Western blot detection of PMI protein in transgenic rice

doi:10.1016/S2095-3119(15)61053-X

Journal of Integrative Agriculture ›› 2016, Vol. 15 ›› Issue (4): 726-734.DOI: 10.1016/S2095-3119(15)61053-X

Western blot detection of PMI protein in transgenic rice

RONG Rui-juan^1*, WU Peng-cheng^2*, LAN Jin-ping¹, WEI Han-fu², WEI Jian¹, CHEN Hao², SHI Jia-nan¹, HAO Yu-jie², LIU Li-juan¹, DOU Shi-juan¹, LI Li-yun¹, WU Lin³, LIU Si-qi³, YIN Chang-cheng², LIU Guo-zhen¹

¹ College of Life Sciences, Hebei Agricultural University, Baoding 071001, P.R.China
² Beijing Protein Innovation Co. Ltd., Beijing 101318, P.R.China
³ Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, P.R.China

收稿日期:2015-03-21 出版日期:2016-04-01 发布日期:2016-04-07
通讯作者: LIU Guo-zhen, Tel: +86-312-7528787, Fax: +86-312-7528250, E-mail: gzhliu@genomics.org.cn; YIN Chang-cheng, Tel: +86-10-80493132, Fax: +86-10-80485325, E-mail: yincc@genomics.cn
基金资助:
This work was supported in part by the Natural Science Foundation of Beijing, China (5121001) and the Cultivate New Varieties of Genetically Modified Organisms Technology Major Projects, the Ministry of Science and Technology of China (2009ZX08012-006B).

Western blot detection of PMI protein in transgenic rice

¹ College of Life Sciences, Hebei Agricultural University, Baoding 071001, P.R.China
² Beijing Protein Innovation Co. Ltd., Beijing 101318, P.R.China
³ Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, P.R.China

Received:2015-03-21 Online:2016-04-01 Published:2016-04-07
Contact: LIU Guo-zhen, Tel: +86-312-7528787, Fax: +86-312-7528250, E-mail: gzhliu@genomics.org.cn; YIN Chang-cheng, Tel: +86-10-80493132, Fax: +86-10-80485325, E-mail: yincc@genomics.cn
Supported by:
This work was supported in part by the Natural Science Foundation of Beijing, China (5121001) and the Cultivate New Varieties of Genetically Modified Organisms Technology Major Projects, the Ministry of Science and Technology of China (2009ZX08012-006B).

摘要/Abstract

摘要： Phosphomannose isomerase (PMI) encoding gene manA is a desirable selective marker in transgenic research. Understanding of its expression patterns in transgenic plant and establishing highly sensitive detection method based on immunoassay have great impacts on the application of PMI. In this study, PMI-specific monoclonal antibodies were generated using recombinant protein as immunogen, and could be used in Western blot to detect as little as 0.5 ng His-tagged PMI protein or rice expressed PMI protein in sample accounted for 0.4% of single rice grain (about 0.08 mg). PMI protein driven by CaMV-35S promoter was detected in dozens of tested tissues, including root, stem, leaf, panicle, and seed at all developmental stages during rice growing, and PMI protein accounted for about 0.036% of total protein in the leaves at seedling stage. The established method potentially can be used to monitor PMI protein in rice grains.

Abstract: Phosphomannose isomerase (PMI) encoding gene manA is a desirable selective marker in transgenic research. Understanding of its expression patterns in transgenic plant and establishing highly sensitive detection method based on immunoassay have great impacts on the application of PMI. In this study, PMI-specific monoclonal antibodies were generated using recombinant protein as immunogen, and could be used in Western blot to detect as little as 0.5 ng His-tagged PMI protein or rice expressed PMI protein in sample accounted for 0.4% of single rice grain (about 0.08 mg). PMI protein driven by CaMV-35S promoter was detected in dozens of tested tissues, including root, stem, leaf, panicle, and seed at all developmental stages during rice growing, and PMI protein accounted for about 0.036% of total protein in the leaves at seedling stage. The established method potentially can be used to monitor PMI protein in rice grains.

Key words: transgenic rice , protein expression , CaMV-35S promoter , phosphomannose isomerase (PMI) , Western blot

RONG Rui-juan, WU Peng-cheng, LAN Jin-ping, WEI Han-fu, WEI Jian, CHEN Hao, SHI Jia-nan, HAO Yu-jie, LIU Li-juan, DOU Shi-juan, LI Li-yun, WU Lin, LIU Si-qi, YIN Chang-cheng, LIU Guo-zhen. Western blot detection of PMI protein in transgenic rice[J]. Journal of Integrative Agriculture, 2016, 15(4): 726-734.

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Western blot detection of PMI protein in transgenic rice

Western blot detection of PMI protein in transgenic rice

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