基于ITS2条形码鉴定酸橙类中药材
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摘要
对正品枳及其5种常见混伪品进行ITS2分析,为制药和临床上鉴别枳实和枳壳类中药材提供一种方法,也为农业上鉴定橘类种子种苗提供一种可靠方法。提取枳及其混伪品DNA,对ITS2序列扩增和双向测序,从GenBank下载其他部分序列。用DNAstar 7.10进行序列长度统计,并计算G+C含量。用MEGA 5.0计算种内种间(K2P)遗传距离,并进行NJ树聚类分析和二级结构分析,最后通过条形码网站鉴定各物种序列。结果发现:枳及其混伪品ITS2序列长度范围为231～279bp,G+C含量范围为69.26%～72.93%;种内最大K2P遗传距离比种间最小遗传距离小;聚类树单系性良好,但二级结构区分不明显,条形码网站鉴定可得到正确物种。ITS2条形码可有效鉴别枳及其混伪品。
We identify Citrus aurantium L. and its adulterants using ITS2 sequence to provide a secure and accurate identification way for the pharmaceutical and clinical use of Citrus aurantium L.,and also to provide a reliable identification method of citrus seeds or seedlings for agriculture.After extracting the DNA of Citrus aurantium L and its adulterants, the ITS2 sequences were amplified by PCR and measured bidirectionally;and the other sequences were downloaded from GenBank. DNAstar 7.10 is used to measure the length of each ITS2 sequence and calculate the G+C content of each ITS2 sequence.The genetic distance(K2 P)and NJ tree analysis were analyzed using MEGA 5.0. Secondary structure analysis and barcode site identification were last done. The sequence lengths of ITS2 were from 231 to 279 bp, and the G+C content were from 69.26%to 72.93%.The maximum K2 P intra-genetic distance in the species was smaller than the minimum inter-genetic distance between species. The NJ tree showed a good mononality, but the differences of the secondary structure of the ITS2 sequences were not very obvious, and the barcode site can correctly identify the species. The ITS2 barcode can be used to identify the Citrus aurantium L and its adulterants.
We identify Citrus aurantium L. and its adulterants using ITS2 sequence to provide a secure and accurate identification way for the pharmaceutical and clinical use of Citrus aurantium L.,and also to provide a reliable identification method of citrus seeds or seedlings for agriculture.After extracting the DNA of Citrus aurantium L and its adulterants, the ITS2 sequences were amplified by PCR and measured bidirectionally;and the other sequences were downloaded from GenBank. DNAstar 7.10 is used to measure the length of each ITS2 sequence and calculate the G+C content of each ITS2 sequence.The genetic distance(K2 P)and NJ tree analysis were analyzed using MEGA 5.0. Secondary structure analysis and barcode site identification were last done. The sequence lengths of ITS2 were from 231 to 279 bp, and the G+C content were from 69.26%to 72.93%.The maximum K2 P intra-genetic distance in the species was smaller than the minimum inter-genetic distance between species. The NJ tree showed a good mononality, but the differences of the secondary structure of the ITS2 sequences were not very obvious, and the barcode site can correctly identify the species. The ITS2 barcode can be used to identify the Citrus aurantium L and its adulterants.
引文
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[10]马敏敏,何芳,杨志刚,等.DNA提取4种中药材方法的筛选[J].中成药,2016,38(8):1776-1781.
[2]蔡逸平,曹岚,范崔生.枳壳、枳实类药材的品种考证和资源应用的调查研究[J].江西中医学院学报,1998,10(4):184-186.
[3]白关亚,孙迎娜,王玉娥,等.“一体二用”中药的比较研究概况及质控思考[J].中药材,2017,40(2):504-508.
[4]辛天怡,李西文,姚辉,等.中药材二维DNA条形码流通监管体系研究[J].中国科学:生命科学,2015,45(7):695-702.
[5]侯典云,杨萌萌,周爽,等.基于ITS2序列的獐牙菜属六种药材的分子鉴定[J].天然产物研究与开发,2018,30(7):1126-1131.
[6]杨美青,杜燕.基于ITS2序列及其二级结构对防己及其混伪品的鉴定[J].中国实验方剂学杂志,2018,24(10):43-48.
[7]辛天怡,雷美艳,宋经元.中药材DNA条形码鉴定研究进展[J].中国现代中药,2015,17(2):170-176,184.
[8]黄林芳,郑司浩,武拉斌,等.基于化学成分及分子特征中药材肉苁蓉生态型研究[J].中国科学:生命科学,2014(3):319-329.
[9]庞文静,郑玉莹,彭维,等.正品枳壳指纹图谱的构建及其与混伪品的聚类分析[J].中南药学,2017(10):1345-1350.
[10]马敏敏,何芳,杨志刚,等.DNA提取4种中药材方法的筛选[J].中成药,2016,38(8):1776-1781.