草鱼肌肉生长抑制素（MSTN）的cDNA克隆及地塞米松对其表达的影响
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摘要
肌肉生长抑制素(myostatin,MSTN)是转化生长因子-β(transforming growth factor-β,TGF-β)超家族的一员,但又与TGF-β超家族其它成员的同源性很低,故又称为GDF-8(growthdifferentiation factor-8),研究证实其功能是抑制肌肉细胞的增殖和分化,在哺乳动物主要分布于肌肉组织,而在鱼类可以分布于各种组织(如肌肉、眼睛、鳃、脑、脾脏、卵巢、肝脏、小肠等)。鱼类与哺乳动物的不同分布、表达方式暗示鱼类MSTN除了抑制肌肉生长外,可能还影响其它组织如神经系统的发育。
本研究以草鱼骨骼肌总RNA为模板,反转录合成cDNA,根据已知鲤科鱼类MSTN cDNA序列,设计3对引物,运用了RT-PCR技术克隆草鱼肌肉生长抑制素(MSTN)基因cDNA的全长序列,并对其核苷酸序列及推导的氨基酸序列以及预测的蛋白质高级结构进行了分析。同时,本实验还检测了注射不同浓度的地塞米松(0μg/kg·d,60μg/kg·d,600μg/kg·d,1200μg/kg·d)以及注射一定浓度(600μg/kg·d)后第1d、第5d、第10d肌肉组织中MSTN表达水平的变化。研究结果表明:
1.克隆草鱼MSTN cDNA长度1764nt,编码区为1178nt,编码的氨基酸多肽链全长375个氨基酸,符合预期结果。同源性分析显示,鲤科鱼类MSTN编码区的氨基酸序列具有较高的同源性,其中草鱼MSTN与黑头软口鲦MSTN之间的同源性最高,氨基酸序列同源性为97.9%;与其他鱼类MSTN的氨基酸序列同源性为78.1%～94.4%。而与鸟类、哺乳类之间的序列同源性相比则相对较低。系统进化分析显示,不同物种间的MSTN序列同源性基本反映了它们的亲缘关系。分析草鱼MSTN蛋白一级结构、二级结构、结构域、三级结构可以看出其蛋白质空间结构以一级结构为基础。
2.地塞米松正向调控草鱼MSTN基因表达。注射不同浓度的地塞米松(0μg/kg·d,60μ/kg·d,600μg/kg·d,1200μg/kg·d),第5d检测草鱼肌肉组织MSTN表达的变化。结果:0μg/kg·d组,60μg/kg·d组,600μg/kg·d组,1200μg/kg·d组相同体积下目的条带与β-actin吸光度的相对比值平均值分别为0.247605,0.247723,0.330207,0.477166。0μg/kg·d组与60μg/kg·d无显著差异(P＞0.05),60μg/kg·d组与600μg/kg·d组有显著差异(P＜0.05),600μg/kg·d组与1200μg/kg·d组差异显著(P＜0.05)。一定浓度(600μg/kg·d)下,第1d、第5d、第10d相同体积下目的条带与β-actin吸光度的相对比值分别为0.54074,0.65098,0.95023,第1d和第5d无显著差异(P＞0.05),第5d和第10d有差异显著性(P＜0.05)。浓度组和时间组实验均表明草鱼MSTN mRNA水平受地塞米松水平影响。
Myostatin(MSTN) is a member of TGF-βsurper family,however,it has relatively low homology with other members of TGF-βsurper family,so myostatin also is named of Gdf-8(growth differentiation factor-8).Myostatin is a negative regulator of development,it supress the development and growth of muscle.In mammal,myostatin abundantly and widely distributed in muscle,however,it almost can supress in each tissues of fish,such as muscle,eyes,gills,brain, spleen,ovary,liver,intestine.Mammal and fish have different distribution and suspression,which suggests that in fish myostatin may affect development of other tissues for a example,nerve.
In this study,the cDNA encoding myostatin(MSTN) was cloned from the skeletal muscle of grass carp by RT-PCR method and analyzed by bioinformatics.At the same time,its potential amino acid sequence was deduced and the corresponding protein structure was predicted and analysed. Meanwhile,the change of expression from muscle tissue of grass carp in the different concentrations (0μg/kg·d,60μg/kg·d,600μg/kg·d,1200μg/kg·d) of injecting grass carp with dexamethasone and in the 1d,5d,10d after injected with dexamethasone(600μg/kg·d),and the results provided the dexamethasone affect expression of myostatin,which provided the scientific approach for advancing fish output.
1.The result showed that the cDNA of grass carp myostatin is 1764nt,which includes an open reading frame of 1128nt coding for a protein of 375 amino acids.Analysis of phylogenetic tree showed that Cyprinidae fishes myostatin proteins are highly homologous.Sequence alignment revealed that the MSTN precursor of grass carp shared high homology of more than 94%with that of pimephales promelas.The precursor sequence alignment of grass carp with other fish species demonstrated moderate degree of homology(78.1～82.3%).Relatively low degree of homology was shown in precursor sequence alignment of grass carp with birds and mammals.Phylogenetic analysis indicated that MSTN sequence homology among vertebrate species correlated primarily to their relationship.Analyzing grass carp MSTN primary structure,secondary structure,domain,tertiary structure,we could consider grass carp MSTN primary structure was base to conformation.
2.Through injected grass carp with dexamethasone in the different concentrations(0μg/kg·d,60μg/ kg·d,600μg/kg·d,1200μg/kg·d),the result showed the expession of grass carp MSTN mRNA (0μg/kg·d,60μg/kg·d,600μg/kg·d,1200μg/kg·d) was 0.247605,0.247723,0.330207,0.477166. There were significant differences(P＜0.05)of grass carp MSTN mRNA between group 60μg/kg·d and group 600μg/kg·d,group 600μg/kg·d and 1200μg/kg·d.in the 1d,5d,10d after injected with dexamethasone(600μg/kg·d),the levels of myostatin mRNA in group(1d,5d,10d) was 0.54074,0.65098,0.95023.There was a significant differences(P＜0.05) of grass carp MSTN mRNA between group in 5d and 10d.Taken together,these results further supported that dexamethasone infected the MSTN mRNA level.
本研究以草鱼骨骼肌总RNA为模板,反转录合成cDNA,根据已知鲤科鱼类MSTN cDNA序列,设计3对引物,运用了RT-PCR技术克隆草鱼肌肉生长抑制素(MSTN)基因cDNA的全长序列,并对其核苷酸序列及推导的氨基酸序列以及预测的蛋白质高级结构进行了分析。同时,本实验还检测了注射不同浓度的地塞米松(0μg/kg·d,60μg/kg·d,600μg/kg·d,1200μg/kg·d)以及注射一定浓度(600μg/kg·d)后第1d、第5d、第10d肌肉组织中MSTN表达水平的变化。研究结果表明:
1.克隆草鱼MSTN cDNA长度1764nt,编码区为1178nt,编码的氨基酸多肽链全长375个氨基酸,符合预期结果。同源性分析显示,鲤科鱼类MSTN编码区的氨基酸序列具有较高的同源性,其中草鱼MSTN与黑头软口鲦MSTN之间的同源性最高,氨基酸序列同源性为97.9%;与其他鱼类MSTN的氨基酸序列同源性为78.1%～94.4%。而与鸟类、哺乳类之间的序列同源性相比则相对较低。系统进化分析显示,不同物种间的MSTN序列同源性基本反映了它们的亲缘关系。分析草鱼MSTN蛋白一级结构、二级结构、结构域、三级结构可以看出其蛋白质空间结构以一级结构为基础。
2.地塞米松正向调控草鱼MSTN基因表达。注射不同浓度的地塞米松(0μg/kg·d,60μ/kg·d,600μg/kg·d,1200μg/kg·d),第5d检测草鱼肌肉组织MSTN表达的变化。结果:0μg/kg·d组,60μg/kg·d组,600μg/kg·d组,1200μg/kg·d组相同体积下目的条带与β-actin吸光度的相对比值平均值分别为0.247605,0.247723,0.330207,0.477166。0μg/kg·d组与60μg/kg·d无显著差异(P＞0.05),60μg/kg·d组与600μg/kg·d组有显著差异(P＜0.05),600μg/kg·d组与1200μg/kg·d组差异显著(P＜0.05)。一定浓度(600μg/kg·d)下,第1d、第5d、第10d相同体积下目的条带与β-actin吸光度的相对比值分别为0.54074,0.65098,0.95023,第1d和第5d无显著差异(P＞0.05),第5d和第10d有差异显著性(P＜0.05)。浓度组和时间组实验均表明草鱼MSTN mRNA水平受地塞米松水平影响。
Myostatin(MSTN) is a member of TGF-βsurper family,however,it has relatively low homology with other members of TGF-βsurper family,so myostatin also is named of Gdf-8(growth differentiation factor-8).Myostatin is a negative regulator of development,it supress the development and growth of muscle.In mammal,myostatin abundantly and widely distributed in muscle,however,it almost can supress in each tissues of fish,such as muscle,eyes,gills,brain, spleen,ovary,liver,intestine.Mammal and fish have different distribution and suspression,which suggests that in fish myostatin may affect development of other tissues for a example,nerve.
In this study,the cDNA encoding myostatin(MSTN) was cloned from the skeletal muscle of grass carp by RT-PCR method and analyzed by bioinformatics.At the same time,its potential amino acid sequence was deduced and the corresponding protein structure was predicted and analysed. Meanwhile,the change of expression from muscle tissue of grass carp in the different concentrations (0μg/kg·d,60μg/kg·d,600μg/kg·d,1200μg/kg·d) of injecting grass carp with dexamethasone and in the 1d,5d,10d after injected with dexamethasone(600μg/kg·d),and the results provided the dexamethasone affect expression of myostatin,which provided the scientific approach for advancing fish output.
1.The result showed that the cDNA of grass carp myostatin is 1764nt,which includes an open reading frame of 1128nt coding for a protein of 375 amino acids.Analysis of phylogenetic tree showed that Cyprinidae fishes myostatin proteins are highly homologous.Sequence alignment revealed that the MSTN precursor of grass carp shared high homology of more than 94%with that of pimephales promelas.The precursor sequence alignment of grass carp with other fish species demonstrated moderate degree of homology(78.1～82.3%).Relatively low degree of homology was shown in precursor sequence alignment of grass carp with birds and mammals.Phylogenetic analysis indicated that MSTN sequence homology among vertebrate species correlated primarily to their relationship.Analyzing grass carp MSTN primary structure,secondary structure,domain,tertiary structure,we could consider grass carp MSTN primary structure was base to conformation.
2.Through injected grass carp with dexamethasone in the different concentrations(0μg/kg·d,60μg/ kg·d,600μg/kg·d,1200μg/kg·d),the result showed the expession of grass carp MSTN mRNA (0μg/kg·d,60μg/kg·d,600μg/kg·d,1200μg/kg·d) was 0.247605,0.247723,0.330207,0.477166. There were significant differences(P＜0.05)of grass carp MSTN mRNA between group 60μg/kg·d and group 600μg/kg·d,group 600μg/kg·d and 1200μg/kg·d.in the 1d,5d,10d after injected with dexamethasone(600μg/kg·d),the levels of myostatin mRNA in group(1d,5d,10d) was 0.54074,0.65098,0.95023.There was a significant differences(P＜0.05) of grass carp MSTN mRNA between group in 5d and 10d.Taken together,these results further supported that dexamethasone infected the MSTN mRNA level.
引文
[1]Mcpherron A C,Lawler A M,Lee S J,et al.Regulation of skeletal muscle mass in mice by a new TGF-β superfamily member J].Nature,1997,387(6):83-90.
[2]Lin J,Arnold H B,Della-fera M A,et al.Myostatin knockout in mice increases myogenesis and decreases adipogenesis[J].Biochem Biophys Res Commun,2002,291(3):701-706.
[3]Whittemore L,Song K,Li X,et al.Inhibition of myostatin in adult mice increases skeletal muscle mass and strength[J].Biochem Biophys Res Commun,2003,300:965-971
[4]Casas E,Keelej W,Fahrenkrugsc,et al.Quantitative analysis of birth,weaning,and yearling weights and calving difficulty in Piedmontese crossbreds segregating an inactive myostatin allele [J].J Anim Sci,1999,77(7):1681-692.
[5]Grobetl,Martin L J R,Poncelet D,et al.A deletion in the bovine myostatin gene causes the double muscled phenotype in cattle[J].Nat Genet,1997,7:71-74.
[6]Kambadur R,Sharma M,Smith T P,et al.Mutations in myostatin in double-muscled Belgian Blue and Piledmontese cattle[J].Genome Res,1997,7(9):910-916.
[7]Mcpherron A C,LEE S J.Double muscling in cattle due to mutations in the myostatin gene[J].Proc Natl Acad Sci,1997,94:12 457-12 461.
[8]赵浩斌,彭扣,王玉凤,等.鱼类肌肉生长抑制素(Myostatin)研究进展[J].水生生物学报,2006,30(2):227-231.
[9]李胜杰,白俊杰,叶星,等.加州鲈(MSTN)cDNA的克隆和序列分析[J].海洋渔业,2007,29(1)13-20.
[10]张利媛,岳文斌.MSTN基因研究进展[J].草食家畜,2006,3:1-4.
[11]Rodgers B D,Weber G M.Sequence conservation among fish myostatin orthologuesand the characterization of two additional cDNA clonesfrom Morone saxatilis and Morone americana[J].Comparative Biochemistry and Physiology,2001,129:597-603.
[12]Maccatrozzo L,Bargelloni L,Radaelli G,et al.Characterization of the myostatin gene in the gilthead seabream(Sparus aurata):sequence,genomic structure,and expression pattern[J].Mar Biotechnol,2001,3:224-230.
[13]Maccatrozzo L,Bargelloni L,Cardazzo B,et al.A novel second myostatin gene is present in teleost fish[J].FEBS Lett,2001,509(1):36-40.
[14]Amali A,Lin C J,Chen Y,et al.Up-regulation of muscle specific transcription factors during embryonic somitogenesis of zebrafish(Danio rerio) by knock-down of myostatin2[J].Dev Dyn,2003,229:847-856.
[15]Roberts S B,Goetz F W.Differential skeletal muscle expression of myostatin across teleost species,and the isolation of multiple myostatin isoforms[J].FEBS Lett,2001,491:212-216.
[16]Roberts S B,Goetz F W.Myostatin protein and RNA transcript levels in adult and developing brook trout[J].Mol Cell Endocrin,2003,210:9-20.
[17]Rescan P Y,Jutel I,Ralliere C.Two myostatin genes are differentially expressed in myotoma 1 muscles of the trout(Oncorhynchus mykiss)[J].J Exp Biol,2001,204:3523-3529.
[18]Ostbye T K,Galloway T F,Nielsen C.The two myostatin genes of Atlantic salmon(Salmo salar)are expressed in a variety of tissues[J].Eur J Bioehem,2001,268((20):5249-5257.
[19]Pelegri.Maternal factors in zebrafish development[J].Dev Dyn,2003,228(3):535-554.
[20]Maccatrozzo L,Bargelloni L,Cardazzo B,et al.A novel second myostatin gene is present in teleost fish[J].FEBS Lett,2001,509(1):36-40.
[21]Maccatrozzo L,Bargelloni L,Patarnello P,et al.Characterization of the myostatin gene and a linked microsatellite marker in shi drum(Umbrina cirrosa Sciaenidae)[J].Aquaculture,2002,205(122):49-60.
[22]Maccatrozzo L,Bargelloni L,Radaelli G,et al.Characterization of the myostatin gene in the gilthead seabream(Sparus aurata):sequence,genomic structure,and expression pattern[J].Mar Biotechnol,2001,3:224-23.
[23]Whittemore L,Song K,Li X,et al.Inhibition of myostatin in adult mice increases skeletal muscle mass and strength[J].Biochem Bio phys Res Comm,2003,300:965-971.
[24]Rios R,Cameiro I.Myostatin is an inhibitor of myogenic differentiation[J].Am J Physiol Cell Physiol,2002,282(5):993-999.
[25]Lin J,Heather B.Myostatin knockout in mice increase myogenesis and decrease adipogenesis[J].Biochem Biophys Res Comm,2002,292(3):701-706.
[26]Xu C,Wu G,Zohar Y,et al.Analysis of myostatin gene structure,expression and function in zebrafish[J].J Exp Biol,2003,206:4067-4079.
[27]Acosta J,arpio Y,Borroto I,et al.Myostatin gene silenced by RNAi show a zebrafish giant phenotype[J].J Biotechnol,2005,19(4):324-331.
[28]Biga P R,Robe rts S B,Iliev D B,et al.The isolation,characterization,and expression of a novel GDF11 gene and a second myostatin form in zebrafish(Danio rerio)[J].Comp Biochem Physiol B,2005,141(2):218-230.
[29]Thomas M,Langley B,Berry C,et al.Myostatin,a negative regulator of muscle growth,unctions by inhibiting myoblast proliferation[J].J Biol Chem,2000,275:40235-40243.
[30]Senna Salerno M,Thomas M,Forbes D,et al.Molecular analysis of fiber-type specific expression of murine myostatin promoter[J].Am J Physiol Cell Physiol,2004,287(4):1031-1040.
[31]Sharma M,R Kambadur,KG Matthews,et al.Myostatin,a transforming growth factor- beta superfamily,is expressed in heart muscle and is upregulated in cardiomyocytes after infarct[J].J Cell Physiol,1999,180(1):1-9.
[32]Iezzi S,Padova M D,Serra C,et al.Deacetylase Inhibitors increase muscle cell size by promoting myoblast recruitment and fusion through induction of follistatin[J].Dev Cell,2004,6:673-684.
[33]Zhu X,Topouzis S,Liang L,et al.Myostatin signaling through Smad2,Smad3 and Smad4is regulated by the inhibitory Smad7 by a negative feedback mechanism[J].Cytokine,2004,26:262-272.
[34]Sharma,Mridula,Langley,et al.Myostatin in Muscle Growth and Repair[J].Exerc Sport Sci Rev,2001,29(4):155-158.
[35]薛鹏.GDF-8基因研究进展[J].医学分子生物学杂志,2005,2(1):74-77.
[36]李兴美,范巍.鲤鱼肌肉生长抑制素(MSTN)克隆及组织表达特征[J].水生生物学报,2007,31(5):643-648.
[37]Peggy R,Steven B,Dimitar B,et al.The isolation,chareterization,and expression of a novel GDFI1 gene and a seeond myostatin form in zebarfish(Danio rerio)[J].Comparative Biochemistry and Physiology,2005,141:218-230.
[38]Kocabas A M,Kucuktas H,Dunham R A,et al.Molecular characterization and differential expression of the myostatin gene in channel catfish(Ictalurus punctatus)[J].Biochim Biophys Acta,2002,1575(123):99-10.
[39]彭扣,陈伟伟,胡炜,等.泥鳅生长肌肉抑制素基因片段的克隆及其表达[J].水产学报, 2007,31(2):145-152.
[40]薛良义,李婷,杨巧一,等.眼斑拟石首鱼肌肉生长抑制基因克隆以及组织表达分析l[J].海洋学报,2008,30(3):67-74.
[41]Roberts S B,Goetz F W.Transgenic salmon overexpressing growth horme exhibit decreased myostatin transcript and protein expression[J].J Exp Bipl,2001,207:3741-3748.
[42]Steven B R,Goetz F W.Myostatin protein and RNA transcript levels in adult and developing brook trout[J]Molecular and Cellular Endocrinologyt,2003,210:9-20.
[43]Biga P R,Cain K D,Hardy R W,et al.Growth hormone differentially regulates muscle myostatin1 and increases circulating cortisol in rainbow trout(Oncorhynchus mykiss)[J].Gen Comp Endocrin,2004,138:32-41.
[44]Roberts S B,Goet Z F W.Myostatin protein and RNA transcript levels in adult and developing brook trout[J].Mol Cell Endocrin,2003,210:9-20.
[45]Weber T E,Small B C,Bosworth B G.Lipopolysaccharide regulates myostatin and MyoD independently of an increase in plasma cortisol in channel catfish(Ictalurus punctatus)[J].Domestic Animal Endocrinology,2005,28:64-73.
[46]Deri L I,Heltedine,Dilip Garikipati,et al.Embryonic and tissue-specific regulation of myostatin-1 and -2 gene expression in zebrafish[J].General and Comparative Endocrinology,2007,151:90-97.
[47]Vianello S,BrazzoduroL.Myostatin expression during development and chronic stress in zebrafish(Danio rerio) Endocrinol[J].J Cell Physiol,2003,176(1):47-59.
[48]Rodgers B D,Weber G M,Kelley K M,et al.Prolonged fasting and cortisol reduce myostatin mRNA levels in tilapia larvae short-term fasting elevates[J].Am J Physiol Regul Integr Comp Physiol,2003,284:1277-1286.
[49]Kun Ma,Con Mallidis,Shalender Bhasin,et al.Glucocorticoid-induced skeletal muscle atrophy is associated with upregulation of myostatin gene expression[J].Biochem Biophys Res Commun,2002,290:1-10.
[50]Wu Gang,Sun Yonghua,Zhu Zuoyang.Growth horme gene transfer in commmon carp Aquat[J].Living Resour1,2003,6:416-420.
[51]刘红云,童富.淡鱼类IGF-I生理功能及其表达调控的研究进展[J].水产科学,2004,23(5):37-40.
[52]Cao Q P.Nucleotide sequence and growth hormone-regulated expression of salmon insulin-like growth factorI mRNA[J].Mol Endocrinol,1989,3:2006-2010.
[53]Chen S L,Sha Z X.Establishment ofa pluripotent embryonic cell line from sea perch (lateolabrax japonidcus) blastula embryo[J],Aquaclture,2003,218:141-154.
[54]Chen S L,Sha Z X.Development of a pluripotent embryonic cell line from red sea bream (Chrysophry Major) blastula[J].J Fish Biol,2003,63:1-11.
[55]Chen S L,Hong Y Schart.Lack of ultraviolet-linght inducibility of the medakafish(Oryzias Latipes) tumor suppressor gene[J].Gene,2001,264:197-203.
[56]Chen S L,Hong Y Schart.Development of a positive-negative selection procedure gene targeting in fish cells[J].aquaculture,2002,214:67-69.
[57]Maclean N.Transgenic fish:an evalution of benefits and risks[J].Fish And Fisher,2000,1(2):146.
[58]李鑫,杨蕊,臧强,等.糖皮质激素的药理作用机制研究进展[J].国际药学研究杂志,2009,36(1):27-30.
[59]赵景鹏,姜克杰,董辉.糖皮质激素对肉鸡骨骼肌糖摄取能力的影响及机制探讨[J].西南农业学报,2008,21(1):170-176.
[60]申传安,柴家科,姚咏明,等.糖皮质激素对大鼠骨骼肌蛋白分解代谢的影响及其机制探讨[J].中国危重病急救医学,2002,14(7):428-431.
[61]徐冲,徐国恒.糖皮质激素与脂肪代谢和胰岛素抵抗[J].生理科学进展,2009,40(1):19-22.
[62]胡新永,吕原,杨华清,等.不同浓度地塞米松对成骨细胞骨钙素基因表达和细胞增殖的影响[J].中国康复医学杂志,2007,22(10):899-903.
[63]郁博,何威,张斌,等.地塞米松对HaCaT细胞糖皮质激素受体α表达的影响[J].临床皮肤科杂志,2008,37(4):216-218.
[64]陈华新,张端莲.地塞米松对大鼠晶状体上皮细胞TGF-β受体表达的影响[J].华南国防医学杂志,2008,22(1):9-14.
[65]秦瑞峰,顾晓明,陈金武,等.鼠肌肉转录调节因子MyoD基因真核表达载体的构建[J].中国修复重建外科杂志,2001,15(5):257-260.
[66]Soheila J,Maleki,Barry K,et al.High-level Expression and Purification of MyoD,Myogenin,and E12[J].Protein ExPression and Purification,1997,9:91-99.
[67]陈云波,侯孟君,王奇,等.半定量RT-PCR检测少量兔血管内皮细胞中特异mRNA的表达[J].广州中医药大学学报,2000,17(1):81-84.
[68]林仁勇,剑冰,温浩,熊海波,等.半定量RT-PCR检测细胞因子表达的研究[J].新疆医科大学学报,2003,26(5):427-429.
[69]陈观水,潘大仁,周以飞,等.甘薯NPR1基因半定量RT-PCR检测方法的建立[J].福建农林大学学报,207,36(1):56-60.
[70]Revillion F,Pawlowski V,Hornez L,et al.Lyceraldehyde phosphate dehydrogenase gene expression in human breast cancer[J].Eur J Cancer,2000,36(8):1038-1042.
[71]Tricarico C,inzani P,Bianchi S,et al.Quantitative real-time reverse transcription polymerase chain reaction:normalization to rRNA or single housekeeping genes is inapp rop riate for human tissue biop sies[J].Anal Biochem,2002,309(2):293-300.
[72]王玉林,史进方,蔡惠芬,等.内参基因β-actin质粒标准品的构建[J].中国组织工程研究与临床康复,2008,12(48):9501-9504.
[73]高海波,张拴林,陈燕红.半定量RT-PCR在基因表达方面的应用[J].畜禽业,2008,226:226-230.
[74]边杉,王颖,于涛,等.聚丙烯酰胺凝胶银染技术在半定量RT-PCR中的应用[J].中国药科大学学报,2004,2:178-182.
[75]奥斯伯 F,金斯顿 R E,等编,马学军等译.精编分子生物学实验指南(第四版)[M].北京:科学出版社,2005,651-686.
[76]C W 迪芬巴赫,G S 德维克斯勒.PCR技术实验指南(第一版)[M].北京:科学出版社,1998,256-280.
[77]王武主编.鱼类增养殖学[M].北京:中国农业出版社,2000,17-20.
[78]朱滨,王国荃,陈坚.用均匀设计优化apo E基因的PCR扩增方案[J].生物化学与生物物理进展,2000,27(3):312-315.
[79]赵晓勤,倪娟,陈立侨,等.日本沼虾基因片段PCR扩增的条件优化[J].华东师范大学学报,2006,2:68-76.
[80]胡根林,吴瑞英.影响RT-PCR产物克隆的因素及其方法的优化[J].生物技术,2008,10(4):11-14.
[81]刘阳,杨淑霞,李敏惠,等.引物浓度与退火温度导致巢式PCR非特异性扩增[J].成都医学院学报,2008,3(2):111-115.
[82]贺福初,孙建中,等主编.蛋白质科学[M].北京:军事医学科学出版社,2002,130-135.
[83]黄卓烈,朱利泉主编.生物化学[M].北京:中国农业出版社,2004,58-60.
[84]芩利娥,刘玉香,秦海峰,等主编.生物化学基础[M].北京:化学工业出版社,2007,30-40.
[85]张洪渊主编.生物化学原理[M].北京:科学出版社,2006,83-88.
[86]胡秀珍.蛋白质规则二级结构中亲-疏水氨基酸的相对使用频率[J].内蒙古工业大学学报,2002,21(1):25-31.
[87]黄敏,沈辉,肖奕.不同结构蛋白质序列的关联特性[J].生物物理学报,2000,16(4):755-760.
[2]Lin J,Arnold H B,Della-fera M A,et al.Myostatin knockout in mice increases myogenesis and decreases adipogenesis[J].Biochem Biophys Res Commun,2002,291(3):701-706.
[3]Whittemore L,Song K,Li X,et al.Inhibition of myostatin in adult mice increases skeletal muscle mass and strength[J].Biochem Biophys Res Commun,2003,300:965-971
[4]Casas E,Keelej W,Fahrenkrugsc,et al.Quantitative analysis of birth,weaning,and yearling weights and calving difficulty in Piedmontese crossbreds segregating an inactive myostatin allele [J].J Anim Sci,1999,77(7):1681-692.
[5]Grobetl,Martin L J R,Poncelet D,et al.A deletion in the bovine myostatin gene causes the double muscled phenotype in cattle[J].Nat Genet,1997,7:71-74.
[6]Kambadur R,Sharma M,Smith T P,et al.Mutations in myostatin in double-muscled Belgian Blue and Piledmontese cattle[J].Genome Res,1997,7(9):910-916.
[7]Mcpherron A C,LEE S J.Double muscling in cattle due to mutations in the myostatin gene[J].Proc Natl Acad Sci,1997,94:12 457-12 461.
[8]赵浩斌,彭扣,王玉凤,等.鱼类肌肉生长抑制素(Myostatin)研究进展[J].水生生物学报,2006,30(2):227-231.
[9]李胜杰,白俊杰,叶星,等.加州鲈(MSTN)cDNA的克隆和序列分析[J].海洋渔业,2007,29(1)13-20.
[10]张利媛,岳文斌.MSTN基因研究进展[J].草食家畜,2006,3:1-4.
[11]Rodgers B D,Weber G M.Sequence conservation among fish myostatin orthologuesand the characterization of two additional cDNA clonesfrom Morone saxatilis and Morone americana[J].Comparative Biochemistry and Physiology,2001,129:597-603.
[12]Maccatrozzo L,Bargelloni L,Radaelli G,et al.Characterization of the myostatin gene in the gilthead seabream(Sparus aurata):sequence,genomic structure,and expression pattern[J].Mar Biotechnol,2001,3:224-230.
[13]Maccatrozzo L,Bargelloni L,Cardazzo B,et al.A novel second myostatin gene is present in teleost fish[J].FEBS Lett,2001,509(1):36-40.
[14]Amali A,Lin C J,Chen Y,et al.Up-regulation of muscle specific transcription factors during embryonic somitogenesis of zebrafish(Danio rerio) by knock-down of myostatin2[J].Dev Dyn,2003,229:847-856.
[15]Roberts S B,Goetz F W.Differential skeletal muscle expression of myostatin across teleost species,and the isolation of multiple myostatin isoforms[J].FEBS Lett,2001,491:212-216.
[16]Roberts S B,Goetz F W.Myostatin protein and RNA transcript levels in adult and developing brook trout[J].Mol Cell Endocrin,2003,210:9-20.
[17]Rescan P Y,Jutel I,Ralliere C.Two myostatin genes are differentially expressed in myotoma 1 muscles of the trout(Oncorhynchus mykiss)[J].J Exp Biol,2001,204:3523-3529.
[18]Ostbye T K,Galloway T F,Nielsen C.The two myostatin genes of Atlantic salmon(Salmo salar)are expressed in a variety of tissues[J].Eur J Bioehem,2001,268((20):5249-5257.
[19]Pelegri.Maternal factors in zebrafish development[J].Dev Dyn,2003,228(3):535-554.
[20]Maccatrozzo L,Bargelloni L,Cardazzo B,et al.A novel second myostatin gene is present in teleost fish[J].FEBS Lett,2001,509(1):36-40.
[21]Maccatrozzo L,Bargelloni L,Patarnello P,et al.Characterization of the myostatin gene and a linked microsatellite marker in shi drum(Umbrina cirrosa Sciaenidae)[J].Aquaculture,2002,205(122):49-60.
[22]Maccatrozzo L,Bargelloni L,Radaelli G,et al.Characterization of the myostatin gene in the gilthead seabream(Sparus aurata):sequence,genomic structure,and expression pattern[J].Mar Biotechnol,2001,3:224-23.
[23]Whittemore L,Song K,Li X,et al.Inhibition of myostatin in adult mice increases skeletal muscle mass and strength[J].Biochem Bio phys Res Comm,2003,300:965-971.
[24]Rios R,Cameiro I.Myostatin is an inhibitor of myogenic differentiation[J].Am J Physiol Cell Physiol,2002,282(5):993-999.
[25]Lin J,Heather B.Myostatin knockout in mice increase myogenesis and decrease adipogenesis[J].Biochem Biophys Res Comm,2002,292(3):701-706.
[26]Xu C,Wu G,Zohar Y,et al.Analysis of myostatin gene structure,expression and function in zebrafish[J].J Exp Biol,2003,206:4067-4079.
[27]Acosta J,arpio Y,Borroto I,et al.Myostatin gene silenced by RNAi show a zebrafish giant phenotype[J].J Biotechnol,2005,19(4):324-331.
[28]Biga P R,Robe rts S B,Iliev D B,et al.The isolation,characterization,and expression of a novel GDF11 gene and a second myostatin form in zebrafish(Danio rerio)[J].Comp Biochem Physiol B,2005,141(2):218-230.
[29]Thomas M,Langley B,Berry C,et al.Myostatin,a negative regulator of muscle growth,unctions by inhibiting myoblast proliferation[J].J Biol Chem,2000,275:40235-40243.
[30]Senna Salerno M,Thomas M,Forbes D,et al.Molecular analysis of fiber-type specific expression of murine myostatin promoter[J].Am J Physiol Cell Physiol,2004,287(4):1031-1040.
[31]Sharma M,R Kambadur,KG Matthews,et al.Myostatin,a transforming growth factor- beta superfamily,is expressed in heart muscle and is upregulated in cardiomyocytes after infarct[J].J Cell Physiol,1999,180(1):1-9.
[32]Iezzi S,Padova M D,Serra C,et al.Deacetylase Inhibitors increase muscle cell size by promoting myoblast recruitment and fusion through induction of follistatin[J].Dev Cell,2004,6:673-684.
[33]Zhu X,Topouzis S,Liang L,et al.Myostatin signaling through Smad2,Smad3 and Smad4is regulated by the inhibitory Smad7 by a negative feedback mechanism[J].Cytokine,2004,26:262-272.
[34]Sharma,Mridula,Langley,et al.Myostatin in Muscle Growth and Repair[J].Exerc Sport Sci Rev,2001,29(4):155-158.
[35]薛鹏.GDF-8基因研究进展[J].医学分子生物学杂志,2005,2(1):74-77.
[36]李兴美,范巍.鲤鱼肌肉生长抑制素(MSTN)克隆及组织表达特征[J].水生生物学报,2007,31(5):643-648.
[37]Peggy R,Steven B,Dimitar B,et al.The isolation,chareterization,and expression of a novel GDFI1 gene and a seeond myostatin form in zebarfish(Danio rerio)[J].Comparative Biochemistry and Physiology,2005,141:218-230.
[38]Kocabas A M,Kucuktas H,Dunham R A,et al.Molecular characterization and differential expression of the myostatin gene in channel catfish(Ictalurus punctatus)[J].Biochim Biophys Acta,2002,1575(123):99-10.
[39]彭扣,陈伟伟,胡炜,等.泥鳅生长肌肉抑制素基因片段的克隆及其表达[J].水产学报, 2007,31(2):145-152.
[40]薛良义,李婷,杨巧一,等.眼斑拟石首鱼肌肉生长抑制基因克隆以及组织表达分析l[J].海洋学报,2008,30(3):67-74.
[41]Roberts S B,Goetz F W.Transgenic salmon overexpressing growth horme exhibit decreased myostatin transcript and protein expression[J].J Exp Bipl,2001,207:3741-3748.
[42]Steven B R,Goetz F W.Myostatin protein and RNA transcript levels in adult and developing brook trout[J]Molecular and Cellular Endocrinologyt,2003,210:9-20.
[43]Biga P R,Cain K D,Hardy R W,et al.Growth hormone differentially regulates muscle myostatin1 and increases circulating cortisol in rainbow trout(Oncorhynchus mykiss)[J].Gen Comp Endocrin,2004,138:32-41.
[44]Roberts S B,Goet Z F W.Myostatin protein and RNA transcript levels in adult and developing brook trout[J].Mol Cell Endocrin,2003,210:9-20.
[45]Weber T E,Small B C,Bosworth B G.Lipopolysaccharide regulates myostatin and MyoD independently of an increase in plasma cortisol in channel catfish(Ictalurus punctatus)[J].Domestic Animal Endocrinology,2005,28:64-73.
[46]Deri L I,Heltedine,Dilip Garikipati,et al.Embryonic and tissue-specific regulation of myostatin-1 and -2 gene expression in zebrafish[J].General and Comparative Endocrinology,2007,151:90-97.
[47]Vianello S,BrazzoduroL.Myostatin expression during development and chronic stress in zebrafish(Danio rerio) Endocrinol[J].J Cell Physiol,2003,176(1):47-59.
[48]Rodgers B D,Weber G M,Kelley K M,et al.Prolonged fasting and cortisol reduce myostatin mRNA levels in tilapia larvae short-term fasting elevates[J].Am J Physiol Regul Integr Comp Physiol,2003,284:1277-1286.
[49]Kun Ma,Con Mallidis,Shalender Bhasin,et al.Glucocorticoid-induced skeletal muscle atrophy is associated with upregulation of myostatin gene expression[J].Biochem Biophys Res Commun,2002,290:1-10.
[50]Wu Gang,Sun Yonghua,Zhu Zuoyang.Growth horme gene transfer in commmon carp Aquat[J].Living Resour1,2003,6:416-420.
[51]刘红云,童富.淡鱼类IGF-I生理功能及其表达调控的研究进展[J].水产科学,2004,23(5):37-40.
[52]Cao Q P.Nucleotide sequence and growth hormone-regulated expression of salmon insulin-like growth factorI mRNA[J].Mol Endocrinol,1989,3:2006-2010.
[53]Chen S L,Sha Z X.Establishment ofa pluripotent embryonic cell line from sea perch (lateolabrax japonidcus) blastula embryo[J],Aquaclture,2003,218:141-154.
[54]Chen S L,Sha Z X.Development of a pluripotent embryonic cell line from red sea bream (Chrysophry Major) blastula[J].J Fish Biol,2003,63:1-11.
[55]Chen S L,Hong Y Schart.Lack of ultraviolet-linght inducibility of the medakafish(Oryzias Latipes) tumor suppressor gene[J].Gene,2001,264:197-203.
[56]Chen S L,Hong Y Schart.Development of a positive-negative selection procedure gene targeting in fish cells[J].aquaculture,2002,214:67-69.
[57]Maclean N.Transgenic fish:an evalution of benefits and risks[J].Fish And Fisher,2000,1(2):146.
[58]李鑫,杨蕊,臧强,等.糖皮质激素的药理作用机制研究进展[J].国际药学研究杂志,2009,36(1):27-30.
[59]赵景鹏,姜克杰,董辉.糖皮质激素对肉鸡骨骼肌糖摄取能力的影响及机制探讨[J].西南农业学报,2008,21(1):170-176.
[60]申传安,柴家科,姚咏明,等.糖皮质激素对大鼠骨骼肌蛋白分解代谢的影响及其机制探讨[J].中国危重病急救医学,2002,14(7):428-431.
[61]徐冲,徐国恒.糖皮质激素与脂肪代谢和胰岛素抵抗[J].生理科学进展,2009,40(1):19-22.
[62]胡新永,吕原,杨华清,等.不同浓度地塞米松对成骨细胞骨钙素基因表达和细胞增殖的影响[J].中国康复医学杂志,2007,22(10):899-903.
[63]郁博,何威,张斌,等.地塞米松对HaCaT细胞糖皮质激素受体α表达的影响[J].临床皮肤科杂志,2008,37(4):216-218.
[64]陈华新,张端莲.地塞米松对大鼠晶状体上皮细胞TGF-β受体表达的影响[J].华南国防医学杂志,2008,22(1):9-14.
[65]秦瑞峰,顾晓明,陈金武,等.鼠肌肉转录调节因子MyoD基因真核表达载体的构建[J].中国修复重建外科杂志,2001,15(5):257-260.
[66]Soheila J,Maleki,Barry K,et al.High-level Expression and Purification of MyoD,Myogenin,and E12[J].Protein ExPression and Purification,1997,9:91-99.
[67]陈云波,侯孟君,王奇,等.半定量RT-PCR检测少量兔血管内皮细胞中特异mRNA的表达[J].广州中医药大学学报,2000,17(1):81-84.
[68]林仁勇,剑冰,温浩,熊海波,等.半定量RT-PCR检测细胞因子表达的研究[J].新疆医科大学学报,2003,26(5):427-429.
[69]陈观水,潘大仁,周以飞,等.甘薯NPR1基因半定量RT-PCR检测方法的建立[J].福建农林大学学报,207,36(1):56-60.
[70]Revillion F,Pawlowski V,Hornez L,et al.Lyceraldehyde phosphate dehydrogenase gene expression in human breast cancer[J].Eur J Cancer,2000,36(8):1038-1042.
[71]Tricarico C,inzani P,Bianchi S,et al.Quantitative real-time reverse transcription polymerase chain reaction:normalization to rRNA or single housekeeping genes is inapp rop riate for human tissue biop sies[J].Anal Biochem,2002,309(2):293-300.
[72]王玉林,史进方,蔡惠芬,等.内参基因β-actin质粒标准品的构建[J].中国组织工程研究与临床康复,2008,12(48):9501-9504.
[73]高海波,张拴林,陈燕红.半定量RT-PCR在基因表达方面的应用[J].畜禽业,2008,226:226-230.
[74]边杉,王颖,于涛,等.聚丙烯酰胺凝胶银染技术在半定量RT-PCR中的应用[J].中国药科大学学报,2004,2:178-182.
[75]奥斯伯 F,金斯顿 R E,等编,马学军等译.精编分子生物学实验指南(第四版)[M].北京:科学出版社,2005,651-686.
[76]C W 迪芬巴赫,G S 德维克斯勒.PCR技术实验指南(第一版)[M].北京:科学出版社,1998,256-280.
[77]王武主编.鱼类增养殖学[M].北京:中国农业出版社,2000,17-20.
[78]朱滨,王国荃,陈坚.用均匀设计优化apo E基因的PCR扩增方案[J].生物化学与生物物理进展,2000,27(3):312-315.
[79]赵晓勤,倪娟,陈立侨,等.日本沼虾基因片段PCR扩增的条件优化[J].华东师范大学学报,2006,2:68-76.
[80]胡根林,吴瑞英.影响RT-PCR产物克隆的因素及其方法的优化[J].生物技术,2008,10(4):11-14.
[81]刘阳,杨淑霞,李敏惠,等.引物浓度与退火温度导致巢式PCR非特异性扩增[J].成都医学院学报,2008,3(2):111-115.
[82]贺福初,孙建中,等主编.蛋白质科学[M].北京:军事医学科学出版社,2002,130-135.
[83]黄卓烈,朱利泉主编.生物化学[M].北京:中国农业出版社,2004,58-60.
[84]芩利娥,刘玉香,秦海峰,等主编.生物化学基础[M].北京:化学工业出版社,2007,30-40.
[85]张洪渊主编.生物化学原理[M].北京:科学出版社,2006,83-88.
[86]胡秀珍.蛋白质规则二级结构中亲-疏水氨基酸的相对使用频率[J].内蒙古工业大学学报,2002,21(1):25-31.
[87]黄敏,沈辉,肖奕.不同结构蛋白质序列的关联特性[J].生物物理学报,2000,16(4):755-760.
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