重组酿酒酵母表达幽门螺杆菌VacA蛋白及其免疫原性分析*

目的:利用酿酒酵母表面展示技术筛选幽门螺杆菌候选疫苗,并分析其免疫原性。方法:以幽门螺杆菌的空泡型细胞毒素A(vacA)基因作为研究对象,构建重组S.cerevisiae EBY100/pYD1-VacA,通过Western blot、免疫荧光标记和流式细胞仪对S.cerevisiae EBY100/pYD1-VacA进行体外表达分析。以PBS和S.cerevisiae EBY100/pYD1为对照组,S.cerevisiae EBY100/pYD1-VacA为实验组,口服免疫SPF级BALB/c小鼠。通过ELISA分析检测口服免疫后小鼠抗VacA特异性IgG及分泌型IgA效价。结果:VacA抗原蛋白被成功地展示在S.cerevisiae EBY100表面。小鼠经口服免疫S.cerevisiae EBY100/pYD1-VacA后可诱导产生较高的VacA特异性抗体。结论:表面展示型酿酒酵母可以作为幽门螺杆菌候选疫苗的递送载体,与此同时,这也为开发其他细菌或病毒疫苗提供新思路。     

A rare case of a natural contact zone in Morocco between an autopolyploid and an allopolyploid of Centaurea aspera with sterile tetraploid hybrids

A new contact zone between Centaurea aspera and Centaurea seridis was found in Morocco. Chromosome counts and flow cytometry showed that both taxa were tetraploid (4x = 44). A literature review and morphometric analysis established that C. aspera corresponds to the autopolyploid C. aspera subsp. gentilii and C. seridis corresponds to the allopolyploid C. seridis var. auriculata. This contact area was compared with the homologous contact zones in Spain formed by the diploid C. aspera subsp. stenophylla and the tetraploid C. seridis subsp. maritima. Natural hybrids between parental species were frequent in both areas. In Spain, hybrids were triploid (from reduced gametes A and gamete AB), highly sterile and exerted a ‘triploid block’. In Morocco, cytometry showed that hybrids were tetraploid and, therefore, probably fertile, but all the capitula lacked achenes. It is likely that the resulting genome of the new tetraploid hybrid (AAAB), through the fusion of reduced gametes AA (from subsp. gentilii) and AB (from var. auriculata), could explain irregularities in meiosis through formation of aneuploid gametes and, therefore, infertility of the hybrid. Moroccan sterile tetraploid hybrids develop, but have the identical irregularities to Spanish triploids, probably due to the odd number of homologous chromosomes. The new hybrid is first described as C. x subdecurrens nothosubsp. paucispinus. In addition, distribution and ecological traits are analysed.     

Isolation of polymorphic microsatellite loci in the clear lake gnat and two other phantom midge species of the genus Chaoborus (Diptera: Chaoboridae)

Chaoborus is of great interest to many freshwater ecologists. The adults can become pests in certain areas in North America and the larvae are an important food source for fish. In this preliminary study, we identified variable microsatellite loci in three species: Chaoborus astictopus (H_E = 0.52–0.76), Chaoborus americanus (H_E = 0.46–0.80) and Chaoborus punctipennis (H_E = 0.66–0.81). Using a biotin/streptavidin capture technique of repetitive sequences in a 96‐well format, we obtained microsatellite‐enriched genomic libraries for all three species and identified six polymorphic microsatellite markers for each species. None of the primers did yield a polymerase chain reaction fragment in a cross‐species test.     

MOLECULAR PHYLOGENY OF THE GENUS KUMANOA (BATRACHOSPERMALES,RHODOPHYTA)1

Species belonging to the newly established genus Kumanoa were sampled from locations worldwide. DNA sequence data from the rbcL gene, cox1 barcode region, and universal plastid amplicon (UPA) were collected. The new sequence data for the rbcL were combined with the extensive batrachospermalean rbcL data available in GenBank. Single gene rbcL results showed the genus Kumanoa to be a well‐supported clade, and there was high statistical support for many of the terminal nodes. However, with this gene alone, there was very little support for any of the internal nodes. Analysis of the concatenated data set (rbcL, cox1, and UPA) provided higher statistical support across the tree. The taxa K. vittata and K. amazonensis formed a basal grade, and both were on relatively long branches. Three new species are proposed, K. holtonii, K. gudjewga, and K. novaecaledonensis; K. procarpa var. americana is raised to species level. In addition, the synonymy of K. capensis and K. breviarticulata is proposed, with K. capensis having precedence. Five new combinations are made, bringing the total number of accepted species in Kumanoa to 31. The phylogenetic analyses did not reveal any interpretable biogeographic patterns within the genus (e.g., K. spermatiophora from the tropical oceanic island Maui, Hawaii, was sister to K. faroensis from temperate midcontinental Ohio in North America). Previously hypothesized relationships among groups of species were not substantiated in the phylogenetic analyses, and no intrageneric classification is recommended based on current knowledge.     

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus

Aims: To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human. Methods and Results: Species‐specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co‐existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples. Significance and Impact of the Study: This simple, rapid and cost‐effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.     

NEW MARINE MEMBERS OF THE GENUS HEMISELMIS (CRYPTOMONADALES,CRYPTOPHYCEAE)1

Cryptomonads are a ubiquitous and diverse assemblage of aquatic flagellates. The relatively obscure genus Hemiselmis includes some of the smallest of these cells. This genus contained only two species until 1967, when Butcher described seven new marine species mainly on the basis of observations with the light microscope. However, from these seven taxa, only H. amylifera and H. oculata were validly published. Additionally, the features Butcher used to distinguish species have since been questioned, and the taxonomy within Hemiselmis has remained clouded due to the difficulty in unambiguously applying his classification and validating many of his species. As a result, marine strains are often placed into one of three species—H. rufescens Parke, H. virescens Droop, or the invalid H. brunnescens Butcher—based on cell color alone. Here we applied microscopic and molecular tools to 13 publicly available Hemiselmis strains in an effort to clarify species boundaries. SEM failed to provide sufficient morphological variation to distinguish species of Hemiselmis, and results from LM did not correlate with clades found using both molecular phylogenetic and nucleomorph genome karyotype analysis, indicating a high degree of morphological plasticity within species. On the basis of molecular characters and collection geography we recognize four new marine species of Hemiselmis—H. cryptochromatica sp. nov., H. andersenii sp. nov., H. pacifica sp. nov., and H. tepida sp. nov.—from the waters around North America.     

人类红细胞膜带3蛋白C端域Lys892～Phe895在Cl-转运过程中作用的研究

采用酵母表面展示系统,表达带3蛋白膜段结构域(Gln404～Val911)至酵母细胞膜,功能研究表明,表达后的膜段结构域具有离子转运的活性,同时,带3蛋白抑制剂4,4′-二异硫氰-2,2′-二黄酸芪(DIDS)能够抑制其离子转运的功能.利用PCR方法,以pFAST-Bac-mdb3为模板扩增出带3蛋白膜段结构域的4种截断突变体,分别去除带3蛋白C端域后4个(Ala908～Val911)、16个(Asp896～Val911)、20个(Lys892～Val911)、32个(Asn880～Val911)氨基酸序列,测序后将其克隆至表达载体pYD1上,构建酵母表达质粒pYD1-Trunc4、pYD1-Trunc16、pYD1-Trunc20和pYD1-Trunc32,诱导4组突变体的蛋白质表达.然后测定Cl^-的转运活性,结果发现去除后20个(Lys892～Val911)氨基酸残基后,离子转运活性明显下降,而去除后32个(Asn880～Val911)后,离子转运没有进一步下降,说明Lys892～Phe895 4个氨基酸残基在带3蛋白的离子转运过程中发挥重要作用.     

An integrative approach to reveal speciation and species richness in the genus Diasporus (Amphibia: Anura: Eleutherodactylidae) in eastern Panama

We have applied an integrative taxonomic approach, including bioacoustics, ecology, morphology, and molecular genetics (barcoding and phylogeography), to explore species richness in the genus Diasporus in eastern Panama, from where only Diasporus quidditus (Lynch, 2001) was previously known. During fieldwork in eastern Panama in 2011 and 2012 we found six additional species, four of which we are describing here as new to science, plus two species that are new for this region. We have evaluated the presence of Diasporus diastema (Cope, 1875) in eastern Panama by comparing morphological, genetic, and bioacoustic characters of specimens from near the type locality in central Panama with specimens from eastern Panama. We further describe and compare male advertisement calls of most Diasporus species. The phylogeographic analysis suggests the allopatric speciation of Diasporus species in eastern Panama following the completion of the Panamanian isthmus in the middle Miocene. Subsequent geological events concur with the vicariant evolution of different lineages in situ, suggesting eastern Panama to be a centre of endemism for this group of frogs. We present an integrative analysis of the species from eastern Panama and include an identification key for all species of the genus.     

Etheliaceae fam. nov. (Gigartinales,Rhodophyta), with a clarification of the generitype of Ethelia and the addition of six novel species from warm waters

Based upon COI‐5P, LSU rDNA, and rbcL sequence data and morphological characteristics, six new members of the noncalcified crustose genus of red algae Ethelia are described in a new family, Etheliaceae (Gigartinales), sister to the recently described Ptilocladiopsidaceae. The novel species are described from subtropical to tropical Atlantic and Indo‐Pacific Ocean basins; E. mucronata sp. nov. and E. denizotii sp. nov. from southern and northern Western Australia respectively, E. wilcei sp. nov. from the Cocos (Keeling) Islands of Australia, E. suluensis sp. nov. from the Philippines, E. umbricola sp. nov. from Bermuda and E. kraftii sp. nov. from Lord Howe Island, Australia. The generitype, Ethelia biradiata, originally reported from the Seychelles, Indian Ocean, is added to the Western Australian flora.     

人类红细胞膜带3蛋白C端域Lys892～Phe895在Cl~-转运过程中作用的研究

采用酵母表面展示系统 ,表达带 3蛋白膜段结构域 (Gln4 0 4～Val911)至酵母细胞膜 ,功能研究表明 ,表达后的膜段结构域具有离子转运的活性 ,同时 ,带 3蛋白抑制剂 4 ,4′ 二异硫氰 2 ,2′ 二黄酸芪 (DIDS)能够抑制其离子转运的功能 .利用PCR方法 ,以 pFAST Bac mdb3为模板扩增出带 3蛋白膜段结构域的 4种截断突变体 ,分别去除带 3蛋白C端域后 4个 (Ala90 8～Val911)、 16个 (Asp896～Val911)、 2 0个 (Lys892～Val911)、 32个(Asn880～Val911)氨基酸序列 ,测序后将其克隆至表达载体 pYD1上 ,构建酵母表达质粒 pYD1 Trunc4、pYD1 Trunc16、pYD1 Trunc2 0和 pYD1 Trunc32 ,诱导 4组突变体的蛋白质表达 .然后测定Cl-的转运活性 ,结果发现去除后 2 0个 (Lys892～Val911)氨基酸残基后 ,离子转运活性明显下降 ,而去除后 32个 (Asn880～Val911)后 ,离子转运没有进一步下降 ,说明Lys892～Phe895 4个氨基酸残基在带 3蛋白的离子转运过程中发挥重要作用     

The monospecific genus Meredithia (Kallymeniaceae,Gigartinales) is species rich and geographically widespread with species from temperate Atlantic,Pacific, and Indian Oceans

Using sequences of 5′ region of the cytochrome oxidase subunit 1 gene, large subunit rDNA, and ribulose‐1,5‐bisphosphate carboxylase/oxygenase large subunit gene as genetic markers to elucidate their phylogenetic positions, six unknown species from Western Australia, Tasmania, Lord Howe Is., and Norfolk Is. cluster with Meredithia in the Kallymeniaceae (Gigartinales), and are described as new members of this previously monospecific genus. Specimens from Bermuda referable to Kallymenia limminghei Mont. in the 20th century also clustered with this genetic grouping, not with the generitype of Kallymenia. The Bermudian specimens are further shown to be morphologically distinct from the type of K. limminghei (Guadeloupe, Caribbean Sea) and are described as a new species, Meredithia crenata. Using these Indo‐Pacific and Bermudian collections, our analyses further show that Psaromenia is closely related to Meredithia, and that Cirrulicarpus nanus sensu stricto should be returned to Meredithia.     

PHYLOGENETIC POSITION AND MORPHOLOGY OF THECAE AND CYSTS OF SCRIPPSIELLA (DINOPHYCEAE) SPECIES IN THE EAST CHINA SEA1

Resting cysts of the marine phytoplanktonic dinoflagellate Scrippsiella spp. are encountered in coastal habitats and shallow seas all over the world. Identification of Scrippsiella species requires information on cyst morphology because the plate pattern of the flagellated cell is conserved. Cysts from sediments of the East China Sea were identified based on traits from both the cysts and the thecal patterns of germinated cells. Calcareous cysts belonged predominantly to S. trochoidea (F. Stein) A. R. Loebl., S. rotunda J. Lewis, and S. precaria Montresor et Zingone. The former two species also produced smooth and noncalcified cysts in the field. A new species, S. donghaienis H. Gu sp. nov, was obtained from six noncalcified cysts with organic spines. These cysts are spherical, full of pale white and greenish granules with a mesoepicystal archeopyle. The vegetative cells consist of a conical epitheca and a round hypotheca with a plate formula of po, x, 4′, 3a, 7′′, 6c (5c + t), 6 s, 5′′′, 2′′′′ and are morphologically indistinguishable from S. trochoidea. Results of internal transcribed spacer (ITS) sequence comparisons revealed that S. donghaienis was distinct from the S. trochoidea complex and appeared nested within the Calciodinellum/Calcigonellum clade. Culture experiments showed that the presence of a red body in the cyst and the shape of the archeopyle were constant within cell lines from one generation to the next, while the morphological features of the cyst wall, such as calcification and spine shape, appeared to be phenotypically plastic.     

A multiplex panel of microsatellite markers for widespread sub‐Saharan rodents of the genus Mastomys

We isolated and characterized 12 polymorphic microsatellite loci in the sub‐Saharan rodent Mastomys huberti. We tested cross‐species amplification of all these loci in three closely related Mastomys species: M. coucha, M. erythroleucus and M. natalensis. Multiplex panels comprising 11 loci were developed and their application to a set of individuals in each species allowed clear and easy characterization of allele sizes. Statistics from 31 M. huberti coming from one locality in Mali showed no deviation from Hardy–Weinberg equilibrium except for one locus, and no significant linkage disequilibria between loci.