马氏珠母贝贝壳基质蛋白基因Pm-PNU7的克隆和功能研究

【目的】研究马氏珠母贝(Pinctada martensii)贝壳基质蛋白(Shell Matrix Proteins, SMPs)基因(Pm-PNU7)表达与功能。【方法】利用马氏珠母贝基因组和蛋白质组信息分析和RACE技术克隆获得壳基质蛋白新基因Pm-PNU7全长序列,通过原位杂交对其进行定位,RNA干扰检测其对贝壳形成的影响。【结果】Pm-PNU7的cDNA全长为797 bp,编码145个氨基酸,具有特殊的"KGG"重复序列和富含天冬氨酸(Asp)序列"DDDDDDHDD"。qRT-PCR分析表明,Pm-PNU7基因在外套膜边缘区和套膜区显著高表达(P<0.05);ISH检测显示,Pm-PNU7主要定位于边缘区外褶外侧和内侧上皮细胞、中褶内侧上皮细胞以及套膜区外侧上皮细胞。RNA干扰后,Pm-PNU7在外套膜边缘区和套膜区的表达量均显著下调,贝壳棱柱层和珍珠层微观结构均出现不同程度的紊乱。【结论】Pm-PNU7参与了贝壳棱柱层和珍珠层的形成。     

Spatial distribution, diversity and composition of bacterial communities in sub-seafloor fluids at a deep-sea hydrothermal field of the Suiyo Seamount

Spatial distribution, diversity, and composition of bacterial communities within the shallow sub-seafloor at the deep-sea hydrothermal field of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific Ocean, were investigated. Fluids were sampled from four boreholes in this area. Each borehole was located near or away from active vents, the distance ranging 2–40 m from active vents. In addition, fluids discharging from a natural vent and ambient seawater were sampled in this area. We extracted DNA from each sample, amplified bacterial 16S rRNA genes by PCR, cloned the PCR products and sequenced. The total number of clones analyzed was 348. Most of the detected phylotypes were affiliated with the phylum Proteobacteria, of which the detection frequency in each clone library ranged from 84.6% to 100%. The bacterial community diversity and composition were different between hydrothermal fluids and seawater, between fluids from the boreholes and the vent, and even among fluids from each borehole. The relative abundances of the phylotypes related to Thiomicrospira, Methylobacterium and Sphingomonas were significantly different among fluids from each borehole. The phylotypes related to Thiomicrospira and Alcanivorax were detected in all of the boreholes and vent samples. Our findings provide insights into bacterial communities in the shallow sub-seafloor environments at active deep-sea hydrothermal vent fields.     

Nematode diversity of Qingdao coast inferred from the 18S ribosomal RNA gene sequence analysis

The 18S ribosomal DNA gene （18S rDNA） sequences （approxtmately 1300 bp in length） were amplified from the DNA extracted from the free-living marine nematodes collected from the inter-tidal sediment of Qingdao coast in bulk with nematode specific primers. The PCR products were cloned, re-amplified, digested with Rsa I and Hin61 restriction endonucleases and separated in agarose gel. Among 17 restriction fragment length types, types 1, 2 and 6 covered 61.2%, 14.4% and 9.3% of the clones analyzed, respectively, while the remaining 14 only covered 21 clones, which accounted for 15.1% of the total. Twenty-four representative clones were sequenced and phylogenetically analyzed by referring to those currently available in RDP and GenBank databases. Although it was hard to assign these sequences to known species or genera due to the lack of the 18S rDNA sequence data of known marine free-living nematodes, the obtained sequences were assigned to the nematodes of Adenophorea. Among them, twelve sequences were close to Pontonema vulgate and Adoncholaimus sp., four to Daptonema procerus and two （identical） to Enoplus brews. Our results showed that free-living marine nematode diversities could be determined by PCR retrieving and analysis of the 18S rDNA sequences and an 18S rDNA sequence could be assigned to a species or a genus only if the 18S rDNA sequences of the free-living marine nematodes were accumulated to some extent.     

Changes in the Chemical Components of Light Crude Oil During Simulated Short Term Weathering

To unambiguously identify spilled oils and to link them to the known sources are extremely important in settling ques-tions of environmental impact and legal liability. The fate and behavior of spilled oils in the environment depend on a number of physicochemical and biological factors. This paper presents the results regarding changes in chemical composition of light crude oil during simulated short-term weathering based on natural environmental conditions. The results show that the saturated hydrocarbons of the light crude oil mainly distribute between n-C8 and n-C23 and the most abundant n-alkanes are found in the n-C10 to n-C16. The main chemical components of the light crude oil are n-alkanes and isoprenoids. The aromatic compounds are subordinate chemical components. Under the conditions of the weathering simulation experiment,n-alkanes less than n-C12,toluene and 1,3-dimethyl ben-zene are lost after 1 d weathering,the n-C13,n-C14,naphthalene and 2-methyl-naphthalene are lost on the fifth day of weathering,and n-C15 alkane components show certain weatherproof capability. The ratios n-C17/pristane and n-C18/phytane are unaltered and can be used to identify the source of the light crude oil during the first 8 d of weathering. After 21 d,the ratio pristine/phytane can not pro-vide much information on the source of the spilled light crude oil. Triterpanes(m/z 191) as biomarker compounds of light crude oil are more valuable.     

Temperature Influences on the Expression of GFP Promoted by the Upstream Sequence of cpcB from Arthrospiraplatensis

In order to investigate the regulation mechanism of the phycocyanin gene,a series of functional analyses of the upstream sequence of cpcB gene from Arthrospira platensis were conducted in E. coli with green fluorescent protein encoding gene(gfp) as the reporter. Results showed that the gfp gene could express at a high level under the promotion of the upstream sequence,suggesting the existence of some strong promoter elements in it. The expression of GFP was influenced by temperature. Higher temperature led to higher expression level. The bioinformatics analyses followed by mutation analyses on the secondary structure of translation ini-tiation region(TIR) revealed that RNA thermosensor might account for the temperature regulation.     

泥蚶(Tegillarca granosa)血细胞转录组序列中免疫相关基因的结构与mRNA表达量分析

贝类血细胞直接参与异物吞噬和包囊作用,还在伤口修复、炎症反应过程中发挥重要作用,是机体免疫防御的承担者。本研究采用RNA-seq测序技术研究了鳗弧菌感染前后泥蚶血细胞的转录组序列,通过序列组装,获得了α-葡萄糖苷酶(GAA)、α-甘露糖苷酶(LAMAN)、芳基硫酸酯酶B(ASB)、天冬酰胺氨基葡萄糖酶(AGA)、整合素β3亚基(ITB3)、热休克蛋白9(HSPA9)和Toll相互作用蛋白(TOLLIP)序列,利用Orffinder获得了其编码蛋白序列,并对蛋白序列进行了理化性质、二级结构、保守结构域和三级结构分析。结果表明:7种蛋白质二级结构均含有α螺旋、β折叠及无规则卷曲,泥蚶和人类ITB3和TOLLIP具有相同的保守结构域, Swiss-model同源建模获得的三级结构与模板同源性均超过35%(TOLLIP除外)。鳗弧菌刺激后,GAA、LAMAN、ASB、AGA和HSPA9的表达量显著增加,ITB3和TOLLIP表达量变化不明显。该研究补充和完善了对泥蚶免疫相关基因的认识,并为进一步开展泥蚶抗病机理研究提供了重要的理论依据。     

基于16S rRNA序列初步探讨贻贝属的系统发育

通过比较贻贝属5个物种包括中国的两种贻贝的线粒体16S rRNA基因部分序列,来初步确定它们的系统发育关系和了解中国沿海两种贻贝的遗传多样性情况.以Perna viridis为外群,采用NJ法和MP法构建分子系统树.系统发育分析表明,5种贻贝(Mytilus californianus, M. corcuscus, M. galloprovincialis, M. edulis, M. trossulus)在系统树上依次进行分叉,呈放射状.M. californianus最为原始,M. corcuscus次之.每一个贻贝物种都形成单系.其中,M. edulis和M. trossulus是非常相似的,M. corcuscus和M. californianus的亲缘关系近.同时发现,我国沿海分布的紫贻贝(M. galloprovincialis)和厚壳贻贝(M. corcuscus)的遗传多样性都较高,但厚壳贻贝的遗传多样性要低于紫贻贝,可能是由于厚壳贻贝过度被渔民开采等导致厚壳贻贝群体大小降低的缘故.这里系统发育分析为将来进行物种进化、迁移和育种方面的比较研究提供理论基础.     

Detection of Prorocentrum donghaiense using sandwich hybridization integrated with nuclease protection assay

Prorocentrum donghaiense is an important harmful algae bloom (HAB) causing creature in China’s seas, and the conventional visual detection can not cope with long-term monitoring and high-throughput sampling projects. An assay for P. donghaiense with sandwich hybridization integrated with nuclease protection assay (NPA-SH) was established. Tests with mixed samples and spiked field ones confirmed its good specificity and sensitivity. The cell number of P. donghaiense correlated well with the optical density, ...