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Published on: March 28, 2017
RT-qPCR analysis of dexB and galE gene expression of Streptococcus alactolyticus in Astragalus membranaceus
Gui J Hao1, Kai Zhang, Jing Y Zhang
1Engineering and Technology Research Center of Traditional Chinese Veterinary Medicine of Gansu Province, Key Lab of New Animal Drug Project of Gansu Province, Lanzhou Institute of Husbandry and Pharmaceutics Sciences of Chinese Academy of Agricultural Sciences, No. 335, Jiangouyan Street, Qilihe District, Lanzhou 730050 Gansu Province, China.
Abstract:
Streptococcus alactolyticus strain FGM, isolated from chicken cecum, was used to increase the extract yield of polysaccharides during Astragalus membranaceus fermentation. It was previously demonstrated that polysaccharides from fermented A. membranaceus by S. alactolyticus had some similar properties to those from A. membranaceus in terms of its ability to help heal hepatic fibrosis in rats and modulate immunopotentiation of broiler chicken. However, methods to increase the yield of the polysaccharides during fermentation of A. membranaceus are not well understood. In this paper, we investigated the involvement of uridine diphosphate (UDP)-glucose 4-epimerase (galE) and glucan-1,6-α-glucosidase (dexB) during A. membranaceus fermentation through real-time reverse transcription quantitative PCR. The galE and dexB genes of S. alactolyticus were cloned by homology-based cloning and the genome walking method for the first time, and the 3D structure of dexB was analyzed by Swiss-PdbViewer 4.0.1 software. The expression of both the galE and dexB genes in A. membranaceus fermentation was studied using the determined ideal reference gene ldh for transcript normalization. The results showed that these two genes were both highly induced and peaked after 12 h of fermentation. The expression level of galE was stepwise increased from 48 to 72 h, while dexB transcripts were markedly increased at 60 h and decreased by 72 h. These data suggested that dexB and galE of S. alactolyticus strain FGM were involved in the regulation of A. membranaceus fermentation and they might play some roles in the increase of polysaccharides.
Insights
Streptococcus alactolyticus fermentation of Astragalus membranaceus increases polysaccharide yield. Key genes, uridine diphosphate (UDP)-glucose 4-epimerase (galE) and glucan-1,6-α-glucosidase (dexB), are highly induced, suggesting their role in polysaccharide production.
Area of Science:
- Microbiology and Biotechnology
- Biochemistry and Molecular Biology
Background:
- Astragalus membranaceus polysaccharides exhibit beneficial properties, including hepatic fibrosis healing and immunomodulation.
- Streptococcus alactolyticus fermentation enhances polysaccharide extraction from A. membranaceus.
- Understanding the molecular mechanisms regulating polysaccharide yield during fermentation is crucial.
Purpose of the Study:
- To investigate the involvement of specific genes in A. membranaceus fermentation by S. alactolyticus.
- To analyze the expression patterns of uridine diphosphate (UDP)-glucose 4-epimerase (galE) and glucan-1,6-α-glucosidase (dexB) genes.
- To explore the potential role of these genes in increasing polysaccharide yield.
Main Methods:
- Isolation and characterization of S. alactolyticus strain FGM from chicken cecum.
- Cloning of galE and dexB genes using homology-based cloning and genome walking.
- Analysis of dexB 3D structure using Swiss-PdbViewer 4.0.1.
- Real-time reverse transcription quantitative PCR to study gene expression during fermentation, with ldh as the reference gene.
Main Results:
- The galE and dexB genes were successfully cloned from S. alactolyticus.
- Both galE and dexB genes showed high induction, peaking at 12 hours of fermentation.
- galE expression increased stepwise from 48 to 72 hours, while dexB expression peaked at 60 hours and decreased by 72 hours.
Conclusions:
- The genes dexB and galE of S. alactolyticus strain FGM are involved in the regulation of A. membranaceus fermentation.
- These genes likely play a significant role in enhancing polysaccharide production during fermentation.
- Further research can leverage these findings to optimize fermentation processes for increased polysaccharide yield.
