenoid production, inhibited the production of spermidine (Spd) and spermine (Spm), and had no effectt on Put content. Under the treatment of 1 mmol/L Put and 0.1 mmol/L H2O2, H2O2 fluorescence intensity and polyamine (PAs) content were between its separated treatment, and triterpenoid content was significantly higher than its separated treatment. Comparing with the fungal elicitor, fungal elicitor and H2O2 scavenger catalase (CAT) induced an increase of 0.62% and 16.05% in Put and Spd content, respectively, while no effect on Spm content. Fungal elicitor and PAs synthesis inhibitor D-arginine (D-arg) decreased the H2O2 fluorescence intensity. Under the treatment of fungal elicitor, the fluorescence intensity of CAT and D-arg, H2O2 and the contents of PAs and triterpenoid were lower than those of fungal elicitor and CAT or D-arg, but triterpenoid content was still higher than that of the control. Conclusion: This can be inferred that the interaction between Put and H2O2 regulates the triterpenoid production in birch suspension cell culture.

Elevated yield of Monacolin K in Monascus purpureus by fungal elicitor and mutagenesis of UV and LiCl

In China, Monascus spp., a traditional fungus used in fermentation, is used as a natural food additive. Monascus spp. can produce a secondary metabolite, monacolin K namely, which is proven to be a cholesterol-lowering and hypotensive agent. Hence, recently, many researchers have begun focusing on how to increase the production of monacolin K by Monascus purpureus. In the present study, we investigated the effect of the fungal elicitor and the mutagenesis of UV & LiCl on the amount of monacolin K produced by Monascus purpureus. The fugal elicitor, Sporobolomyces huaxiensis, was isolated from tea leaves and its filtrate was added into the culture filtrate of Monascus purpureus during growth to induct the production of monacolin K. The results showed that the highest amount of monacolin K produced by the liquid fermentation was 446.92 mg/mL, which was produced after the fungal elicitor was added to the culture filtrate of Monascus purpureus on the day 4; this amount was approximately 6 times greater than that of the control culture filtrate, whereas the highest amount of monacolin K produced by the mutated strain was 3 times greater than the control culture after the irradiation of UV light in the presence of 1.0 % LiCl in the medium.

Effects of fungal elicitors on accumulation of indole alkaloids in Catharanthus roseus calli / 中草药

Object To investigate the effects of fungal elicitors derived from the fungi Fusarium solani and Aspergillum niger on the accumulation of indole alkaloids in Catharanthus roseus calli. Methods The total indole alkaloid was extracted after the calli were treated with fungal elicitors. Then, the determination of ajmalicine and catharanthine was carried out by RP HPLC. Results The two fungal elicitors stimulate the accumulation not only the total indole alkaloid but the ajmalicine and catharanthine. The optimal exposure time of the two fungal elicitors for different kinds of indole alkaloid was investigated. Conclusion The two fungal elicitors have obvious effect on the accumulation of indole alkaloid in C. roseus calli.

Establishment of suspension cell line of Atractylodes lancea and effect of endophytic fungal elicitors on its essential oil accumulation / 中草药

Objective To establish the suspension cell line of Atractylodes lancea and to study the effect of two endophytic fungal elicitors on its essential oil production.Methods The essential oil was extracted by using ultrasonic wave after suspension cell was treated with endophytic fungal elicitors.Then,the determination of four compounds(atractylone,hinesol,?-eudesmol,and atractylodin) was carried out by gas chromatography.Results By testing in various conditions,the suspension cell line with a rapid growth rate was established.Its highest biomass(6.95 g/L) was obtained on day 21.?-Eudesmol was the only detection in the control suspension cell,and its highest content(17.469 ?g/g) was also reached on day 21.The effect of crude elicitors of two endophytic fungi(belong to Cunninghamella sp.and Gilmaniella sp.respectively,named AL4 and AL12) on the cell growth and the production of essentia1 oil were investigated.Overall AL4 elicitor got better effect.When suspension cell of 14-day-old cultures was exposed to AL4 elicitor(carbohydrate 20 mg/L medium) for 7 d,the biomass increased 3.31% over the control,and the four compounds(atractylone: 14.715 ?g/g,hinesol: 28.395 ?g/g,?-eudesmol: 38.794 ?g/g,and atractylodin: 8.310 ?g/g) were all detected.Among them,the content of ?-eudesmol reached 2.22 times as much as the control.Conclusion The cell growth and the accumulation of essential oil of A.lancea could also be promoted by adding crude elicitors of the endophytic fungi AL4 and AL12.

Effects of fungal elicitors on growth of Dendrobium candidum protocorms / 中草药

Objective To study the effects of two fungal elicitors on the growth of Dendrobium candidum protocorms cultured on the solid medium.Methods The medium for propagation of protocorms was selected and the growth curve on that medium was obtained.According to the curve,the two fungal elicitors were added at the different growth stages of protocorms.The fresh weight(FW) and dry weight(DW) of protocorms were measured.Results The medium for propagation of protocorms was 1/2MS+20% potato extract+3% sucrose+0.8% agar.Compared with the control MF23 elicitor added at weeks 11 and 13 could significantly increase the FW by 16.4%(P