中药材苦地胆及其混淆品的DNA指纹鉴定

用聚合酶链式反应方法，包括臆断引物聚合酶链式反应技术和随意引物扩增的多态性ＤＮＡ技术，鉴定中药材苦地胆及其混淆品，结果表明：用六个长的和一个短的引物扩增的苦地胆及其混淆品基因组ＤＮＡ指纹和多态性，可在琼脂糖凝胶上明显地区别开来。根据这些指纹图谱及由此估算的相似度值，证实从福建，台湾，香港以及澳门获得的商品药材苦地胆的基原为菊科植物地胆草。     

不同产地白及遗传多态性的RAPD分析

目的 研究国内不同产地白及的遗传多样性和亲缘关系。方法 利用随机扩增多态DNA （random amplified polymorphic DNA,RAPD）引物,RAPD分子标记技术分析国内50个不同产地的白及。结果 RAPD技术扩增产物经琼脂凝胶电泳检测,从50条引物中筛选出10条（S8,S9,S14,S19,S23,S25,S28,S29,S30,S31）条带清晰的引物,10条引物共扩增出88条DNA条带,其中多态性的DNA条带数目为81条,占总数的92.05%。每个引物能扩增出5~11条DNA条带,平均可扩增出8.8条;扩增最少的引物为S19,扩增出5条DNA条带;扩增最多的引物为S29,扩增出11条DNA条带。而每个引物能扩增的多态性DNA条带数为3~11条,平均可扩增出8.6条。结论 不同产地的白及具有较丰富的遗传多样性,RAPD可有效应用于白及的遗传多样性研究。     

Genetic Ecotoxicology III: the Relationship Between DNA Strand Breaks and Genotype in Mosquito Fish Exposed to Radiation

DNA polymorphism in mosquito fish (Gambusia affinis), as revealed by RAPD (randomly amplified polymorphic DNA) and allozyme analysis, was compared to relative amounts of DNA strand breakage in blood and liver tissues. Mosquito fish were exposed to radionuclide contamination in situ and to X-rays in the laboratory. The types of RAPD metrics used were the number of RAPD bands per individual and the frequency of certain RAPD bands. In a previous study, it was noted that in some instances the number of RAPD bands and the frequency of certain RAPD bands were elevated in radionuclide-contaminated sites relative to reference sites. In the present study, it was found that the median molecular length (MML) of the DNA (which is inversely proportional to the amount of DNA strand breakage) was correlated in several cases to the number of RAPD bands per individual. In addition, for those RAPD bands that occurred at a higher frequency in mosquito fish from radionuclide-contaminated sites, DNA strand breakage was often lower for those fish with than without these RAPD bands. RAPD data obtained on mosquito fish exposed to X-rays in the laboratory paralleled those from the field. Furthermore, analysis showed that heterozygotes for the allozyme locus nucleoside phosphorylase were more prevalent in radionuclide-contaminated sites and had fewer DNA strand breaks than did homozygotes. These results provide additional evidence that changes in population genetic structure of mosquito fish exposed to a genotoxicant (radiation) can be detected at the DNA level.     

不同居群白芍的DNA随机扩增多态性研究

目的分析6个不同居群白芍的遗传多样性,为白芍的种质鉴定及遗传多样性分析提供依据。方法运用随机扩增多态性DNA(RAPD)技术,对浙江磐安、四川中江、安徽亳州、上海崇明、江苏宿迁和山东荷泽居群白芍的基因组DNA进行随机扩增,利用NTsys2.10e软件计算遗传相似性,运用UPGMA法进行聚类分析并构建树状图。结果共筛选了70个随机引物,从中挑选出8条多态性强、重复性好的引物,共检测出215个位点,多态性位点137个,多态位点比率为63.7%,UPGMA聚类可以将不同来源的白芍很好地区分开。结论不同产地间的白芍存在丰富的遗传多样性,RAPD分子标记方法可以用来鉴定不同产地的白芍。     

利用随机扩增多态性DNA技术鉴定五种药用八角莲

现有的传统中药材鉴定方法有时很难可靠地用于药用植物八角莲的鉴定研究。本文在利用作者新建立的从干燥材料中分离高质量DNA的技术和比较不同聚合酶链反应（PCR）模板浓度的基础上,利用随机扩增多态性DNA（RAPD）技术对五种药用八角链的7个样品进行了分子鉴定。在筛选的42个引和中,12个引物产生的多态性条带,可靠地区分了这7个八角莲样品,包括3个不同地区的D.versipellis,1个D.majorense,1个D.pleiantha,1个D.furfuracea和1个D.veitchii。每个样品都找到了1－3个稳定可靠的特异分子标记。此外,本文发现D.furfuracea与D.versipellis的亲缘关系最远,与D.veitchii较近,而与D.makjorense最近。D.veitchii与D.majorense的亲缘关系比与D.versipellis,D.furfuracea和D.pleiantha更近。结果表明RAPD技术能够有效地用于这7个药用八角莲样品的分子鉴定。     

中药材蒲公英及其混淆品的DNA指纹鉴别研究

采用分子生物技术包括任意引物聚合酶链式反应（AP－PCR）和随意扩增多态性DNA（RAPD）方法扩增蒲公英及其六种土公英混淆品的基因组DNA，获得清晰可靠的DNA指纹图谱。根据琼脂糖胶上显示的DNA带型差异可鉴别蒲公英和土公英混淆品。     

Molecular authentication of Panax ginseng species by RAPD analysis and PCR-RFLP

In order to develop convenient and reproducible methods for the identification of ginseng drugs at a DNA level, randomly amplified polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses were applied within Panax species. To authenticate Panax ginseng among ginseng populations, RAPD analysis was carried out using a 20 mer-random primer. The similarity coefficients among the DNA of ginseng plants analyzed were low, ranging from 0.197 to 0.491. In addition, by using PCR-RFLP analysis, very different fingerprints were obtained within Korean ginseng plants. These results suggest that these methods are able to authenticate the concerned Panax species. Broader application of this approach to authenticate other morphologically similar medicinal materials is rationalized.     

Application of random amplified polymorphic DNA (RAPD) to detect genotoxic effect of trifluralin on maize (Zea mays)

Trifluralin is a widely used dinitroaniline herbicide throughout the world. However, limited efforts have been made to study its genotoxic effects on different plants. The present study aimed to evaluate the herbicide’s genotoxic potential on maize (Zea mays) by using the random amplified polymorphic DNA (RAPD) technique. For this purpose, maize seedlings were treated with aqueous solutions of trifluralin at concentrations ranging from 0.5 to 3 ppm for 7 days. In the RAPD analyses, 15 primers were used and 91 bands were obtained, with an average of 6.06 bands per primer in the control seedlings. After trifluralin treatment, significant changes were observed in RAPD profiles. These changes included loss of normal bands and appearance of new bands, in comparison to the control group, and they were dose dependent. In addition, root growth and total soluble protein level in trifluralin-treated seedlings were analyzed and compared for genomic template stability (GTS), which was performed for the qualitative measurement of changes in randomly amplified polymorphic DNA (RAPD) profiles. The results showed that GTS, root growth, and total soluble protein content of the seedlings gradually decreased with an increase in trifluralin concentration. These findings suggest that the RAPD technique is a useful biomarker assay to evaluate the genotoxic effects of herbicides on plants.     

人参和其他中草药的遗传学鉴定(英文)

The main objective of this paper is to review the chemical and genetic methods used in authentication of ginseng, especially the recent advances in microsatellite genotyping and its application to the authentication of other traditional Chinese medicines (TCM). The standardization and modernization of TCM hinge on the authentication of their botanical identities. Analysis of well-characterized marker compounds is now the most popular method for identifying the herbal materials and quality control of TCM, eg, ginsenoside profiling for authentication of Panax species. However, in many herbal species the chemical composition of the plant changes with the external environment and processing conditions, which lowers the reliability of these authentication methods. In the light of the advances in molecular biotechnology in the past few decades, genetic tools are now considered to provide more standardized and reliable methods for authentication of herbal materials at the DNA level. These genetic tools include     

Differentiation of Lycium barbarum from its related Lycium species using random amplified polymorphic DNA.

The RAPD (random amplified polymorphic DNA) technique was applied for the first time to distinguish Lycium barbarum from other closely related species of the same genus. In this study, eight samples were collected, including five species, two varieties and one cultivated variety. A total of fifty arbitrary primers were used in the RAPD analysis. Distinctive DNA fingerprints corresponding to different Lycium species were successfully obtained from ten primers. Similarity index (S.I.) analysis revealed that the values are higher between intraspecies than interspecies. These results confirmed that the RAPD technique can be employed for distinguishing closely related species of Lycium.     

铁皮石斛野生居群遗传多样性的RAPD分析与鉴别

目的采用RAPD分子标记技术，对铁皮石斛8个野生居群的遗传多样性、亲缘关系以及分子鉴别等进行研究。方法筛选随机引物进行RAPD分析，通过UPGMA聚类，研究铁皮石斛各居群间的遗传关系，构建居群亲缘关系的分子系统树；利用特异性条带对铁皮石斛野生居群进行指纹分析鉴别。结果共筛选出10个有效引物，在8个野生居群材料的RAPD扩增中共得到439个位点，平均每个引物扩增出43.9个位点，每个居群扩增出54.9个位点；在所获得的104条可重现谱带中，9条是单态的，95条是多态的，多态性程度达91.35%，遗传距离在0.590～0.727之间，平均为0.686。结论铁皮石斛居群间遗传差异明显，具有较丰富的遗传多样性；RAPD可以作为铁皮石斛野生居群遗传多态性、居群亲缘关系和分子鉴别研究的有效手段；引物S412可以有效鉴别铁皮石斛的8个野生居群。     

Use of RAPD to detect DNA damage induced by nitrofurazone in marine ciliate, Euplotes vannus (Protozoa, Ciliophora)

The random amplified polymorphic DNA (RAPD) assay was evaluated as a potential tool to detect the ecotoxicity induced by nitrofurazone in marine ciliate, Euplotes vannus. The data revealed a reduction in viability of the test ciliates with increasing nitrofurazone concentration in the range of 0-24 mgl(-1) and time of exposure from 24 to 96 h. The nitrofurazone treated ciliates were subjected to DNA damage analysis by RAPD assay. Among the 33 test RAPD primers used in this study, 11 primers with 60-70% GC content produced unique polymorphic band patterns. A total of 213 bands of 155-3317 bp in molecular size range were observed in the untreated cells. In comparison with the control ciliates, the nitrofurazone treated groups showed differences in RAPD profiles with respect to the band intensity, disappearance of bands and appearance of new bands of amplified DNA. The variation of RAPD profiles showed both the time- and concentration-dependent relationships. The data suggested significant genomic template instability, which corresponds well with the viability of the test ciliates. Thus the results demonstrated the potential of the RAPD assay for application as a powerful tool for detecting genotoxicity induced by fishy drugs in aquatic environment.     

Molecular Characterization of Contaminant-Indicative RAPD Markers

This paper describes genetic markers which can be used to study selection and genetic adaptation of organisms to radionuclide and other types of contaminant stress. Previous research using the randomly amplified polymorphic DNA (RAPD) technique has identified several markers which revealed genetic differences between contaminated and reference western mosquitofish (Gambusia affinis) populations. Experimental evidence suggested that these markers may be associated with loci involved in determining relative fitness in radionuclide-contaminated environments ("contaminant-indicative markers"). In the present study, Southern blot analyses show these markers to be highly conserved in DNA sequence and molecular length in sea urchins, mosquitofish, herring gulls and humans. Such conservation is thought to be rare among RAPD bands. Results of DNA sequencing efforts did not provide definitive evidence as to the identity of these loci, but indicated that short segments (<40 bp) of known DNA sequences were homologous to various regions of the RAPD sequences. Furthermore, the regions of homology seemed to be non-randomly distributed along the length of the RAPD markers. Although the identity of these bands is still unknown, the high degree of conservatism suggests that these loci might play an important role in molecular processes.     

中药材分子鉴别新方法:锚定引物扩增多态性DNA的研究

为了寻找稳定性好、可操作性强的分子鉴定新方法，在充分吸取RAPD优势的基础上，对其引物和退火温度进行了改进。本文以人参、西洋参为例进行了方法的探索和各种验证，并推广应用到天花粉以及白芷类药材的鉴别。结果显示引物Pg-q36F得到人参、西洋参及其9种伪品的多态性条带。对于人参、西洋参的鉴别结果与文献鉴别方法结果一致，并且具有更高的稳定性。引物TkS1-64F得到了天花粉及其11种伪品的多态性条带，引物AfS1-100F得到白芷及其3种伪品的多态性条带，均能准确鉴别各种药材。实验结果证明本方法具有简单易行、稳定性和重复性好、提供的信息量大等优点，是一种极具前途的中药材分子鉴定新方法，被命名为锚定引物扩增多态性DNA(anchored primer amplification polymorphism DNA，APAPD)。     

Genetic and metabolic diversity in Stevia rebaudiana using RAPD and HPTLC analysis

Abstract

Context: Stevia rebaudiana Bertoni (Asteraceae) is an important medicinal plant and is much used due to its zero calories sweetening property. Stevia leaves as well as its extracts and pure compounds are currently used in the preparation of several medicines, food products and neutraceuticals.

Objective: To study the genetic and metabolic variability in S. rebaudiana among accessions of different geographical regions of India using random amplified polymorphic DNA (RAPD) markers and high-performance thin layer chromatography (HPTLC) analysis.

Materials and methods: The RAPD analysis of Stevia rebaudiana (11 accessions) was carried out using 20 random operon primers. Dendrogram was constructed for cluster analysis based on the unweighted pair group method with arithmetic means (UPGMA) using Winboot. The HPTLC analysis of all samples was carried out on silica using acetone:ethyl acetate:water (5:4:1, v/v/v) for fingerprinting and quantification of stevioside and rebaudioside A at 360?nm after spraying with anisaldehyde sulphuric acid.

Results: Ten out of 20 primers screened were found most informative; amplification products of the genotypes yielded a total of 87 scorable bands (67 polymorphic), whereas genetic similarity (GS) coefficient (0.01–0.08) and polymorphism (67.24–92.40%) showed huge variability. Similarly, HPTLC analysis showed large variation among different samples with respect to their presence or absence of metabolite and their concentration.

Conclusion: Out of the 11 Stevia accessions, Delhi and Mohali varieties showed much relatedness with each other and were concluded to be the superior genotype in context to RAPD and HPTLC analysis. The information obtained here could be valuable for devising strategies for cultivating this medicinal plant.     

Genotoxic effects of 8-hydroxylquinoline in loach (Misgurnus anguillicaudatus) assessed by the micronucleus test,comet assay and RAPD analysis

This study was a preliminary step in evaluating the genotoxic effects of 8-hydroxylquinoline (8-HOQ) in loach (Misgurnus anguillicaudatus) using the micronucleus, comet and RAPD assays. In the micronucleus test and comet assay, the micronuclei rate (%) and three comet parameters (trailing rate, tail length and tail moment) in treated fish increased with increasing 8-HOQ concentration and treatment time. These results showed that exposure to 8-HOQ significantly induced genetic toxicity in loach blood cells. A subsequent RAPD assay also showed that 8-HOQ induced a genotoxic effect in loach. Among the 23 tested RAPD primers, 11 primers produced unique polymorphic band patterns and generated RAPD profile variations that displayed the band intensity, disappearance of bands and appearance of new bands of amplified DNA in the 8-HOQ-treated genomic DNA samples. In addition, the variation in RAPD profiles was time- and concentration-dependent. These results suggested that 8-HOQ is potentially harmful to fish and may be a toxic contaminant in the aquatic environment.     

Assessing phylogenetic relationships of Lycium samples using RAPD and entropy theory

AIM: To evaluate the phylogenetic relationships among related species of Lycium samples. METHODS: Random amplified polymorphic DNA (RAPD) fingerprinting and lab-on-a-chip electrophoresis techniques were used to analyze the characteristics of Lycium species. Seven species and 3 varieties of Lycium were studied. Based on RAPD fingerprint data obtained from 11 primers, we proposed a new index, called dispersivity, using entropy theory and projection methods to depict the diversity of the DNA fingerprints. RESULTS: Using the proposed dispersivity, primers were sorted and the dendrograms of the 7 species and 3 varieties of Lycium were constructed synthetically by merging primer information. CONCLUSION: Phylogenetic relationships among Lycium samples were constructed synthetically based on RAPD fingerprint data generated from 11 primers.     

Genetic ecotoxicology II: population genetic structure in mosquitofish exposed in situ to radionuclides

In 1977, approximately 250 mosquitofish (Gambusia affinis) from a relatively uncontaminated site (Crystal Springs) were transplanted into a small pond on the Department of Energy Oak Ridge Reservation which is heavily contaminated with radionuclides (Pond 3513). Starting in 1992, DNA polymorphism was evaluated using the RAPD (Randomly Amplified Polymorphic DNA) and allozyme genotype techniques to determine if genetic differentiation had occurred between the two populations. Fish from a second radionuclide-contaminated population (White Oak Lake) and another unrelated non-contaminated population (Wolf Creek) were also examined. For the RAPD analyes, 15 RAPD primers (from a total of 40) were found to produce polymorphic banding patterns in at least two of the four populations and subsequently were used to produce a total of 142 bands. Data generated by these RAPD primers indicated an increased genetic diversity in radionuclide-contaminated sites relative to reference sites. Furthermore, the patterns from six RAPD primers produced a higher average number of bands when using DNA from radionuclide- contaminated populations than from non-contaminated, and for three RAPD primers the average number of bands from radionuclide- contaminated populations was lower. In addition, 17 bands occurred at a higher frequency in the radionuclide-contaminated compared to the non-contaminated populations. For the allozyme analyses, it was found that there was a higher percentage of polymorphism and heterozygosity in the radionuclide-contaminated relative to non-contaminated sites. These findings contribute to our understanding of the evolutionary effects of contaminant exposure as well as to the development of population-level biomarkers     

鱼腥草种质资源的RAPD分析

目的分析鱼腥草种质资源在分子水平上的遗传多样性。方法应用RAPD技术对92份鱼腥草种质资源进行检测。结果34个随机引物中有32个引物(94.1%)扩增产物具多态性。34个引物共得到200条扩增DNA片段,其中93.5%的片段具多态性。每个多态性引物平均可扩增出5.8个多态性片段。峨眉蕺菜和蕺菜种内平均遗传相似系数(genetic similarity,GS)分别为0.521和0.572,二者种间GS值为0.517。峨眉蕺菜与蕺菜中染色体数目为36的细胞型间相似程度最高,其平均GS值达0.530。栽培蕺菜类群比其野生类群遗传多样性相对较高。聚类分析表明,利用RAPD技术可将全部供试材料区分开,所有材料共划分为14类。其中,绝大多数(62个)聚为一类,且根据RAPD遗传相似系数划分的类群同地理分布有一定关系。结论(1) 鱼腥草种质资源在分子水平上确实存在较大遗传差异。(2) RAPD标记可作为构建鱼腥草DNA指纹图谱的有效工具。(3) 鱼腥草药材道地性与环境因素有关,但更大程度上由其遗传因素所决定。     

Use of RAPD to detect sodium arsenite-induced DNA damage in human lymphoblastoid cells

Inorganic arsenic is a known human carcinogen, yet its mechanism of action remains unclear. Our previous study showed that arsenite significantly induces oxidative DNA adducts and DNA-protein cross-links in several mammalian cell lines. In the present study, we used the random amplified polymorphic DNA (RAPD) assay to evaluate the possible target in the genomic DNA of human lymphoblastoid cells that were exposed to sodium arsenite. Treatment with both 10 and 80 microM arsenite for 4h induced significant changes in RAPD profiles compared with the control pattern. Two 10-mer RAPD primers (D11 and F1) produced the most distinguishable banding profiles between arsenite-treated and control genomic DNA. The sequencing of four arsenite-sensitive RAPD bands showed that the RB1CC1 and PACE4 genes might be the DNA targets of sodium arsenite treatment. We propose that arsenite may induce sequence- or gene-specific damage and then change the RAPD profile in human lymphoblastoid cells. The results of our study also show that RAPD combined with other techniques is a good tool for detecting alterations in genomic DNA and for the direct screening of new molecular markers related to arsenite-induced carcinogenesis.