Association Between Polymorphisms Of Fads2 And Elovl5 And Levels Of Unsaturated Fatty Acids In Carp

Fish is the main food source for human body to obtain n-3 HUFAs.It is of great significance to improve the ability of fish to synthesize n-3 HUFAs.Desaturase gene(fads2)and elongase gene(elovl5)are essential enzymes for controlling PUFAs synthesis in fish,and their catalytic efficiency and expression level directly affect PUFA synthesis ability.In the preliminary study of this project,two fads2 and elovl5 genes,fads2a/2b and elovl5a/5b,were identified,and PUFA content of different varieties of carp was significantly different.In order to elucidate the genetic mechanism of PUFA synthesis in carp,association analysis of fads2 and elovl5 gene polymorphisms and PUFA content in different breeds of carp was carried out in this study to identify the effects of related genetic loci on the expression regulation of fads2 and elovl5.Based on the polymorphism of CDS and promoter sequences of fads2 and elovl5 genes in three carp populations,this project will clarify the genetic basis and molecular mechanism of PUFA synthesis ability differences,so as to provide molecular basis for improving PUFA content of carp and breeding new varieties of carp.In this paper,the content of PUFA in three carp species was determined,and the data of 12 PUFAs traits were obtained.Then the CDS region and promoter region of fads2a/2b and elovl5a/5b genes were sequenced and genotyped to obtain the SNP loci of their genes.Finally,the correlation analysis between the genotypes and 12 PUFAs traits was carried out,and the key loci affecting PUFA synthesis were found.The specific experimental results are as follows:1.Determination and analysis of PUFAs content in muscle of Furui carp(FC),Huanghe carp(HHC)and Jian carp(JC)A total of 269 carp were selected from three populations with large differences in PUFA content: Furui carp,Huanghe carp and Jian carp.Fatty acid content in muscle of carp was determined by fatty acid methylation experiment and gas chromatography.The results showed that there was no significant difference in the distribution of PUFA content in the three carp populations,and the data distribution of Yellow River carp was more diffuse,but most of the data distribution of the three carp populations still overlain.Therefore,we analyze the three groups as one group.On the whole,the content of n-6 PUFAs in carp was significantly higher than that of n-3 PUFA.The content of C18:2n-6 was 25.24±10.13%,and decreased with the LC-PUFA synthesis pathway.2.Detection and analysis of SNP in CDS region and promoter region of fads2a/2b and elovl5a/5b genes in 3 populations of carpThe CDS fragments and promoter fragments of fads2 a,fads2b,elovl5 a and elovl5 b genes were amplified and sequenced.After Blast confirmation analysis,SNP typing was performed.A total of 34 c SNPs were found in the CDS region of the 4genes,including 5 fads2 a c SNPs,11 fads2 b c SNPs,10 elovl5 a c SNPs,and 8 elovl5 b c SNPs.Fads2 a included 1 non-synonymous mutation(F2a.502),and fads2 b included6 non-synonymous mutations(F2b.128,F2 b.168,F2 b.288,F2 b.291,F2 b.552 and F2 b.751).Elovl5 a included one non-synonymous mutation(E5a.29),and elovl5 b included three non-synonymous mutations(E5b.172,E5 b.174,and E5 b.182).Nineteen effective SNPs loci have been detected in the promoter regions of the four genes,of which 10 are fads2 b promoters,indicating that fads2 b has higher genetic diversity than the other three genes.In order to improve the accuracy of association analysis,the SNPs of the four genes were used to analyze the population structure of the three carp,and it was found that there was no significant difference in the distribution of SNPs among the three carp populations.Therefore,the three populations were regarded as one population in the subsequent association analysis.3.Correlation analysis between SNP of fads2a/2b and elovl5a/5b genes and PUFAs content in carpThe SNPs of fads2 a,fads2b,elovl5 a and elovl5 b genes were correlated with PUFAs content in carp.In order to improve the accuracy of the analysis,the combined analysis of GLM,MLM and ANOVA was carried out,and a total of 6 significantly associated loci(E5a.87,E5 b.172,E5 b.782,F2 a.624,F2 b.751 and F2 b.1197)were detected in the CDS region of 4 genes.E5 b.172,E5 b.782,F2 b.751 and F2 b.1197were all significantly correlated with multiple PUFAs,and the PUFA content in heterozygous was higher than that in homozygous,suggesting that elovl5 b and fads2 b may be more susceptible to genetic factors,thus affecting the synthesis of PUFA.Three significant loci(E5b.-932,F2 b.-397 and F2 b.-664)were detected in the core promoter regions of the four genes,but these loci were only associated with partial PUFA.We hypothesized that CDS protein coding region plays a leading role in fatty acid synthesis and promoter region plays a synergistic regulatory role.4.c SNP analysis of fads2 b and elovl5 b genes in Cyprinus carpioBased on the fact that fads2 b and elovl5 b are the main genes regulating PUFA synthesis in carp,four significant associated loci E5 B.172,E5 B.782,F2 b.751 and F2 b.1197 were analyzed.In the association analysis,the genetic effect of single locus on PUFA content was less than 10%,but in the aggregation analysis,the explanatory degree of four genes was significantly improved,among which C18:2n-6 reached54.95%.In summary,the association analysis between SNPs and fatty acids showed that fads2 b and elovl5 b played a major regulatory role in the process of PUFA extension and dehydrogenation,while fads2 b and elovl5 b played a relatively minor role.SNPs in different regions of the genome also have significant differences in effect,and SNPs in protein coding region is the core factor leading to the difference in PUFA content between individuals.The results showed that E5 b.172,E5 b.782,F2 b.751 and F2 b.1197 were effective molecular markers for screening PUFA in carp.