Phylogenetic relationship in the genus Panax: inferred from chloroplast trnK gene and nuclear 18S rRNA gene sequences

Chloroplast trnK gene and nuclear 18S rRNA gene sequences of 13 Panax taxa, collected mainly from Sino-Japanese floristic region, were investigated in order to construct phylogenetic relationship and to assist taxonomic delimitation within this genus. The length of trnK gene sequence varied from 2537 bp to 2573 bp according to the taxa, whereas matK gene sequences, embedded in the intron of trnK gene, were of 1512 bp in all taxa. Species-specific trnK/ matK sequence provided much insight into phylogeny and taxonomy of this genus. 18S rRNA gene sequences were of 1808 or 1809 bps in length, only 9 types of 18S rRNA sequences were observed among 13 taxa. Parsimony and neighbor-joining analyses of the combined data sets of trnK-18S rRNA gene sequences yielded a well-resolved phylogeny within genus Panax, where three main clades were indicated. P. pseudoginseng and P. stipuleanatus formed a sister group located at a basal position in the phylogenetic tree, which suggested the relatively primitive position of these two species. Monophyly of P. ginseng, P. japonicus (Japan) and P. quinquefolius, which are distributed in northern parts of Asia or America, was well supported (Northern Clade). The remaining taxa distributed in southern parts of Asia formed a relatively large clade (Southern Clade). The taxonomic debated taxa traditionally treated as subspecies or varieties of P. japonicus or P. pseudoginseng showed various nucleotide sequences, but all fell into one cluster. It might suggest these taxa are differentiated from a common ancestor and are in a period of high variation, which is revealed not only on morphological appearance, but also on molecular divergence. By comparing trnK and 18S rRNA gene sequences among 13 Panax taxa, a set of valuable molecular evidences for identification of Ginseng drugs was obtained.     

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…     

Detection of genetic homogeneity of Panax notoginseng cultivars by sequencing nuclear 18S rRNA and plastid matK genes

The nuclear 18S rRNA and chloroplast MATK genes of 18 samples of Panax notoginseng and its processed material Sanqi (Radix Notoginseng) were analyzed. The two genes, regardless of cultivar origin, were found to be identical to genotype R1 and M1, respectively, of the published sequences (GenBank accession no. D85171 and AB027526). This phenomenon implies that the species is highly conserved, which is probably caused by the use of the same strain in cultivation and the lack of active mutation in these two genes.     

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。     

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。     

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.     

基于COⅠ与16S rRNA基因对广地龙的DNA分子鉴定研究

目的:建立一种快速、准确和标准化的广地龙DNA分子标记鉴别方法。方法:测定了5个不同居群广地龙的线粒体细胞色素酶亚单位(CO)Ⅰ和16S rRNA基因序列,采用CodonCode Aligner进行序列拼接,通过下载GenBank地龙原动物的COⅠ与16S rRNA序列,采用MEGA4.1计算广地龙及其伪品地龙的种内、种间的K2P遗传距离,并基于K2P模型构建NJ和MP树。结果:COⅠ变异位点、信息位点均高于16SrRNA,COⅠ基因无插入和缺失,16S rRNA存在4个插入和缺失。COⅠ和16S rRNA序列种间遗传距离均明显大于种内,COⅠ和16S rRNA基因均能将广地龙从其他地龙或蚯蚓物种鉴别开来。结论:获得的广地龙COⅠ和16S rRNA序列可为动物性中药材地龙的分子水平鉴定提供参考,为动物性中药材DNA条形码数据库积累了相关信息数据。     

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.     

DNA sequencing analysis of ITS and 28S rRNA of Poria cocos

We determined the DNA sequences of the internal transcribed spacer 1 and 2 (ITS 1 and 2), the 5.8S rRNA gene and most of the 28S rRNA gene of Poria cocos for the first time, and conducted analysis of 20 samples including cultured mycelias and crude drug materials obtained from various localities and markets. Direct sequencing of the ITS 1 and 2 regions of the samples, except for four wild samples, showed that they had identical DNA sequences for ITS 1 and 2 with nucleotide lengths of 997 bps and 460 bps, respectively. By cloning, the four wild samples were found to have combined sequences of common ITS sequences with 1 or 2-base-pair insertions. Altogether both ITS 1 and 2 sequences were substantially longer than those of other fungal crude drugs such as Ganoderma lucidum and Polyporus umbellatus. Thus, Poria cocos could be distinguished from these crude drugs and fakes by comparing the nucleotide length of PCR products of ITS 1 and 2. Contrary to the basic homogeneity in ITS 1 and 2, three types (Group 1, 2, 3) of the 28S rRNA gene with distinctive differences in length and sequence were found. Furthermore, Group 1 could be divided into three subgroups depending on differences at nucleotide position 690. Products with different types of 28S rRNA gene were found in crude drugs from Yunnan and Anhui Provinces as well as the Korean Peninsula, suggesting that the locality of the crude drugs does not guarantee genetic uniformity. The result of DNA typing of Poria cocos may help discrimination of the quality of the crude drug by genotype.     

Genetic profiling of Sasa species by analysis of chloroplast intron between rbcL and ORF106 and partial ORF106 regions

Kuma-zasa is Japanese folk medicine derived from plants of genus Sasa, family Bambusaceae. Although the plants of origin of Kuma-zasa were reported to be Sasa palmata, S. senanensis, S. yahikoensis, and S. kurilensis, authentication of those plants was difficult because of similarity in morphology. Several methods for the classification of genus Sasa are available, but none involve a genetic approach. Here, we performed the genetic profiling of genus Sasa, including the four species used medicinally. Thirteen sequences were observed in chloroplast DNA intron between rbcL and ORF106 and partial ORF106 regions of 34 specimens of 16 Sasa species and one specimen of Phyllostachys pubescens. We observed differences in alignment in this region among the specimens. The analyzed lengths varied from 759 to 821 bp depending on the specimen. There were nine base substitutions, eight successive thymines or adenines, and one to three repeat units of 31 bp. Moreover, we could not find species-specific alignment: different alignments were observed in specimens of the same species, while the same alignment was observed in specimens of different species. In the phylogenetic tree reconstructed by maximum parsimony analysis, medicinally used species did not form a cluster, although most of them were positioned close to each other. The genetic profiling of Sasa species would be of use in determining the botanical origin of the herbal medicine derived from the leaves of Sasa plants.     

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.     

台湾海峡海洋沉积物放线菌的多样性

目的探究台湾海峡海洋沉积物中放线菌的多样性及发现合成药物先导化合物的新菌源。方法采用6种选择性培养基分离15份来自台湾海峡沉积物样品中含有的放线菌。挑选不同培养特征的放线菌进行初步分类鉴别、16S rRNA基因序列系统进化分析及基于PCR的烯二炔抗生素基因筛选。结果共分离到497株放线菌,挑选的95株放线菌分别属于放线菌7个科,11个属。16S rRNA基因序列分析结果提示分离到的小单孢菌科菌种存在数个潜在新种,95株菌中有27%的菌株含有烯二炔抗生素核弹头的生物合成基因片段。结论海洋环境蕴含丰富的放线菌资源,具有产生烯二炔类抗生素的潜能。     

Correlation of camptothecin-producing ability and phylogenetic relationship in the genus Ophiorrhiza

Camptothecin (CPT) is an essential precursor of semisynthetic chemotherapeutic agents for cancers throughout the world. In spite of the rapid growth of market demand, CPT raw material is still harvested by extraction from Camptotheca acuminata and Nothapodytes foetida because its total synthesis is not cost-effective. In this study, we examined eight species of the genus Ophiorrhiza (Rubiaceae) from Thailand as novel alternative sources of CPT. CPT and/or 9-methoxy camptothecin (9-MCPT) were detected at different amounts in the leaf and root extracts of five species. We found that the CPT production ability of Ophiorrhiza spp. in Thailand was related mainly to species, not habitat. Chloroplast MATK and nuclear TOPI genes of eight species were investigated and compared with those of other Ophiorrhiza sequences from GenBank in order to classify and study the evolution in this genus. The molecular phylogenetic trees of both separated and combined MATK and TOPI nucleotide sequences revealed a major clade of Ophiorrhiza taxa correlated with production of CPT and its derivatives. Several amino acid markers of CPT- or 9-MCPT-producing Ophiorrhiza plants were also suggested from the alignment of TopI amino acid sequences. Our findings suggest that genetic factors play an important role in determining the CPT- and 9-MCPT-producing properties of Ophiorrhiza plants. Consequently, MATK and TOPI gene sequences could be utilized for the prediction of CPT and 9-MCPT production ability of members of Ophiorrhiza.     

东南亚燕窝的12S rRNA和Cytb基因序列分析

目的 对实地采集的25份未进行任何加工的东南亚燕窝样品的12SrRNA和cytb基因序列进行分析,确定东南亚地区燕窝来源物种的真实身份,其中包括两份珍贵的采集自马来西亚燕洞的洞燕和血燕标本。方法 25份未经过任何加工的样品采集自马来西亚、新加坡和越南地区的燕洞和燕屋,以直接提取自燕窝的基因组DNA为模板,利用重新设计的引物扩增得到12SrRNA和cytb 基因序列,并进行克隆测序。结果 测序结果与GenBank中的同源序列进行BLAST比对分析并构建亲缘关系树,结合样品采集地的金丝燕种类信息初步确定爪哇金丝燕(Aerodramus fuciphagus)和大金丝燕(Aerodramus maximus)。结论 分析结果证实扩增的均为正确的来自对应金丝燕属的线粒体基因序列,结合金丝燕种源地的物种分布资料和基因序列信息,为制定进口燕窝的质量标准提供了依据。     

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。     

中药材龟甲的分子鉴定研究

用PCR产物直接测序法对中药材龟甲(板)进行鉴别。从乌龟 Chinemys revesii 和其他20种产地为中国或东南亚国家的龟类的组织材料中提取DNA,扩增约110bp的线粒体12SrRNA,基因片段并进行序列分析,构建了21种龟类的12SrRNA基因片段序列数据库。序列比较的结果表明乌龟与其它20种龟类的这段序列均有差别,序列差异在3.7～15.7%之间。从江苏省药品检验所提供的19块龟甲检品上各取样0.1～0.5g提取 DNA,扩增与上述相同的基因片段,与构建的数据库进行比较,结果表明19块龟甲中只有3块的原动物为乌龟,其余的龟甲均为混淆品。本文的结果为药材龟甲的鉴定找到了有效、可靠的分子遗传标记方法。     

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.     

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.     

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。     

北柴胡、紫花阔叶柴胡叶绿体全基因组解析及柴胡属药用植物叶绿体基因组比较与系统发育分析

柴胡属(Bupleurum L.)是伞形科(Apiaceae)中具有重要经济价值的药用类群。本研究利用Illumina HiSeq X Ten平台测序获得北柴胡(B.chinense DC.)和紫花阔叶柴胡(B.boissieuanum H.Wolff)的叶绿体全基因组序列,对其进行了组装、注释和特征分析,并与同属已发表的叶绿体全基因组进行了比较和系统发育分析。北柴胡和紫花阔叶柴胡叶绿体全基因组大小分别为155458、155800 bp,均为由一个大单拷贝区(large single copy,LSC;85343、85804 bp)、一个小单拷贝区(small single copy,SSC;17495、17410 bp)和一对反向重复区(inverted repeat,IRa/IRb;26310、26293 bp)构成的环状四分体结构;两者分别注释得到129个基因,包括84个蛋白编码基因、37个tRNA基因和8个rRNA基因;此外,两者重复序列的类型与分布模式相似,但数量有所差异。比较基因组学分析结果表明,柴胡属植物叶绿体全基因组大小、结构、GC含量及基因组成和排列顺序等在种内、种间均高度保守,IRs区未出现明显扩张或收缩;序列的种间变异高于种内,非编码序列(包括基因间区和内含子)变异高于编码基因序列,LSC和SSC区序列变异高于IRs区;此外,筛选到11条核苷酸多样性较高的种间高变异序列,分别位于LSC和SSC区。系统发育分析结果强烈支持柴胡属为单系,其中,北柴胡同种不同个体聚为一支,并与紫花鸭跖柴胡(B.commelynoideum H.Boissieu)亲缘关系最近,而紫花阔叶柴胡与三岛柴胡(B.falcatum L.)亲缘关系更近。本研究将为柴胡属药用植物的分类鉴定、系统发育及资源开发利用等相关研究提供基础。