DGGE和T-RFLP在堆肥微生物群落结构研究中的应用

对堆肥微生物种群分布及其动态变化的研究进行了分析,论述了分子生物技术中的变性梯度凝胶电泳和末端标记限制性片段长度多态性的原理和特点,以及用于研究堆肥微生物的群落结构演变规律,为分析和筛选堆肥中的微生物提供更加有效、快速的信息,促进堆肥技术的发展。     

稀土尾矿土壤细菌群落结构对植被修复的响应

选用赣州-安远稀土弃废尾矿及其不同植被修复的堆浸田为研究对象,调查废弃尾矿及6种不同植被修复方案下土壤理化性质的变化,并利用变性凝胶梯度电泳(DGGE)技术,分析土壤微生物群落结构对植被修复的响应。结果表明:与未修复尾矿土壤相比,经不同植被修复后的土壤理化性质均得到明显改良,其中土壤含水量、有机质含量均比未修复尾矿土壤增加2—3倍。微生物群落结构分析表明,植被修复后土壤微生物群落与废弃尾矿土壤微生物群落亲缘度仅为0.21,表明植被修复后,土壤微生物群落结构发生了明显变化,且微生物多样性、均匀度、丰富度与未修复尾矿土壤相比均有了明显的提高。而在不同植被修复方案中,以湿地松和山胡椒为优势群落的两种植被修复方案对土壤改良效果最为明显,这两种修复方案不仅能显著改善土壤的固水性、有机质含量,并且对微生物群落的改善作用也最为显著。典范对应分析表明,废弃尾矿土壤微生物群落结构受土壤p H影响最为显著,而植被修复后土壤微生物群落的环境影响因子则转变为含水量、有机质、有机碳及总磷含量。进一步揭示了微生物在植被修复过程中所起到的重要作用,并为矿山生态重建过程中的土壤改良工作提供了丰富的理论依据。     

Proteome analysis of leaves of the desiccation-tolerant grass, Sporobolus stapfianus, in response to dehydration

Drought and its affects on agricultural production is a serious issue facing global efforts to increase food supplies and ensure food security for the growing world population. Understanding how plants respond to dehydration is an important prerequisite for developing strategies for crop improvement in drought tolerance. This has proved to be a difficult task as all of the current research plant models do not tolerate cellular dehydration well and, like all crops, they succumb to the effects of a relatively small water deficit of −4 MPa or less. For these reasons many researchers have started to investigate the usefulness of resurrection plants, plants that can survive extremes of dehydration to the point of desiccation, to provide answers as to how plants tolerate water loss. We have chosen to investigate the leaf proteome response of the desiccation-tolerant grass Sporobolus stapfianus Gandoger to dehydration to a water content that encompasses the initiation of the cellular protection response evident in these plants. We used a combination of two-dimensional Difference Gel Electrophoresis (2D-DIGE) and liquid chromatography-tandem-mass spectrometry to compare the proteomes of young leaves from hydrated plants to those dehydrated to approximately 30% relative water content. High-resolution 2D-DIGE revealed 96 significantly different proteins and 82 of these spots yielded high-quality protein assignments by tandem-mass spectrometry. Inferences from the bioinformatic annotations of these proteins revealed the possible involvement of protein kinase-based signaling cascades and brassinosteroid involvement in the regulation of the cellular protection response. Enzymes of glycolysis, both cytoplasmic and plastidic, as well as five enzymes of the Calvin cycle increased in abundance. However, the RuBisCO large subunit and associated proteins were reduced, indicating a loss of carbon fixation but a continued need to supply the necessary carbon skeletons for the constituents involved in cell protection. Changes in abundance of several proteins that appear to have a function in chromatin structure and function indicate that these structures undergo significant changes as a result of dehydration. These observations give a unique “snap-shot” of the proteome of S. stapfianus at a critical point in the passage towards desiccation.     

应用TGGE技术分析人肠道中双歧杆菌的组成

用温度梯度凝胶电泳(TGGE)技术结合16SrDNA克隆、测序,对健康人肠道中双歧杆菌的组成进行了分析。10例健康人肠道双歧杆菌的TGGE分析显示:人肠道内双歧杆菌的组成具有宿主特异性,不同人肠道双歧杆菌种的多样性和种类不同。通过对一健康儿童肠道双歧杆菌属特异性PCR扩增产物的测序及TGGE电泳行为的分析发现,该个体双歧杆菌TGGE图谱中的条带分别代表Bifidobacteriumbifidum、B.infantis、B.longum、B.adolescentis、B.pseudocatenulatum等种和一新种,其中B.pseudocatenulatum(假小链双歧杆菌)是大多数个体共有且较优势的种类。用传统培养方法只检出B.pseudocatenulatum和B.longum两种。基于双歧杆菌属特异性PCR基础上的TGGE方法结合16SrDNA克隆文库分析可较灵敏、直观地反映人肠道中双歧杆菌的组成。     

Structure and dynamics of an amphibian metacommunity in two regions

1. The concept of metacommunity is based on the hypothesis that species occurrence depends on species dynamics and interactions on local and regional scales via the movements of individuals between localities. Metacommunity approaches are currently being applied to pond breeding taxa such as amphibians. 2. Given that animal movement is also influenced by the physical quality of the matrix to be crossed to reach a breeding habitat and by the affinity of the species for specific terrestrial habitats, matrix characteristics may enhance or hinder dispersal success. These characteristics would, in turn, affect the composition of larval assemblages at local level and, consequently, determine metacommunity structure and dynamics. 3. Here we compared the structures and dynamics of two metacommunities with the same pool of anurans along similar freshwater gradients in two regions that are well differentiated in terms of their respective terrestrial matrix. 4. Abundance of tadpole species and species assemblage in the two regions were determined principally by local processes (at pond level); however, the structure and dynamics of the communities differed. In one region species abundance was explained in part by landscape factors and consequently showed lower co-occurrence and lower colonization rates (species sorting models) indicating that terrestrial habitat could restrict animal movements, whereas in the other region higher co-occurrence and higher colonization rates (mass effect models) indicated low dispersal limitations.     

The distribution of HIV-1 subtypes in Fukuoka, Japan.

To determine the incidence of human immunodeficiency virus type-1 (HIV-1) subtypes in Fukuoka, Japan, viruses from 41 HIV-1 infected individuals were subtyped. Subtyping by V3-loop enzyme-linked immunosorbent assay (ELISA) showed 31 of the 41 subjects as subtype B (MN type), one as subtype A, one as subtype C, and eight untypable. The subject infected with subtype C was identified as a foreigner; the subtype A subject was Japanese. A phylogenetic analysis of nucleic acid sequences from the env C2-V3 region was also conducted. Genetic subtyping was successful for 25 samples: 23 samples were determined as subtype B, one subtype A and one subtype E. One of the individuals infected with subtype B, as well as the subtype A and subtype E subjects, were not Japanese. This study indicated that subtype B (USA and European type) is still dominant among HIV-1 infections in Fukuoka. Further, no Japanese were subtype E positive, which is increasing in the Kanto region. It is notable, however, that subtype A and subtype C infections, which are rare in Japan, were found in Fukuoka, located far from the metropolitan area of Tokyo.     

Phylogenetic analysis of spotted fever group rickettsiae based on gltA, 17-kDa, and rOmpA genes amplified by nested PCR from ticks in Japan

In order to understand the natural situation of rickettsiae in the ticks in Japan, the rickettsial genes, gltA gene, rOmpA gene, and 17-kDa gene, were amplified from the ticks by nested PCR. The prevalences of rickettsial gltA genes among Haemaphysalis formosensis, H. longicornis, H. megaspinosa, Ixodes ovatus, H. flava, H. kitaokai, and I. persulcatus were 62, 57, 24, 24, 19, 13, and 10%, respectively; 26% (186/722) being the average. The gltA genes amplified from the ticks were classified into 9 genotypes (I to IX) by the difference in nucleotide sequences. Genotype I was detected from 7 species of ticks. Genotype II mainly was detected from H. longicornis and H. formosensis. Genotypes III and VII mainly were detected from H. flava and I. ovatus. The polarization in the distribution of genotypes among regions where the ticks were collected was not clear. Based on the phylogenetic analysis of the three genes presented here, genotypes I, III, and IV (detected from H. formosensis, H. hystricia, and I. ovatus ) are genetically close with each other, but rickettsiae of the same property still have not been isolated from ticks anywhere in the world. These genotypes should be considered as new species among SFG rickettsiae. Genotype II was identical with strain FUJ-98, genetically close to R. japonica which has been isolated from ticks in China. Genotype V was identical with R. felis and strain California 2 isolated from the cat flea. This is the first report on the detection of R. felis from ticks. Genotype VI detected from Ixodes sp. did not seem to belong to genus Rickettsia. Based on the previous antigenic data and the phylogenetic analysis presented here, Genotype VII should be considered a variant of R. helvetica and genotype VIII detected from I. ovatus and I. persulcatus were identical with R. helvetica. Genotype IX detected from I. nipponensis was genetically close to the strains IRS3, IRS4, and IrR/Munich isolated from I. ricinus in Slovakia and German.     

Continental‐extent patterns in amphibian malformations linked to parasites,chemical contaminants,and their interactions

Widespread observations of malformed amphibians across North America have generated both concern and controversy. Debates over the causes of such malformations—which can affect >50% of animals in a population—have continued, likely due to involvement of multiple causal factors. Here, we used a 13‐year dataset encompassing 53,880 frogs and toads from 422 wetlands and 42 states in the conterminous USA to test hypotheses relating abnormalities and four categories of potential drivers: (i) chemical contaminants, (ii) land use practices, (iii) parasite infection, and (iv) targeted interactions between parasites and pesticides. Using a hierarchically nested, competing‐model approach, we further examined how these associations varied spatially among geographic regions. Although malformations were rare overall (average = 1.6%), we identified 96 hotspot sites with 5%–25% abnormal individuals. Using the full dataset of 934 collections (without data on parasite infection), malformation frequency was best predicted by the presence of oil and gas wells within the watershed. Among collections also examined for parasite infection (n = 154), average parasite load and its interaction with pesticide application positively predicted malformations: wetlands with a greater abundance of the trematode Ribeiroia ondatrae were more likely to have malformed amphibians, but these effects were strongest when pesticide application was also high, consistent with prior experimental research. Importantly, however, the influence of these factors also varied regionally, helping explain divergent results from previous studies at local scales; parasite infection was more influential in the West and Northeast, whereas pesticide application and oil/gas wells correlated with abnormalities in the Northeast, Southeast, and western regions of the USA. These results, based on the largest systematic sampling of amphibian malformations, suggest that increased observations of abnormal amphibians are associated with both parasite infection and chemical contaminants, but that their relative importance and interaction strength varied with the spatial extent of the analysis.     

Characterization of spotted fever group rickettsiae detected in dogs and ticks in Okinawa,Japan

Spotted fever group (SFG) rickettsial DNAs were detected in 2.4% of 340 canine blood samples and a pool of 84 tick pool samples (229 ticks) collected in Okinawa, Japan by PCR using a citrate synthase and an SFG rickettsial 190-kDa surface antigen gene primer pair. The sequences of both genes from canine blood and tick samples showed high levels of similarity with those of Rickettsiajaponica and several SFG rickettsiae (R. aeschlimannii, R. massiliae, R. rhipicephali and Bar-29 strain). Phylogenesis of canine blood and tick samples was closely related to that of reference SFG rickettsiae. Serological evidence of SFG rickettsial infection in dogs and humans in Okinawa, where no clinical human cases have been reported, has been obtained. In this study, genetical characterization of SFG rickettsia in Okinawa was investigated phylogenetically.     

Cytological,genetic, and biochemical characteristics of an unusual non‐Chlorella photobiont of Stentor polymorphus collected from an artificial pond close to the shore of Lake Biwa,Japan

Stentor polymorphus (Müller) Ehrenberg (Heterotrichea, Ciliophora) is a trumpet‐form ciliate with endosymbiotic green algae (photobionts). Previous reports have indicated that this photobiont has a form typical of Chlorella Beijerinck (Trebouxiophyceae), with one chloroplast and a distinct pyrenoid. We collected S. polymorphus from an artificial pond on the shore of Lake Biwa in Japan. Microscopic examination demonstrated that there was no pyrenoid in the photobiont, and thus we analyzed the cytological, genetic, and biochemical characteristics of these algal cells. Cultured photobionts were smaller than those in hospite. Phylogenetic analyses placed the photobiont within the genus Mychonastes Simpson and Van Valkenburg, a chlorophycean alga occurring in fresh/brackish waters that lacks pyrenoids. Mychonastes provided little photosynthate to its host; the quantity of carbohydrate amounted to only 2% of that provided to Paramecium bursaria (Ehrenberg) Focker by its photobiont.     

Development of a broadly reactive nested reverse transcription-PCR assay to detect murine noroviruses, and investigation of the prevalence of murine noroviruses in laboratory mice in Japan

A broadly reactive nested RT-PCR assay to detect MNV was developed and subsequently used to investigate the prevalence of MNV in laboratory mice in Japan. MNV were detected in 8 (22%) of 37 murine stool specimens by second-round PCR, although no positive band was obtained from any specimen by first-round PCR. Genetic analysis of the second round PCR products showed that MNV sequences detected in this study were closely matched (97.2 ∼ 99.1%) to that of MNV-3 (DQ223042). This is the first report demonstrating the prevalence of MNV in Japan.     

Phylogenetic placement,taxonomic revision and a new species of Nothostele (Orchidaceae), an enigmatic genus endemic to the cerrado of central Brazil

Nothostele is a rare genus endemic to central Brazil. The taxonomy of the genus is controversial and almost every taxonomist has had a different point of view regarding its generic and subtribal classification. After the first collection 138 years ago, N. acianthiformis has been collected again and we report here a phylogenetic analysis of the genus based on nuclear ribosomal internal transcribed spacer and plastid matK and trnL–trnF sequence data. Our results show that Nothostele belongs to subtribe Spiranthinae and is sister to Eltroplectris within the Stenorrhynchos clade. A reanalysis of gynostemium morphology shows that the presence of a hamulus was misinterpreted in Nothostele and that the placement of the genus in Cranichidinae based on morphology of the pollinarium is unwarranted. Furthermore, the flattened, sessile, prostrate leaves of N. acianthiformis, which are described here for the first time, occur in some Spiranthinae but are atypical in Cranichidinae. Specimens from Brasília in the Brazilian central plateau are a distinct new species, which is described here as N. brasiliaënsis. Currently, the genus comprises two disjunct species restricted to the cerrado and rocky field vegetation of central Brazil. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 165 , 348–363.     

Origin of the plastid in the anomalously pigmented dinoflagellate Gymnodinium mikimotoi (Gymnodiniales, Dinophyta) as inferred from phylogenetic analysis based on the gene encoding the large subunit of form I-type RuBisCO

The dinoflagellate Gymnodinium mikimotoi Miyake et Kominami ex Oda possesses an anomalously pigmented plastid which contains 19′‐hexanoyloxyfucoxanthin, 19′‐butanoyloxyfucoxanthin and fucoxanthin instead of peridinin as the major carotenoids. Previously, we have shown that the plastid of G. mikimotoi belongs to the rhodoplast lineage as inferred from phylogenetic analyses based on the amino acid sequences deduced from psbA and psaA and the nucleotide sequence of the plastid small subunit ribosomal RNA. Furthermore, in the present study, we cloned and sequenced an additional representative plastid gene, rbcL, encoding the large subunit of ribulose 1–5 bisphosphate carboxylase/oxygenase (RuBisCO LSU) from G. mikimotoi. The amino acid sequence deduced from the rbcL gene of G. mikimotoi apparently revealed the conventional form I RuBisCO LSU, which is present in most photosynthetic organisms, and not the divergent form II existing in typically pigmented dinofl age Nates with plastids containing peridinin as the main carotenoid. This finding supports the hypothesis that the origins of the plastids in G. mikimotoi and peridinin‐type dinoflagellates are not related to each other. Molecular phylogenetic analysis based on the amino acid sequence deduced from the rbcL gene further showed that the plastid of G. mikimotoi belongs to the rhodoplast lineage. In particular, G. mikimotoi clustered with haptophytes in the phylogenetic tree. From this result, two hypotheses with respect to the origin of the plastid in G. mikimotoi can be proposed: G. mikimotoi may have engulfed a haptophyte‐like cell (tertiary symbiosis) or englulfed a rhodophyte‐like cell that was closely related to the origin of the plastid in the haptophyte (secondary symbiosis).     

Population dynamics and production of Jesogammarus annandalei, an endemic amphipod, in Lake Biwa, Japan

SUMMARY 1. Population dynamics and production of Jesogammarus annandalei , an endemic amphipod in Lake Biwa, were examined from April 1997 to June 1998. The life cycle of this species was 1 year with the new generation beginning in early autumn. They preferred low temperature (<12 °C) and their spatial distribution varied seasonally and accordingly.
2. In deep water, the abundance of J. annandalei ranged from 200 to 63 000 m⁻² and decreased towards summer and the biomass (0.01∼3.6 g C m⁻²) was on average comparable that of zooplankton. The density was much higher than that recorded by a study conducted 35 years ago.
3. Individual growth rate of this amphipod was high in winter and spring but decreased in summer. Annual production of J. annandalei (6.2 g C m⁻² year⁻¹) was only 2% of primary production but was at the higher end of the range reported for amphipods in oligo- and mesotrophic lakes.
4. These results are consistent with the view that Lake Biwa is becoming more eutrophic, with a consequent decrease in the abundance of predatory fish in the profundal zone.     

Ecology,conservation status,and phylogenetic placement of endemic Pristimantis frogs (Anura: Craugastoridae) in Trinidad and Tobago and genetic affinities to northern Venezuela

Trinidad and Tobago are home to three endemic species in the anuran genus Pristimantis, of which two (Pristimantis charlottevillensis and Pristimantis turpinorum) occur in Tobago alone and the third (Pristimantis urichi) is present on both islands. Earlier, the IUCN assessed the conservation status of these species as: P. urichi, Endangered (EN); P. charlottevillensis, Least Concern (LC); P. turpinorum, Vulnerable (VU). However, these assessments were based on very little field-based evidence. Here, we present survey results which contributed to reassessments as LC, VU and Data Deficient for these three species, respectively. Despite the close proximity of Trinidad to northern Venezuela, the islands do not share any Pristimantis species with the mainland, which holds a rich endemicity of Pristimantis regionally. In this study, we used genetic sequencing from several island populations and compared them to northern Venezuelan endemics to assess genetic divergence for the first time. The time tree analyses found that only the northern Tobago species P. turpinorum is closely related to mainland Pristimantis, with a genetic split dating to the Late Miocene, suggesting a vicariant event of mainland and island species. Pristimantis urichi, although identical between the two islands, remains highly divergent from the mainland species. Similar results were found for P. charlottevillensis. In addition, there was a high level of divergence between P. urichi and P. charlottevillensis. These findings indicate different island colonization events by different lineages. Sequencing other Venezuelan species remains pivotal to unravel the complexity of the colonization episodes in the region, likely influenced by the changing topography and multiple connection and isolation episodes of the islands by eustatic sea-level changes.     

Climatic preferences and frequent co-occurrence of boreal and temperate plants in Hokkaido Island,northern Japan

Effects of climatic factors on the forest plant distribution were examined in Hokkaido Island, northern Japan, where boreal and temperate plants frequently co-occur, and the relationship of floral make-up with phytogeography and life form was analysed. From the climatic preference of tree species consisting of mixed forest, the co-occurrence of boreal and temperate plants was found over a wider thermal range in regions of little rainfall. However, co-occurrence in a certain forest site was more notable in the understory than in the crown. A particular case of this involves southern evergreen shrubs and herbs in a boreal coniferous forest. Another is the presence of northern summergreen herbs in a temperate hardwood forest. It is speculated that the co-occurrence dates back at least to the late Quaternary, when a decrease in temperature associated with the glacial period forced understory plants to adapt their life form or leaf habit to snowcover and the light conditions of interior forests. Recent geographical evidence suggests that the island has been isolated from the southern island for at least 70000 years. Thus, the postglacial re-expansion of southern species does not appear to be due to immigration but rather due to their expansion from refuges on the island such as Oshima Peninsula for snow-tolerant species and Hidaka District for snow-intolerant species.     

Molecular epidemiology of Coxsackievirus A16 strains isolated from children in Yamagata,Japan between 1988 and 2011

To clarify the longitudinal molecular epidemiology of coxsackievirus A16, phylogenetic analysis based on the VP1 region of 220 isolates in Yamagata, Japan was performed. The resultant phylogenetic tree indicates that the Yamagata isolates and reference strains can be readily genotyped into three genogroups, and 0, 12 and 208 isolates belonged to the first, second, and third genogroups, respectively. The first genogroup includes only the prototype strain, the second strains that had disappeared by the end of the 20th century and the third comprises those that have been circulating since then in local communities, such as Yamagata.     

Structure and composition of riparian forests with special reference to geomorphic site conditions along the Tokachi River,northern Japan

The structure and composition of riparian forests were examined along the Tokachi River, northern Japan. Both the hydrogeomorphic gradient and the temporal gradient were analyzed in attempt to explain the present pattern of riparian forests. The stability of floodplain surfaces was estimated on the basis of the elevation above the riverbed and the distance from the river channel. The characteristics of the substratum on which trees were established were also examined by excavation of buried sediment. The results indicated that soil moisture and organic content increased while the size of particles in the substratum decreased with increased elevation and distance from the river channel. Gradient analysis was employed to examine the distribution of dominant species, such as Alnus hirsuta, Toisusu urbaniana, Populus maximowiczii, Picea jezoensis and Abies sachalinensis. The relative dominance of each could be arrayed across the elevation gradient. Although the three broad-leaved pioneers dominated bars and floodplains near the river channel, their modes shifted from lower to higher elevation and amplitudes of distribution curves decreased in the following order: A. hirsuta, T. urbaniana, P. maximowiczii and conifers, which were located on the highest floodplains. Sites could be divided into three classes in terms of stability. There were fewer species at active sites, which favored the three pioneer species, but species richness and diversity increased with stand age. Semi-active and stable sites were more diverse with the establishment of conifers and other broad-leaved trees, which included upland species. However, species richness peaked and then decreased after trees reached 50 to 60 years of age. The growth of dwarf bamboo and the development of conifer-dominant stands impeded the establishment of other species, thereby reducing species richness and diversity in mature stands. Chronologically, floodplains could be differentiated into high- and low-frequency zones of flood disturbance, with pioneer species occupying the former, and late successional species found largely in the latter.     

Genotyping and quantitation of noroviruses in oysters from two distinct sea areas in Japan

Norovirus (NV) is a causative agent of acute gastroenteritis in humans, and shellfishes including oysters act as major vehicles of the virus. To investigate the genetic characteristics of NVs, we collected 1,512 oysters for raw consumption between October 2002 and March 2005 from two distinct areas (area A: the Sanriku Sea area; area B: the Setouchi Sea area). We detected the capsid gene and subjected it to phylogenetic analysis. By further quantification of the copy number of the genome by using real-time PCR, the NV capcid gene was detected in approximately 5% of the oysters, and they showed wide diversity. Two percent of the oysters from area B showed relatively large number of NVs, i.e., over 100 copies of capsid gene/oyster, whereas this was not observed in area A. Most of the detected NVs from oysters and humans were genetically related when the capsid region was compared. These results suggested that NVs obtained from humans and those obtained from oysters showed a potential relationship to each other and that some populations of Japanese oysters accumulated a relatively large number of NVs.