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。     

细茎石斛叶绿体全基因组序列特征及系统发育分析

细茎石斛是药用石斛的重要来源,也是正品枫斗的主要来源之一。本研究利用Illumina高通量测序技术对细茎石斛叶绿体全基因组进行测序,完成了其物理图谱绘制和基因组结构特征解析,并与近缘物种进行了比较和系统发育分析。细茎石斛叶绿体基因组全长为150 754 bp,两个反向互补重复区(IRs)长25 906 bp,大单拷贝区(LSC)和小单拷贝区(SSC)长度分别为84 818 bp和14 124 bp。共注释到123个基因,包括77个蛋白编码基因、38个tRNA基因和8个rRNA基因,其中17个基因含有内含子。生物信息学分析获得53个SSR位点,大多数位点具有A-T碱基偏好性。采用33条石斛属植物的叶绿体基因组序列构建系统发育树,结果显示细茎石斛复合种中的物种聚在同一个大支,说明其亲缘关系较近。本研究为细茎石斛的鉴定、细茎石斛复合种系统发育关系及其本草基因组学研究提供理论基础。     

石斛属27种药用植物的性状鉴定特征比较

目的 比较中国西南地区产27种石斛属药用植物药材(茎)的性状特征,为石斛药材质量标准的制定提供依据.方法 考察药材来源,并比较性状鉴定.结果 描述了27种石斛属药用植物药材的性状鉴别特征.结论 可为石斛属药用植物药材的准确鉴定提供参考.     

铁皮石斛与几种常用混淆品的红外光谱鉴别

目的 寻找能够鉴别铁皮石斛与几种常用混淆品的新方法．为进一步探索石斛类药材、饮片快速、有效的鉴别方法。方法 采用药材原粉末加KBr直接压片法测定铁皮石斛与几种常用混淆品的红外光谱．所获得的指纹图谱进行特征峰指认和对比分析。结果 铁皮石斛与几种常用混淆品的红外吸收频率、吸收峰的峰形和相对强度都存在较显著的差异。结论 首次采用红外光谱法鉴别铁皮石斛与几种常用混淆品,结果快速、准确,为控制铁皮石斛商品提供了可靠的依据。     

铁皮石斛的PCR-RFLP鉴别方法

目的:建立铁皮石斛专有的分子鉴别方法。方法:通过对铁皮石斛及其近缘种石斛的叶绿体matk基因序列进行对比分析,根据鉴别位点设计特异的铁皮石斛鉴别引物,通过聚合酶链反应-限制性片段长度多态性方法(PCR-RFLP)技术进行鉴别。结果:本试验中鉴别引物可成功扩增铁皮石斛及其近缘种石斛,PCR产物为331 bp,铁皮石斛的PCR产物可被Tru9 Ⅰ限制性内切酶切开,而近缘种石斛不能被切开,可通过琼脂糖凝胶电泳成功鉴别。结论:本试验建立的PCR-RFLP鉴别方法可以鉴别铁皮石斛及其近缘种石斛。     

霍山地区4种石斛属植物的遗传多态性分析

利用AP-PCR方法对4种霍山来源的石斛属植物基因组DNA进行了多态性分析，从75种随机引物中筛选出10种扩增条带清晰、稳定的随机引物，通过PCR扩增共获得250条DNA片段，其中呈多态性的片段数为165条，占扩增条带数的66％，显示了霍山来源的石斛植物具有较高的遗传多态性；利用NT-syspc分析软件对不同石斛品种间的平均遗传距离进行了分析，并利用UPGMA聚类分析方法构建它们的亲缘关系聚类图，结果表明4种霍山来源的石斛品种具有较近的亲缘关系，它们之间的平均遗传相似系数为0．63，其中铁皮石斛和铜皮石斛的亲缘关系最近，两者间的遗传相似系数为0．78，它们与串珠石斛的遗传相似系数分别为0．6和0．61，与霍山石斛的遗传相似系数分别为0．63和0．58；但是，相对于形态接近的铜皮石斛，铁皮石斛与霍山石斛的亲缘关系更近。     

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。     

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。     

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     

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.     

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.     

Enzymatic fingerprints of polysaccharides of Dendrobium officinale and their application in identification of Dendrobium species

Enzymatic fingerprinting of polysaccharides from Dendrobium officinale was studied and applied to authenticate Dendrobium species. Results showed that Dendrobium officinale species from Anhui province, Fujian province, Yunnan province, Guangdong province and Guangxi province of China, could be identified by polysaccharide analysis using carbohydrate gel electrophoresis (PACE). However, the fingerprints of Dendrobium officinale from Jiangxi province, Hu'nan province and Wenzhou, Yandangshan and Fuyang in Zhejiang province were very similar. As far as the fingerprints of different Dendrobium species were concerned, the differences between Dendrobium officinale, Dendrobium huoshanense, Dendrobium moniliforme, Dendrobium devonianum, Dendrobium aphyllum, Dendrobium wilsonii and Dendrobium crystallinum were obvious. Moreover, the genetic relationships between different samples were analyzed by using principal component analysis and unweighted pair group method with arithmetic mean cluster analysis. Results suggested that polysaccharide fingerprint analysis by PACE has the potential to become a valuable new method for the identification and control of quality of herbal medicines in future.     

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).     

铁皮石斛内生真菌的鉴定及菌株205509的次级代谢产物研究

摘要：目的 鉴定铁皮石斛内生真菌及从中发现活性菌株,探究菌株205509产生的次级代谢产物。 方法 利用分子生物学方法并结合形态特征,对9株铁皮石斛内生真菌进行鉴定;采用琼脂扩散法和MTT法,筛选出次级代谢产物丰富,且具有抑制病原指示菌和人乳腺癌MCF-7细胞系的活性菌株;利用大米固体培养基对活性菌株进行发酵,乙酸乙酯萃取;采用硅胶、反相硅胶、葡聚糖凝胶Sephadex LH-20和HPLC色谱技术进行分离纯化,得到化合物;通过质谱(MS)和核磁共振(1H NMR和13C NMR)等波谱技术并结合文献比对,确定化合物结构。结果 9株铁皮石斛内生真菌分属3个科的3个属,初步判定其中1株为枝顶孢霉属(Acremonium)的一个新种,2株为树粉孢属(Oidiodendron)的一个新种,2株为Acremoniopsis属的未确定种;获得抗菌活性和细胞毒活性较好的菌株205509,并从中分离得到了4个化合物,其中化合物HXW-3浓度为3 μg/mL时对嗜水气单胞菌(Aeromonas hydrophila)、鼠伤寒沙门菌(Salmonella typhimurium)和蜡样芽胞杆菌(Bacillus cereus)表现出抑制活性。结论 铁皮石斛内生真菌物种较新颖,其次级代谢产物具有较好的生理活性,值得进一步研究。