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。     

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。     

中药黄草石斛rDNA ITS序列分析

目的　研究黄草石斛ITS片段遗传多样性,分析该片段在黄草药材DNA分子鉴别和石斛属植物系统学研究中的意义。方法　用一对引物进行PCR扩增,扩增产物纯化后用双脱氧终止法(Sangerdideoxy)测序。结果　获得核糖体DNA中ITS和5 8SrDNA完整序列,14个石斛类群的ITS 1与ITS 2序列的长度分别为228-233bp和242-247 bp。石斛种间ITS 1序列的差异百分率为11.79% - 31.58% ,ITS 2序列的差异百分率为10.29% - 25.30% ,金钗石斛种内ITS 1序列的差异百分率为0.87% ,ITS 2序列无差异。石斛各类群与外类群的差异百分率ITS 1序列为23.56% - 36.89% ,ITS 2序列为26.5.2 % - 33.31%。用NJ法根据ITS 1与ITS 2序列数据重建系统发生树。结论　两段序列在石斛种内保守,在种间有较大的差异,与外类群的差异最大,可作为中药黄草石斛分子鉴定的标记,而石斛属的系统发生关系尚须进一步研究     

花椒及其混淆品的rDNA ITS区序列分析与鉴别

目的研究不同居群的花椒及其混淆品的rDNA ITS区碱基序列的特征及其差异，为花椒的鉴别提供可靠的分子标记。方法运用PCR产物直接测序和克隆测序法对甘肃、陕西、四川、河北等7个花椒居群及3个混淆种的rDNA ITS区(包括ITS1，5.8S，ITS2)碱基序列进行序列测定。结果首次报道花椒ITS区的碱基序列，序列总长度为619-620 bp，长度变异较少，与混淆种长度仅相差4 bp。花椒各居群中，rDNA ITS区碱基序列有15个变异位点、12个信息位点、3个特异性识别位点。与混淆品间的碱基差异则较为显著，多达71个变异位点，有4个花椒特异性识别位点。结论依据花椒ITS区的序列特征可准确鉴别各居群的花椒及其混淆品；亲缘关系密切的花椒居群在地理位置上也非常靠近；rDNA ITS序列特征可作为花椒种内和种间鉴别的有效分子标记。     

中药“枫斗”的性状鉴别研究

目的:研究同属不同种植物来源的枫斗性状,寻找鉴别特征。方法:收集石斛属植物20种并进行物种鉴定,将茎加工枫斗,对各种枫斗性状特征进行观察并描述。结果:20种枫斗在性状特征上有较大的差别。结论:研究结果可为20种枫斗供鉴别依据。     

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。     

药用植物束花石斛、流苏石斛及其形态相似种的PCR-RFLP鉴别研究药用植物束花石斛、流苏石斛及其形态相似种的PCR-RFLP鉴别研究

目的建立简易的采用rDNA ITS区作为分子标记对中药石斛的基源植物进行分子鉴定的技术。方法 用聚合酶链反应—限制性片段长度多态性(PCR-RFLP)方法获得ITS片段的限制性图谱。束花石斛及其形态相似种的PCR扩增产物用Cla I和Apa LI酶切,流苏石斛及其形态相似种的PCR扩增产物用Sph I酶切。结果根据预测的PCR-RFLP图谱,可以鉴别药用植物束花石斛、流苏石斛及它们的形态相似种。用这种方法鉴定了市场上收集的25件商品束花石斛、流苏石斛新鲜药材的原植物。结论rDNA ITS的PCR-RFLP可用于鉴定石斛药材的原植物,具有简便、费用低的优点。     

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。     

鼠尾草属药用植物及其近缘种的ITS序列分析

采用分子系统学方法分析鼠尾草属药用植物及其近缘种的遗传多样性，为准确进行基源鉴定、阐明本属内的种间关系及发现新的药用资源提供分子证据。本文从野外采集的27个鼠尾草属植物叶片样品中分离提取DNA，PCR扩增ITS区及5.8S rDNA完整序列并测序，采用Mega 3.1软件进行系统学分析。27个鼠尾草样品的ITS及5.8S rDNA区序列全长为612～617 bp，邻接法(neighbor-Joining)构建的系统发生树部分支持了形态学的属下划分，但对部分种的系统位置特别是三叶鼠尾草和黄花鼠尾草两个亚种的处理上与形态学划分存在明显的分歧。序列分析显示5.8S rDNA序列相当保守，而ITS区段则在亚属间差异明显，且原产我国的该属植物与欧美引进种明显具有不同起源。ITS系统树对于亚属和组的处理较为合理，但对组下的划分则表现出了信息量不足，需要其他相关证据的支持。ITS分析支持了丹参组内其他近缘种作为丹参类药材替代资源的合理性，同时也揭示了甘西鼠尾类高山丹参在遗传上与丹参类药材的显著不同。     

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rDNAITS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rDNAITS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rDNAITS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 )rDNAITS区的差异较大 ,绝对遗传距离分别为 4 0和 4 2。在rDNAITS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rDNAITS区碱基序列差异 ,可准确鉴别曲茎石斛及其近似种药材。     

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.     

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.     

Allele-specific primers for diagnostic PCR authentication of Dendrobium officinale

Based on rDNA ITS sequences of D. officinale and the other 37 species of Dendrobium, a pair of allele-specific diagnostic primers, TP-JB01S and TP-JB01X, were designed to authenticate D. officinale from the other species. Before the diagnostic PCR, the primer pair, P1 and P2, for amplifying the whole ITS region was used to validate template DNA and to obtain the appropriate template DNA for the diagnostic PCR. Diagnostic PCRs were performed using the diagnostic primers with the total DNAs of the original plants as a template. When the annealing temperature was raised to 66 degrees C, only the template DNA of D. officinale could be amplified whereas the diagnostic PCRs of the other Dendrobium species were all negative. The diagnostic PCRs have been repeated many times and have played an important role in authenticating the stems of D. officinale in China. Compared with the authentication method by sequencing DNA fragments, the allele-specific diagnostic PCR is not only simpler and time-saving but also practical and effective.     

SNP, ARMS and SSH authentication of medicinal Dendrobium officinale KIMURA et MIGO and application for identification of Fengdou drugs

Dried stems of Dendrobium officinale have been used as crude drugs in traditional Chinese medicine (TCM) with good tonic efficacy. Sequences of chloroplast, nuclear and mitochondria genes and the method of genomic DNA (gDNA) suppression subtraction hybridization (SSH) were used to authenticate different populations during the process of good agriculture practice (GAP) and crude drug quality control. Six populations could be authenticated successfully by nine single sucleotide polymorphism (SNP) sites and six pairs of diagnostic primers for amplification refractory mutation system (ARMS) were also designed to identify six populations on the basis of single nucleotide polymorphism (SNPs). The remainder two populations (JSR, GGL) with the same sequences could be authenticated by SSH. One population-specific fragment was obtained by SSH and a pair of specific primers (SSH-JB01, SSH-JB02) on the specific sequence was designed to authenticate GGL population from the other populations tested. As the resultants were population-specific, the botanic origins of fifty "Fengdou" drug samples from markets could be classified. It is evident that the combined methods provide a high throughput and reliable approach for identification of D. officinale plants and "Fengdou" drugs.     

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).