猪MYNN基因可变剪接体的克隆及表达特性研究
|
摘要
旨在克隆猪MYNN基因的可变剪接体,并预测编码蛋白的结构与功能,研究各转录本的时空表达特性。本研究以马身猪为试验动物,采用RT-PCR技术对猪MYNN基因全长CDS区进行克隆,并采用生物信息学技术分析其编码蛋白的生物学特性;在心、肝、脾、肺、肾、小脑、小肠、胰、胃、肌肉和脂肪组织中,采用实时荧光定量PCR技术对MYNN基因的表达谱进行研究,并在胃和肌肉组织中进行发育性表达规律的研究。结果表明,本研究成功克隆出猪MYNN基因的两个转录本MYNN-1(GenBank登录号:KY470829)和MYNN-2(GenBank登录号:KY670835)。MYNN-1的CDS区全长1 830bp,编码609个氨基酸,编码的蛋白质属于碱性可溶性稳定蛋白质;MYNN-2的CDS区全长1 746bp,编码581个氨基酸,编码的蛋白质属于碱性可溶性不稳定蛋白质;与MYNN-1相比,MYNN-2少了84bp,缺失第6外显子,且通过功能结构域预测发现,MYNN-2比MYNN-1少一个C2H2类型的锌指蛋白结构域;同源性及进化树分析发现,猪MYNN基因的两个转录本所编码的氨基酸序列与北极熊、山羊、马、家犬等物种的同源性较高,遗传距离较近,说明MYNN基因在进化过程中十分保守。MYNN-1和MYNN-2在马身猪的各个组织中均有表达,且各组织之间表达差异显著(P<0.05);在马身猪胃、小肠和胰中高表达,脂肪组织中表达量最低。在各组织(除肾)中MYNN-1的表达量均显著或极显著高于MYNN-2(P<0.05;P<0.01),说明MYNN-1为主要亚型;随着日龄的增加,MYNN-1和MYNN-2在胃中的表达量均呈现逐渐降低的趋势,在背最长肌中,呈现先上升后下降的趋势。本试验成功克隆了猪MYNN基因的两个可变剪接体,并推测MYNN在猪的消化吸收以及骨骼肌的生长发育过程中有重要作用,但其作用的具体机制还需进一步研究。
The aim of this study were to clone alternative splicing isoforms of pig MYNN gene,predict the structures and functions of their coding proteins,and investigate the temporal-spatial expression characteristics of each transcript.The full-length CDS of MYNN was cloned by RT-PCRand the biological characteristics of MYNN protein was analyzed by bioinformatics in Mashen pig.Quantitative real-time PCR was employed to detect the expression patterns of MYNN gene in heart,liver,spleen,lung,kidney,cerebellum,small intestine,stomach,pancreas,longissimus dorsi and fat tissues of Mashen pig,and to study the developmental expression patterns in stomach and longissimus dorsi tissues.Two transcripts of MYNN gene were successfully cloned in present study,and named MYNN-1(GenBank accession number:KY470829)and MYNN-2(GenBank accession number:KY670835),respectively.MYNN-1 CDS was composed of 1 830 bp encoding 609 amino acids,which belonged to the stable alkaline soluble protein.Whereas MYNN-2 CDS was composed of 1 746 bp encoding 581 amino acids,which belonged to the unstable alkaline soluble protein.MYNN-2 was 84 bp less than MYNN-1,and lacked the exon6.MYNN-2 was found a C2 H2 type zinc finger protein domain less than MYNN-1 by the prediction of function domain.The analysis of homology and phylogenetic tree showed that the two transcripts amino acid sequences of pig MYNNgene had high homology and close genetic distance with polar bear,goat,horse,dog,and so on,which proved that the pig MYNNgene was very conservative during evolutionary process.MYNN-1 and MYNN-2 were universally expressed in all pig tissues detected,and there were significant differences in expression among different tissues(P<0.05).The two variants had higher expression levels in stomach,small intestine and pancreas of Mashen pig,and their expression were the lowest in fat tissue.The expression level of MYNN-1 was significantly or extremely significantly higher than that of MYNN-2 in all tissues except kidney(P<0.05,P<0.01),which testified that MYNN-1 was the main variant in pig.The expression of MYNN-1 and MYNN-2 decreased gradually with the increase of age in the stomach of Mashen pig.In longissimus dorsi,the expression of MYNN-1 and MYNN-2 increased firstly and then decreased with the increase of age.In this study,two transcripts of pig MYNN gene were successfully cloned.And it was speculated that MYNN play important role in the process of digestion and absorption,as well as growth and development of skeletal muscle in pig,whereas the specific mechanisms were still remaining to be further elucidated.
The aim of this study were to clone alternative splicing isoforms of pig MYNN gene,predict the structures and functions of their coding proteins,and investigate the temporal-spatial expression characteristics of each transcript.The full-length CDS of MYNN was cloned by RT-PCRand the biological characteristics of MYNN protein was analyzed by bioinformatics in Mashen pig.Quantitative real-time PCR was employed to detect the expression patterns of MYNN gene in heart,liver,spleen,lung,kidney,cerebellum,small intestine,stomach,pancreas,longissimus dorsi and fat tissues of Mashen pig,and to study the developmental expression patterns in stomach and longissimus dorsi tissues.Two transcripts of MYNN gene were successfully cloned in present study,and named MYNN-1(GenBank accession number:KY470829)and MYNN-2(GenBank accession number:KY670835),respectively.MYNN-1 CDS was composed of 1 830 bp encoding 609 amino acids,which belonged to the stable alkaline soluble protein.Whereas MYNN-2 CDS was composed of 1 746 bp encoding 581 amino acids,which belonged to the unstable alkaline soluble protein.MYNN-2 was 84 bp less than MYNN-1,and lacked the exon6.MYNN-2 was found a C2 H2 type zinc finger protein domain less than MYNN-1 by the prediction of function domain.The analysis of homology and phylogenetic tree showed that the two transcripts amino acid sequences of pig MYNNgene had high homology and close genetic distance with polar bear,goat,horse,dog,and so on,which proved that the pig MYNNgene was very conservative during evolutionary process.MYNN-1 and MYNN-2 were universally expressed in all pig tissues detected,and there were significant differences in expression among different tissues(P<0.05).The two variants had higher expression levels in stomach,small intestine and pancreas of Mashen pig,and their expression were the lowest in fat tissue.The expression level of MYNN-1 was significantly or extremely significantly higher than that of MYNN-2 in all tissues except kidney(P<0.05,P<0.01),which testified that MYNN-1 was the main variant in pig.The expression of MYNN-1 and MYNN-2 decreased gradually with the increase of age in the stomach of Mashen pig.In longissimus dorsi,the expression of MYNN-1 and MYNN-2 increased firstly and then decreased with the increase of age.In this study,two transcripts of pig MYNN gene were successfully cloned.And it was speculated that MYNN play important role in the process of digestion and absorption,as well as growth and development of skeletal muscle in pig,whereas the specific mechanisms were still remaining to be further elucidated.
引文
[1]ARAVIND L,KOONIN E V.Fold prediction and evolutionary analysis of the POZ domain:structural and evolutionary relationship with the potassium channel tetramerization domain[J].J Mol Biol,1999,285(4):1353-1361.
[2]LI X M,PENG H Z,SCHULTZ D C,et al.Structure-function studies of the BTB/POZ transcriptional repression domain from the promyelocytic leukemia zinc finger oncoprotein[J].Cancer Res,1999,59(20):5275-5282.
[3]TAKENAGA M,HATANO M,TAKAMORI M,et al.Bcl6-dependent transcriptional repression by BAZF[J].Biochem Biophys Res Common,2003,303(2):600-608.
[4]PHAN R T,DALLA-FAVERA R.The BCL6protooncogene suppresses p53expression in germinal-centre B cells[J].Nature,2004,432(7017):635-639.
[5]KELLY K F,OTCHERE A A,GRAHAM M,et al.Nuclear import of the BTB/POZ transcriptional regulator Kaiso[J].J Cell Sci,2004,117(25):6143-6152.
[6]HUYNH K D,BARDWELL V J.The BCL-6POZ domain and other POZ domains interact with the corepressors N-CoR and SMRT[J].Oncogene,1998,17(19):2473-2484.
[7]DAVID G,ALLAND L,HONG S H,et al.Histone deacetylase associated with mSin3A mediates repression by the acute promyelocytic leukemia-associated PLZF protein[J].Oncogene,1998,16(19):2549-2556.
[8]MELNICK A,CARLILE G,AHMAD K F,et al.Critical residues within the BTB domain of PLZF and Bcl-6modulate interaction with corepressors[J].Mol Cell Biol,2002,22(6):1804-1818.
[9]AHMAD K F,MELNICK A,LAX S,et al.Mechanism of SMRT corepressor recruitment by the BCL6BTB domain[J].Mol Cell,2003,12(6):1551-1564.
[10]LI X C,ZHANG D,HANNINK M,et al.Crystal structure of the Kelch domain of human Keap1[J].J Biol Chem,2004,279(52):54750-54758.
[11]LEDEE D R,GAO C Y,SETH R,et al.A specific interaction between muskelin and the cyclin-dependent kinase 5activator p39promotes peripheral localization of muskelin[J].J Biol Chem,2005,280(22):21376-21383.
[12]PRAG S,COLLETT G D M,ADAMS J C.Molecular analysis of muskelin identifies a conserved discoidin-like domain that contributes to protein self-association[J].Biochem J,2004,381(2):547-559.
[13]周畅,李麓芸.C2H2型锌指蛋白的研究进展[J].生命科学研究,2004,8(3):215-220.ZHOU C,LI L Y.Advances in C2H2zinc finger proteins[J].Life Science Research,2004,8(3):215-220.(in Chinese)
[14]VENTER J C,ADAMS M D,MYERS E W,et al.The sequence of the human genome[J].Science,2001,291(5507):1304-1351.
[15]ALLIEL P M,SEDDIQI N,GOUDOU D,et al.Myoneurin,a novel member of the BTB/POZ-Zinc Finger family highly expressed in human muscle[J].Biochem Biophys Res Common,2000,273(1):385-391.
[16]CIFUENTES-DIAZ C,BITOUN M,GOUDOU D,et al.Neuromuscular expression of the BTB/POZ and zinc finger protein myoneurin[J].Muscle Nerve,2004,29(1):59-65.
[17]ALLIEL P M,CIFUENTS-DIAZ C,BITOUN M,et al.Expression of the transcriptional repressor gene myoneurin at the neuromuscular junction,during mouse development and in the adult[J].J Physiol-Paris,2006,99(2-3):1.
[18]MITCHELMORE C,KJAERULFF K M,PEDERSEN H C,et al.Characterization of two novel nuclear BTB/POZ domain zinc finger isoforms.Association with differentiation of hippocampal neurons,cerebellar granule cells,and macroglia[J].J Biol Chem,2002,277(9):7598-7609.
[19]SAKASHITA C,FUKUDA T,OKABE S,et al.Cloning and characterization of the human BAZF gene,a homologue of the BCL6oncogene[J].Biochem Biophys Res Common,2002,291(3):567-573.
[20]TRAPPE R,BUDDENBERG P,UEDELHOVEN J,et al.The murine BTB/POZ zinc finger gene Znf131:predominant expression in the developing central nervous system,in adult brain,testis,and thymus[J].Biochem Biophys Res Common,2002,296(2):319-327.
[21]孟莹莹,许兴智,廖蓟.转录因子ZBTB家族与肿瘤[J].生命科学,2015,27(4):477-485.MENG Y Y,XU X Z,LIAO J.ZBTB transcription factors and tumorigenesis[J].Chinese Bulletion of Life Sciences,2015,27(4):477-485.(in Chinese)
[22]RAMAKERS C,RUIJTER J M,DEPREZ R H L,et al.Assumption-free analysis of quantitative realtime polymerase chain reaction(PCR)data[J].Neurosci Lett,2003,339(1):62-66.
[23]张天,李祥龙,周荣艳,等.不同毛色山羊皮肤组织Agouti基因剪接体类型研究[J].畜牧兽医学报,2015,46(11):1934-1943.ZHANG T,LI X L,ZHOU R Y,et al.Study on Agouti spliceosome types in goat skin with different coat color[J].Acta Veterinaria et Zootechnica Sinica,2015,46(11):1934-1943.(in Chinese)
[24]MODREK B,LEE C.A genomic view of alternative splicing[J].Nat Genet,2002,30(1):13-19.
[25]FENG H J,QIN Z Y,ZHANG X G.Opportunities and methods for studying alternative splicing in cancer with RNA-Seq[J].Cancer Lett,2013,340(2):179-191.
[26]LIAO X B,CLEMENS K R,TENNANT L,et al.Specific interaction of the first three zinc fingers of TFIIIA with the internal control region of the Xenopus 5SRNA gene[J].J Mol Biol,1992,223(4):857-871.
[27]BRAYER K J,SEGAL D J.Keep your fingers off my DNA:protein-protein interactions mediated by C2H2 zinc finger domains[J].Cell Biochem Biophys,2008,50(3):111-131.
[28]NOLTE R T,CONLIN R M,HARRISON S C,et al.Differing roles for zinc fingers in DNA recognition:structure of a six-finger transcription factor IIIA complex[J].Proc Natl Acad Sci U S A,1998,95(6):2938-2943.
[29]NEELY L,TRAUGER J W,BAIRD E E,et al.Importance of minor groove binding zinc fingers within the transcription factor IIIA-DNA complex[J].J Mol Biol,1997,274(4):439-445.
[30]贾浩,张小白,宋晓峰.人类胞内蛋白半衰期与其亚细胞定位的相关性研究[J].计算机与应用化学,2011,28(4):411-414.JIA H,ZHANG X B,SONG X F.Relationship between intracellular protein half-life and subcellular localization in human cells[J].Computers and Applied Chemistry,2011,28(4):411-414.(in Chinese)
[31]牛姣艳.Pax7、MyoD和MyoG基因在猪背最长肌中的发育性表达研究[D].太谷:山西农业大学,2015.NIU J Y.Study on the developmental expression of Pax7,MyoDand MyoGgenes in longissimus dorsi of pigs[D].Taigu:Shanxi Agricultural University,2015.(in Chinese)
[2]LI X M,PENG H Z,SCHULTZ D C,et al.Structure-function studies of the BTB/POZ transcriptional repression domain from the promyelocytic leukemia zinc finger oncoprotein[J].Cancer Res,1999,59(20):5275-5282.
[3]TAKENAGA M,HATANO M,TAKAMORI M,et al.Bcl6-dependent transcriptional repression by BAZF[J].Biochem Biophys Res Common,2003,303(2):600-608.
[4]PHAN R T,DALLA-FAVERA R.The BCL6protooncogene suppresses p53expression in germinal-centre B cells[J].Nature,2004,432(7017):635-639.
[5]KELLY K F,OTCHERE A A,GRAHAM M,et al.Nuclear import of the BTB/POZ transcriptional regulator Kaiso[J].J Cell Sci,2004,117(25):6143-6152.
[6]HUYNH K D,BARDWELL V J.The BCL-6POZ domain and other POZ domains interact with the corepressors N-CoR and SMRT[J].Oncogene,1998,17(19):2473-2484.
[7]DAVID G,ALLAND L,HONG S H,et al.Histone deacetylase associated with mSin3A mediates repression by the acute promyelocytic leukemia-associated PLZF protein[J].Oncogene,1998,16(19):2549-2556.
[8]MELNICK A,CARLILE G,AHMAD K F,et al.Critical residues within the BTB domain of PLZF and Bcl-6modulate interaction with corepressors[J].Mol Cell Biol,2002,22(6):1804-1818.
[9]AHMAD K F,MELNICK A,LAX S,et al.Mechanism of SMRT corepressor recruitment by the BCL6BTB domain[J].Mol Cell,2003,12(6):1551-1564.
[10]LI X C,ZHANG D,HANNINK M,et al.Crystal structure of the Kelch domain of human Keap1[J].J Biol Chem,2004,279(52):54750-54758.
[11]LEDEE D R,GAO C Y,SETH R,et al.A specific interaction between muskelin and the cyclin-dependent kinase 5activator p39promotes peripheral localization of muskelin[J].J Biol Chem,2005,280(22):21376-21383.
[12]PRAG S,COLLETT G D M,ADAMS J C.Molecular analysis of muskelin identifies a conserved discoidin-like domain that contributes to protein self-association[J].Biochem J,2004,381(2):547-559.
[13]周畅,李麓芸.C2H2型锌指蛋白的研究进展[J].生命科学研究,2004,8(3):215-220.ZHOU C,LI L Y.Advances in C2H2zinc finger proteins[J].Life Science Research,2004,8(3):215-220.(in Chinese)
[14]VENTER J C,ADAMS M D,MYERS E W,et al.The sequence of the human genome[J].Science,2001,291(5507):1304-1351.
[15]ALLIEL P M,SEDDIQI N,GOUDOU D,et al.Myoneurin,a novel member of the BTB/POZ-Zinc Finger family highly expressed in human muscle[J].Biochem Biophys Res Common,2000,273(1):385-391.
[16]CIFUENTES-DIAZ C,BITOUN M,GOUDOU D,et al.Neuromuscular expression of the BTB/POZ and zinc finger protein myoneurin[J].Muscle Nerve,2004,29(1):59-65.
[17]ALLIEL P M,CIFUENTS-DIAZ C,BITOUN M,et al.Expression of the transcriptional repressor gene myoneurin at the neuromuscular junction,during mouse development and in the adult[J].J Physiol-Paris,2006,99(2-3):1.
[18]MITCHELMORE C,KJAERULFF K M,PEDERSEN H C,et al.Characterization of two novel nuclear BTB/POZ domain zinc finger isoforms.Association with differentiation of hippocampal neurons,cerebellar granule cells,and macroglia[J].J Biol Chem,2002,277(9):7598-7609.
[19]SAKASHITA C,FUKUDA T,OKABE S,et al.Cloning and characterization of the human BAZF gene,a homologue of the BCL6oncogene[J].Biochem Biophys Res Common,2002,291(3):567-573.
[20]TRAPPE R,BUDDENBERG P,UEDELHOVEN J,et al.The murine BTB/POZ zinc finger gene Znf131:predominant expression in the developing central nervous system,in adult brain,testis,and thymus[J].Biochem Biophys Res Common,2002,296(2):319-327.
[21]孟莹莹,许兴智,廖蓟.转录因子ZBTB家族与肿瘤[J].生命科学,2015,27(4):477-485.MENG Y Y,XU X Z,LIAO J.ZBTB transcription factors and tumorigenesis[J].Chinese Bulletion of Life Sciences,2015,27(4):477-485.(in Chinese)
[22]RAMAKERS C,RUIJTER J M,DEPREZ R H L,et al.Assumption-free analysis of quantitative realtime polymerase chain reaction(PCR)data[J].Neurosci Lett,2003,339(1):62-66.
[23]张天,李祥龙,周荣艳,等.不同毛色山羊皮肤组织Agouti基因剪接体类型研究[J].畜牧兽医学报,2015,46(11):1934-1943.ZHANG T,LI X L,ZHOU R Y,et al.Study on Agouti spliceosome types in goat skin with different coat color[J].Acta Veterinaria et Zootechnica Sinica,2015,46(11):1934-1943.(in Chinese)
[24]MODREK B,LEE C.A genomic view of alternative splicing[J].Nat Genet,2002,30(1):13-19.
[25]FENG H J,QIN Z Y,ZHANG X G.Opportunities and methods for studying alternative splicing in cancer with RNA-Seq[J].Cancer Lett,2013,340(2):179-191.
[26]LIAO X B,CLEMENS K R,TENNANT L,et al.Specific interaction of the first three zinc fingers of TFIIIA with the internal control region of the Xenopus 5SRNA gene[J].J Mol Biol,1992,223(4):857-871.
[27]BRAYER K J,SEGAL D J.Keep your fingers off my DNA:protein-protein interactions mediated by C2H2 zinc finger domains[J].Cell Biochem Biophys,2008,50(3):111-131.
[28]NOLTE R T,CONLIN R M,HARRISON S C,et al.Differing roles for zinc fingers in DNA recognition:structure of a six-finger transcription factor IIIA complex[J].Proc Natl Acad Sci U S A,1998,95(6):2938-2943.
[29]NEELY L,TRAUGER J W,BAIRD E E,et al.Importance of minor groove binding zinc fingers within the transcription factor IIIA-DNA complex[J].J Mol Biol,1997,274(4):439-445.
[30]贾浩,张小白,宋晓峰.人类胞内蛋白半衰期与其亚细胞定位的相关性研究[J].计算机与应用化学,2011,28(4):411-414.JIA H,ZHANG X B,SONG X F.Relationship between intracellular protein half-life and subcellular localization in human cells[J].Computers and Applied Chemistry,2011,28(4):411-414.(in Chinese)
[31]牛姣艳.Pax7、MyoD和MyoG基因在猪背最长肌中的发育性表达研究[D].太谷:山西农业大学,2015.NIU J Y.Study on the developmental expression of Pax7,MyoDand MyoGgenes in longissimus dorsi of pigs[D].Taigu:Shanxi Agricultural University,2015.(in Chinese)