峨嵋山区姜科药用植物ITS2序列分析
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摘要
目的:评价以ITS2序列作为DNA条形码对峨眉山地区姜科药用植物的鉴定。方法:于四川省峨眉山地区采集姜科药用植物样本43个,提取其基因组DNA,进行转录间隔区(ITS) 2序列PCR扩增、测序,从Gen Bank上下载40条姜科植物ITS2序列;运用MEGA6. 0软件对所有样本序列种间和种内遗传距离进行计算分析,构建neighbor joining(NJ)系统进化树;利用TAXON DNA软件分析序列种内、种间变异并作barcoding gap分析;从ITS2数据库中预测并比较样本ITS2序列的二级结构差异。结果:姜科样本种间最小距离大于种内最大距离,有较为明显的barcoding gap; NJ进化树中各属样本分别聚山姜属(Alpinia),姜黄属(Curcuma),舞花姜属(Globba),姜花属(Hedychium),姜属(Zingiber)五大支,种内之间于各属分支中各聚为小支;各属及各种样本ITS2二级结构存在差异明显。结论:以ITS2序列作为DNA条形码,能够对姜科药用植物进行准确、快速的识别和鉴定,准确地弄清物种之间的系统进化关系,为该属药用植物的分布、种群及种群量研究提供指导,对其质量控制、安全用药及资源保护及合理开发利用提供理论依据。
Objective: To evaluate the use of ITS2 sequences as DNA barcode to identify the Zingiberaceae medicinal plants from E'mei area. Method: The genomic DNAs were extracted from 43 Zingiberaceae medicinal plant samples from Sichuan E'mei area. The ITS2 sequences of these samples were amplified and bidirectionally sequenced by PCR. 40 ITS2 sequences were downloaded from the GenBank,and then the interspecific and intraspecific genetic distances were calculated and analyzed by using MEGA 6. 0 to construct Neighbor-joining(NJ) tree; TAXON DNA software was also used to analyze intraspecific and interspecific variations and barcoding gaps. The differences in secondary structure of the ITS2 sequences were predicted and compared. Result: The minimum interspecific distance in Zingiberaceae samples was greater than the maximum intra specific distance,with obvious barcoding gap. The NJ tree showed that the samples were clustered into five different branches,Alpinia,Curcuma,Globba,Hedychium,and Zingiber respectively,and further cluster into sub-branches. Significant differences were also present in the secondary structures of ITS2 between different samples. Conclusion: ITS2 sequences as DNA barcode can be used to conduct accurate and rapid identification of the Zingiberaceae plants and clearly figure out the phylogenetic relationship among them,providing guidance for the study of the distribution of medicinal plants of this genus,as well as theoretical basis for the quality control,medication safety and rational development of Zingiberaceae medicinal plants in E'mei area.
Objective: To evaluate the use of ITS2 sequences as DNA barcode to identify the Zingiberaceae medicinal plants from E'mei area. Method: The genomic DNAs were extracted from 43 Zingiberaceae medicinal plant samples from Sichuan E'mei area. The ITS2 sequences of these samples were amplified and bidirectionally sequenced by PCR. 40 ITS2 sequences were downloaded from the GenBank,and then the interspecific and intraspecific genetic distances were calculated and analyzed by using MEGA 6. 0 to construct Neighbor-joining(NJ) tree; TAXON DNA software was also used to analyze intraspecific and interspecific variations and barcoding gaps. The differences in secondary structure of the ITS2 sequences were predicted and compared. Result: The minimum interspecific distance in Zingiberaceae samples was greater than the maximum intra specific distance,with obvious barcoding gap. The NJ tree showed that the samples were clustered into five different branches,Alpinia,Curcuma,Globba,Hedychium,and Zingiber respectively,and further cluster into sub-branches. Significant differences were also present in the secondary structures of ITS2 between different samples. Conclusion: ITS2 sequences as DNA barcode can be used to conduct accurate and rapid identification of the Zingiberaceae plants and clearly figure out the phylogenetic relationship among them,providing guidance for the study of the distribution of medicinal plants of this genus,as well as theoretical basis for the quality control,medication safety and rational development of Zingiberaceae medicinal plants in E'mei area.
引文
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[6]石林春,金钺,赵春颖,等.基于DNA条形码技术的知母种子基原鉴定[J].中国实验方剂学杂志,2018,24(12):21-27.
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[14]任瑶瑶,张良,谢博文,等.峨眉山区桑科药用植物ITS2条形码序列鉴定[J].世界科学技术—中医药现代化,2018,20(1):140-145.
[15]王孟虎,康廷国,许亮,等.基于COI序列的哈蟆油基原动物DNA条形码鉴定研究[J].中国中药杂志,2017,42(8):1572-1577.
[16]樊丛照,徐建国,李亚伟,等.基于DNA条形码技术的维吾尔药材桑葚基原鉴定研究[J].中国中药杂志,2017,42(16):3219-3224.
[17]许亮,刘春生,杨燕云,等. DNA条形码技术鉴定一种植物样品的研究[J].中国实验方剂学杂志,2014,20(7):127-129.
[18]朱英杰,陈士林,姚辉.重楼属药用植物DNA条形码鉴定研究[J].药学学报,2010,45(3):376-382.
[19]祝正银.峨眉山姜花属二新种[J].云南植物研究,1984,6(1):63-66.
[20]祝正银.四川山姜属一新种[J].广西植物,1987,7(4):295-296.
[21]吴荭,庄平,张超,等.峨眉山资源植物研究[J].资源开发与市场,2011,27(4):347-351.
[22]秦运潭,王书林,王金鹏.峨眉山珍稀濒危药用植物保护研究[J].中国医药指南,2011,9(32):279-280.
[2]蒋虎臣.峨眉山志.卷2[M].壁经堂,1672.
[3]李萍,何丽华,魏华,等.姜科植物精油抑菌作用的进展研究[J].食品工业科技,2014,35(6):377-382.
[4]中国科学院《中国植物志》编辑委员会.中国植物志.16(2)卷[M].北京:科学出版社,1981.
[5]鲁松,谢孔平,李策宏.峨眉山区野生濒危药用植物资源评价体系的初步研究[J].广西植物2013,33(2):229-235.
[6]石林春,金钺,赵春颖,等.基于DNA条形码技术的知母种子基原鉴定[J].中国实验方剂学杂志,2018,24(12):21-27.
[7]王孟虎,许亮,康廷国,等.动物类中药DNA条形码鉴定研究进展[J].中国实验方剂学杂志,2016,22(15):227-234.
[8] CHEN S L,YAO H,HAN J P,et al. Validation of the ITS2 regionas a novel DNA barcode for identifying medicinal plant species[J]. PLo S One,2010,5(1):e8613.
[9]任瑶瑶,江南屏,刘睿颖,等.藏药臭蒿及其近缘种药材的ITS2 DNA条形码鉴别[J].中国中药杂志,2017,42(7):1395-1400.
[10]陈士林.中药DNA条形码分子鉴定[M].北京:人民卫生出版社,2012.
[11] Keller A,Schleicher T,Schultz J,et al. 5. 8S-28S rRNAinteraction and HMM-based ITS2 annotation[J].Gene,2009,430(1):50-57.
[12] Tamura K,Peterson D,Peterson N,et al. MEGA5:molecularevolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Mol Biol Evol,2011,28(10):2731-2739.
[13] Koetschan C,F9rster F,Keller A,et al. The ITS2database III-sequences and structures for phylogeny[J].Nucleic Acids Res,2010,38(suppl 1):D275-D279.
[14]任瑶瑶,张良,谢博文,等.峨眉山区桑科药用植物ITS2条形码序列鉴定[J].世界科学技术—中医药现代化,2018,20(1):140-145.
[15]王孟虎,康廷国,许亮,等.基于COI序列的哈蟆油基原动物DNA条形码鉴定研究[J].中国中药杂志,2017,42(8):1572-1577.
[16]樊丛照,徐建国,李亚伟,等.基于DNA条形码技术的维吾尔药材桑葚基原鉴定研究[J].中国中药杂志,2017,42(16):3219-3224.
[17]许亮,刘春生,杨燕云,等. DNA条形码技术鉴定一种植物样品的研究[J].中国实验方剂学杂志,2014,20(7):127-129.
[18]朱英杰,陈士林,姚辉.重楼属药用植物DNA条形码鉴定研究[J].药学学报,2010,45(3):376-382.
[19]祝正银.峨眉山姜花属二新种[J].云南植物研究,1984,6(1):63-66.
[20]祝正银.四川山姜属一新种[J].广西植物,1987,7(4):295-296.
[21]吴荭,庄平,张超,等.峨眉山资源植物研究[J].资源开发与市场,2011,27(4):347-351.
[22]秦运潭,王书林,王金鹏.峨眉山珍稀濒危药用植物保护研究[J].中国医药指南,2011,9(32):279-280.