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Effect of Signaling Molecules on Secondary Metabolites in Tissue Culture of Dendrobium Nobile

Author: ZhuShengNan
Tutor: WangHuiZhong
School: Hangzhou Normal University
Course: Botany
Keywords: Dendrobium nobile secondary metabolic signaling molecules High Performance Liquid Chromatography Gas Chromatography Real-time quantitative PCR
CLC: S567.239
Type: Master's thesis
Year: 2011
Downloads: 78
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Abstract

Dendrobium nobile is a traditional precious herbs, as Blanco (orchidaceae) perennial herb with naturally rich in polysaccharides, alkaloids, sesquiterpenes and other active ingredients, with the anti-tumor, anti-aging, lowering blood sugar, etc. It is very important to improve its medicinal value by increasing the effective content of active ingredients.Using the inducers, such as JA, SA etc, to improve the active ingredients in Dendrobium nobile had been studied well. However, such kinds of inducers had not been applied for improving the Dendrobium nobile-based active ingredients of secondary metabolites in Dendrobium nobile. In this study, we investigated the inducing ability of secondary metabolites of tissue culture in Dendrobium nobile with three signal molecules, including salicylic acid (SA), Nitric oxide (NO) and methyl jasmonate (MeJA). The detail processing as following:The experimental materials of seedling of Dendrobium nobile was acquired by tissue culture. The young seedling from Dendrobium nobile were cultured on B5 media applied with or without signal molecules SA, sodium nitroprusside (SNP, a NO donor) and MeJA, to produce tissue culture seedling, and measure water-soluble polysaccharide, Dendrobin and P-carotene content with intervals of time. We found that polysaccharide content of tissue culture seedling of Dendrobium nobile did not increase after 28 days inducing with adding of any of inducers in media, the content of polysaccharide is stable on 12%, which indicate that the addition of signaling molecules did not affect the content of polysaccharides. To measure the Dendrobin content in seedlings, we used gas chromatograph approach. We found that with the extension of tissue culture time, the Dendrobin content increased gradually and reached a peak in 28 days. Compared to the control, Dendrobin content were increased 3.2%,2.7% and 5.1% when the culture medium were supplied with SA, SNP and MeJA respectively. This result indicates that MeJA is the most efficient inducer on Dendrobin production compared with SA and SNP. To determine the content ofβ-carotene, we used HPLC approach. The results showed that similar to Dendrobin,β-carotene also accumulated with the peak in 28 days culture andβ-carotene content increased 21.1% by MeJA,11.9% by SA, and 3.9% by SNP. Those results show that MeJA has a highly inducing feature in Dendrobin and P-carotene accumulation than SA and SNP.To explore the inducing mechanism of signaling molecules in secondary metabolic, we constructed a cDNA libray from Dendrobium nobile and selected several key genes controlling carbohydrate metabolism, terpenoid metabolism, alkaloid metabolism and pigment metabolism. By designing the gene-specific primers, we detected the gene expression differences by real-time fluorescence quantitative PCR within 24 h of 1% salicylic acid,100μM sodium nitroprusside solution and 100μM methyl jasmonate solution treatment on the tissue culture of Dendrobium nobile. The results showed that each of three signaling molecules can induce phytoene genes expression significantly; 1% salicylic acid treatment can induce the maximal expression of carRP in 8 h, about 12.2 times related to the control; 100μM sodium nitroprusside solution treatment made the gene expression level reached the maximum at 16h,4.4 times compared to control; 100μM MeJA solution treatment made the gene expression level reaching to maximum at 24 h,4.2 times increasing than control.DnTR I and DnTR II are key enzyme genes in the process of alkaloid synthesis. To test if those genes expression can be affected by signaling molecules, we detected their expression after inducer treatment. It is significant effect of 1% salicylic acid on DnTR I gene expression. After 8 h treatment, the gene expression level reached 7.21 times compared with control; 100μM MeJA solution treatment on DnTR II gene expression have a significant impact,24 h reaching to 6.68 times higher than control, which is consistent with previous secondary metabolic content assay that three types of signal molecules were favorable to the accumulation of Dendrobin.This is the first report that the content of secondary metabolites were tested by liquid chromatography and gas chromatography after the tissue cultured seedling induced by MeJA, SA and NO. This study showed that MeJA is very important inducer for the synthesis of Dendrobine. We also test the possible molecular mechanism of Dendrobin synthesis during MeJA etc inducing. We found that MeJA increased the content of Dendrobin may through alter the expression level of Tropinone reductase gene, which is the key gene in Dendrobin biosynthesis.

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