驴Zfx基因CDS序列克隆及序列分析
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摘要
本研究旨在克隆驴Zfx基因CDS序列,并对其进行生物信息学分析。根据GenBank中公布的牛Zfx基因mRNA序列(登录号:D84097.1)设计特异性引物,利用RT-PCR技术获取驴Zfx基因CDS序列,并对驴Zfx基因CDS区核苷酸序列和蛋白质结构进行生物信息学分析。结果表明,驴Zfx基因CDS区序列长度为2 403 bp,编码800个氨基酸;驴与马、牛、家犬、白犀牛、马鹿、猫、人、绵羊、黑猩猩的Zfx基因CDS区核苷酸序列同源性分别为99.6%、95.0%、95.3%、97.9%、93.5%、94.9%、95.0%、95.7%和94.9%。对驴及其他9种哺乳动物Zfx基因CDS区的核苷酸序列构建系统进化树,结果显示,驴与马亲缘关系很近,与白犀牛的亲缘关系次之;同时又与聚在一类的家犬、猫亲缘关系较近,而与绵羊、牛的亲缘关系稍远。Zfx蛋白理论等电点(pI)为5.19,不稳定系数为42.94,消光系数为35 810,属于不稳定的亲水性蛋白,含有60个磷酸化位点,无信号肽及跨膜结构,属于非分泌蛋白。Zfx蛋白α-螺旋、延伸链和无规则卷曲分别为23.12%、20.25%和56.63%,三级结构主要为α-螺旋和无规则卷曲。本试验成功克隆获得驴Zfx基因CDS序列,为后期深入研究该基因及其编码蛋白的结构功能提供依据。
This study was aimed to clone the CDS sequence of Zfx gene in Equus asinus and analyze the sequence by bioinformatics softwares.Specific primers were designed according to the mRNA sequence of Zfx gene in Bos taurus published in GenBank(accession No.:D84097.1).The CDS sequence of Zfx gene in Equus asinus was gained by RT-PCR,and the nucleotide sequence and protein structure prediction of Zfx gene CDS in Equus asinus were analyzed.The results showed that the CDS sequence of Zfx gene in Equus asinus was 2 403 bp,encoding 800 amino acids.The nucleotide sequence homology of Zfx gene CDS sequence in Equus asinus with Equus caballus,Bos taurus,Canis lupus familiaris,Ceratotherium simum,Cervus elaphus,Feils catus,Homo sapiens,Ovis aries and Pan troglodytes were 99.6%,95.0%,95.3%,97.9%,93.5%,94.9%,95.0%,95.7% and 94.9%,respectively.Phylogenetic tree was constructed for nucleotide sequences of Zfx gene CDS in Equus asinuss and other 9 mammals,which showed that Equus asinus were closely related to Equus caballus,followed by genetic relationship with Ceratotherium simum,and they were closely related to Cants lupus familiaris and Feils catus that were in the same class,and the genetic relationship was week with Ovis aries and Bos taurus.The theoretical isoelectric point(pI) of Zfx protein was 5.19,the instability coefficient was 42.94,and the extinction coefficient was 35 810.Zfx protein belonged to unstable hydrophilic protein and non-secreted protein,contained 60 phosphorylation sites,no signal peptide and transmembrane structure.The alpha helix,extended chain and random coil of Zfx protein were 23.12%,20.25% and 56.63%,respectively,and the tertiary structure was mainly alpha helix and random coil.In this experiment,the CDS sequence of Zfx gene in Equus asinus was successfully cloned,which provided a basis for further study on the structural function of Zfx gene and its enconded protein.
This study was aimed to clone the CDS sequence of Zfx gene in Equus asinus and analyze the sequence by bioinformatics softwares.Specific primers were designed according to the mRNA sequence of Zfx gene in Bos taurus published in GenBank(accession No.:D84097.1).The CDS sequence of Zfx gene in Equus asinus was gained by RT-PCR,and the nucleotide sequence and protein structure prediction of Zfx gene CDS in Equus asinus were analyzed.The results showed that the CDS sequence of Zfx gene in Equus asinus was 2 403 bp,encoding 800 amino acids.The nucleotide sequence homology of Zfx gene CDS sequence in Equus asinus with Equus caballus,Bos taurus,Canis lupus familiaris,Ceratotherium simum,Cervus elaphus,Feils catus,Homo sapiens,Ovis aries and Pan troglodytes were 99.6%,95.0%,95.3%,97.9%,93.5%,94.9%,95.0%,95.7% and 94.9%,respectively.Phylogenetic tree was constructed for nucleotide sequences of Zfx gene CDS in Equus asinuss and other 9 mammals,which showed that Equus asinus were closely related to Equus caballus,followed by genetic relationship with Ceratotherium simum,and they were closely related to Cants lupus familiaris and Feils catus that were in the same class,and the genetic relationship was week with Ovis aries and Bos taurus.The theoretical isoelectric point(pI) of Zfx protein was 5.19,the instability coefficient was 42.94,and the extinction coefficient was 35 810.Zfx protein belonged to unstable hydrophilic protein and non-secreted protein,contained 60 phosphorylation sites,no signal peptide and transmembrane structure.The alpha helix,extended chain and random coil of Zfx protein were 23.12%,20.25% and 56.63%,respectively,and the tertiary structure was mainly alpha helix and random coil.In this experiment,the CDS sequence of Zfx gene in Equus asinus was successfully cloned,which provided a basis for further study on the structural function of Zfx gene and its enconded protein.
引文
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[13] HAWKINS J R.Mutational analysis of SRY in XY females[J].Human Mutation,1993,2(5):347-350.
[14] REYNOLDS R,VARLARO J.Gender determination of forensic samples using PCR amplification of ZFX/ZFY gene sequences[J].Journal of Forensic Sciences,1996,41(2):279-286.
[15] VERNRT N,MAHADEVAIAH S K,DE ROOIJ D G,et al.Zfy genes are required for efficient meiotic sex chromosome inactivation (MSCI) in spermatocytes[J].Human Molecular Genetics,2016,25(24):5300-5310.
[16] 李超程.马鹿Zfy基因干扰载体筛选及性控效果的研究[D].石河子:石河子大学,2017.LI C C.Study on the screening of Zfy interference vector and the effect of sexual control in Cervus elaphus’s gene[D].Shihezi:Shihezi University,2017.(in Chinese)
[17] ZHANG Y S,XI J F,JIA B,et al.RNAi as a tool to control the sex ratio of mouse offspring by interrupting Zfx/Zfy genes in the testis[J].Mammalian Genome,2017,28(3-4):100-105.
[18] 侯孟典,王会,柴志欣,等.牦牛RETN基因克隆及组织表达分析[J].黑龙江畜兽医,2018,7:123-128.HOU M D,WANG H,CHAI Z X,et al.Molecular cloning and tissue expression of yak RETN gene[J].Heilongjiang Animal Science and Veterinary Medicine,2018,7:123-128.(in Chinese)
[19] 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):29-32.JIA H,ZHANG X B,SONG X F.Relationship between intracellular protein half-life and subcellular localization in human cells[J].Computer and Applied Chemistry,2011,28(4):29-32.(in Chinese)
[20] 营瑞文,张永生,夏欢,等.驴Zfy基因cDNA克隆及生物信息学分析[J].中国畜牧兽医,2018,45(1):39-46.YING R W,ZHANG Y S,XIA H,et al.cDNA cloning and bioinformatics analysis of Zfy gene in Equus asinus[J].China Animal Husbandry & Veterinary Medicine,2018,45(1):39-46.(in Chinese)
[21] SKOLNICK J,FETROW J S.From genes to protein structure and function:Novel applications of computational approaches in the genomic era[J].Trends in Biotechnology,2000,18(1):34-39.
[22] 王亚琪,郭敏,刘铮铸,等.美洲水貂TYRP1基因编码蛋白质结构及功能的生物信息学分析[J].江苏农业科学,2018,46(20):28-32.WANG Y Q,GUO M,LIU Z Z,et al.Bioinformatics analysis of the structure and function of TYRP1 gene encoding protein in mink[J].Jiangsu Agricultural Science,2018,46(20):28-32.(in Chinese)
[2] 张院萍.驴产业成为中国畜牧业发展新动力——第三届(2017)中国驴业发展大会暨第七届驴产业发展高峰论坛举办[J].中国畜牧业,2017,17:10-12.ZHANG Y P.Donkey industry has become a new driving force for the development of animal husbandry in China-The third (2017) China donkey industry development conference and the seventh donkey industry development summit forum[J].Chinese Animal Industry,2017,17:10-12.(in Chinese)
[3] LAITY J H,LEE B M,WRIGHT P E.Zinc finger proteins:New insights into structural and functional diversity[J].Current Opinion in Structural Biology,2001,11(1):39-46.
[4] WU J,WEI B,QIAN W,et al.ZFX facilitates cell proliferation and imatinib resistance in chronic myeloid leukemia cells[J].Cell Biochemistry & Biophysics,2016,74(2):277-283.
[5] ZHU Z,LI K,XU D,et al.ZFX regulates glioma cell proliferation and survival in vitro and in vivo[J].Journal of Neurooncology,2013,112(1):17-25.
[6] 唐璐.干扰猪Zfx基因性控效果及对母猪繁殖性能影响[D].石河子:石河子大学,2017.TANG L.The effects of sexual control and breeding performance for swine by interrupting Zfx gene[D].Shihezi:Shihezi University,2017.(in Chinese)
[7] 纪俊明.犬Zfx/Zfy基因干扰载体构建及性控效果的观察[D].石河子:石河子大学,2015.JI J M.The RNAi vecter construction and verification of the effects of sexual control from dog Zfx/Zfy gene[D].Shihezi:Shihezi University,2015.(in Chinese)
[8] 马世俊,马军德,贾斌,等.高效抑制小鼠精子zfx基因shRNA分子的筛选[J].石河子大学学报(自然科学版),2013,31(4):443-448.MA S J,MA J D,JIA B,et al.The shRNA screen of high effective inhibition zfx from type X sperm of mice[J].Journal of Shihezi University (Natural Science),2013,31(4):443-448.(in Chinese)
[9] XI J F,WANG X Z,ZHANG Y S,et al.Sex control by Zfy siRNA in the dairy cattle[J].Animal Reproduction Science,2019,200:1-6.
[10] ARENZANA T L,SMITH-RASKA M R,REIZIS B.Transcription factor Zfx controls BCR-induced proliferation and survival of B lymphocytes[J].Blood,2009,113(23):5857-5867.
[11] BANKS K G,JOHONSON K A,LERNER C P,et al.Retroposon compensatory mechanism hypothesis not supported:Zfa knockout mice are fertile[J].Genomics,2003,82(3):254-260.
[12] 马世俊.小鼠zfx基因RNAi片段的筛选及性控效果的观察[D].石河子:石河子大学,2013.MA S J.The RNAi fragments screening and observation of the effects of sexual control from Mus zfx gene[D].Shihezi:Shihezi University,2013.(in Chinese)
[13] HAWKINS J R.Mutational analysis of SRY in XY females[J].Human Mutation,1993,2(5):347-350.
[14] REYNOLDS R,VARLARO J.Gender determination of forensic samples using PCR amplification of ZFX/ZFY gene sequences[J].Journal of Forensic Sciences,1996,41(2):279-286.
[15] VERNRT N,MAHADEVAIAH S K,DE ROOIJ D G,et al.Zfy genes are required for efficient meiotic sex chromosome inactivation (MSCI) in spermatocytes[J].Human Molecular Genetics,2016,25(24):5300-5310.
[16] 李超程.马鹿Zfy基因干扰载体筛选及性控效果的研究[D].石河子:石河子大学,2017.LI C C.Study on the screening of Zfy interference vector and the effect of sexual control in Cervus elaphus’s gene[D].Shihezi:Shihezi University,2017.(in Chinese)
[17] ZHANG Y S,XI J F,JIA B,et al.RNAi as a tool to control the sex ratio of mouse offspring by interrupting Zfx/Zfy genes in the testis[J].Mammalian Genome,2017,28(3-4):100-105.
[18] 侯孟典,王会,柴志欣,等.牦牛RETN基因克隆及组织表达分析[J].黑龙江畜兽医,2018,7:123-128.HOU M D,WANG H,CHAI Z X,et al.Molecular cloning and tissue expression of yak RETN gene[J].Heilongjiang Animal Science and Veterinary Medicine,2018,7:123-128.(in Chinese)
[19] 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):29-32.JIA H,ZHANG X B,SONG X F.Relationship between intracellular protein half-life and subcellular localization in human cells[J].Computer and Applied Chemistry,2011,28(4):29-32.(in Chinese)
[20] 营瑞文,张永生,夏欢,等.驴Zfy基因cDNA克隆及生物信息学分析[J].中国畜牧兽医,2018,45(1):39-46.YING R W,ZHANG Y S,XIA H,et al.cDNA cloning and bioinformatics analysis of Zfy gene in Equus asinus[J].China Animal Husbandry & Veterinary Medicine,2018,45(1):39-46.(in Chinese)
[21] SKOLNICK J,FETROW J S.From genes to protein structure and function:Novel applications of computational approaches in the genomic era[J].Trends in Biotechnology,2000,18(1):34-39.
[22] 王亚琪,郭敏,刘铮铸,等.美洲水貂TYRP1基因编码蛋白质结构及功能的生物信息学分析[J].江苏农业科学,2018,46(20):28-32.WANG Y Q,GUO M,LIU Z Z,et al.Bioinformatics analysis of the structure and function of TYRP1 gene encoding protein in mink[J].Jiangsu Agricultural Science,2018,46(20):28-32.(in Chinese)