动物性中药材地龙DNA条形码的初步研究
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摘要
目的：通过不同方法提取不同来源中药材地龙的基因组总DNA并进行比较,以确定提取中药材地龙DNA提取的最佳方法。以通用引物对不同来源中药材地龙DNA中线粒体细胞色素c氧化酶亚基Ⅰ基因(COⅠ)、16S rRNA基因(16S rDNA)序列进行PCR扩增、测序、序列比对及数据分析,并构建系统进化树,为中药材地龙的种属鉴定提供可参考的分子标记,初步建立动物性中药材地龙的DNA条形码。
方法：用三种不同方法对不同来源中药材地龙的基因组总DNA进行提取,筛选出在中药材地龙中最合适的DNA提取方法。用通用引物对中药材地龙的COⅠ、16S rDNA片断进行PCR扩增、测序；运用DNAstar、DNAman、Geneious、Clustal X、MEGA等软件对测序所得碱基序列进行分析,并根据获得的序列构建系统进化树；将获得的中药材地龙CO I、16S rDNA序列提交GenBank中的DNA Barcoding数据库。
结果：确定了提取中药材地龙DNA的一种合适方法；优化了中药材地龙DNA中COⅠ和16S rDNA序列PCR扩增的条件;获得了不同来源中药材地龙DNA中COⅠ和16S rDNA碱基序列；并经构建系统进化树,对不同来源的中药材地龙进行了聚类分析；将获得的中药材地龙DNA中COⅠ和16S rDNA序列提交GenBank中的DNA条形码数据库,获得了序列号。
结论：所获得的中药材地龙CO I、16S rDNA序列可为动物性中药材地龙的分子水平鉴定提供参考；中药材地龙DNA中COⅠ序列基因多态性比16S rDNA序列更显著,适合做为中药材地龙的DNA条形码；为动物性中药材DNA条形码数据库积累了相关信息数据。
Objective:To extract genomic DNA from different sources Chinese traditional medicine earthworm in different methods,and compared these methods to determine which is the best method.Through PCR amplification,sequencing,sequence analysis and data analysis of different sources Chinese traditional medicine earthworm DNA Cytochrome c oxidase I gene(COI)、16S rRNA gene(16S rDNA) sequences with universal primers, and construct phylogenetic tree,to provide reliable molecular markers of Chinese traditional medicine earthworm species identification and preliminary establishment of animal Chinese traditional medicine earthworm's DNA barcode.
Methods:To Extract genomic DNA from different sources Chinese traditional medicines earthworm and select the most appropriate DNA exaction method of Chinese traditional medicine.Different sources Chinese traditional medicine earthworm DNA CO I,16S rDNA sequences with universal primers were PCR amplified and sequenced.Using DNAstar,DNAman,Geneious,Clustal X,MEGA softwares to analysis these DNA sequences, and construct phylogenetic tree.To submit the different sources Chinese traditional medicine earthworm DNA CO I,16S rDNA sequences to GenBank database DNA Barcoding database.
Results:The suitable DNA extraction method of Chinese traditional medicine earthworm was determine.PCR amplification of different sources Chinese traditional medicine earthworm DNA CO I and 16S rDNA fragments was optimized,and obtain the different sources Chinese traditional medicine earthworm DNA COI,16S rDNA sequence and construct phylogenetic tree, different sources Chinese traditional medicine earthworm were clustered and analysised;then submit these sequences to GenBank database DNA Barcoding database.
Conclusion:The obtained Chinese traditional medicine earthworm DNA COI、16S rDNA sequences can provide the molecular identification basis;the Chinese traditional medicine earthworm DNA sequence Co I polymorphism are more significant than the 16S rDNA sequence and are more suitable to be the Chinese traditional medicine earthworm DNA barcoding; to accumulated data.to the DNA barcode database of animal Chinese traditional medicine.
方法：用三种不同方法对不同来源中药材地龙的基因组总DNA进行提取,筛选出在中药材地龙中最合适的DNA提取方法。用通用引物对中药材地龙的COⅠ、16S rDNA片断进行PCR扩增、测序；运用DNAstar、DNAman、Geneious、Clustal X、MEGA等软件对测序所得碱基序列进行分析,并根据获得的序列构建系统进化树；将获得的中药材地龙CO I、16S rDNA序列提交GenBank中的DNA Barcoding数据库。
结果：确定了提取中药材地龙DNA的一种合适方法；优化了中药材地龙DNA中COⅠ和16S rDNA序列PCR扩增的条件;获得了不同来源中药材地龙DNA中COⅠ和16S rDNA碱基序列；并经构建系统进化树,对不同来源的中药材地龙进行了聚类分析；将获得的中药材地龙DNA中COⅠ和16S rDNA序列提交GenBank中的DNA条形码数据库,获得了序列号。
结论：所获得的中药材地龙CO I、16S rDNA序列可为动物性中药材地龙的分子水平鉴定提供参考；中药材地龙DNA中COⅠ序列基因多态性比16S rDNA序列更显著,适合做为中药材地龙的DNA条形码；为动物性中药材DNA条形码数据库积累了相关信息数据。
Objective:To extract genomic DNA from different sources Chinese traditional medicine earthworm in different methods,and compared these methods to determine which is the best method.Through PCR amplification,sequencing,sequence analysis and data analysis of different sources Chinese traditional medicine earthworm DNA Cytochrome c oxidase I gene(COI)、16S rRNA gene(16S rDNA) sequences with universal primers, and construct phylogenetic tree,to provide reliable molecular markers of Chinese traditional medicine earthworm species identification and preliminary establishment of animal Chinese traditional medicine earthworm's DNA barcode.
Methods:To Extract genomic DNA from different sources Chinese traditional medicines earthworm and select the most appropriate DNA exaction method of Chinese traditional medicine.Different sources Chinese traditional medicine earthworm DNA CO I,16S rDNA sequences with universal primers were PCR amplified and sequenced.Using DNAstar,DNAman,Geneious,Clustal X,MEGA softwares to analysis these DNA sequences, and construct phylogenetic tree.To submit the different sources Chinese traditional medicine earthworm DNA CO I,16S rDNA sequences to GenBank database DNA Barcoding database.
Results:The suitable DNA extraction method of Chinese traditional medicine earthworm was determine.PCR amplification of different sources Chinese traditional medicine earthworm DNA CO I and 16S rDNA fragments was optimized,and obtain the different sources Chinese traditional medicine earthworm DNA COI,16S rDNA sequence and construct phylogenetic tree, different sources Chinese traditional medicine earthworm were clustered and analysised;then submit these sequences to GenBank database DNA Barcoding database.
Conclusion:The obtained Chinese traditional medicine earthworm DNA COI、16S rDNA sequences can provide the molecular identification basis;the Chinese traditional medicine earthworm DNA sequence Co I polymorphism are more significant than the 16S rDNA sequence and are more suitable to be the Chinese traditional medicine earthworm DNA barcoding; to accumulated data.to the DNA barcode database of animal Chinese traditional medicine.
引文
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