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[汞(Ⅱ)诱导型发光报告基因系统的构建及其在红壤汞检测中的应用]

[Construction of Hg(2+)-induced luminescent reporter gene system and its application to detect mercury in red soil].

作者信息

He Wei, Han Cheng, Zhong Wen-Hui, Lin Xian-Gui

机构信息

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China.

出版信息

Huan Jing Ke Xue. 2011 Oct;32(10):3045-52.

PMID:22279922
Abstract

A luminescent reporter gene system was constructed by fusing the mercury-inducible promoter, P(merT), and its regulatory gene, merR with a promoterless reporter gene EGFP. A stable whole-cell reporter was created by mini-Tn5 and introducing the merR-egfp system cassette into the chromosome of Pseudomonas putida strain, then applied it for mercury detection in the red soil of Jiangxi province, the fluorescence density of the sensor strain was confirmed in soil extraction and fluorescence intensity was quantified by flow cytometry. The results showed positive correlation with the mercury pollutant in the concentration range of 0.04-50 mg x kg(-1). The background heavy metal irons such as Cr2+, Zn2+, Co2+, Cu2+, Pb2+, Ag+ at certain level did not interfere with the measurement. The key factor for detecting the fluorescence density was the induction time and the optimal temperature for EGFP expression was 30-35 degrees C. Spiked with 0.1 mg x kg(-1) Hg2+ and after 15 and 30 days incubation, red soil samples were extracted and evaluated water soluble, bioavailable, organic matter bound and residual fractions of mercury by both sensor strain and analytical way. The sensor strain appeared to have a detection range similar to that of atomic absorption spectroscopy (AAS) method and the effective detection ratio was 35%-64%.

摘要

通过将汞诱导型启动子P(merT)及其调控基因merR与无启动子的报告基因EGFP融合,构建了一个发光报告基因系统。利用mini-Tn5并将merR-egfp系统盒导入恶臭假单胞菌菌株的染色体中,创建了一个稳定的全细胞报告基因,然后将其应用于江西省红壤中的汞检测,在土壤提取物中确认了传感器菌株的荧光密度,并通过流式细胞术对荧光强度进行了定量。结果表明,在0.04 - 50 mg x kg(-1)的浓度范围内,与汞污染物呈正相关。一定水平的背景重金属离子如Cr2+、Zn2+、Co2+、Cu2+、Pb2+、Ag+不干扰测量。检测荧光密度的关键因素是诱导时间,EGFP表达的最佳温度为30 - 35℃。在添加0.1 mg x kg(-1) Hg2+并孵育15天和30天后,提取红壤样品,通过传感器菌株和分析方法评估汞的水溶性、生物有效性、有机质结合态和残留态部分。传感器菌株的检测范围似乎与原子吸收光谱法(AAS)相似,有效检测率为35% - 64%。

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