基于四环素调控系统的叶绿体启动子在大肠杆菌中活性检测

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

旨在研究四环素调控系统在叶绿体基因工程中调控启动子活性。以四环素基因及四环素特异性识别序列的核心操控区为基础,首先合成带有四环素核心操控区的prrn O1O2启动子,通过酶切连接的方法连接到表达载体Bio3-GFP,并在大肠杆菌中验证该启动子的活性。结果表明,在该启动子的驱动下GFP基因表达使菌落呈明亮绿色;构建四环素诱导下GFP基因的表达载体Bio3-Tet R-prrn O1O2-GFP,筛选出适应大肠杆菌生长的最高四环素使用浓度为5μg/m L;然后构建四环素调控系统下的GFP表达载体,在未加入四环素时,prrn O1O2启动子的功能被抑制,加入四环素后,GFP基因表达出绿色荧光蛋白。说明利用四环素调控系统可以在大肠杆菌中控制叶绿体启动子prrn O1O2的活性,从而避免了核基因组对质体基因组的调控干扰,为进一步利用质体基因工程育种提供有效的方法和途径。

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 prrnO102 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-prmO102-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 prrnO102 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 prrnO102 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.