Transcriptional Regulation And Preparation Of Polyclonal Antibodies Of Several Copper Transporters In Yellow Catfish Pelteobagrus Fulvidraco

Copper(Cu)is an essential micronutrient and an indispensable cofactor.Cu can drive a range of biochemical processes that are essential to fish.Studies have shown that Cu deficiency or excess in waters and diets will adversely affect the growth,development and metabolism of fish,and even induce oxidative stress and the occurrence of fatty liver.The absorption of Cu by fish is mainly regulated by a series of related Cu transporters.Therefore,it is of great significance to study the mechanism of Cu absorption,transport and homeostasis for maintaining fish growth and preventing disease.Yellow catfish(Pelteobagrus fulvidraco)was selected as the research object in this study.Promoter sequences of Cu transport-related genes ctr1,ctr2 and atox1 were obtained by promoter cloning technology,and the regulatory effects of NRF2 and SREBP1 transcription factors on ctr1,ctr2 and atox1 promoters in response to Cu were studied.The effects of different levels of Cu on the expression levels of CTR1,CTR2 and ATOX1 genes and proteins in liver tissues and primary hepatocytes of yellow catfish were investigated.Polyclonal antibodies against CCS and COX17 proteins of yellow catfish were successfully prepared.The specific results are as follows:1.Transcriptional regulation of ctr1,ctr2 and atox1 genes in yellow catfishWe obtained ctr1(1359 bp),ctr2(1842 bp)and atox1(1825 bp)promoter sequences of yellow catfish,and predicted the presence of transcription factor binding sites(TFBS)on these promoters,such as NRF2 and SREBP1,etc.It was found that Cu incubation had different effects on the activities of ctr1,ctr2 and atox1 promoters with different fragment lengths by dual luciferase activity assay and EMSA assay.In addition,NRF2 could directly bind to the-326/-334 bp and-1232/-1240 bp sites in the atox1 promoter and mediated transcriptional regulation of the atox1 promoter through these two sites.SREBP1 could directly bind to the-91/-100 bp site in the ctr1 promoter and the-232/-241 bp and-699/-708 bp sites in the atox1 promoter and mediated transcriptional regulation of ctr1 and atox1 promoters through these sites.Cu inhibited the binding ability of NRF2 to atox1 promoter,but promoted the binding ability of SREBP1 to ctr1 and atox1 promoters.This study preliminarily revealed the potential mechanism of Cu regulating the transcriptional activities of ctr1,ctr2 and atox1 promoters through NRF2 and SREBP1 transcription factors,providing a new idea for studying Cu homeostasis regulation.2.Effects of different Cu levels on the expression levels of CTR1,CTR2 and ATOX1 genes and proteins in liver tissues and primary hepatocytes of yellow catfishOur in vivo experiment found that the expression levels of CTR1,CTR2 and ATOX1 m RNA and proteins in liver tissues were significantly reduced in the diet supplemented with excess Cu(CE)group compared with the diet supplemented with adequate Cu(AC)group.Primary hepatocytes of yellow catfish were incubated with different concentrations of Cu for 48 h.The results were similar to in vivo experiment,high concentration of Cu(10 μM)significantly decreased the m RNA and protein expression levels of CTR1,CTR2,and ATOX1.Intracellular co-localization indicated that CTR1 is mainly located in the cell membrane,CTR2 in the cell membrane and lysosome,and ATOX1 in the cytoplasm.This study laid a theoretical foundation for further study of the regulation mechanism of Cu homeostasis.3.Preparation of polyclonal antibodies against CCS and COX17 proteins of yellow catfishWe successfully expressed the ccs and cox17 genes of yellow catfish in the prokaryotic system by constructing the fusion plasmids of the target genes and the prokaryotic expression vector.The expressions of CCS and COX17 proteins in yellow catfish were determined by IPTG induction.CCS was purified in the form of inclusion body and COX17 in the form of soluble protein.Polyclonal antibodies against CCS and COX17 proteins of yellow catfish were successfully obtained by immunizing mice three times.Through SDS-PAGE identification,it was finally proved that these two antibodies can be used in the follow-up study.This study provided a powerful research tool to clarify the regulation mechanism of Cu homeostasis from the perspective of Cu chaperone.