The Influence of Aerial Exposure on Sea Anemones Aulactinia veratra Mucin Genes Expression Using the RNA Sequencing

Mucin genes are the main component of mucus. The sea anemone species, Aulactinia veratra (Phylum Cnidaria) contains different types of mucin genes. In the intertidal zone, A. veratra is found to be exposed to air during the low tide and produces large quantities of mucus as an external covering. The relation between low tide and mucus secretion is still unclear, and what is the role of mucin during arial exposure is not yet investigated. This study hypothesised that the mucin genes in A. veratra would have significantly high expression in response to aerial exposure. Therefore, the aim of current study was to examine and analyses the response of A. veratra mucins in response to an experiment involving three hours of aerial exposure. To achieve this, aim the RNA-sequencing and bioinformatics analyses were used to examine the expression profile of A. veratra mucin genes in response to aerial exposure. The generated results have shown that, Mucin4-like and mucin5B-like were up-regulated in response to the three hours of aerial exposure in A. veratra. This finding shows a significant role of mucin5B-like and mucin4-like genes in response to air stress at low tide. The data generated from this study could be used in conjunction with future mucin gene studies of sea anemones and other cnidarians to compare A. veratra mucin gene expression results across time, and to extend our understanding of mucin stress response in this phylum.

The Characteristics of vasa Gene from Japanese Sea Bass(Lateolabrax japonicas) and Its Response to the External Hormones

The RNA helicase Vasa is an important regulator of primordial germ cell development. Its function in mature fish, especially the hormone-related differences in maturing male fish has seldom been documented. In this study, a full length c DNA sequence of the vasa gene was cloned from Japanese sea bass, Lateolabrax japonicas, and it was named jsb-vasa. Homology analysis showed that jsb-vasa was closely related to its teleost homologs. The spatial distribution of jsb-vasa indicated that it was only highly expressed in testis, showing its germ cell-specific expression pattern. During the testicular development cycle, jsb-vasa was highly expressed during early period of spermatogenesis, and reduced when spermatogenesis advanced. In addition, the jsb-vasa gene expression was significantly inhibited at 6 h, 12 h and 24 h after injecting h CG(human chorionic gonadotropin) and Gn RHa(Gonadotropin-releasing hormone analogue), indicating that jsb-vasa gene may play an important role in spermatogenesis of Japanese sea bass, and be under the regulation of external sex hormones.

Gene cloning and sequence analysis of the cold-adapted chaperones DnaK and DnaJ from deep-sea psychrotrophic bacterium Pseudoalteromonas sp. SM9913

Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR （GenBank accession Nos DQ640312, DQ504163 ）. The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.

Differential gene expression in the body wall of the sea cucumber(Apostichopus japonicus)under strong lighting and dark conditions

Sea cucumber, Apostichopus japonicus is very sensitive to light changes. It is important to study the influence of light on the molecular response of A. japonicus. In this study, RNA-seq provided a general overview of the gene expression profiles of the body walls of A. japonicus exposed to strong light（＂light＂）, normal light（＂control＂） and fully dark（＂dark＂） environment. In the comparisons of ＂control＂ vs. ＂dark＂, ＂control＂ vs. ＂light＂ and ＂dark＂ vs.＂light＂, 1 161, 113 and 1 705 differentially expressed genes（DEGs） were identified following the criteria of｜log2 ratio｜≥1 and FDR≤0.001, respectively. Gene ontology analysis showed that ＂cellular process＂ and ＂binding＂enriched the most DEGs in the category of ＂biological process＂ and ＂molecular function＂, while ＂cell＂ and ＂cell part＂ enriched the most DEGs in the category of ＂cellular component＂. And the DEGs were mapped to 214, 41 and229 pathways in the Kyoto Encyclopedia of Genes and Genomes database, and 51, 2 and 57 pathways were significantly enriched, respectively. Light-specific DEGs identified in this study will be important targets for further investigation to establish the biochemical mechanisms involved in the adaption of this sea cucumber to changes in the level of environmental light.

Microbial community structure and nitrogenase gene diversity of sediment from a deep-sea hydrothermal vent field on the Southwest Indian Ridge

A sediment sample was collected from a deep-sea hydrothermal vent field located at a depth of 2 951 m on the Southwest Indian Ridge. Phylogenetic analyses were performed on the prokaryotic community using polymerase chain reaction（PCR） amplification of the 16 S rRNA and nifH genes. Within the Archaea, the dominant clones were from marine benthic group E（MBGE） and marine group I（MGI） belonging to the phyla Euryarchaeota and Thaumarchaeota, respectively. More than half of the bacterial clones belonged to the Proteobacteria, and most fell within the Gammaproteobacteria. No epsilonproteobacterial sequence was observed. Additional phyla were detected including the Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Nitrospirae, Chloroflexi, Chlorobi, Chlamydiae, Verrucomicrobia, and candidate divisions OD1, OP11, WS3 and TM6, confirming their existence in hydrothermal vent environments. The detection of nifH gene suggests that biological nitrogen fixation may occur in the hydrothermal vent field of the Southwest Indian Ridge. Phylogenetic analysis indicated that only Clusters I and III NifH were present. This is consistent with the phylogenetic analysis of the microbial 16 S rRNA genes, indicating that Bacteria play the main role in nitrogen fixation in this hydrothermal vent environment.

Spatial distribution characteristics of bacterial community structure and gene abundance in sediments of the Bohai Sea

This study investigated differences in the community structure and environmental responses of the bacterial community in sediments of the Bohai Sea.Illumina high-throughput sequencing technology and real-time PCR were used to assay the bacterial 16S rRNA genes in the surface sediments of 13 sampling stations in the Bohai Sea.The results showed that sediments at the majority of the 13 sampling stations were contaminated by heavy metal mercury.The main phyla of bacteria recorded included Proteobacteria(52.92%),Bacteroidetes(11.76%),Planctomycetes(7.39%),Acidobacteria(6.53%)and Chloroflexi(4.97%).The genus with the highest relative abundance was Desulfobulbus(4.99%),which was the dominant genus at most sampling stations,followed by Lutimonas and Halioglobus.The main factors influencing bacterial community structure were total organic carbon,followed by depth and total phosphorus.The content of lead,cadmium,chromium,copper and zinc had a consistent effect on community structure.Arsenic showed a negative correlation with bacterial community structure in most samples,while the impact of mercury on community structure was not significant.The bacterial community in sediment samples from the Bohai Sea was rich in diversity and displayed an increase in diversity from high to low latitudes.The data indicated that the Bohai Sea had abundant microbial resources and was rich in bacteria with the potential to metabolize many types of pollutants.

Phylogenetic diversity of ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit genes of bacterioplankton in the East China Sea

Phylogenetic diversity of Form I and Form II ribulose1, 5-bisphosphate carboxylase/oxygenase (RubisCO) largesubunit (rbcL) genes in the inshore and offshore areas of the East China Sea were investigated. Two new primer setswere designed for amplifying partial sequences of rbcL genes from Proteobacteria. Four rbcL gene clone libraries wereconstructed by amplification and cloning of approximately 640~800 bp sequences of bacterioplankton populations.The method of screening library by denaturing gradient gel electrophoresis (DGGE) was introduced. The resultsshow that the diversity of Form I is higher in offshore waters with higher salinity and lower productivity, while thatof Form II is higher at the inshore station where salinity is lower and productivity is higher. Several clusters ofsequences obtained are deeply rooted and show low similarity (60%~78%) to the known rbcL in existing databases.The degree of diversity of rbcL genes is directly related to environmental variables, including temperature, salinity,pH, dissolved oxygen, etc. These results indicate that rbcL gene can be used as an effective indicator for geneticdiversity and population variability of bacterioplankton with the ability of carbon dioxide fixation in the sea.

Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

Quantitative real-time reverse transcription-polymerase chain reaction(qRT-PCR) is a technique that is widely used for gene expression analysis,and its accuracy depends on the expression stability of the internal reference genes used as normalization factors.However,many applications of qRT-PCR used housekeeping genes as internal controls without validation.In this study,the expression stability of eight candidate reference genes in three tissues(intestine,respiratory tree,and muscle) of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm,NormFinder,delta CT,and RefFinder algorithms.The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation.In general,the P-tubulin(TUBB) gene was relatively stable in the intestine and respiratory tree tissues.The optimal reference gene combination for intestine was 40 S ribosomal protein S18(RPS18),TUBB,and NADH dehydrogenase(NADH);for respiratory tree,it was P-actin(ACTB),TUBB,and succinate dehydrogenase cytochrome B small subunit(SDHC);and for muscle it was a-tubulin(TUBA) and NADH dehydrogenase[ubiquinone]1 a subcomplex subunit 13(NDUFA13).These combinations of internal control genes should be considered for use in further studies of gene expression in A.japonicus during aestivation.

SYBR Green I荧光定量PCR检测乳中携带sea基因金黄色葡萄球菌的研究

金黄色葡萄球菌是引起食物中毒的一种常见的致病菌,而其产生的肠毒素A是乳及乳制品食物中毒事件的主要原因。本研究建立了SYBR Green I荧光定量PCR方法对乳中携带sea基因的金黄色葡萄球菌进行检测。该方法能快速、稳定地在8h内完成对乳中金黄色葡萄球菌的检测,对人工污染乳中金黄色葡萄球菌检测的最低检出限为83CFU/ml。

B7.1及SEA基因共转染肝癌细胞的初步研究

目的获得可表达共刺激分子B7.1和超抗原分子SEA的肝癌细胞株，检测其表达。方法用免疫组织化学方法，筛选表达B7.1及SEA的肝癌细胞阳性克隆。用流式细胞仪检测B7.1表达的阳性率，ELISA检测上清中SEA的含量。结果获得表达B7.1和SEA的肝癌细胞株，培养上清中SEA的含量达10～14×10-8g/L。结论建立了免疫细胞在肝癌细胞上的SEA和B7.1联合识别效应系统。

花鲈半乳糖凝集素基因家族的鉴定及在不同环境因子胁迫下表达响应

为探究在花鲈(Lateolabrax maculatus)响应环境因子胁迫中半乳糖凝集素(Galectins)发挥的作用,本研究通过全基因组水平鉴定获得了花鲈的14个Galectin基因(LGALS1、LGALS2a、LGALS2b、LGALS3a、LGALS3ba、LGALS3bb、LGALS4、LGALS8a、LGALS8b、LGALS9、LGALS17、GRPa、GRPb和GRPc)。通过系统发育、拷贝数、共线性和基因结构分析证实了基因注释的准确性和结构上的保守性。利用转录组测序数据进行Galectin基因的组织表达及在多种环境因子胁迫下的表达模式分析。研究表明,Galectin家族各基因在花鲈的7个组织(肝、鳃、脾、胃、脑、精巢和卵巢)中广泛表达,且有6个基因(LGALS3ba、LGALS4、LGALS8a、LGALS9、LGALS17和GRPa)表现出明显的组织特异性。Galectin家族的不同成员在4种环境因子胁迫下(低氧、碱度、高温及盐度适应)表现不同程度的表达响应,其中LGALS3a、LGALS3ba、LGALS4、LGALS17和GRPb在4种环境胁迫中表现出显著的差异表达,表明其广泛参与了对环境胁迫的响应与调控。

BCR/ABL和SEA双基因重组载体的构建和表达

目的:构建和表达含BCR/ABL融合基因和葡萄球菌肠毒素A(SEA)的真核双表达质粒。方法:利用RT-PCR技术从K562细胞中扩增出含BCR/ABL融合位点基因片段,提取金黄色葡萄球菌基因组DNA扩增出SEA基因,分别将两基因片段连在pIRES载体的多克隆位点A和B上,构建BCR/ABL-pIRES-SEA、SEA-pIRES-BCR/ABL重组质粒。将重组质粒转染K293细胞,RT-PCR鉴定重组质粒在真核细胞的转录情况,SDS-PAGE电泳鉴定目的蛋白在真核细胞的表达。结果:成功扩增出BCR/ABL和SEA基因片段;双酶切鉴定BCR/ABL-pIRES-SEA、SEA-pIRES-BCR/ABL重组质粒中含有BCR/ABL和SEA基因,测序证实完全正确;将重组质粒转染K293细胞后,经RT-PCR扩增鉴定,插入到重组质粒的BCR/ABL和SEA基因能在真核细胞正常转录,经SDS-PAGE电泳鉴定重组质粒能够在真核细胞中表达BCR/ABL和SEA蛋白。结论:成功构建BCR/ABL-pIRES-SEA、SEA-pIRES-BCR/ABL真核双表达质粒,可在真核细胞中正常转录并表达BCR/ABL和SEA蛋白。

中国当代文学作品海外传播的文化基因——以徐则臣《北上》为例

作为从小生长在水边的当代作家,徐则臣自20世纪末就开始创作有关运河的作品。他用20余年的时间欣赏运河、感受运河,最终凭借《北上》这部作品获得第十届茅盾文学奖,成为“70后”首位茅盾文学奖得主。新时代下,中国当代文学已进入全球化语境,受世界瞩目。随着中国文学“走出去”的步伐逐渐加快,中国文学作品中的文化基因是什么,中国当代文学作品海外传播中的文化使命是什么,都值得我们深思。《北上》是徐则臣创作的一部文学作品,其以程裕祯对文化分层理论为依据,探索中国当代文学作品中的文化基因,并进一步思考中国当代文学作品海外传播中文化基因的使命,力图为中国文学作品海外传播提供思路。

EGF-SEA融合蛋白在大肠杆菌中的表达和纯化

根据基因库中查到的金黄色葡萄球菌肠毒素A(SEA)基因序列和人体表皮生长因子(EGF)基因序列进行密码子优化,以适于大肠杆菌表达。人工合成SEA基因与EGF基因。将两目的基因克隆至原核表达载体pET22b中,经测序验证表明成功构建了重组表达质粒pET22b-EGF-SEA。将构建好的pET22b-EGF-SEA质粒转化大肠杆菌BL21(DE3),经IPTG诱导进行表达;SDS-PAGE分析表明融合基因EGF-SEA在大肠杆菌BL21(DE3)中以包涵体的形式得到了高效表达,产物相对分子质量约为44kDa,与理论值大小一致。包涵体经洗涤,变性、复性后用His.BindKit进行分离纯化,所得蛋白纯度≥95%。高纯度EGF-SEA融合蛋白的获得为进一步研究其生物学活性及肿瘤治疗奠定了基础。

Identification of Five Sea Cucumber Species Through PCR-RFLP Analysis

Sea cucumbers are traditional marine food and Chinese medicine in Asia. The rapid expansion of sea cucumber market has resulted in various problems, such as commercial fraud and mislabeling. Conventionally, sea cucumber species could be distinguished by their morphological and anatomical characteristics; however, their identification becomes difficult when they are processed. The aim of this study was to develop a new convenient method of identifying and distinguishing sea cucumber species. Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) analysis of mitochondrial cytochrome oxidase I gene(COI) was used to identifing five sea cucumber species(Apostichopus japonicus, Cucumaria frondosa, Thelenota ananas, Parastichopus californicus and Actinopyga lecanora). A 692 bp fragment of COI was searched for BamHI, KpnI, PstI, XbaI and Eco31 I restriction sites with DNAMAN 6.0, which were then used to PCR-RFLP analysis. These five sea cucumber species can be discriminated from mixed sea cucumbers. The developed PCR-RFLP assay will facilitate the identification of sea cucumbers, making their source tracing and quality controlling feasible.

The first record of Pavlova pinguis (Pavlovophyceae, Haptophyta) in China seas

One strain of unicellular flagellated yellow-green algae was successlully isolated lrom the coastal area near Tianjin in the Bohai Sea in May 2015. The strain ranged from round to elongated in shape. Most of the cells possessed active motility, and some cells formed non-motile aggregation. Based on evidences from morphology, ultrastructure and molecular analysis, we identified the strain as Pavlova pinguis which belonged to Pavlovophyceae, Haptophyta. For Pavlovophyceae, only Pavlova viridis （Diacronema viridis） was reported in China seas prior to this study and it played an important role in aquaculture. This is the first record of Pavlova pinguis in Chinese waters.

High-throughput sequencing of 16S r RNA amplicons characterizes gut microbiota shift of juvenile sea cucumber Apostichopus japonicus feeding with three antibiotics

Sea cucumber Apostichopus japonicus is an important marine economic species in Asian countries due to its profound nutritional and medicinal value. So far, with the rapid development of intensifi ed artifi cial aquaculture of sea cucumbers, the use of antibiotics is still an inexpensive and dispensable way to treat pathogenic infections, especially during the nursery phase. However, there is little information on the eff ects of antibiotics on the intestinal microbiota of sea cucumber. Therefore an Illumina based sequencing method was used to examine the intestinal bacterial composition of juvenile A . japonicas following diets with three typical antibiotics (tetracycline, erythromycin, and norfl oxacin) under 15, 30, and 45 d. The fi ndings reveal that diff erent antibiotics have distinct eff ects on the growth performance of juvenile sea cucumbers. However, the richness and diversity of microbiota were barely aff ected by antibiotics but the community composition alterations indicated that the three antibiotics exhibited their respective patterns of reshaping the intestinal bacteria of juvenile sea cucumbers. In common, the abundance of some sensitive genera with helpful functions, such as Thalassotalea , Shewanella , Sulfi tobacter , and Halomonas decreased signifi cantly with exposure to antibiotics and the abundance of multiple potential pathogenic- and suspected antibiotic-resistant microorganisms like Arcobacter , Leucothrix , and Clostridium_sensu_stricto_1 was found increased signifi cantly in the antibiotic groups. These results suggest that low doses of antibiotics could aff ect the composition of the intestinal microbiota of sea cucumbers and might increase the risk of infection of the hosts. This study could help us to explore how antibacterial compounds modify the gut microbiota of sea cucumbers and provide theoretical guidance in hatchery management by scientifi c antibiotic use in sea cucumber mariculture.

Triterpene-Enriched Olive Extract as an Immunopotentiator in Black Sea Bream(Acanthopagrus schlegelii)

Recently,the use of natural immunopotentiators,such as plant extracts,is predicted to offer high disease-prevention potential in aquaculture.However,few reports on the immunomodulatory impacts of olive extract(OE)on teleost are available.Therefore,the effects of dietary intake of triterpene-enriched OE on black sea bream(Acanthopagrus schlegelii)were investigated in this study.Our data showed that total blood cell counts,in vivo content of lysozyme,activities of antiprotease and myeloperoxidase,and contents of IL-4 and IL-6 were significantly up-regulated by dietary intake of triterpene-enriched OE.Additionally,the hampered immune response induced by cadmium exposure was significantly mitigated by the administration of OE as were indicated by partially or completely rescued immune-relating parameters.Furthermore,the expressions of immune-related genes encoding NF-κB inhibitor alpha(IkBα),tumor necrosis factorα(TNFα),cyclooxygenase-2(COX-2),and proto-oncogene protein c-fos(FOS)were found to be significantly up-regulated by the dietary intake of OE.In general,the results suggested that the dietary intake of triterpene-enriched OE has immune enhancing effect in black sea bream.Such effect may be realized by 1)increasing the total counts of diverse blood cells;2)activating nonspecific immune biomolecules;and 3)affecting signaling pathways such as IKK and ERK and subsequently inducing IL-4 and IL-6 biosyntheses.

Molecular cloning and expression analysis of interleukin - 1β from Japanese sea perch(Lateolabrax japonicus )

The technology of homology cloning and anchored PCR was used to clone the IL-1β gene from the Japanese sea perch （Lateolabrax iaponicus）. The full-length cDNA of sea perch IL-1β was 1 310 bp, including a 5＇ untranslated regiop （UTR） of 136 bp, a 3＇ UTR ot 430 bp, and an ORF of 774 bp encoding a polypeptide of 258 amino acids with an estimated molecular mass of 29.31 kDa. The searches for nucleotides and protein sequence similarities with the BLAST analysis indicated that the deduced amino acid sequence of sea perch IL-1β was homological to the IL-1β in other fish species and even the mammalian. Conserved signature sequences of the IL-1β gene family were found in the sea perch IL-1β deduced amino acid sequence. Temporal expressions of the IL-1β gene in LPS or iridovirus challenged group and in control group were measured by the semi-quantitative RT-PCR. The mRNA transcripts of IL-1β could be detected in head-kidney, spleen, liver, gill and heart of the healthy individuals, and the expression level of IL-1β in head-kidney, spleen and gill was higher than that in liver and heart, but it was hard to be detected in the brain. After being stimulated by the LPS or iridovirus, the IL-1β expression in most of examined tissues was up-regulated, and also could be detected in the brain. These results indicated that the expression of sea perch IL-1β was constitutive and could be up-regulated by immune effector stimulation. Therefore the sea perch IL-1β could play a critical role in the host-pathogen interaction.

Microbial community structure and diversity in deep-sea hydrothermal vent sediments along the Eastern Lau Spreading Centre

The aim of this study is to investigate microbial structures and diversities in five active hydrothermal fields＇ sediments along the Eastern Lau Spreading Centre （ELSC） in the Lau Basin （southwest Pacific）. Microbial communities were surveyed by denatured gradient gel electrophoresis （DGGE） and clone library analysis of 16S rRNA genes. The differences in microbial community structures among sediment samples from the five deep-sea hydrothermal sites were revealed by DGGE profiles. Cluster analysis of DGGE profiles sepa- rated the five hydrothermal samples into two groups. Four different 16S rRNA gene clone libraries, repre- senting two selected hydrothermal samples （19-4TVG8 and 19-4TVG11）, were constructed. Twenty-three and 32 phylotypes were identified from 166 and 160 bacterial clones respectively, including Proteobacteria, Bacteroidetes, Firmicutes, Nitrospirae and Planctomycetes. The phylum Proteobacteria is dominant in both bacterial libraries with a predominance of Gamma-Proteobacteria. A total of 31 and 25 phylotypes were obtained from 160 and 130 archaeal clones respectively, including Miscellaneous Crenarchaeotic Group, Marine Group Ⅰ and Ⅲ, Marine Benthic Group E, Terrestrial Hot Spring Crenarchaeota and Deep-sea Hy- drothermal Vent Euryarchaeota. These results show a variety of clones related to those involved in sulfur cycling, suggesting that the cycling and utilization of sulfur compounds may extensively occur in the Lau Basin deep-sea hydrothermal ecosystem.