家兔矮小同源盒基因(Shox2)的克隆和表达分析

doi:10.11843/j.issn.0366-6964.2019.01.008

Abstract

Abstract:

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 4 different developmental phases(E20.5 d, E28.5 d, 10 d and adult) of 3 animals for each phase. Shox2 gene was successfully cloned in present study (GenBank accession number:KP726285), CDS of Shox2 was composed of 666 bp encoding 221 amino 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 E20.5 d, in heart and spleen in embryo for E28.5 d, in dermis and liver for 10 d, 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.

Key words: Oryctolagus cuniculus, short stature homeobox 2(Shox2), cloning, bioinformatics analysis, expression pattern

CLC Number:

S829.12

CHEN Rui, ZHU Zifeng, HUANG Jian, YANG Xiaonong. Molecular Cloning and Expression Analysis of Short Stature Homeobox 2 (Shox2) in Oryctolagus cuniculus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(1): 61-70.

References

[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,ROVESCALLI A C,et al.Homeobox gene Prx3 expression 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.SEPT9 and SHOX2 DNA 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 SHOX2 and RASSF1A in bronchoalveolar lavage fluid for early lung cancer diagnosis[J].Ann Diagn Pathol,2017,27:57-61.
[6] SCHNEIDER K U,DIETRICH D,FLEISCHHACKER M,et al.Correlation of SHOX2 gene amplification and DNA methylation in lung cancer tumors[J].BMC Cancer,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 Prx1 protein 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.A mouse model for human short-stature syndromes identifies Shox2 as an upstream regulator of Runx2 during long-bone development[J].Proc Natl Acad Sci U S A,2006,103(12):4511-4515.
[16] BOBICK B E,COBB J.Shox2 regulates 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.Shox2 mediates Tbx5 activity by regulating Bmp4 in 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 Shox2 and 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.Shox2 function 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.Islet1 is a direct transcriptional target of the homeodomain transcription factor Shox2 and 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 Shox2 in 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 Shox2 is 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 SHOX2 methylation in plasmatic circulating cell-free DNA as auxiliary staging parameter in colorectal cancer:a prospective 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 SHOX2 and RASSF1A panel 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 homeobox 2 methylation 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 SHOX2 and SEPT9 is a potential 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.SHOX2 is 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 SHOX2 and SEPT9 DNA methylation and cytology in benign,paramalignant,and malignant ascites[J].Clin Epigenetics,2016,8:24.

Molecular Cloning and Expression Analysis of Short Stature Homeobox 2 (Shox2) in Oryctolagus cuniculus

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	OUYANG Qingyuan, WANG Yushi, HU Shenqiang, LI Liang, LIU Hehe, HE Hua, WANG Jiwen. Cloning of GATA4/5/6 Genes and Their Expression Patterns in Duck Hearts and Livers during Postnatal Development [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(9): 1775-1786.
[2]	ZHAO Xueliang, WANG Shuyi, SUN Ke, SU Qian, WANG Wenlong, LIU Chunxia. Comparative Transcriptome Analysis of Albendazole-susceptible and Resistant Strains of Haemonchus contortus by RNA-Seq [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(9): 1940-1944.
[3]	LI Chunyan, LIU Qiuyue, HU Wenping, WANG Xiangyu, MA Lin, ZHANG Xiaosheng, ZHANG Jinlong, DI Ran, CHU Mingxing. Expression Pattern of GnIH and VIP Genes in Different Reproductive States of Sheep [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(5): 930-938.
[4]	LI Chunyan, LIU Qiuyue, HU Wenping, WANG Xiangyu, MA Lin, ZHANG Xiaosheng, ZHANG Jinlong, DI Ran, CHU Mingxing. Expression Pattern of GnIH and VIP Genes in Different Reproductive States of Sheep [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(5): 930-938.
[5]	CHEN Rui, ZHU Zifeng, HUANG Jian, YANG Xiaonong. Molecular Cloning and Expression Analysis of Short Stature Homeobox 2 (Shox2) in Oryctolagus cuniculus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(1): 61-70.
[6]	GAN Xin-meng, CHEN Kai-wen, LIU He-he, LI Yan-ying, WANG Ji-wen, LI Liang, BAI Li-li. The Expression Patterns of TLRs Gene Family during Duck Bursa of Fabricius Development and Their Molecular Evolutionary Analysis [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(9): 1851-1860.
[7]	GAN Xin-meng, CHEN Kai-wen, LIU He-he, LI Yan-ying, WANG Ji-wen, LI Liang, BAI Li-li. The Expression Patterns of TLRs Gene Family during Duck Bursa of Fabricius Development and Their Molecular Evolutionary Analysis [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(9): 1851-1860.
[8]	GUO Xiao-hong, LI Meng, GAO Peng-fei, CAO Guo-qing, CHENG Zhi-min, ZHANG Ning-fang, LE Bao-yu, LIU Jian-feng, LIU Xiao-jun, LI Bu-gao. Molecular Cloning on Alternative Splice Variants of Pig MYNN Gene and Their Expression Patterns [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(3): 477-487.
[9]	GUO Xiao-hong, LI Meng, GAO Peng-fei, CAO Guo-qing, CHENG Zhi-min, ZHANG Ning-fang, LE Bao-yu, LIU Jian-feng, LIU Xiao-jun, LI Bu-gao. Molecular Cloning on Alternative Splice Variants of Pig MYNN Gene and Their Expression Patterns [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(3): 477-487.
[10]	LI Xiao-yu, HE Xiao-yun, LIU Qiu-yue, WANG Xiang-yu, GUO Xiao-fei, XIA Qing, HU Wen-ping, ZHANG Xiao-sheng, ZHANG Jin-long, CHU Ming-xing, DI Ran. Expression Pattern Analysis of TAC1 and PRLR Genes in Different Reproductive States of Sheep [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(2): 253-262.
[11]	XIA Qing, ZHANG Xiao-sheng, LIU Qiu-yue, WANG Xiang-yu, HE Xiao-yun, GUO Xiao-fei, HU Wen-ping, ZHANG Jin-long, CHU Ming-xing, DI Ran. Expression Patterns of EYA 3 and TSH β in Seasonal Estrous and Year-round Estrous Sheep [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(2): 263-269.
[12]	LI Xiao-yu, HE Xiao-yun, LIU Qiu-yue, WANG Xiang-yu, GUO Xiao-fei, XIA Qing, HU Wen-ping, ZHANG Xiao-sheng, ZHANG Jin-long, CHU Ming-xing, DI Ran. Expression Pattern Analysis of TAC1 and PRLR Genes in Different Reproductive States of Sheep [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(2): 253-262.
[13]	XIA Qing, ZHANG Xiao-sheng, LIU Qiu-yue, WANG Xiang-yu, HE Xiao-yun, GUO Xiao-fei, HU Wen-ping, ZHANG Jin-long, CHU Ming-xing, DI Ran. Expression Patterns of EYA 3 and TSH β in Seasonal Estrous and Year-round Estrous Sheep [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(2): 263-269.
[14]	JIA Zhi-peng, PENG Xiao-bin, LUO Cha-gan, HASI Su-rong. The Full-length Cloning, Sequence Information and Tissues Expression Analysis of CYP2J cDNA in Bactrian Camel [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(10): 2133-2144.
[15]	JIA Zhi-peng, PENG Xiao-bin, LUO Cha-gan, HASI Su-rong. The Full-length Cloning, Sequence Information and Tissues Expression Analysis of CYP2J cDNA in Bactrian Camel [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(10): 2133-2144.