应用MSAP技术研究扇贝全基因组DNA甲基化水平

DNA甲基化作为真核生物基因组重要的表观遗传学修饰,对生物体基因的表达有重要的调控作用。为获得扇贝基因组DNA甲基化修饰水平及模式等表观遗传学信息,以栉孔扇贝(Chlamys farreri)、海湾扇贝(Argopecten irradians)、虾夷扇贝(Mizuhopecten yessoensis)以及本课题组培育的"海大金贝"为材料,建立了甲基化敏感扩增多态性方法(Methylation-sensitive amplified polymorphism,MSAP)的反应体系,利用该方法对其基因组DNA CCGG区域的甲基化水平进行分析。结果表明,本研究筛选得到的引物组合可用于贝类DNA甲基化的研究,在栉孔扇贝、海湾扇贝、普通虾夷扇贝和"海大金贝"的甲基化比例分别为32.08%、25.99%、32.88%和34.97%。几种扇贝基因组CCGG序列中,胞嘧啶的全甲基化率要高于半甲基化率,推测扇贝基因组中主要的甲基化模式是CpG型。通过对"海大金贝"和普通虾夷扇贝闭壳肌甲基化谱进行比较,筛查得到46个差异位点,这些位点可能参与"海大金贝"闭壳肌积累类胡萝卜素的调控。     

立体选择性半合成全甲基化没食子儿茶素没食子酸酯

为了立体选择性合成全甲基化没食子儿茶素没食子酸酯(GCG),本文以全甲基化表没食子儿茶素没食子酸酯(EGCG)为原料,通过酯水解和Mitsunobu反应立体选择性地合成了构型C-3位翻转的目标产物全甲基化GCG。全甲基化GCG经水解得到5,7,3′,4′,5′-五氧甲基没食子儿茶素(GC),再经Mitsunobu反应得到全甲基化EGCG。经核磁和旋光验证,所合成的全甲基化EGCG与原料全甲基化EGCG相同。核磁测试证实两次Mitsunobu反应产物C-3均发生立体构型翻转。初步细胞实验表明1μmol/L全甲基化GCG与紫杉醇共同作用于P-gp高表达LCC6/MDR乳腺癌耐药细胞株时,紫杉醇IC50为10.1nmol/L,全甲基化GCG逆转倍数(RF)达到13.9。     

基于MethylRAD-Seq技术对仿刺参DNA甲基化图谱的研究

全基因组DNA甲基化作为重要的表观遗传学现象,其在生物体诸多生理生化过程中起了重要作用。研究表明全基因组DNA甲基化在基因的表达调控、维持胚胎正常发育、染色体结构稳定等方面发挥了重要作用。DNA甲基化也成为当今的研究热点之一,目前在脊椎动物领域研究相对较多,但是对无脊椎动物的甲基化规律的研究比较匮乏,因此本研究以棘皮动物门中仿刺参作为研究对象,采用最新的全基因组DNA甲基化检测方法 MethylRAD-Seq技术探究了仿刺参三种组织的甲基化状况,得到了甲基化标签数量、甲基化位点分布以及甲基化标签密度等信息;此外通过整合仿刺参甲基化文库数据和表达谱数据发现基因甲基化水平与表达水平之间存在弱的正相关性,这暗示了无脊椎动物基因组DNA甲基化可能起到促进基因表达的作用。     

单甲基化表没食子儿茶素没食子酸酯的化学合成

表没食子儿茶素没食子酸酯(EGCG)经甲基化后在抗过敏、降血脂等方面具有更高的生物活性。但茶叶中甲基化EGCG含量较低,为进一步研究其生物活性,本文对其半合成路线进行了优化。先以EGCG为原料制得五苄基化的表没食子儿茶素(EGC),然后将没食子酸丙酯经4-甲氧基苯甲醛二甲缩醛保护,甲基化,脱保护,苄基化,水解得到3,4-二苄氧基-5-甲氧基苯甲酸,再与苄基化EGC酯化和脱苄基得到表没食子儿茶素3-O-甲基没食子酸酯(3″-Me-EGCG);同样将没食子酸丙酯经全乙酰化、选择性甲基化、脱除乙酰基、苄基化、水解得到3,5-二苄氧基-4-甲氧基苯甲酸,再与苄基化EGC酯化和脱苄基得到表没食子儿茶素4-O-甲基没食子酸酯(4″-Me-EGCG);总产率分别为53%和57%。     

海带中岩藻黄素异构体的分离纯化及其体外抗肿瘤活性研究

海带采用乙醇提取色素,通过柱层析分离纯化其中的岩藻黄素,并最终通过高效制备液相色谱制备了三种岩藻黄素的异构体。通过紫外光谱(UV)、核磁共振氢谱(1H-NMR)以及质谱(MS)对岩藻黄素及其异构体进行了结构表征和鉴定。采用乳腺癌细胞MDA-MB-231模型,对岩藻黄素的体外抗肿瘤活性进行了研究。结果表明,岩藻黄素对乳腺癌细胞生长有显著的抑制作用,其中13-顺和13′-顺岩藻黄素组分对癌细胞的抑制活性最强, IC50值达到23.71μg/mL。全反式岩藻黄素和9′-顺岩藻黄素两种异构体IC50值分别为45.30和27.09μg/mL。qPCR试验表明岩藻黄素各异构体可以通过抑制癌细胞NF-κB家族基因表达而抑制乳腺癌细胞生长。     

虾夷扇贝选育群体与野生群体基因组DNA甲基化的MSAP分析

基因组DNA甲基化作为表观遗传学研究的重要手段之一,在水产科学领域得到越来越广泛的应用。为从表观遗传学角度探讨虾夷扇贝选育群体-"玉贝"和普通虾夷扇贝群体的DNA甲基化差异,本文运用甲基化敏感扩增多态性(Methylation-sensitive amplification polymorphism,MSAP)技术分析了两群体的DNA序列中CCGG位点的甲基化情况。结果表明,筛选得到11对引物组合能够在两个群体中得到稳定扩增、清晰且多态性丰富的片段;11对引物在"玉贝"和对照群体中分别共检测到1432和1442个位点,其中发生甲基化的位点分别为306和341个,总甲基化率分别为21.5%和23.65%,"玉贝"总甲基化率下降2.15%;全甲基化位点分别为152和236个,全甲基化率为10.68%和16.37%,"玉贝"全甲基化率下降5.69%;半甲基化位点分别为154和105个,半甲基化率为10.82%和7.28%,"玉贝"半甲基化率上升3.54%;"玉贝"群体的甲基化多态性条带所占比例高于对照群体,分别为31%和29.167%,尤其是B类型条带链,"玉贝"群体明显高于对照群体。这说明,通过对虾夷扇贝生长和耐热性状的累代选育导致了DNA甲基化水平和模式的改变,本研究为进行虾夷扇贝抗逆基因的筛查提供了参考数据,丰富了表观遗传学在扇贝中的研究资料。     

O-羧甲基壳聚糖的制备及其结构表征

本文以壳聚糖为原料,采用苯甲醛选择性的保护壳聚糖C2位氨基的方法,制备了O-羧甲基壳聚糖(O-CMC)。采用红外光谱及核磁共振波谱法对产物结构进行了表征,确定了羧甲基化反应只发生在壳聚糖的O位。该方法制备得到的O-CMC的羧甲基度为42.2%,反应选择性好,步骤简单,产率高达85%。     

L-半胱氨酸缩邻香草醛Co(Ⅱ)配合物的合成、表征及与DNA的作用研究

制备了L-半胱氨酸缩邻香草醛配体C11H13NO4S,将其与Co(Ⅱ)作用合成了一种新型的配合物.通过元素分析、核磁共振、红外光谱、紫外-可见光谱、摩尔电导率分析及TG-DTG等手段对合成的配合物进行了表征.双核配合物的组成为[Co2(C11H12NO4S)2 (CH3COO)2],配体中的酚羟基的氧原子,氨基上的氮原子分别参与配位,羧基以双齿形式参与配位,Co(Ⅱ)的配位数为6.利用紫外-可见光谱,荧光发射光谱,离子强度改变,[Fe(CN)6]4荧光猝灭及黏度测定等方法对Co(Ⅱ)配合物与小牛胸腺DNA(CT-DNA)的结合方式进行了研究.实验结果表明,Co(Ⅱ)配合物与CT-DNA的结合方式为插入结合.     

北极海洋细菌Pseudoalteromonas sp.2018抑菌活性及其代谢产物研究

从2014年第六次中国北极科学考察采集的海洋沉积物中,分离获得了具有抑制农作物病原真菌活性的菌株Pseudoalteromonas sp.2018。测定了该菌株对病原真菌的抑制范围和对黄瓜白粉病的大棚防治效果,并对该菌株代谢产物通过硅胶色谱、凝胶色谱及半制备HPLC等进行分离纯化,通过质谱(MS)、氢谱(1 H NMR)和碳谱(13 C NMR)等进行结构鉴定。研究结果表明,Pseudoalteromonas sp.2018菌株对辣椒疫霉(Phytophthora capsici)、褐孢霉(Fulvia fulva)、链格孢(Alternaria alternata)、黄瓜专化型尖孢镰孢(Fusarium oxysporum f.sp.cucumerinum)、番茄专化型尖孢镰孢(Fusarium oxysporumf.sp.lycopersici)和细极链格孢(Alternaria tenuissima)等具有抑制作用,对大棚黄瓜白粉病的防治效果达66.7%;研究分离获得了环(脯-酪)二肽、环(脯-异亮)二肽、环(脯-亮)二肽和环(脯-苯丙)二肽,其中环(脯-酪)二肽和环(脯-亮)二肽对测试病原真菌的最小抑制浓度为125~500μg/mL。该研究为极地微生物农用资源的开发利用提供理论参考。     

改进的P—M谱

本文提出如下改进的无因次P—M谱 S(ω)=Aω~(-5)exp(-Bω~(-4-(?))并给出确定参数A、B、e的方法.改进P—M谱满足下述条件:谱峰通过给定点,谱面积等于1.     

Microbial Biomass and Organic Nutrients in the Deep-Sea Sediments of the Central Indian Ocean Basin

In order to assess the impact of deep-sea mining on the in situ benthic life, we measured the microbial standing stock and concentration of organic nutrients in the deep-sea sediments of the Central Indian Ocean Basin in the Indian pioneer area. Sediments were collected using box core and grab samples during September 1996. The total bacterial numbers ranged from 10 10 -10 11 cells per g -1 dry weight sediment. There was a marginal decrease in the number of bacteria from surface to 30 cm depth, though the subsurface section registered a higher number than did the surface. The highest numbers were encountered at depths of 4-8 cm. The retrievable number of bacteria were two orders less in comparison with the direct total counts of bacteria. An almost homogeneous distribution of bacteria, total organic carbon, living biomass, and lipids throughout the depth of cores indicates active microbial and benthic processes in the deep sea sediments. On the other hand, a uniform distribution of total counts of bacteria, carbohydrates, and total organic carbon in all the cores indicates their stable nature and suggests that they can serve as useful parameters for long-term monitoring of the area after the benthic disturbance. Further studies on temporal variability in this region would not only verify the observed norms of distribution of these variables but would also help to understand restabilization processes after the simulated benthic disturbance.     

海上大直径钢管桩打桩过程中桩周土强度弱化研究

海上大直径钢管桩打桩过程中,桩周土体受到强烈扰动而发生强度弱化,掌握桩周土体强度弱化规律对于准确预测打桩过程、保证工程安全具有重要意义。为研究土体强度弱化规律,开展了环剪试验模拟打桩对桩周土体的扰动,测试土体强度随剪切速率的变化规律,建立了描述土体强度弱化规律的拟合公式,引入到打桩分析软件中。研究结果表明：土体的强度折减程度不仅与土体本身的性质有关还受到土体的埋深和剪切速率的影响,埋深越深土体强度折减程度越低,剪切速率越高土体强度折减越高,在打桩分析中可采用这里推荐的线性折减方法来模拟不同深度处土体强度的折减规律。     

Geoacoustic Inversion via Genetic Algorithm and Its Application to Manganese Sediment Identification

An acoustic inversion method using a wide-band signal and two near field receivers is proposed and applied to multiple layered seabed models including a manganese sediment. The inversion problem can be formulated into a probabilistic model comprised of signals, a forward model, and additive noise. The forward model simulates wide-band signals, such as chirp signals, and is chosen to be the source-waveletconvolution plane wave modeling method. The wavelet matching technique, using weighted least-squares fitting, estimates the sediment sound-speed and thickness on which determination of the possible numerical ranges for a priori uniform distribution is based. The genetic algorithm is applied to a global optimization problem to find a maximum a posteriori solution for determined a priori search space. Here the object function is defined by an L 2 norm of the difference between measured and modeled signals. Not only the marginal pdf but also its statistics are calculated by numerical evaluation of integrals using the samples selected during importance sampling process of the genetic algorithm.     

Phylogenetic diversity and phenotypic characterization of cultivable bacterioplankton isolated from polar oceans

A set of 27 marine planktonic bacteria isolated from the polar regions was characterized by 16S rDNA sequencing and physiological and biochemical testing. More than half of these bacteria were positive for caseinase, gelatinase and 13-glucosidase, and could utilize glucose, maltose or malic acid as carbon source for cell growth. Twelve isolates expressed nitrate reduction activities. Except for one antarctic isolate BSwlO175 belonging to Actinobacteria phylum, these isolates were classified as γ-Proteobacteria, suggesting that γ-Proteobacteria dominated in cultivable marine bacterioplankton at both poles. Genus Pseudoalteromonas was the predominant group in the Chukchi Sea and the Bering Sea, and genus ShewaneUa dominated in cultivable bacterioplankton in the Prydz Bay. With sequence similarities above 97%, genus Psychrobacter was found at both poles. These 27 isolates were psychrotolerant, and significant 16S rDNA sequence similarities were found not only between arctic and antarctic marine bacteria （ 〉 99% ）, but also between polar marine bacteria and bacteria from other aquatic environments （ ≥ 98.8% ）, including temperate ocean, deep sea, pond and lake, suggesting that in the polar oceans less temperature-sensitive bacteria may be cosmopolitan and have a bipolar, even global, distribution at the species level.     

Deep-Sea Sediment Compression Curves: Some Controlling Factors, Spurious Overconsolidation, Predictions, and Geophysical Reproduction

Oedometer tests have been carried out on 70 undisturbed surficial clays (at approximately 250 mm below the mudline), mostly collected by free-fall corers from sites widely scattered throughout the deep-sea North Atlantic. Acoustic measurements were also made, initially on contiguous samples and ultimately on the same sample using a geophysically instrumented oedometer which also collected electrical resistivity data. Apart from those quiescent areas below the carbonate compensation depth, such as north of the West Indies where very fine clays exist, most of the samples are silty clays whose geotechnical-geophysical properties are dependent on the type of clay minerals present (and their ability to take in moisture), the sand-size fraction, and the quantity of carbonate present. Thus the pure clays have high compressibilities which decrease on the addition of coarse particles, while the converse is true for the acoustic parameters, these increasing with the sand fraction. Using the notion of the intrinsic compression line for all samples, and comparison to it of the measured compression curves, it is clear that, contrary to some previously held ideas, most deep-sea clays are normally consolidated; the addition of carbonate has the effect of creating an open, stronger sediment skeleton. Interestingly, where information is available, the variation with depth of a sample's acoustic velocity follows the void ratio pressure relationship of the compression curve. This allows the construction of an in-situ sediment compression curve using the in-situ geophysical observations.     

INFORMATION FOR AUTHORS

<正>Acta Oceanologica Sinica(AOS)is a comprehensive academic journal edited by the Editorial Committee of Acta Oceanologica Sinica and is designed to provide a forum for important research papers of the marine scientific community which reflect the information on a worldwide basis.The journal publishes scholarly papers on marine science and technology,including physics,chemistry,biology,     

Estimation of Sediment Properties during Benthic Impact Experiments

Sediment properties, such as water content and density, have been used to estimate the dry and wet weights, as well as the volume of sediment recovered and discharged, during benthic impact experiments conducted in the Pacific and Indian Oceans. The estimates show that the weights of dry (355-1332 t) and wet sediment (1651-4888 t) recovered during the experiments, are not only a function of the total duration and distance covered during the experiment, but also depend on water content and density of the sediment. Estimation of dry sediment and its volumetric ratio in the discharge, are key parameters for calculating the discharged volume. The estimated volume of wet sediment recovered (1427-4049 m 3 ) as well as discharged (2693-6951 m 3 ) during the experiments, provide important inputs to evaluate the sediment resettlement and migration. Using these estimates, the average depth of excavation on the seafloor, can also been calculated. However, the stages of sediment recovery and discharge are expressed differently in some of the experiments. In order to standardize these, different stages have been identified, and definitions of certain terms have been suggested, for use in the future. The methods of calculating different properties, as well as weight and volume of discharged sediment are described in the paper for use in other applications concerning deep-sea discharges.