Phylogenetic analysis based on 18S rRNA gene and matK gene sequences of Panax vietnamensis and five related species

Panax vietnamensis was discovered recently in Vietnam. Its bamboo-like rhizomes, called Vietnamese Ginseng, have attracted considerable attention because of their specific pharmacological activities. In order to define the taxonomic position of this new species and include it in the molecular authentication of Ginseng drugs, the 18S ribosomal RNA gene and matK gene sequences of P. vietnamensis were determined and compared with those of its related taxa, P. japonicus var. major and P. pseudo-ginseng subsp. himalaicus, besides previously reported P. ginseng, P. japonicus and P. quinquefolius. The 18S rRNA gene sequences were found to be 1809 bps in length. The sequence of P. vietnamensis was identical to that of P. quinquefolius, and presented one base substitution from those of both P. japonicus var. major and P. pseudo-ginseng subsp. himalaicus. The matK gene sequences of 6 taxa were found to be 1509 bps in length. The sequence of P. vietnamensis differed from those of P. japonicus var. major, P. pseudo-ginseng subsp. himalaicus, P. ginseng, P. japonicus and P. quinquefolius at 4, 5, 9, 9 and 10 nucleotide positions, respectively. The phylogenetic tree reconstructed by the combined 18S rRNA-matK gene analysis using the maximum parsimony method showed that P. vietnamensis was sympatric with other Panax species and had a close relationship with P. japonicus var. major and P. pseudo-ginseng subsp. himalaicus.     

Genetic heterogeneity of ribosomal RNA gene and matK gene in Panax notoginseng

Previously, 185 ribosomal RNA gene and matK gene sequences of Chinese herbal medicines, Ginseng Radix, Panacis Japonici Rhizoma and Panacis Quinquefolli Radix were shown to correspond with those of the original plants, Panax ginseng, P. japonicus and P. quinquefolius, respectively, with the species-specific sequences especially for 18S rRNA gene sequences. In P. notoginseng and its derivative, Notoginseng Radix, however, we found two genetic groups with respect to both gene sequences. Five base substitutions were detected on both gene sequences and the homology between two groups was 99.7% for the 18S rRNA gene and 99.6% for the matK gene, respectively. One genetic group was found to have the identical sequences as those of P. ginseng.     

Species identification from Ginseng drugs by multiplex amplification refractory mutation system (MARMS)

The multiplex amplification refractory mutation system (MARMS) was applied to the identification of 5 Panax species ( P. ginseng, P. japonicus, P. quinquefolius, P. notoginseng and P. vietnamensis). A set of specific primers, including 2-pair primers on chloroplast trnK gene and nuclear 18S rRNA gene regions, respectively, was designed and synthesized for each species on the basis of species-specific sequences of the 2 genes. By using 5 sets of specific primers, in turn, PCR amplifications were performed with total DNA extracted from 5 Panax species as template under appropriate condition, and each resulting product was detected by agarose gel electrophoresis. The results showed that two expected fragments, one from trnK gene and another from 18S rRNA gene regions, were observed simultaneously only when the set of species-specific primers encountered template DNA of the corresponding species. This assay could give more reliable results for identification of not only 5 Panax species but also corresponding Ginseng drugs by simultaneous detection of 4-site nucleotide differences on 2 completely different genes.     

Molecular analysis of medicinally-used Chinese and Japanese Curcuma based on 18S rRNA gene and trnK gene sequences.

Curcuma drugs have been used discriminatingly for invigorating blood circulation, promoting digestion, and as a cholagogic in China. However, there is confusion about the drug's botanical origins and clinical uses because of morphological similarity of Curcuma plants and drugs. In order to develop an ultimate identification, molecular analysis based on 18S rRNA gene and trnK gene sequences were performed on 6 Curcuma species used medicinally in China and Japan. The 18S rRNA gene sequences were found to be of 1810 bps in length. In comparison with the common sequence of C. longa, C. phaeocaulis, C. wenyujin and C. aromatica, that of C. kwangsiensis had one base substitution, and the same base difference was observed between the Chinese and the Japanese populations of C. zedoaria. The trnK gene sequences were found to span 2698-2705 bps. There were base substitutions, small deletions or insertions at some sites between the trnK coding region and matK region among each species. Based on the base substitutions, C. zedoaria and C. kwangsiensis specimens were divided into two groups, respectively. An identical sequence was detected in C. phaeocaulis and in the Chinese population of C. zedoaria, as well as in the Japanese population of C. zedoaria and in one group of C. kwangsiensis with a purple-colored band in leaves. New taxonomic information to be used for authenticating Curcuma drugs was obtained.     

Authentication of Curcuma species (Zingiberaceae) based on nuclear 18S rDNA and plastid trnK sequences

Curcuma drugs have been used discriminatingly for invigorating blood circulation, promoting digestion, and as a cholagogic in China. However, there is confusion about the drug's botanical origins and clinical uses because of morphological similarity of Curcuma plants and drugs. Comparative sequencing of the 18S rRNA gene in nuclear ribosomal DNA (rDNA) and trnK gene in chloroplast DNA (cpDNA) was carried out in order to examine interspecies phylogeny and to identify ultimately Curcuma species. A total of a hundred of accessions of eighteen species were analyzed. This resulted in an aligned matrix of 1810 bp for 18S rDNA and 2 800 bp for trnK. 18S rDNA sequence divergence within the ingroup ranged from 0-0.05%, trnK ranged from 0-0.19%. One base transversion-substituted site (from cytosine to thymine) was observed from the upstream of 18S rDNA at nucleotide position 234 in C. kwangsiensis and Japanese population of C. zedoaria which have separated genetic distance to other Curcuma taxa. Two noncoding regions embedded in trnK intron showed higher variability, including nucleotide substitutions, repeat insertion and deletions. Based on consensus of relationship, eighteen major lineages within Curcuma are recognized at the species level. The results suggest that Curcuma is monophyletic with 100% bootstrap support and sister to the genera Hedychium and Zingiber. The trnK sequences showed considerable variations between Curcuma species and thus were revealed as a promising candidate for barcoding of Curcuma species, which provide valuable characters for inferring relationship within species but are insufficient to resolve relationships among closely related taxa.     

广藿香与土藿香的DNA序列分析及其分子鉴别

目前市场上藿香类商品药材有两种 ,一种为唇形科刺蕊草属 (Ｐｏｇｏｓｔｅｍｏｎ)植物广藿香Ｐｏｇｏｓｔｅｍｏｎｃａｂｌｉｎ (Ｂｌａｎｃｏ)Ｂｅｎｔｈ.的干燥地上部分 ,主产广东、海南 ,习称“广藿香”,均为栽培品 ,有芳香化浊、开胃止呕、发表解暑的功效 ,是中成药“藿香正气水”的主要原料。据我们分析广州市郊黄村产“石牌广藿香”药材茎枝挥发油成分 ,其中 71 %为广藿香酮(ｐｏｇｏｓｔｏｎｅ) [1 ] ;另一种来源于同科另一属 ,即藿香属(Ａｇａｓｔａｃｈｅ)植物藿香Ａｇａｓｔａｃｈｅｒｕｇｏｓａ (Ｆｉｓｃｈ.ｅｔＭｅｙ.)Ｏ .Ｋ…     

6种川产姜黄属药用植物叶绿体trnK基因序列变异分析及其分子鉴定

目的建立6种川产姜黄属(Curcuma)药用植物快速简单的分子鉴定方法.方法采用叶绿体赖氨酸tRNA基因(trnK)测序与序列变异分析方法.结果 6种姜黄属药用植物(包括姜黄C. longa、莪术C. phaeocaulis、川郁金C. sichuanensis、川郁金C. chuanyujin、川黄姜C. chuanhuangjiang、川莪术C. chuanezhu)完整trnK基因长度在2699～2705 bp.序列可变区包括matK基因编码区和trnK外显子与matK内含子之间区域,共有6个单核苷酸多态性(SNPs)位点、1个9-bp的缺失重复序列和2个4-bp、14-bp插入重复序列.结论 trnK基因序列可变位点可以作为6种川产姜黄属药用植物快速简单的分子鉴定标记,并为它们之间种的归并提供了分子依据.     

6种川产姜黄属药用植物叶绿体trnK基因序列变异分析及其分子鉴定

目的建立6种川产姜黄属（Curcuma）药用植物快速简单的分子鉴定方法。方法采用叶绿体赖氨酸tRNA基因（trnK）测序与序列变异分析方法。结果6种姜黄属药用植物（包括姜黄C. longa、莪术C. phaeocaulis、川郁金C. sichuanensis、川郁金C. chuanyujin、川黄姜C. chuanhuangjiang、川莪术C. chuanezhu）完整trnK基因长度在2699～2705 bp。序列可变区包括matK基因编码区和trnK外显子与matK内含子之间区域，共有6个单核苷酸多态性（SNPs）位点、1个9-bp的缺失重复序列和2个4-bp、14-bp插入重复序列。结论trnK基因序列可变位点可以作为6种川产姜黄属药用植物快速简单的分子鉴定标记，并为它们之间种的归并提供了分子依据。     

广藿香的基因序列与挥发油化学型的相关性分析

目的探讨“南药”广藿香Pogostemon cablin (Blanco) Benth.不同产地间的叶绿体和核基因组的基因型与挥发油化学型的关系，为广藿香道地性品质评价、规范化种植提供分子依据。方法用PCR直接测序技术对广藿香6个产地样本的叶绿体matK基因和核18S rRNA基因核苷酸序列进行测序分析研究。结果广藿香6个样本的matK基因序列长均为1 245 bp，编码415个氨基酸成熟酶。18S rRNA基因序列长为1 803～1 805 bp。根据排序比较，广藿香6个样本间的matK基因序列存在47个变异位点，18S rRNA基因存在17个变异位点，非加权组平均法构建的系统分支树表明广藿香基因序列分化与其产地、所含挥发油化学变异类型呈良好的相关性。结论结合挥发油分析数据，基因测序分析技术可作为广藿香道地性品质评价方法这一以及规范化种植过程关键技术“物种鉴定”的强有力工具。     

Sequence analysis of Chinese and Japanese Curcuma drugs on the 18S rRNA gene and trnK gene and the application of amplification-refractory mutation system analysis for their authentication

The botanical origins of Chinese and Japanese Curcuma drugs were determined to be Curcuma longa, C. phaeocaulis, the Japanese population of C. zedoaria, C. kwangsiensis, C. wenyujin, and C. aromatica based on a comparison of their 18S rRNA gene and trnK gene sequences with those of six Curcuma species reported previously. Moreover, to develop a more convenient identification method, amplification-refractory mutation system (ARMS) analysis of both gene regions was performed on plants. The ARMS method for the 18S rRNA gene was established using two types of forward primers designed based on the nucleotide difference at position 234. When DNAs of four Curcuma species were used as templates, PCR amplification with either of the two primers only generated a fragment of 912 base pairs (bp). However, when DNAs of the purple-cloud type of C. kwangsiensis and C. wenyujin were used, PCR amplifications with both primers unexpectedly generated the fragment, suggesting that these two were heterozygotes. The ARMS method for the trnK gene was also established using a mixture of four types of specific reverse primers designed on the basis of base substitutions and indels among six species, and common reverse and forward primers. C. phaeocaulis or the Chinese population of C. zedoaria, the Japanese population of C. zedoaria or the purple-cloud type of C. kwangsiensis, the pubescent type of C. kwangsiensis or C. wenyujin, and C. aromatica were found to show specific fragments of 730, 185, 527 or 528, and 641 or 642 bp, respectively. All species including C. longa also showed a common fragment of 897-904 bp. Using both ARMS methods, together with information on producing areas, the identification of Curcuma plants was achieved. Moreover, the ARMS method for the trnK gene was also useful for authentication of Curcuma drugs.     

三七的18S rRNA,matK基因序列和HPLC化学指纹图谱分析研究

目的分析中药三七Panaxnotoginseng的18SrRNA和matK基因的分子特征和三七的化学指纹特征,为三七的正品药材基原鉴定提供分子和化学依据。方法采用PCR直接测序技术测定三七及其7种伪品的18SrRNA和matK基因部分核苷酸序列以及不同产地三七的DNA分子特征。利用HPLC的化学分析技术,明确产地对三七化学成分的影响,以及三七不同部位的化学指纹特征。结果(1)三七及其7种常见伪品的核糖体18SrRNA基因序列存在很大的差异。(2)不同产地的三七的核糖体18SrRNA和叶绿体matK基因序列特征完全一致,分别与GenBank上已报道的R1型(D85171)和M1型(AB027526)序列吻合。(3)不同产地的三七HPLC指纹图谱相似。(4)三七不同部位均具有其相对稳定的HPLC指纹特征,其中花、叶具有特有的指纹区,根、须根、剪口、筋条等不同商品规格的HPLC指纹图谱比较相似。结论基因序列标记能从分子水平定性分辨三七及其伪品的遗传背景差异,为中药品种标准化提供了先进可行、稳定可靠的分子标准;HPLC指纹图谱分析可以直观地为三七的化学成分定性,三七不同商品规格的特征性指纹有望成为以其为原材料的各种产品的质控标准,而三七不同部位(尤其是花和叶)的HPLC指纹图谱将有望成为制定三七花、三七叶新药用资源质控标准的依据。     

Genetic variation and identification of cultivated Fallopia multiflora and its wild relatives by using chloroplast matK and 18S rRNA gene sequences

FALLOPIA MULTIFLORA (Thunb.) Harald . has been widely and discriminatingly used in China for the study and treatment of anemia, swirl, deobstruent, pyrosis, insomnia, amnesia, atheroma and also for regulating immune functions. However, there is still confusion about the herbal drug's botanical origins and the phylogenetic relationship between the cultivars and the wild relatives. In order to develop an efficient method for identification, a molecular analysis was performed based on 18 S rRNA gene and partial MATK gene sequences. The 18 S rRNA gene sequences of F. MULTIFLORA were 1809 bp in length and were highly conserved, indicating that the cultivars and the wild F. MULTIFLORA have the same botanical origin. Based on our 18 S rRNA gene sequences analysis, F. MULTIFLORA could be easily distinguished at the DNA level from adulterants and some herbs with similar components. The MATK gene partial sequences were found to span 1271 bp. The phylogenetic relation of F. MULTIFLORA based on the MATK gene showed that all samples in this paper were divided into four clades. The sequences of the partial MATK gene had many permutations, which were related to the geographical distributions of the samples. MATK gene sequences provided valuable information for the identification of F. MULTIFLORA. New taxonomic information could be obtained to authenticate the botanical origin of the F. MULTIFLORA, the species and the medicines made of it.     

Molecular analysis of Rheum species used as Rhei Rhizoma based on the chloroplast matK gene sequence and its application for identification

Rhei Rhizoma (Dahuang in Chinese) is widely known as a purgative and antiinflammatory agent. In the Japanese Pharmacopoeia, Rhei Rhizoma is prescribed for four Rheum species, Rheum palmatum, R. tanguticum, R. officinale, and R. coreanum, while the first three species are prescribed for Dahuang in the Chinese Pharmacopoeia. Due to the morphologic similarity of the aerial parts and frequent occurrence of intermediate forms, the taxonomy of this genus and the correct identification of Rheum species and their derivative drugs are very difficult. To resolve taxonomic problems of the genus Rheum and develop an ultimate identification method for plants and drugs, molecular analysis of the chloroplast matK gene and nuclear 18S ribosomal RNA gene were performed on nine species. The sequence comparison of the matK gene revealed that most species had variable sequences not only inter- but also intraspecies. However, the specimens of the same species belonged to the same subclade in the phylogenetic tree constructed based on matK gene sequences, except for R. palmatum, in which specimens belonged to three subclades related to their production areas. The nucleotide differences at positions 587, 707, and 838 distinguished official species from others, while specific nucleotides at positions 367 and 937 became identification markers for R. palmatum, R. tanguticum, and R. officinale (or R. coreanum). Moreover, three groups of R. palmatum, each belonging to three subclades, were characterized by the nucleotides at positions 619, 769, 883, and 1061. By detecting marker nucleotides, the botanical origins of Rhei Rhizoma were determined.     

中日产川芎的matK、ITS基因序列及其物种间的亲缘关系

目的分析中国产川芎Ligusticum chuanxiong Hort.及日本产川芎Cnidium officinale Makino的核基因组ITS和叶绿体基因组matK序列,为探讨中日产川芎物种间的亲缘关系提供分子依据。方法采用PCR直接测序技术测定川芎和日本川芎的ITS基因和matK基因核苷酸序列并作序列变异分析。结果川芎和日本川芎的matK序列长度均为1268 bp,编码422个氨基酸。ITS1-5.8S-ITS2序列长度均为699 bp,其中18S rRNA基因3′端序列54 bp,ITS1序列215 bp,5.8S rRNA基因序列162 bp,ITS2序列222 bp,26S rRNA基因5′端序列46 bp。根据排序比较,川芎原植物与其商品药材间的matK基因和ITS基因序列完全相同,而川芎与日本川芎间matK基因则仅有1个变异位点,即在上游959 nt处1个转换替代(T→C),反映在氨基酸序列则发生一个非同义取代V(GTG)→A(GCG);ITS基因也仅有1个变异位点,即在ITS1上游54 nt处1个转换替代(T→C)。结论通过进化速率较快的基因序列同源性分析,基本可以认为中日所产川芎基原一致,日本川芎学名似应改为Ligusticum chuanxiong Hort.。     

Development of species specific AFLP-derived SCAR marker for authentication of Panax japonicus C. A. MEYER

Panax japonicus is an important medicinal plant. The aim of this study was to develop species-specific molecular markers for P. japonicus. Amplified fragment length polymorphism (AFLP) was compared among P. japonicus, P. ginseng and P. quinquefolius. A clear species-specific AFLP marker for P. japonicus was generated. After isolation and sequencing of the AFLP fragment, a DNA sequence (293 bp) was obtained and named JG14. Oligonucleotide primer (23 mer) was designed for amplifying 191 bp of the sequence of JG14. PCR analysis revealed a clear amplified band for P. japonicus but not in 3 other Panax species (P. ginseng, P. quinquefolius and P. notoginseng). This sequence characterized amplified regions (SCAR) marker will be used for rapid authentication of P. japonicus among other related Panax species. This is the first report of species-specific SCAR marker development in P. japonicus.     

基因测序技术在中药质量研究中的应用（Ⅰ）——山东郓城猴头半夏基原的DNA测序鉴别

目的：分析半夏Pinellia ternata(Thunb.)Breit.及其伪品虎常南星Pinellia pedatisecta Schott的核基因组序列,为半夏正品基原鉴别提供分子依据。方法：采用PCR直接测序技术测定半夏及其伪品的18S rRNA基因核苷酸序列并作序列变异和选择性内切酶谱（PCR－SR）分析。结果：半夏和伪品的18S rRNA序列长度均为1805bp,根据排序比较,半夏原植物与商品药材间的序列完全相同,虎掌南星亦如此。而半夏与其伪品虎掌南星间则存在序列差异（有4个变异位点）。在半夏18S rRNA序列中有一个限制性内切酶Ase Ⅰ识别位点,通过PCR－SR图谱显示800bp和900bp2个酶切片断,而虎掌南星则无此位点,PCR－SR图谱显示1个未消化的1800bp片断。结论：通过核基因组序列和PCR－SR图谱差异DNA测序技术可成为半夏正品基原鉴别准确而有效的分子方法。     

Rapid detection of Panax ginseng by loop-mediated isothermal amplification and its application to authentication of Ginseng

We have developed a novel method called loop-mediated isothermal amplification (LAMP) to detect Panax ginseng, the botanical source of Ginseng (Ginseng Radix), and to distinguish P. ginseng from Panax japonicus. Six allele-specific primers (two outer primers, two inner primers, and two loop primers) were designed based on the 18S ribosomal RNA gene sequence of P. ginseng, and LAMP was performed using those primers and total DNA extracted from P. ginseng as template. Amplifications were observed from approximately 30 min onwards at DNA concentrations of 0.5 to 10.0 ng. The presence of loop primers shortened the reaction time considerably. In contrast, in the reactions using total DNA from P. japonicus as template, no amplifications were observed. LAMP also enabled us to distinguish Ginseng from Japanese Ginseng (Panacis Japonici Rhizoma). LAMP was proven to be a rapid, highly sensitive, and specific method for the detection of P. ginseng and Ginseng.     

Isolation of actinomycetes from the root of the plant, Ophiopogon japonicus, and proposal of two new species, Actinoallomurus liliacearum sp. nov. and Actinoallomurus vinaceus sp. nov

Actinomycete strains K10-0485(T) and K10-0528(T) were isolated from the roots of Ophiopogon japonicus collected in Yokohama, Kanagawa Prefecture, Japan. The 16S ribosomal RNA (rRNA) gene sequences, morphological characteristics and chemotaxonomic data indicated that these strains belonged to the genus Actinoallomurus. Strain K10-0485(T) showed high similarity of the 16S rRNA gene sequence with A. luridus TT02-15(T) (99.1%), but the DNA-DNA hybridization relatedness values between strain K10-0485(T) and A. luridus TT02-15(T) were below 70%. Three species showed similarities of 16S rRNA gene sequences with K10-0528(T), namely A. spadix JCM 3146(T) (98.0%), A. purpureus TTN02-30(T) (98.0%) and A. luridus TT02-15(T) (97.9%), but all similarity values of the 16S rRNA gene sequences were lower than the boundary value (98.7%) for distinguishing as different species. Based on phylogenetic analyses, DNA-DNA hybridization relatedness and physiological characteristics, the two isolated strains should be classified as two new species in the genus Actinoallomurus, and we propose the names Actinoallomurus liliacearum sp. nov. and Actinoallomurus vinaceus sp. nov. The type strain of Actinoallomurus liliacearum is K10-0485(T) (=JCM 17938(T), BCC 49424(T), NBRC 108672(T)) and that of Actinoallomurus vinaceus is K10-0528(T) (=JCM 17939(T), BCC 49425(T), NBRC 108763(T)).     

Authentication of Panax notoginseng by 5S-rRNA spacer domain and random amplified polymorphic DNA (RAPD) analysis

The great majority of Panax species are well-known herbal medicines in the Orient, and many of them share a close resemblance in appearance and chemical composition. Among these Panax species, the root of P. notoginseng (Sanqi) is a unique herb that has distinct clinical usage. Here, the 5S-rRNA spacer domains were isolated from P. notoginseng, P. japonicus var. major, P. stipuleanatus, P. quinquefolius, P. ginseng, P. zingiberensis, and P. wangianus, and four common adulterants of P. notoginseng including Curcuma wenyujin, Curcuma longa, Bletilla striata and Gynura segetum. The spacer domains were sequenced and compared, which showed over 75 % DNA identity among all Panax species, but not for the adulterants. In addition, random amplification of polymorphic DNA (RAPD) analysis was used to distinguish different members of Panax genus as well as the morphological variants of P. notoginseng. These molecular methods could be used in the authentic identification of P. notoginseng from other Panax species.     

湖泊放线菌SIIA-A16124的分类鉴定及基因组挖掘

目的 对湖泊放线菌SIIA-A16124进行分类鉴定和基因组挖掘。方法 采用多相分类法对菌株的形态学、生理生化、细胞壁化学组分、16S rRNA基因序列进行测定和分类鉴定,采用基因组挖掘分析生物合成基因簇,经发酵培养、抗菌活性检测及活性产物质谱分析,推测其活性产物的化合物结构类别。结果 菌株SIIA-A16124的16S rRNA基因与菌株Actinokineospora inagensis NRRL B-24050T同源性最高为99%;菌株SIIA-A16124在ISP3培养基上中等产孢和水解淀粉的特性与模式菌株存在明显差异;鉴定菌株SIIA-A16124为动孢菌Actinokineospora sp.,具有羊毛硫肽生物合成基因簇,其活性次级代谢产物属于羊毛硫肽类抗生素。结论 首次发现动孢菌属放线菌具有生物合成羊毛硫肽类抗生素的能力。