Differential CTX-M Expression from a Conserved Promoter: Role of Promoter-Associated Spacer Sequences Downstream of the blaCTX-M Regulon
Differential CTX-M Expression from a Conserved Promoter: Role of Promoter-Associated Spacer Sequences Downstream of the blaCTX-M Regulon
复制标题
保守启动子的差异 CTX-M 表达：blaCTX-M 调节子下游启动子相关间隔序列的作用
DOI：
10.1159/000445950
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发表时间：
2016-01-01
影响因子：
1.2
通讯作者：
Gao, Zhancheng
中科院分区：
文献类型：
--
作者：
Liu, Lin;Zhang, Xiangyan;Gao, Zhancheng
目的：该项目的目的是探索具有不同间隔序列的单个保守启动子产生的不同 CTX-M 表达水平，假设其变异是 CTX-M 水平波动的关键因素。方法：对具有5种不同间隔序列的blaCTX-M启动子片段进行扩增、测序并克隆至携带绿色荧光蛋白（GFP）基因的pUA66表达载体中。使用荧光显微镜、流式细胞术和qRT-PCR分析转接合子中blaCTX-M的表达。结果：所有blaCTX-M基因的启动子均由ISEcp1提供，且极其保守。启动子相关的间隔序列从 42 bp 到 127 bp 不等，并且观察到 5 个转接合子中 GFP 表达的变化。通过影响blaCTX-M表达的变化，在间隔序列中检测到核酸缺失和点突变。结论：不同的间隔序列对保守启动子的活性有显着影响。与之前的报道相反，保守启动子和 blaCTX-M 基因之间较短的间隔序列不会特异性增强 blaCTX-M 的表达。 blaCTX-M 的表达可能受到不同间隔序列引起的启动子活性变化的调节。
Aims: The aim of this project was to explore the different CTX-M expression levels occurring from a single conserved promoter with different spacer sequences, the variation of which is hypothesized to be a key factor in fluctuating levels of CTX-M. Methods: The blaCTX-M promoter fragments with five different spacer sequences were amplified, sequenced and cloned into the pUA66 expression vector carrying the green fluorescent protein (GFP) gene. The expression of blaCTX-M in the transconjugants was analyzed using fluorescence microscopy, flow cytometry and qRT-PCR. Results: The promoters of all the blaCTX-M genes were provided by ISEcp1 and were extremely conserved. The promoter-associated spacer sequences varied from 42 to 127 bp and variations in GFP expression in the five transconjugants were observed. A nucleic acid deletion and point mutation were detected in the spacer sequences by variations in which the expression of blaCTX-M was influenced. Conclusion: The different spacer sequences have a significant impact on the activity of the conserved promoter. The shorter spacer sequence between the conserved promoter and the blaCTX-M gene does not specifically enhance the expression of blaCTX-M, contrary to previous reports. The expression of blaCTX-M may be regulated by changes in promoter activity caused by diverse spacer sequences.