中华鳖(Pelodiscus sinensis)MITF基因的生物信息学及表达差异研究
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摘要
小眼畸形相关转录因子(microphthalmia-associated transcription factor,MITF)是一种黑色素细胞重要的转录因子。本研究通过中华鳖(Pelodiscus sinensis)的肝脏组织进行转录组测序,获得MITF基因的全长序列,生物信息学分析结果显示中华鳖MITF基因5’和3’非编码区分别为32bp和175bp,开放阅读框1512bp,编码503个氨基酸,预测蛋白分子质量为56kDa,理论等点电为6.06。中华鳖MITF蛋白二级结构预测显示,α-螺旋(Alpha helix)占25.37%,延伸链(Extended strand)占20.90%,β-转角(Beta-turn)占7.76%,无规则卷曲(Random coil)占45.97%。氨基酸序列多重比对分析显示,中华鳖MITF基因核苷酸序列与绿海龟、西部锦龟、美国短吻鳄、非洲爪蟾和人的同源性分别为93%、93%、85%、58%和76%。构建进化树分析表明,中华鳖与西部锦龟、绿海龟MITF基因分子进化距离最近,亲缘关系最密切。实时荧光定量PCR对中华鳖8个组织MITF基因表达差异性研究结果表明,该基因在中华鳖的肌肉表达量最高,心脏次之,二者存在显著性差异,肝脏、脾脏、肺、肾脏、眼、皮肤等组织表达量相对较少,显著低于肌肉和心脏。这些研究结果不仅为研究中华鳖MITF基因在黑色素合成过程中的重要作用提供理论基础,还有助于了解龟鳖类体色调控的内在机理,同时也可为中华鳖等爬行动物的遗传育种提供参考。
To understand the molecular mechanism of MITF(microphthalmia-associated transcription factor) and the body color change of Chinese soft-shelled turtle Pelodiscus sinensis, we built the c DNA library of P. sinensis, obtained the full-length sequence of the MITF gene by transcriptome sequencing, and analyzed the tissues expression of the MITF. Bioinformatics analysis showed that the 5' and 3' non-encoding regions were 32 bp and 175 bp, with an open reading frame of 1512 bp, encoding a putative 503 aa. The deduced molecular weight of MITF protein was 56 kDa and the theoretical isoelectric point is 6.06. The secondary structure showed that a-helix(Alpha helix) accounted for 25.37%, extension chain(Extended strand) for 20.90%, a beta angle(Beta-turn) for 7.76%, and a random coil(Random coil) for 45.97%. Alignment analysis indicated that the nucleotide sequence showed a high homology with the MITF sequences of Chrysemys picta bellii(93%), Chelonia mydas(93%), Alligator mississippiensis(85%), Xenopus laevis(58%), and Homo sapiens(76%). In the phylogenetic tree, the molecular evolutionary distance of MITF gene among P. sinensis, Chrysemys picta bellii and Chelonia mydas was the closest, and the genetic relationship was most closely related. Real-time PCR revealed the expression of MITF mRNA in the muscle of P. sinensis was significantly higher than in heart, and the expression of MITF mRNA in heart was significantly higher than in the liver, but there was no significant difference between liver, spleen, lung, kidney, eye, and skin. These results are helpful to provide a reference for genetic breeding of P. sinensis and other reptiles.
To understand the molecular mechanism of MITF(microphthalmia-associated transcription factor) and the body color change of Chinese soft-shelled turtle Pelodiscus sinensis, we built the c DNA library of P. sinensis, obtained the full-length sequence of the MITF gene by transcriptome sequencing, and analyzed the tissues expression of the MITF. Bioinformatics analysis showed that the 5' and 3' non-encoding regions were 32 bp and 175 bp, with an open reading frame of 1512 bp, encoding a putative 503 aa. The deduced molecular weight of MITF protein was 56 kDa and the theoretical isoelectric point is 6.06. The secondary structure showed that a-helix(Alpha helix) accounted for 25.37%, extension chain(Extended strand) for 20.90%, a beta angle(Beta-turn) for 7.76%, and a random coil(Random coil) for 45.97%. Alignment analysis indicated that the nucleotide sequence showed a high homology with the MITF sequences of Chrysemys picta bellii(93%), Chelonia mydas(93%), Alligator mississippiensis(85%), Xenopus laevis(58%), and Homo sapiens(76%). In the phylogenetic tree, the molecular evolutionary distance of MITF gene among P. sinensis, Chrysemys picta bellii and Chelonia mydas was the closest, and the genetic relationship was most closely related. Real-time PCR revealed the expression of MITF mRNA in the muscle of P. sinensis was significantly higher than in heart, and the expression of MITF mRNA in heart was significantly higher than in the liver, but there was no significant difference between liver, spleen, lung, kidney, eye, and skin. These results are helpful to provide a reference for genetic breeding of P. sinensis and other reptiles.
引文
李思发,蔡完其,刘至治等,2004.中华鳖七群体体形和腹部黑斑图案的差异比较.水产学报,28(1):15-22
何中央,张海琪,蔡引伟等,2016.中华鳖高效养殖致富技术与实例.北京:中国农业出版社,28-35
张浩,徐超,宋兴超等,2016.黑色素细胞调控小眼畸形相关转录因子的研究进展.畜牧与兽医,48(8):113-117
张永正,张海琪,何中央等,2008.中华鳖(Pelodiscus sinensis)5个不同地理种群细胞色素b基因序列变异及种群遗传结构分析.海洋与湖沼,39(3):234-239
周泓燕,2015.MITF基因对动物毛色中黑色素细胞的作用.中国畜牧兽医文摘,31(3):46
郑嫩珠,辛清武,朱志明等,2015.白绒乌鸡MITF基因的c DNA克隆、表达及其对黑色素沉积的影响.中国农业科学,48(18):3711-3718
Arnheiter H,2010.The discovery of the microphthalmia locus and its gene,Mitf.Pigment Cell&Melanoma Research,23(6):729-735
Bertolotto C,Abbe P,Hemesath T J et al,1998.Microphthalmia gene product as a signal transducer in c AMP-induced differentiation of melanocytes.The Journal of Cell Biology,142(3):827-835
Carreira S,Goodall J,Aksan I et al,2005.MITF cooperates with Rb1 and activates p21Cip1 expression to regulate cell cycle progression.Nature,433(7027):764-776
Gao E W,Wang Y,Alcorn J L et al,1997.The basic helix-loop-helix-zipper transcription factor USF1 regulates expression of the surfactant protein-A gene.Journal of Biological Chemistry,272(37):23398-23406
Hallsson J H,Haflidadóttir B S,Stivers C et al,2004.The basic helix-loop-helix leucine zipper transcription factor Mitf is conserved in Drosophila and functions in eye development.Genetics,167(1):233-241
Han J L,Yang M,Guo T T et al,2015.Molecular characterization of two candidate genes associated with Coat color in Tibetan sheep(Ovis arise).Journal of Integrative Agriculture,14(7):1390-1397
Hou L,Pavan W J,2008.Transcriptional and signaling regulation in neural crest stem cell-derived melanocyte development:do all roads lead to MITF?Cell Research,18(12):1163-1176
Lang D,Lu M M,Huang L et al,2005.Pax3 functions at a nodal point in melanocyte stem cell differentiation.Nature,433(7028):884-887
Lee H D,Lee W H,Roh E et al,2011.Manassantin A inhibits c AMP-induced melanin production by down-regulating the gene expressions of MITF and tyrosinase in melanocytes.Experimental Dermatology,20(9):761-763
Livak K J,Schmittgen T D,2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-(35)(35)CT method.Methods,25(4):402-408
Opdecamp K,Nakayama A,Nguyen M T et al,1997.Melanocyte development in vivo and in neural crest cell cultures:crucial dependence on the Mitf basic-helix-loop-helix-zipper transcription factor.Development,124(12):2377-2386
Osawa M,Egawa G,Mak S S et al,2005.Molecular characterization of melanocyte stem cells in their niche.Development,132(24):5589-5599
Steingrímsson E,Copeland N G,Jenkins N A,2004.Melanocytes and the microphthalmia transcription factor network.Annual Review of Genetics,38:365-411
Steingrímsson E,Moore K J,Lamoreux M L et al,1994.Molecular basis of mouse microphthalmia(mi)mutations helps explain their developmental and phenotypic consequences.Nature Genetics,8(3):256-263
Wellbrock C,Marais R,2005.Elevated expression of MITFcounteracts B-RAF-stimulated melanocyte and melanoma cell proliferation.The Journal of Cell Biology,170(5):703-708
Xu J H,Zhao J,Li Y Q et al,2016.Evaluation of differentially expressed immune-related genes in intestine of Pelodiscus sinensis after intragastric challenge with lipopolysaccharide based on transcriptome analysis.Fish&Shellfish Immunology,56:417-426
Yasumoto K I,Yokoyama K,Shibata K et al,1994.Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene.Molecular and Cellular Biology,14(12):8058-8070
Zhang C,Xu X J,Zhang H Q et al,2015a.PCR-RFLPidentification of four Chinese soft-shelled turtle Pelodiscus sinensis strains using mitochondrial genes.Mitochondrial DNA,26(4):538-543
Zhang H Q,Xu X J,He Z Y et al,2017.De novo transcriptome analysis reveals insights into different mechanisms of growth and immunity in a Chinese soft-shelled turtle hybrid and the parental varieties.Gene,605:54-62
Zhang H Q,Zhang C,Xu X J et al,2015b.Differentiation of four strains of Chinese soft-shelled turtle(Pelodiscus sinensis)based on high-resolution melting analysis of single nucleotide polymorphism sites in mitochondrial DNA.Genetics and Molecular Research,14(4):13144-13150
Zhao H B,Li M Y,Purwanti Y I et al,2012a.MITF is a transcriptional activator of medaka germ genes in culture.Biochimie,94:759-767
Zhao H L,Yang H S,Zhao H et al,2012b.Differences in MITFgene expression and histology between albino and normal sea cucumbers(Apostichopus japonicus Selenka).Chinese Journal of Oceanology and Limnology,30(1):80-91
何中央,张海琪,蔡引伟等,2016.中华鳖高效养殖致富技术与实例.北京:中国农业出版社,28-35
张浩,徐超,宋兴超等,2016.黑色素细胞调控小眼畸形相关转录因子的研究进展.畜牧与兽医,48(8):113-117
张永正,张海琪,何中央等,2008.中华鳖(Pelodiscus sinensis)5个不同地理种群细胞色素b基因序列变异及种群遗传结构分析.海洋与湖沼,39(3):234-239
周泓燕,2015.MITF基因对动物毛色中黑色素细胞的作用.中国畜牧兽医文摘,31(3):46
郑嫩珠,辛清武,朱志明等,2015.白绒乌鸡MITF基因的c DNA克隆、表达及其对黑色素沉积的影响.中国农业科学,48(18):3711-3718
Arnheiter H,2010.The discovery of the microphthalmia locus and its gene,Mitf.Pigment Cell&Melanoma Research,23(6):729-735
Bertolotto C,Abbe P,Hemesath T J et al,1998.Microphthalmia gene product as a signal transducer in c AMP-induced differentiation of melanocytes.The Journal of Cell Biology,142(3):827-835
Carreira S,Goodall J,Aksan I et al,2005.MITF cooperates with Rb1 and activates p21Cip1 expression to regulate cell cycle progression.Nature,433(7027):764-776
Gao E W,Wang Y,Alcorn J L et al,1997.The basic helix-loop-helix-zipper transcription factor USF1 regulates expression of the surfactant protein-A gene.Journal of Biological Chemistry,272(37):23398-23406
Hallsson J H,Haflidadóttir B S,Stivers C et al,2004.The basic helix-loop-helix leucine zipper transcription factor Mitf is conserved in Drosophila and functions in eye development.Genetics,167(1):233-241
Han J L,Yang M,Guo T T et al,2015.Molecular characterization of two candidate genes associated with Coat color in Tibetan sheep(Ovis arise).Journal of Integrative Agriculture,14(7):1390-1397
Hou L,Pavan W J,2008.Transcriptional and signaling regulation in neural crest stem cell-derived melanocyte development:do all roads lead to MITF?Cell Research,18(12):1163-1176
Lang D,Lu M M,Huang L et al,2005.Pax3 functions at a nodal point in melanocyte stem cell differentiation.Nature,433(7028):884-887
Lee H D,Lee W H,Roh E et al,2011.Manassantin A inhibits c AMP-induced melanin production by down-regulating the gene expressions of MITF and tyrosinase in melanocytes.Experimental Dermatology,20(9):761-763
Livak K J,Schmittgen T D,2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-(35)(35)CT method.Methods,25(4):402-408
Opdecamp K,Nakayama A,Nguyen M T et al,1997.Melanocyte development in vivo and in neural crest cell cultures:crucial dependence on the Mitf basic-helix-loop-helix-zipper transcription factor.Development,124(12):2377-2386
Osawa M,Egawa G,Mak S S et al,2005.Molecular characterization of melanocyte stem cells in their niche.Development,132(24):5589-5599
Steingrímsson E,Copeland N G,Jenkins N A,2004.Melanocytes and the microphthalmia transcription factor network.Annual Review of Genetics,38:365-411
Steingrímsson E,Moore K J,Lamoreux M L et al,1994.Molecular basis of mouse microphthalmia(mi)mutations helps explain their developmental and phenotypic consequences.Nature Genetics,8(3):256-263
Wellbrock C,Marais R,2005.Elevated expression of MITFcounteracts B-RAF-stimulated melanocyte and melanoma cell proliferation.The Journal of Cell Biology,170(5):703-708
Xu J H,Zhao J,Li Y Q et al,2016.Evaluation of differentially expressed immune-related genes in intestine of Pelodiscus sinensis after intragastric challenge with lipopolysaccharide based on transcriptome analysis.Fish&Shellfish Immunology,56:417-426
Yasumoto K I,Yokoyama K,Shibata K et al,1994.Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene.Molecular and Cellular Biology,14(12):8058-8070
Zhang C,Xu X J,Zhang H Q et al,2015a.PCR-RFLPidentification of four Chinese soft-shelled turtle Pelodiscus sinensis strains using mitochondrial genes.Mitochondrial DNA,26(4):538-543
Zhang H Q,Xu X J,He Z Y et al,2017.De novo transcriptome analysis reveals insights into different mechanisms of growth and immunity in a Chinese soft-shelled turtle hybrid and the parental varieties.Gene,605:54-62
Zhang H Q,Zhang C,Xu X J et al,2015b.Differentiation of four strains of Chinese soft-shelled turtle(Pelodiscus sinensis)based on high-resolution melting analysis of single nucleotide polymorphism sites in mitochondrial DNA.Genetics and Molecular Research,14(4):13144-13150
Zhao H B,Li M Y,Purwanti Y I et al,2012a.MITF is a transcriptional activator of medaka germ genes in culture.Biochimie,94:759-767
Zhao H L,Yang H S,Zhao H et al,2012b.Differences in MITFgene expression and histology between albino and normal sea cucumbers(Apostichopus japonicus Selenka).Chinese Journal of Oceanology and Limnology,30(1):80-91