华贵栉孔扇贝MSTN基因启动子的功能分析
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摘要
为了解肌肉生长抑制素myostatin (MSTN)在华贵栉孔扇贝(Chalmys nobilis)闭壳肌肌肉生长和发育过程中所起的负调控作用,对华贵栉孔扇贝的MSTN启动子序列进行了生物信息学分析。结果显示,MSTN启动子序列长1 358 bp,有4个转录起始位点。核心启动子区为–100～–51 bp。有1个TATA-box (–92～–86 bp)和2个Ebox等顺式作用元件;潜在的转录因子结合位点有MEF2、MEF3、FoxO、MTBF和MyoD等;启动子区域无CpG岛。成功构建了6个MSTN启动子不同长度片段的荧光素酶表达载体,瞬时转染到293T细胞并进行双荧光素酶报告基因活性检测,表明6个启动子片段均有转录活性,PGL-534的活性最高,其次为PGL-274、PGL-22和PGL-102,最低的为PGL-995。–216～–364区域可能存在负调控基因表达的转录因子结合位点,–364～–825区域可能存在正调控基因表达的转录因子结合位点。
In order to investigate the negative regulater role of myostatin(MSTN) in growth and development of adductor of Chalmys nobilis, we analyzed the promoter sequence of MSTN of C. nobilis. The results show that the promoter sequence was 1 358 bp in length, including four transcription start sites(TSS). The core promoter region was located from –100 bp to –51 bp. Two kinds of cis-regulatory element, a TATA-box(located from –92 bp to –86 bp) and two E-boxes, were detected in promoter. Potential transcription factor binding sites including MEF2, MEF3, FoxO, MTBF, MyoD and so on were found in promoter. No CpG island was found. Six luciferase expression vectors with different lengths of MSTN promoter were successfully constructed and transiently transfected into 293 T cells for an analysis of the activity of dual luciferase reporter gene. It is shown that all the six promoter sequences had transcriptional activity, with PGL-534 the highest, followed by PGL-274, PGL-22 and PGL-102, and PGL-995 the lowest. There might be some binding sites of potential negative transcription factors from –216 bp to –364 bp, and potential positive transcription factors from –364 bp to –825 bp.
In order to investigate the negative regulater role of myostatin(MSTN) in growth and development of adductor of Chalmys nobilis, we analyzed the promoter sequence of MSTN of C. nobilis. The results show that the promoter sequence was 1 358 bp in length, including four transcription start sites(TSS). The core promoter region was located from –100 bp to –51 bp. Two kinds of cis-regulatory element, a TATA-box(located from –92 bp to –86 bp) and two E-boxes, were detected in promoter. Potential transcription factor binding sites including MEF2, MEF3, FoxO, MTBF, MyoD and so on were found in promoter. No CpG island was found. Six luciferase expression vectors with different lengths of MSTN promoter were successfully constructed and transiently transfected into 293 T cells for an analysis of the activity of dual luciferase reporter gene. It is shown that all the six promoter sequences had transcriptional activity, with PGL-534 the highest, followed by PGL-274, PGL-22 and PGL-102, and PGL-995 the lowest. There might be some binding sites of potential negative transcription factors from –216 bp to –364 bp, and potential positive transcription factors from –364 bp to –825 bp.
引文
[1]杨世平,刘慧玲,劳赞,等.墨西哥湾扇贝经济性状对闭壳肌重决定效应分析[J].渔业现代化,2013, 40(3):41-45.
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[13]GUO L, LI L, ZHANG S D, et al. Molecular cloning and characterization of the myostatin gene in a cultivated variety of bay scallop, Argopecten irradians[J]. Aquaculture, 2012, 350(2):192-199.
[14]MORELOS R M, RAMIREZ J L, GARCIA-GASCA A, et al. Expression of the myostatin gene in the adductor muscle of the Pacific lion-paw scallop Nodipecten subnodosus in association with growth and environmental conditions[J]. J Exp Zool A, 2015,323(4):239-255.
[15]NIU D, WANG L,BAI Z,et al. Identification and expression characterization of the myostatin(MSTN)gene and association analysis with growth traits in the razor clam Sinonovacula constricta[J]. Gene, 2015, 555(2):297-304.
[16]FAN S, XU Y, LIU B, et al. Molecular characterization and expression analysis of the myostatin gene and its association with growth traits in Noble scallop(Chlamys nobilis)[J]. Comp Biochem Physiol B, 2017, 212:24-31.
[17]WANG X, MENG X, SONG B, et al. SNPs in the myostatin gene of the mollusk Chlamys farreri:association with growth traits[J].Comp Biochem Physiol B, 2010, 155(3):327-330.
[18]GUO L, LI L, ZHANG S, et al. Novel polymorphisms in the myostatin gene and their association with growth traits in a variety of bay scallop, Argopecten irradians[J]. Anim Genet, 2011,42(3):339-340.
[19]GONZALEZ-CADAVID N F, TAYLOR W E, YARASHESKI K, et al. Organization of the human myostatin gene and expression in healthy men and HIV-infected men with muscle wasting[J]. Proc Natl Acad Sci USA, 1998, 95(25):14938-14943.
[20]JEANPLONG F, SHARMA M, SOMERS W G, et al. Genomic organization and neonatal expression of the bovine myostatin gene[J]. Mol Cell Biochem, 2001, 220(1/2):31-37.
[21]BONGIORNI S, TILESI F, BICORGNA S, et al. Promoter polymorphisms in genes involved in porcine myogenesis influence their transcriptional activity[J]. BMC Genet,2014, 15:119.
[22]XUE L, DONG X, ZHANG X, et al. Organization and functional analysis of the 5'flanking regions of myostatin-1 and 2 genes from Larimichthys crocea[J]. DNA Cell Biol, 2012, 31(5):845-855.
[23]FUNKENSTEIN B, BALAS V, REBHAN Y, et al. Characterization and functional analysis of the 5'flanking region of Sparus aurata myostatin-1 gene[J]. Comp Biochem Physiol A, 2009,153(1):55-62.
[24]杜荣,安晓荣,陈永福,等.绵羊Myostatin基因启动子的功能分析[J].中国科学C辑,2007, 37(5):551-557.
[25]SPILLER M P, KAMBADUR R, JEANPLONG F, et al. The myostatin gene is a downstream target gene of basic helix-loophelix transcription factor MyoD[J]. Mol Cell Biol, 2002, 22(20):7066-7082.
[26]夏丹.关岭牛MSTN基因启动子相关转录因子的筛选、克隆及表达纯化[D].贵阳:贵州大学,2016:23.
[27]LI M, CHAN K, CAI D, et al. Identification and purification of an intrinsic human muscle myogenic factor that enhances muscle repair and regeneration[J]. Arch Biochem Biophys, 2000, 384(2):263-268.
[28]李佳,邓捷,张军林,等.肌肉增强子因子2对猪肌肉生长抑制素启动子活性的调节[J].生物工程学报,2012, 28(8):918-926.
[29]OLSON E N, PERRY M, SCHULZ R A. Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors[J]. Dev Biol, 1995, 172(1):2-14.
[2]刘志刚,章启忠,王辉.华贵栉孔扇贝主要经济性状对闭壳肌重的影响效果分析[J].热带海洋学报,2009, 28(1):61-66.
[3]张存善,常亚青,曹学彬,等.虾夷扇贝体形性状对软体重和闭壳肌重的影响效果分析[J].水产学报,2009, 33(1):87-94,
[4]刘志刚,王辉,吕文刚,等.华贵栉栉孔扇贝(Chlamys nobilis)壳色与闭壳肌颜色遗传规律的研究[J].海洋与湖沼,2012, 43(2):237-243.
[5]宋扬,张晴,周晏琳,等.虾夷扇贝横纹肌和平滑肌的蛋白分布及理化性质[J].水产学报,2017, 41(11):1798-1805.
[6]MCPHERRON A C, LEE S J. Double muscling in cattle due to mutations in the myostatin gene[J]. Proc Natl Acad Sci USA,1997, 94(23):12457-12461.
[7]DUSHYANTH K, BHATTACHARYA T K, SHUKLA R, et al.Gene expression and polymorphism of myostatin gene and its association with growth traits in chicken[J]. Anim Biotechnol, 2016,27(4):269-277.
[8]QIAN Z, MI X, WANG X, et al. cDNA cloning and expression analysis of myostatin/GDF11 in shrimp, Litopenaeus vannamei[J]. Comp Biochem Physiol A, 2013, 165(1):30-39.
[9]DALL'OLIO S, FONTANESI L, NANNI COSTA L, et al. Analysis of horse myostatin gene and identification of single nucleotide polymorphisms in breeds of different morphological types[J]. J Biomed Biotechnol, 2010, 2010:542945.
[10]NUNEZ-ACUNA G, GALLARDO-ESCARATE C. The myostatin gene of Mytilus chilensis evidences a high level of polymorphism and ubiquitous transcript expression[J]. Gene, 2014, 536(1):207-212.
[11]HU X,GUO H,HE Y,et al. Molecular characterization of myostatin gene from Zhikong scallop Chlamys farreri(Jones et Preston 1904)[J]. Genes Genet Syst, 2010, 85(3):207-218.
[12]KIM H W,MYKLES D L,GOETZ F W,et al. Characterization of a myostatin-like gene from the bay scallop, Argopecten irradians[J]. Biochim Biophys Acta, 2004, 1679(2):174-179.
[13]GUO L, LI L, ZHANG S D, et al. Molecular cloning and characterization of the myostatin gene in a cultivated variety of bay scallop, Argopecten irradians[J]. Aquaculture, 2012, 350(2):192-199.
[14]MORELOS R M, RAMIREZ J L, GARCIA-GASCA A, et al. Expression of the myostatin gene in the adductor muscle of the Pacific lion-paw scallop Nodipecten subnodosus in association with growth and environmental conditions[J]. J Exp Zool A, 2015,323(4):239-255.
[15]NIU D, WANG L,BAI Z,et al. Identification and expression characterization of the myostatin(MSTN)gene and association analysis with growth traits in the razor clam Sinonovacula constricta[J]. Gene, 2015, 555(2):297-304.
[16]FAN S, XU Y, LIU B, et al. Molecular characterization and expression analysis of the myostatin gene and its association with growth traits in Noble scallop(Chlamys nobilis)[J]. Comp Biochem Physiol B, 2017, 212:24-31.
[17]WANG X, MENG X, SONG B, et al. SNPs in the myostatin gene of the mollusk Chlamys farreri:association with growth traits[J].Comp Biochem Physiol B, 2010, 155(3):327-330.
[18]GUO L, LI L, ZHANG S, et al. Novel polymorphisms in the myostatin gene and their association with growth traits in a variety of bay scallop, Argopecten irradians[J]. Anim Genet, 2011,42(3):339-340.
[19]GONZALEZ-CADAVID N F, TAYLOR W E, YARASHESKI K, et al. Organization of the human myostatin gene and expression in healthy men and HIV-infected men with muscle wasting[J]. Proc Natl Acad Sci USA, 1998, 95(25):14938-14943.
[20]JEANPLONG F, SHARMA M, SOMERS W G, et al. Genomic organization and neonatal expression of the bovine myostatin gene[J]. Mol Cell Biochem, 2001, 220(1/2):31-37.
[21]BONGIORNI S, TILESI F, BICORGNA S, et al. Promoter polymorphisms in genes involved in porcine myogenesis influence their transcriptional activity[J]. BMC Genet,2014, 15:119.
[22]XUE L, DONG X, ZHANG X, et al. Organization and functional analysis of the 5'flanking regions of myostatin-1 and 2 genes from Larimichthys crocea[J]. DNA Cell Biol, 2012, 31(5):845-855.
[23]FUNKENSTEIN B, BALAS V, REBHAN Y, et al. Characterization and functional analysis of the 5'flanking region of Sparus aurata myostatin-1 gene[J]. Comp Biochem Physiol A, 2009,153(1):55-62.
[24]杜荣,安晓荣,陈永福,等.绵羊Myostatin基因启动子的功能分析[J].中国科学C辑,2007, 37(5):551-557.
[25]SPILLER M P, KAMBADUR R, JEANPLONG F, et al. The myostatin gene is a downstream target gene of basic helix-loophelix transcription factor MyoD[J]. Mol Cell Biol, 2002, 22(20):7066-7082.
[26]夏丹.关岭牛MSTN基因启动子相关转录因子的筛选、克隆及表达纯化[D].贵阳:贵州大学,2016:23.
[27]LI M, CHAN K, CAI D, et al. Identification and purification of an intrinsic human muscle myogenic factor that enhances muscle repair and regeneration[J]. Arch Biochem Biophys, 2000, 384(2):263-268.
[28]李佳,邓捷,张军林,等.肌肉增强子因子2对猪肌肉生长抑制素启动子活性的调节[J].生物工程学报,2012, 28(8):918-926.
[29]OLSON E N, PERRY M, SCHULZ R A. Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors[J]. Dev Biol, 1995, 172(1):2-14.