利用多功能裂合酶CpcE/F合成非天然重组藻胆蛋白

藻胆蛋白裂合酶是催化藻胆色素与脱辅基藻胆蛋白亚基共价连接的裂合酶,在藻胆蛋白的生物合成途径中发挥着重要作用。本实验以藻蓝蛋白裂合酶Cpc E/F为研究对象,采用基因工程组合表达技术,构建了pCDFDuet-apcA-cpcE/F-hox1-pebS、pCDFDuet-pecA-cpcE/F-hox1-pebS等重组质粒,并导入大肠杆菌中,在IPTG的诱导下,成功表达得到两种非天然藻胆蛋白ApcA-PEB和PecA-PEB,这表明Cpc E/F不仅可以催化藻蓝胆素和脱辅基藻蓝蛋白的结合,还可以催化藻红胆素(PEB)与别藻蓝蛋白α-亚基(ApcA)和藻红蓝蛋白α-亚基(PecA)的结合,因此是一种多功能的裂合酶。根据重组产物的光谱特征峰分析发现,ApcA-PEB和PecA-PEB作为两种非天然存在的重组藻胆蛋白,具有与对应天然藻胆蛋白有着相近的特征吸收光谱和荧光发射峰,但同时具有明显的波长偏移并伴有色素异化现象,其光谱学性质主要由其所携带的色素基团决定。另外,荧光寿命衰减分析发现,不同脱辅基蛋白对于重组藻胆蛋白的光谱稳定性具有重要的影响。     

坛紫菜R-藻蓝蛋白和变藻蓝蛋白的亚基组成和发色团含量

坛紫菜(Porphyra haitanensis T.J.Chang et B.F.Zheng)中的R-藻蓝蛋白在Bio-Rex 70柱上用脲溶液(pH3.0)解离和层析,得到了α和β两个亚基,用SDS-PAGE测定α和β亚基的分子量分别为18 400和20 500。变藻蓝蛋白的α和β两亚基均分子量经测定,分别为18 800和19 700。R-藻蓝蛋白和变藻蓝蛋白的分子量分别确定为117 000和112 000,两藻胆蛋白中α和β两个亚基的摩尔比都为1:1,确定两藻胆蛋白的亚基组成都为(αβ)_3。各亚基的发色团含量为:R-藻蓝蛋白中,α亚基含1个藻蓝胆素,β亚基含1个藻蓝胆素和1个藻红胆素。变藻蓝蛋白中,α和β亚基各含1个藻蓝胆素。     

坛紫菜藻红蛋白α,β亚基基因的克隆和序列分析

藻胆蛋白(Phycobiliprotein,PBP)是原核藻类和真核藻类进行光合作用的捕光色素蛋白,占藻体干重的2%左右,主要分为3类:藻红蛋白(phycoer-ythrin,PE)、藻蓝蛋白(phycocyanin,PC)和别藻蓝蛋白(allophycocyanin,APC)[1].藻胆蛋白一般由α亚基和β亚基组成,部分藻胆蛋白中还存在γ亚基.一般由α和β亚基形成稳定的单体(αβ),再由单体聚合为多聚体(αβ)_n.从蓝藻和红藻中分离的藻胆蛋白为三聚体(αβ)₃或六聚体(αβ)₆[2].     

钝顶节旋藻别藻蓝蛋白基因克隆、分析及重组表达蛋白荧光活性的研究

为探究蓝藻藻胆体核心色素蛋白-别藻蓝蛋白单个亚基的光学活性和功能,本研究克隆了钝顶节旋藻(Arthrospira platensis FACHB314)中的脱辅基别藻蓝蛋白基因apcAB及其亚基基因apcA和apcB。节旋藻基因组中apcAB全长共1 056 bp,由apcA和apcB串联组成,中间间隔84 bp。其中,apcA和apcB基因的全长均为486 bp,且均编码161个氨基酸,预测分子量分别约为17.39和17.33 kDa,它们分别编码的α和β亚基均属于Globin＿like超级家族,系统进化树分析显示其氨基酸序列在蓝藻和节旋藻中较为保守。将克隆出的亚基基因apcA、apcB分别同前期A.platensis FACHB314中获得的藻蓝胆素合成相关基因ho和pcyA以及色基裂合酶基因(cpcE、cpcF、cpcU、cpcS、cpcT)共同转化至大肠杆菌中,获得重组别藻蓝蛋白亚基菌株E.coli HPEFUSTA、E.coli HPEFUSTB。SDS-PAGE和Western Blot均显示重组菌株中已成功表达别藻蓝蛋白单亚基。荧光检测结果显示,在600 nm波长激发下,...     

光学活性藻蓝蛋白β亚基的体内重组

在Synechococcus sp.PCC 7002中,首先发现了特异性催化脱辅基藻蓝蛋白β亚基(153位)与藻蓝胆素(phycocyanobiling, PCB)连接的裂合酶基因cpcT.本实验在Synechocystis sp.PCC 6803中找到与cpcT具有高度同源性的基因slr1649.构建2组相容的重组质粒pCDF-cpcB(C82I)或(C153I)-ho1-pcyA和pRSF-slr1649,cpcB(C82I)和cpcB(C153I)分别编码82和153位被突变的脱辅基藻蓝蛋白β亚基,slr1649编码可能的特异性裂合酶,在大肠杆菌体内生成PCB必需的2个基因(ho1编码血红素氧化酶,pcyA编码藻蓝胆素合成酶),将这些基因共同转化到大肠杆菌诱导表达,利用亲和层析柱对可溶性蛋白进行纯化,产物进行SDS-PAGE、色素蛋白锌电泳和光谱检测.结果表明基因slr1649能够特异性催化CpcB(153位)与PCB的连接,产生了具有光学活性的CpcB(C82I)-PCB,为进一步研究螺旋藻藻蓝蛋白的生物活性奠定了基础.     

别藻蓝蛋白基因在毕赤酵母(Pichia pastoris)中的表达

采用亚克隆方法,将别藻蓝蛋白(allophycocyanin,APC)基因插入到毕赤酵母整合型表达载体pPIC6αA中,再用SacⅠ线性化,纯化后,用氯化锂法转化毕赤酵母菌株X-33,筛选表达APC的重组子。PCR和序列分析表明,APC基因已整合到酵母染色体中。重组子经甲醇诱导,用Western blotting在培养基上清液检测到APC,表明别藻蓝蛋白(APC)在毕赤酵母中可以表达。APC由两种亚基组成,分子量分别为19kDa和21kDa。重组子经摇瓶培养48h表达量达4.4mg/L。     

雨生红球藻虾青素合成关键酶的原核表达及纯化

雨生红球藻是天然虾青素的重要来源,在胁迫条件下积累虾青素含量能达到细胞干重的5%。八氢番茄红素合成酶(PSY)、β-胡萝卜素酮化酶(CRTW)和β-胡萝卜素羟化酶(CRTZ)是雨生红球藻虾青素合成的关键酶,本研究分别以雨生红球藻的DNA和cDNA为模板,克隆了这三个基因,并构建不同的原核表达载体pET-24a-crtw,pET-24a-crtz,pET-24a-psy。转化大肠杆菌Rosetta感受态,经IPTG诱导后,SDS-PAGE和western blotting结果显示三种重组蛋白都能够正常表达,利用6×His标签对重组蛋白进行纯化能得到相对单一的条带,相对于纯化前目的条带明显加深。ELISA检测重组菌株E.coli RZ,E.coli RW,E.coli RP中的酶活性分别为3.0,10.5,10.0 IU/L,纯化后酶活性显著提高,分别为125.5,124.7,118.0 IU/L。本研究获得了这三种酶的活性重组蛋白,为其功能分析和抗体制备奠定了基础。     

条斑紫菜中R-藻红蛋白的纯化及其α和β亚基的分离与发色团含量的测定

本文从条斑紫菜(Porphyra yezoensis)中提取了藻胆蛋白,找到了一种新的简便有效的提纯R-藻红蛋白的方法。用Bio-GelP300柱进一步纯化后,得到了高纯度的R-藻红蛋白。用Bio-Rex 70柱分离R-藻红蛋白,得到了它的α和β亚基溶液。对亚基溶液进行的一些光谱分析表明亚基溶液具有R-藻红蛋白的一些性质。本文对α和β亚基的发色团含量进行了测定:α亚基含2个藻红胆素,β亚基含3个藻红胆素和1个藻尿胆素。     

坛紫菜别藻蓝蛋白α,β亚基基因的克隆和序列分析

以野生坛紫菜色素体DNA为模板, 通过PCR扩增获得编码坛紫菜别藻蓝蛋白α亚基和β亚基的序列及两者之间的间隔序列,该序列全长为1 055 bp,其中编码α和β亚基的序列均为486 bp,间隔序列为83 bp.该序列与GenBank收录的其他4种红藻相关序列的同源性在75.6%~87.4%,与2种蓝藻的同源性分别为66.9%,68.5%,其中编码区同源性更高.     

别藻蓝蛋白β亚基基因在莱茵衣藻中重组表达及其对光能传递的影响研究

为了探究别藻蓝蛋白与类囊体膜光合系统之间的光能传递规律,及其与异源类囊体膜光合系统之间光能转移的可能性,本研究构建了含有钝顶节旋藻(Arthrospira platensis)的别藻蓝蛋白β亚基基因(apcB),及合成藻蓝胆素的血红素氧化酶基因(ho)和铁氧还蛋白氧化还原酶基因(pcyA)的转化载体pHyg3-apcB-ho-pcyA,利用玻璃珠转化法将其导入莱茵衣藻中。聚合酶链式反应筛选验证阳性转化藻株,Southern Blot结果表明基因apcB、ho、pcyA成功转化入莱茵衣藻基因组中。而后将转化藻株与对照藻株均置于高光(50μmol·m^-2·s^-1)和低光(25μmol·m^-2·s^-1)条件、白光和绿光条件中培养,并检测别藻蓝蛋白β亚基的表达、总叶绿素含量、77 K低温荧光、光系统表观电子传递效率、生物量变化。结果显示：实时定量PCR分析和Western Blot检测表明外源基因apcB,pcyA和ho均在转基因藻株Cr-ApcBHP中得到了转录表达。高光和低光条件下光系统Ⅱ(PSⅡ)和光...     

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.     

A Review of Some Environmental Issues Affecting Marine Mining

This article reviews information recently available from existing marine and coastal mining for responses to environmental issues affecting marine mining at different depths. It is particularly but not exclusively concerned with those issues affecting seabed biodiversity impact and recovery. Much information has been gathered in the past 10 years from shallow mining operations for construction aggregate, diamonds, and gold, from coastal mines discharging tailings to shallow and deep water, and from experimental deep mining tests. The responses to issues identified are summarized in a series of eight tables intended to facilitate site-specific consideration. Since impacts can spread widely in the surface mixing layer SML, and can affect the biologically productive euphotic zone, the main issues considered arise from the depth of mining relative to the SML of the sea. Where mining is below the SML, the issue is whether it is environmentally better to bring the extraction products to the surface vessel for processing (and waste discharge), or to process the extraction products as much as possible on the seabed. Responses to the issues need to be sitespecific, and dependent on adequate preoperational environmental impact and recovery prediction. For deep tailings disposal from a surface vessel, there are four important environmental unknowns: (1) the possible growth of "marine snow" (bacterial flocs) utilizing the enormous quantities of fine tailings particles (hundreds or thousands of metric tons per day) as nuclei for growth, (2) the possibility that local keystone plankton and nekton species may migrate diurnally down to and beyond the depth of deep discharge and hence be subjected to tailings impact at depth, (3) the burrow-up capability of deep benthos and their ability to survive high rates of tailings deposition, and (4) the pattern and rate of dispersion of a tailings density current through the deep water column from discharge point to seabed. Actions to obtain relevant information in general and site-specifically are suggested.     

Increased Particle Fluxes at the INDEX Site Attributable to Simulated Benthic Disturbance

Particle fluxes were measured 7 m above the sea bottom during the predisturbance, disturbance, and postdisturbance periods by using time series sediment traps attached to seven deep-sea moorings deployed in the INDEX experiment site in the Central Indian Basin. The predisturbance particle fluxes varied between 22.3 to 55.1 mg m -2 day -1 . Increased and variable particle fluxes were recorded by the sediment traps during the disturbance period. The increase observed was 0.5 to 4 times more than the background predisturbance fluxes. The increases in particle fluxes (~4 times) recorded by the sediment trap located in the southwestern direction (DMS-1) were the greatest, which could be the result of preferential movement of resuspended particles generated during the deep-sea benthic disturbance along the general current direction prevailing in this area during the experimental period. Also, the traps located closer to the disturbance area recorded greater fluxes than did the traps far away, across the Deep Sea Sediment Resuspension System path. This variability in recorded particle fluxes by the traps around the disturbance area clearly indicates that physical characteristics such as grain size and density of the resuspended particles produced during the disturbance had an important effect on particle movement. The postdisturbance measurements during ~5 days showed a reduction in particle fluxes of ~50%, indicating rapid particle settlement.     

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.     

Determination of In Situ Sediment Shear Strength from Advanced Piston Corer Pullout Forces

The advanced piston corer (APC) has been used by the Ocean Drilling Program since 1985 for recovering soft sediments from the ocean floor. The pullout force measured on extracting the core barrel from the sediment is shown to correlate with the average shear strength of the sediment core measured in the ship's laboratory. A simple rule of thumb is derived relating the shear strength of the sediment to the pullout force. Multiple APC holes at individual sites allow the consistency of the pullout measurements to be assessed. The effects of different operational procedures during APC coring are also explored. Although generally applicable, the correlation between pullout force and laboratory measurements of shear strength breaks down for some APC holes, possibly because of the disturbance of some sediment types during the APC coring process. A better understanding of the physical process of APC coring, and its effect on the properties of the sediment both inside and immediately outside the core barrel, would indicate what confidence can be put on the measurement of pullout force as a way of evaluating the in situ shear strength of deep sea sediments.     

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.