枫斗类石斛rDNA ITS区的全序列数据库及其序列分析鉴别

目的建立枫斗类石斛的rDNA ITS区碱基全序列数据库,利用该数据库对枫斗类石斛待检种进行准确鉴别。方法对枫斗类石斛的rDNA ITS区进行了PCR扩增、测序,运用CLUSTRAL,MEGA等软件以及枫斗类石斛rDNA ITS区全序列数据库对待检种rDNA ITS区进行序列分析鉴别。结果建立了21种枫斗类石斛的rDNA ITS区全序列数据库, 枫斗类石斛在该区的种间差异显著而稳定,转换和颠换总数为11～122,变异位点数为341,信息位点数为195。与外类群植物云南石仙桃间的差异较大,转换和颠换总数为131～161。枫斗类石斛居群间的差异较小,转换和颠换总数为0～6。结论利用枫斗类石斛的全序列数据库及遗传分析软件,通过对待检种rDNA ITS区进行序列测定,可以成功鉴别属于数据库中枫斗类石斛的待检种。     

球花石斛的位点特异性PCR鉴别研究

为建立球花石斛的DNA分子标记鉴别方法，本文根据测定及GenBank上登录的109种共计164个石斛样本的rDNA ITS序列，设计了特异性鉴别引物QH-JB1 和QH-JB2，并对球花石斛进行了位点特异性PCR鉴别研究。结果表明，当复性条件为63.5 ℃，1 min时，只有球花石斛的模板DNA能被扩增出约300 bp的阳性扩增带，而其他种石斛均为阴性。证明用本法鉴别球花石斛简便、省时，也适用于干燥球花石斛药材的鉴别，具有广泛的应用前景。     

重楼属药用植物DNA条形码鉴定研究

为评价DNA条形码候选序列对重楼属药用植物的鉴定作用, 探讨重楼属药用植物鉴定新方法, 本研究对重楼属11个物种17份样品的psbA-trnH、rpoB、rpoC1、rbcL、matK和核ITS2序列进行PCR扩增和测序, 比较各序列扩增和测序效率、种内和种间变异, 进行barcoding gap分析, 采用BLAST1和Nearest Distance方法评价不同序列的鉴定能力。结果显示, ITS2序列在所研究的重楼属药用植物中的扩增和测序效率均为100%, 其种内种间变异、barcoding gap与其他DNA条形码候选序列相比具有明显的优势, ITS2序列在重楼属中的鉴定成功率达到100%, 而生物条形码协会 (CBOL) 植物工作组推荐的matK和rbcL序列的鉴定成功率分别为52.9% 和5.9%, 二者联合鉴定能力没有提高, 对于ITS2序列扩大至29个物种67份样品依然具有100%的鉴定成功率。实验结果表明, ITS2序列能够准确鉴定重楼属药用植物, 可以作为潜在的药用植物通用条形码序列。     

基于DNA条形码序列分析的5种菖蒲属药用植物鉴定

目的构建菖蒲属药用植物的鉴别方法。方法采用DNA条形码序列技术,对5种菖蒲属药用植物的4条候选DNA条形码序列(ITS2、rbc L、mat K、psb A-trn H)进行PCR扩增和测序,比较各序列的扩增和测序效率,对比分析种内、种间的遗传变异、分析barcoding Gap并构建聚类分析树。结果 4条候选DNA条形码序列PCR扩增和测序效率均为100%,ITS2序列对菖蒲属药用植物的鉴定成功率最高。结论基于ITS2序列的DNA条形码技术可以鉴定菖蒲属药用植物。     

基于ITS序列分析技术鉴定川牛膝与常见伪品麻牛膝

目的:比较川牛膝与其常见伪品麻牛膝之间的ITS序列差异及规律。为川牛膝与麻牛膝的DNA条形码鉴别提供适合的分子标记。方法:收集川牛膝及麻牛膝成品药材并提取纯化其基因组DNA,经PCR扩增得到ITS序列(包括ITS1、5.8S nrDNA、ITS2)并进行T-A克隆后测序,分析两者序列差异。结果:PCR扩增获得两者ITS序列,经多序列对比分析得出川牛膝与伪品麻牛膝的ITS序列存在明显差异。结论:ITS序列分析可以用作鉴定川牛膝与麻牛膝药材。     

紫苏属药用植物的rDNA ITS区SNP分子标记与位点特异性PCR鉴别

目的分析单种属——紫苏属各变种间rDNA ITS区的序列以及存在的单核苷酸多态性(SNP)现象，设计出位点特异性PCR引物，用于紫苏属各变种间的分子标记鉴别。方法对紫苏属各变种多个体的rDNA ITS区全序列进行了准确测定，运用Clustral X 1.8，MEGA 3.0进行排序并进行SNP分析，从而设计出鉴别各变种的等位基因位点特异性PCR鉴别引物。结果紫苏属各变种(紫苏、白苏、鸡冠苏和耳齿紫苏等)的rDNA ITS区全序列共有615～618 bp的长度，ITS1为233～235 bp，5.8S为179 bp，ITS2为203～204 bp，GC含量为61.5%～61.9%。从rDNA ITS区碱基变异的整体情况来看，紫苏属各变种间不仅在非编码的转录间隔区ITS1和ITS2内存在非编码区单核苷酸多态性(ncSNP)，而且在保守的5.8S编码区内也存在3个位点的单核苷酸多态性，即编码区SNP(cSNP)，所有的SNP均只具2等位多态性。5.8S区cSNP的出现与产生该变异的变种出现的显著形态差异关联。本文还利用这些SNP位点设计出了鉴别紫苏属各变种的位点特异性PCR引物，无需测序即可对紫苏属的原植物及“苏子”、“苏叶”等药材进行有效准确的分子鉴别。结论紫苏属药用植物rDNA ITS区存在的SNP可用作紫苏属各变种鉴别的分子标记。     

齿瓣石斛的位点特异性PCR鉴别

目的设计出齿瓣石斛的位点特异性鉴别引物,仅通过PCR就能完成对齿瓣石斛真伪进行准确鉴别。方法根据齿瓣石斛及其他枫斗类、黄草类石斛的rDNA ITS序列数据库,设计了齿瓣石斛的位点特异性PCR鉴别引物JB-Chiban-01S和JB-Chiban-01X。然后,对38种石斛属植物模板DNA进行了PCR扩增,阳性者即为齿瓣石斛正品。结果当退火温度设定为58℃时,只有齿瓣石斛的模板DNA能被扩增出来,而其他的37种石斛属植物均为阴性。该鉴别反应重复性好,已在鉴别齿瓣石斛时发挥重要作用。结论运用位点特异性鉴别引物能成功地对齿瓣石斛进行PCR鉴别,与DNA测序鉴别方法相比,位点特异性PCR具有高效、准确、简便、省时等优点。     

曲茎石斛及其近似种鉴别的形态和DNA分子证据

目的　探讨曲茎石斛(南阳居群)与同属近似种:细茎石斛(南阳居群)、霍山石斛(霍山居群)、铁皮石斛(天台居群)药材鉴别的形态及DNA分子证据。方法　对曲茎石斛(南阳居群)及其近似种的药材进行了外部形态和rDNA ITS序列比较。结果　曲茎石斛及其近似种药材的外部形态虽无明显区别,但在rDNA ITS序列上却存在着显著而稳定的差异。曲茎石斛(南阳居群)与铁皮石斛(天台居群)的rDNA ITS序列的差异最小,绝对遗传距离为7;但与细茎石斛(南阳居群)及霍山石斛(霍山居群)rDNA ITS区的差异较大,绝对遗传距离分别为40和42。在rDNA ITS区域中,作者共挑选了7个碱基位点用作鉴别曲茎石斛(南阳居群)及其近似种的DNA证据。结论　根据药材的形态特征及rDNA ITS区碱基序列差异,可准确鉴别曲茎石斛及其近似种药材。     

基于ITS2序列的柴胡属植物种子的鉴定及遗传多样性分析

目的:本研究旨在对国家中药种质资源库收集的41份柴胡属植物种子进行分子鉴定,验证ITS2序列对柴胡属植物种子的鉴定能力,并分析柴胡属植物的遗传多样性。方法:应用ITS2序列测定样品种子的DNA序列,从分子角度鉴定柴胡属种子的物种,并构建系统聚类树,分析其遗传多样性。结果:鉴定出8种柴胡属植物的种子,2种非柴胡属植物的种子,测得的所有种子DNA序列的GC量在53.9%-57.0%,平均为55.7%,变异位点数202个,K2P遗传距离为0-1.460,构建出UPGMA聚类树。结论:基于ITS2序列的分子鉴定方法可成功鉴别柴胡属药用植物及其混淆品的种子,是一种操作方便,准确性高、鉴定效果好的鉴定方法。柴胡属植物物种内及物种间具有丰富的遗传多样性,相同物种间的遗传变异相对稳定,种质及生态环境对柴胡属药用植物的遗传变异有较大的影响。     

羌活药材ITS/ITS2条形码鉴定及其稳定性与准确性研究

为验证DNA条形码鉴定的稳定性与准确性,本文选用羌活药材作为研究对象,对31份样本提取基因组DNA,通过聚合酶链式反应(polymerase chain reaction,PCR)扩增内部转录间隔区(internal transcribed spacer,ITS)序列并进行双向测序,所得序列经CodonCode Aligner拼接后,采用MEGA5.0软件与其混伪品进行序列比对,计算种内和种间距离,构建邻接树(neighbor-joining tree,NJ Tree)。ITS2序列采用基于隐马尔可夫模型(hidden Markov model,HMMer)的注释方法获得。结果表明,羌活药材ITS序列长度为603～604 bp,ITS2序列长度均为228 bp,羌活药材ITS/ITS2序列单倍型与其基原植物叶片序列一致。两种基原植物ITS/ITS2序列种内平均kimura 2-parameter(K2P)遗传距离均远远小于其与混伪品的种间平均K2P遗传距离;NJ树结果显示羌活、宽叶羌活与其混伪品均可明显区分,表现出良好单系性。因此ITS/ITS2序列作为DNA条形码能稳定、准确鉴别羌活药材,为保障临床安全用药提供了新的技术手段。     

DNA microarray for identification of the herb of dendrobium species from Chinese medicinal formulations

A DNA microarray for detecting processed medicinal Dendrobium species (Herba Dendrobii) was constructed by incorporating the ITS1-5.8S-ITS2 sequences of 16 Dendrobium species on a glass slide. Using fluorescence-labeled ITS2 sequences as probes, distinctive signals were obtained for the five medicinal Dendrobium species listed in the Chinese Pharmacopoeia. The established microarray was able to detect the presence of D. nobile in a Chinese medicinal formulation containing nine herbal components.     

Authentication of medicinal Dendrobium species by the internal transcribed spacer of ribosomal DNA

Herba Dendrobii (Shihu) is a commonly used Chinese medicine derived from the stem of several orchid species belonging to the genus Dendrobium. It is rather expensive and adulteration is frequent. Proper authentication of the medicinal species is necessary to protect consumers and support conservation measures. DNA sequences of the internal transcribed spacer 2 (ITS 2) of 16 Dendrobium species were shown to be significantly different from one another by an average of 12.4% and from non-orchids and Pholidota (an adulterant of Shihu) by 29.8% and 18.8%, respectively. The intra-specific variation among the Dendrobium species studied was only about 1%. Therefore, ITS 2 regions could be adopted as a molecular marker for differentiating medicinal Dendrobium species from one another and also from non-orchids and adulterants.     

Identification of dendrobium species used for herbal medicines based on ribosomal DNA internal transcribed spacer sequence

Stems of genus Dendrobium (Orchidaceae) have been traditionally used as an herbal medicine (Dendrobii Herba) in Eastern Asia. Although demand for Dendrobium is increasing rapidly, wild resources are decreasing due to over-collection. This study aimed to identify plant sources of Dendrobii Herba on the market based on sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. We constructed an ITS1-5.8S-ITS2 sequence database of 196 Dendrobium species, and the database was employed to identify 21 herbal samples. We found that 13 Dendrobium species (D. catenatum, D. cucullatum, D. denudans, D. devonianum, D. eriiflorum, D. hancockii, D. linawianum, D. lituiflorum, D. loddigesii, D. polyanthum, D. primulinum, D. regium, and D. transparens) were possibly used as plant sources of Dendrobii Herba, and unidentified species allied to D. denudans, D. eriiflorum, D. gregulus, or D. hemimelanoglossum were also used as sources. Furthermore, it is clear that D. catenatum is one of the most important sources of Dendrobii Herba (5 out of 21 samples).     

Differentiation of Dendrobium species used as "Huangcao Shihu" by rDNA ITS sequence analysis

The genus Dendrobium Sw. is composed of 74 species and two varieties in China, and 32 species carry the name "Huangcao Shihu" on the herbal medicine market, making the identification of the origin of "Huangcao Shihu" difficult for consumers. Here, the ITS regions were sequenced and evaluated to differentiate the 18 Dendrobium species used as "Huangcao Shihu". Diversity in DNA sequences among various species was found with the inter-specific sequence divergence ranging from 3.2% to 37.9% in ITS1 and 5.0% to 26.6% in ITS2. Moreover, the variations within species were very low, ranging in sequence divergence from 0 to 3.0% in ITS1 and 0 to 4.0% in ITS2. Therefore, these species could be easily distinguished at the DNA level. Furthermore, based on the divergent ITS regions, five pairs of species-specific primers were designed and used for the rapid PCR identification of five Dendrobium species listed in the Chinese Pharmacopoeia.     

中药材乌梢蛇及其混淆品的DNA序列分析鉴别

ＤＮＡ序列分析鉴别是中药材品种鉴定的新方法［１］,已应用于海马、龟甲、鳖甲等中药材的鉴定［２～７］。目前商品流通中乌梢蛇（ＺＡＯＣＹＳ）药材的混淆品种类较多,游蛇科（Ｃｏｌｕｂｒｉｄａｅ）的常见种类都有可能被当作乌梢蛇制成药材,品种鉴定困难［８］。而...     

基于matK和ITS2及二级结构对药材香薷及其混伪品的鉴别研究

目的 快速、准确鉴别药材香薷及其混伪品，保障香薷的药材质量和用药安全。方法 收集石香薷、江香薷和香薷植物材料分别进行matK和ITS2序列的扩增与测序，测序结果经Codon Code Aligner软件校对，同时从GenBank下载石香薷、江香斋及其易混品种海州香薷、香薷、密花香薷、牛至等物种的matK和ITS序列。其中，ITS序列经隐马尔可夫模型去除两端的5.8S和28S序列，共得到16个物种的ITS2序列50条；经Clustal软件校对共获得9个物种的matK序列28条。通过Mega7.0软件分析matK和ITS2序列，计算所有物种种内和种间遗传距离，构建邻接法(neighbor joining，NJ)聚类树，通过ITS2 Database预测ITS2二级结构，采用4Sale软件比对二级结构，通过ProfDistS软件构建基于联合ITS2一级序列及其二级结构的剖面邻接(profile neighbor-joining，PNJ)系统发育树。结果 基于matK和ITS2序列的遗传距离均表明香薷正品与其各种混伪品之间存在明显barcoding gap。NJ和PNJ进化树的拓扑关系一致，可以区分药材香薷及其混伪品。香薷的ITS2二级结构与其各混伪品具有显著差异。结论 建议matK和ITS2序列均可以作为鉴别香薷与其混伪品的DNA条形码，ITS2二级结构信息的加入可丰富鉴定结果，为香薷药材的准确鉴别、香薷属与石荠苎属植物的科学分类提供参考。     

Authentication of stems of Dendrobium officinale by rDNA ITS region sequences

The rDNA ITS regions of five Dendrobium species were sequenced. Each Dendrobium species was found to have a unique sequence in the ITS region, so that they could be easily distinguished at the DNA level. The aligned 644 bp of the ITS region includes 235 bp ITS1, 163 bp 5.8S, and 246 bp ITS2. One hundred and eighty-nine sites are variable. The sequences of D. officinale could be easily distinguished from the other four adulterant species according to the sequence variation at 11 sites, 7 in ITS1, 1 in 5.8S, and 3 in ITS2. These could be used as molecular characters to distinguish the stems of D. officinale from the adulterants.     

Authentication of medicinal herbs using PCR-amplified ITS2 with specific primers

Different parts of medicinal herbs have long been used as traditional Chinese drugs for treating many diseases, whereas materials of similar morphology and chemical fingerprints are often misidentified. Analyses of sequence variations in the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) have become a valid method for authentication of medicinal herbs at the intergenic and interspecific levels. DNA extracted from processed materials is usually severely degraded or contaminated by microorganisms, thus generates no or unexpected PCR products. The goal of this study is to apply the ITS fragments selectively amplified with two designed primer sets for efficient and precise authentication of medicinal herbs. The designed primers led to an accurate PCR product of the specific region in ITS2, which was confirmed with DNA extracted from 55 processed medicinal herbs belonging to 48 families. Moreover, the selectively amplified ITS2 authenticated five sets of easily confusable Chinese herbal materials. The designed primers were proven to be suitable for a broad application in the authentication of herbal materials.     

石斛中微量元素的含量测定及其安全性评价

目的：建立测定石斛中微量元素含量的方法,从微量元素层面分析铁皮石斛、金钗石斛、流苏石斛、鼓槌石斛、球花石斛、迭鞘石斛的安全性,并探讨微量元素与石斛抗白内障、降血糖作用的关系。方法：采用电感耦合等离子体质谱（ICP-MS）法测定各石斛样品中6种必需微量元素镁、铁、锌、锰、铬、硒和5种重金属元素铜、砷、铅、汞、镉的含量,并对微量元素进行安全性评价和药效关系探讨。结果：所测样品中6种必需微量元素含量丰富,5种重金属元素中铅、镉、汞均有不同程度的超标现象。结论：《中国药典》中规定的12g·d-1石斛服用量所包含的微量元素符合美国食品和药物管理局每日微量元素推荐摄入量,不会引起必需微量元素中毒症。石斛中有害重金属含量不容乐观,建议加强中药的无公害种植。石斛中丰富的镁、锌、铁、锰、硒、铬微量元素可能与其抗白内障、降血糖作用有关。