The Response And The Molecular Regulation Mechanism Of Calcineurin In Gymnocypris Eckloni Under Cu²⁺ Stress

Gymnocypris eckloni,which is a typical species distributing in the upper region of the Yellow River and is an important component of biodiversity in the upper region of the Yellow River,not only has the economic value of exploitation and utilization,but also the important ecological protection value.In the past dozen years,with the development of industry and agriculture and the enhancement of man activities on Tibet plateau,the monitoring results of heavy metals in Qinghai Province fishery waters shows that it has been close to or even exceed the standard of water quality at some point in some monitoring sections,which has a potential impact on wild Gymnocypris eckloni.Therefore,it is very necessary to learn the response mechanism of Gymnocypris eckloni under heavy metal stress,and to find some biomolecules as biomarkers to monitor the waters quality accurately and quickly,providing a theoretical basis for the protection and restoration of fish in the upper region of the Yellow River.In this study,six genes were cloned by reverse transcriptase polymerase chain reaction?RT-PCR?and rapid amplification of c DNA ends?RACE?.Subsequently,the expression distribution of these genes were analyzed.Besides,the transcription levels of interleukin-1??IL-1??,tumor necrosis factor ??TNF-??and metallothionein?MT?in liver,kidney and gill tissues were measured by real-time fluorescence quantitative PCR?Real time PCR?when the concentration of Cu²⁺ is 0.01 mg/L.In addition,Real time PCR and Western blot were used to study the transcription and protein patterns of Calcineurin?CN?.It is concluded that CN and its signal path are involved in the immune regulation of Gymnocypris eckloni unde Cu²⁺ stress.The results were as follows:?1?The c DNA sequences of Calcineurin?CNA?,CNA? and CNB?,MT,IL-1? and TNF-? were obtained,which were named as Ge-CNA??KX772783?,Ge-CNA??KX772785?,Ge-CNB?KX772784?,Ge-MT,Ge-IL-1??KX772786?,and Ge-TNF-??KX772787?respectively.Besides,we determined the expression of IL-1?,TNF-?,MT,CNA?,CNA? and CNB by RT-PCR,showing that the expression level of genes was various in different tissues.?2?The result of Real time PCR showed that the expression of MT in liver,kidney and gill tissues were higher than that in the control group within72 h after Cu²⁺ stress.And the expression of MT in the kidney was the highest,followed by the liver and gills.In addition,IL-1? and TNF-? levels showed a trend of decreasing first and then increasing.It is concluded that MT,IL-1?and TNF-? play a role in immune response when the concentration of Cu²⁺ is0.01 mg/L.?3?The expression of CN in liver,kidney and gill,which all showed the trend of increasing,were similar within 72 h after Cu²⁺ stress.However,the expression of CNA? protein increased first and then decreased,indicating that CN responses to Cu²⁺ stress.Then it is speculated that CN may regulate the expression of IL-1?,TNF-? and MT through NF-AT/NF-?B pathway or combinating with CN dependent response element?CDRE?of MT upstream regulatory element.The results show that CN and related genes may play a key role in response to heavy metal stress and can be used as potential biomarkers to monitor and evaluate heavy metal pollution in waters.The results will provide some clues to further understand the function of CN,and provide a technical basis for the water quality evaluation in the upper region of the Yellow River.