中国现代中药
中國現代中藥
중국현대중약
MODERN CHINESE MEDICINE
2015年
7期
668-672,682
,共6页
张乐%朱虹%韦坤华%张春红%李辉
張樂%硃虹%韋坤華%張春紅%李輝
장악%주홍%위곤화%장춘홍%리휘
沙苑子%ITS 序列%分子鉴别
沙苑子%ITS 序列%分子鑒彆
사원자%ITS 서렬%분자감별
Astragalus complanatus%ITS sequences%molecular identification
目的：研究沙苑子与其混伪品之间的 DNA 分子鉴别方法。方法：采集不同产地的沙苑子药材10份，基原植物2份，达乌里黄芪3份，蒙古黄芪4份，直立黄芪3份，混伪品猪屎豆和凹叶野百合各1份，所有样品进行总 DNA 的提取，PCR 扩增，并对扩增产物进行测序得到相应的序列，用 MEGA 计算其种间的 K-2-P 距离，最后利用 ITS 序列构建其系统发育树。结果：测得了24份样品的 ITS 序列全长，分别为沙苑子644～688 bp，蒙古黄芪为646～673 bp；直立黄芪685～687 bp；达乌里黄芪为676～688 bp；猪屎豆为785 bp；凹叶野百合为837 bp。通过以ITS 序列重建系统进化树进行的聚类分析可以将沙苑子与其混伪品有效的区分开。用 MEGA 软件基于 ITS 序列构建的沙苑子与其混伪品间的遗传距离分析得到了沙苑子与达乌里黄芪的种间 K-2-P 距离0.0802；蒙古黄芪为0.0812，与直立黄芪之间的遗传距离为0.0824，凹叶野百合和猪屎豆与沙苑子的遗传距离稍大，达到0.2362和0.2299。用 MEGA 软件测定沙苑子种内遗传距离为0.0010。此外，对通过测定种间的遗传距离发现，蒙古黄芪与直立黄芪种间的遗传距离最小为0.0009，与猪屎豆种间的遗传距离最大为0.2362。结论：ITS 序列能够成功鉴定沙苑子及其混伪品，可以作为沙苑子与其混伪品的分子鉴别方法。
目的：研究沙苑子與其混偽品之間的 DNA 分子鑒彆方法。方法：採集不同產地的沙苑子藥材10份，基原植物2份，達烏裏黃芪3份，矇古黃芪4份，直立黃芪3份，混偽品豬屎豆和凹葉野百閤各1份，所有樣品進行總 DNA 的提取，PCR 擴增，併對擴增產物進行測序得到相應的序列，用 MEGA 計算其種間的 K-2-P 距離，最後利用 ITS 序列構建其繫統髮育樹。結果：測得瞭24份樣品的 ITS 序列全長，分彆為沙苑子644～688 bp，矇古黃芪為646～673 bp；直立黃芪685～687 bp；達烏裏黃芪為676～688 bp；豬屎豆為785 bp；凹葉野百閤為837 bp。通過以ITS 序列重建繫統進化樹進行的聚類分析可以將沙苑子與其混偽品有效的區分開。用 MEGA 軟件基于 ITS 序列構建的沙苑子與其混偽品間的遺傳距離分析得到瞭沙苑子與達烏裏黃芪的種間 K-2-P 距離0.0802；矇古黃芪為0.0812，與直立黃芪之間的遺傳距離為0.0824，凹葉野百閤和豬屎豆與沙苑子的遺傳距離稍大，達到0.2362和0.2299。用 MEGA 軟件測定沙苑子種內遺傳距離為0.0010。此外，對通過測定種間的遺傳距離髮現，矇古黃芪與直立黃芪種間的遺傳距離最小為0.0009，與豬屎豆種間的遺傳距離最大為0.2362。結論：ITS 序列能夠成功鑒定沙苑子及其混偽品，可以作為沙苑子與其混偽品的分子鑒彆方法。
목적：연구사원자여기혼위품지간적 DNA 분자감별방법。방법：채집불동산지적사원자약재10빈，기원식물2빈，체오리황기3빈，몽고황기4빈，직립황기3빈，혼위품저시두화요협야백합각1빈，소유양품진행총 DNA 적제취，PCR 확증，병대확증산물진행측서득도상응적서렬，용 MEGA 계산기충간적 K-2-P 거리，최후이용 ITS 서렬구건기계통발육수。결과：측득료24빈양품적 ITS 서렬전장，분별위사원자644～688 bp，몽고황기위646～673 bp；직립황기685～687 bp；체오리황기위676～688 bp；저시두위785 bp；요협야백합위837 bp。통과이ITS 서렬중건계통진화수진행적취류분석가이장사원자여기혼위품유효적구분개。용 MEGA 연건기우 ITS 서렬구건적사원자여기혼위품간적유전거리분석득도료사원자여체오리황기적충간 K-2-P 거리0.0802；몽고황기위0.0812，여직립황기지간적유전거리위0.0824，요협야백합화저시두여사원자적유전거리초대，체도0.2362화0.2299。용 MEGA 연건측정사원자충내유전거리위0.0010。차외，대통과측정충간적유전거리발현，몽고황기여직립황기충간적유전거리최소위0.0009，여저시두충간적유전거리최대위0.2362。결론：ITS 서렬능구성공감정사원자급기혼위품，가이작위사원자여기혼위품적분자감별방법。
Objective:To explore a new method for identification of Astragalus complanatus from its adulterants by using ITS sequences.Methods:10 samples of the different A.complanatus and 2 samples of original plant,3 samples of A. dahuricus,4 samples of the A.membranaceus var.mongholicus,3 samples of A.adsurgens,1 sample of the Crotalaria pallida and C.retusa were collected.ITS sequence was amplified by PCR and sequenced unidirectionally.The interspecific genetic distances among 6 species of A.complanatus and its adulterants were calculated,and NJ tree and UPGMA tree were constructed by MEGA 5.Among them,A.membranaceus var.mongholicus and A.dahuricus interspecific K-2-P minimum distance was 0.080 2,and then followed by A.membranaceus var.mongholicus.A.adsurgens interspecific was 0.082 4,C.pallida and C.retusa interspecific was 0.236 2 and 0.229 9,respectively.In addition,A.complanatus intraspecific genetic distance was 0.003 0 computed by MEGA 5.0.Accoding to determination of A.complanatus adulterants,it showed that A. membranaceus var.mongholicus and A.dahuricus interspecific K-2-P minimum distance was 0.000 9 and C.pallida was 0.236 2.Results:ITS sequences were obtained from 24 samples respectively, there were A. complanatus 644-688 bp, A.membranaceus var.mongholicus 664-673 bp,A.adsurgens 685-687 bp,A.dahuricus 676-688 bp,C.pallida 785 bp and C.retusa 837 bp.Phylogeny tree reconstruction using NJ and UPGMA analysis based on ITS nucleotide sequences can effectively distinguish A.complanatus from adulterants.Conclusion:ITS sequences can be used to identify A.complanatus from its adulterants successfully and is an efficient molecular marker for authentication of A.complanatus and its adulterants.
