Analysis Of The Response Of Hepatic CYP1A In Sebastiscus Marmoratus To Marine Pollution Using Loop-mediated Isothermal Amplification

Induction of cytochrome P450enzymes has been proposed as one of the most sensitive biological response to environmental pollution. CYP1A (Cytochrome P4501A) gene is sensitive to the pollutants and it can be used as a biomarker to reflect the oil and other organic pollution in water. Therefore, it is important to establish a rapid and simple method to detect CYP1A to monitor environmental pollution.Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology. LAMP has been widely used in the qualitative detection of bacteria, parasites and virus because of its high sensitivity, specificity, convenient, efficiency, etc.In this study, LAMP technology for detecting the hepatic CYP1A gene in Sebastiscus marmoratus was established and optimized. Feasibility of LAMP in the quantification of CYP1A can be confirmed by comparing the results detected by real-time PCR. After alone or co-exposure to water soluble fraction (WSF) of oil, Cd and Pb, hepatic CYP1A mRNA in S. marmoratus was detected by quantitative LAMP to analysis the response of CYP1A to these contaminants.The main findings are as follows:(1) Establishment and optimization of the LAMP technology for the detection of CYP1A in the liver of S. marmoratus. The results showed that the best ratio of outer primers to inner primers was1:6. LAMP reaction was carried out in a total reaction volume of25μL containing6mmol-L-1Mg2+,0.4mmmol-L-1betaine and1.4mmol-L’1dNTP, the optimal amplification condition was at64℃for50min.(2) Expression levels of hepatic CYP1A mRNA in S. marmoratus were detected by LAMP and real-time PCR simultaneously after exposure to WSF, both of the results were highly consistent with each other. These results confirmed the feasibility of LAMP for the quantification of gene in marine fish.(3) Expression levels of CYP1A mRNA in the liver of S. marmoratus were detected by quantitative LAMP after exposure to WSF, Cd and Pb alone. The results showed hepatic CYP1A in S. marmoratus was sensitive to WSF and Pb exposure alone, however, there’s no significant response for Cd exposure. After exposure to WSF, CYPIA mRNA expressions increased first with increasing concentration of WSF, and reached the maximum at50μg·L-1group, then showed a downtrend when WSF concentrations were greater than50μg·L-1, but it was still higher than control. Pb exposure can significantly induce the expression of CYPIA mRNA in S. marmoratus. However, Cd did not affect the expression of CYPIA mRNA.(4) Expression levels of CYPIA mRNA in the liver of S. marmoratus after co-exposure to WSF and Cd or Pb were tested by quantitative LAMP. The results showed that hepatic CYPIA in S. marmoratus was not sensitive to co-exposure of WSF and Cd. In contrast, hepatic CYPIA in S. marmoratus was very sensitive to co-exposure of WSF and Pb. Pb strongly stimulated the induction of CYPIA by WSF.