Study Of Gene Cloning,Expression,Polymorphism And Regulation Of Key Gene Of Hufa Synthesis In Common Carp (Cyprinus Carpio Var Jian)

Fish are the most important source of n-3 highly unsaturated fatty acids(HUFA),which have beneficial health effects on humans.A6-desaturase and Elovl5 elongase are the critical enzyme in the biosynthetic pathway of HUFA in fish.Common carp(C.carpio var.Jian)is a freshwater,omnivorous and bottomliving specie,and part of the food come from the benthicanimal.Common carp(C.carpio)is commonly regarded as tetraploid due to its high chromosome number and DNA content.This has led to the hypothesis that common carp may have more complicated mechanism in the regulation in HUFA biosynthesis than other freshwater fish.In the paper,for the first time,the present work successfully cloned two A6-desaturase-like and two Elovl5-like elongase genes from common carp,and investigated the gene structure,spatial and temporal expression of two A6-desaturase-like and two Elovl5-like elongase genes cDNAs and genome.To find the SNP loci relative to the growth of common carp in two ?6-desaturase-like and two Elovl5-like elongase genes,that will in favour of doing the molecular marker-assisted breeding in common carp.By regulated the EFA in feed and the water temperature of common carp,the pathway and mechanism of HUFA synthesis have been systematic studied.Trying to find a reasonable ways of producing the rich in HUFA common carp from the nutrition,environment,ecology and melocular level.The results will be benefit to reveal the melocular mechanism of HUFA biosynthesis in polyploid fish.1.Cloning and sequence analysis of two ?6 fatty acid desaturase genes of common carp(Cyprinus carpio var Jian)Using a combination of RT-PCR and RACE,two ?6-desaturase-like cDNAs(termed ?6-Fad-a and ?6_Fad-b)were cloned from common carp.The results show that the full-length cDNAs of?6-Fad-a and ?6-Fad-b were 1996bp,1931 bp,respectively,and both have ORFs of 1335bp encoding proteins of 444 amino acids.The two proteins sequences included all the characteristic features of microsomal fatty acyl desaturases,including three histidine boxes,two transmembrane regions,and one cytochrome b5 domain.Multiple sequences alignment analysis revealed that the deduced amino acid sequences of A6-Fad-a and A6-Fad-b share high percentage of identity with those of other fish.The identity between A6-Fad-a and A6-Fad-b is 90.5%.Phylogenetic analysis by the neighbor-joining method showed that the common carp A6-desaturase-like genes were clustered in the fish A6-Fad groups.PCR was performed to amplify genomic DNA fragment of A6-desaturase using primers,which were designed on the full-length of A6-desaturase cDNA and the intronic sequences cloned.Spliced the isolated the DNA fragment sequences,the A6-Fad-a and A6-Fad-b DNA sequences cloned both being comprised of 13 exons and 12 introns.Comparison of the cDNA and genomic sequences for A6-Fad-a and A6-Fad-b enabled the identification of an upstream 5' non-coding exon.The two fad genes had identical coding exon structures,being comprised of 12 coding exons,with a high degree of sequence identity.Identification of these as separate genes was confirmed as each had introns of distinct size and sequence composition.Clearly,they were not allelic variants of a single gene.Intron regions could be aligned with sequences of RepBase entries for various DNA transposons.2.Cloning and sequence analysis of two fatty acid elongase(Elovl5)genes of common carp(Cyprinus carpio var Jian)Using a combination of RT-PCR and RACE,two Elovl5-like elongase cDNAs(termed Elovl5-a and Elovl5-b)were cloned from common carp.The putative two Elovl5-like elongase were shown that the full-length cDNAs of Elovl5-a and Elovl5-b were 1225bp,1167bp,respectively,and both have ORFs of 876bp encoding proteins of 291 amino acids.The protein sequences included characteristic features of microsomal fatty acyl elongases,both including a single histidine box redox centre motif(HXXHH),an endoplasmic reticulum retention(ER)signal and multiple transmembrane regions.Multiple sequences alignment analysis revealed that the deduced amino acid sequences share high identity with elongase of other teleosts.The identity between Elovl5-a and Elovl5-b is 94.1%.Phylogenetic analysis by the neighbor-joining method showed that the common carp Elovl5-like elongase genes were clustered in the fish elovl5 groups.PCR was performed to amplify genomic DNA fragment of fatty acid elongases(Elovl5)using primers,which were designed on the full-length of fatty acid elongases(Elovl5)cDNA and the intronic sequences cloned.Spliced the isolated the DNA fragment sequences,the Elovl5_a and Elovl5-b DNA sequences cloned both being comprised of 8 exons and 7 introns.Comparison of the cDNA and genomic sequences for Elovl5-a and Elovl5-b enabled the identification of an upstream 5' non-coding exon.The two elongases genes had identical coding exon structures,being comprised of 7 coding exons,with a high degree of sequence identity.Identification of these as separate genes was confirmed as each had introns of distinct size and sequence composition.Clearly,they were not allelic variants of a single gene.Intron regions could be aligned with sequences of RepBase entries for various DNA transposons.3.The spatial and temporal expression of ?6-desaturase and Elovl5 elongase genes and the correlation analysis fatty acid composition with the genes.In the paper,the spatial and temporal expression of ?6-Fad-a and A6-Fad-b,Elovl5-a and Elovl5-b genes of fertilized eggs,larvae,young and adult fish were determined using real-time PCR.The results shows that ?6-Fad-a and ?6-Fad-b,Elovl5-a and Elovl5-b have distinct difference in gene expression in different organs,respectively.The gene expression quantity of ?6-Fad-a and Elovl5-a gene in the liver and intestine is higher than that of A6-Fad-b and Elovl5-b gene of young and adult common carp,respectively.The highest expression of A6-Fad and Elovl5 genes was in the liver,secondly is in the intestine.There is a dinstict change of ?6-Fad and Elovl5 genes during fertilized eggs,larvae and young carp.The gene expression level of ?6-Fad and Elovl5 reached the highest level at the 20th larvae,and the gene expression level of?6-Fad-a and Elovl5-a genes are higher than that of A6-Fad-a and Elovl5-a genes,respectively.The results shows that the fatty acid composition of carp fertilized eggs and larvaes is also changing with the ?6-Fad and Elovl5 genes,the content of total products and DHA from 1h to 48h of carp embryos shows downward trend,then rise and fluctuation.The correlation coefficient of the change of fatty acid composition with the?6-Fad and Elovl5 gene expression during carp fertilized eggs and larvae.The ?6-Fad gene expression maily be effected by the total products and DHA.However,the Elovl5 gene expression maily be effected by the C18 substracts.The ?6-Fad-a and A6-Fad-b,Elovl5-a and Elovl5-b gene may exist functional difference,respectively,which laid a foundation for further study carp jian HUFA synthesis pathway and regulation mechanism.4.Study on the regulation of ?6-desaturase and Elovl5 elongase genes in common carp(Cyprinus carpio var Jian)?6-desaturase and Elovl5 elongase genes are the critical enzyme in the biosynthetic pathway of highly unsaturated fatty acids(HUFA).The extent to which fish can convert C18 polyunsaturated fatty acids(PUFA)to C20/22HUFA varies with species.In this study,the effects of dietary fatty acids on fatty acid composition and expression of?6-desaturase-like and Elovl5-like elongase were investigated in common carp(Cyprinus carpio var.Jian).After the juveniles were fed three semi-purified diets(D1-3)for 6 weeks with different lipid source:D1,fish oil with high highly unsaturated fatty acids(HUFA);D2,corn oil with high linoleic acid(18:2n-6,LA),but no HUFA;D3,linseed oil with high a-linolenic acid(18:3n-3,LNA),but no HUFA.Comparing muscle fatty acids with fish fed D1-3,the content of LA and arachidonic acid(20:4n-6,AA)in common carp fed D2 was higher than that at initial,fed D1 and D3(P<0.05),but the content of LNA,eicosapentaenoic acid(20:5n-3,EPA)and docosahexaenoic acid(22:6n-3,DHA)were lower(P<0.05).However,the content of LA and AA in common carp fed D3 was lower than that at initial and fed D2(P<0.05),but the content of LNA,EPA and DHA were higher(P<0.05).The liver transcript abundance of Fad6-a and Elovl5-a in fish fed D2 and D3 for 6 weeks were generally higher than that at initial(P<0.05),but ?6-Fad-b and Elovl5-b were seldom effected by the diets.The results suggest that there are two different A6-desaturase-like and two different Elovl5-like elongase genes in common carp(C.carpio var.Jian).Common carp could synthesize HUFA from their precursors in vegetable oil.The dietary fatty acids affect the HUFA biosynthesis and are involved in regulation of A6-desaturase-like and Elovl5-like elongase genes expression of common carp.The transcriptional control of ?6-Fad-a and Elovl5-a genes expression maybe one mechanism whereby dietary fatty acids in regulation the HUFA biosynthesis,further study should be given to A6-Fad-a and Elovl5-a in future.This study investigated the mode of gene changes of two ?6 fatty acid desaturase and two fatty acid elongase(Elovl5)genes under the high-temperature and low-temperature stress.The results show that the carp larvae in the high temperature(32°C)for 96h,?6-Fad-a gene was significantly elevated from 12h to 48h,and then markedly decreased.The ?6-Fad-b is not obvious under the high-temperature and low-temperature state,Elovl5-a Elovl5-b genes trend occurred under the high-temperature and low-temperature environment.There are significant differences of the two A6 fatty acid desaturase and two fatty acids elongase(Elovl5)genes in the stress involved in the regulation of temperature,respectively,in the liver,brain and gut.Carp jian at low temperature,?6-Fad-a gene in the liver first 4d expression levels highest and in the brain and intestinal 7d of expression highest.In addition,?6-Fad-b gene in the liver did not change significantly,while in the brain and the gut rendered obvious changes.Similarly,Elovl5-a gene in the liver,brain and intestinal temperature stress,among the amount of gene expression,there are obvious changes in the timing differences;Elovl5-b is not obvious in the liver and brain changes,and presented clear trend in the gut.5.Single Nucleotide Polymorphisms(SNP)of ?6-Fad and Elovl5 segments and their association with the growth traits of common carp(Cyprinus carpio var Jian)Highly unsaturated fatty acids(HUFA)are crucial to the nutritional health,physiology and reproductivity of vertebrates.A6-desaturase and Elovl5 elongase are the critical enzyme in the biosynthetic pathway of HUFA.Single nucleotide polymorphism(SNP)analysis of genes functionally related to the growth traits of common carp(Cyprinus carpio var Jian)can provide useful information for common carp molecular breeding.We isolated two ?6-Fad genes and two Elovl5 genes from common carp in the experiment.Thirty-six SNPs loci were identified on ?6-Fad and Elovl5 respectively.PCR-RFLP was applied to detect genotypes of three SNPs(A6-Fad-a intron10_C73T??6-Fad-b intron 10 A56G and Elovl5-a intron 5_C64A)in 615 individuals(female:male,335:280).Correlation analysis between genotypes and weight gain showed that?6-Fad-a intron10_C73T was associated with common carp weight gain significantly(P<0.05),and female juvenile with TT genotype higher in weight gain significantly(P<0.01)than that with CC genotype.But the adult common carp with TT genotype higher in weight gain significantly(P<0.05)than that with CC genotype.Correlation analysis between genotypes and weight gain showed that ?6-Fad-b intron 10_A56G was associated with common carp weight gain significantly(P<0.01),and female juvenile with GG genotype higher in weight gain significantly(P<0.01)than that with AA genotype.But the adult common carp with GG genotype higher in weight gain significantly(P<0.01)than that with AA genotype.But to the Elovl5-a intron 5_C64A,the male juvenile with CC genotype higher in weight gain significantly(P<0.05)than that with AA genotype,and the female adult common carp with CC genotype higher in weight gain significantly(P<0.05)than that with AA genotype.In addition,weight gain increased with the enrichment of molecular SNPs makers,consistent with the characteristics of quantitative traits.These results indicate that the?6-Fad and Elovl5 genes can be a candidate gene that can be useful for common carp molecular breeding.