中药地枫皮及其伪品假地枫皮的DNA条形码鉴定
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摘要
目的:评价和比较几个常用DNA条形码候选序列对地枫皮及伪品假地枫皮的鉴定作用。方法:采集不同产地的地枫皮及假地枫皮样品,进行总DNA提取,选取核基因内转录间隔区2(ITS2)序列、叶绿体rbcL,mat K基因序列进行聚合酶链式反应(PCR)扩增,纯化产物测序,利用Condon Code Aligner V3. 7. 1校对拼接。结果:地枫皮及假地枫皮rbcL序列进行多次PCR扩增及测序结果均不理想,初步推测地枫皮及假地枫皮rbcL序列太长,进化较慢,不适合作为地枫皮及假地枫皮种间鉴别;地枫皮及假地枫皮的matK基因序列测序成功率分别为0和76. 8%,可能是不同类群的植物matK序列的引物标准不一;对地枫皮及假地枫皮的ITS2序列PCR扩增及测序结果最为理想,测序的成功率分别为89. 3%及91. 2%,对测序结果序列进行分析,地枫皮的ITS2序列总长度均为268个碱基,存在2个变异位点;假地枫皮的ITS2序列总长度均为430个碱基,存在4个或3个变异位点。实验结果说明地枫皮及假地枫皮ITS2序列较短,有明显的变异性,便于扩增,表明ITS2序列用于地枫皮及假地枫皮的分子鉴定优于rbcL和matK序列。结论:ITS2序列作为标准的DNA条形码能够有效地鉴定地枫皮及其伪品假地枫皮。
Objective: To evaluate and compare the identification of several DNA barcoding candidate sequences on Illicium difengpi and its fake I. jiadifengpi. Method: Samples from different origins of I. difengpi and I. jiadifengpi,were collect extraction of total DNA,nuclear gene ITS2 sequence,chloroplast rbc L,mat K gene sequence were selected for PCR amplification,product purification and sequencing,and CondonCode Aligner V3. 7. 1 was used to proofread stitching. Result: PCR amplification and sequencing of rbc L sequences of I. difengpi and I. jiadifengpi were not satisfactory. It is assumed that their rbc L sequences were too long with slow evolution,which was unsuitable for interbreeding. The success rate of mat K sequencing of I. difengpi and I. jiadifengpi was 0 and 76. 8%,which may be because primer standards were different for mat K sequences of different groups. The results of PCR amplification and sequencing of ITS2 on I. difengpi and I. jiadifengpi were successful,with the success rate of sequencing was 89. 3% and 91. 2%. Analysis sequencing results,the total length of ITS2 sequences was 268 bases,and there were 2 variation sites of I. difengpi. The total length of ITS2 sequences was 430 bases,and there were 4 or 3 variation sites of I. jiadifengpi. It shows that ITS2 sequences of I. difengpi and I. jiadifengpi were short and has obvious variability and can be amplify,that ITS2 sequence was better than rbc L and mat K sequence in molecular identification of I. difengpi and I. jiadifengpi. Conclusion:DNA barcoding based on ITS2 sequence was a powerful and efficient tool for identification of I. difengpi and its fake I. jiadifengpi.
Objective: To evaluate and compare the identification of several DNA barcoding candidate sequences on Illicium difengpi and its fake I. jiadifengpi. Method: Samples from different origins of I. difengpi and I. jiadifengpi,were collect extraction of total DNA,nuclear gene ITS2 sequence,chloroplast rbc L,mat K gene sequence were selected for PCR amplification,product purification and sequencing,and CondonCode Aligner V3. 7. 1 was used to proofread stitching. Result: PCR amplification and sequencing of rbc L sequences of I. difengpi and I. jiadifengpi were not satisfactory. It is assumed that their rbc L sequences were too long with slow evolution,which was unsuitable for interbreeding. The success rate of mat K sequencing of I. difengpi and I. jiadifengpi was 0 and 76. 8%,which may be because primer standards were different for mat K sequences of different groups. The results of PCR amplification and sequencing of ITS2 on I. difengpi and I. jiadifengpi were successful,with the success rate of sequencing was 89. 3% and 91. 2%. Analysis sequencing results,the total length of ITS2 sequences was 268 bases,and there were 2 variation sites of I. difengpi. The total length of ITS2 sequences was 430 bases,and there were 4 or 3 variation sites of I. jiadifengpi. It shows that ITS2 sequences of I. difengpi and I. jiadifengpi were short and has obvious variability and can be amplify,that ITS2 sequence was better than rbc L and mat K sequence in molecular identification of I. difengpi and I. jiadifengpi. Conclusion:DNA barcoding based on ITS2 sequence was a powerful and efficient tool for identification of I. difengpi and its fake I. jiadifengpi.
引文
[1]王洪禄,何永志,史利利,等.地枫皮研究进展[J].实用中医药杂志,2011,27(8):577-578.
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[4]唐辉,史艳财,孔德鑫,等.岩溶特有植物地枫皮的种质资源调查及地理分布[J].广东农业科学,2011,38(12):113-117.
[5]梁惠凌,王满莲,孔德鑫,等.地枫皮及其伪品假地枫皮的研究进展[J].广西科学院学报,2013,29(3):164-168.
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[11]梅学伟,刘艳新,门婧,等.地枫皮与伪品地枫皮的UV光谱鉴定[J].中医药信息,1995(3):40.
[12]Schmidt G J,Schilling E E.Phylogeny and biogeography of Eupatorium(As terceae:eupat orieae)based on nuclear ITS sequence date[J].Amer J Bot,2000,87:716-726.
[13]CHEN S L,YAO H,HAN J P,et al.Validation of the ITS2 region as a novel DNA bar code for identifying medicinal plant species[J].PLo S One,2010,5(1):e8613.
[14]任瑶瑶,江南屏,张良,等.峨嵋山区姜科药用植物ITS2序列分析[J].中国实验方剂学杂志,2019,25(1):217-223.
[15]LI X K,WANG B,HAN R C,et al.Identification of medicinal plant Schisandra chinensis using a potential DNA barcode ITS2[J].Acta Soc Bot Pol,2013,82(4):283-288.
[16]张忠廉,宋美芳,李海涛,等.千斤拔属药用植物DNA条形码鉴定研究[J].中草药,2015,46(1):118-122.
[17]冯杉杉,郑司浩,李亚康,等.中药材威灵仙及其伪品DNA条形码鉴别研究[J].药学学报,2014,49(2):260-266.
[18]夏至,冯翠元,高致明,等.黄芩及其同属近缘种的DNA条形码鉴定研究[J].中草药,2014,45(1):107-112.
[19]方强强,王燕,彭春,等.中药DNA条形码分子鉴定技术的应用与展望[J].中国实验方剂学杂志,2018,24(22):197-205.
[20]王海燕,刘石生,文明富,等.基于mat K序列的几种重要大戟科植物系统发育初步研究[J].热带作物学报,2011,32(4):715-719.
[21]黄琼林,马新业,詹若挺,等.基于rbc L条形码的鸡血藤真伪鉴别[J].江苏农业科学,2016,44(6):57-60.
[22]夏至,王璐静,李贺敏,等.基于psb A-trn H基因序列对中药材茜草的分子鉴定[J].时珍国医国药,2016,27(2):374-376.
[23]杨慧洁,杨世海,张淑丽,等.药用植物DNA条形码研究进展[J].中草药,2014,45(18):2581-2587.
[24]刘义梅.杜鹃花属药用植物DNA条形码的鉴定研究[D].武汉:湖北中医药大学,2011.
[25]李保进.叶籽银杏mat K和ITS序列分析及系统发育研究[D].泰安:山东农业大学,2008.
[26]Kress W J,Wurdack K J,Zimmer E A,et al.Use of DNAbarcodes to identify flowering plant[J].PNAS,2005(102):8369-8374.
[27]谢伟玲,邹蓉,杨雪,等.珍稀濒危植物DNA条形码研究进展[J].北方园艺,2015(4):178-183.
[28]费希同,巨苗苗,林源,等.ITS2序列在植物DNA条形码鉴定中的应用(综述)[J].亚热带植物科学,2014,43(4):339-342.
[2]黄宝优,吴庆华,柯芳.中药地枫皮的研究概况[J].大众科技,2008(1):126,117.
[3]刘元,韦焕英,姚树汉,等.地枫皮类药理作用研究[J].湖南中医药导报,1997(Z1):72-75.
[4]唐辉,史艳财,孔德鑫,等.岩溶特有植物地枫皮的种质资源调查及地理分布[J].广东农业科学,2011,38(12):113-117.
[5]梁惠凌,王满莲,孔德鑫,等.地枫皮及其伪品假地枫皮的研究进展[J].广西科学院学报,2013,29(3):164-168.
[6]陈瑞生,陈相银,张露露.地枫皮真伪鉴别[J].首都医药,2013,20(17):41.
[7]孔德鑫,李雁群,梁惠凌,等.地枫皮营养器官解剖结构特征及其叶片结构的生态适应性[J].基因组学与应用生物学,2012,31(3):282-288.
[8]孔德鑫,唐辉,韦霄,等.中药地枫皮及其伪品的FTIR分析与鉴定[J].光谱实验室,2010,27(6):2417-2421.
[9]高秀清,刘敏,李永升.地枫皮与其伪品的鉴别[J].时珍国医国药,2002,13(11):665-666.
[10]赖茂祥,饶伟源,杨敏,等.地枫皮及其混伪品的生药鉴别[J].中药材,1997(12):601-604.
[11]梅学伟,刘艳新,门婧,等.地枫皮与伪品地枫皮的UV光谱鉴定[J].中医药信息,1995(3):40.
[12]Schmidt G J,Schilling E E.Phylogeny and biogeography of Eupatorium(As terceae:eupat orieae)based on nuclear ITS sequence date[J].Amer J Bot,2000,87:716-726.
[13]CHEN S L,YAO H,HAN J P,et al.Validation of the ITS2 region as a novel DNA bar code for identifying medicinal plant species[J].PLo S One,2010,5(1):e8613.
[14]任瑶瑶,江南屏,张良,等.峨嵋山区姜科药用植物ITS2序列分析[J].中国实验方剂学杂志,2019,25(1):217-223.
[15]LI X K,WANG B,HAN R C,et al.Identification of medicinal plant Schisandra chinensis using a potential DNA barcode ITS2[J].Acta Soc Bot Pol,2013,82(4):283-288.
[16]张忠廉,宋美芳,李海涛,等.千斤拔属药用植物DNA条形码鉴定研究[J].中草药,2015,46(1):118-122.
[17]冯杉杉,郑司浩,李亚康,等.中药材威灵仙及其伪品DNA条形码鉴别研究[J].药学学报,2014,49(2):260-266.
[18]夏至,冯翠元,高致明,等.黄芩及其同属近缘种的DNA条形码鉴定研究[J].中草药,2014,45(1):107-112.
[19]方强强,王燕,彭春,等.中药DNA条形码分子鉴定技术的应用与展望[J].中国实验方剂学杂志,2018,24(22):197-205.
[20]王海燕,刘石生,文明富,等.基于mat K序列的几种重要大戟科植物系统发育初步研究[J].热带作物学报,2011,32(4):715-719.
[21]黄琼林,马新业,詹若挺,等.基于rbc L条形码的鸡血藤真伪鉴别[J].江苏农业科学,2016,44(6):57-60.
[22]夏至,王璐静,李贺敏,等.基于psb A-trn H基因序列对中药材茜草的分子鉴定[J].时珍国医国药,2016,27(2):374-376.
[23]杨慧洁,杨世海,张淑丽,等.药用植物DNA条形码研究进展[J].中草药,2014,45(18):2581-2587.
[24]刘义梅.杜鹃花属药用植物DNA条形码的鉴定研究[D].武汉:湖北中医药大学,2011.
[25]李保进.叶籽银杏mat K和ITS序列分析及系统发育研究[D].泰安:山东农业大学,2008.
[26]Kress W J,Wurdack K J,Zimmer E A,et al.Use of DNAbarcodes to identify flowering plant[J].PNAS,2005(102):8369-8374.
[27]谢伟玲,邹蓉,杨雪,等.珍稀濒危植物DNA条形码研究进展[J].北方园艺,2015(4):178-183.
[28]费希同,巨苗苗,林源,等.ITS2序列在植物DNA条形码鉴定中的应用(综述)[J].亚热带植物科学,2014,43(4):339-342.