基于涉案兽类12S rRNA基因的NCBI数据库序列可靠性分析
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摘要
本研究以常用于动物种属鉴定的12S rRNA基因位点为研究对象,利用所测得的17种常见涉案兽类12S rRNA基因部分片段序列及NCBI数据库中下载的该物种DNA序列及其近缘物种DNA序列,构建系统进化树。根据进化树的聚类情况,判断NCBI数据库中的相关基因序列或物种名称的正确性,并对其中错误序列的登陆号进行标记,以防对后续涉案动物的准确鉴定造成影响。分别从17种常见涉案兽类(共26份样本)中提取线粒体DNA,并利用通用引物扩增线粒体DNA上的12S rRNA基因部分片段并进行测序分析。通过NCBI数据库的Blast比对功能,筛选出与本研究物种同源性由高到低的物种,并从NCBI基因数据库中下载此类近缘物种的12S rRNA基因序列共351条,利用MEGA7.0软件构建该物种及其近缘物种系统进化树。通过比对发现NCBI中登录号为KP202279等3个序列所对应物种拉丁名错误。登录号为AY184436等11个序列所对应物种拉丁名可能存在疑问。GenBank中某些物种拉丁名有同种异名现象。因此,NCBI数据库数据可靠性有待进一步验证,只能作为涉案物种鉴定的参考数据之一,可借助构建系统进化树等方法来确认其结果的准确性。
A common barcoding gene for animal species identification, 12 S rRNA, was used to investigate 17 common mammals and their close species. Combining directly sequencing and downloading reference sequence from NCBI database for the 12 S rRNA gene fragment, phylogenetic tree was constructed. According to the evolutionary tree, reliability of gene sequences or species naming in NCBI database was evaluated. Moreover,accession numbers of related error sequence were marked to prevent further wrong identification of the animals.Mitochondrial DNA was extracted from 17 kinds of mammals(a total of 26 samples), and the 12 S rRNA gene fragment of mitochondrial DNA was amplified by universal primers and then sequenced. Through comparison function built in Blast module, the sequences of the same or close species of the tested species were downloaded from the NCBI database. For each species, at least two sequences were derived from different laboratories.Meanwhile, DNA sequences from related close relative species were downloaded as much as possible. MEGA7.0 software was used to construct phylogenetic tree of the species. By comparison, we found that the Latin names of 3 species with corresponding register numbers including KP202279 were wrong in NCBI. There were some doubts about the Latin name of 11 species with corresponding login numbers such as AY184436. Additionally, Latin name of some species had senior synonym phenomenon in GenBank. So the reliability of NCBI database needs to be further verified, and can only be used as one of the reference for species identification. The phylogenetic tree can be added to confirm the accuracy of the result.
A common barcoding gene for animal species identification, 12 S rRNA, was used to investigate 17 common mammals and their close species. Combining directly sequencing and downloading reference sequence from NCBI database for the 12 S rRNA gene fragment, phylogenetic tree was constructed. According to the evolutionary tree, reliability of gene sequences or species naming in NCBI database was evaluated. Moreover,accession numbers of related error sequence were marked to prevent further wrong identification of the animals.Mitochondrial DNA was extracted from 17 kinds of mammals(a total of 26 samples), and the 12 S rRNA gene fragment of mitochondrial DNA was amplified by universal primers and then sequenced. Through comparison function built in Blast module, the sequences of the same or close species of the tested species were downloaded from the NCBI database. For each species, at least two sequences were derived from different laboratories.Meanwhile, DNA sequences from related close relative species were downloaded as much as possible. MEGA7.0 software was used to construct phylogenetic tree of the species. By comparison, we found that the Latin names of 3 species with corresponding register numbers including KP202279 were wrong in NCBI. There were some doubts about the Latin name of 11 species with corresponding login numbers such as AY184436. Additionally, Latin name of some species had senior synonym phenomenon in GenBank. So the reliability of NCBI database needs to be further verified, and can only be used as one of the reference for species identification. The phylogenetic tree can be added to confirm the accuracy of the result.
引文
Alacs E.A.,Geo rges A.,Fitz Simmons N.N.,and Robertson J.,2010,DNA detective:a review of molecular approaches to wildlife forensics,Forensic Sci.Med.Pathol.,6:180-194
Bellis C.,Ashton K.J.,Freney L.,Blair B.,and Griffiths L.R.,2003,A molecular genetic approach for forensic animal species identification,Forensic Sci.Int.,134:99-108
Dalton D.L.,and Kotze A.,2011,DNA barcoding as a tool for species identification in three forensic wildlife cases in South Africa,Forensic Sci.Int,207:e51-e54
Feng H.,Wang X.Y.,Feng C.L.,Huang Y.,Ren Y.,and Wang L.,2016,Molecular identification and gene barcode construction of illegal trading animal in Qinling Mountains of Shaanxi Province,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(3):568-572(冯慧,汪晓阳,冯成利,黄原,任轶,王璐,2016,陕西秦岭非法贸易动物(兽类)基因条码构建及分子鉴定,基因组学与应用生物学,35(3):568-572)
Huang Y.L.,2005,Identification of animal putrid intramuscular swatch by sequencing of patial 12S r RNA gene,Xingshi Jishu(Forensic Science and Technology),(4):10-11(黄娅琳,2005,mt DNA 12S r RNA基因序列测定在腐烂动物肌肉样本鉴定中的应用,刑事技术,(4):10-11)
Huang Y.L.,2008,Application of sequencing 12S r RNA and Cyt b gene to identification of dairy products of hydropotes iner mis,Sichuan Dongwu(Sichuan Journal of Zoology),27(6):1111-1114(黄娅琳,2008,12S r RNA和Cytb基因序列测定在獐乳制品鉴定中的应用,四川动物,27(6):1111-1114)
Huang Y.L.,2017,The differences of 12S r RNA and Cyt b gene marker in wildlife species identification,Sichuan Nongyedaxue Xuebao(Journal of Sichuan Agricultural University),35(2):273-279(黄娅琳,2017,12S r RNA,Cyt b基因标记在几类野生动物检材鉴定中的差异,四川农业大学学报,35(2):273-279)
Iyengar A.,2014,Forensic DNA analysis for animal protection and biodiversity conservation:a review,J.Nat.Conservat.,22(3):195-205
Johnson R.N.,Wilsonwilde L.,and Linacre A.,2014,Current and future directions of DNA in wildlife forensic science,Forensic Sci.Int:Genet.,(10):1-11
Kocher T.D.,Thomas W.K.,Meyer A.,Edwards S.V.,Villablanca F.X.,and Wilson A.C.,1989,Dynamics of mitochondrial DNA evolution in animals:amplification and sequencing with conserved primers,Proc.Natl.Acad.Sci.USA,86:6196-6200
Saitou N.,and Nei M.,1987,The neighbor-joining method:Anew method for reconstructing phylogenetic trees,Molecular Biology and Evolution,4:406-425
Tobe S.S.,and Linacre A.,2010,DNA typing in wildlife crime:recent developments in species identification,Forensic Sci.Med.Pathol.,6:195-206
Yang G.,Cai Y.,and Liu H.,2004,Identification of several imported and exported animal products by mitochondrial DNA,Dongwuxue Zazhi(Chinese Journal of Zoology),39(5):40-43(杨光,蔡垚,刘海,2004,mt DNA标记在几种海关进出口动物产品鉴定中的应用,动物学杂志,39(5):40-43)
Bellis C.,Ashton K.J.,Freney L.,Blair B.,and Griffiths L.R.,2003,A molecular genetic approach for forensic animal species identification,Forensic Sci.Int.,134:99-108
Dalton D.L.,and Kotze A.,2011,DNA barcoding as a tool for species identification in three forensic wildlife cases in South Africa,Forensic Sci.Int,207:e51-e54
Feng H.,Wang X.Y.,Feng C.L.,Huang Y.,Ren Y.,and Wang L.,2016,Molecular identification and gene barcode construction of illegal trading animal in Qinling Mountains of Shaanxi Province,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(3):568-572(冯慧,汪晓阳,冯成利,黄原,任轶,王璐,2016,陕西秦岭非法贸易动物(兽类)基因条码构建及分子鉴定,基因组学与应用生物学,35(3):568-572)
Huang Y.L.,2005,Identification of animal putrid intramuscular swatch by sequencing of patial 12S r RNA gene,Xingshi Jishu(Forensic Science and Technology),(4):10-11(黄娅琳,2005,mt DNA 12S r RNA基因序列测定在腐烂动物肌肉样本鉴定中的应用,刑事技术,(4):10-11)
Huang Y.L.,2008,Application of sequencing 12S r RNA and Cyt b gene to identification of dairy products of hydropotes iner mis,Sichuan Dongwu(Sichuan Journal of Zoology),27(6):1111-1114(黄娅琳,2008,12S r RNA和Cytb基因序列测定在獐乳制品鉴定中的应用,四川动物,27(6):1111-1114)
Huang Y.L.,2017,The differences of 12S r RNA and Cyt b gene marker in wildlife species identification,Sichuan Nongyedaxue Xuebao(Journal of Sichuan Agricultural University),35(2):273-279(黄娅琳,2017,12S r RNA,Cyt b基因标记在几类野生动物检材鉴定中的差异,四川农业大学学报,35(2):273-279)
Iyengar A.,2014,Forensic DNA analysis for animal protection and biodiversity conservation:a review,J.Nat.Conservat.,22(3):195-205
Johnson R.N.,Wilsonwilde L.,and Linacre A.,2014,Current and future directions of DNA in wildlife forensic science,Forensic Sci.Int:Genet.,(10):1-11
Kocher T.D.,Thomas W.K.,Meyer A.,Edwards S.V.,Villablanca F.X.,and Wilson A.C.,1989,Dynamics of mitochondrial DNA evolution in animals:amplification and sequencing with conserved primers,Proc.Natl.Acad.Sci.USA,86:6196-6200
Saitou N.,and Nei M.,1987,The neighbor-joining method:Anew method for reconstructing phylogenetic trees,Molecular Biology and Evolution,4:406-425
Tobe S.S.,and Linacre A.,2010,DNA typing in wildlife crime:recent developments in species identification,Forensic Sci.Med.Pathol.,6:195-206
Yang G.,Cai Y.,and Liu H.,2004,Identification of several imported and exported animal products by mitochondrial DNA,Dongwuxue Zazhi(Chinese Journal of Zoology),39(5):40-43(杨光,蔡垚,刘海,2004,mt DNA标记在几种海关进出口动物产品鉴定中的应用,动物学杂志,39(5):40-43)