The Effect Of High Fat Diet On The Expressions Of The Clock Related Genes In The Skeletal Muscle And Liver Of Chinese Soft-shelled Turtle

The Chinese soft-shelled turtles were used in this study to explore the effect of high fat diet on daily rhythm expressions of the core clock genes and important functional genes and microRNA in the skeletal muscle and liver.The methods and results were as follows:Using cloning,sequencing and bioinformatics analysis method,we obtained ORF sequences sizes of three core clock genes including Clock,Bmal2 and Per2 in the Chinese soft-shelled turtles were 2553 bp,1902 bp and 3888 bp,which encoded 850,633,1295 amino acides,respectively.The two predicted proteins of Clock and Bmal2 possessed three function domains: a b-HLH DNA-binding(BHLH)domain,two conservative PAS domains,while Per2 protein possessed only a conservative PAS domain.Phylogenetic tree analyses of the three genes as Clock,Bmal2 and Per2 indicated that the Chinese soft-shelled turtles had a close relationship with the other reptiles and birds,but they were far separated from teleostean and mammals.The optimal reference genes were detected from six candidate reference genes such as GAPDH,18 S rRNA,RPL13,RPL19,RPS2 and ?-actin by using qRT-PCR method and three softwares analyses of geNorm,NormFinder and BestKeeper.The resulted indicated that the most stable reference genes in the skeletal muscl and liver of the Chinese soft-shelled turtle were RPL13 and RPL19,respectively.The 180 juvenile Chinese soft-shelled turtles were randomly divided into two groups.They were fed for six weeks by a control fat diet(the CON group,7.98% lipid)and a high fat diet(the HFD group,13.86% lipid),respectively.They were also administrated by the photophase regimen of 24 h light/dark(12L:12D)cycle.At the end of the experiment,the tissue samples of muscle and liver were collected from 9 turtles of each group every 3 h(Zeitgeber time: ZT0,3,6,9,12,15,18,21 and 24,it was 9 times for 24 h).The influence of the high fat diet on daily rhythm expressions of the core clock genes,important functional genes and microRNA in the skeletal muscle and liver were investigated using RT-qPCR technology,SPSS significant analysis and cosine analysis methods.The results were as follows:(1)The expression patterms of 13 core clock genes(Clock,Bmal2,Tim,Cry1/2,Per1,DBP,AANAT,NIFL3,BHLHE40,NR1D2 and RORA/C)and 9 important muscle functional genes(FBXO32,MSTN,Myf5,Myf6,MyoD,MyoG,PCNA,Trim63 and UCP3)exhibited circadian oscillations in the skeletal muslcle of the CON group.Among these genes,they showed positive or negative correlation with each other.However,in the HFD group,the circadian rhythm expression patterns of 8 core clock genes(Clock,Bmal2,Cry2,Per1,DBP,NFIL3,BHLHE40 and RORA)and 6 important muscle functional genes(MSTN,Myf5,MyoD,MyoG,PPARa and Trim63)were disrupted.The positive or negative correlationship with each gene were also changed by HFD.(2)Similarly,the expression characters of 11 core clock genes(Clock,Bmal1/2,Tim,Cry2,Per2,NFIL3,NR1D1/2 and RORB/C)and 16 lipid metabolism genes(FAS,DGAT1,HMGCR,LDLR1,LIPIN1,CPT1 A,PPAR?,PPAR?,PPAR?,Sirt1,APOA1,APOB,PDK4,ACSL1,Lxr? and RXRA)exhibited circadian oscillations in the liver of the CON group.Among these genes,they also showed positive or negative correlations with each other.However,in the HFD group,the circadian rhythms expression patterns of 8 core clock genes(Clock,Cry2,Per2,NFIL3,NR1D1/2 and RORB/C)and 7 lipid metabolism genes(FAS,CPT1 A,Sirt1,APOA1,APOB,PDK4 and ACSL1)were destroyed,and the other genes showed the advanced or delayed expression peaks compared to the CON group.The positive or negative correlationship of these genes were also altered by HFD.(3)Three important microRNAs exhibited circadian oscillations in the liver of the CON group.They were strong positive or negative correlationship with both the core clock genes(Clock?Bmal1 ? Bmal2)and lipid metabolism genes(Lxr??PPAR? and Sirt1).However,in the HFD group,their circadian rhythm patterns and expression levels were altered to go along with their positive or negative correlations being changed.In summary,these results demonstrated that feeding high fat diet could altere the circadian rhythm of the core clock genes,important functional genes and microRNA in the skeletal muscle and liver of Chinese soft-shelled turtles,and also change the correlationship with each gene.These obtained results would play important roles in accumulating data to reveal the relationship between circadian clock and related gene expression by HFD in the Chinese turtle,and have some theoretical value and production guidance significance.