藏绵羊FAM134B基因克隆与表达分析
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摘要
采用RT-PCR技术克隆了藏绵羊FAM134B(family with sequence similarity 134,member B)基因,利用生物学软件进行相关生物信息学分析,同时用实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)研究组织表达水平。克隆获得FAM134B基因全长1 144 bp(Gen Bank登录号:KX580309),开放阅读框1 071 bp,编码356个氨基酸。系统进化树表明,藏绵羊FAM134B和绵羊、山羊关系较近。藏绵羊FAM134B基因编码蛋白的分子式为C_(1717)H_(2696)N_(448)O_(572)S_(12),分子量为39 151.72 u,理论等电点(pI)为4.49,消光系数为24 450,不稳定系数为43,疏水指数为80.31,平均亲水性为-0.422,属不稳定可溶性酸性蛋白质。二级结构主要是α-螺旋(45.51%)、无规卷曲(41.85%)和延伸链(12.64%),属于混合类蛋白质。亚细胞定位和功能分析结果显示,FAM134B编码的蛋白在内质网、细胞质膜概率分别为30.4%和21.7%,主要在蛋白质运输和结合(77.3%)方面起作用。系统发育树表明,藏绵羊FAM134B氨基酸序列与绵羊、山羊同源性非常高。qRT-PCR结果表明,FAM134B基因网胃中表达最高,十二指肠表达量最低。研究结果可为进一步研究藏绵羊FAM134B基因结构和生理功能及其遗传特性提供理论依据。
The gene of FAM134B( family with sequence similarity 134,member B) was cloned by RT-PCR. The bioinformatics analysis was carried out by using biological software. The expression level of FAM134B was also studied by real-time fluorescence quantitative PCR. The full length of FAM134B gene was 1 144 bp( Gen Bank accession number: KX580309),open reading frame 1 071 bp,encoding 356 amino acids. The phylogenetic tree showed that the Tibetan sheep FAM134B had a close relationship with the sheep and goat. The molecular formula of FAM134B protein in Tibetan sheep was C_(1717)H_(2696)N_(448)O_(572)S_(12),its molecular weight was 39151. 72 u,the theory isoelectric point was 4. 49,the extinction coefficient was 24 450. The instability index was 43,the aliphatic index was 80. 31,and the grand average hydropathicity was-0. 422. It was unstable and soluble acidic protein. The secondary structure ofFAM134B was mainly composed of α-helices and random coil and extended chain,with α-helices was 45. 51%,random coil was 41. 85% and extension chains was 12. 64%,which belongs to mixed type of protein. The results of subcellular localization and function showed that the frequencies of FAM134B protein in endoplasmic reticulum and cytoplasmic membrane were 30. 4% and 21. 7%. It mainly plays a role in protein transport and binding( 77. 3%).Phylogenetic tree showed that the amino acid sequence of FAM134B showed high homology with sheep and goat.qRT-PCR results showed that the expression of FAM134B gene was the highest in the stomach and lowest in the duodenum. The results can provide a theoretical basis for further study on the structure and physiological functions and genetic characteristics of FAM134B gene.
The gene of FAM134B( family with sequence similarity 134,member B) was cloned by RT-PCR. The bioinformatics analysis was carried out by using biological software. The expression level of FAM134B was also studied by real-time fluorescence quantitative PCR. The full length of FAM134B gene was 1 144 bp( Gen Bank accession number: KX580309),open reading frame 1 071 bp,encoding 356 amino acids. The phylogenetic tree showed that the Tibetan sheep FAM134B had a close relationship with the sheep and goat. The molecular formula of FAM134B protein in Tibetan sheep was C_(1717)H_(2696)N_(448)O_(572)S_(12),its molecular weight was 39151. 72 u,the theory isoelectric point was 4. 49,the extinction coefficient was 24 450. The instability index was 43,the aliphatic index was 80. 31,and the grand average hydropathicity was-0. 422. It was unstable and soluble acidic protein. The secondary structure ofFAM134B was mainly composed of α-helices and random coil and extended chain,with α-helices was 45. 51%,random coil was 41. 85% and extension chains was 12. 64%,which belongs to mixed type of protein. The results of subcellular localization and function showed that the frequencies of FAM134B protein in endoplasmic reticulum and cytoplasmic membrane were 30. 4% and 21. 7%. It mainly plays a role in protein transport and binding( 77. 3%).Phylogenetic tree showed that the amino acid sequence of FAM134B showed high homology with sheep and goat.qRT-PCR results showed that the expression of FAM134B gene was the highest in the stomach and lowest in the duodenum. The results can provide a theoretical basis for further study on the structure and physiological functions and genetic characteristics of FAM134B gene.
引文
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[10]李盛杰,杜晓华,罗玉柱,等.天祝白牦牛NGB基因的克隆及生物信息学分析[J].畜牧兽医学报,2013,44(3):395-398.LI S J,DU X H,LUO Y Z,et al.Cloning and bioinformatics analysis of NGB gene of Tianzhu white yak[J].Chinese Journal of Animal and Veterinary Sciences,2013,44(3):395-398.(in Chinese with English abstract)
[11]魏琳琳,高晋生,王晋霖,等.绵羊LPIN1基因的克隆和m RNA表达研究[J].畜牧兽医学报,2013,44(9):1371-1379.WEI L L,GAO J S,WANG J L,et al.Study on the cloning and ontogenetic m RNA expression of ovine LP1N1 gene[J].Acta Veterinaria et Zootechnica Sinica,2013,44(9):1371-1379.(in Chinese with English abstract)
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[14]孙伟,李达,苏锐,等.绵羊YAP1基因全长c DNA克隆及生物信息学分析[J].中国农业科学,2013,46(8):1725-1735.SUN W,LI D,SU R,et al.Cloning and bioinformatics analysis of full-length c DNA sequence of YAP1 gene in sheep[J].Scientia Agricultura Sinica,2013,46(8):1725-1735.(in Chinese with English abstract)
[15]JENSEN L J,GUTA R,BLOM N,et al.Prediction of human protein function from post-translational modifications and localization features[J].Journal of Molecular Biology,2002,319:1257-1265.
[16]陈书霞,王晓武,房玉林.单菌落PCR法直接快速鉴定重组克隆[J].微生物学通报,2006,33(3):52-56.CHEN S X,WANG X W,FANG Y L.Rapid characterization of recombination clone by PCR screening of individual bacterial colonies[J].Microbiology,2006,33(3):52-56.(in Chinese with English abstract)
[17]KOZAK M.Structural features in eukaryotic RNAs that modulate the initiation of translation[J].Biological Chemistry,1991,266(30):19867-19870.
[18]KYTE J,DOOLITTLE R F.A simple method for displaying the hydropathic character of a protein[J].Journal of Molecular Biology,1982,157(1):105-132.
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[20]KYTE J,DOOLITTLE R F.A simple method for displaying the hydropathic character of a protein[J].Journal of Molecular Biology,1982,157:105-132.
[21]刘芳,格日力.藏羚羊组蛋白去乙酰化酶1基因编码区的克隆与序列分析[J].兽类学报,2011,31(4):396-340.LIU F,GE R L.Cloning and sequencing of HDAC1 coding c DNA sequence from Tibetan antelope(Pantholops hodgsonii)[J].Acta Theriologica Sinica,2011,31(4):396-340.(in Chinese with English abstract)
[22]徐飞,成述儒,罗玉柱.绵羊DRB1基因生物信息学分析[J].生物技术通报,2011(1):113-118.XU F,CHENG S R,LUO Y Z.Bioinformatics analysis of sheep DRB1 gene[J].Biotechnology Bulletin,2011(1):113-118.(in Chinese with English abstract)
[23]ROST B,SANDER C.Combining evolutionary information and neural networks to predict protein secondary strucure[J].Proteins,1994,19(1):55-72.
[24]贾浩,张小白,宋晓峰.人类胞内蛋白半衰期与其亚细胞定位的相关性研究[J].计算机与应用化学,2011,28(4):411-414.JIA H,ZHANG X B,SONG X F.Relationship between intracellular protein half-life and subcellular localization in human cells[J].Computers and Applied Chemistry,2011,28(4):411-414.(in Chinese with English abstract)
[25]BENNETT M K,SEO Y K,DATTA S,et al.Selective binding of sterol regulatory element-binding protein isoforms and co-regulatory proteins to promoters for lipid metabolic genes in liver[J].Journal of Biological Chemistry,2008,283(23):15628-15637.
[26]GRAUGNARD D E,PIANTONI P,BIONAZ M,et al.Adipogenic and energy metabolism gene networks in longissimus lumborum during rapid post-weaning growth in Angus and Angus×Simmental cattle fed high-starch or low-starch diets[J].BMC Genomics,2009,10:142.
[27]DA COSTA A S,PIRES V M,FONTES C M,et al.Expression of genes controlling fat deposition in two genetically diverse beef cattle breeds fed high or low silage diets[J].BMC Veterinary Research,2013,9(1):1-16.
[28]XU H,XU G,WANG D,et al.Molecular cloning and tissue distribution of the phosphotyrosine interaction domain containing 1(PID1)gene in Tianfu goat[J].Gene,2013,515(1):71-77.
[2]TANG W K,CHUI C H,FATIMA S,et al.Oncogenic properties of a novel gene JK-1 located in chromosome 5p and its overexpression in human esophageal squamous cell carcinoma[J].International Journal of Molecular Medicine,2007,19(6):915-923.
[3]袁章琴,任阳,汪以真.猪新基因FAM134B的克隆及稳定干扰肌内前体脂肪细胞阳性克隆的筛选[J].中国畜牧杂志,2013,49(9):10-14.YUAN Z Q,REN Y,WANG Y Z.The new gene cloning of pig FAM134B and stable interference intramuscular preadipocytes positive clones screening[J].Chinese Journal of Animal Science,2013,49(9):10-14.(in Chinese with English abstract)
[4]朱武政,江明锋,唐璐,等.山羊FAM134B基因克隆、序列分析及其m RNA表达与肌内脂肪含量的相关性[J].农业生物技术报,2014,22(10):1277-1285.ZHU W Z,JIANG M F,TANG L,et al.Cloning,sequence analysis of FAM134B and correlation analysis between its m RNA expression and intramuscular fat content in goat(Capra hirus)[J].Journal of Agricultural Biotechnology,2014,22(10):1277-1285.(in Chinese with English abstract)
[5]曾国敏,杜晓华,杨阳,等.牦牛FAM134B基因CDS区克隆与生物信息学分析[J].华北农学报,2015,30(6):77-83.ZENG G M,DU X H,YANG Y,et al.Cloning and bioinformatics analysis on CDS of FAM134B gene in yak[J].Acta Agriculturae Boreali-Sinica,2015,30(6):77-83.(in Chinese with English abstract)
[6]季舒涵,昝林森,王洪宝,等.秦川牛A-FABP基因的生物信息学分析[J].西北农林科技大学学报(自然科学版),2010,38(6):77-81.JI S H,ZAN L S,WANG H B,et al.Biological information analysis of A-FABP gene in Qinchuan cattle[J].Journal of Northwest A&F University(Natural Science Edition),2010,38(6):77-81.(in Chinese with English abstract)
[7]SAMBOOK J,FRITSCH E F,MANLATIS T.Molecular cloning:A laboratory manual[M].2nd ed.New York:Cold Spring Harbor Laboratory,1989.
[8]罗轶.鸡FATP1基因c DNA的克隆、组织表达及其生物信息学分析[D].雅安:四川农业大学,2008.LUO Y.c DNA cloning,tissue expression and bioinformatic analysis of fatty acid transport protein 1 in chicken[D].Ya'an:Sichuan Agricultural University,2008.(in Chinese with English abstract)
[9]孙雪婧,杜晓华,杨孝朴,等.牦牛CYGB基因CDS区克隆与生物信息学分析[J].中国农业科学,2014,47(13):2690-2698.SUN X J,DU X H,YANG X P,et al.Cloning and bioinformatics analysis on CDS of CYGB gene in yak[J].Scientia Agricultura Sinica,2014,47(13):2690-2698.(in Chinese with English abstract)
[10]李盛杰,杜晓华,罗玉柱,等.天祝白牦牛NGB基因的克隆及生物信息学分析[J].畜牧兽医学报,2013,44(3):395-398.LI S J,DU X H,LUO Y Z,et al.Cloning and bioinformatics analysis of NGB gene of Tianzhu white yak[J].Chinese Journal of Animal and Veterinary Sciences,2013,44(3):395-398.(in Chinese with English abstract)
[11]魏琳琳,高晋生,王晋霖,等.绵羊LPIN1基因的克隆和m RNA表达研究[J].畜牧兽医学报,2013,44(9):1371-1379.WEI L L,GAO J S,WANG J L,et al.Study on the cloning and ontogenetic m RNA expression of ovine LP1N1 gene[J].Acta Veterinaria et Zootechnica Sinica,2013,44(9):1371-1379.(in Chinese with English abstract)
[12]陶士珩.生物信息学[M].1版.北京:科学出版社,2007:177-179.
[13]石宁宁,杜晓华,罗玉柱,等.甘南牦牛NGB基因克隆及序列分析[J].西北农林科技大学学报(自然科学版),2013,41(4):1-7.SHI N N,DU X H,LUO Y Z,et al.Cloning and sequence analysis of NGB gene in Gannan yak[J].Journal of Northwest A&F University(Natural Science Edition),2013,41(4):1-7.(in Chinese with English abstract)
[14]孙伟,李达,苏锐,等.绵羊YAP1基因全长c DNA克隆及生物信息学分析[J].中国农业科学,2013,46(8):1725-1735.SUN W,LI D,SU R,et al.Cloning and bioinformatics analysis of full-length c DNA sequence of YAP1 gene in sheep[J].Scientia Agricultura Sinica,2013,46(8):1725-1735.(in Chinese with English abstract)
[15]JENSEN L J,GUTA R,BLOM N,et al.Prediction of human protein function from post-translational modifications and localization features[J].Journal of Molecular Biology,2002,319:1257-1265.
[16]陈书霞,王晓武,房玉林.单菌落PCR法直接快速鉴定重组克隆[J].微生物学通报,2006,33(3):52-56.CHEN S X,WANG X W,FANG Y L.Rapid characterization of recombination clone by PCR screening of individual bacterial colonies[J].Microbiology,2006,33(3):52-56.(in Chinese with English abstract)
[17]KOZAK M.Structural features in eukaryotic RNAs that modulate the initiation of translation[J].Biological Chemistry,1991,266(30):19867-19870.
[18]KYTE J,DOOLITTLE R F.A simple method for displaying the hydropathic character of a protein[J].Journal of Molecular Biology,1982,157(1):105-132.
[19]GURUPRASAD K,REDDY B V B,PANDIT M W.Correlation between stability of a protein and its dipeptide composition:a novel approach for predicting in vivo stability of a protein from its primary sequence[J].Protein Engineering,1990,4(2):155-161.
[20]KYTE J,DOOLITTLE R F.A simple method for displaying the hydropathic character of a protein[J].Journal of Molecular Biology,1982,157:105-132.
[21]刘芳,格日力.藏羚羊组蛋白去乙酰化酶1基因编码区的克隆与序列分析[J].兽类学报,2011,31(4):396-340.LIU F,GE R L.Cloning and sequencing of HDAC1 coding c DNA sequence from Tibetan antelope(Pantholops hodgsonii)[J].Acta Theriologica Sinica,2011,31(4):396-340.(in Chinese with English abstract)
[22]徐飞,成述儒,罗玉柱.绵羊DRB1基因生物信息学分析[J].生物技术通报,2011(1):113-118.XU F,CHENG S R,LUO Y Z.Bioinformatics analysis of sheep DRB1 gene[J].Biotechnology Bulletin,2011(1):113-118.(in Chinese with English abstract)
[23]ROST B,SANDER C.Combining evolutionary information and neural networks to predict protein secondary strucure[J].Proteins,1994,19(1):55-72.
[24]贾浩,张小白,宋晓峰.人类胞内蛋白半衰期与其亚细胞定位的相关性研究[J].计算机与应用化学,2011,28(4):411-414.JIA H,ZHANG X B,SONG X F.Relationship between intracellular protein half-life and subcellular localization in human cells[J].Computers and Applied Chemistry,2011,28(4):411-414.(in Chinese with English abstract)
[25]BENNETT M K,SEO Y K,DATTA S,et al.Selective binding of sterol regulatory element-binding protein isoforms and co-regulatory proteins to promoters for lipid metabolic genes in liver[J].Journal of Biological Chemistry,2008,283(23):15628-15637.
[26]GRAUGNARD D E,PIANTONI P,BIONAZ M,et al.Adipogenic and energy metabolism gene networks in longissimus lumborum during rapid post-weaning growth in Angus and Angus×Simmental cattle fed high-starch or low-starch diets[J].BMC Genomics,2009,10:142.
[27]DA COSTA A S,PIRES V M,FONTES C M,et al.Expression of genes controlling fat deposition in two genetically diverse beef cattle breeds fed high or low silage diets[J].BMC Veterinary Research,2013,9(1):1-16.
[28]XU H,XU G,WANG D,et al.Molecular cloning and tissue distribution of the phosphotyrosine interaction domain containing 1(PID1)gene in Tianfu goat[J].Gene,2013,515(1):71-77.