Shaolin Xie,Bing Yang,Siying Li,Liangjun Ge,Min Li,Qingshi Chen,Xian Qing,Jixing Zou
CYP1A is the most commonly used biomarker and transgenic fish which carrying a cyp1a promoter to drive a reporter gene can be used as reliable way to monitor dioxin/dioxin-like compounds (DLCs) in the environment. Here, we cloned the cyp1a promoter of Gambusia affinis and this promoter showed stronger transcriptional activity than that of zebrafish. Then, a Tg(GAcyp1a:eGFP/Luc) transgenic zebrafish line was first constructed with the G. affinis cyp1a promoter driving eGFP expression using meganuclease I-SceI mediated transgenesis technology. The Tg(GAcyp1a:eGFP/Luc) larvae at 72 h post-fertilization (hpf) were tested by exposing to TCDD for 72 h, and induced GFP was mainly expressed in the liver with low background. The Tg(GAcyp1a:eGFP/Luc) zebrafish showed high sensitivity (limit of detection of 0.322 ng/L TCDD and 0.7 TEQ-ng/L PCDD/Fs) and specificity (insensitive to responses to PAHs and PCBs). In addition, the transgenic line showed a low detection concentration of the DLCs contaminated environmental samples (as low as 1.8 TEQ-ng/L), and the eGFP fluorescence intensity and the chemical-TEQ values were closely correlated. In conclusion, a sensitively and specifically transgenic zebrafish line was established to convenient and effective to detect DLCs in the environment.
Dioxin-like compounds (DLCs) are environmental xenobiotics that can activate the aryl hydrocarbon receptors (AhR), thereby imposing a significant threat to human health through biomagnifications processes. In this study, a dioxin-activated nano-luminescent Saccharomyces cerevisiae bioreporter, called DnaSc, was developed for simple and rapid detection of DLCs and AhR agonists. The bioreporter used nano-luciferase (NLuc) as a signal generator to emit bioluminescent signals in response to DLCs without cell lysis. Through optimizing AhR nuclear translocator (ARNT) expression and engineering the AhR, the yeast-based bioassay exhibited a detection limit of 10 fM for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) within 6 h, making it the most sensitive whole-cell biosensor reported to date. Furthermore, the detection capacity of the dioxin-activated nano-luminescent S. cerevisiae bioreporter (DnaSc) bioassay for DLCs and AhR agonists was characterized. In summary, the yeast-based bioreporter developed in this study provided a simple, sensitive, and cost-effective method for DLCs detection.
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