Activity Detection of Chloroplast Promoter Based on Tetracycline Regulatory System in Escherichia coli

doi:10.13560/j.cnki.biotech.bull.1985.2017.02.022

Abstract

Abstract: In order to study tetracycline regulatory system regulating the activity of promoter in chloroplast genetic engineering,having the tetracycline（Tet）gene and the core regulatory area of TetR（Tet repressor）specific recognition sequences as the basis,the promoter prrnO1O2 with tetracycline core regulatory area was synthetized,then ligated to expression vector Bio3-GFP,and the activity of the promoter was validated in Escherichia coli. The expression of the GFP gene driven by the promoter led colonies to be bright green. The expression vector Bio3-TetR-prrnO1O2-GFP for GFP gene induced by Tet was constructed,and screening experiment confirmed that the highest concentration of tetracycline for the proper growth of E. coli was 5 μg/mL. Finally,GFP expression vector based on tetracycline regulatory system was constructed,the function of prrnO1O2 promoter was suppressed while without adding tetracycline. GFP gene expressed to be green fluorescent protein after adding tetracycline. These results indicated that tetracycline regulatory system controlled the activity of chloroplasts promoter prrnO1O2 in E. coli,thus avoiding the regulation interference of nuclear genome to plastid genome,and which provides effective ways and methods to plastid genetic engineering breeding further.

Key words: tetracycline regulatory system, chloroplast, promoter, prrnO1O2, Escherichia coli

YU Xiao-jun, CAO Shao-yu, DONG Yu-mei, BI Bao-liang, ZHANG Ying-hua, XU Jun-qiang. Activity Detection of Chloroplast Promoter Based on Tetracycline Regulatory System in Escherichia coli[J]. Biotechnology Bulletin, 2017, 33(2): 149-154.

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