| Haliotis diversicolor supertexta is a commercially valuable mollusc species for both fisheries and aquaculture,its has a wide geographical distribution in warm water regions of southern provinces of China.However,disease outbreaks and substantial mortality of H.diversicolor supertexta have occurred at the postlarval and grow-out stages in southern China since 1990 s,the production of H.diversicolor supertexta has dramatically decreased and currently,the industry is close to collapse.genetic problems are considered to be the causes of the dramatic decrease in H.diversicolor supertexta production.and Selection a species which increasing the growth rate and adaptability to environmental conditions may be the major solutions for the mortality event.Understanding the gene growth-related traits of H.diversicolor supertexta will be extremely useful for the ongoing breeding programs,future Marker-Assisted Selection(MAS)and management of this species.The transforming growth factor(TGF)family which comprises a large number of structurally related multifunctional cytokines,control cell proliferation,cell differentiation and cell growth.Myostatin is an important member of the transforming growth factor(TGF)family that functions to regulate muscle devopment and growth in animals,and has become one of the most important taget genens for genetic improvement of the production of farmed animals.The bone morphogenentic protein(BMP)constitute a subfamily of the transforming growth factor type beta(TGF-?)supergene family,which palys an important role in animal organogenesis and bone development.BMP-2 plays an importantrole not only in osteoblast differentiation but also in pattern formation during development.The purpose of this study was to characterize and predict function of the myostatin and BMP-2 gene of the H.diversicolor supertexta which the important aquaculture shellfish.and the myostatin and BMP-2 gene of the H.diversicolor supertexta were cloned and characterized by rapid amplification c DNA ends(RACE)methods,and in silico the nucleotide and deduced amino acid sequcence of each gene are analyzed.the expression levels of these gene in embryos/lavae and adult tisues are analyzed by Quantitative real-time PCR.To analysis correlation single nucleotide polymorphisms of the myostatin and BMP-2 gene and growth traits of the H.diversicolor supertexta,the single nucleotide polymorphisms of the myostatin and BMP-2 gene were identified by direct sequencing of PCR products,and the association these SNPs with H.diversicolor supertexta growth traits,including shell length,shell height and body weight were analysed.Association analysis of SNPs with H.diversicolor supertexta growth traits of Hs-MSTN expression level by Quantitative real-time PCR.This study will be extremely useful for understanding the gene growth-related traits of H.diversicolor supertexta and future Marker-Assisted Selection(MAS).The major results are as follows.1.Molecular cloning and expression analysis of the myostatin cDNA in Haliotis diversicolor supertexta In this study,the myostatin(Hs-MSTN)cDNA of the H.diversicolor supertexta were cloned and characterized by rapid amplification c DNA ends(RACE)methods,The full length of Hs-MSTN cDNA sequence consists of 3755 bp contained a 5' untranslated region(UTR)of 324 bp,a 3' UTR of 1985 bp,and an open reading frame consists of 1446 bp encoding for a protein with 481 amino acid residues,with a calculated molecular mass of 54.96 ku,and the theoretical isoelectric point of 9.41,The structure of Hd-MSTN included a putative signal peptide(1-17 aa),a TGF-? propeptide domain(157-367 aa)and a conserved TGF-? domain(379-481 aa),Multiple sequence alignment results revealed conservation of the RRPR proteolytic site and nine conserved cysteines of the Hs-MSTN with MSTN from other animals,and two propeptide proteolytic sites RQRR(120-124 aa),RYRR(235-239 aa)were found.Phylogenetic analysis showed that the Hs-MSTN gene was clustered in the same subgroup with the Haliotis rufescens,Quantitative real-time PCR detection results indicated that the Hs-MSTN gene expressed widely in addutor musle,mantle,gonad,liver,gill,foot and the highest expression level was observed in the addutor musle,mantle,foot,and Hs-MSTN transcript was widely detected in early developmental stages unfertilized egg,fertilized egg,4-cell embryos,8-cell embryos,gastrulae,larvae,juvenile stage,and the higher in fertilized egg,in 4-cell embryos and 8-cell embryos,increases in gastrulae,larvae and decreases in juvenile stage.Our results indicate that MSTN is involved in musle growth rgulation of H.diversicolor supertexta.2.Molecular cloning and expression analysis of the BMP-2 cDNA in Haliotis diversicolor supertexta In this study,the Bone morphogenetic protein 2(Hs-BMP-2)cDNA of the H.diversicolor supertexta were cloned and characterized by rapid amplification cDNA ends(RACE)methods,The full length of Hs-BMP-2 cDNA sequence consists of 2572 bp contained a 5' untranslated region(UTR)of 123 bp,a 3' UTR of 1150 bp,and an open reading frame consists of 1299 bp encoding for a protein with 432 amino acid residues,with a calculated molecular mass of 48.59 ku,and the theoretical isoelectric point of 9.84,The structure of Hs-BMP-2 included a putative signal peptide(1-39 aa),a TGF-? propeptide domain(63-294 aa)and a conserved TGF-? domain(331-432 aa).Multiple sequence alignment results revealed conservation of the RLRR(272-275 aa)proteolytic site and seven conserved cysteines of the Hs-BMP-2 with BMP-2 from other animals.Phylogenetic analysis showed that the Hs-BMP-2 gene was clustered in the same subgroup with the Haliotis asinina.Quantitative real-time PCR detection results indicated that the Hs-BMP-2 gene expressed widely in addutor musle,mantle,gonad,liver,gill,foot and the highest expression level was observed in the addutor musle,mantle,foot,liver and Hs-BMP-2 transcript was widely detected in early developmental stages unfertilized egg,fertilized egg,4-cell embryos,8-cell embryos,gastrulae,larvae,juvenile stage,and the higher in fertilized egg,4-cell embryos,8-cell embryos,larvae,juvenile stage.Our results indicate that BMP-2 is involved in shell growth rgulation of H.diversicolor supertexta.3.Single nucleotide polymorphisms of myostation gene and its association with growth traits in Haliotis diversicolor supertexta In this study,the single nucleotide polymorphisms of the myostatin gene(Hs-MSTN)of H.diversicolor supertexta were identified by direct sequencing of PCR products,and association analysis of SNPs with H.diversicolor supertexta growth traits Hs-MSTN expression level by Quantitative real-time PCR.Nine SNPs(5 ?UTR g252 G>A;ORF g414 A>C,g558 C>A,g909 C>T and g960 A>T;3 ?UTR g2148 G>A,g2164 A>G,g2420 C>G and g2520 C>G)were identified in cDNA of Hs-MSTN,including 4 synonymous mutations.The results of genetic diversity showed that the polymorphism information content(PIC)value ranged from 0.1913 to 0.3621,and the expected heterozygosity and observed heterozygosity ranged from 0.2155 to 0.4778,0.2195 to 0.4471,respectively.The association these SNPs with H.diversicolor supertexta growth traits,including shell length,shell height and body weight were analysed.The g909 C>T SNP locus was found associate with shell length,shell heightand body weight.The TT type H.diversicolor supertexta showed significantly higher traits values than those of TC and CC type in body weight,and the TT type H.diversicolor supertexta also showed significantly higher traits values than CC type in shell length and shell height(P(27)0.05).On the contrary,significantly more Hs-MSTN transcripts were detccted in the addutor musle of those of TC and CC type than in TT type ones(P(27)0.05).Our results suggested that Hs-MSTN might regulate the addutor musle growth negatively,and SNP g909C>T can be used as a candidate marker for the slective breeding of H.diversicolor supertexta.4.Single nucleotide polymorphisms of BMP-2 gene and its association with growth traits in in Haliotis diversicolor supertexta In this study,the single nucleotide polymorphisms of the bone morphogenetic protein 2 gene(Hs-BMP-2)of H.diversicolor supertexta were identified by direct sequencing of PCR products,and association analysis of SNPs with H.diversicolor supertexta growth traits.Four SNPs(ORF g1176G>A,g1275C>T;3?UTR g1436G>T,g1506G>T)were identified in cDNA of Hs-BMP-2 including 2 synonymous mutations.The results of genetic diversity showed that the polymorphism information content(PIC)value ranged from 0.1560 to 0.3615,and the expected heterozygosity and observed heterozygosity ranged from 0.1287 to 0.3895,0.1713 to 0.4762,respectively.The association these SNPs with H.diversicolor supertexta growth traits,including shell length,shell height and body weight were analysed.SNP locus was not found associate with shell length,shell heightand body weight.Our results suggested that Hs-BMP-2 of SNP provide knowledge for the marker-assisted breeding of H.diversicolor supertexta. |
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