Regulatory Mechanism Of Cadmium Stress And MTF1 On Zebrafish Danio Rerio Id1 Gene

This study is to investigate the regulatory mechanism of MTF1?metal transcription factor?on id1 gene in zebrafish?Danio rerio?under acute CdCl₂ stimulation.Real-time PCR ? EPC cell transfection assay and dual-luciferase report experiments were proceeded over the course of the study.In addition,we assessed id1 promoter activity through in vitro and in vivo experiments.There are three contents in our study:Part One.Zebrafish id1 gene and mtf expression levels after CdCl₂ stimulation.Zebrafish were respectively exposed to 0?control?,1/25?low dose group?and 1/5?high dose group?LC50 CdCl₂ for 96 h.Five organs?liver,gill,intestine,spleen and kidney?were collected for RNA extraction and reverse transcription.The expression of id1,mtf1 and mtf2 were determined by quantitative PCR?qPCR?.In liver,all of them were up-regulated after CdCl₂ stimulation.Specifically,id1 mRNA level increased significantly in high dose group,while mtf1 and mtf2 mRNA level increased significantly in low dose group compared to control group.In gills,the expression of id1 in high dose group was significantly higher than that in control group,but no significant difference was observed between low dose group and the control one;mtf1 expression significantly decreased in low dose group in comparison with control group,then recovered in high dose group.In intestine,spleen and kidney,the expression of the three detected genes were down-regulated in low dose group and then be up-regulated in high dose group,although no significant difference was observed.Part Two.Dual-luciferase report analysis for id1 regulatory mechanism study.We analyzed the id1 promoter sequence by bioinformatic methods and found that,there are 7 potential MRE?metal responsive elements?within the ³kb fragment before the transcription start site,and they were named MRE1-7.A series of plasmids with individual MRE site mutated and the MTF1 eukaryotic expression plasmids were constructed.Subsequently,id1 promoter activity in EPC cell with dual-luciferase report assay was carried out.The results showed that MTF1 can affect id1 expression in a dose dependent manner,and 7 MREs contribute differently for id1 promoter activity: MRE1 and MRE5 can promote the activity,while MRE3 and MRE7 repress it.Part Three.id1 promoter activity analysis though in vivo and in vitro studies.To explore the biological activity of zebrafish id1?inhibitor of differentiation/DNA binding protein 1?promoter,a set of zebrafish id1 promoter with different length were amplified from genomic DNA and inserted to luciferase vector PGL3-basic,and subsequently were transfected into EPC cell line and microinjected into zebrafish embryo at 1-2 cell stage.The promoter activity was determined with the dual-luciferase reporter system assay.We found that the id1 promoter activity varied when the length change,and the in vivo and in vitro studies revealed that id1 expression presented different patterns.Conclusions:1.CdCl₂ exposure can up-regulate id1 and mtf expression,which indicates their significant role in Cd stress.The similar expression patterns between id1 and mtf in liver may suggest there are signaling pathways between them.2.The results obtained from the transfection in EPC cells with dual-luciferase reporter assay indicated that the expression of id1 is,at least partly,regulated by MTF1 via MREs in id1 promoter.3.The results of in vitro and in vivo studies for id1 promoter activity showed that the id1 promoter activity varied when the length changed,and presented different patterns in vitro and in vivo,which indicated that the cis-element located in the id1 promoter were selectively used in different types of cells.