中间球海胆smad2/3基因克隆、组织表达及其脂多糖刺激响应
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摘要
为明确中间球海胆Strongylocentrotus intermedius smad2/3基因(命名为Si-smad2/3)信息,初步研究了该基因的序列特征、组织表达模式及脂多糖对其表达的影响,采用RACE技术克隆获得了成体中间球海胆Si-smad2/3基因的全长cDNA序列。结果表明:Si-smad2/3基因的cDNA全长为2146 bp,共编码446个氨基酸;生物信息学分析发现,Si-smad2/3基因所编码的蛋白相对分子质量为50 300,等电点为6.93,属于亲水性非跨膜蛋白;通过与9种已公布物种的smad2/3蛋白氨基酸序列进行多重序列比对和系统进化分析发现,中间球海胆Si-smad2/3蛋白的氨基酸序列与其他真核生物smad2/3蛋白序列具有较高的相似性,与紫球海胆Strongylocentrotus purpuratus smad2/3蛋白的一致性高达96%,符合中间球海胆的分类和进化地位;实时定量PCR(qRT-PCR)检测结果显示,Si-smad2/3基因在中间球海胆不同组织中均有表达,其相对表达量从高到低为体腔细胞>管足>性腺>围口膜>肠>齿间肌;利用脂多糖(LPS,0.1 mg/mL)对中间球海胆进行免疫刺激发现,与对照组相比,LPS刺激后Si-smad2/3基因在中间球海胆体腔细胞、管足和围口膜中均呈先升高后降低的表达趋势,其中,Si-smad2/3基因在中间球海胆体腔细胞中的相对表达量在LPS刺激9 h时达到峰值,管足中表达量在LPS刺激6 h时达到峰值,而围口膜中的表达量在LPS刺激72 h时达到峰值。研究表明,Si-smad2/3可能参与中间球海胆的免疫应答过程且免疫响应具有组织特异性。
A novel smad2/3 gene was cloned in sea urchin Stronglocentrotus intermedius(here designated Si-smad2/3)by rapid amplification of cDNA ends(RACE) and Si-smad2/3 gene was expressed in various tissues of the sea urchin injected individually with 100 mL of lipopolysaccharides(LPS, 0.1 mg/mL) for 3, 6, 9, 12, 24, 48, and 72 h to investigate the function of the smad gene in echinoderms. It was found that Si-smad2/3 had full-length cDNA sequence of 2146 base pairs(bp), with a 1341 bp open reading frame encoding 446 amino acid residues. Bioinformatics analysis including multiple sequence alignment and systematic evolution revealed that the predicted Si-smad2/3 protein belonged to the hydrophilic non-transmembrane protein with a molecular weight of 50 300 and an isoelectric point of 6.93, similar to the smad2/3 homolog(96% of homology) in sea urchin S.purpuratus, and a relative high sequence identity with the smad2/3 proteins from other eukaryotic species. Quantitative Real-time PCR(qRT-PCR) analysis indicated that Si-smad2/3 was expressed in all of the examined tissues of healthy adult sea urchin S.intermedius, with the highest level of expression detected in coelomocytes. Significant changes in Si-smad2/3 gene expression in the coelomocytes were observed in vivo at 3, 6, 9, 12, 24, 48, and 72 h after 0.1 mg/mL LPS challenge. The findings provide more knowledge of the characteristics of the smad homolog in sea urchin, and association with immune function.
A novel smad2/3 gene was cloned in sea urchin Stronglocentrotus intermedius(here designated Si-smad2/3)by rapid amplification of cDNA ends(RACE) and Si-smad2/3 gene was expressed in various tissues of the sea urchin injected individually with 100 mL of lipopolysaccharides(LPS, 0.1 mg/mL) for 3, 6, 9, 12, 24, 48, and 72 h to investigate the function of the smad gene in echinoderms. It was found that Si-smad2/3 had full-length cDNA sequence of 2146 base pairs(bp), with a 1341 bp open reading frame encoding 446 amino acid residues. Bioinformatics analysis including multiple sequence alignment and systematic evolution revealed that the predicted Si-smad2/3 protein belonged to the hydrophilic non-transmembrane protein with a molecular weight of 50 300 and an isoelectric point of 6.93, similar to the smad2/3 homolog(96% of homology) in sea urchin S.purpuratus, and a relative high sequence identity with the smad2/3 proteins from other eukaryotic species. Quantitative Real-time PCR(qRT-PCR) analysis indicated that Si-smad2/3 was expressed in all of the examined tissues of healthy adult sea urchin S.intermedius, with the highest level of expression detected in coelomocytes. Significant changes in Si-smad2/3 gene expression in the coelomocytes were observed in vivo at 3, 6, 9, 12, 24, 48, and 72 h after 0.1 mg/mL LPS challenge. The findings provide more knowledge of the characteristics of the smad homolog in sea urchin, and association with immune function.
引文
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[17] Lan Debin,Lu Min,Sharma S,et al.Trans-resveratrol inhibits phosphorylation of Smad2/3 and represses FSHβ gene expression by a SirT1-independent pathway in LβT2 gonadotrope cells[J].Reproductive Toxicology,2011,32(1):85-92.
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[19] Kumar S,Pan C C,Shah N,et al.Activation of mitofusin2 by Smad2-RIN1 complex during mitochondrial fusion[J].Molecular Cell,2016,62(4):520-531.
[20] Finnson K W,Parker W L,Chi Y,et al.Endoglin differentially regulates TGF-β-induced Smad2/3 and Smad1/5 signalling and its expression correlates with extracellular matrix production and cellular differentiation state in human chondrocytes[J].Osteoarthritis and Cartilage,2010,18(11):1518-1527.
[21] Blaney Davidson E N,Van Caam A P M,Vitters E L,et al.TGF-β is a potent inducer of nerve growth factor in articular cartilage via the ALK5-Smad2/3 pathway.Potential role in OA related pain?[J].Osteoarthritis and Cartilage,2015,23(3):478-486.
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[23] 贾顺姬.Smad2/3在斑马鱼胚胎早期发育中的作用[D].北京:清华大学,2008.
[24] Thien A,Prentzell M T,Holzwarth B,et al.TSC1 activates TGF-β-Smad2/3 signaling in growth arrest and epithelial-to-mesenchymal transition[J].Developmental Cell,2015,32(5):617-630.
[25] Murai M,Tsuji G,Hashimoto-Hachiya A,et al.An endogenous tryptophan photo-product,FICZ,is potentially involved in photo-aging by reducing TGF-β-regulated collagen homeostasis[J].Journal of Dermatological Science,2018,89(1):19-26.
[26] Jung H Y,Shin J C,Park S M,et al.Pinus densiflora extract protects human skin fibroblasts against UVB-induced photoaging by inhibiting the expression of MMPs and increasing type I procollagen expression[J].Toxicology Reports,2014,1:658-666.
[27] 程志芬,孙凤丹,张金子,等.胃癌组织中TGF-β1、TGF-βRⅡ、Smad2/3和CDC25蛋白的表达及其临床意义[J].临床与实验病理学杂志,2011,27(10):1044-1047.
[28] 韩奋杰,张伟杰,秦宇博,等.中间球海胆幼体及稚海胆生长性状的遗传参数估计[J].大连海洋大学学报,2017,32(2):145-149.
[29] 常亚青,封妮莎,王轶南,等.虾夷马粪海胆致病菌强壮弧菌的PCR检测方法[J].大连海洋大学学报,2012,27(4):289-293.
[30] 李磊,杨丹,齐守冰,等.中间球海胆繁殖前后性腺中脂类和脂肪酸含量变化[J].大连海洋大学学报,2018,33(4):423-429.
[31] 姬南京,杨芸菲,丁君,等.虾夷马粪海胆溶菌酶基因全长cDNA的克隆与表达分析[J].中国水产科学,2013,20(5):950-957.
[32] Shi Yigong,Massagué J.Mechanisms of TGF-β signaling from cell membrane to the nucleus[J].Cell,2003,113(6):685-700.
[33] 孟繁伊,麦康森,马洪明,等.棘皮动物免疫学研究进展[J].生物化学与生物物理进展,2009,36(7):803-809.
[34] 常亚青,丁君,宋坚,等.海参、海胆生物学研究与养殖[M].北京:海洋出版社,2004.
[35] 李太武,徐善良,王仁波,等.虾夷马粪海胆黑嘴病的初步研究[J].海洋科学,2000,24(3):41-44.
[2] Range R,Lapraz F,Quirin M,et al.Cis-regulatory analysis of nodal and maternal control of dorsal-ventral axis formation by univin,a TGF-β related to Vg1[J].Development,2007,134(20):3649-3664.
[3] Yaguchi S,Yaguchi J,Burke R D.Sp-Smad2/3 mediates patterning of neurogenic ectoderm by nodal in the sea urchin embryo[J].Developmental Biology,2007,302(2):494-503.
[4] Zito F,Costa C,Sciarrino S,et al.Expression of univin,a TGF-β growth factor,requires ectoderm-ECM interaction and promotes skeletal growth in the sea urchin embryo[J].Developmental Biology,2003,264(1):217-227.
[5] Zhao Jingsong,Crowe D L,Castillo C,et al.Smad7 is a TGF-β-inducible attenuator of Smad2/3-mediated inhibition of embryonic lung morphogenesis[J].Mechanisms of Development,2000,93(1-2):71-81.
[6] Datto M,Wang X F.The Smads:transcriptional regulation and mouse models[J].Cytokine & Growth Factor Reviews,2000,11(1-2):37-48.
[7] Yoshida K,Matsuzaki K,Mori S,et al.Transforming growth factor-β and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury[J].The American Journal of Pathology,2005,166(4):1029-1039.
[8] Gao Sheng,Alarcón C,Sapkota G,et al.Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-β signaling[J].Molecular Cell,2009,36(3):457-468.
[9] Jia Shunji,Wu Di,Xing Cencan,et al.Smad2/3 activities are required for induction and patterning of the neuroectoderm in zebrafish[J].Developmental Biology,2009,333(2):273-284.
[10] Preisser F,Giehl K,Rehm M,et al.Inhibitors of oxygen sensing prolyl hydroxylases regulate nuclear localization of the transcription factors Smad2 and YAP/TAZ involved in CTGF synthesis[J].Biochimica et Biophysica Acta (BBA) - Molecular Cell Research,2016,1863(8):2027-2036.
[11] García R,Nistal J F,Merino D,et al.p-SMAD2/3 and DICER promote pre-miR-21 processing during pressure overload-associated myocardial remodeling[J].Biochimica et Biophysica Acta (BBA) - Molecular Cell Research,2015,1852(7):1520-1530.
[12] Jung K J,Kim J,Park Y K,et al.Wen-pi-tang-Hab-Wu-ling-san reduces ureteral obstructive renal fibrosis by the reduction of oxidative stress,inflammation,and TGF-β/Smad2/3 signaling[J].Food and Chemical Toxicology,2010,48(2):522-529.
[13] Ruetz T,Pfisterer U,Di Stefano B,et al.Constitutively active SMAD2/3 are broad-scope potentiators of transcription-factor-mediated cellular reprogramming[J].Cell Stem Cell,2017,21(6):791-805.
[14] Singh A M,Reynolds D,Cliff T,et al.Signaling network crosstalk in human pluripotent cells:a Smad2/3-regulated switch that controls the balance between self-renewal and differentiation[J].Cell Stem Cell,2012,10(3):312-326.
[15] Coutts S M,Childs A J,Fulton N,et al.Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression[J].Developmental Biology,2008,314(1):189-199.
[16] Will A E,Baker J C.E2a is necessary for Smad2/3-dependent transcription and the direct repression of lefty during gastrulation[J].Developmental Cell,2015,32(3):345-357.
[17] Lan Debin,Lu Min,Sharma S,et al.Trans-resveratrol inhibits phosphorylation of Smad2/3 and represses FSHβ gene expression by a SirT1-independent pathway in LβT2 gonadotrope cells[J].Reproductive Toxicology,2011,32(1):85-92.
[18] Lapraz F,Duboc V,Lepage T.A genomic view of TGF-β signal transduction in an invertebrate deuterostome organism and lessons from the functional analyses of Nodal and BMP2/4 during sea urchin development[J].Signal Transduction,2007,7(2):187-206.
[19] Kumar S,Pan C C,Shah N,et al.Activation of mitofusin2 by Smad2-RIN1 complex during mitochondrial fusion[J].Molecular Cell,2016,62(4):520-531.
[20] Finnson K W,Parker W L,Chi Y,et al.Endoglin differentially regulates TGF-β-induced Smad2/3 and Smad1/5 signalling and its expression correlates with extracellular matrix production and cellular differentiation state in human chondrocytes[J].Osteoarthritis and Cartilage,2010,18(11):1518-1527.
[21] Blaney Davidson E N,Van Caam A P M,Vitters E L,et al.TGF-β is a potent inducer of nerve growth factor in articular cartilage via the ALK5-Smad2/3 pathway.Potential role in OA related pain?[J].Osteoarthritis and Cartilage,2015,23(3):478-486.
[22] Miosge N.Chondrogenic progenitor cells are key players in the regeneration of cartilage tissue[J].Osteoarthritis and Cartilage,2015,23 (Suppl 2):A381-A382.
[23] 贾顺姬.Smad2/3在斑马鱼胚胎早期发育中的作用[D].北京:清华大学,2008.
[24] Thien A,Prentzell M T,Holzwarth B,et al.TSC1 activates TGF-β-Smad2/3 signaling in growth arrest and epithelial-to-mesenchymal transition[J].Developmental Cell,2015,32(5):617-630.
[25] Murai M,Tsuji G,Hashimoto-Hachiya A,et al.An endogenous tryptophan photo-product,FICZ,is potentially involved in photo-aging by reducing TGF-β-regulated collagen homeostasis[J].Journal of Dermatological Science,2018,89(1):19-26.
[26] Jung H Y,Shin J C,Park S M,et al.Pinus densiflora extract protects human skin fibroblasts against UVB-induced photoaging by inhibiting the expression of MMPs and increasing type I procollagen expression[J].Toxicology Reports,2014,1:658-666.
[27] 程志芬,孙凤丹,张金子,等.胃癌组织中TGF-β1、TGF-βRⅡ、Smad2/3和CDC25蛋白的表达及其临床意义[J].临床与实验病理学杂志,2011,27(10):1044-1047.
[28] 韩奋杰,张伟杰,秦宇博,等.中间球海胆幼体及稚海胆生长性状的遗传参数估计[J].大连海洋大学学报,2017,32(2):145-149.
[29] 常亚青,封妮莎,王轶南,等.虾夷马粪海胆致病菌强壮弧菌的PCR检测方法[J].大连海洋大学学报,2012,27(4):289-293.
[30] 李磊,杨丹,齐守冰,等.中间球海胆繁殖前后性腺中脂类和脂肪酸含量变化[J].大连海洋大学学报,2018,33(4):423-429.
[31] 姬南京,杨芸菲,丁君,等.虾夷马粪海胆溶菌酶基因全长cDNA的克隆与表达分析[J].中国水产科学,2013,20(5):950-957.
[32] Shi Yigong,Massagué J.Mechanisms of TGF-β signaling from cell membrane to the nucleus[J].Cell,2003,113(6):685-700.
[33] 孟繁伊,麦康森,马洪明,等.棘皮动物免疫学研究进展[J].生物化学与生物物理进展,2009,36(7):803-809.
[34] 常亚青,丁君,宋坚,等.海参、海胆生物学研究与养殖[M].北京:海洋出版社,2004.
[35] 李太武,徐善良,王仁波,等.虾夷马粪海胆黑嘴病的初步研究[J].海洋科学,2000,24(3):41-44.