Cloning and Expression Analysis of Furostanol Glycoside 26-O-<i>β</i>-glucosidase Gene in <i>Anoectochilus roxburhii</i>

LIN Jiang-bo; WANG Wei-ying; ZOU Hui; DAI Yi-min

doi:10.19303/j.issn.1008-0384.2020.04.009

Volume 35 Issue 4

Apr. 2020

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Article Navigation > Fujian Journal of Agricultural Sciences > 2020 > 35(4): 422-428

LIN J B, WANG W Y, ZOU H, et al. Cloning and Expression Analysis of Furostanol Glycoside 26-O- β-glucosidase Gene in Anoectochilus roxburhii [J]. Fujian Journal of Agricultural Sciences，2020，35（4）：422−428 doi: 10.19303/j.issn.1008-0384.2020.04.009

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LIN J B, WANG W Y, ZOU H, et al. Cloning and Expression Analysis of Furostanol Glycoside 26-O- β -glucosidase Gene in Anoectochilus roxburhii [J]. Fujian Journal of Agricultural Sciences，2020，35（4）：422−428 doi: 10.19303/j.issn.1008-0384.2020.04.009

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Cloning and Expression Analysis of Furostanol Glycoside 26-O-β-glucosidase Gene in Anoectochilus roxburhii

doi: 10.19303/j.issn.1008-0384.2020.04.009

Subtropical Agriculture Research Institute, Fujian Academy of Agricultural Sciences, Zhangzhou, Fujian　363005, China

Received Date: 2019-12-24
Rev Recd Date: 2020-02-01
Publish Date: 2020-04-01

Abstract

Abstract

Objective In order to further understand the regulatory mechanism of steroidal saponins biosynthesis and metabolism of Anoectochilus roxburhii, the full-length cDNA of ArF26G gene was cloned and its expression pattern in the stem and leaf was analyzed. Method The full-length cDNA of ArF26G gene was cloned by RACE. Prokaryotic expression vector was constructed with pET-28a (+), and induced by IPTG. The expression levels of ArF26G gene in stem and leaf of Anoectochilus roxburhii at different temperatures and planting times were analyzed by fluorescence quantitative PCR. Result The full-length cDNA of ArF26G gene is 1 982 bp with an 1 764 bp ORF, which encoded a protein of 587 amino acids containing a conservative domain of Glycosyl hydrolase 1 (GH1) superfamily. ArF26G is located in chloroplast and a putative chloroplast transit peptide of 33 amino acid residues at the N-terminus. The molecular weight of ArF26G protein is 66.48 kD, and the theoretical isoelectric (pI) is 5.31, and the instability coefficient is 37.92, it is a stable protein. Prokaryotic expression vector pET-28a-ArF26G was constructed and induced successfully by IPTG in E. coli BL21 (DE3). The results of fluorescence quantitative PCR analysis showed that the expression of ArF26G in stem was significantly higher than that in leaf, and the highest expression of tissue culture seedlings was at 25℃. When the planting time was up to 4 months and 5 months, the expression of ArF26G gene in the stem was 11.9 times and 23.3 times as much as that in the stem of tissue culture seedlings treated at 25℃ respectively. Conclusion In this study, the full-length cDNA of ArF26G gene was cloned. The expression of ArF26G gene was the highest at 25℃ or planting for 5 months.
- Anoectochilus roxburhii,
- Furostanol glycoside 26-O-β-glucosidase,
- steroidal saponin,
- gene expression

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References(23)

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