冬虫夏草人工繁育品与野生冬虫夏草DNA条形码比较研究
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摘要
目的:从分子生物学的角度比较分析冬虫夏草人工繁育品和野生冬虫夏草的核糖体DNA内部转录间隔区(ITS)和细胞色素C氧化酶亚基1(COⅠ)条形码序列,并验证冬虫夏草人工繁育品与野生冬虫夏草虫和菌的来源是否符合《中华人民共和国药典》规定。方法:通过聚合酶链式反应(PCR)对冬虫夏草人工繁育品和野生冬虫夏草的ITS和COⅠ序列进行扩增,利用邻接法比较冬虫夏草人工繁育品与野生冬虫夏草的亲缘关系;通过与GenBank数据库进行Blast比对,确定冬虫夏草虫与菌的科属。结果:冬虫夏草人工繁育品与野生冬虫夏草ITS和COⅠ序列的电泳结果均显示为明亮单一条带,且条带大小介于500 bp和750 bp之间;COⅠ序列的系统树结果中冬虫夏草人工繁育品和野生冬虫夏草除野生冬虫夏草-18、野生冬虫夏草-20这2个样品均各自聚为一支,且支持率为99%,呈现良好的单系性,ITS序列的系统树结果中冬虫夏草人工繁育品和野生冬虫夏草混聚在一起,没有明确的分支;冬虫夏草人工繁育品的COⅠ序列经GenBank数据库比对结果均为小金蝠蛾(Hepialus xiaojinensis),为蝙蝠蛾科Hepialidae sp.昆虫,野生冬虫夏草的COⅠ序列经GenBank数据库比对结果均为蝙蝠蛾科Hepialidae sp.昆虫,其中野生冬虫夏草-8为人支蝠蛾(Thitarodes renzhiensis),野生冬虫夏草-12为贡嘎蝠蛾(Thitarodes gonggaensis),冬虫夏草人工繁育品及野生冬虫夏草的ITS序列与GenBank数据库比对后,相似度达99%以上,比对结果显示20批冬虫夏草人工繁育品和26批野生冬虫夏草的虫菌均为冬虫夏草菌(Ophiocordyceps sinensis)。Ophiocordyceps sinensis和Cordyceps sinensis为同物异名。结论:20批冬虫夏草人工繁育品的虫体及虫菌来源与26批野生冬虫夏草的虫体及虫菌来源均符合《中华人民共和国药典》2015年版中的描述:冬虫夏草为麦角菌科真菌冬虫夏草菌Cordyceps sinensis(Berk.)Sacc.寄生在蝙蝠蛾科昆虫幼虫上的子座和幼虫尸体的干燥复合体。
Objective:To compare the sequences of internal transcribed spacer(ITS)and cytochrome C oxidase subunit 1(CO Ⅰ)of cultivated and natural Cordyceps sinensis by the method of molecular biology,and to conform whether the two kinds of samples meet the requirement of ChP.Methods:ITS and CO Ⅰ sequences of cultivated and natural Cordyceps sinensis were amplified with polymerase chain reaction(PCR).The genetic relationship between cultivated and natural Cordyceps sinensis was evaluated by the method of neighbor-joining.The sequences were blasted with GenBank database to determine the family and genus of the worm and fungi of the samples.Results:The PCR products amplified from the ITS region and CO Ⅰ region of cultivated and natural Cordyceps sinensis displayed as a single bright stripe and the size was between 500 bp and 750 bp in the result of gel imaging.The phylogenetic tree results of CO Ⅰ sequences of cultivated and natural Cordyceps sinensis(except samples No.18 and No.20)revealed better monophyly,and the approval rate was 99%.Phylogenetic tree results of ITS sequences of cultivated and natural Cordyceps sinensis were mixed together and there was no clear branch.Blasting the CO Ⅰ sequences of cultivated Cordyceps sinensis in the GenBank database,the results revealed that all the samples were identified as Hepialus xiaojinensis,the insects of Hepialidae sp.On the other hand,all the samples of natural Cordyceps sinensis were identified as the insects of Hepialidae sp.Among them,sample No.8 was identified as Thitarodes renzhiensis and No.12 was identified as Thitarodes gonggaensis.In all,20 batches of cultivated samples and 26 batches of natural samples were all identified as Ophiocordyceps sinensis,and the similarities were above 99%,which indicated that Ophiocordyceps sinensis and Cordyceps sinensis were synonyms.Conclusion:All the samples conformed to the description of Pharmacopoeia of the People's Republic of China(2015 edition):Cordyceps sinensis is the dry complex of Cordyceps sinensis(Berk.)Sacc.from Clavicipitaceae parasitized on the insect larvae from Hepialidae.
Objective:To compare the sequences of internal transcribed spacer(ITS)and cytochrome C oxidase subunit 1(CO Ⅰ)of cultivated and natural Cordyceps sinensis by the method of molecular biology,and to conform whether the two kinds of samples meet the requirement of ChP.Methods:ITS and CO Ⅰ sequences of cultivated and natural Cordyceps sinensis were amplified with polymerase chain reaction(PCR).The genetic relationship between cultivated and natural Cordyceps sinensis was evaluated by the method of neighbor-joining.The sequences were blasted with GenBank database to determine the family and genus of the worm and fungi of the samples.Results:The PCR products amplified from the ITS region and CO Ⅰ region of cultivated and natural Cordyceps sinensis displayed as a single bright stripe and the size was between 500 bp and 750 bp in the result of gel imaging.The phylogenetic tree results of CO Ⅰ sequences of cultivated and natural Cordyceps sinensis(except samples No.18 and No.20)revealed better monophyly,and the approval rate was 99%.Phylogenetic tree results of ITS sequences of cultivated and natural Cordyceps sinensis were mixed together and there was no clear branch.Blasting the CO Ⅰ sequences of cultivated Cordyceps sinensis in the GenBank database,the results revealed that all the samples were identified as Hepialus xiaojinensis,the insects of Hepialidae sp.On the other hand,all the samples of natural Cordyceps sinensis were identified as the insects of Hepialidae sp.Among them,sample No.8 was identified as Thitarodes renzhiensis and No.12 was identified as Thitarodes gonggaensis.In all,20 batches of cultivated samples and 26 batches of natural samples were all identified as Ophiocordyceps sinensis,and the similarities were above 99%,which indicated that Ophiocordyceps sinensis and Cordyceps sinensis were synonyms.Conclusion:All the samples conformed to the description of Pharmacopoeia of the People's Republic of China(2015 edition):Cordyceps sinensis is the dry complex of Cordyceps sinensis(Berk.)Sacc.from Clavicipitaceae parasitized on the insect larvae from Hepialidae.
引文
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[3]郭慧,王谦博,贾力维,等.中药材DNA条形码技术研究进展[J].中国药师,2016,19(3):566GUO H,WANG QB,JIA LW,et al.Study progress in DNA barcode of traditional Chinese medicine[J].China Pharm,2016,19(3):566
[4]HEBERT PD,CYWINSKA A,BALL SL.Biological identifications through DNA barcodes[J].Proc R Soc Biol Sci Ser B,2003,270(1512):313
[5]陈士林,庞晓慧,姚辉,等.中药DNA条形码鉴定体系及研究方向[J].世界科学技术-中医药现代化,2011,13(5):747CHEN SL,PANG XH,YAO H,et al.Identification system and perspective for DNA barcoding traditional Chinese materia medica[J].World Sci Technol-Mod Tradit Chin Med Mater Med,2011,13(5):747
[6]陈士林,姚辉,宋经元,等.基于DNA barcoding(条形码)技术的中药材鉴定[J].世界科学技术-中医药现代化,2007,9(3):7CHEN SL,YAO H,SONG JY,et al.Use of DNA barcoding to identify Chinese medicinal materials[J].World Sci Technol Mod Tradit Chin Med Mater Med,2007,9(3):7
[7]LI DZ,LIU JQ,CHEN ZD,et al.Plant DNA barcoding in China[J].J Syst Evol,2011,49(3):165
[8]LI M,CAO H,BUT PPH,et al.Identification of herbal medicinal materials using DNA barcodes[J].J Syst Evol,2011,49(3):271
[9]梁宗琦.中国真菌志[M].北京:科学出版社,2007:125LIANG ZQ.Fungi of China[M].Beijing:Science Press,2007:125
[10]中华人民共和国药典2015年版.四部[S].2015:419.ChP 2015.VolⅣ[S].2015:419
[11]董彩虹,李文佳,李增智,等.我国虫草产业发展现状、问题及展望—虫草产业发展金湖宣言[J].菌物学报,2016,35(1):1DONG CH,LI WJ,LI ZZ,et al.Cordyceps industry in China:current status,challenges and perspectives—Jinhu declaration for Cordyceps industry development[J].Mycosystema,2016,35(1):1
[12]SUNG GH,HYEEL-JONES NL,SUNG JM,et al.Phylogenetic classification of Cordyceps and the clavicipitaceous fungi[J].Stud Mycol,2008,57:5
[13]姚艺桑,朱佳石.中药冬虫夏草和所含多种冬虫夏草菌拉丁名混用的历史和现状[J].中国中药杂志,2016,41(7):1361YAOYS,ZHUJS.IndiscriminateuseofLatinnamefor natural Cordyceps sinensis insect-fungi complex and multiple Ophiocordyceps sinensis fungi[J].China J Chin Mater Med,2016,41(7):1361
[14]张姝,张永杰,SHRESTHA Bhushan,等.冬虫夏草菌和蛹虫草菌的研究现状、问题及展望[J].菌物学报,2013,32(4):577ZHANG S,ZHANG YJ,SHRESTHA Bhushan,et al.Ophiocordyceps sinensis and Cordyceps militaris:research advances,issues and perspectives[J].Mycosystema,2013,32(4):577