| MSTN gene is a typical negative regulatory factor that inhibit skeletal muscle growth and development in vertebrates,some studies found that loss of the function of MSTN induced by nucleic acid mutation can produce "double-muscled" animal,which showed a high application prospect in animal husbandry.Therefore,this gene has been considered a promising gene and researched for a long time in meat animal genetics breeding.Chinese mitten crab(Eriocheir sinensis)is a typical crustacean with high economic value.Es-MSTN gene has been studied and few phenotypic correlation SNPs has been obtained in our preliminary study.This aroused our interest and confusion,Is the synonymous mutation site of Es-MSTN associated with growth traits? Is there any production application value and breeding potential? Are there differences in transcriptional and translational activity at the synonymous mutation sites? All these questions need to be verified by experiments.In view of the above problems,more in-depth and systematic research has been carried out.Relevant research results are presented as follows.1.Gene structure and SNP analysis of Es-MSTN gene in Chinese mitten crab(E.sinensis)In this chapter,we cloned and analyzed the gene sequence and structure of Es-MSTN by gene molecular cloning technique and sequence homologous alignment in E.sinensis,the results found that Es-MSTN gene of E.sinensis has a high homology with vertebrates,gene structure analysis showed that Es-MSTN is made up of three exons,two introns and the non-translation region at the end of the sequence.Fortunately,we found two alleles of Es-MSTN,there were 10224 bp and 10220 bp respectively with the 24 bp insertion or deletion in the 598 th base of the first exon,and encoding 476 and 468 amino acids,respectively.The phenotypic association analysis found no correlation between the two alleles and the phenotype(P>0.05).In addition,we found two new synonymous SNPS nearby in the previously discovered SNP sites(C714T,A729 G and T753G),which was named C712 T and C717 T.The results showed that Es-MSTN gene of E.sinensis were highly conserved in the genome structure and functional domain with vertebrates,and this coding region is rich in SNP locus variation,which provided basic data for the subsequent studies on gene function and molecular-assisted marker breeding.2.Verification and correlation analysis of phenotypic association SNP of Es-MSTN gene in Chinese mitten crab(E.sinensis)In this chapter,we verified the SNP2(C714T)locus with significant correlation with phenotype(body weight)found in previous studies by 2873 samples.The results showed that the distribution frequencies of TT,TC and CC of the three genotypes of the SNP locus in 2873 large samples were 56.84%,37.52% and 5.64%,respectively.The corresponding mean body weight was 212.23±74.11 g,214.94±75.14 g and 208.46±79.72 g,respectively.There was no significant difference among the three genotypes(P > 0.05).Then we established self-fertilization family(TC×TC)of this locus to compare the growth dynamics difference among three genotypes(TT,TC,CC)in the first filial generation,and no significant correlation between genotype and phenotype was found in the phenotypic growth dynamics of the self-crossbred offspring(P>0.05).In addition,association analysis of 5 SNP loci with different genotypes were conducted and showed there were a total of 73 genotypes in 2873 samples,and two genotype combinations(CC/TT/AA/TT and CC/TT/AA/TG)with the highest proportion and high statistical significance.,and the analysis found two genotypes in female individuals had no significant genotype and phenotype correlation(P>0.05),however,the body weight of CC/TT/AA/TT genotype is significantly higher than CC/TT/AA/TG in male individuals(P < 0.05).Further analysis found that these two genotypes are subject to selection pressure,the genotype of CC/TT/AA/TT was positively selected,while the CC/TT/AA/TG was negatively selected(P < 0.05).The results indicated that the two genotypes(CC/TT/AA/TT and CC/TT/AA/TG)could be used as new marker locus in molecular marker assisted breeding of E.sinensis in future.3.Effects of synonymous mutations on gene transcription and translation in Chinese mitten crab(E.sinensis)Synonymous mutation can affect the transcription,translation and post-transcription levels in gene function.In this chapter,the total of 14 genotypes(GC,AT,1T,2T,3T,4A,5T,1C,2C,3C,4G,5G,GG,TT)that combined with the five SNP loci(T/C(C712T),T/C(C714T),T/C(C717T),A/G(A729G),and T/G(T753G))were employed by the means of site-directed mutagenesis,in vitro transcription,and gene overexpression.The result showed that the 14 alleles displayed obviously different transcriptional efficiency in vitro,there was the highest transcriptional level in 1C allele and the lowest in the GC and AT alleles(P<0.05),in which the AT allele exhibited significantly higher transcriptional level than the GC allele(P<0.05),however,the transcriptional intensity of GG was slightly higher than that of TT type,but there was no significant difference(P>0.05).The overexpression in 293 T cells found that expression intensity of different mutant genotypes was significantly different at different time(24 h,36 h,48 h,60 h,72 h,84 h,96 h,108 h)after transfection(P<0.05),and the AT allele exhibited significantly higher expression level than the GC allele(P<0.05),however,no significant differences were detected between the expression intensity of phenotypic association mutants(GG and TT)(P>0.05).Generally,the present study indicated that these five synonymous mutations have important effects on transcription and translation of Es-MSTN gene in E.sinensis,suggesting that synonymous mutations would play an important role in biological process.However,there was no significant difference in the molecular function of the gene coding region between the two phenotypic association mutations(GG and TT)(P>0.05),indicating that its influence on the phenotype may be affected by other regulatory regions or genes linked to this locus.4.RNA interference provides insights into the multi-functional profiles of Es-MSTN gene in Chinese mitten crab(E.sinensis)Myostatin(MSTN)is a negative regulation factor involved in skeletal muscle growth and development in vertebrates.In order to understand the multi-functional roles of MSTN in crustaceans,we assessed the molting / growth characters,analyzed the tissues composition and investigated the expression levels of lipid metabolism and immune related genes in Chinese mitten crab(E.sinensis)after injection of Es-MSTN ds RNA during one molt cycle of 49 days.We found the RNAi group displayed shorter molting interval,higher specific growth rate,and lower hepatosomatic index compared with the control group,evidencing MSTN may also be a negative regulator of muscle growth and development in E.sinensis.In the RNAi group,the expression levels were upregulated for lipid metabolism-related genes of pancreatic lipase(LP),carnitine palmitoyl transferases 1β(CPT1β)and fatty acid-binding protein 3(FABP3)in muscles,but were downregulated for these three genes and fatty acid-binding protein 9(FABP9)in hepatopancreas.Meanwhile,the expression levels were all upregulated for immunerelated genes of prophenoloxidase(PHE),anti-lipopolysaccharide factor(ALF),Lectin H(Lec H),and prophenoloxidase-activating factor(PPAF)in both muscle and hepatopancreas in the RNAi group.Further tissue components analysis showed that there were no significant differences in amino acid composition between the RNAi and control groups in muscle and hepatopancreas,but most of significant differences in fatty acid composition were detected between the two groups in both muscle and hepatopancreas.This study demonstrated that Es-MSTN is a vital muscle growth inhibition factor and displayed diverse function in participating in energy metabolism and immune response in E.sinensis. |
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