The Mining And Functional Study Of Muscle Fat Trait-related Genes In Common Carp

Common carp(Cyprinus carpio)is one of the most widely cultured fish species.It is very important in global aquaculture,as its annual production exceeded 4.45 million tons,accounting for 10% of the freshwater aquaculture production.Common carp is not only important in global aquaculture industry,but also a traditional food in China.In the past decades,most of the excellent common carp species were cultured in China,which could supply animal protein for consumers to satisfy the market demand.With the development of consumer’s rising living standards,increasingly importance had been attached to aquatic product quality.It was continually met the growing of market demand for “quality”,such as meat quality,mouth feel,nutrient value of common carp.One of the effective ways to solve the problem is to develop good quality and high nutritional value of breeds using genes closely linked to flesh quality traits.Flesh quality traits were a series of complex traits influenced by various factors,which in fish,were mainly fat content,fatty acid content and composition.In this study,we selected F2 family of the mirror carp as subjects,and 250 K SNP assay of common carp was used to perform a genome-wide association study(GWAS).Thus,the SNP loci significantly related to muscular fat traits were identified,and candidate genes associated with muscle fatty acid and fat content traits were obtained.Candidate genes associated to fat traits were blasted to the whole genome annotation information of common carp.To validate whether genes were the crucial or not,partial genes were verified using cloning techniques,correlation analysis and RT-qPCR.To explore the mechanism action on how to effect between genes and fat traits,gene knock-out technique was the most direct and effective method on the basis of CRISPR/Cas9.These results offer valuable insights into the complex genetic basis for molecular breeding of fat metabolism and flesh quality improvement.The main results were listed as follows:1)To identify loci and candidate genes associated with muscle fatty acid and muscle fat content traits,genome-wide association study(GWAS)designed by full-sib family were performed using mixed linear model(MLM).Fifty-four loci surpassing the 5% genome-wide significant level were determined for 10 traits:C22:0,C24:1n9,C18:3n6,C20:3n3,C22:2n6,EPA,DHA,EPADHA,HUFA and n-3PUFA.Using common carp annotation information,34 loci were found in or near known genes.A total of 18 loci surpassing the genome-wide suggestive significance level were detected for 4 traits:fat content in dorsal muscle(MFdo),fat content in abdominal muscle(MFab),abdominal fat weight(AbFW),and AbFW as a percentage of eviscerated weight(AbFP),and 10 loci were harbored in or near known genes.Among them,one SNP(carp089419)affecting both AbFW and AbFP reached the 5% genome-wide significant level.Furthermore,the relative expressions of 8genes(ANKRD10A,TANC2,FJX1,CHKA,ADAM8 A,FASN,LPL-a and LPL-b)were detected by real-time PCR in dorsal muscle samples with high and low fat content phenotypic values,the results showed that 7 genes were significantly different.The expression of ANKRD10 A and TANC2 in the samples with high fat content was lower than that in low fat samples,and the difference was extremely significant(P<0.01).The expression of FJX1,CHKA,FASN,LPL-a and LPL-b in the high fat content samples was significantly higher than that of low fat content samples,among them,CHKA,FASN,LPL-a and LPL-b showed extreme significant differences(P<0.01).Therefore,ANKRD10 A,TANC2,FJX1,CHKA,FASN,LPL-a and LPL-bmight be the prospective candidate genes for further research.2)Three candidate genes(FASN,LPL-a and LPL-b)were selected for further study.The full-length cDNA of the three genes was cloned,and gene sequences,biological information and gene expression patterns were analyzed.The FASN cDNA(KY378913)was 8927 bp,which encoded 2511 amino acid,with 5’ untranslated region(UTR)of 202 bp and 3’ untranslated region(UTR)of 1192 bp.The full length of LPL-a cDNA(KM213240.1)was 2631 bp,encoding a protein of 507 amino acids,with 5’-UTR 162 bp and 3’-UTR 945 bp,moreover LPL-b cDNA(KM213241.1)was 2413 bp,encoding a protein of 507 amino acids,with 5’-UTR 158 bp and 3’-UTR 732 bp.Sequence homology and phylogentic analysis showed that the homology of FASNand other fish amino acids was 68%-96%,and it clustered closely with Sinocyclocheilus graham.The homology of LPL-a and LPL-bamino acids was 95%,and they shared 61%-93% sequence identity with other fish and were closest toCarassius auratus.Bioinformatics analysis of proteins revealed that the molecular weight of FASN was 274.1456 kD,isoelectric point was 6.10 and instability coefficient was 41.14,suggesting it an unstable protein.FASN protein was located in the cytoplasm and cell nucleus,without a signal peptide,and its secondary structure mainly included irregular coils,accounting for 44.66%.The molecular weight of LPL-a and LPL-b protein was 57.6688 kD and 57.5877 kD,respectively.LPL-a and LPL-b proteins were both located in the cell cytoplasm,with signal peptide of 23 amino acids,their secondary structures mainly included irregular coils,accounting for 50.10% and 51.87%,respectively.They were organised into two structurally distinct regions,consisting of a pancreat-lipase-like domain and a carboxy-terminal PLAT/LH2 domain connected by a flexible peptide.It was indicated that three genes were expressed in various tissues of common carp by using RT-qPCR analysis,FASN was expressed highly in brain,LPL-a and LPL-b were separately expressed highly in abdominal muscle and adipose tissue.We also detected three genes expression pattern in different development periods of common carp through RT-qPCR technique.The results showed FASN was expressed highly from zygote period rang to late gastrula period but the highest was in late blastula period,LPL-a and LPL-b which had the similar expression pattern were the highest at period of feeding.3)The correlation between candidate gene polymorphism loci and fat-related traits was analyzed in this study.22 SNPs were found in the three genes,FASN,LPL-a and LPL-b,according to the location of the genome and SNP database.Genotype data was obtained about SNPs in common carp by sequencing,and among those,17 SNPs were polymorphic across all the genotype and the remaining amplified identical fragment in size,respectively.On 17 polymorphic SNPs,the effective number of alleles(Ne)was 1.0195-1.9998,the average was 1.8094;the Observed heterozygosity(Ho)was 0.0145-0.7374,the average was 0.5070;the expected heterozygosity(He)was 0.0192-0.5012,the average was 0.44216.Correlation analysis showed that8 SNPs were significantly correlated with fat-related traits(P<0.05).In FASN gene,Carp165088(A>T)was significantly correlated with AbFW and SFA;carp165093(G>A)was significantly correlated with MFdo and n-3PUFA;carp165096(A>T)and carp005641(G>A)was significantly associated with EPA;carp005638(C>T)was significantly correlated with MFdo and n-6PUFA.The SNP,carp076703(T>C),located in LPL-a gene was significantly correlated with MFab.Two SNPs,carp 076738(T>G)and carp076740(T>C),harbored in LPL-b gene was significantly correlated with MFab,and carp076740 also was significantly correlated with SFA.From the above results,FASN,LPL-a and LPL-b might be the major candidate genes of fat-related traits.4)Here we generated LPL mutant common carp via the CRISPR/Cas9 technique.The number of F0 mutated individuals of LPL-a and LPL-b were 203 and 197,respectively.Eeffect of LPL mutation on fat deposition was studied and zebrafish was used as model.Two zebrafishLPL homozygous mutant lines were obtained which were named the NO.1 line(-13bp)and NO.2 line(+11/-7bp),meanwhile performed morphological and functional characterizations of those mutants.We observed that there was no significant phenotypic difference between homozygous mutants(LPL-/-)and wild-type(LPL+/+)20 days post-fertilization(dpf),and the growth rate was significantly slower by measured body weight(P<0.05).Compared withLPL+/+,abdominal mesenteric fat deposition was decreased and even abolished through anatomical observation at the age of 2 months post-fertilization(mpf).The content of saturated fatty acid(SFA),monounsaturated fatty acid(MUFA)and polyunsaturated fatty acid(PUFA)of LPL-/-were decreased,and SFA and PUFA were significantly different(P<0.05),while the content of triacylglycerol(TG)increased and the difference was extremely significant(P<0.01).Real time PCR results reveled the expression of LPL gene was significantly lower in LPL-/-zebrafish.In addition,the mutation of LPL gene leads to the expression of its upstream and downstream genes were down regulated in different degrees in the Glycerolipid metabolism and PPAR pathway.These results will provide a reference for the study of the mechanism of LPL in the fat deposition of common carp.