Bioinformatics Analysis Of Selenoprotein Gene In Ctenopharyngodon Idella And Its Expression Response To High Lipid And Selenium

In grass carp breeding,feeding high-fat diet(HFD)will cause liver lipid excessive accumulation,oxidative stress and inflammation,which will affect the health and quality of grass carp.It was found that selenium can improve the liver lipid accumulation of grass carp caused by HFD.Selenium(Se)plays a role in humans and animals through selenoprotein,but the research on selenoprotein in fish is not deep,especially in grass carp.Therefore,in this study,the molecular characteristics,tissue expression profile,and expression response of selenoprotein genes to high lipid and Se in grass carp were investigated by gene identification,bio-confidence analysis,in vivo feeding and liver cells culture.The main conclusions are as follows:Experiment 1.The cloning and bioinformatics analysis of 9 selenoprotein genes in grass carp.Complete CDS of 9 grass carp selenoprotein genes were obtained by PCR,gene sequencing and other methods.The length of complete CDS sequence of SELENOF(MW987785),SELENOM(MW987789),SELENOS(MW987793),SELENOP1(MW987794),SELENOP2(MW987787),SELENOE(MW987786),SELENOL(MW987792),SELENOU1a(MW987791),SELENOU1b(MW987788)is 462,465,567,1131,678,417,909,639 and 657 bp,respectively.Through amino acid sequence alignment and phylogenetic tree construction,it was found that these 9 selenoprotein genes were highly conserved in evolution,and had close genetic relationship with zebrafish and other cyprinids.The expression profile analysis showed that the 9 selenoprotein genes had high expression levels in hepatopancreas,fat and muscle tissues of grass carp.Experiment 2.Study on the expression response of selenoprotein genes of grass carp to High-fat and Se in diets.Grass carp were fed with control diet(C),HFD,HFD supplemented with 0.3 and 0.6mg/kg Se(HSe 0.3 and HSe 0.6)for 8 weeks.The hepatopancreas,adipose and muscle tissues were collected.The RNA was extracted.The triglyceride levels in hepatopancreas and the transcription levels of 9 selenoprotein genes in three tissues were detected.The results showed that HFD significantly increased triglyceride level(P < 0.05),while Se significantly decreased triglyceride level in hepatopancreas(P < 0.05).In hepatopancreas,compared with the group C,the transcription levels of SELENOF and SELENOS in the group HFD were significantly increased(P < 0.05),and SELENOU1 b was significantly decreased(P < 0.05)in the group HFD.However,the transcription levels of SELENOF,SELENOM,SELENOS and SELENOP2 were significantly decreased(P < 0.05),and SELENOE,SELENOL and SELENOU1 b were significantly increased(P < 0.05)in the group HSe compared with the group HFD.In adipose tissue,compared with the group C,the transcription levels of SELENOP2,SELENOE,SELENOL and SELENOU1 a in the group HFD were significantly increased(P < 0.05),while the levels of SELENOP2,SELENOEM,SELENOU1 a were significantly decreased(P < 0.05).Compared with the group HFD,the transcription levels of SELENOF,SELENOP2,SELENOE,SELENOL,SELENOUla and SELENOU1 b in the group HSe were significantly increased(P < 0.05).In muscle tissues,the transcription level of SELENOM in the group HFD was significantly decreased compared with the group C(P < 0.05);The transcription levels of SELENOP1 and SELENOU1a in the group HSe were significantly increased(P < 0.05),while SELENOP2 was significantly decreased(P < 0.05).The results showed that feeding HFD and HFD supplemented with Se affected the expression levels of these 9 selenoprotein genes in hepatopancreas,adipose and muscle tissues of grass carp.It is suggesting that selenoprotein genes play a certain role in the regulation of lipid metabolism in the tissues of grass carp.Experiment 3.Study on the expression response of selenoprotein genes in liver cells of grass carp to high lipid and Se.Compared with normal cultured liver cells of grass carp(group C),liver cells of grass carp were treated with oleic acid-induced lipid storage(group OA)and sodium selenite after oleic acid-induced lipid storage(group OS).The treated cell samples were collected,and the triglyceride content and selenoprotein genes transcription level were detected.The results showed that OA significantly increased triglyceride content in liver cells(P < 0.05),while sodium selenite significantly reduced the increase of triglyceride content caused by OA(P < 0.05).The m RNA level of SELENOW1 in liver cells of group OA was significantly lower than that of group C(P < 0.05),and the m RNA levels of SELENOF and SELENOS decreased after OA treatment(P > 0.05).Compared with group C,the m RNA levels of SELENOM,SELENOP1,SELENOE,SELENOL,SELENOU1 a and SELENOU1 b in group OA increased,but there was no significant difference(P > 0.05).Compared with group OA,the m RNA levels of SELENOS,SELENOP2,SELENOE and SELENOW1 in group OS increased significantly(P < 0.05),while SELENOF,SELENOM,SELENOU1 a and SELENOU1 b decreased(P > 0.05).These results indicate that sodium selenite can regulate the expression level of selenoprotein genes in fatty liver cells of grass carp induced by OA,and further verify that selenoprotein genes play an important role in lipid metabolism of grass carp liver in vitro.To sum up,the selenoprotein genes of grass carp is highly conserved in evolution and highly expressed in tissues related to lipid metabolism(hepatopancreas,fat and muscle tissues).High lipid and high lipid supplemented Se treatment can regulate the expression levels of selenoprotein genes in hepatopancreas,adipose tissues,muscle tissues and liver cells of grass carp,suggesting that selenoprotein genes play an important role in the process of Se reducing lipid accumulation in grass carp.This study provides basic data for revealing the mechanism of selenoproteins in the regulation of lipid metabolism in fish by Se.