猪TIMP-2基因克隆、序列特征和表达分析  

Cloning,Sequence Characterization and Expression Analysis of Porcine TIMP-2 Gene

作　　者：黄宏刚[1] 肖邦[1,2] 任红艳[1,2] 唐中林[1] 杨述林[1] 崔文涛[1] 牟玉莲[1] 储明星[1] 李奎[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所农业部畜禽遗传资源与利用重点开放实验室,北京100193 [2]华中农业大学农业动物遗传育种与繁殖教育部重点实验室,武汉430070

出　　处：《畜牧兽医学报》2009年第12期1727-1734,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(30700569);国家863计划(2006AA10Z135);国家973计划(2006CB102105);欧盟第六框架合作项目(036245-2);国家支撑计划;中国农业科学院北京畜牧兽医研究所科技创新团队基金项目资助

摘　　要：本研究分析了五指山试验用小型猪TIMP-2基因的分子特性和可能的生物学功能。将人TIM-P2 mR-NA全长序列在猪ESTs数据库中检索获得同源性较高的ESTs,用电子克隆结合RT-PCR方法克隆获得包含完整CDS区的猪TIMP-2基因cDNA序列,应用生物信息学方法分析了其核苷酸序列及其编码蛋白质分子结构特性,应用RT-PCR研究该基因在猪不同组织和发育时期的特异表达情况。结果,猪TIM-P2 cDNA序列全长790 bp,包括663 bp的完整开放阅读框,编码221个氨基酸。多序列比对和系统进化分析表明,该基因编码蛋白质与人(97%)、大鼠(97%)、小鼠(97%)等物种TIMP-2蛋白质具有较高的同源性。蛋白质序列预测分析发现,猪TIMP-2氨基酸序列理论等电点(pI)为7.65,相对分子质量(MW)为24.5 ku,它包括27AA的前导序列和194AA的成熟肽段,其前导序列比其它物种多1个亮氨酸(Leu)。结构功能预测发现其具有1个在种间高度保守的NTR_TIMP结构域和N端VIRAK保守序列,序列中存在的12个半胱氨酸(Cys)可以形成6对二硫键结构。表达谱分析表明TIMP-2基因在猪的多个组织和不同发育时期的表达量存在较大差异,在睾丸、垂体、胃、大肠、卵巢、子宫和90 d胚胎骨骼肌中高表达;而在心脏、小肠、脑、肝脏、成年猪骨骼肌中表达量极低;在肾脏、胸腺、脾、甲状腺3、3 d胚胎中中度表达。结果表明该基因在发生组织发育和重构活动相对活跃的组织器官和时期表达水平较高,亦符合其固有生物学功能。In this study,the molecular properties and potential functions of porcine TIMP-2 gene were analyzed and characterized in Wuzhishan laboratory miniature pig.Porcine TIMP-2 cDNA was obtained by reverse transcription polymerase chain reaction（RT-PCR）,using in silico cloning strategy based on porcine ESTs database,the nucleotide and amino acid sequences were characterized with bioinformatics methods,and its expression patterns in different tissues and development stages were also studied by RT-PCR.The length of the cloned porcine TIMP-2 cDNA was 909 bp including a 663 bp entire open reading frame（ORF）,which encodes a 221 amino acids protein.Multiple sequences alignment and phylogeny analysis indicated that porcine TIMP-2 protein shared high similarity with that of other species,such as Homo sapiens（97%）,Mus musculus（97%） and Rattus norvegicus（97%）.Protein sequence analysis showed that the deduced porcine TIMP-2 protein sequence（pI=7.65,MW= 24.5 ku） contained 27AA leader sequence and 194 AA mature peptide,the leader sequence in pig has one more amino acid（Leu residue） than that in other 6 species.A NTR-TIMP function domain and a conserved VIRAK motif at N-terminus were predicted by bioinformatic analysis and homologous comparison,and the 12 Cys residues in the sequence were predicted to constitute 6 disulphide bonds.The expression of porcine TIMP-2 mRNA varied greatly in different tissues as well as different developmental stages,it was highly expressed in testis,pituitary body,stomach,large intestine,ovary,uterus and 90 days embryonic longissimus muscle,but lowly in heart,small intestine,brain,liver and adult longissimus muscle,and a moderate expression level was detected in kidney,thymus,spleen,thyroid and 33 days embryonic longissimus muscle.The result indicated that the porcine TIMP-2 mRNA was highly expressed in organs and phases with relatively high tissue growth and/or remodelling activities,and it was in accordance with the innate biological function of TIMP-2.

关 键 词：猪 TIMP-2 电子克隆 序列特征 表达谱 

分 类 号：S828[农业科学—畜牧学] Q523.8[农业科学—畜牧兽医]