小克银汉霉核糖体DNA PCR扩增产物的限制性片段长度多型性

应用一对寡核苷酸引物ITSl与ITS4对核DNA G+C百分数小于30％的小克银汉霉(Cunninghamella)属的核糖体DNA(rDNA)内转录间区(ITS)进行了扩增。测试的属于10个种和变种的22株菌都得到了扩增产物。在同属不同种之间扩增的ITS片段长度有巨大差别。据此可分为三组。这三组所含分类群数及其DNA长度分别为：第一组4种1变种,小于 764碱基对(bp);第二组2种,765～824 bp;第三组2种l变种,大于825 bp。所研究的许多种中,特别是第三组,单凭其PCR产物的长度就能区分开来。但在第一、二组就需借助限制性内切酶的分析才能予以区分。我们选用了Rsa I,Tru 9 I,和Hinf I 4种限制性内切酶,对所有扩增产物进行了限制性片段长度多型性(RFLPs)的分析。在雅致小克银汉霉(C elegans),巴西玉蕊小克银汉霉(C.bertholletiae)、和布拉氏霉(C. blakesleeana )各种的限制性酶切图谱,种内非常一致而种与种之间有差别。反之,在某些种其限制性酶切图谱不仅种间互不相同,在种内不同株之间也出现1～3种酶切图型的差异。在这种情况下,只能综合各种资料才能将它们区分开来。本研究肯定了PCR-RFLP在区分小克银汉霉种和变种上的意义,并发现种内个体的差异。这在我们后来所进行的序列分析研究中得到了进一步的证实。     

小克银汉霉核糖体DNAPCR扩增产物的限制性片段长度多型性

应用一对寡苷酸引物ＩＴＳ１与ＩＴＳ４对核ＤＮＡＧ＋Ｃ百分数小于３０％的小克银汉霉属的核糖体ＤＮＡ（ｒＤＮＡ）内转录间区（ＩＴＳ）进行了扩增。测试的属于１０个种和变种的２２株菌都得到了扩增产物,在同属不同种之间扩增的ＩＴＳ片段长度有巨大差别。据此可分为三组。这三组所含分类群数及其ＤＮＡ长度 ：第一组４种１变种,小于７６４碱基对;第二组２种,７６５－８２４ｂｐ;第三组２种１变种,大于８２５ｂｐ。所研究     

中国大麦叶绿体DNA和核糖体RNA基因限制性片段长度多型性

本文报道了我们对我国不同大麦区80份大麦品种叶绿体DNA和核糖体RNA基因限制性片段长度多型性的研究。结果表明:rDNA间隔序列长度存在丰富的多样性,80份材料中出现了8种长度变异炎型共组成8种表现型。长度变异类型及其表现型在地理分布上存在着明显的区域性。推测这种分布上的地区性与植物对环境的适应性有关。所用的两个叶绿体DNA克隆片段未检测到限制性片段长度多型性,说明栽培大麦叶绿体DNA变异程度低。     

小鼠DNA的限制性片段长度多态性分析

用人类肌红蛋白基因内含子中３３ｂｐ的小卫星ＤＮＡ重复序列为探针,经ｐＢＲ３２２的ＥｃｏＲ Ｉ正向测序引物和α－３２Ｐ－ｄＣＴＰ在Ｔａｑ ＤＮＡ聚合酶作用下做合成标记,与６种小鼠的ＤＮＡ限制性片段杂交。结果显示,每个品系平均出现可分辨带纹在１３条以上,不同品系小鼠间的杂交带纹表现出高度的多态性。多态性带纹主要分布在６￣２３ｋｂ区段;同一近交系的不同个体间的带纹特点及分布完全一致;ＣＳ７与ＡＫＲ及二者     

用于扩增较大片段DNA的PCR方法

用于扩增较大片段ＤＮＡ的ＰＣＲ方法方向东,陈淳（中科院上海生物化学研究所国家分子生物学实验室,上海２０００３１）诸江（上海第二医科大学上海血液学研究所,上海２０００２５）关键词ＤＮＡ扩增,ＰＣＲ自从Ｔａｑ（Ｔｈｅｒｍｕｓａｑｕａｔｉｃｕｓ）ＤＮＡ聚合...     

水环境细菌16S rNDA限制性片段长度多型性及群落结构分析

用细菌16S核菌体RNA基因（rDNA）限制性片段长度多型性描述了水环境细菌群落结构。从环境水样中直接分离DNA,以细菌特异的引物扩增16S rDNA。构建质粒文库,随机分离重组质粒,用限制性内切酶消化获得16S rDNA基因型,用基因型的种类及频率描述特定水体生境的细菌群落结构,该方法在分析水体隐含遗传多样性、揭示污染的生物学效应和评价水环境质量等方面具有重要的应用价值。     

荧光标记DNA扩增片段长度多态性方法的改进

采用常规PCR试剂和合成的接头和引物,其中MseⅠ引物为荧光标记物FAM标记,并对扩增片段长度多态性(AFLP)程序进行了改进,优化了PCR反应和电泳条件,建立了一个新的、有效的反应体系,降低了实验费用,经比较实验结果与AFLP荧光标记试剂盒的实验效果一致.     

影响籼稻DNA限制性片段长度多态性检测的因素分析

对窄叶青（籼稻）和京系１７（粳稻）的ＲＦＬＰ进行了系统分析。结果表明,某种酶多态性检测能力与同时检测到此多态性的其余酶的数目之间存在极显著的正相关（ｒ＝０．９６２＊＊）。由此推论,籼粳稻大部分ＲＦＬＰ可能来自大范围ＤＮＡ的结构变化而不是碱基取代。ｃＤＮＡ克隆虽然具有高度的保守性,但却具有比基因组克隆更高的多态性检测能力。在实验使用的探针范围内,探针长度和检测到多态性无相关性。由探针检测到的多态性位     

黑叶猴和灰叶猴的线粒体DNA限制性片段长度多态研究

本文以15种限制性内切酶分析黑叶猴和灰叶猴种内及种间mtDNA多态。从各个样品中分别检出了41—50个酶切位点。综合15种限制内酶的酶切类型,在2只黑叶猴和2只灰叶猴中分别检出了两种限制性类型,并与其4个地理来源相对应。结合恒河猴和红面猴的资料,构建了4种猴科动物的分子系统树。结果表明,黑叶猴和灰叶猴种内的分歧分别始于30和35万年以前,两种叶猴的分离始于190万年以前,猴亚科和疣猴亚科的分离应早于1100万年。叶猴属在中国的扩散不是很晚才发生的。     

RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER AND 5.8S REGIONS IN JAPANESE ALEXANDRIUM SPECIES (DINOPHYCEAE)1

The 5.8S ribosomal RNA (rDNA) gene and flanking internal transcribed spacers (ITS1 and ITS2)from 9 isolates of Alexandrium catenella (Whedon and Kofoid) Taylor, 11 isolates of A. tamarense (Lebour) Taylor, and single isolates of A. affine (Inoue et Fukuyo) Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from various locations in Japan were amplified using the polymerase chain reaction (PCR) and subjected to restriction fragment-length polymorphism (RFLP) analysis. PCR products from all strains were approximately 610 bp, inclusive of a limited region of the 18S and 28S rRNA coding regions. RFLP analysis using four restriction enzymes revealed six distinct classes of rDNA (“ITS types”). Restriction patterns of A. catenella were uniform at the intra-specific level and clearly distinguishable from those of A. tamarense. The patterns associated with A. tamarense (“tamarense group”) were also uniform except for one strain, WKS-1. Some restriction fragments from WKS-1 were in common with those of A. catenella or A. tamarense, whereas some were distinct from all Alexandrium species tested. Alexandrium affine, A. insuetum, and A. pseudogonyaulax carry unique ITS types. The ITSs of the “tamarense group” exhibit sequence heterogeneity. In contrast, the ITSs of all other isolates (including WKS-1) appear homogeneous. RFLP analysis of the 5.8S rDNA and flanking ITSs regions from Alexandrium species reveals useful taxonomic and genetic markers at the species and/or population levels.     

RIBOSOMAL DNA LENGTH POLYMORPHISMS WITHIN POPULATIONS OF XYLARIA MAGNOLIAE (ASCOMYCOTINA)

Xylaria magnoliae forms abundant conidial and sexual fruiting structures on the fallen strobili of Magnolia spp. throughout the southeastern United States. Populations of X. magnoliae may be identified as isolates originating from a single strobilus, among different strobili from different host trees, and different host tree localities. We examined the stratified structure of X. magnoliae populations by analysis of their ribosomal DNA restriction patterns. A sample of 48 single-conidial isolates was obtained from strobili of both Magnolia grandiflora and M. fraseri in the piedmont and mountains of North Carolina and Georgia. Southern blotting experiments revealed considerable variation in rDNA lengths between and within different isolates. Although a single rDNA phenotype usually dominates within a particular collecting locality, different phenotypes were also observed within a locality and even between isolates obtained from a single strobilus. Some rDNA restriction phenotypes are distributed over localities separated by up to 400 km. These results suggest a fine-grained population structure for X. magnoliae, together with the potential for genetic exchange and dispersal between widely separated localities.     

木耳属真菌rDNA特异性扩增片段的RFLP研究

对木耳属8个种25个菌株的ITS和28SrDNA5’端两个区域分别进行了PCR扩增和限制酶切片段长度多态性（RFLP）研究。ITS－RFMP研究结果表明,HaeⅢ可将黑木耳与其它种区分开,MspⅠ可将盾形木耳、角质木耳、琥珀木耳和黑木耳4个种区分开,而供试的HaeⅢ、TaqⅠ、HinfⅠ和MaPⅠ这四种限制酶均不能将皱木耳、大木耳、网脉木耳及毛木耳4个种区分开,表明它们之间的亲缘关系较近;结果还表明,ITS—rDNA拷贝在毛木耳和琥珀木耳种内是异质性的,而在黑木耳种内是同质性的。285rDNA-RFLP研究结果表明,供试的4种限制酶中,仅MspⅠ可将盾形木耳和角质木耳区分开,而不能将其它种区分开,这显示了28SrDNA序列在木耳属不同种间的保守性。     

木耳属真菌rDNA特异性扩增片段的RFLP研究*

对木耳属8个种25个菌株的ITS和28SrDNA5’端两个区域分别进行了PCR扩增和限制酶切片段长度多态性（RFLP）研究。ITS－RFMP研究结果表明,HaeⅢ可将黑木耳与其它种区分开,MspⅠ可将盾形木耳、角质木耳、琥珀木耳和黑木耳4个种区分开,而供试的HaeⅢ、TaqⅠ、HinfⅠ和MaPⅠ这四种限制酶均不能将皱木耳、大木耳、网脉木耳及毛木耳4个种区分开,表明它们之间的亲缘关系较近;结果还表明,ITS—rDNA拷贝在毛木耳和琥珀木耳种内是异质性的,而在黑木耳种内是同质性的。285rDNA-RFLP研究结果表明,供试的4种限制酶中,仅MspⅠ可将盾形木耳和角质木耳区分开,而不能将其它种区分开,这显示了28SrDNA序列在木耳属不同种间的保守性。     

中国脊髓灰质炎Ⅰ型野毒株的PCR－RFLP法分析

近年来我国流行地区分离的脊髓灰质炎病毒多为Ⅰ型野毒。本文报导对我国一些省、自治区防疫站从急性驰缓性麻痹病人粪便中分离的７７株Ⅰ型野毒株,经ＰＣＲ－ＲＦＬＰ法分析结果,发现不同地区分离的野毒株在电泳图像上有明显差异。根据电泳图像所显示的三种酶切条带的数目,可分为１４个类型,而且同一省、自治区内不同地区（如新疆）,或同一地区不同年份（如广东）的毒株也存在着明显差异。但在某些相邻省份如广东省与海南省,９２年流行毒株电泳图像完全一致。说明本法虽不如核酸序列分析精确,但在一定程度上也能反映毒株间的差异,有助于说明流行株的亲疏关系,可用于研究毒株的分子流行病学。     

EVOLUTION OF RIBOSOMAL DNA: FIFTY MILLION YEARS OF RECORDED HISTORY IN THE FROG GENUS RANA

Evolution of nuclear ribosomal DNA (rDNA) arrays of frogs of the genus Rana was examined among 32 species that last shared a common ancestor approximately 50 million years ago. Extensive variation in restriction sites exists within the transcribed and nontranscribed rDNA spacer regions among the species, whereas rDNA coding regions exhibit comparatively little interspecific variation in restriction sites. The most parsimonious phylogenetic hypothesis for the evolution of the group was constructed based on variation in restriction sites and internal spacer lengths among the 32 species of Rana and one species of Pyxicephalus (examined for outgroup comparison). This analysis suggests that R. sylvatica of North America is more closely related to the R. temporaria group of Eurasia than to other North American Rana. The hypothesized phylogeny also supports the monophyly of the R. boylii group, the R. catesbeiana group, the R. palmipes group, the R. tarahumarae group, and the R. pipiens complex. Furthermore, the restriction site data provide information about the evolution within and among these species groups. This demonstrates that restriction site mapping of rDNA arrays provides a useful molecular technique for the examination of historical evolutionary questions across considerable periods of time.     

PHYLOGENETICS OF THE LISIANTHIUS SKINNERI (GENTIANACEAE) SPECIES COMPLEX IN PANAMA UTILIZING DNA RESTRICTION FRAGMENT ANALYSIS

The well-delimited and evolutionary interesting tropical shrub group, the Lisianthius skinneri (Gentianaceae) species complex, was analyzed for variation in nuclear ribosomal DNA and chloroplast DNA by restriction endonuclease fragment analysis. A most parsimonious tree using variations in both DNAs was constructed for seven populations in the group by including an appropriate outgroup. This phylogeny is significantly more compatible with the DNA data than most, but not all, less parsimonious phylogenies. At least two distinct lineages have independently evolved geographically restricted, cloud forest species from the putative ancestral, widespread, and lower elevation L. skinneri. Lisianthius skinneri itself is shown to be paraphyletic with populations derived separately from the two distinct lineages. Except for a switch in the placement of two populations, this DNA-based phylogeny is congruent with an isozyme-based Wagner network depicting relationships in the species complex. Relative rates of divergence, in terms of nuclear ribosomal DNA, chloroplast DNA, isozymes, and morphology, differ markedly within and between lineages. The non-transcribed spacer region of ribosomal DNA is shown to evolve in a manner that is not in accord with a molecular clock hypothesis. Small population sizes, restricted and isolated nature of populations, and probable founder events are suggested as instrumental in causing this lack of concerted divergence within and between lineages of the L. skinneri species complex.     

RESTRICTION ENDONUCLEASE ANALYSIS OF RIBOSOMAL DNA SEQUENCE VARIATION IN LAMINARIA (PHAEOPHYTA)1

The taxonomy and evolutionary relationships of species in the genus Laminaria are poorly understood. Previous studies have demonstrated significant plasticity of morphological characters used to describe taxa, and interfertility has been reported among putative species. We analyzed nuclear ribosomal DNA (rDNA) sequence variation in eight species of Laminaria (L. agardhii Kjell., L. digitata (Huds.) Lamour., L. groenlandica Rosenv. [sensu Druehl 1968], L. longicruris De la Pyl., L. longipes Bory, L. saccharina (L.) Lamour., L. setchellii Silva, and L. yezoensis Miyabe) to elucidate evolutionary relationships in this genus. Restriction maps were constructed using a small subunit rDNA probe from Costaria costata (Turn.) Saunders, an rDNA repeat from the nematode Caenorhabditis elegans, and 11 hexameric restriction endonucleases in an annealing analysis of genomic DNA. Laminaria rDNA restriction maps were compared to each other and to that of the outgroup taxon, C. costata. rDNA restriction maps of Laminaria species and C. costata were similar. Restriction fragment length polymorphisms mapped to both the coding regions and the nontranscribed spacer of rDNA. Laminaria species were distinguished with this method. The restriction maps of L. agardhii, L. saccharina, and L. longicruris were identical, supporting a previous hypothesis that these species are conspecific. Comparison of restriction maps of Laminaria species suggested that the generic subdivision of Sections Simplices and Digitatae may be invalid.