槟榔江水牛LALBA基因克隆及序列特征分析
|
摘要
【目的】α-乳清蛋白由LALBA基因编码,在乳糖合成中起着重要作用。近年在普通奶牛中发现该基因与泌乳性状关系密切,但在水牛中研究较少。为了揭示水牛LALBA基因的序列特征。【方法】本研究采用RT-PCR测序法对槟榔江水牛LALBA基因的编码区序列进行了克隆和生物信息学分析。【结果】槟榔江水牛LALBA基因的编码区全长429 bp,编码蛋白含142个氨基酸。其α-LA含有1个C型溶菌酶/乳蛋白家族结构域(AA20~138)和1个信号肽序列(AA19~20),无跨膜结构,是胞外分泌的亲水蛋白;其N末端疏水,C末端亲水;有4类功能活性位点。基于LALBA基因编码区序列的系统发育分析表明,槟榔江水牛与牛亚科物种聚在一起,揭示它们LALBA基因功能的相似性。在LALBA基因编码区共检测到5个SNP位点:c.63A>G、c.107A>G、c.147A>G、c.249C>T、c.291T>C。这些变异位点全部处于Hardy-Weinberg不平衡状态,只有SNP107为异义替换,导致编码氨基酸由天冬氨酸变为甘氨酸。功能分析显示SNP107对槟榔江水牛α-LA功能没有影响。【结论】水牛LALBA基因的序列与其他牛科物种长度相同,其编码产物的结构、功能与其他牛科物种相似。本研究可为进一步研究LALBA基因在水牛泌乳过程中的作用机制奠定基础。
[Purpose]α-lactlbumin which is encoded by LALBA gene plays an important role in biosynthesis of lactose. In recent years,LALBA is found to have close relationship with milk production traits of Holsteins cows,but few studies are reported in buffalo. To elucidate the sequence characteristics of buffalo LALBA gene. [Method]the complete coding sequence of the Binglangjiang buffalo LALBA gene was cloned using RT-PCR sequencing and the data obtained were analyzed by functionalbioinformatics method in this study. [Results] Whole coding region of the buffalo LALBA gene in length is 429 bp which encodes a polypeptide composed of 142 amino acids. Buffalo LALBA contains a C-type lysozyme/alpha-lactalbumin family domain( AA20-138) and a signal peptide sequence( AA19-20),without transmembrane domains. This protein has 4 kinds of functional active site. It is an extracellular secreted and hydrophilic protein with a hydrophobic N-terminal and a hydrophilic C-terminal.Phylogenesis analysis based on the sequences of LALBA coding region shows that Binglangjiang buffalo clustered with other bovine species,which indicated that the functional similarity of LALBA gene in the family of Bovidae. Five SNPs were identified in Binglangjiang buffalo LALBA gene,viz. c. 63 A > G,c. 107 A > G,c. 147 A > G,c. 249 C > T and c. 291 T > C. All these variants were in Hardy-Weinberg disequilibrium and only the SNP107 was a nonsynonymous substitution leading to corresponding coded amino acid change from aspartic acid to glycine,but this change has no effect on the function of buffalo LALBA protein by functional analysis. [Conclusion]The results revealed that the buffalo LALBA gene is the same in length with other bovine and its coding. Polypeptide has similar structure and function with other bovine species. This study will lay a foundation for further study on the functional mechanism of buffalo LALBA in the process of lactation.
[Purpose]α-lactlbumin which is encoded by LALBA gene plays an important role in biosynthesis of lactose. In recent years,LALBA is found to have close relationship with milk production traits of Holsteins cows,but few studies are reported in buffalo. To elucidate the sequence characteristics of buffalo LALBA gene. [Method]the complete coding sequence of the Binglangjiang buffalo LALBA gene was cloned using RT-PCR sequencing and the data obtained were analyzed by functionalbioinformatics method in this study. [Results] Whole coding region of the buffalo LALBA gene in length is 429 bp which encodes a polypeptide composed of 142 amino acids. Buffalo LALBA contains a C-type lysozyme/alpha-lactalbumin family domain( AA20-138) and a signal peptide sequence( AA19-20),without transmembrane domains. This protein has 4 kinds of functional active site. It is an extracellular secreted and hydrophilic protein with a hydrophobic N-terminal and a hydrophilic C-terminal.Phylogenesis analysis based on the sequences of LALBA coding region shows that Binglangjiang buffalo clustered with other bovine species,which indicated that the functional similarity of LALBA gene in the family of Bovidae. Five SNPs were identified in Binglangjiang buffalo LALBA gene,viz. c. 63 A > G,c. 107 A > G,c. 147 A > G,c. 249 C > T and c. 291 T > C. All these variants were in Hardy-Weinberg disequilibrium and only the SNP107 was a nonsynonymous substitution leading to corresponding coded amino acid change from aspartic acid to glycine,but this change has no effect on the function of buffalo LALBA protein by functional analysis. [Conclusion]The results revealed that the buffalo LALBA gene is the same in length with other bovine and its coding. Polypeptide has similar structure and function with other bovine species. This study will lay a foundation for further study on the functional mechanism of buffalo LALBA in the process of lactation.
引文
[1]PERMYAKOV E A,BERLINER L J.Alpha-lactalbumin:structure and function[J].FEBS Letters,2000,473(3):269.DOI:10.1016/S0014-5793(00)01546-5.
[2]BREW K,HILL R L.Lactose biosynthesis[M]//Reviews of Physiology,Biochemistry and Pharmacology:V01.72.Heidelberg:Springer,1975:105.
[3]BLECK G T,BREMEL R D.Correlation of the alphalactalbumin(+15)polymorphism to milk production and milk composition of Holsteins[J].Journal of Dairy Science,1993,76(8):2292.DOI:10.3168/jds.S0022-0302(93)77566-9.
[4]VOELKER G R,BLECK G T,WHEELER M B.Singlebase polymorphisms within the 5'flanking region of the bovine alpha-lactalbumin gene[J].Journal of Dairy Science,1997,80(1):194.DOI:10.3168/jds.S0022-0302(97)75927-7.
[5]DETTORI M L,PAZZOLA M,PASCHINO P,et al.Variability of the caprine whey protein genes and their association with milk yield,composition and renneting properties in the Sarda breed.1.The LALBA gene[J].Journal of Dairy Research,2015,82(4):434.DOI:10.1017/S0022029915000461.
[6]GARCA-GMEZ E,GUTIRREZ-GIL B,SAHANA G,et al.GWA analysis for milk production traits in dairy sheep and genetic support for a QTN influencing milk protein percentage in the LALBA gene[J].PLo S One,2012,7(10):e47782.DOI:10.1371/journal.pone.0047782.
[7]BREW K.Secretion of alpha-lactalbumin into milk and its relevance to the organization and control of lactose synthetase[J].Nature,1969,222(5194):671.DOI:10.1038/222671a0.
[8]杨永强,龚俞,焦仁刚,等.牛α-乳清蛋白基因5'调控区多态性研究[J].中国畜牧兽医,2013,40(1):1671.DOI:10.3969/j.issn.1671-7236.2013.01.035.
[9]ZIDI ALI,CASAS E,AMILLS M,et al.Genetic variation at the caprine lactalbumin,alpha(LALBA)gene and its association with milk lactose concentration[J].Animal Genetics,2014,45(4):612.DOI:10.1111/age.12152.
[10]刘建忠,田为宇,敖敬,等.水牛α-乳清蛋白基因的克隆及序列分析[J].西北农业学报,2009,18(5):31.DOI:10.3969/j.issn.1004-1389.2009.05.007.
[11]樊宝良,张庆桥,李宁,等.水牛α-乳清蛋白基因部分序列的测定与分析[J].中国畜牧杂志,2005,41(7):21.DOI:10.3969/j.issn.0258-7033.2005.07.008.
[12]章纯熙,苗永旺,李大林.我国首例本土河流型水牛——槟榔江水牛的种质特征[J].家畜生态学报,2011,32(6):39.DOI:10.3969/j.issn.1673-1182.2011.06.008.
[13]HAYSSEN V,BLACKBURN D G.α-lactalbumin and the origins of lactation[J].Evolution,1985,39(5):1.DOI:10.2307/2408741.
[14]QASBA P K,KUMAR S.Molecular divergence of lysozymes and alpha-lactalbumin[J].Critical Reviews in Biochemistry and Molecular Biology,1997,32(4):255.DOI:10.3109/10409239709082574.
[15]DAYAL S,BHATTACHARYA T K,VOHRA V,et al.Genetic polymorphism of alpha-lactalbumin gene in riverine Buffalo[J].DNA Sequence:the Journal of DNA Sequencing and Mapping,2005,16(3):173.DOI:10.1080/10425170500088205.
[16]CAFFIN J P,POUTREL B,RAINARD P.Physiological and pathological factors influencing bovine alpha-lactalbumin and beta-lactoglobulin concentrations in milk[J].Journal of Dairy Science,1985,68(5):1087.DOI:10.3168/jds.S0022-0302(85)80933-4.
[17]杨玉红,林海.乳清蛋白的组成及生物学活性[J].生物学教学,2011,36(4):5.DOI:10.3969/j.issn.1004-7549.2011.04.003.
[18]梁贤威,杨炳壮,邹彩霞,等.水牛奶在乳品加工中的优势[J].广西畜牧兽医,2004,20(3):125.DOI:10.3969/j.issn.1002-5235.2004.03.015.
[2]BREW K,HILL R L.Lactose biosynthesis[M]//Reviews of Physiology,Biochemistry and Pharmacology:V01.72.Heidelberg:Springer,1975:105.
[3]BLECK G T,BREMEL R D.Correlation of the alphalactalbumin(+15)polymorphism to milk production and milk composition of Holsteins[J].Journal of Dairy Science,1993,76(8):2292.DOI:10.3168/jds.S0022-0302(93)77566-9.
[4]VOELKER G R,BLECK G T,WHEELER M B.Singlebase polymorphisms within the 5'flanking region of the bovine alpha-lactalbumin gene[J].Journal of Dairy Science,1997,80(1):194.DOI:10.3168/jds.S0022-0302(97)75927-7.
[5]DETTORI M L,PAZZOLA M,PASCHINO P,et al.Variability of the caprine whey protein genes and their association with milk yield,composition and renneting properties in the Sarda breed.1.The LALBA gene[J].Journal of Dairy Research,2015,82(4):434.DOI:10.1017/S0022029915000461.
[6]GARCA-GMEZ E,GUTIRREZ-GIL B,SAHANA G,et al.GWA analysis for milk production traits in dairy sheep and genetic support for a QTN influencing milk protein percentage in the LALBA gene[J].PLo S One,2012,7(10):e47782.DOI:10.1371/journal.pone.0047782.
[7]BREW K.Secretion of alpha-lactalbumin into milk and its relevance to the organization and control of lactose synthetase[J].Nature,1969,222(5194):671.DOI:10.1038/222671a0.
[8]杨永强,龚俞,焦仁刚,等.牛α-乳清蛋白基因5'调控区多态性研究[J].中国畜牧兽医,2013,40(1):1671.DOI:10.3969/j.issn.1671-7236.2013.01.035.
[9]ZIDI ALI,CASAS E,AMILLS M,et al.Genetic variation at the caprine lactalbumin,alpha(LALBA)gene and its association with milk lactose concentration[J].Animal Genetics,2014,45(4):612.DOI:10.1111/age.12152.
[10]刘建忠,田为宇,敖敬,等.水牛α-乳清蛋白基因的克隆及序列分析[J].西北农业学报,2009,18(5):31.DOI:10.3969/j.issn.1004-1389.2009.05.007.
[11]樊宝良,张庆桥,李宁,等.水牛α-乳清蛋白基因部分序列的测定与分析[J].中国畜牧杂志,2005,41(7):21.DOI:10.3969/j.issn.0258-7033.2005.07.008.
[12]章纯熙,苗永旺,李大林.我国首例本土河流型水牛——槟榔江水牛的种质特征[J].家畜生态学报,2011,32(6):39.DOI:10.3969/j.issn.1673-1182.2011.06.008.
[13]HAYSSEN V,BLACKBURN D G.α-lactalbumin and the origins of lactation[J].Evolution,1985,39(5):1.DOI:10.2307/2408741.
[14]QASBA P K,KUMAR S.Molecular divergence of lysozymes and alpha-lactalbumin[J].Critical Reviews in Biochemistry and Molecular Biology,1997,32(4):255.DOI:10.3109/10409239709082574.
[15]DAYAL S,BHATTACHARYA T K,VOHRA V,et al.Genetic polymorphism of alpha-lactalbumin gene in riverine Buffalo[J].DNA Sequence:the Journal of DNA Sequencing and Mapping,2005,16(3):173.DOI:10.1080/10425170500088205.
[16]CAFFIN J P,POUTREL B,RAINARD P.Physiological and pathological factors influencing bovine alpha-lactalbumin and beta-lactoglobulin concentrations in milk[J].Journal of Dairy Science,1985,68(5):1087.DOI:10.3168/jds.S0022-0302(85)80933-4.
[17]杨玉红,林海.乳清蛋白的组成及生物学活性[J].生物学教学,2011,36(4):5.DOI:10.3969/j.issn.1004-7549.2011.04.003.
[18]梁贤威,杨炳壮,邹彩霞,等.水牛奶在乳品加工中的优势[J].广西畜牧兽医,2004,20(3):125.DOI:10.3969/j.issn.1002-5235.2004.03.015.