An Efficient Procedure for Isolating Microsatellite DNAs from Sea Cucumber(Apostichopus japonicus)

1 Introduction Microsatellite DNAs, also called simple sequence re-peats (SSRs), are stretches of DNA, consisting of tan-demly repeating mono-, di-, tri-, tetra- or penta-nucleotide units. They are evenly distributed throughout the ge-nomes of most eukaryotic species (Tohn et al., 2000). Their high information content about the divergence of genome, which is directly related to the number of alleles at each locus, and the ease of PCR assays make them ex-cellent molecular markers (Powell …     

An efficient procedure for isolating microsatellite DNAs from sea cucumber (Apostichopus japonicus)

The construction of enrichment library proves to be one of the efficient approaches for isolating microsatellites in this study. The genomic DNA of sea cucumber was digested with HaeIII and size-selected DNA fragments (250–700 bp) were ligated to an adaptor. Microsatellite-containing sequences were captured by using a combination of GA and CA probes, which were attached to a nylon membrane. The microsatellite enrichment library constructed in this study consisted of approximately 700 clones. Two hundred and thirty-two clones reacted positively after the library screening procedure. Of the 50 clones sequenced, all contained at least one microsatellite and one duplicate clone was found. Approximately 86% of the sequenced fragments permitted to design primers for sequence tagged microsatellite site (STMS).     

Construction of bacterial artificial chromosome libraries for Zhikong Scallop <Emphasis Type="Italic">Chlamys farreri</Emphasis>

Two Large-insert genomic bacterial artificial chromosome （BAC） libraries of Zhikong scallop Chlamys farreri were constructed to promote our genetic and genomic research. High-quality megabase-sized DNA was isolated from the adductor muscle of the scallop and partially digested by BamH I and Mbo I, respectively. The BamH I library consisted of 53 760 clones while the Mbo I library consisted of 7 680clones. Approximately 96 % of the clones in BamH I library contained nuclear DNA inserts in average size of 100 kb, providing a coverage of 5.3 haploid genome equivalents. Similarly, the Mbo I library with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1 haploid genome equivalents.     

Antimicrobial activities of novel cultivable bacteria isolated from marine sponge Tedania anhelans

Marine sponge Tedania anhelans distributes throughout the intertidal zone of Fujian, southeastern China, and is a potential source of natural bioactive products. The sponge harbors a large number of bacterial groups that have been identified using various techniques, including fluorescent in situ hybridization (FISH). Fractionation of dissociated sponge allowed isolation of 25 bacterial species. Based on 16S rRNA gene sequencing, phylogenetic analysis attributed most of these eubacteria to α-Proteobacteria, γ-Proteobacteria, Cytophaga/Flavobacterium/Bacteroidetes (CFB group), and the family Bacillaceae of Gram-positive bacteria. In sequence similarity, five putatively novel species were identified with less than 98% similarity to other strains in the NCBI database. Tests for antimicrobial activities were performed against Gram-positive bacteria, Gram-negative bacteria, fungi, antitumor indicators Escherichia coli 343/591 (with DNA repair deficiency), regular E. coli 343/636 (with different DNA repair capacity), and 10 bacterial isolates exhibited inhibitory bioactivities. Among these strains, three isolates were detected involving function gene NRPS-A domains, which were most closely related to the amino acid sequences of linear gramicidin synthetase and pyoverdine synthetase. These results contribute to our knowledge of the microbes associated with marine sponges and further reveal novel bacterial resources for the screening of bioactive marine natural products.     

Recent researches of bioactive metabolites in marine organisms-associated microorganisms

1　Introduction　Sincetheearly 196 0s ,oceanhasbeenconsideredasanewanduntouchedsourceofpotentiallyusefulcom pounds .Theresultsfromtheresearchesontheorgan ismssuchassponges,softcorals ,algae ,ascidians ,bryozoansandmollusks ,havedemonstratedthatma rineorga…     

盐酸普鲁卡因诱导下海洋真菌Hypocrea lixii次级代谢化学成分

【目的】通过化学诱导方法从一株海洋真菌Hypocrea lixii中获得多样化的活性小分子化合物。【方法】对菌株在盐酸普鲁卡因的化学诱导作用下进行固体发酵,代谢产物采用硅胶减压柱层析、Sephadex LH-20以及pHPLC等手段分离纯化,对化合物运用NMR、MS等波谱学技术并比对文献进行结构鉴定,采用Ellman比色法及DPPH自由基清除法对化合物进行初步抗乙酰胆碱酯酶(AChE)和抗氧化活性测试。【结果】从海洋真菌Hypocrea lixii在盐酸普鲁卡因诱导下的发酵提取物中分离得到4个异黄酮类化合物,包括6'-O-crotonylgenistin(1)、Genistein(2)、Daidzein(3)和Genistin(4),以及对氨基苯甲酸甲酯(5)、对羟基苯丙酸(6)和胞嘧啶(7),其中化合物1和6为该种中首次报道,化合物5为生物转化来源首次报道,化合物2~4可能源自培养基。在100μmol/L剂量浓度下,化合物1~4对AChE的抑制以及DPPH自由基的清除活性均较弱,化合物5和6显示了相对强的AChE抑制活性,抑制率分别为40%和38%。【结论】盐酸普鲁卡因对该菌次级代谢产生了显著的影响,为深入研究该菌株化学诱导次级代谢产物提供基础。     

Analysis of SSRs Derived from an EST Library of Half-Smooth Tongue Sole Cynoglossus semilaevis

A complementary DNA (cDNA) library was constructed from half-smooth tongue sole spleen. A long-read expressed sequence tag (EST) database was generated, containing 3100 cDNA clones, of which 220 clones were fully sequenced. A total of 1060 non-redundant simple sequence repeats (SSRs) were obtained from the cDNA library. An average of 5 kb sequence generates 1 SSR in the half-smooth tongue sole spleen cDNA library. The proportion of the SSR unit size was different in the cDNA library. The monomeric repeats (51.4%) are the most abundant class of SSR in the dataset. The dimeric, trimeric, tetrameric and hexameric re- peats are represented in decreasing proportions of 27.2%, 16.0%, 2.8% and 1.9%, respectively. The frequency of pentameric repeats was observed the least (only 0.7%). Most of the monomeric and dimeric repeats are distributed in 3' and 5' un-translation region. If translation regions are considered merely, trimeric repeats are the highest, accounting for 57% of the total microsatellites.     

Analysis of SSRs derived from an EST library of half-smooth tongue sole <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

A complementary DNA (cDNA) library was constructed from half-smooth tongue sole spleen. A long-read expressed sequence tag (EST) database was generated, containing 3100 cDNA clones, of which 220 clones were fully sequenced. A total of 1060 non-redundant simple sequence repeats (SSRs) were obtained from the cDNA library. An average of 5 kb sequence generates 1 SSR in the half-smooth tongue sole spleen cDNA library. The proportion of the SSR unit size was different in the cDNA library. The monomeric repeats (51.4%) are the most abundant class of SSR in the dataset. The dimeric, trimeric, tetrameric and hexameric repeats are represented in decreasing proportions of 27.2%, 16.0%, 2.8% and 1.9%, respectively. The frequency of pentameric repeats was observed the least (only 0.7%). Most of the monomeric and dimeric repeats are distributed in 3′ and 5′ un-translation region. If translation regions are considered merely, trimeric repeats are the highest, accounting for 57% of the total microsatellites.     

Isolation and characterization of bioactive fungi from shark Carcharodon carcharias' gill with biopharmaceutical prospects

Until recently,little was known about the fungi found in shark gills and their biomedicinal potential. In this article,we described the isolation,bioactivity,diversity,and secondary metabolites of bioactive fungi from the gill of a shark(Carcharodon carcharias). A total of 115 isolates were obtained and grown in 12 culture media. Fifty-eight of these isolates demonstrated significant activity in four antimicrobial,pesticidal,and cytotoxic bioassay models. Four randomly selected bioactive isolates inhibited human cancer cell proliferation during re-screening. These active isolates were segregated into 6 genera using the internal transcribed spacer-large subunit(ITS-LSU) rDNA-sequence BLAST comparison. Four genera,Penicillium,Aspergillus,Mucor,and Chaetomium were the dominant taxa. A phylogenic tree illustrated their intergenera and intragenera genetic diversity. HPLC-DAD-HRMS analysis and subsequent database searching revealed that nine representative strains produced diverse bioactive compound profiles. These results detail the broad range of bioactive fungi found in a shark's gills,revealing their biopharmaceutical potential. To the best of our knowledge,this is the first study characterizing shark gill fungi and their bioactivity.     

Antimicrobial Activity of Ulva reticulata and Its Endophytes

Seaweeds are known to exhibit various antimicrobial properties,since it harbours an enormous range of indigenous bioactive compounds.The emergence of drug resistant strains has directed to the identification of prospective metabolites from seaweed and its endophytes,thereby exploiting the properties in resisting bacterial diseases.The current study was aimed to assess the antimicrobial activity of extracts obtained from Ulva reticulate,for which metabolites of Ulva reticulata and its endophytes were extracted and assessed against human pathogens like Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa,Salmonella typhi,and Bacillus subtilis.It was observed that the hexane extract of isolate VITDSJ2 was effective against all the tested pathogens but a significant inhibition was observed for Staphylococcus aureus and Escherichia coli.Further,Gas chromatography coupled with Mass spectroscopy(GC-MS) revealed the existence of phenol,3,5-bis(1,1-dimethylethyl) in the crude hexane extract which is well-known to possess antibacterial activity.The effective isolate VITDSJ2 was identified to be the closest neighbour of Pseudomonas stutzeri by phenotypic and genotypic methods.The crude extracts of the seaweed Ulva reticulata was also screened for antibacterial activity and the hexane extract was effective in showing inhibition against all the tested pathogens.The compound in the crude extract of Ulva reticulata was identified as hentriacontane using GC-MS.The extracts obtained from dichloromethane did not show significant activity in comparison with the hexane extracts.Hence the metabolites of Ulva reticulata and the bacterial secondary metabolites of the endophytes could be used in the treatment of bacterial infections.     

Phylogenetic diversity and bioactivity of culturable deep-sea-derived fungi from Okinawa Trough

Deep-sea sediments are now recognized as a home for rich and largely microbial community. Recently, it has been believed in an increasing number of studies that bacteria could be abundant in deepsea sediments of many types; however, fungi in deep-sea sediments remain relatively unknown. The phylogenetic diversity and bioactivity of culturable deep-sea-derived fungi from Okinawa Trough sediments were investigated in traditional method combined with fungal identification of molecular biology in this study. A total of 76 isolates belonged to 15 fungal taxa were recovered in a harsh condition of low nutrient and low temperature, indicating that the fungal communities in deep-sea sediments from Okinawa Trough were relatively abundant and diversified. Aspergillus, Cladosporium, and Penicillium were the dominant fungal genera, while Mycosphaerella, Purpureocillium, and Schizophyllum were relatively rare in the deep-sea sediments from Okinawa Trough. Among the six genera recovered, Mycosphaerella was firstly recovered from deep-sea sediments in this study. Moreover, about 75% of the extracts from the 15 fungal representative isolates displayed distinct bioactivity against at least one indicator bacterium or marine macrofouler, emphasizing the potentials of these deep-sea-derived fungi from Okinawa Trough as producers of bioactive metabolites. Notably, isolates Cladosporium oxysporum SCSIO z001 and Penicillium citrinum SCSIO z049 displayed a wide spectrum of bioactivities, isolates Cladosporium cladosporioides SCSIO z015, Cladosporium sphaerospermum SCSIO z030, and Penicillium verruculosum SCSIO z007 exhibited a strong anti-bacterial-growth activity, and isolate Penicillium chrysogenum SCSIO z062 displayed a strong anti-larval-settlement activity. These results suggest that these isolates deserved further study as potential sources of novel bioactive metabolites.     

Construction of cDNA Library of Pyrocystis lunula（Pyrophyta）

1　Introduction　Photosyntheticdinoflagellatesareimportantprima ryproducersandcausesoftoxic‘redtide’ .Theyarefoundinmostaquaticenvironmentsandformamajorpartofthemodernplankton .Dinoflagellatesmaybeimportantindicatorsofenvironmentalstatus (Dale ,1996 ) .…     

Construction of a Muscle cDNA Library of Chinese Shrimp Fenneropenaeus chinensis and Sequence Analysis of the Troponin Ⅰ Gene

A muscle cDNA library of Chinese shrimp (Fenneropenaeus chinensis) was constructed with the SMARTTM cDNA Li- brary Construction Kit. The titer of optimal primary library was 7.7×105 pfu mL-1 and that of the amplified library was 3.0×109 pfu mL-1. The percentages of the recombinant clones of primary and amplified libraries were over 98%. The insert sizes were longer than 400 bp with an average of 1000 bp. A positive clone containing a 794 bp insert was sequenced and identified encoding fast skeletal troponin Ⅰ gene. This library provided a useful resource for the functional genomic research ofE chinensis.     

Sequences Characterization of Microsatellite DNA Sequences in Pacific Abalone (Haliotis discus hannat)

The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the microsatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber(1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats(13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (＜ 20 repeats) were most abundant,accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatetlite isolation in other abalone species.     

Sequences characterization of microsatellite DNA sequences in Pacific abalone (Haliotis discus hannai)

The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the microsatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber(1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats(13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (＜ 20 repeats) were most abundant,accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatetlite isolation in other abalone species.     

Sequences Characterization of Microsatellite DNA Sequences in Pacific Abalone(Haliotis discus hannai)

1 Introduction Microsatellites or simple sequence repeats (SSRs) are tandemly repeated motifs of one to six bases found in all prokaryotic and eukaryotic genomes analysed to date (Zane et al., 2002). Due to their hyper-variable and co-dominant nature, relatively high abundance and random distribution in the genome, microsatellites are among the most efficient class of molecular markers. Such repeats display high polymorphism because of variation in repeat length and can be rapidly analysed t…     

Isolation and characterization of bioactive fungi from shark <Emphasis Type="Italic">Carcharodon carcharias</Emphasis>’ gill with biopharmaceutical prospects

Until recently, little was known about the fungi found in shark gills and their biomedicinal potential. In this article, we described the isolation, bioactivity, diversity, and secondary metabolites of bioactive fungi from the gill of a shark (Carcharodon carcharias). A total of 115 isolates were obtained and grown in 12 culture media. Fifty-eight of these isolates demonstrated significant activity in four antimicrobial, pesticidal, and cytotoxic bioassay models. Four randomly selected bioactive isolates inhibited human cancer cell proliferation during re-screening. These active isolates were segregated into 6 genera using the internal transcribed spacer-large subunit (ITS-LSU) rDNA-sequence BLAST comparison. Four genera, Penicillium, Aspergillus, Mucor, and Chaetomium were the dominant taxa. A phylogenic tree illustrated their intergenera and intragenera genetic diversity. HPLC-DAD-HRMS analysis and subsequent database searching revealed that nine representative strains produced diverse bioactive compound profiles. These results detail the broad range of bioactive fungi found in a shark’s gills, revealing their biopharmaceutical potential. To the best of our knowledge, this is the first study characterizing shark gill fungi and their bioactivity.     

Construction of a muscle cDNA library of Chinese shrimp <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> and sequence analysis of the troponin I gene

A muscle cDNA library of Chinese shrimp (Fenneropenaeus chinensis) was constructed with the SMART™ cDNA Library Construction Kit. The titer of optimal primary library was 7.7×10⁵ pfu mL⁻¹ and that of the amplified library was 3.0×10⁹ pfu mL⁻¹. The percentages of the recombinant clones of primary and amplified libraries were over 98%. The insert sizes were longer than 400 bp with an average of 1000 bp. A positive clone containing a 794 bp insert was sequenced and identified encoding fast skeletal troponin I gene. This library provided a useful resource for the functional genomic research of F. chinensis.     

Identification and characterization of endosymbiosis-related immune genes in deep-sea mussels Gigantidas platifrons

Deep-sea mussels of the subfamily Bathymodiolinae are common and numerically dominant species widely distributed in cold seeps and hydrothermal vents.During long-time evolution,deep-sea mussels have evolved to be well adapted to the local environment of cold seeps and hydrothermal vents by various ways,especially by establishing endosymbiosis with chemotrophic bacteria.However,biological processes underlying the establishment and maintenance of symbiosis between host mussels and symbionts are largely unclear.In the present study,Gigantidasplatifrons genes possibly involved in the symbio sis with methane oxidation symbionts were identified and characterized by Lipopoly saccharide(LPS) pull-down and in situ hybridization.Five immune related proteins including Toll-like receptor 2(TLR2),integrin,vacuolar sorting protein(VSP),matrix metalloproteinase 1(MMP1),and leucine-rich repeat(LRR-1) were identified by LPS pull-down assay.These five proteins were all conserved in either molecular sequences or functional domains and known to be key molecules in host immune recognition,phagocyto sis,and ly so some-mediated digestion.Furthermore,in situ hybridization of LRR-1,TLR2 and VSP genes was conducted to investigate their expression patterns in gill tissues of G.platifrons.Consequently,LRR-1,TLR2,and VSP genes were found expressed exclusively in the bacteriocytes of G.platifrons.Therefore,it was suggested that TLR2,integrin,VSP,MMP1,and LRR-1 might be crucial molecules in the symbiosis between G.platifrons and methane oxidation bacteria by participating in symbio sis-related immune processes.     

Construction and characterization of a fosmid library for pathogenic bacterium <Emphasis Type="Italic">Vibrio anguillarum</Emphasis>

Vibrio anguillarum is a common bacterial pathogen in fish.However,little is known about its pathogenic mechanism,in part,because the entire genome has not been completely sequenced.We constructed a fosmid library for V.anguillarum containing 960 clones with an average insert size of 37.7 kb and 8.6-fold genome coverage.We characterized the library by end-sequencing 50 randomly selected clones.This generated 93 sequences with a total length of 57 485 bp covering 1.4% of the whole genome.Of these sequences,58...