人参细胞器核糖体小亚单位DNA的PCR扩增与序列测定

采用CTAB法提取了人参(Panax ginseng)根基因组DNA,根据植物叶绿体16S rDNA和线粒体18SrDNA与细菌16S rDNA序列具有高度同源性,用扩增细菌16S rDNA的一对通用引物(8f,1492r)扩增了人参细胞器核糖体小亚单位DNA.对扩增产物进行了克隆与测序,经多序列比对,扩增片段分别与已知植物叶绿体16S rDNA和线粒体18S rDNA具高度同源性,表明该对引物可以用来扩增绝大多数植物细胞器核糖体小亚单位DNA,可以作为鉴定植物叶绿体16S rDNA和线粒体18S rDNA的一种基本实验技术.     

蓖麻蚕核糖体核糖核酸基因上18S、28S和5.8S核糖体核糖核酸基因的定位

蓖麻蚕的核糖体核糖核酸(rRNA)的基因(rDNA)是多拷贝基因,其重复单位成线性方向排列。在每一重复单位中含有18S、28S和5.8S rRNA基因各一个。了解它们的排列状况是认识rDNA结构的基础。本文将无性繁殖的该rDNA用各种限制性内切酶水解后,制成Southern转移膜与放射性同位素标记的18S、28S和5.8S rRNA杂交;又将18S和28S rRNA制成Northern转移膜与放射性同位素标记的rDNA片段杂交,从而排出18S、28S和5.8S rRNA基因在rDNA上面的相对位置。     

棉铃虫18S核糖体RNA基因的序列分析及其分子系统学

克隆并分析了鳞翅目棉铃虫Helicoverpa armigera (Hübner)18S核糖体RNA基因(18S rDNA)的全序列,将该序列与鞘翅目、膜翅目、同翅目、双翅目、捻翅目和弹尾目各一种昆虫的同源保守区进行了比较。序列分析结果显示：鳞翅目和双翅目昆虫在18S rDNA结构上彼此较为相似,捻翅目昆虫的18S rDNA分子结构表现出与其它目昆虫有较大的差异,但相对与弹尾目昆虫的18S rDNA较为接近。该结果支持了有关捻翅目属于一个独立的目级分类阶元的论点。     

褐飞虱不同致害性种群体内共生菌18S rDNA部分序列比较

分离纯化了褐飞虱3种不同致害性种群体内类酵母共生菌 (yeast-like symbionts, YLS),并对其18S rDNA基因序列进行了比较。结果表明,褐飞虱3种不同致害性种群体内类酵母共生菌18S rDNA均扩增出600 bp左右的片断。依据获得的18S rDNA特异性序列,结合已知真菌的18S rDNA部分序列,构建了不同宿主的YLS分子系统树。结果显示, 褐飞虱3种不同致害性种群体内的YLS同属子囊菌亚门（Ascomycotina）的核菌纲（Pyrenomycetes）,并与此纲中的Hypomyces chrysospermus亲缘关系相对最近。     

基于18S rDNA遗传距离与GC含量对游走类纤毛虫的分子系统学研究

为了揭示游走类纤毛虫的系统发生,对寄生于淡水鱼类的车轮虫科中的6种车轮虫进行了18S rDNA的测序并获得了9个序列。采用了最大似然法(ML)与贝叶斯法(BI)对GenBank中所有游走类纤毛虫的18S rDNA序列进行了系统树的构建,并首次将SPSS与18S rDNA遗传距离结合分析了游走类纤毛虫的系统发生。研究结果进一步证实了车轮虫属(Trichodina)的非单系发生与小车轮虫属 (Trichodinella) 的有效性。此外,研究结合18S rDNA 的GC含量与遗传距离分析提出了游走类纤毛虫科属及种间新的鉴定依据: 18S rDNA 的GC含量可用于游走类纤毛虫的科属区分,且与游走类纤毛虫的分化密切相关; 18S rDNA的遗传距离在游走类纤毛虫的不同阶元中具有一定的阈值范围,即通常种内遗传距离阈值范围为0.000-0.005,属种间阈值范围为0.005-0.150,当遗传距离大于0.150时,则达到了科间水平。     

基于18S rDNA遗传距离与GC含量对游走类纤毛虫的分子系统学研究（英文）

为了揭示游走类纤毛虫的系统发生,对寄生于淡水鱼类的车轮虫科中的6种车轮虫进行了18S rDNA的测序并获得了9个序列。采用了最大似然法(ML)与贝叶斯法(BI)对GenBank中所有游走类纤毛虫的18S rDNA序列进行了系统树的构建,并首次将SPSS与18S rDNA遗传距离结合分析了游走类纤毛虫的系统发生。研究结果进一步证实了车轮虫属(Trichodina)的非单系发生与小车轮虫属(Trichodinella)的有效性。此外,研究结合18S rDNA的GC含量与遗传距离分析提出了游走类纤毛虫科属及种间新的鉴定依据:18S rDNA的GC含量可用于游走类纤毛虫的科属区分,且与游走类纤毛虫的分化密切相关;18S rDNA的遗传距离在游走类纤毛虫的不同阶元中具有一定的阈值范围,即通常种内遗传距离阈值范围为0.000—4).005,属种间阈值范围为0.005—0.150,当遗传距离大于0.150时,则达到了科间水平。     

核糖体小亚基V4和V9区引物扩增大亚湾真核浮游生物的比较研究

对大亚湾水体的环境DNA分别进行18S rDNA的V4和V9区的引物扩增,通过高通量测序技术进行测序,并比较分析二者浮游真核生物基因多样性和相对丰度。18S rDNA V4区引物扩增共检测出浮游动物56纲, 101目,浮游植物52纲, 69目; 18S rDNA V9区引物扩增共检测出浮游动物47纲, 81目,浮游植物56纲, 101目。两对引物对浮游真核生物扩增都具有较高覆盖度,在纲级别上二者的结果相近:颚足纲(Maxillopoda)是浮游动物优势类群;甲藻纲(Dinophyceae)、圆筛藻纲(Coscinodiscophyceae)、小豆藻纲(Mamiellophyceae)是浮游植物优势类群,其中甲藻纲多样性与丰度的结果相近,而18S rDNA V9区引物扩增得到的圆筛藻纲丰度高于18S rDNA V4区引物。分析结果表明, 18S rDNAV4区引物扩增的浮游动物多样性比18SrDNAV9区引物高,而18SrDNAV9区引物扩增的浮游植物多样性比18S rDNA V4区引物高。同时,通过高通量测序技术首次确定大亚湾海区大量存在着寄生型甲藻(Syndiniales),小豆藻目...     

基于荧光原位杂交的藜属植物核型分析

为精确地识别藜属植物染色体组的核型特征,该文研究了4种来自青海高原的野生藜属植物(灰绿藜、藜、菊叶香藜及杂配藜)和1种从美国引进的栽培藜麦品种PI614932-HX(3)基于染色体荧光原位杂交(rDNA FISH)的核型。利用5S rDNA和45S rDNA对5种藜属植物有丝分裂中期的染色体进行FISH研究。藜属植物的核型分析结果表明:(1)藜属植物中存在二倍体(2n=2x=18)和四倍体(2n=4x=36)两种倍性,藜麦和灰绿藜为四倍体,其余3种为二倍体。(2)藜麦、灰绿藜、藜、菊叶香藜及杂配藜的核型公式分别为2n=4x=36=34m(2AST)+2sm,2n=4x=36=32m(4AST)+4sm,2n=2x=18=16m(4AST)+2sm,2n=2x=18=18m及2n=2x=18=16m+2sm。(3)染色体由大部分的中部着丝粒染色体(m)和少部分近中部着丝粒染色体(sm)组成。(4)核型类型除了菊叶香藜为1B以外,其余均属于2B类型。(5)在藜麦、灰绿藜及藜中具有分布位置不同、数量不等的双随体。5S rDNA、45S rDNA FISH结果表明:(1)藜麦和灰绿藜的染色体上存在2对5S rDNA位点和1对45S rDNA位点,藜、杂配藜的染色体上存在1对5S rDNA位点和1对45S rDNA位点,菊叶香藜的染色体上只存在1对5S rDNA位点。(2)5S rDNA和45S rDNA位点均位于染色体的短臂上。该研究首次获得了藜属植物基于5S rDNA和45S rDNA荧光原位杂交核型,为藜属植物亲缘关系研究和细胞生物学研究提供了分子细胞遗传学依据。     

烟夜蛾18S rDNA的克隆及序列分析

利用PCR方法克降得到了烟夜蛾18S rDNA全基因序列,基因全长1904bp;构建了其全长、保守区和非保守区的系统发育树,比较了与其他已知蛾类昆虫18S rDNA全序列的同源性.结果表明,蛾类之间该基因的同源性达到92%以上,利用其多变区构建的发育树更能反映蛾类昆虫的亲缘关系;比较烟夜蛾与棉铃虫的18S rDNA序列发现,两个近缘种之间仅有lO个核苷酸的差异.     

蓖麻蚕18S rRNA基因3′末端的顺序特征

本文测定了蓖麻蚕18S rRNA基因(rDNA) 3′末端及其外侧的DNA顺序。将这一顺序与家蚕、果蝇、大鼠 18S rDNA 3′末端顺序以及大肠杆菌16 S rDNA 3′末端顺序作了比较,发现它们间有惊人的同源性。不仅如此,这些基因的3′末端所形成的茎环结构也十分相似,在3′末端还有保守的EcoRI切点。这些研究结果对了解18S rRNA 3′末端在蛋白质合成中的功能及在rRNA前体加工成熟中的作用;对于了解rRNA基因的进化打下了基础。     

Phylogenetic position of endosymbiotic green algae in Paramecium bursaria Ehrenberg from Japan

Endosymbiotic green algae of Japanese Paramecium bursaria were phylogenetically analyzed based on DNA sequences from the ribosomal DNA operon (18S rDNA, ITS1, 5.8S rDNA, and ITS2). Phylogenetic trees constructed using 18S rDNA sequences showed that the symbionts belong to the Chlorella sensu stricto (Trebouxiophyceae) group. They are genetically closer to the C. vulgaris Beijerinck group than to C. kessleri Fott et Nováková as proposed previously. Branching order in C. vulgaris group was unresolved in 18S rDNA trees. Compared heterogeneities of 18S rDNA, ITS1, 5.8S r, and ITS2 among symbionts and two Chlorella species, indicated that the ITS2 region (and probably also ITS1) is better able to resolve phylogenetic problems in such closely related taxa. All six symbiotic sequences obtained here (approximately 4000-bp sequences of 18S rDNA, ITS1, 5.8S rDNA, and ITS2) were completely identical in each, strongly suggesting a common origin.     

Chromosomal mapping of 18S-28S and 5S rRNA genes by two-colour fluorescent in situ hybridization in six sturgeon species.

The number and distribution of the 18S-28S and 5S rRNA (rDNA) gene sequences were examined on mitotic chromosomes of six sturgeon species by two-colour in situ hybridization. Four of the six species, Huso huso, Acipenser stellatus, Acipenser sturio, and Acipenser ruthenus, with about 120 chromosomes, showed from six to eight 18S-28S rDNA signals, while 5S rDNA signals were on only one chromosome pair. The two species with 250-270 chromosomes, Acipenser baerii and Acipenser transmontanus, showed from 10 to 12 18S-28S sites and two chromosome pairs bearing 5S rDNA signals. In all examined species, the rather intense 5S rDNA signals apparently overlapped those of 18S-28S rDNA. These data support the diploid-tetraploid relationships between the two chromosome groups of sturgeons. The close association between the two rDNA families in species belonging to an ancestral fish order, such as Acipenseriformes, supports the hypothesis that the association represents a primitive condition.     

18S coding sequences in amplified ribosomal DNA from Xenopus laevis oocytes are highly homogeneous, unmethylated, and lack major open reading frames.

We have used two approaches to search for sequence variants in the 18S coding region of amplified ribosomal DNA (rDNA) from Xenopus laevis oocytes. First, using clones derived from amplified rDNA, we compared the equivalent of a complete 18S coding region from two clones and short regions from two other clones with the 18S sequence previously determined from a "reference" clone. The respective sequences in all the clones were identical. Secondly, we examined greater than 60% of the 18S sequence in "pooled 18S genes" in uncloned amplified rDNA. The predominant sequence corresponded to that in the reference clone and no heterogeneities were apparent. Since many chromosomal rDNA units contribute to rDNA amplification the findings indicate that 18S coding sequences in X. laevis are largely homogeneous. The previously established sequence is the predominant one, thus providing a reliable basis for studies on 18S rRNA. Sequencing gels on uncloned amplified rDNA confirmed the absence of methylated cytosine in this DNA. The 18S sequence lacks major open reading frames.     

Segmented Gene Conversion as a Mechanism of Correction of 18S rRNA Pseudogene Located Outside of rDNA Cluster in D. melanogaster

The peculiarities of the sequences of 18S rDNA included in a 90-kb DNA segment cloned in YAC vector are described. This heterochromatic segment is situated on the X chromosome distal to the main rDNA cluster. The pseudo 18S rDNA sequence comprised undamaged stretches of rDNA interspersed with segments characterized by high density of nucleotide substitutions and insertions/deletions. The observed patchwork arrangement of unaltered rDNA sequences was considered as evidence of segmented gene conversion events between the normal and damaged genes which are thought to constitute one of the mechanisms of rDNA array homogenization. The 18S rDNA fragment (510 bp) located nearby, homologous to the internal, undamaged part of pseudo 18S rDNA, carries comparable density of randomly distributed nucleotide substitutions with no evidence of correction. Received: 8 August 1996 / Accepted: 7 December 1996     

Mapping of rDNA on the chromosomes of Eleusine species by fluorescence in situ hybridization

Mapping of rDNA sites on the chromosomes of four diploid and two tetraploid species of Eleusine has provided valuable information on genome relationship between the species. Presence of 18S-5.8S-26S rDNA on the largest pair of the chromosomes, location of 5S rDNA at four sites on two pairs of chromosomes and presence of 18S-5.8S-26S and 5S rDNA at same location on one pair of chromosomes have clearly differentiated E. multiflora from rest of the species of Eleusine. The two tetraploid species, E. coracana and E. africana have the same number of 18S-5.8S-26S and 5S rDNA sites and located at similar position on the chromosomes. Diploid species, E. indica, E. floccifolia and E. tristachya have the same 18S-5.8S-26S sites and location on the chromosomes which also resembled with the two pairs of 18S-5.8S-26S rDNA locations in tetraploid species, E. coracana and E. africana. The 5S rDNA sites on chromosomes of E. indica and E. floccifolia were also comparable to the 5S rDNA sites of E. africana and E. coracana. The similarity of the rDNA sites and their location on chromosomes in the three diploid and two polyploid species also supports the view that genome donors to tetraploid species may be from these diploid species.     

Chromosomal Mapping and Molecular Characterization of Ribosomal RNA Genes in Lebias fasciata (Teleostei, Cyprinodontidae)

Chromosome location of major (18S, 5.8S and 28S) and 5S ribosomal RNA genes (rDNAs) was examined in Lebias fasciata collected from different Italian blackish-waters, using silver (Ag)- and chromomycin A3 (CMA3)-staining and/or fluorescence in situ hybridization (FISH). Both 18S and 5S rDNA probes for FISH were obtained with polymerase chain reaction-directed cloning from genomic DNA of the examined species. Nucleolar organizer regions (NORs) containing the major rDNAs showed intraspecific polymorphism in number as detected by Ag-and CMA3-staining and FISH with the 18S rDNA probe. On the other hand, 5S rDNA loci constantly occurred on one chromosome pair and co-localized with a pair of the major rDNA loci as evidenced by two-color FISH using the 5S and 18S rDNA probes. Sequential CMA3- and Ag-NOR staining and FISH revealed apparent inactivation of some NORs. The cloned 5S rDNA was found to contain some TATA-like sequences that might play an important role in the regulation of gene expression.     

rDNA (18S-28S and 5S) colocalization and linkage between ribosomal genes and (TTAGGG)(n) telomeric sequence in the earthworm,Octodrilus complanatus (Annelida: Oligochaeta: Lumbricidae), revealed by single- and double-color FISH

Spermatogonial and metaphase I chromosomes of the lumbricid earthworm Octodrilus complanatus (Annelida: Oligochaeta) were examined using fluorescent in situ hybridization (FISH) with three repetitive DNA probes-5S rDNA, 18S-26S rDNA, and (TTAGGG)(n). Single-color FISH consistently mapped one chromosome pair per spread using either 5S rDNA or 18S-26S rDNA as probes. Simultaneous (18S-26S)-5S and (18S-26S)-(TTAGGG)(n) FISH demonstrated that repeated units of the two ribosomal families were overlapped and closely associated with telomeric sequences.     

Trends in site-number change of rDNA loci during polyploid evolution in Sanguisorba (Rosaceae)

To elucidate the evolutionary dynamics of rDNA site number in polyploid plants, we determined 5S and 18S-5.8S-26S rDNA sites for ten species of Sanguisorba (2n=14, 28, 56) and a single species of each of three outgroup genera, Agrimonia (2n=28), Rosa (2n=14), and Rubus (2n=14) by the fluorescence in situ hybridization (FISH) method. We also estimated phylogenetic relationships among these species using matK chloroplast DNA (cpDNA) sequences, and reconstructed the evolutionary history of rDNA site number based on the maximum parsimony method. The 2n=14 and 2n=28 plants of all genera except Rosa carried two 5S rDNA sites, whereas Rosa and 2n=56 plants carried four sites. The 2n=14 plants had two 18S-5.8S-26S rDNA sites, whereas Sanguisorba annua and 2n=28 plants had four or six sites. Phylogenetic analysis showed that polyploidization from 2n=14 to 2n=28 has occurred once or three times in Sanguisorba and Agrimonia. The 5S rDNA sites duplicated during each ancestral polyploidization were evidently lost after each polyploidization. However, the duplicated 18S-5.8S-26S rDNA sites were all conserved after each polyploidization. Thus, the duplicated 5S rDNA sites tend to have been eliminated, whereas those of 18S-5.8S-26S rDNA tend to have been conserved in Sanguisorba. In the most parsimonious hypothesis, 2n=14 in S. annua is a secondary, putatively dysploid state, reduced from 2n=28.     

Comparative physical mapping of the 18S-5.8S-26S rDNA in three sorghum species.

The physical locations of the 18S-5.8S-26S rDNA sequences were examined in three sorghum species by fluorescence in situ hybridization (FISH) using biotin-labeled heterologous 18S-5.8S-26S rDNA probe (pTa71). Each 18S-5.8S-26S rDNA locus occurred at two sites on the chromosomes in Sorghum bicolor (2n = 20) and S. versicolor (2n = 10), but at four sites on the chromosomes of S. halepense (2n = 40) and the tetraploid S. versicolor (2n = 20). Positions of the rDNA loci varied from the interstitial to terminal position among the four accessions of the three sorghum species. The rDNA data are useful for investigation of chromosome evolution and phylogeny. This study excluded S. versicolor as the possible progenitor of S. bicolor.