家兔矮小同源盒基因(Shox2)的克隆和表达分析
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摘要
为了研究新西兰白兔矮小同源盒基因(Shox2)序列,预测其结构和功能,以及研究Shox2基因的时空表达特征,本研究克隆了新西兰白兔Shox2基因全长CDS区,分析其编码蛋白的同源性、亲疏水性、二级和三级结构特点、系统进化树等生物信息学特征,并用Western blot法验证融合蛋白TrxA-Shox2。分别在胚胎中期(E20.5d)、胚胎后期(E28.5d)、幼龄期(出生后10d)、成年期(A)4个时间点采集3只动物的肾、大血管、真皮、心、大脑、肝、肺、脾、肌肉、胆囊共10种组织,采用实时荧光定量PCR技术研究Shox2基因的时空表达图谱。结果,成功克隆了新西兰白兔Shox2基因(登录号:KP726285)。Shox2基因CDS区全长666bp,翻译221个氨基酸。生物信息学分析显示,Shox2为碱性、不稳定蛋白,Shox2蛋白氨基酸二级结构中α-螺旋区域占61.99%,无规则卷曲区域占33.48%,延伸链占4.52%。Western blot验证了融合蛋白TrxA-Shox2的表达。家兔Shox2蛋白氨基酸序列与眼镜猴、猩猩、褐家鼠、人的氨基酸序列同源性较高,说明Shox2蛋白在进化中较保守。Shox2基因mRNA在很多器官都有表达,在E20.5d的家兔胚胎中,Shox2mRNA在肾、肌肉和胆囊的表达量高,在E28.5d时表达量较高的是脾、心,幼龄期时表达量较高的是真皮、肝,成年期时表达量较高的是真皮、脾。本研究成功克隆了新西兰白兔Shox2基因,预测了其结构和功能。Shox2基因mRNA在很多器官都有表达,不同的组织和不同时间特征的表达量变化可能与Shox2功能高度相关,并受到严格的调控。
To clone and characterize short stature homeobox 2 (Shox2)gene in New Zealand rabbit,predict its structure and function,and to investigate its expression pattern,the full-length CDS sequence of Shox2 was cloned,and the biological characteristics of Shox2 protein were analyzed in this study,such as homology,hydrophilicity,secondary and tertiary structure characteristics and phylogenetic tree.Western blot was employed to verify fusion protein TrxA-Shox2 .Quantitative real-time RT-PCR(RT-qPCR)was employed to detect the expression patterns of Shox2 in kidney,aorta,heart,dermis,lung,brain,liver,spleen,gallbladder and muscle tissues at 4different developmental phases(E20.5d,E28.5d,10dand adult)of 3animals for each phase.Shox2 gene was successfully cloned in present study(GenBank accession number:KP726285),CDS of Shox2 was composed of 666bp encoding 221amino acids.According to the results of bioinformatics analysis,Shox2 belonged to the unstable alkaline protein,61.99%of which amino acids was alpha helix,33.48%for random coil,4.52%for extended strand.The result of Western blot verified the expression of fusion protein TrxA-Shox2 .The analysis of homology and phylogenetic tree showed that the amino acid sequence of Oryctolagus cuniculus Shox2 protein had high homology and close genetic distance with tarsier,gorilla,rattus norvegicus and human,which meant that Shox2 gene was conservative during evolutionary process.According to the result of RT-qPCR,Shox2 gene universally expressed in most tissues detected in this study.A relatively higher Shox2 mRNA expression level was detected in kidney,gallbladder and muscle tissues in embryo for E20.5d,in heart and spleen in embryo for E28.5d,in dermis and liver for 10d,and in derminds and spleen for adult.In this study,Shox2 gene was successfully cloned and characterized.Shox2 gene universally expressed in all tissues detected,its expression level was related to the function of Shox2 in different tissues and at different developmental stages,which was strictly regulated.
To clone and characterize short stature homeobox 2 (Shox2)gene in New Zealand rabbit,predict its structure and function,and to investigate its expression pattern,the full-length CDS sequence of Shox2 was cloned,and the biological characteristics of Shox2 protein were analyzed in this study,such as homology,hydrophilicity,secondary and tertiary structure characteristics and phylogenetic tree.Western blot was employed to verify fusion protein TrxA-Shox2 .Quantitative real-time RT-PCR(RT-qPCR)was employed to detect the expression patterns of Shox2 in kidney,aorta,heart,dermis,lung,brain,liver,spleen,gallbladder and muscle tissues at 4different developmental phases(E20.5d,E28.5d,10dand adult)of 3animals for each phase.Shox2 gene was successfully cloned in present study(GenBank accession number:KP726285),CDS of Shox2 was composed of 666bp encoding 221amino acids.According to the results of bioinformatics analysis,Shox2 belonged to the unstable alkaline protein,61.99%of which amino acids was alpha helix,33.48%for random coil,4.52%for extended strand.The result of Western blot verified the expression of fusion protein TrxA-Shox2 .The analysis of homology and phylogenetic tree showed that the amino acid sequence of Oryctolagus cuniculus Shox2 protein had high homology and close genetic distance with tarsier,gorilla,rattus norvegicus and human,which meant that Shox2 gene was conservative during evolutionary process.According to the result of RT-qPCR,Shox2 gene universally expressed in most tissues detected in this study.A relatively higher Shox2 mRNA expression level was detected in kidney,gallbladder and muscle tissues in embryo for E20.5d,in heart and spleen in embryo for E28.5d,in dermis and liver for 10d,and in derminds and spleen for adult.In this study,Shox2 gene was successfully cloned and characterized.Shox2 gene universally expressed in all tissues detected,its expression level was related to the function of Shox2 in different tissues and at different developmental stages,which was strictly regulated.
引文
[1]KRUMLAUF R,HOLLAND P W,MCVEY J H,et al.Developmental and spatial patterns of expression of the mouse homeobox gene,Hox 2.1[J].Development,1987,99(4):603-617.
[2]VAN SCHAICK H S A,SMIDT M P,ROVESCAL-LI A C,et al.Homeobox gene Prx3expression in rodent brain and extraneural tissues[J].Proc Natl Acad Sci U S A,1997,94(24):12993-12998.
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[5]REN M,WANG C,SHENG D,et al.Methylation analysis of SHOX2and RASSF1Ain bronchoalveolar lavage fluid for early lung cancer diagnosis[J].Ann Diagn Pathol,2017,27:57-61.
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[7]陈瑞,杨晓农,曾婉秋,等.家兔不同发育阶段和组织中内参基因的稳定性分析[J].畜牧兽医学报,2016,47(3):477-483.CHEN R,YANG X N,ZENG W Q,et al.Expression stability analysis of reference genes in different development periods and tissues in oryctolagus cuniculus[J].Acta Veterinaria et Zootechnica Sinica,2016,47(3):447-483.(in Chinese)
[8]CHESTERMAN E S,GAINEY G D,VARN A C,et al.Investigation of Prx1protein expression provides evidence for conservation of cardiac-specific posttranscriptional regulation in vertebrates[J].Dev Dyn,2001,222(3):459-470.
[9]SMITH T M,LOZANOFF S,IYYANAR P P,et al.Molecular signaling along the anterior-posterior axis of early palate development[J].Front Physiol,2013,3:488.
[10]CENSANI M,ANYANE-YEBOA K,WAPNER R,et al.Rare inheritance of Leri-Weill Syndrome due to crossover of short stature Homeobox Gene(SHOX)deletions between X and Y Chromosomes:a case report[J].Int J Pediatr Endocrinol,2013(1):11.
[11]GROSSCHEDL R,GIESE K,PAGEL J.HMG domain proteins:architectural elements in the assembly of nucleoprotein structures[J].Trends Genet,1994,10(3):94-100.
[12]YU L,GU S P,ALAPPAT S,et al.Shox2-deficient mice exhibit a rare type of incomplete clefting of the secondary palate[J].Development,2005,132(19):4397-4406.
[13]HILLIARD S A,YU L,GU S P,et al.Regional regulation of palatal growth and patterning along the anterior-posterior axis in mice[J].J Anat,2005,207(5):655-667.
[14]LI Q,DING J X.Gene expression analysis reveals that formation of the mouse anterior secondary palate involves recruitment of cells from the posterior side[J].Int J Dev Biol,2007,51(2):167-172.
[15]COBB J,DIERICH A,HUSS-GARCIA Y,et al.Amouse model for human short-stature syndromes identifies Shox2as an upstream regulator of Runx2during long-bone development[J].Proc Natl Acad Sci U S A,2006,103(12):4511-4515.
[16]BOBICK B E,COBB J.Shox2regulates progression through chondrogenesis in the mouse proximal limb[J].J Cell Sci,2012,125:6071-6083.
[17]PUSKARIC S,SCHMITTECKERT S,MORI A D,et al.Shox2mediates Tbx5activity by regulating Bmp4in the pacemaker region of the developing heart[J].Hum Mol Genet,2010,19(23):4625-4633.
[18]NEUFELD S J,WANG F,COBB J.Genetic interactions between Shox2and Hox genes during the regional growth and development of the mouse limb[J].Genetics,2014,198(3):1117-1126.
[19]VICKERMAN L,NEUFELD S,COBB J.Shox2function couples neural,muscular and skeletal development in the proximal forelimb[J].Dev Biol,2011,350(2):323-336.
[20]YE W D,SONG Y N,HUANG Z,et al.A unique stylopod patterning mechanism by Shox2-controlled osteogenesis[J].Development,2016,143(14):2548-2660.
[21]GU S P,WEI N,YU L,et al.Shox2-deficiency leads to dysplasia and ankylosis of the temporomandibular joint in mice[J].Mech Dev,2008,125(8):729-742.
[22]LIN D H,HUANG Y D,HE F L,et al.Expression survey of genes critical for tooth development in the human embryonic tooth germ[J].Dev Dyn,2007,236(5):1307-1312.
[23]HOFFMANN S,BERGER I M,GLASER A,et al.Islet1is a direct transcriptional target of the homeodomain transcription factor Shox2and rescues the Shox2-mediated bradycardia[J].Basic Res Cardiol,2013,108(2):339.
[24]HU W Y,XIN Y G,ZHAO Y N,et al.Shox2:The role in differentiation and development of Cardiac conduction system[J].Tohoku J Exp Med,2018,244(3):177-186.
[25]BLASCHKE R J,HAHURIJ N D,KUIJPER S,et al.Targeted mutation reveals essential functions of the homeodomain transcription factor Shox2in sinoatrial and pacemaking development[J].Circulation,2007,115(14):1830-1838.
[26]LIU H B,CHEN C H,YE W D,et al.Phosphorylation of Shox2is required for its function to control sinoatrial node formation[J].J Am Heart Assoc,2014,3(3):e000796.
[27]YAMAMOTO Y J,GESTA S,LEE K Y,et al.Adipose depots possess unique developmental gene signatures[J].Obesity,2010,18(5):872-878.
[28]WEISS G,SCHLEGEL A,KOTTWITZ D,et al.Validation of the SHOX2/PTGER4 DNA methylation marker panel for plasma-based discrimination between patients with malignant and nonmalignant lung disease[J].J Thorac Oncol,2017,12(1):77-84.
[29]BERGHEIM J,SEMAAN A,GEVENSLEBEN H,et al.Potential of quantitative SEPT9 and SHOX2methylation in plasmatic circulating cell-free DNA as auxiliary staging parameter in colorectal cancer:aprospective observational cohort study[J].Br J Cancer,2018,118(9):1217-1228.
[30]ZHANG C Z,YU W J,WANG L,et al.DNA methylation analysis of the SHOX2and RASSF1Apanel in bronchoalveolar lavage fluid for lung cancer diagnosis[J].J Cancer,2017,8(17):3585-3591.
[31]NI S M,YE M,HUANG T.Short stature homeobox2methylation as a potential noninvasive biomarker in bronchial aspirates for lung cancer diagnosis[J].Oncotarget,2017,8(37):61253-61263.
[32]BRANCHI V,SCHAEFER P,SEMAAN A,et al.Promoter hypermethylation of SHOX2and SEPT9is apotential biomarker for minimally invasive diagnosis in adenocarcinomas of the biliary tract[J].Clin Epigenetics,2016,8:133.
[33]ZHANG Y A,ZHOU Y,LUO X,et al.SHOX2is a potent independent biomarker to predict survival of WHO Grade II-III diffuse gliomas[J].EBioMedicine,2016,13:80-89.
[34]JUNG M,PUTZER S,GEVENSLEBEN H,et al.Diagnostic and prognostic value of SHOX2and SEPT9DNA methylation and cytology in benign,paramalignant,and malignant ascites[J].Clin Epigenetics,2016,8:24.
[2]VAN SCHAICK H S A,SMIDT M P,ROVESCAL-LI A C,et al.Homeobox gene Prx3expression in rodent brain and extraneural tissues[J].Proc Natl Acad Sci U S A,1997,94(24):12993-12998.
[3]SEMAAN A,VAN ELLEN A,MELLER S,et al.SEPT9and SHOX2DNA methylation status and its utility in the diagnosis of colonic adenomas and colorectal adenocarcinomas[J].Clin Epigenetics,2016,8:100.
[4]ROVESCALLI A C,ASOH S,NIRENBERG M.Cloning and characterization of four murine homeobox genes[J].Proc Natl Acad Sci U S A,1996,93(20):10691-10696.
[5]REN M,WANG C,SHENG D,et al.Methylation analysis of SHOX2and RASSF1Ain bronchoalveolar lavage fluid for early lung cancer diagnosis[J].Ann Diagn Pathol,2017,27:57-61.
[6]SCHNEIDER K U,DIETRICH D,FLEISCHHACK-ER M,et al.Correlation of SHOX2gene amplification and DNA methylation in lung cancer tumors[J].BMCCancer,2011,11:102.
[7]陈瑞,杨晓农,曾婉秋,等.家兔不同发育阶段和组织中内参基因的稳定性分析[J].畜牧兽医学报,2016,47(3):477-483.CHEN R,YANG X N,ZENG W Q,et al.Expression stability analysis of reference genes in different development periods and tissues in oryctolagus cuniculus[J].Acta Veterinaria et Zootechnica Sinica,2016,47(3):447-483.(in Chinese)
[8]CHESTERMAN E S,GAINEY G D,VARN A C,et al.Investigation of Prx1protein expression provides evidence for conservation of cardiac-specific posttranscriptional regulation in vertebrates[J].Dev Dyn,2001,222(3):459-470.
[9]SMITH T M,LOZANOFF S,IYYANAR P P,et al.Molecular signaling along the anterior-posterior axis of early palate development[J].Front Physiol,2013,3:488.
[10]CENSANI M,ANYANE-YEBOA K,WAPNER R,et al.Rare inheritance of Leri-Weill Syndrome due to crossover of short stature Homeobox Gene(SHOX)deletions between X and Y Chromosomes:a case report[J].Int J Pediatr Endocrinol,2013(1):11.
[11]GROSSCHEDL R,GIESE K,PAGEL J.HMG domain proteins:architectural elements in the assembly of nucleoprotein structures[J].Trends Genet,1994,10(3):94-100.
[12]YU L,GU S P,ALAPPAT S,et al.Shox2-deficient mice exhibit a rare type of incomplete clefting of the secondary palate[J].Development,2005,132(19):4397-4406.
[13]HILLIARD S A,YU L,GU S P,et al.Regional regulation of palatal growth and patterning along the anterior-posterior axis in mice[J].J Anat,2005,207(5):655-667.
[14]LI Q,DING J X.Gene expression analysis reveals that formation of the mouse anterior secondary palate involves recruitment of cells from the posterior side[J].Int J Dev Biol,2007,51(2):167-172.
[15]COBB J,DIERICH A,HUSS-GARCIA Y,et al.Amouse model for human short-stature syndromes identifies Shox2as an upstream regulator of Runx2during long-bone development[J].Proc Natl Acad Sci U S A,2006,103(12):4511-4515.
[16]BOBICK B E,COBB J.Shox2regulates progression through chondrogenesis in the mouse proximal limb[J].J Cell Sci,2012,125:6071-6083.
[17]PUSKARIC S,SCHMITTECKERT S,MORI A D,et al.Shox2mediates Tbx5activity by regulating Bmp4in the pacemaker region of the developing heart[J].Hum Mol Genet,2010,19(23):4625-4633.
[18]NEUFELD S J,WANG F,COBB J.Genetic interactions between Shox2and Hox genes during the regional growth and development of the mouse limb[J].Genetics,2014,198(3):1117-1126.
[19]VICKERMAN L,NEUFELD S,COBB J.Shox2function couples neural,muscular and skeletal development in the proximal forelimb[J].Dev Biol,2011,350(2):323-336.
[20]YE W D,SONG Y N,HUANG Z,et al.A unique stylopod patterning mechanism by Shox2-controlled osteogenesis[J].Development,2016,143(14):2548-2660.
[21]GU S P,WEI N,YU L,et al.Shox2-deficiency leads to dysplasia and ankylosis of the temporomandibular joint in mice[J].Mech Dev,2008,125(8):729-742.
[22]LIN D H,HUANG Y D,HE F L,et al.Expression survey of genes critical for tooth development in the human embryonic tooth germ[J].Dev Dyn,2007,236(5):1307-1312.
[23]HOFFMANN S,BERGER I M,GLASER A,et al.Islet1is a direct transcriptional target of the homeodomain transcription factor Shox2and rescues the Shox2-mediated bradycardia[J].Basic Res Cardiol,2013,108(2):339.
[24]HU W Y,XIN Y G,ZHAO Y N,et al.Shox2:The role in differentiation and development of Cardiac conduction system[J].Tohoku J Exp Med,2018,244(3):177-186.
[25]BLASCHKE R J,HAHURIJ N D,KUIJPER S,et al.Targeted mutation reveals essential functions of the homeodomain transcription factor Shox2in sinoatrial and pacemaking development[J].Circulation,2007,115(14):1830-1838.
[26]LIU H B,CHEN C H,YE W D,et al.Phosphorylation of Shox2is required for its function to control sinoatrial node formation[J].J Am Heart Assoc,2014,3(3):e000796.
[27]YAMAMOTO Y J,GESTA S,LEE K Y,et al.Adipose depots possess unique developmental gene signatures[J].Obesity,2010,18(5):872-878.
[28]WEISS G,SCHLEGEL A,KOTTWITZ D,et al.Validation of the SHOX2/PTGER4 DNA methylation marker panel for plasma-based discrimination between patients with malignant and nonmalignant lung disease[J].J Thorac Oncol,2017,12(1):77-84.
[29]BERGHEIM J,SEMAAN A,GEVENSLEBEN H,et al.Potential of quantitative SEPT9 and SHOX2methylation in plasmatic circulating cell-free DNA as auxiliary staging parameter in colorectal cancer:aprospective observational cohort study[J].Br J Cancer,2018,118(9):1217-1228.
[30]ZHANG C Z,YU W J,WANG L,et al.DNA methylation analysis of the SHOX2and RASSF1Apanel in bronchoalveolar lavage fluid for lung cancer diagnosis[J].J Cancer,2017,8(17):3585-3591.
[31]NI S M,YE M,HUANG T.Short stature homeobox2methylation as a potential noninvasive biomarker in bronchial aspirates for lung cancer diagnosis[J].Oncotarget,2017,8(37):61253-61263.
[32]BRANCHI V,SCHAEFER P,SEMAAN A,et al.Promoter hypermethylation of SHOX2and SEPT9is apotential biomarker for minimally invasive diagnosis in adenocarcinomas of the biliary tract[J].Clin Epigenetics,2016,8:133.
[33]ZHANG Y A,ZHOU Y,LUO X,et al.SHOX2is a potent independent biomarker to predict survival of WHO Grade II-III diffuse gliomas[J].EBioMedicine,2016,13:80-89.
[34]JUNG M,PUTZER S,GEVENSLEBEN H,et al.Diagnostic and prognostic value of SHOX2and SEPT9DNA methylation and cytology in benign,paramalignant,and malignant ascites[J].Clin Epigenetics,2016,8:24.
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