九孔鲍MSTN基因cSNP多态性及与生长性状关联分析
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摘要
九孔鲍(Haliotisdiversicolorsupertexta)是中国南方具有较高经济价值的养殖贝类。肌肉生长抑制素(Myostatin,MSTN)是转化生长因子β超家族(TGF-β)中参与动物肌肉生长的重要调控因子,因此MSTN基因是动物遗传改良的重要候选基因。为探究九孔鲍MSTN基因的多态性及其生长相关性,实验采用PCR产物直接测序方法对九孔鲍MSTN基因进行单核苷酸多态性(SNP)筛选,对MSTN基因SNP与体质量、壳长、壳宽进行关联性分析,运用实时荧光定量PCR(qRT-PCR)技术检测九孔鲍MSTN基因不同基因型的表达水平。结果显示:在九孔鲍MSTN基因中共筛选出9个SNP位点。SNP位点与生长性状关联分析发现,九孔鲍MSTN基因编码区SNP位点g909C>T发生C/T同义突变与九孔鲍体质量、壳长、壳宽显著相关。TT基因型九孔鲍的体质量显著高于CC基因型和TC基因型个体;TT基因型的壳长、壳宽显著高于CC基因型(p<0.05)。通过qRT-PCR检测显示,在九孔鲍MSTN基因SNP位点g909C>T的3种基因型中, TC和CC基因型的基因表达量显著高于TT基因型(p?0.05)。研究结果表明MSTN基因SNP位点g909C>T可作为九孔鲍标记辅助选择育种重要候选标记。
Myostatin is an important member of the transforming growth factor(TGF) family that functions to regulate muscle development and growth in animals. It has become one of the most important target genes for genetic improvement of the production of farmed animals. The purpose of this study was to analyze correlation between single nucleotide polymorphisms of the myostatin gene and growth traits of the Haliotis diversicolor supertexta, which is an important aquaculture shellfish.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 and Hs-MSTN expression level. Nine SNPs were identified in cDNA of Hs-MSTN, including four synonymous mutations. The associations of these SNPs were analyzed with H. diversicolor supertexta growth traits, including shell length, shell height, and body weight.The g909 C>T SNP locus was found to be associated with shell length, shell height, and body weight. The TT type H.diversicolor supertexta showed significantly higher traits values than those of TC and CC types 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?0.05). On the contrary, Hs-MSTN transcripts in the musle of those of TC and CC types significantly more than in that of TT type(p?0.05). The results suggested that Hs-MSTN might regulate the addutor musle growth negatively, and SNP g909 C>T could be used as a candidate marker for slective breeding of H. diversicolor supertexta.
Myostatin is an important member of the transforming growth factor(TGF) family that functions to regulate muscle development and growth in animals. It has become one of the most important target genes for genetic improvement of the production of farmed animals. The purpose of this study was to analyze correlation between single nucleotide polymorphisms of the myostatin gene and growth traits of the Haliotis diversicolor supertexta, which is an important aquaculture shellfish.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 and Hs-MSTN expression level. Nine SNPs were identified in cDNA of Hs-MSTN, including four synonymous mutations. The associations of these SNPs were analyzed with H. diversicolor supertexta growth traits, including shell length, shell height, and body weight.The g909 C>T SNP locus was found to be associated with shell length, shell height, and body weight. The TT type H.diversicolor supertexta showed significantly higher traits values than those of TC and CC types 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?0.05). On the contrary, Hs-MSTN transcripts in the musle of those of TC and CC types significantly more than in that of TT type(p?0.05). The results suggested that Hs-MSTN might regulate the addutor musle growth negatively, and SNP g909 C>T could be used as a candidate marker for slective breeding of H. diversicolor supertexta.
引文
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BROOKES A J,1999.The essence of SNPs[J].Gene,234(2):177-186.
CAI JUNPENG,HAN YUN,WANG ZHI,2006.Isolation of Vibrio parahaemolyticus from abalone(Haliotis diversicolor supertexta L.)postlarvae associated with mass mortalities[J].Aquaculture,257(1-4):161-166.
CHIANG Y A,KINOSHITA M,MAEKAWA S,et al,2016.TALENs-mediated gene disruption of myostatin produces a larger phenotype of medaka with an apparently compromised immune system[J].Fish&Shellfish Immunology,48:212-220.
CLOP A,MARCQ F,TAKEDA H,et al,2006.A mutation creating a potential illegitimate micro RNA target site in the myostatin gene affects muscularity in sheep[J].Nature Genetics,38(7):813-818.
COLLINS F S,BROOKS L D,CHAKRAVARTI A,1998.ADNA polymorphism discovery resource for research on human genetic variation[J].Genome Research,8(12):1229-1231.
FAN SIGANG,XU YOUHOU,LIU BAOSUO,et al,2017.Molecular characterization and expression analysis of the myostatin gene and its association with growth traits in Noble scallop(Chlamys nobilis)[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,212:24-31.
FU QIANG,GUO HUIHUI,FENG LIYING,et al,2016.Association of myostatin variants with growth traits of Zhikong scallop(Chlamys farreri)[J].Journal of Ocean University of China,15(1):145-151.
GABILLARD J-C,BIGA P R,RESCAN P-Y,et al,2013.Revisiting the paradigm of myostatin in vertebrates:insights from fishes[J].General and Comparative Endocrinology,194:45-54.
GAO FENG,SUN BOXING,XING SHENYANG,et al,2014.The effect of leader peptide mutations on the biological function of bovine myostatin gene[J].Gene,540(2):171-177.
GRISOLIA A B,D’ANGELO G T,PORTO NETO L R,et al,2009.Myostatin(GDF8)single nucleotide polymorphisms in Nellore cattle[J].Genetics and Molecular Research,8(3):822-830.
HEMMER-HANSEN J,NIELSEN E E,MELDRUP D,et al,2011.Identification of single nucleotide polymorphisms in candidate genes for growth and reproduction in a nonmodel organism;the Atlantic cod,Gadus morhua[J].Molecular Ecology Resources,111(S1):71-80.
HILL E W,GU JINGJING,EIVERS S S,et al,2010.A sequence polymorphism in MSTN predicts sprinting ability and racing stamina in thoroughbred horses[J].PLoS One,5(1):e8645.
JIANG HONGLIN,LUCY M C,2001.Variants of the5?-untranslated region of the bovine growth hormone receptor m RNA:isolation,expression and effects on translational efficiency[J].Gene,265(1-2):45-53.
KAMBADUR R,SHARMA M,SMITH T P L,et al,1997.Mutations in myostatin(GDF8)in double-muscled Belgian blue and piedmontese cattle[J].Genome Research,7(9):910-915.
KINGSLEY D M,1994.The TGF-βsuperfamily:new members,new receptors,and new genetic tests of function in different organisms[J].Genes&Development,8(2):133-146.
LEE C-Y,HU SHAO-YANG,GONG HONGYI,et al,2009.Suppression of myostatin with vector-based RNA interference causes a double-muscle effect in transgenic zebrafish[J].Biochemical and Biophysical Research Communications,387(4):766-771.
LI QIN,LI YAOGUO,HUANG XIANDE,et al,2014.Molecular cloning and single nucleotide polymorphisms identification of myostatin c DNA in the pearl oyster,Pinctada fucata[J].Journal of the World Aquaculture Society,45(6):638-651.
LI SHILEI,ZHOU ZUNCHUN,DONG YING,et al,2016.Molecular characterization,expression analysis of the myostatin gene and its association with growth traits in sea cucumber(Apostichopus japonicus)[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,201:12-20.
MCPHERRON A C,LAWLER A M,LEE S-J,1997.Regulation of skeletal muscle mass in mice by a new TGF-βsuperfamily member[J].Nature,387(6628):83-90.
MIRETTI S,MARTIGNANI E,ACCORNERO P,et al,2013.Functional effect of mir-27b on myostatin expression:a relationship in piedmontese cattle with double-muscled phenotype[J].BMC Genomics,14:194.
NIU DONGHONG,WANG LIE,BAI ZHIYI,et al,2015.Identification and expression characterization of the myostatin(MSTN)gene and association analysis with growth traits in the razor clam Sinonovacula constricta[J].Gene,555(2):297-304.
Nú?EZ-ACU?A G,GALLARDO-ESCáRATE C,2014.The myostatin gene of Mytilus chilensis evidences a high level of polymorphism and ubiquitous transcript expression[J].Gene,536(1):207-212.
SáNCHEZ-RAMOS I,CROSS I,MáCHA J,et al,2012.Assessment of tools for marker-assisted selection in a marine commercial species:significant association between MSTN-1gene polymorphism and growth traits[J].The Scientific World Journal,2012:369802.
SHIN S,SONG YAN,AHN J,et al,2015.A novel mechanism of myostatin regulation by its alternative splicing variant during myogenesis in avian species[J].American Journal of Physiology-Cell Physiology,309(10):C650-C659.
SUN YANHONG,LI QING,WANG GUIYING,et al,2017.Polymorphisms in the myostatin-1 gene and their association with growth traits in Ancherythroculter nigrocauda[J].Chinese Journal of Oceanology and Limnology,35(3):597-602.
THOMAS M,LANGLEY B,BERRY C,et al,2000.Myostatin,a negative regulator of muscle growth,functions by inhibiting myoblast proliferation[J].The Journal of Biological Chemistry,275(51):40235-40243.
WANG XIULI,MENG XIANGYING,SONG BIN,et al,2010.SNPs in the myostatin gene of the mollusk Chlamys farreri:association with growth traits[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,155(3):327-330.
WENNE R,2018.Single nucleotide polymorphism markers with applications in aquaculture and assessment of its impact on natural populations[J].Aquatic Living Resources,31(2):17.
ZHUO RUIQUN,ZHOU TINGTING,YANG SHIPING,et al,2017.Characterization of a molt-related myostatin gene(FmMstn)from the banana shrimp Fenneropenaeus merguiensis[J].General and Comparative Endocrinology,248:55-68.
ACOSTA J,CARPIO Y,BORROTO I,et al,2005.Myostatin gene silenced by RNAi Show a zebrafish giant phenotype[J].Journal of Biotechnology,119(4):324-331.
BROOKES A J,1999.The essence of SNPs[J].Gene,234(2):177-186.
CAI JUNPENG,HAN YUN,WANG ZHI,2006.Isolation of Vibrio parahaemolyticus from abalone(Haliotis diversicolor supertexta L.)postlarvae associated with mass mortalities[J].Aquaculture,257(1-4):161-166.
CHIANG Y A,KINOSHITA M,MAEKAWA S,et al,2016.TALENs-mediated gene disruption of myostatin produces a larger phenotype of medaka with an apparently compromised immune system[J].Fish&Shellfish Immunology,48:212-220.
CLOP A,MARCQ F,TAKEDA H,et al,2006.A mutation creating a potential illegitimate micro RNA target site in the myostatin gene affects muscularity in sheep[J].Nature Genetics,38(7):813-818.
COLLINS F S,BROOKS L D,CHAKRAVARTI A,1998.ADNA polymorphism discovery resource for research on human genetic variation[J].Genome Research,8(12):1229-1231.
FAN SIGANG,XU YOUHOU,LIU BAOSUO,et al,2017.Molecular characterization and expression analysis of the myostatin gene and its association with growth traits in Noble scallop(Chlamys nobilis)[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,212:24-31.
FU QIANG,GUO HUIHUI,FENG LIYING,et al,2016.Association of myostatin variants with growth traits of Zhikong scallop(Chlamys farreri)[J].Journal of Ocean University of China,15(1):145-151.
GABILLARD J-C,BIGA P R,RESCAN P-Y,et al,2013.Revisiting the paradigm of myostatin in vertebrates:insights from fishes[J].General and Comparative Endocrinology,194:45-54.
GAO FENG,SUN BOXING,XING SHENYANG,et al,2014.The effect of leader peptide mutations on the biological function of bovine myostatin gene[J].Gene,540(2):171-177.
GRISOLIA A B,D’ANGELO G T,PORTO NETO L R,et al,2009.Myostatin(GDF8)single nucleotide polymorphisms in Nellore cattle[J].Genetics and Molecular Research,8(3):822-830.
HEMMER-HANSEN J,NIELSEN E E,MELDRUP D,et al,2011.Identification of single nucleotide polymorphisms in candidate genes for growth and reproduction in a nonmodel organism;the Atlantic cod,Gadus morhua[J].Molecular Ecology Resources,111(S1):71-80.
HILL E W,GU JINGJING,EIVERS S S,et al,2010.A sequence polymorphism in MSTN predicts sprinting ability and racing stamina in thoroughbred horses[J].PLoS One,5(1):e8645.
JIANG HONGLIN,LUCY M C,2001.Variants of the5?-untranslated region of the bovine growth hormone receptor m RNA:isolation,expression and effects on translational efficiency[J].Gene,265(1-2):45-53.
KAMBADUR R,SHARMA M,SMITH T P L,et al,1997.Mutations in myostatin(GDF8)in double-muscled Belgian blue and piedmontese cattle[J].Genome Research,7(9):910-915.
KINGSLEY D M,1994.The TGF-βsuperfamily:new members,new receptors,and new genetic tests of function in different organisms[J].Genes&Development,8(2):133-146.
LEE C-Y,HU SHAO-YANG,GONG HONGYI,et al,2009.Suppression of myostatin with vector-based RNA interference causes a double-muscle effect in transgenic zebrafish[J].Biochemical and Biophysical Research Communications,387(4):766-771.
LI QIN,LI YAOGUO,HUANG XIANDE,et al,2014.Molecular cloning and single nucleotide polymorphisms identification of myostatin c DNA in the pearl oyster,Pinctada fucata[J].Journal of the World Aquaculture Society,45(6):638-651.
LI SHILEI,ZHOU ZUNCHUN,DONG YING,et al,2016.Molecular characterization,expression analysis of the myostatin gene and its association with growth traits in sea cucumber(Apostichopus japonicus)[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,201:12-20.
MCPHERRON A C,LAWLER A M,LEE S-J,1997.Regulation of skeletal muscle mass in mice by a new TGF-βsuperfamily member[J].Nature,387(6628):83-90.
MIRETTI S,MARTIGNANI E,ACCORNERO P,et al,2013.Functional effect of mir-27b on myostatin expression:a relationship in piedmontese cattle with double-muscled phenotype[J].BMC Genomics,14:194.
NIU DONGHONG,WANG LIE,BAI ZHIYI,et al,2015.Identification and expression characterization of the myostatin(MSTN)gene and association analysis with growth traits in the razor clam Sinonovacula constricta[J].Gene,555(2):297-304.
Nú?EZ-ACU?A G,GALLARDO-ESCáRATE C,2014.The myostatin gene of Mytilus chilensis evidences a high level of polymorphism and ubiquitous transcript expression[J].Gene,536(1):207-212.
SáNCHEZ-RAMOS I,CROSS I,MáCHA J,et al,2012.Assessment of tools for marker-assisted selection in a marine commercial species:significant association between MSTN-1gene polymorphism and growth traits[J].The Scientific World Journal,2012:369802.
SHIN S,SONG YAN,AHN J,et al,2015.A novel mechanism of myostatin regulation by its alternative splicing variant during myogenesis in avian species[J].American Journal of Physiology-Cell Physiology,309(10):C650-C659.
SUN YANHONG,LI QING,WANG GUIYING,et al,2017.Polymorphisms in the myostatin-1 gene and their association with growth traits in Ancherythroculter nigrocauda[J].Chinese Journal of Oceanology and Limnology,35(3):597-602.
THOMAS M,LANGLEY B,BERRY C,et al,2000.Myostatin,a negative regulator of muscle growth,functions by inhibiting myoblast proliferation[J].The Journal of Biological Chemistry,275(51):40235-40243.
WANG XIULI,MENG XIANGYING,SONG BIN,et al,2010.SNPs in the myostatin gene of the mollusk Chlamys farreri:association with growth traits[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,155(3):327-330.
WENNE R,2018.Single nucleotide polymorphism markers with applications in aquaculture and assessment of its impact on natural populations[J].Aquatic Living Resources,31(2):17.
ZHUO RUIQUN,ZHOU TINGTING,YANG SHIPING,et al,2017.Characterization of a molt-related myostatin gene(FmMstn)from the banana shrimp Fenneropenaeus merguiensis[J].General and Comparative Endocrinology,248:55-68.
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