锯缘青蟹(Scylla serrata)呼肠孤病毒5个结构蛋白互作分析

采用酵母双杂交系统,对青蟹呼肠孤病毒(Scylla serrata reovirus)5个结构蛋白(VP3,VP6,VP9,VP11,VP12)间的双向互作进行了分析。将5个结构蛋白对应的ORF分别亚克隆至诱饵载体p GBKT7和猎物载体p GADT7,成功构建了10个酵母双杂交重组载体。将重组诱饵载体或重组捕获载体转化至酵母菌Y2H Gold中进行自激活检测,结果显示5个结构蛋白均不能激活酵母菌报告基因HIS3的表达,表明5个病毒蛋白均不具有自激活活性。通过酵母双杂交实验,从5个结构蛋白25个可能性互作对中,共筛选出2个双向互作对(VP11VP6,VP11VP12)和3个自身互作对(VP3VP3,VP11VP11,VP12VP12)。本研究是有关Ss RV结构蛋白互作的首次报道。     

利用酵母双杂交研究铜绿微囊藻生物钟蛋白KaiB的自身相互作用

从铜绿微囊藻(Microcystis aeruginosa)PCC7820中克隆了生物钟基因kaiB,并将其克隆入酵母双杂交系统的两个质粒pGADT7和pGBKT7中。经测序验证后,将重组质粒pGADT7-kaiB和pGBKT7-kaiB转化酵母菌AH109进行自激活性和自身相互作用的检测和验证。结果表明,铜绿微囊藻生物钟蛋白KaiB无自激活性,KaiB蛋白自身能发生相互作用。因此,可用KaiB蛋白为诱饵利用酵母双杂交系统筛选文库钓取与KaiB发生相互作用的蛋白。     

酵母双杂交系统用于WSSV结构蛋白VP39相互作用的宿主蛋白基因的初步研究

为研究WSSV的结构蛋白VP39相关蛋白的基因,应用酵母双杂交系统筛选中国明对虾(Fenneropenaeus chinensis)鳃细胞全长cDNA文库。将构建好的VP39基因的重组载体pGBKT7-VP39转化酵母Y187后,与含文库质粒的酵母株AH109融合,在营养缺陷培养基上进行反复筛选,得到1个阳性克隆,测序并进行生物信息学分析。结果显示,此cDNA片段在数据库中是新序列,该蛋白在WSSV识别并结合宿主的过程中所起的作用还有待于进一步研究。本实验成功筛选了VP39相关蛋白的cDNA片段,为进一步研究WSSV的致病机理奠定了基础。     

对虾传染性皮下及造血组织坏死病毒编码蛋白的相互作用研究

采用Matchmaker酵母双杂交系统,将对虾传染性皮下及造血组织坏死病毒(Infection hypodermal and hematopoietic necrosis virus,IHHNV)编码蛋白NS1、NS2和CP序列分别构建到酵母猎物载体p GADT7和诱饵载体p GBKT7上,分别转化至酵母AH109以检测重组猎物载体和诱饵载体的自激活作用及对宿主的毒性作用,发现无自激活作用和毒性作用,随后将重组猎物载体和诱饵载体两两共转至酵母AH109中,涂布于SD/-Leu/-Trp固体培养基上,再点种至SD/-Ade/-His/-Leu/-Trp/X-α-gal固体培养基以鉴定编码蛋白间的相互作用。表型鉴定结果显示,只有共转化有p GADT7-CP/p GBKT7-CP的酵母重组子能够在SD/-Ade/-His/-Leu/-Trp/X-α-gal上生长并显蓝,而其它重组子均不能在其上生长,表明病毒的CP能够自身互作,而其他编码蛋白间无相互作用。为了进一步研究病毒CP自身互作的作用位点,分别从CP的N端和C端截短若干个氨基酸序列,结果发现CP的自身互作是高度敏感的,任何较少氨基酸序列的缺失都将导致其自身互作的丧失。本研究为深入探讨病毒的组装机制和致病机理奠定了理论基础。     

应用酵母双杂交系统筛选虾夷扇贝FOXL2的相互作用蛋白

FOXL2是脊椎动物的雌性决定基因,在多种无脊椎动物中也被认为是雌性决定与分化的关键基因,但其在无脊椎动物中的作用机制目前尚不清晰。本文利用Clontech酵母双杂交系统筛查虾夷扇贝FOXL2的相互作用蛋白。研究首先构建诱饵质粒pGBKT7-FOXL2,并检测诱饵蛋白的毒性及自激活活性,结果显示诱饵蛋白对酵母菌株无毒,也无自激活活性。利用该诱饵质粒,本研究从虾夷扇贝酵母双杂交cDNA文库筛选得到17个阳性克隆,测序验证得到12个读码框正确的阳性克隆,它们来自4种蛋白:COP9信号复合体亚基5、钠/钾ATP酶β3、哺乳动物室管膜相关蛋白1和胰脂肪酶相关蛋白2。研究进一步将诱饵质粒与4种蛋白的猎物质粒进行共转化验证,结果均为阳性,表明这4种蛋白与虾夷扇贝FOXL2都可能存在相互作用。本文为研究FOXL2在无脊椎动物性别决定与分化中的作用提供了参考。     

草鱼(Ctenopharyngodon idellus)呼肠孤病毒(GCRV)VP6相互作用蛋白的筛选

为筛选与草鱼呼肠孤病毒GCRV096 VP6发生相互作用的宿主蛋白,先将VP6基因的PCR扩增产物,克隆至酵母表达载体p GBKT7以构建其诱饵质粒(p GBKT7-VP6);再将酵母菌Y2HGold(含p GBKT7-VP6)与酵母菌Y187[含草鱼肾脏细胞(CIK)c DNA文库质粒]进行人工诱导融合,然后经选择培养筛选阳性克隆。结果共筛选到4株阳性克隆,经测序、生物信息学分析,分别属于两条不同的核苷酸序列,其中之一所编码的产物与GAPDH(3-磷酸甘油醛脱氢酶)同源性较高。可见,该酶极可能在GCRV096侵染宿主的初期发挥着重要作用。     

凡纳滨对虾桃拉综合征病毒主要结构蛋白基因的克隆及原核表达

根据GenBank中桃拉病毒的全基因组序列,分别设计扩增该病毒主要结构蛋白基因vp1、vp2、vp3的相应引物,以发病的凡纳滨对虾中提取的总RNA为模板,利用RT-PCR技术对目的基因进行了扩增,分别获得分子量为1 164、849、507bp的基因片段,与预期一致.再将目的基因与pET-28a载体连接,分别构建重组表达载体pET-VP1、pET-VP2和pET-VP3,重组子经酶切和测序鉴定后导入大肠杆菌BL21(DE3),IPTG诱导表达后进行SDS-PAGE检测.SDS-PAGE电泳的结果表明,在37℃下,0.5mmol/dm3的IPTG诱导表达的重组蛋白均以包涵体的形式存在,重组蛋白VP1、VP2、VP3的大小分别为48、36、24KDa.本研究的结果为制备桃拉病毒主要结构蛋白的特异性抗体,进而研制桃拉病毒快速检测试剂盒奠定了基础.     

中国明对虾血蓝蛋白C末端片段在毕赤酵母中的表达及其抗菌活性

为研究中国明对虾(Fenneropenaeus chinensis)血蓝蛋白C末端(FcHC-C)的抗菌功能,将血蓝蛋白基因FcHC的2个C末端基因片段连接到毕赤酵母表达载体pPIC9K中,构建酵母表达载体pPIC9K/FcHC-C。该载体经SalI酶切后,采用PEG法转化毕赤酵母(Pichia pastoris GS115)。转化子经过PCR鉴定后,阳性克隆通过含有G418的YPD平板筛选,获得高拷贝重组子。重组毕赤酵母利用甲醇诱导表达目的基因。经Tricine-SDS-PAGE和Western blot分析结果表明,利用酵母工程菌成功表达了血蓝蛋白C末端片段(rFcHC-C1和rFcHC-C2)。抑菌活性鉴定实验结果显示,重组蛋白rFcHC-C1和rFcHC-C2作为阴离子抗菌肽具有抗真菌和抗细菌的活性。     

以rDNA为同源重组位点酵母表达鲑鱼降钙素基因多拷贝整合载体的构建

根据酵母整合质粒的设计要求,PCR扩增2.2 kb rDNA片段,导入酿酒酵母(Saccharomyces cerevisiae)整合载体pYD1中,设计并合成适合在酵母中表达的鲑鱼降钙素(Salmon Calcitonin),以此为目标基因,构建适合酿酒酵母多拷贝整合的表达载体p-r-sCT.通过载体p-r-sCT引入到酿酒酵母菌株EBY100中,得到酵母重组菌株yAGA2-r-sCT.流式细胞检测结果表明,目标基因鲑鱼降钙素在重组菌株的yAGA2-r-sCT中得到了表达,实现了外源基因在酿酒酵母菌株中的稳定表达.     

大黄鱼抗菌肽hepcidin在巴斯德毕赤酵母中的表达及其产物的抑菌活性

Hepcidin是一类具有独特性质的小分子抗菌肽,克隆得到大黄鱼(Pseudosciana crocea)的hepcidin基因(Lyc-Hepc),cDNA序列全长585个核苷酸,编码一个由85个氨基酸组成的蛋白,包含24个氨基酸的信号肽、35个氨基酸的前导肽和26个氨基酸的成熟肽,成熟肽具有8个保守的半胱氨酸.将Lyc-Hepc 183bp的编码前导肽和成熟肽的前体肽(25aa-85aa)基因片段克隆到毕赤酵母表达载体pPICZαA中,然后电击转化巴斯德毕赤酵母SMD1168,经高浓度Zeocin筛选获得多个单克隆.挑取4个克隆进行培养,提取酵母基因组进行PCR检测,结果表明目的基因片段已成功整合到酵母菌SMD1168基因组中,获得酵母工程菌株SMD1168/pP1CZα-Lyc-Hepc.SDS-PAGE和WesternBlot分析表明酵母工程菌株在0.5％甲醇诱导培养72h后,Lyc-Hepc在培养上清中得到了分泌型表达.体外抑菌实验表明重组Lyc-Hepc蛋白能够抑制2株水产养殖病害菌嗜水气单胞菌和副溶血弧菌的生长.研究结果表明大黄鱼hepcidin可能在抗细菌免疫中发挥了作用.     

中国鱼类名录Ⅻ--鳚亚目、(鱼衔)亚目、喉盘鱼亚目、鰕虎鱼亚目、微体鱼亚目、刺尾鱼亚目、带鱼亚目、鲭亚目、鲳亚目、金枪鱼亚目、攀鲈亚目、刺鳅亚目

鳚亚目 4 科 33 属 95 种，鰕虎鱼亚目 5 科 98 属 259 种，刺尾鱼亚目 5 科 11 属 65 种，鲈形目 19亚目 104 科 535 属 1799 种。     

The distribution of Mn,Fe, Cu,Ni, Co,Ga, Cr,V, Ba,Sr, Sn,Zn, and Pb,in some soil-sized particulates from the lower troposphere over the world ocean

Soil-sized particulates have been collected on board ship by a mesh technique from the lower troposphere of the North, Equatorial and South Atlantic Ocean, northern and southern Indian Ocean, South and East China Sea and various coastal localities.Spectrographic analysis reveals that, on average, the particulates have concentrations of Mn, Ni, Co, Ga, Cr, V, Ba, and Sr which are of the same order of magnitude as those in average crustal material. In contrast, the average concentrations of Pb, Sn, and Zn are one order of magnitude higher than those in average crustal material.Within this “world-wide” average there are significant geographical variations in the distributions of Pb, Sn, and Zn which may be related to anthropogenic sources.On the basis of trace-element distributions lower tropospheric soil-sized marine particulates have been divided into four genetic components; local, zonal, inter-zonal, and global. The proportions of these components vary geographically, and each component may have both a natural and an anthropogenic fraction.     

Distribution, abundance, and growth of larval tautog, Tautoga onitis, in Buzzards Bay, Massachusetts, USA

Tautog, Tautoga onitis, is an abundant species of fish in estuaries of the northeastern United States. Planktonic tautog larvae are abundant in summer in these estuaries, but there is little information on rates of growth of tautog larvae feeding on natural assemblages of food in the plankton. We examined abundance and growth of larval tautog and environmental factors during weekly sampling at three sites along a nearshore‐to‐offshore transect in Buzzards Bay, Massachusetts, USA during summer 1994. This is the first study of a robust sample size (336 larvae) to estimate growth rates of field‐caught planktonic tautog larvae feeding on natural diets, using the otolith daily‐growth‐increment method. The study was over the entire summer period when tautog larvae were in the plankton. The sampling sites contrasted in several environmental variables including temperature, dissolved oxygen (DO), and chlorophyll a concentration. There was a temporal progression in the abundance of tautog larvae over the summer, in relation to location and temperature. Tautog larvae were first present nearshore, with a pronounced peak in abundance occurring at the nearshore sites during the last 2 weeks in June. Larvae were absent at this time further offshore. From late June through August, larval abundance progressively decreased nearshore, but increased offshore although never approaching the abundance levels observed at the nearshore sites. The distribution and abundance of tautog larvae appeared to be related to a nearshore‐to‐offshore seasonal warming trend and a nearshore decrease in DO. Otoliths from 336 larvae ranging from 2.3 to 7.7 mm standard length had otolith increment counts ranging from 0 to 19 increments. Growth of larval tautog was estimated at 0.23 mm·day^?1, and length of larvae prior to first increment formation was estimated at 2.8 mm indicating that first increment formation occurs 3–4 days after hatching at 2.2 mm. Despite spatial and temporal differences in environmental factors, there were no significant differences in growth rates at any of three given sites over time, or between sites. Because larval presence only occurred at a narrow range of temperature (17–23.5 °C) and DO (6.5–9.3 mg·l^?1), in situ differences in growth did not appear to be because of differences in larval distribution and abundance patterns relative to these parameters.     

Cadmium,cobalt, copper,iron, lead,nickel and zinc in Indian Ocean water

Results of trace-metal analyses of water samples obtained during a cruise with the Soviet R.V. “Akademik Kurchatov” in the Indian Ocean are presented. The determinations were performed on board with atomic absorption spectrophotometry after a two-stage dithiocarbamate—Freon extraction procedure. Trace-metal concentrations found are in the same range as those found recently for similar open-ocean areas by other workers. The values for lead and zinc are probably high due to contamination. Vertical profiles indicate biogenic processes as controlling factors for the increase of cadmium, copper and nickel concentrations with depth. Iron shows an irregular depth distribution as a result of large random variations in concentration.     

The behavior of Zn,Cd, Cu,Ni, Co,Fe, and Mn in anoxic baltic waters

In June 1981, dissolved Zn, Cd, Cu, Ni, Co, Fe, and Mn were determined from two detailed profiles in anoxic Baltic waters (with extra data for Fe and Mn from August 1979). Dramatic changes across the O₂H₂S interface occur in the abundances of Cu, Co, Fe, and Mn (by factors of ?100). The concentrations of Zn, Cd, and Ni at the redox front decrease by factors between 3 to 5.Equilibrium calculations are presented for varying concentrations of hydrogen sulfide and compared with the field data. The study strongly supports the assumption that the solubility of Zn, Cd, Cu, and Ni is greatly enhanced and controlled by the formation of bisulfide and(or) polysulfide complexes. Differences between predicted and measured concentrations of these elements are mainly evident at lower ΣH₂S concentrations.Cobalt proved to be very mobile in anoxic regions, and the results indicate that the concentrations are limited by CoS precipitation. The iron (Fe²⁺) and manganese (Mn²⁺) distribution in sulfide-containing waters is controlled by total flux from sediment-water interfaces rather than by equilibrium concentrations of their solid phases (FeS and MnCO₃). The concentrations of these metals are therefore expected to increase with prolonged stagnation periods in the basin.     

Arsenic,barium, germanium,tin, dimethylsulfide and nutrient biogeochemistry in Charlotte Harbor,Florida, a phosphorus-enriched estuary

Concentrations of dissolved nutrients (NO₃, PO₄, Si), germanium species, arsenic species, tin, barium, dimethylsulfide and related parameters were measured along the salinity gradient in Charlotte Harbor. Phosphate enrichment from the phosphate industry on the Peace River promotes a productive diatom bloom near the river mouth where NO₃ and Si are completely consumed. Inorganic germanium is completely depleted in this bloom by uptake into biogenic opal. The $\text{Ge}\text{Si}$ ratio taken up by diatoms is about 0·7 × 10^?6, the same as that provided by the river flux, confirming that siliceous organisms incorporate germanium as an accidental trace replacement for silica. Monomethylgermanium and dimethylgermanium concentrations are undetectable in the Peace River, and increase linearly with increasing salinity to the seawater end of the bay, suggesting that these organogermanium species behave conservatively in estuaries, and are neither produced nor consumed during estuarine biogenic opal formation or dissolution. Inorganic arsenic displays slight removal in the bloom. Monomethylarsenic is produced both in the bloom and in mid-estuary, while dimethylarsenic is conservative in the bloom but produced in mid-estuary. The total production of methylarsenicals within the bay approximately balances the removal of inorganic arsenic, suggesting that most biological arsenic uptake in the estuary is biomethylated and released to the water column. Dimethylsulfide increases with increasing salinity in the estuary and shows evidence of removal, probably both by degassing and by microbial consumption. An input of DMS is observed in the central estuary. The behavior of total dissolvable tin shows no biological activity in the bloom or in mid-estuary, but does display a low-salinity input signal that parallels dissolved organic material, perhaps suggesting an association between tin and DOM. Barium displays dramatic input behavior at mid-salinities, probably due to slow release from clays deposited in the harbor after catastrophic phosphate slime spills into the Peace River.     

Northstar-Geology,Exploration History,and Development Status,Beaufort Sea,Alaska

Exploration for oil at Northstar has been long and costly. Northstar leases were first acquired in 1979 at a joint state and federal sale by Shell Oil, Amerada Hess, and Texas Eastern. The Northstar Unit is 6 mi offshore and about 4 mi northeast of the Point McIntyre Field. Oil was first discovered in Shell's Seal Island 1 in 1983. Five additional appraisal wells were drilled (1983-1986) from two man-made gravel islands in 40 ft of water. Early engineering estimates put the cost of development at $ 1.6 billion. In February 1995, BP Exploration (Alaska) acquired a 98 % interest in the Northstar Unit from Amerada Hess and Shell Oil. When developed by BP, Northstar will be the first oil produced from federal leases in Alaska. To date, the oil industry has invested in excess of $ 140 million in exploration and appraisal operations. An additional $ 90 million was spent on lease bonus bids. The giant Prudhoe Bay and Kuparuk Fields lie along the Barrow Arch. This arch is bounded to the north by a rift margin that deepens into the present-day offshore region. Northstar is located among a series of down-stepping faults off this northern rift margin of the Prudhoe Kuparuk high. The structure is a gently south-dipping northwest-trending faulted anticline. The crest of the structure is located near 10,850 ft subsea. The primary reservoir is the Ivishak Formation (325 ft thick) of the Sadlerochit Group. This is the same primary reservoir at Prudhoe Bay, approximately 12 mi to the south. At Northstar the Ivishak is a high-energy, coarse-grained conglomeratic facies of the Ivishak Formation. The primary lithology is a pebbly chert to quartz conglomerate with occasional sandstone. This very high net to gross reservoir appears to contain no regionally continuous permeability barriers. Cementation has reduced primary porosity to less than 15 %. Accurate porosity estimates are difficult to make due to the coarse-grained nature of the lithology and the presence of kaolinite and microporous chert. Permeability is highly variable, but averages 10 to 100 mDarcies. Oil is a very light and volatile 42 API crude with approximately 2,100 ft3 of gas per stock tank barrel of oil. This oil is very different from the heavier oils (26) found to the south in Prudhoe Bay. Estimated recoverable oil reserves range from 100 to 160 million barrels. A free-standing drilling rig is required at Northstar because the reserves are beyond extended-reach drilling techniques from shore-based facilities. The current development plan is to expand the existing Seal Island to about 5 acres. This is significantly less than Endicott's 40-acre island. The proposed drilling and produc tion island will be accessed by summer barges and winter ice roads. Oil, gas, and water will be processed at a stand-alone facility and then sent to shore via a subsurface pipeline. Northstar will have the first Arctic subsea pipeline in Alaska to transport oil to shore facilities (TAPS). Preliminary tests in Spring 1996 were very successful in demonstrating the technology to successfully bury a subsea pipeline safely in the Arctic.