哈萨克羊INHβA基因克隆及生物信息学分析
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摘要
试验旨在克隆哈萨克羊抑制素βA(inhibin beta A,INHβA)基因序列,并探究其序列特征及编码蛋白的结构和功能。根据GenBank中绵羊INHβA基因序列(登录号:NM_001009458.1)设计1对特异性引物,通过RT-PCR方法对哈萨克羊INHβA基因进行扩增,将获得的INHβA基因片段插入到pMD19-T载体中进行克隆测序,并结合生物信息学方法预测和分析其核苷酸序列、氨基酸序列、蛋白跨膜、蛋白修饰位点及二级结构、三级结构模型等。结果显示,哈萨克羊INHβA基因长1 278 bp,编码425个氨基酸。哈萨克羊INHβA基因与绵羊、牛、野猪、小鼠、人、大鼠、猫、兔、马的同源性分别为98.6%、97.7%、90.4%、87.9%、91.1%、88.2%、91.8%、89.8%和91.8%,表明INHβA基因在不同物种之间具有较高的保守性。INHβA蛋白分子式为C_(2072)H_(3325)N_(603)O_(628)S_(26),分子质量为47.57 ku,理论等电点(pI)为7.87,不稳定系数为66.16,脂溶指数为78.47,亲水值为-0.507。INHβA是一种碱性不稳定的亲水性脂溶性蛋白,没有跨膜结构,含有信号肽。二级结构预测显示,INHβA蛋白α-螺旋、β-转角、无规则卷曲、延伸链分别占22.59%、4.47%、54.12%和18.82%。三级结构预测显示,INHβA蛋白以α-螺旋为主,是含有8种蛋白修饰位点的β-桶状蛋白。本试验结果为深入研究哈萨克羊INHβA蛋白功能及探讨INHβA基因对提高哈萨克羊繁殖力的影响提供参考数据。
This study was aimed to clone the sequence of inhibin beta A(INHβA) in Kazakh sheep,and explore the sequence characteristics of INHβA gene and the structure and function of its encoded protein.One pair of specific primers was disigned based on the sequence of sheep INHβA gene(accession No.:NM_001009458.1) in GenBank,the sequence of INHβA gene in Kazakh sheep was amplified by RT-PCR.INHβA gene was inserted into pMD19-T vector for cloning and sequencing,its nucleotide sequence,amino acids sequence,protein transmembrane,protein modification site and secondary structure,tertiary structure model were predicted and analyzed by bioinformatics methods.The results showed that INHβA gene in Kazakh sheep was 1 278 bp in length and encoded 425 amino acids.The homology of INHβA gene in Kazakh sheep with Ovis aries,Bos taurus,Sus scrofa,Mus musculus,Homo sapiens,Rattus norvegicus,Felis catus,Rytolagus cuniculus and Equus caballus was 98.6%,97.7%,90.4%,87.9%,91.1%,88.2%,91.8%,89.8% and 91.8%,respectively,indicating that INHβA gene was highly conserved among different species.The molecular formula of INHβA protein was C_(2072)H_(3325)N_(603)O_(628)S_(26),the molecular weight was 47.57 ku,the theoretical isoelectric point(pI) was 7.87,the instability coefficient was 66.16,the fat solubility index was 78.47,and the hydrophilic value was-0.507.The INHβA protein was an alkaline labile hydrophilic liposoluble protein containing a signal peptide,with no transmembrane structure.The secondary structure prediction showed that alpha helix,beta turn,random coil,and extended chain of INHβA protein accounted for 22.59%,4.47%,54.12% and 18.82%,respectively.The tertiary structure prediction showed that the spatial structure of the protein was mainly alpha helix,and contained β-barrel structures of 8 protein modification sites.This results provided a data reference for the later study of the function of INHβA protein and the effect of INHβA gene on improving the fecundity in Kazakh sheep.
This study was aimed to clone the sequence of inhibin beta A(INHβA) in Kazakh sheep,and explore the sequence characteristics of INHβA gene and the structure and function of its encoded protein.One pair of specific primers was disigned based on the sequence of sheep INHβA gene(accession No.:NM_001009458.1) in GenBank,the sequence of INHβA gene in Kazakh sheep was amplified by RT-PCR.INHβA gene was inserted into pMD19-T vector for cloning and sequencing,its nucleotide sequence,amino acids sequence,protein transmembrane,protein modification site and secondary structure,tertiary structure model were predicted and analyzed by bioinformatics methods.The results showed that INHβA gene in Kazakh sheep was 1 278 bp in length and encoded 425 amino acids.The homology of INHβA gene in Kazakh sheep with Ovis aries,Bos taurus,Sus scrofa,Mus musculus,Homo sapiens,Rattus norvegicus,Felis catus,Rytolagus cuniculus and Equus caballus was 98.6%,97.7%,90.4%,87.9%,91.1%,88.2%,91.8%,89.8% and 91.8%,respectively,indicating that INHβA gene was highly conserved among different species.The molecular formula of INHβA protein was C_(2072)H_(3325)N_(603)O_(628)S_(26),the molecular weight was 47.57 ku,the theoretical isoelectric point(pI) was 7.87,the instability coefficient was 66.16,the fat solubility index was 78.47,and the hydrophilic value was-0.507.The INHβA protein was an alkaline labile hydrophilic liposoluble protein containing a signal peptide,with no transmembrane structure.The secondary structure prediction showed that alpha helix,beta turn,random coil,and extended chain of INHβA protein accounted for 22.59%,4.47%,54.12% and 18.82%,respectively.The tertiary structure prediction showed that the spatial structure of the protein was mainly alpha helix,and contained β-barrel structures of 8 protein modification sites.This results provided a data reference for the later study of the function of INHβA protein and the effect of INHβA gene on improving the fecundity in Kazakh sheep.
引文
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[7] BUKHARI I,LI G,WANG L,et al.Effects of androgen receptor mutation on testicular histopathology of patient having complete androgen insensitivity[J].Journal of Molecular Histology,2017,48(3):159-167.
[8] FRAGOSO M C,KOHEK M B,MARTIN R M,et al.An inhibin B and estroger-secreting adrenocortical carcinoma leading to selective FSH suppression[J].Hormone Research in Paediatrics,2007,67(1):7-11.
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[11] DAN X,LIU X,HAN Y,et al.Effect of the novel DNA vaccine fusing inhibin a (1-32) and the RF-ama ide related peptide-3 genes on immune response,hormone levels and fertility in Tan sheep[J].Animal Reproduction Science,2016,164(1):105-110.
[12] LI W,CHEN L,CHEN S,et al.Polymorphisms in inhibin A gene promoter associated with male infertility[J].Gene,2015,559(2):172-176.
[13] KROGH A,LARSSON B,VON HEIJINE G,et al.Predicting transmembrane protein topology with a hidden Markov model:Application to complete genomes[J].Journal of Molecular Biology,2001,305(3):567-580.
[14] SONNHAMMER E,VON HEIJINE G,KROGH A.A hidden Markov model for predicting transmembrane helices in protein sequences[J].Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology,1998,6:175-182.
[15] YACHDAV G,KLOPPMANN E,KAJAN L,et al.Predict protein——An open resource for online prediction of protein structural and functional features[J].Nucleic Acids Research,2014,42(1):337-343.
[16] THOMAS N P,S?REN B,GUNNARVON H,et al.SignalP 4.0:Discriminating signal peptides from transmembrane regions[J].Nature Methods,2011,8(10):785-786.
[17] ZHANG Y.I-TASSER server for protein 3D structure prediction[J].BMC Bioinformatics,2008,9:40.
[18] ROY A,KUCUKURAL A,ZHANG Y.I-TASSER:A unified platform for automated protein structure and function prediction[J].Nature Protocols,2010,5(4):725-738.
[19] YANG J,YAN R,ROY A,et al.The I-TASSER Suite:Protein structure and function prediction[J].Nature Methods,2015,12(1):7-8.
[20] 肖朝庭.影响绵羊高繁殖力和非季节性发情基因的研究[D].杭州:浙江大学,2007.XIAO C T.Study on genes related to ovine prolificacy and year-round estrus[D].Hangzhou:Zhejiang University,2007.(in Chinese)
[21] DAS N,RAJENDRA K.Molecular regulation of follicle-stimulating hormone synthesis,secretion and action[J].Journal of Molecular Endocrinology,2018,60(3):R131-R155.
[22] MAKANJI Y,ZHU J,MISHRA R,et al.Inhibin at 90:From discovery to clinical application,a historical review[J].Endocrine Reviews,2014,35(5):747-794.
[23] WALTON K L,MAKAN Y,ROBERTSON D M,et al.The synthesis and secretion of inhibins[J].Vitamins and Hormones-Advances in Research and Applications,2011,85:149-184.
[24] VITT U A,HSU S Y,HSUEH A J.Evolution and classification of cystine knot-containing hormones and related extracellular signaling molecules[J].Molecular Endocrinology,2001,15(5):681-694.
[25] ZHU J,BRAUN E L,KOHNO S,et al.Phylogenomic analyses reveal the evolutionary origin of the inhibin ar subunit,a unique TGF superfamily antagonist[J].PLoS One,2010,5(3):e9457.
[26] 张宇飞,李晓霞,王士勇,等.北极狐及乌苏里貉抑制素α亚基基因克隆及生物信息学分析[J].中国畜牧兽医,2017,44(12):3391-3400.ZHANG Y F,LI X X,WANG S Y,et al.Cloning and bioinformatics analysis of inhibin α subunit gene of arctic fox and Wusuli raccoon dog[J].China Animal Husbandry & Veterinary Medicine,2017,44(12):3391-3400.(in Chinese)
[2] LI W H,CHEN S X,LI H J I,et al.A new insertion/deletion fragment polymorphism of inhibinα gene associated with follicular cysts in Large White sows[J].Journal of Veterinary Medical Science,2016,78(3):473-476.
[3] 李婷,马爱团,张英杰,等.中国内动物抑制素研究现状与热点透析[J].中国畜牧杂志,2016,52(22):28-32.LI T,MA A T,ZHANG Y J,et al.Research status and hotspot analysis of animal inhibin in China[J].Chinese Journal of Animal Science,2016,52(22):28-32.(in Chinese)
[4] NAMWANJE M,BROWN C W.Activins and inhibins:Roles in development,physiology and disease[J].Cold Spring Harbor Perspectives in Biology,2016,8(7):a021881.
[5] SUSAN M G,KENNETH P M,LISA M C,et al.Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner[J].Nature Genetics,2000,25(3):279-283.
[6] NICOLAS N,MICHEL V,BHUSHAN S,et al.Testicular activin and follistatin levels are elevated during the course of experimental autoimmune epididymo-orchitis in mice[J].Scientific Reports,2017,7:42391.
[7] BUKHARI I,LI G,WANG L,et al.Effects of androgen receptor mutation on testicular histopathology of patient having complete androgen insensitivity[J].Journal of Molecular Histology,2017,48(3):159-167.
[8] FRAGOSO M C,KOHEK M B,MARTIN R M,et al.An inhibin B and estroger-secreting adrenocortical carcinoma leading to selective FSH suppression[J].Hormone Research in Paediatrics,2007,67(1):7-11.
[9] 黄元射,毛景欣,胡煜,等.山羊多胎主效基因的研究进展[J].四川畜牧兽医,2018,45(3):35-37.HUANG Y S,MAO J X,HU Y,et al.The research progress of goat multiplets traits[J].Sichuan Animal & Veterinary Sciences,2018,45(3):35-37.(in Chinese)
[10] 薛昱,储明星,周忠孝.抑制素基因的研究进展[J].遗传,2004,26(5):749-755.XUE Y,CHU M X,ZHOU Z X.Advances on inhibin genes[J].Hereditas,2004,26(5):749-755.(in Chinese)
[11] DAN X,LIU X,HAN Y,et al.Effect of the novel DNA vaccine fusing inhibin a (1-32) and the RF-ama ide related peptide-3 genes on immune response,hormone levels and fertility in Tan sheep[J].Animal Reproduction Science,2016,164(1):105-110.
[12] LI W,CHEN L,CHEN S,et al.Polymorphisms in inhibin A gene promoter associated with male infertility[J].Gene,2015,559(2):172-176.
[13] KROGH A,LARSSON B,VON HEIJINE G,et al.Predicting transmembrane protein topology with a hidden Markov model:Application to complete genomes[J].Journal of Molecular Biology,2001,305(3):567-580.
[14] SONNHAMMER E,VON HEIJINE G,KROGH A.A hidden Markov model for predicting transmembrane helices in protein sequences[J].Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology,1998,6:175-182.
[15] YACHDAV G,KLOPPMANN E,KAJAN L,et al.Predict protein——An open resource for online prediction of protein structural and functional features[J].Nucleic Acids Research,2014,42(1):337-343.
[16] THOMAS N P,S?REN B,GUNNARVON H,et al.SignalP 4.0:Discriminating signal peptides from transmembrane regions[J].Nature Methods,2011,8(10):785-786.
[17] ZHANG Y.I-TASSER server for protein 3D structure prediction[J].BMC Bioinformatics,2008,9:40.
[18] ROY A,KUCUKURAL A,ZHANG Y.I-TASSER:A unified platform for automated protein structure and function prediction[J].Nature Protocols,2010,5(4):725-738.
[19] YANG J,YAN R,ROY A,et al.The I-TASSER Suite:Protein structure and function prediction[J].Nature Methods,2015,12(1):7-8.
[20] 肖朝庭.影响绵羊高繁殖力和非季节性发情基因的研究[D].杭州:浙江大学,2007.XIAO C T.Study on genes related to ovine prolificacy and year-round estrus[D].Hangzhou:Zhejiang University,2007.(in Chinese)
[21] DAS N,RAJENDRA K.Molecular regulation of follicle-stimulating hormone synthesis,secretion and action[J].Journal of Molecular Endocrinology,2018,60(3):R131-R155.
[22] MAKANJI Y,ZHU J,MISHRA R,et al.Inhibin at 90:From discovery to clinical application,a historical review[J].Endocrine Reviews,2014,35(5):747-794.
[23] WALTON K L,MAKAN Y,ROBERTSON D M,et al.The synthesis and secretion of inhibins[J].Vitamins and Hormones-Advances in Research and Applications,2011,85:149-184.
[24] VITT U A,HSU S Y,HSUEH A J.Evolution and classification of cystine knot-containing hormones and related extracellular signaling molecules[J].Molecular Endocrinology,2001,15(5):681-694.
[25] ZHU J,BRAUN E L,KOHNO S,et al.Phylogenomic analyses reveal the evolutionary origin of the inhibin ar subunit,a unique TGF superfamily antagonist[J].PLoS One,2010,5(3):e9457.
[26] 张宇飞,李晓霞,王士勇,等.北极狐及乌苏里貉抑制素α亚基基因克隆及生物信息学分析[J].中国畜牧兽医,2017,44(12):3391-3400.ZHANG Y F,LI X X,WANG S Y,et al.Cloning and bioinformatics analysis of inhibin α subunit gene of arctic fox and Wusuli raccoon dog[J].China Animal Husbandry & Veterinary Medicine,2017,44(12):3391-3400.(in Chinese)