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Aspergillus niger Enhance Bioactive Compounds Biosynthesis As Well As Expression of Functional Genes in Adventitious Roots of Glycyrrhiza uralensis Fisch

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Abstract

In the present study, the culture conditions for the accumulation of Glycyrrhiza uralensis adventitious root metabolites in balloon-type bubble bioreactors (BTBBs) have been optimized. The results of the culture showed that the best culture conditions were a cone angle of 90° bioreactor and 0.4–0.6–0.4-vvm aeration volume. Aspergillus niger can be used as a fungal elicitor to enhance the production of defense compounds in plants. With the addition of a fungal elicitor (derived from Aspergillus niger), the maximum accumulation of total flavonoids (16.12 mg g−1) and glycyrrhetinic acid (0.18 mg g−1) occurred at a dose of 400 mg L−1 of Aspergillus niger resulting in a 3.47-fold and 1.8-fold increase over control roots. However, the highest concentration of polysaccharide (106.06 mg g−1) was achieved with a mixture of elicitors (Aspergillus niger and salicylic acid) added to the medium, resulting in a 1.09-fold increase over Aspergillus niger treatment alone. Electrospray ionization tandem mass spectrometry (ESI-MSn) analysis was performed, showing that seven compounds were present after treatment with the elicitors, including uralsaponin B, licorice saponin B2, liquiritin, and (3R)-vestitol, only identified in the mixed elicitor treatment group. It has also been found that elicitors (Aspergillus niger and salicylic acid) significantly upregulated the expression of the cinnamate 4-hydroxylase (C4H), β-amyrin synthase (β-AS), squalene epoxidase (SE) and a cytochrome P450 monooxygenase (CYP72A154) genes, which are involved in the biosynthesis of bioactive compounds, and increased superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activity.

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Abbreviations

A. niger :

Aspergillus niger

SA:

Salicylic acid

SODs:

Superoxide dismutases

CATs:

Catalases

PODs:

Peroxidases

G. uralensis Glycyrrhiza uralensis :

Glycyrrhiza uralensis Fisch

ROS:

Reactive oxygen species

C4H:

Cinnamate 4-hydroxylase

SE:

Squalene epoxidase

β-AS:

β-Amyrin synthase

BTBB:

Balloon-type bubble bioreactor

MS:

Murashige and Skoog (1962)

IBA:

Indole-3-butric acid

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Acknowledgments

This research was funded by the 863 Program (2014AA022201-04), Central Significant Increase or Decrease Program, China (2060302), and National Science and Technology Support Program (2012BAI29B02).

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Authors and Affiliations

  1. Tianjin Key Laboratory for Modern Drug Delivery and High Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, People’s Republic of China

    Jing Li, Juan Wang, Jinxin Li, Hongfa Li & Wenyuan Gao

  2. State Key Laboratory Breeding Base of Dao-di Herbs, China Academy of Chinese Medical Sciences, Beijing, 100700, China

    Jing Li

  3. Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences, Yunnan, 650231, China

    Dahui Liu

  4. Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and Technology, Tianjin, 300457, China

    Jianli Li & Shujie Liu

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  1. Jing Li
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  2. Juan Wang
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  3. Jinxin Li
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Correspondence to Wenyuan Gao.

Electronic supplementary material

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Table S1

The primers of genes used in RT-PCR (DOC 46 kb)

Table S2

HPLC–MSn data of triterpenoid saponins and flavonoids in adventitious root culture of G. uralensis (DOC 51 kb)

Fig. S1

General phenylpropanoid and flavonoid biosynthetic pathway (DOC 268 kb)

Fig. S2

Biosynthetic pathways of glycyrrhizic acid in G. uralensis (DOC 319 kb)

Fig. S3

LC-MSn spectra of liquiritin (a, b), isoliquiritin (c, d), glycyrrhizic acid (e, f), and (3R)-vestitol (g, h) (DOC 277 kb)

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Li, J., Wang, J., Li, J. et al. Aspergillus niger Enhance Bioactive Compounds Biosynthesis As Well As Expression of Functional Genes in Adventitious Roots of Glycyrrhiza uralensis Fisch. Appl Biochem Biotechnol 178, 576–593 (2016). https://doi.org/10.1007/s12010-015-1895-5

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