Molecular Characteristics,Transcriptional Regulation And Role Of Zinc Transporter In Lipid Metabolism Pathway Of Yellow Catfish

Zinc is an essential trace element for vertebrates and participates in a large number of physiological and biochemical reactions in fish.Excess or deficiency of zinc will cause disease in the fish body,so it is very important to maintain the zinc homeostasis in the fish body.Studies have shown that there are two ways for fish to absorb zinc,that is,from water through the gills or from food through the intestine.The ZIP family of zinc transporters plays an important part in these processes.Current research mainly focuses on the effect of zinc on the growth performance,tissue structure and some physiological and biochemical processes of fish such as oxidative stress,fat metabolism,etc.Few studies have focused on the transcriptional regulation of the ZIP family of zinc transporters.And the absorption pathway of different dietary zinc sources,such as nano-sized zinc oxide,and the mechanism of action on lipid metabolism are seldomly studied.In this paper,the yellow catfish was used to explore the transcriptional regulatory mechanism of the ZIP family of zinc transporters,the absorption of nano-zinc oxide and the influence mechanism on fat metabolism.The following are the main results obtained from our experiment:1.Molecular characteristics and tissue expression analysis of key genes in the ZIP family of Pelteobagrus fulvidracoOur research used RT-PCR and RACE technology to obtain 10 full-length cDNAs of zinc transport genes ZIP1,ZIP3,ZIP6,ZIP7,ZIP8,ZIP9,ZIP10,ZIP11,ZIP13 and ZIP14 from yellow catfish.Subsequently,through the analysis of the basic characteristics of molecular biology of their cDNA sequences and protein sequences,it was found that the total length of the base sequences of these zinc transport genes was 1070bp～3420bp,the ORF was 864bp～2685bp,and they encoded 287bp～894bp of amino acids.Domain analysis showed that all the putative proteins of zinc transport genes have similar domains to mammals.Our phylogenetic tree based on the putative amino acid sequence further verified these genes and inferred their evolutionary relationship.The phylogenetic tree results showed that in each ZIP branch,the yellow catfish gathers with other teleosts to form a unique branch,but was separated from the mammals.We detected the mRNA expression of 10 ZIP genes in 9 tissues and found that their expression in these tissues was different.These results indicated that the zinc transporter gene of Pelteobagrus fulvidraco was very conserved in evolution,which provides important evidence for its important role in lower vertebrate fish.The differences in tissue expression indicated the uniqueness of each zinc transport gene,and provided theoretical support for subsequent separate studies of its transcriptional regulation.2.Transcriptional regulation of the ZIP family of zinc transportersZIP3 and ZIP8 are widely expressed in many tissues and participate in the process of obtaining zinc from many cells of the body.However,ZIP3 and ZIP8 have many differences in tissue distribution,expression,subcellular localization and regulation.These differences are of great significance to their function of maintaining zinc balance in the body.In the second part of our research,we obtained the promoter sequence of ZIP3with a length of 2027 bp and ZIP8 with a length of 1664 bp,and predicted several key elements on the promoter,such as CREB,KLF4,MTF-1 And RREB1 and other binding sites.After constructing a plasmid with missing fragments,luciferase analysis confirmed the missing fragments that may have regulatory sites.In different deletion plasmids,the luciferase activities of ZIP3 and ZIP8 promoters varied with the zinc concentration.Site mutation and EMSA found that the 1327bp/1343bp MTF-1 binding site on the ZIP3promoter,the 248 bp/267 bp RREB1 binding site and the 1543bp/1557bp MTF-1 binding site on the ZIP8 promoter are functional sites point.Our results showed that low zinc increased the binding ability of MTF-1 and its response sites on the ZIP3 promoter,and high zinc increased the transcriptional activation of ZIP3 by RREB1.Zinc also promoted the ability of MTF-1 to bind to its response elements on the ZIP8 promoter.This study provides direct evidence for the response elements of MTF-1 and RREB1 on ZIP3 and MTF-1 on ZIP8 to Zn for the first time,which is very important for evaluating the nutrition and toxicity of Zn in vertebrates.3.Absorption patterns of dietary nano zinc oxide and its effect mechanism on lipid metabolismThe present study was conducted to explore the mechanism of nano-ZnO absorption and its influence on lipid metabolism in the intestine of yellow catfish Pelteobagrus fulvidraco.The experimental results showed that 100mg/kg dietary nano-ZnO（H-Zn group）significantly increased the intestinal Zn content of yellow catfish.ZIP6 and ZIP10mRNA expression levels were significantly higher in the H-Zn group than control group.ZIP1 and ZIP14 had less mRNA expression in H-Zn group than in 0mg/kg group（the control）and 50mg/kg group（L-Zn group）,and ZIP4 mRNA was more highly expressed in the control than in the L-Zn and H-Zn groups.With the addition of dietary nano-ZnO,Eps15,Dynamin1,Dynamin2,Caveolin1 and Caveolin2 mRNA expression levels showed a downward trend.Meanwhile,the protein expression level of Dynamin1 was significantly decreased in H-Zn group.When dietary nano-ZnO reached 100mg/kg,TG content increased significantly.The enzyme activity of 6PGD in the H-Zn group was significantly higher than that in the control.With the increase of dietary nano-ZnO,the enzyme activities of G6PD and ICDH had a rising trend.The mRNA expression of 6PGD,FAS,ATGL and SREBP1 increased with the addition of dietary nano-ZnO.The protein expression level of MTF-1 in the H-Zn group was significantly higher than that in the control and the L-Zn group.The mRNA expression of L-Zn and H-Zn groups of FXR was significantly lower than that of the control.The mRNA expression of small heterodimer partner（SHP）decreased significantly with the addition of dietary nano-ZnO,and the results of western blot showed that the protein expression level of SHP in H-Zn group was significantly higher than that of the control and L-Zn group.Our further in vitro study found that nano-ZnO can enter cells through encodytosis,while Zn²⁺absorption was dependent on ZIP pathway.Lipogenesis effects of nano-ZnO were mainly caused by nanoparticles rather than inorganic form and were probably dependent on key gene SHP which speculatively regulated by MTF1 and/or FXR.