Production and transport of long-chain alkenones and alkyl alkenoates in a sea water column in the northwestern Pacific off central Japan

Sinking particles collected from year-long time-series sediment traps at 1674, 4180, 5687 and 8688 m depths, the underlying bottom sediment at 9200 m depth, and suspended particles from surface and subsurface waters in the northwestern North Pacific off Japan were analyzed for long-chain alkenones and alkyl alkenoates (A&A) which are derived mainly from Gephyrocapsacean algae, especially Emiliania huxleyi and Gephyrocapsa oceanica. Alkenone temperature records in sediment trap samples at 1674 m were almost similar to observed sea surface temperatures (SST) with a time delay of one half to one full month. However, alkenone temperatures in trap samples were about slightly lower than measured SST in late spring to early fall. The lowering might be caused by formation of the seasonal thermocline. Nevertheless, these temperature drops observed in trap samples were smaller than those actually observed in a subsurface layer off central Japan. Vertical profiles of A&A concentrations and alkenone temperatures in suspended particles collected from the subsurface waters in early fall indicated that these compounds were produced mostly in a surface mixed layer above the depth of the chlorophyll maximum even in warm seasons. These results suggested that alkenone temperatures strongly reflected SST rather than the temperatures of thermocline waters in these study areas even in such a warm season. Pronounced maxima in A&A fluxes found in sediment trap samples at 1674 m in late spring to summer showed that A&A productions were highest during the periods of spring bloom, according to a time delay between alkenone temperatures and observed SST. Seasonal patterns of alkenone records in trap samples at 4180 and 5687 m could also preserve SST signals well, suggesting that A&A in deep sea waters were mainly derived from primary products in the surface layer. A&A fluxes tended to decrease with water depth, and the ratios of A&A to particulate organic carbon (POC) rapidly decreased in underlying bottom sediment. This clearly indicates that A&A were decomposed and diluted by other refractory organic materials in either the water column or the sediment–water interface. However, A&A compositions were consistently uniform between the trap samples and the underlying bottom sediments, so that A&A could not qualitatively alter during early diagenetic processes.     

DNA/RNA提取及处理方法对评价沉积物中纤毛虫分子多样性的影响

环境DNA(eDNA)技术结合高通量测序已广泛应用于评价微型生物多样性与群落构成。相较于eDNA,RNA在环境中易降解,环境RNA(eRNA)可更准确地反映群落近期生命活性状态。理论上,基于eRNA检获的生物多样性要低于eDNA检获的总体多样性。但前期已发表研究显示同一站点eDNA获得的多样性低于eRNA检获量,推测可能原因包括:1)样本量不同;2)RNA中存在DNA污染。为验证假设,本研究以陆架区2个站位沉积物中的纤毛虫为实验对象,系统性比较4种DNA/RNA提取方法:DNA直接提取(0.9 g沉积物),大样本量DNA直接提取(2 g), DNA洗脱法(2 g), RNA直接提取法(2g);以及3种RNA提取后的处理方法:无DNA酶反转录,含DNA酶反转录,先纯化RNA再反转录。研究结果表明,大样本量(2 g)DNA直接提取法所检获的可操作分类单元(OTUs)约为小样本量(0.9 g)的2倍。相同样本量下, DNA洗脱法检获的OTUs最多,而DNA直接提取检获的OTUs低于有/无DNA酶反转检获的数量,先纯化再反转录所获得OTUs最少。就群落构成而言, DNA洗脱法和RNA纯化可有效...     

胶州湾浮游桡足类18S核糖体RNA基因(18S rDNA)扩增及序列变异初步研究

采用PCR扩增、文库构建、限制性片段长度多态性分析、序列分析和系统学分析等方法，初步研究了夏季胶州湾上层海水浮游桡足类核糖体小亚基RNA基因(18S rDNA)约1．5kb片段的序列变异。从浮游生物混合DNA中选择性扩增桡足类18S rDNA，建立桡足类18S rDNA变异类型文库，并从文库中随机挑选的30个克隆进行分析。结果表明，Vsp Ⅰ限制性内切酶能将这些克隆分成频率分别为0．17、0．23和0．6的3种操作分类单元(OTUs)，遗传多样性指数达到0．95。3条OTU代表克隆序列与甲壳纲桡足亚纲核苷酸差异数在75．4—97．8之间，而与其他亚纲的差异都高于100。3条OTU代表克隆序列均属于桡足亚纲，其中，AY437861和AY437862属于哲水蚤目。3条OTU代表克隆序列可分为2个高变异区和3个相对保守区，其GC％分别为47．37％、48．16％和48．57％。研究结果表明，混合DNA提取方法简单，设计的引物可选择性地扩增浮游桡足类18S rDNA，根据18S rDNA序列序列变异描述浮游桡足类多样性是可行的。研究结果也为在浮游桡足类分类中引入18S rDNA序列奠定了基础。     

Flow cytometric detection and enumeration of DNA and RNA viruses infecting marine eukaryotic microalgae

Sample preparation protocols for flow cytometry (FCM) analysis with five algal viruses were optimized: Heterocapsa circularisquama virus (HcV), Heterosigma akashiwo virus (HaV), Chaetoceros salsugineum nuclear inclusion virus (CsNIV), Rhizosolenia setigera RNA virus (RsRNAV) and H. circularisquama RNA virus (HcRNAV). The optimum staining protocols differed significantly among the viruses tested. FCM counts for the large DNA algal viruses HaV and HcV (∼0.2 μm in diameter) were similar to numbers determined by epifluorescence microscopy (EFM). In contrast, FCM counts of viruses smaller than 40 nm that harbor DNA (CsNIV) or RNA genomes (RsRNAV, HcRNAV) were comparable to or lower than most probable number (MPN) values, which indicate only infectious virus number, suggesting that the FCM counts were underestimates. This is presumably because their single particle fluorescence signals were below the detection limit for the flow cytometer. These results indicate that a large portion of the smaller viruses in the aquatic plankton virus community may be overlooked by FCM.     

下调心肌CPT1b的表达对肥胖小鼠心肌细胞钙调控的影响

目的 研究干扰心肌组织中肉毒碱脂酰转移酶-1b（CPT1b）的表达对高脂饮食诱导的肥胖小鼠心肌细胞钙调控的影响。方法 4周龄的雄性C57小鼠,随机分为正常对照组（N-mock）、肥胖对照组（O-mock）及肥胖干预组（O-CPT1b）,采用高脂饮食诱导肥胖。6周龄时,经心肌分别注射靶向CPT1b（O-CPT1b）或靶向无关基因（N-mock、O-mock）的重组慢病毒,以下调目标基因的表达。10周后,取小鼠左心室组织检测RNA干扰的效率;采用Western blot法检测左心室组织中肌浆网钙泵（SERCA2a）的蛋白表达;并分离单个心室肌细胞,使用活细胞工作站检测心肌细胞钙瞬变。结果 肥胖小鼠心肌组织中CPT1b的表达增加,慢病毒介导的RNA干扰显著下调其表达。肥胖引起心肌细胞SERCA2a的蛋白表达降低及肌质网钙处理能力的下降,下调CPT1b的表达增加了SERCA2a的含量,并改善了钙调控异常。结论 下调心肌组织中CPT1b的表达可改善肥胖所导致的心肌细胞钙调控异常。     

泰国近海习见有毒立方水母和钵水母的遗传分析

本研究利用线粒体16S rDNA和核基因18S rDNA片段,对泰国沿海常见的有毒水母进行遗传分析,并比较了2个基因片段作为通用分子标记,在研究水母类多个纲的遗传多样性中的应用。研究发现,泰国近海的有毒水母存在较高的遗传多样性,所获得的32个样品可以分为9个种,包括4种钵水母、4种立方水母和1种水螅水母。然而,完全确定各种的分类地位,还需要更多的形态、生活史等方面信息。两个基因片段均能明确区分各种类,但核基因18S序列比线粒体基因片段更为保守。根据16S基因片段序列计算水母种内和种间的K2P(Kimura 2-parameter)遗传距离,发现所研究的9个水母种类,种内遗传距离在0~0.050之间,其中94%的种内遗传距离小于0.040,同纲种间的遗传距离为0.204~0.474,其中91%的种间遗传距离大于0.250;而利用18S基因,种内距离在0~0.002之间,同纲种间距离为0.008~0.066(平均为0.038,SE=0.006)。16S的AT碱基含量明显高于核基因18S,且16S的碱基含量在不同纲之间有显著差异,进一步表明水母线粒体16S基因的突变率相对较高,适合研究水母较低分类阶元以及种下的遗传差异。     

单条固定线虫基因组DNA提取及18S rRNA基因PCR扩增

根据线虫18S核糖体RM基因PCB扩增效果比较了丙酮、乙醇、乙醇 0．05mol／L FDTA(pH8．0)和5％海水福尔马林4种固定剂,碱裂解和蛋白酶K处理2种单条线虫基因组DNA提取方法的优劣。用乙醇固定的样品最适合制备RR模板DNA,而5％海水福尔马林固定的样品能最完整地保持样品形态。蛋白酶K处理获得的DNA较碱裂解获得的更适合PCR扩增。结果有助于分子生物学方法在海洋线虫分类、多样性和生态学研究中的应用。     

红砂正常和脱水组织中总RNA提取的改进CTAB法

红砂属于柽柳科超旱生小灌木,是荒漠半荒漠地区植被中的优势种。为了研究促分裂原激活的蛋白激酶(MAPK)信号传导通路在这类植物抗旱中的作用,我们尝试了很多RNA提取的方法都没有获得完整的RNA。在脱水组织中由于多糖、多酚以及其他与核酸能结合或共沉淀的化合物的含量升高而更不容易得到高质量的RNA。以CTAB为基础的改进的RNA提取方法,能成功地从少量的红砂植物的正常组织和脱水组织中获得高质量的RNA。这种方法经济且操作方便,提取的RNA纯度和完整性都很好,可用于RT-PCR反应并成功克隆了550 bp 左右的MAPK基因片段。     

Novel active kinetoplastids associated with hypersaline anoxic basins in the Eastern Mediterranean deep-sea

The combination of nearly saturated salt concentration and corresponding high density, high hydrostatic pressure, absence of light, anoxia, and a sharp chemocline make the deep hypersaline anoxic basins in the Eastern Mediterranean Sea some of the most polyextreme habitats on Earth. Using kinetoplastid-specific primers, we detected kinetoplastid flagellates in some of the harshest deep-sea environments known to date, including some whose small subunit ribosomal RNA gene sequences are not closely related to cultured representatives. Kinetoplastids, including presumably novel representatives appear to be specialists of halocline environments in the Eastern Mediterranean, and to comprise a significant fraction of the protist communities in the brines and haloclines of several basins. Fluorescent in situ hybridization data indicate a novel ‘unidentified’ sequence clade of kinetoplastids related to bodonids represents as much as 10% of the total protist community in the Discovery Basin halocline. Different kinetoplastid groups are unevenly represented in the different basins and habitats we sampled, which we discuss as a result of environmental selection.