Design and testing of a plant-specific PCR primer for ecological and evolutionary studies

A polymerase chain reaction (PCR) primer, 28KJ (5-GGCGGTAAATTCCGTCC-3), was developed to specifically amplify plant DNA. This primer is located approximately 250 bases downstream of the 5′ end of the 28S ribosomal RNA gene, and it was used in combination with the universal primer 28C. The specificity of this primer combination was tested against 31 angiosperms, 9 conifers, 1 alga and 30 fungi (21 basidiomycetes and 9 ascomycetes). Both herbarium specimens and fresh samples were tested. The 28KJ/28C primer combination successfully amplified all angiosperm and conifer DNAs, but no fungal or algal DNAs. Plant DNA was amplified from plant/fungal symbioses (ectomycorrhizae of conifers and ericoid mycorrhizae of Ericaceous plants), and the plants involved in these symbioses were identified by comparing DNA sequences or restriction enzyme digest patterns of the mycorrhizal DNAs to those of known plant samples. These methods allow rapid and accurate identification of plant associates in complex plant/ fungal systems when the identity of the roots is unclear.     

Molecular phylogeny of Collembola inferred from ribosomal RNA genes

The classification of taxa within Collembola (Springtails, Hexapoda) has been controversial. In this study, we combined complete 18S rRNA gene with partial 28S rRNA gene (D7-D10) sequences to investigate the phylogeny of Collembola. About 2500 aligned sites of thirty species representing 29 genera from14 families of Collembola were analyzed, including one species of Neelipleona from which no sequence has been reported previously.The phylogenetic trees were obtained by different methods (maximum parsimony, maximum likelihood, and Bayesian analysis). Our results supported the monophyly of two of the four taxonomic groups of Collembola summarized by Deharveng [Deharveng, L., 2004. Recent advances in Collembola systematics. Pedobiologia 48, 415–433.], namely of Poduromorpha and of Symphypleona. Within Poduromorpha, Neanuridae was monophyletic with high support, but Hypogastruridae was not. Entomobryomorpha was paraphyletic, as the Tomoceroidea (Tomoceridae and Oncopoduridae) was found to be apart from the other entomobryomorphs. In the latter Isotomoidea and Entomobryoidea joined into a group with moderate support. Within Symphypleona, the phylogenetic relationship [(Sminthuridae + Bourletiellidae) + Sminthurididae] was consistent with traditional morphological studies. Neelipleona grouped with Symphypleona in all trees, with moderate support in the ML and Bayesian analyses.     

小鼠胚胎核移植实验的初步报告

Abstract:Oocytes recovered from Fallopian tubes of female mice of Kun-Ming-Bai strain were enucleated by microsurgery and a single blastomere aspirated from 2-cell stage embryo derived from C57 was injected into the perivitelline space of earh enucleated oocyte.Then those coupled cells were exposed to PEG solution and cultured in an atmosphere of 5% Co2 at 37℃ for four hours.After that,couples which had been fused to one cell were transferred to the oviducts of pseudopregnant foster mothers.Six offsprings were obtained from this transplantation.Key words:Mouse,Embryo,Nuclear transplantation     

伪指环虫、异钩虫和三钩虫的系统位置

采用单个虫体PCR扩增、序列测定与分析的方法,对锚首虫科中后吸器形态较特殊的3个属:伪指环虫属、异钩虫属和三钩虫属的28S rRNA基因5'端序列进行了研究,并采用PAUP软件构建了分子系统树.结果显示,异钩虫属、三钩虫属和伪指环虫属明显地嵌合于其他锚首虫属之间,进而明确了这3个属的分类地位,应归属锚首虫科.锚首虫科和指环虫科之间的关系则有待今后进一步研究.     

中国沿海蛾螺科5属10种28S rRNA基因的系统学分析

目前已报道在我国分布的蛾螺科种类有13个属,约31个种,系统学和分类地位仍存在较大的争议.本研究利用核糖体大亚基28S rRNA的部分序列对我国辽宁、山东、福建沿海蛾螺科5属10个种的系统发生进行了分析.通过PCR获得了大约1400 bp的片段,测序之后,通过遗传分析软件对序列进行了比对分析,以骨螺科的脉红螺作为外群,利用Neighbor-Joining (NJ)法和Minimum Evolution (ME)法建立了系统树.结果显示,所研究的蛾螺科5属10个种可以被分为5个亚群:第一大分支为香螺亚群,包括Neptunea属的香螺、新英格兰蛾螺、Neptunea eulimata Dall、和一个未知种以及Siphonalia属的略胀管蛾螺;第二个分支为侧平肩螺亚群;第三个分支为荻曷莺突坪6曷菅侨海坏谒母龇种止芏曷菅群;第五个分支为方斑东风螺亚群.由系统学分析可知,香螺是较为进化的种;未知种为香螺属内的种;略胀管蛾螺与Neptunea属的种类亲缘关系较近,序列相似系数为0.9%-1.4%,已经达到了属内水平,建议将略胀管蛾螺归为Neptunea属.     

蓖麻蚕核糖体大亚基RNA基因3‘—端序列分析及进化研究

测定了蓖麻蚕核糖体大亚基ＲＮＡ编码区３’－端ＤＮＡ序列,分析了其二级结构,并与昆虫伊蚊、果蝇;线虫;脊椎动物人、小鼠、爪蟾;低等脊索动物海鞘以及真菌酵母、毛霉相应的保守区段进行了同源比较。邻接法分析表明,昆虫核糖体大亚基ＲＮＡ在进化上与５ＳｒＲＮＡ相似,有加快的趋势。     

Diversity of commensal bacteria from mid-gut of pink stem borer (Sesamia inferens [Walker])-Lepidoptera insect populations of India

Sesamia inferens (W.) is polyphagous agricultural pests and prevalent in the India, China, South Asia and South East Asia. Insecticides is not recommended because, apart from the hazardous effects of chemicals, its larvae tunnel throughout the stem from first instar. Associated bacteria with insects provide several benefits to their host, revealing the types of bacteria associated with S. infersns will give basic information, which may throw light on management of this noxious pest. The culture dependant, 16S rRNA gene technique revealed thirty two bacterial isolates from gut of S. inferens from different region of India and host, comprising phyla Proteobacteria, Firmicutes and Bacteroidetes. Among which proteobacteria phyla was dominant with families and genus like Enterobacteriaceae (Citrobacter, Enterobacter, Serratia, Klebsiella and Xenorhabdus), Pseudomonadaceae (Pseudomans), Moraxellaceae (Acinetobactor) and Comamonadaceae (Comamonas). The phyla Firmicutes less dominant with four families and one genus each viz., Staphylococcaceae (Staphylococcus), Bacillaceae (Lysinibacillus), Streptococcaceae (Lactococcus) and Enterococcaceae (Enterococcus). Third phyla had only one family viz., Flavobacteriaceae (Chryseobacterium). The bacterial diversity varied greatly among insects that were from different host plants than those from the same host plant of different locations. This result suggested that the type of host plant greatly influences the mid-gut bacterial diversity more than the location of the host plant of S.inferens. These bacterial populations may have a key role in digestion, as well as other benefits to the S. inferens larvae. Determination of the bacterial community and its biological functions within the insect could provide us with basic information for future pest control research.     

毛头鬼伞中一条新28S rRNA的发现及其同源性分析

本研究从担子菌毛头鬼伞(Coprinus comatus)菌丝中分离获得一条新的28S rRNA序列,序列长度为906bp(GenBank accession No.GU568178)。该序列是我们前期在从毛头鬼伞中克隆一种烟草花叶病毒(TMV)的抗性蛋白基因y3时意外获得的一条非目的条带。将此获得的序列通过NCBI的BLAST,以及与其同源序列进行Clustal w和MEGA聚类分析,证实该序列是28S rRNA,同时还发现毛头鬼伞的系统进化关系比较离散。此外,在这一新28S rRNA与TMV的抗性蛋白基因y3之间发现有两个同源区段有可能是PCR扩增y3基因时出现非目的条带的原因。在这两个同源区段中,其一区段与克隆y3基因时所用的PCR引物之一有较高的相似性,另一区段也是一般PCR引物的类似物。本研究中新28S rRNA序列的获得是PCR扩增中出现非目的条带的新例,该序列的发现及聚类分析的结果有助于真菌基因组学研究及真菌生物分子分类系统的建立。     

Screening for Booroola (FecB) and Galway (FecX) mutations in Indian sheep

This study reports the status of the Booroola (FecB) and Galway (FecX^G) mutations in Indian sheep breeds. The Kendrapada sheep (n = 46) was genotyped for the presence of FecB and FecX^G mutations, while the Garole (n = 34), Malpura (n = 30), and Decanni sheep (n = 15) for the FecX^G mutation. The FecB and FecX^G genotyping was carried out by forced restriction fragment length polymorphism PCR technique. In the present study, FecB mutation was discovered in the Kendrapada sheep of Orissa, which is now the second prolific sheep of India after the Garole. Out of 46 individuals of Kendrapada sheep, 26 were homozygous (BB), 15 heterozygous (B+) and 5 non-carriers (++) for the FecB mutation. The frequency of the FecB allele in this sample was about 0.73. Results indicated that the frequency of the FecB mutation is high, but the gene is not fixed in the population as reported in Garole sheep. None of sheep breeds carried the FecX^G mutation. The discovery of the FecB mutation in Kendrapada sheep will facilitate the use of FecB allele in improving the prolificacy of non-prolific sheep breeds of India.     

Lateral transfer of eukaryotic ribosomal RNA genes: an emerging concern for molecular ecology of microbial eukaryotes

Ribosomal RNA (rRNA) genes are widely utilized in depicting organismal diversity and distribution in a wide range of environments. Although a few cases of lateral transfer of rRNA genes between closely related prokaryotes have been reported, it remains to be reported from eukaryotes. Here, we report the first case of lateral transfer of eukaryotic rRNA genes. Two distinct sequences of the 18S rRNA gene were detected from a clonal culture of the stramenopile, Ciliophrys infusionum. One was clearly derived from Ciliophrys, but the other gene originated from a perkinsid alveolate. Genome-walking analyses revealed that this alveolate-type rRNA gene is immediately adjacent to two protein-coding genes (ubc12 and usp39), and the origin of both genes was shown to be a stramenopile (that is, Ciliophrys) in our phylogenetic analyses. These findings indicate that the alveolate-type rRNA gene is encoded on the Ciliophrys genome and that eukaryotic rRNA genes can be transferred laterally.     

Staphylotrichum boninense, a new hyphomycete (Chaetomiaceae) from soils in the Bonin Islands, Japan

Staphylotrichum boninense, a new hyphomycete classified in the Chaetomiaceae (Ascomycota), was isolated from soils in the Bonin Islands, Japan. It is characterized morphologically by the production of yellow-orange colonies and subglobose holoblastic conidia. Morphologically the species is similar to S. coccosporum, but it is significantly different from S. coccosporum in phylogeny and also differs with respect to its secondary metabolite profile.     

DNA barcodes for marine fungal identification and discovery

We employed DNA barcodes for identification of fungal species in marine sediments. Sediments were collected seasonally along the Southeast coast of India from which a culturable fungal library was constructed. All cultured species were morphologically documented using microscopical analysis. A maximum population density of 19.3 × 10³ CFU/g was recorded in monsoon and minimum of 3 × 10³ CFU/g in premonsoon season. Two-way analysis of variance suggests that the fungal community varied significantly between the seasons (F = 9.543, P < 0.001) and at various depths sampled (F = 4.655, P < 0.05). In total, 54 fungal species belonging to 13 different families were documented and all species were sequenced for internal transcribed spacer genes. Each species was represented by at least two specimens constituting a total of 171 specimens for DNA barcoding. Twelve species of a marine fungi were sequenced for the first time. Branching patterns of phylogenetic tree strongly supported the sequence variations within and between all species barcoded. Based on the pairwise distance model we suggest barcode gaps of 15 %, 21 %, 30 %, 35 % and 51 % for genera, family, order, class and phyla respectively.     

Conformation change of cytochrome c. II. Ferricytochrome c refinement at 1.8 A and comparison with the ferrocytochrome structure

The X chromosomal nucleolus organizer of Drosophila hydei contains about 500 ribosomal RNA genes. The 28 S rRNA coding region of about 50% of these genes is interrupted by an intervening sequence of 6.0 × 10³ base-pairs. Restriction enzyme analysis revealed that more than 90% of the rRNA genes with intervening sequences are present as one or a few clusters within the X chromosomal nucleolus organizer. Furthermore, even though X chromosomal rRNA genes show several distinct size classes of non-transcribed spacers, the cluster of repeating units containing an intervening sequence has major spacer lengths of 4.4 × 10³ and 4.6 × 10³ base-pairs; spacers 5.1 × 10³ base-pairs in length are mainly linked with genes lacking the intervening sequence.