两种缘毛类纤毛虫胞质Hsp70基因序列的克隆及其系统发育分析

克隆得到2种缘毛类纤毛虫——钟形钟虫(Vorticella campanula)和螅状独缩虫(Carchesium polypinum)的胞质Hsp70基因部分序列,长度均为438bp,编码146个氨基酸。以细菌为外类群,利用最大似然法和邻接法构建包括其他5种纤毛虫在内的共26个物种的Hsp70基因氨基酸序列系统发育树,其拓扑结构显示：V．campanula和C．polypinum聚在一起,并与另2种寡膜纲的嗜热四膜虫(Tetrahymena thermophila)及草履虫(Paramecium tetraurelia)聚为姊妹枝,提示了缘毛类纤毛虫为单系,且隶属于寡膜纲的系统发育地位。     

五株纤毛虫遗传关系的ISSR分析

采用ISSR分子标记技术,尝试对四种五株纤毛虫(褶累枝虫(Epistylis plicatilis)、绿草履虫(Paramecium bursaria)、多态喇叭虫(Stentor polymorphus)、嗜热四膜虫BF1株(Tetrahymena thermophilaBF1)和嗜热四膜虫BF5株(T.ther-mophilaBF5))进行遗传关系研究。用13个ISSR引物对五株纤毛虫进行扩增,六个ISSR引物获得多态片段。根据Nei s遗传距离矩阵构建了五株纤毛虫的遗传关系树状图。UPGMA,NJ聚类图表明:两株嗜热四膜虫最先聚在一起;其次是褶累枝虫和多态喇叭虫聚在一起,然后再与嗜热四膜虫聚在一起;咽膜亚纲的绿草履虫形成独立的一枝。结果显示:①缘毛亚纲纤毛虫可能是寡膜纲中较独特的一个类群,建议提升缘毛亚纲纤毛虫的分类地位;②缘毛亚纲褶累枝虫与膜口亚纲嗜热四膜虫的亲缘关系近于咽膜亚纲绿草履虫,在寡膜纲中绿草履虫处于原始地位;③五株纤毛虫基因组中均含有微卫星DNA序列:(GTG)4(、GACA)4(、AG)8(、CAA)6和(GAA)6。     

利用三种分子标记研究缘毛类纤毛虫的系统发育地位

为了探讨缘毛类纤毛虫的系统发育地位 ,利用RAPD方法得到了 9种缘毛类纤毛虫、 1种四膜虫和1种喇叭虫的 3个随机引物的电泳带谱 ;测定了 7种缘毛类纤毛虫rRNA基因中的间隔区 1(ITS1)和小亚基核糖体核糖核酸 (SSrRNA)基因序列 ,并构建了相应的系统树。在比较和分析RAPD、ITS1和SSrRNA基因序列在缘毛类纤毛虫系统发育研究中的适用范围的基础上 ,以SSrRNA基因序列为分子标记研究了缘毛类纤毛虫系统发育地位 ,结果表明 :①缘毛亚纲是单系的 ,作为寡膜纲中一个亚纲的分类地位是合理的 ;②缘毛类纤毛虫可能是寡膜纲中较高等的一个类群。     

6种累枝虫(Epistylis)rRNA基因18S-ITS1序列及其分子系统发育关系

建立了由采自自然界中的样品、不经实验室培养而直接用于大核DNA提取和PCR反应的原位(in situ)方法; 在此基础上, 测定并比较了6种累枝虫(Epistylis wenrichi, E. urceolata, E. chrysemydis, E. plicatilis, E. hentscheli, E. galea)的18S-ITS1序列, 结果显示: E. wenrichi, E. urceolata, E. chrysemydis, E. plicatilis和E. hentscheli间18S和ITS1区序列的碱基相似性很高, 而它们与E. galea间碱基变异位点在18S区和ITS1区均高达33个以上, 遗传变异度分别大于15%和23%. 以多态喇叭虫(Stentor polymorphus)为外类群, 利用最大简约法、最大似然法和邻接法构建了它们的系统发育树.分析指出: E. galea和其他5种累枝虫分属为两个类群, 与其他5种累枝虫相比, E. galea可能较早从祖先种中分化出来; 相比其他特征, 大核和口围区可能与累枝虫进化的关系更为密切, 是重要的系统发育特征.     

Phylogenetic relationships among six species of Epistylis inferred from 18S-ITS1 sequences

Phylogenetic relationships among six species of Epistylis (i. e. E. plicatilis, E. urceolata, E. chrysemydis, E. hentscheli, E. wenrichi, and E. galea) were investigated using sequences of the first internal transcribed spacer region (ITS-1) of ribosomal DNA (rDNA). Amplified rDNA fragment sequences consisted of 215 or 217 bases of the flanking 18S and 5.8S regions, and the entire ITS-1 region (from 145 to 155 bases). There were more than 33 variable bases between E. galea and the other five species in both the 18S region and the ITS-1 region. The affiliation of them was assessed using Neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) analyses. In all the NJ, MP and ML analyses E. galea, whose macronucleic position and shape are distinctly different from those of the other five species, was probably diverged from the ancestor of Epistylis earlier than the other five species. The topology in which E. plicatilis and E. hentscheli formed a strongly supported sister clade to E. urceol     

四种淡水缘毛类纤毛虫的形态学研究

通过活体显微观察及银染法对采自武汉东湖的4种固着缘毛类纤毛虫: 粗茎睫纤虫Ophrydium crassicaule Penard, 1922, 念珠伪钟虫Pseudovorticella monilata (Tatem, 1870) Foissner & Schiffmann, 1974, 垂盖虫Opercularia nutans (Ehrenberg, 1831) Stein, 1854和一个鞘居虫未定种Vaginicola sp.的活体、纤毛图式、银线系以及细胞核的形态学特征进行详细的描述, 补充和完善了缘毛类纤毛虫的形态学资料, 为纤毛虫分类和系统发育学的研究提供重要信息。此外, 首次在伪钟虫中观察到有性生殖现象, 为纤毛虫生殖进化的研究提供了基础资料。     

利用RAPD技术对三种伪角毛虫及五种全列虫(原生动物,纤毛虫)的属间及属内关系探讨

利用11种随机引物对腹毛类纤毛虫中两个相近属内8个海洋种(3种伪角毛虫和5种全列虫)进行了种间及种内关系的RAPD分析,所得清晰条带显示种间均具有明显的差异.计算和比较了种群间、同属种间及不同属间的相似性指数.利用RAPDistance软件构建的邻接(NJ)树表明:伪角毛虫属与全列虫属之间能很好地分开,而不同种群间表现了很好的同源性.本工作表明RAPD技术可以很可靠的用来区分形态相似种,同时也表明RAPD分析在纤毛虫属间水平的系统关系推定中存在一定的适用性.     

缘毛类纤毛虫伸缩泡收缩频率的研究

纤毛虫伸缩泡的主要功能是排出进入体内的多余水分及部分代谢废物(Kudo,1966)。Kitching(1948),Tsukuda et al.(1984)等曾对纤毛虫伸缩泡的收缩频率进行研究,但国内尚未见这方面的报道。鉴于缘毛类纤毛虫对环境的生物监测及污染水体的生物治理中具有重要作用,为探索有关规律,我们测定了53种缘毛类纤毛虫伸缩泡的收缩频率以及盐度和温度对伸缩泡收缩频率的影响。材料和方法缘毛类纤毛虫(简称缘纤虫)多固着在水生动植物及杂质上,可通过采集水生动植物、水中杂质或水中挂片得到。将采得的被固着物直接放于显微镜下观察,鉴定种类,以秒表记录每…     

家蚕胚胎细胞系的DNA指纹图谱分析

在建立可靠的家蚕细胞系基因组DNA制备和PCR扩增技术体系的基础上,筛选具有稳定多态性位点的RAPD和ISSR引物,建立家蚕细胞系基因组DNA的ISSR和RAPD分子标记技术体系,检测家蚕细胞系的DNA分子标记多态性,构建细胞系的DNA指纹图谱。筛选出了26个ISSR引物和43个RAPD引物,通过PCR扩增在家蚕胚胎细胞系和传代昆虫细胞系等9个样品中分别获得了797条和1205条多态性条带,多态性达到89．9%和76．6%,不同细胞系的DNA多态性有较大差异,三个家蚕胚胎细胞系具有各自特有的DNA标记。测定了9个样品间的Nei's相似系数和遗传距离,构建了系统发育树,结果表明本实验室建立的3个家蚕胚胎细胞系和家蚕“夏芳×秋白”聚为一簇,亲缘关系较近,而来自不同物种的五个传代昆虫细胞系聚为一簇,它们之间的遗传距离比3个家蚕胚胎细胞系之间的遗传距离更小。     

中国对虾体表共栖纤毛虫七新种(缘毛目:聚缩虫科,累枝虫科)

我国海洋生纤毛虫,除三、四十年代王家楫、倪达书,五十年代尹光德曾有过研究外,至今报道甚少。对于北方重要养殖品种——中国对虾(Penaeus orientalis)致病性缘毛类纤毛虫的研究尚属空白。1984年4月至10月,作者在对黄渤海沿岸养殖对虾体表纤毛虫的调查中,共检得缘毛类三十八种,分属四科九属,包括七新种,二十一种国内新记录,兹记述如下。     

Phylogenetic relationships of the subclass peritrichia (Oligohymenophorea, Ciliophora) with emphasis on the genus Epistylis, inferred from small subunit rRNA gene sequences

The peritrichs have been recognized as a higher taxon of ciliates since 1968. However, the phylogenetic relationships among them are still unsettled, and their placement within the class Oligohymenophorea has only been supported by the analysis of the small subunit rRNA gene sequence of Opisthonecta henneguyi. DNA was isolated directly from field-sampled species for PCR, and was used to resolve relationships within the genus Epistylis and to confirm the stability of the placement of peritrichs. Small subunit rRNA gene sequences of Epistylis plicatilis, Epistylis urceolata, Epistylis chrysemydis, Epistylis hentscheli, Epistylis wenrichi, and Vorticella campanula were sequenced and analyzed using both distance-matrix and maximum-parsimony methods. In phylogenetic trees, the monophyly of both the genus Episrylis and the subclass Peritrichia was strongly supported, while V. campanula clustered with Vorticella microstoma. The topology in which E. plicatilis and E. hentscheli formed a strongly supported sister clade to E. urceolata, E. chrysemydis, and E. wenrichi was consistent with variations in the thickness of the peristomial lip. We concluded that the peristomial area, especially the peristomial lip, might be the important phylogenetic character within the genus Epistylis.     

Phylogenetic relationships among six species of Epistylis inferred from 18S-ITS1 sequences

Phylogenetic relationships among six species of Epistylis (i. e. E. plicatilis, E. urceolata, E. chrysemydis, E. hentscheli, E. wenrichi, and E. galea) were investigated using sequences of the first internal transcribed spacer region (ITS-1) of ribosomal DNA (rDNA). Amplified rDNA fragment sequences consisted of 215 or 217 bases of the flanking 18S and 5.8S regions, and the entire ITS-1 region (from 145 to 155 bases). There were more than 33 variable bases between E. galea and the other five species in both the 18S region and the ITS-1 region. The affiliation of them was assessed using Neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) analyses. In all the NJ, MP and ML analyses E. galea, whose macronucleic position and shape are distinctly different from those of the other five species, was probably diverged from the ancestor of Epistylis earlier than the other five species. The topology in which E. plicatilis and E. hentscheli formed a strongly supported sister clade to E. urceolata, E. chrysemydis, and E. wenrichi was consistent with variations in the thickness of the peristomial lip. We concluded that the macronucleus and peristomial lip might be the important phylogenetic characteristics within the genus Epistylis.     

Characterization and phylogenetic relationships among microsporidia infecting silkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis.

This study is the first report on the genetic characterization and relationships among different microsporidia infecting the silkworm, Bombyx mori, using inter simple sequence repeat PCR (ISSR-PCR) analysis. Six different microsporidians were distinguished through molecular DNA typing using ISSR-PCR. Thus, ISSR-PCR analysis can be a powerful tool to detect polymorphisms and identify microsporidians, which are difficult to study with microscopy because of their extremely small size. Of the 100 ISSR primers tested, only 28 primers had reproducibility and high polymorphism (93%). A total of 24 ISSR primers produced 55 unique genetic markers, which could be used to differentiate the microsporidians from each other. Among the 28 SSRs tested, the most abundant were (CA)n, (GA)n, and (GT)n repeats. The degree of band sharing was used to evaluate genetic similarity between different microsporidian isolates and to construct a phylogenetic tree using Jaccard's similarity coefficient. The results indicate that the DNA profiles based on ISSR markers can be used as diagnostic tools to identify different microsporidia with considerable accuracy. In addition, the small subunit ribosomal RNA (SSU-rRNA) sequence gene was amplified, cloned, and sequenced from each of the 6 microsporidian isolates. These sequences were compared with 20 other microsporidian SSU-rRNA sequences to develop a phylogenetic tree for the microsporidia isolated from the silkworms. This method was found to be useful in establishing the phylogenetic relationships among the different microsporidians isolated from silkworms. Of the 6 microsporidian isolates, NIK-1s revealed an SSU-rRNA gene sequence similar to Nosema bombycis, indicating that NIK-1s is similar to N. bombycis; the remaining 5 isolates, which differed from each other and from N. bombycis, were considered to be different variants belonging to the species N. bombycis.     

Phylogenetic relationships of the subclass Peritrichia (Oligohymenophorea, Ciliophora) inferred from small subunit rRNA gene sequences

The phylogenetic relationships among peritrichs remain unresolved. In this study, the complete small subunit rRNA (SSrRNA) gene sequences of seven species (Epistylis galea, Campanella umbellaria, Carchesium polypinum, Zoothamnium arbuscula, Vaginicola crystallina, Ophrydium versatile, and Opercularia microdiscum) were determined. Trees were constructed using distance-matrix, maximum-likelihood and maximum-parsimony methods, all of which strongly supported the monophyly of the subclass Peritrichia. Within the peritrichs, 1) E. galea grouped with Opercularia microdiscum and Campanella umbellaria but not the other Epistylis species, which indicates that the genus Epistylis might not be monophyletic; 2) the topological position of Carchesium and Campanella suggested that Carchesium should be placed in the family Zoothamniidae, or be elevated to a higher taxonomic rank, and that Campanella should be independent of the family Epistylididae, and probably be given a new rank; and 3) Opisthonecta grouped strongly with Astylozoon, which suggested that Opisthonecta species were not the ancestors of the stalked peritrichs.     

蚬壳花椒ISSR-PCR反应体系的建立及优化

通过单因素试验及正交设计方法对影响蚬壳花椒ISSR-PCR扩增的主要因素(模板DNA、Mg2+的浓度、引物、dNTPs,TaqDNA聚合酶的用量以及退火温度)进行优化,以建立蚬壳花椒ISSR-PCR反应的最佳体系。结果表明:最佳反应体系(20μL)为模板DNA 60ng、MgCl2 2.5mmol/L、dNTPs 0.15mmol/L、引物0.6μmol/L、TaqDNA聚合酶2.4U。在此基础上,从100条引物中筛选出18条扩增稳定、多态性好的ISSR引物并经过10份蚬壳花椒种质检验,证明该体系具有扩增条带清晰、稳定、重复性好等优点。该反应体系的建立为蚬壳花椒种质资源分类、遗传多样性分析提供了更客观可靠的方法。     

六种淡水纤毛虫形态学研究

借助活体观察和银染法对6种淡水纤毛虫(纵长板壳虫Coleps elongatus、瓜形原膜袋虫Protocyclidiumcitrullus、亨氏累枝虫Epistylis hentscheli、钟形钟虫Vorticella campanula、长柄球吸管虫Metacineta macrocaulis、四分锤吸管虫Tokophrya quadripatita)的形态和纤毛图式进行了详尽研究。本工作首次描述了亨氏累枝虫、四分锤吸管虫、纵长板壳虫的中国种群,同时揭示了亨氏累枝虫长期未知的纤毛图式特征并给出该种类的新定义: 虫体高钟形,活体大小约(100-130) m(60-65) m; 伸缩泡1个,腹位; 大小核各1枚,大核C型; 群体为规则对称二叉分枝; 口区三片小膜均由三列动基列组成,第三小膜由三列等长的动基列组成,其终止处高于第一小膜下端; 虫体前端到反口纤毛环间的银线约71-74条,反口纤毛环到帚胚间的银线约35-47条; 淡水生境。此外,本研究对长柄球吸管虫、钟形钟虫、瓜形原膜袋虫进行了重新描述,补充了显微照片。     

Genetic Analyses of Casuarinas Using ISSR and FISSR Markers

Inter simple sequence repeat polymerase chain reaction (ISSR-PCR) was used for the genetic analysis of the six species of Allocasuarina, five species of Casuarina and 12 superior performing selections of C. equisetifolia L. We also fingerprinted C. equisetifolia L. selections using Fluorescent-ISSR-PCR (FISSR-PCR), an improvised ISSR-PCR assay. The ISSR analysis provided information on the frequency of various simple sequence repeats in the casuarina genome. The di-nucleotide repeats were more common, among which (CA)n and its complementary nucleotide (GT),, repeat motifs amplified relatively higher number of bands with an average of 6.0+/-3.5 and 6.3+/-1.8 respectively. Eleven species of casuarinas were amplified with 10 primers anchored either at 5' or 3' end. A total of 253 PCR products were obtained and all were polymorphic, out of which 48 were specific to Allocasuarina and 36 were specific to Casuarina genus. Genetic similarity among the species was 0.251. A UPGMA dendrogram grouped all the Casuarina species together. The 12 superior performing selections of C. equisetifolia L. produced 57 polymorphic ISSR markers while the FISSR assay revealed 105 polymorphic markers. The primer CRR(ATT)4 distinguished all the selections. DNA profiles obtained with ISSR and FISSR assays would serve as a reference library for the establishment of clonal identity in casuarinas.