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Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker |
GU Xin-xi, TAN Jian-xin, TIAN Hong-tao, ZHANG Yu-lan, LUO Yun-bo , GUO Xing-hua |
1、College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001, P.R.China
2、College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, P.R.China
3、Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, P.R.China |
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摘要 Construction of a food-grade expression vector for application to lactic acid bacteria (LAB) is of importance for dairy fermentation system. An α-galactosidase (aga) gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid pRAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36e to substitute the primary antibiotic selectable marker of pMG36e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase (amy) gene from Bacillus licheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. The selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU mg-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putative molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.
Abstract Construction of a food-grade expression vector for application to lactic acid bacteria (LAB) is of importance for dairy fermentation system. An α-galactosidase (aga) gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid pRAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36e to substitute the primary antibiotic selectable marker of pMG36e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase (amy) gene from Bacillus licheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. The selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU mg-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putative molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.
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Received: 17 April 2013
Accepted:
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Fund: This work was supported by the National High-Tech R&D Program of China (863 Program, 2006AA10Z317). |
Corresponding Authors:
TIAN Hong-tao, Mobile: 13126883987, E-mail: tht631022@163.com
E-mail: tht631022@163.com
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About author: GU Xin-xi, Tel: +86-312-7528423, E-mail: helloguxinxi@163.com; TAN Jian-xin, Tel: +86-312-7528180, E-mail: jianxintan@sina.com |
Cite this article:
GU Xin-xi, TAN Jian-xin, TIAN Hong-tao, ZHANG Yu-lan, LUO Yun-bo , GUO Xing-hua.
2014.
Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker. Journal of Integrative Agriculture, 13(8): 1802-1808.
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