Screening and molecular classification of low-temperature protease from Antarctic microorganism and its characterization

107 strains producing protease were screened from 260 strains of Antarctic psychrophilic bacteria, among which proteolytic activity of five strains was more than 45 U ml^-1. The 16S rRNA gcne sequences homology and phylogcnetic analysis of five Antarctic psychrophillc bacteria showed that NJ276, NJS-9, NJ16-70,NJ345 belonged tO the described genus Pseudoalteromonas and NJ341 belonged to the genus Colwellia. The growth and the protease characteristic of four Antarctic psychrophilic bacteria had been studied, and the result showed that the 6ptimal temperature for growth and protease-produeing of four strains was about 10℃. Their growth and protease-produeing were still high during incubatlng 2-5 days. The maximum proteolytic activity occurred at pH 9 for four Antarctic psychrophilic bacteria. The optimal temperature of protease action of both strains NJ276 and NJ5-9 was about 50℃, however, the optimal temperature of protease aetlon of both strains NJ341 and NJ345 was about 40 ℃, and their proteolytic activity under 0℃ exhibited nearly 30% of the maximum activity, but their thermal stabilities were weaker. These results indicated that proteases from NJ341 and NJ345 were low-temperature proteases.     

Phylogenetic analysis of bacteria in sea ice brine sampled from the Canada Basin, Arctic Ocean

Bacterial diversity in sea ice brine samples which collected from four stations located at the Canada Basin, Arctic Ocean was analyzed by PCR-DGGE. Twenty-three 16S rDNA sequences of bacteria obtained from DGGE bands were cloned and sequenced. Phylogenetic analysis clustered these sequences within γ-proteobacteria, Cytophaga-Flexlbacter-Bacteroides （CFB） group, Firmicutes and Actinobacteria. The phylotype of Pseudoalteromonas in the γ-proteobacteria was predominant and members of the CFB group and γ-proteobacteria were highly abundant in studied sea ice brine samples.     

Isolation and identification of bacteria associated with the surfaces of several algal species

We conducted this study to assess the diversity of bacteria associated with the surfaces of algae based on 16S rDNA sequence analyses.Twelve strains of bacteria were obtained from the surfaces of the following four species of algae:Gracilaria textorii,Ulva pertusa,Laminaria japonica,and Polysiphonia urceolata.The isolated strains of bacteria can be divided into two groups:Halomonas and Vibrio,in physiology,biochemical characteristics and 16S rDNA sequence analyses.The phylogenetic tree constructed based on ...     

Relationships between two Pseudoalteromonas strains isolated from the Canada Basin and the Southern Ocean using a polyphasic approach

The evolutionary relationships and taxonomic position of two marine planktonic bacterial strains BSw20211 and BSw10014, isolated from the Canada Basin and from the Southern Ocean, respectively, were determined using a polyphasic taxonomic approach. There was a close phylogenetic relationship between the two strains and most phenotypic properties were shared. Nonetheless, they were found to belong to different species of the genus Pseudoalteromonas on the basis of genotypic analyses. Findings were consistent with the suggestion that gyrB gene sequence comparison and DNA-DNA relatedness might better define phylogenetic relationships of bacteria at the species level. However, a cut-off value of 90% gyrB gene sequence similarity was not reliable for the differentiation of species within the genus Pseudoalteromonas.     

Studies on culture condition and extracellular hydrolase of psychrophilic bacteria from Arctic sea ice

Arctic sea ice in the polar region provides a cold habitat for microbial community. Arctic sea ice microorganisms are revealed to be of considerable importance in basic research and potential in biotechnological application. This paper investigated the culture condition and extraceIlular hydrolase of 14 strains of different Arctic sea ice bacteria. The results showed that optimal growth temperature of strains is 15 ℃ or 20 ℃. The optimal pH is about 8.0. They hardly grow at acid condition. 3 % NaCl is necessary for better growth. These strains have different abilities in producing amylase, protease, eellulase and lipase. Pseudoalteronomas sp. Bsi429 and Pseudoalteronomas sp. Bsi539 produced both cellulose, protease and lipase. These results provide a basis for further developing and exploiting the cold adapted marine enzyme resources.     

Cold-adaptive alkaline protease from the psychrophilic Planomicrobium sp.547: enzyme characterization and gene cloning

A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA analysis. The optimal and highest growth temperatures for strain 547 were 15℃ and 30℃, respectively. The extracellular protease was purified by ammonium sulfate precipitation and DEAE cellulose-52 chromatography. The optimal temperature and pH for the activity of the purified enzyme were 35 ℃ and pH 9.0, respectively. The enzyme retained approximately 40% of its activity after 2 h of incubation at 50℃. The enzymatic activity was inhibited by 1 mmol/L phenylmethyl sulfonylfluoride (PMSF) and hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), indicating that it was a serine protease. The presence of Ca2+ and Mn2+ increased the activity of the enzyme. The protease gene with a size of 1 269 bp was cloned from Planomicrobium sp. 547 using primers designed based on the conserved sequences of proteases in GenBank. The Planomicrobium sp. 547 protease contained a domain belonging to the peptidase S8 family, which has a length of 309 amino acid (AA) residues. The alignment and phylogenetic analysis of the AA sequence indicated that the protease belonged to the subtilisin family.     

广东省罗非鱼主养区无乳链球菌的分离、鉴定与致病性

从广东省5个罗非鱼主养区患暴发性的罗非鱼中分离到15株菌,挑选其中的5株(不同地域、不同组织分离的菌株)进行人工感染试验,表现出自然发病的症状,确定此5株分离菌为导致罗非鱼暴发病的主要病原。通过形态学观察和生理生化实验,并结合16SrRNA基因序列分析,5株分离菌株均为无乳链球菌(Streptococcus agalactiae)。5株分离菌在形态学和生理生化实验结果上保持一致,16SrRNA基因序列同源性达98.6%～99.8%,但在致病性和对药物的敏感性上却呈现一定的差异。另外10株分离菌株通过生理生化实验鉴定也为无乳链球菌。研究表明,广东省罗非鱼流行性暴发病的病原菌主要为无乳链球菌。     

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

1　Introduction　PorphyraisthemainobjectforalgafarmingandplaysaveryimportantroleinChinesemarineindus tries.Recently ,therehasbeenagreatlossinPor phyracultivationduetothedegenerationoftheculti var,sothereisanincreasingdemandforgoodPor phyracultivars .Theprerequisiteofthetraditionalbreed ingandbioengineeringresearchofPorphyraistheconstructionofpurelines .Traditionally ,theclassifi cationofPorphyrawasaccordingtotheirmorphologi calcharacteristics .However ,mostmorphologicalfea turesofPorphyraar…     

2015—2020年辽东湾海冰冰情时空特征及其影响因素

受冬季强寒潮侵袭,辽东湾会出现大范围结冰现象。为了分析2015—2020年辽东湾海冰冰情的变化规律与影响因素,本文选取Sentinel-1A/B数据开展辽东湾海冰监测。首先,采用巴氏距离选择最优纹理特征组合,再利用最大似然方法实现海冰分类;然后,根据上述海冰分类结果,分析海冰冰情等级、海冰外缘线、海冰面积、海冰类型和海冰结冰概率等冰情特征的变化规律;最后,研究海水深度、海温、气温和风速与海冰冰情的关系。主要结论如下：① 采用不同纹理特征组合方法和本文方法对2020年2月1日Sentinel-1B影像进行实验,结果表明本文方法的总体分类精度和Kappa系数分别为93.16%和0.85,分类精度最高。② 11月末到12月海冰类型以初生冰为主,间有灰冰;1月到2月中上旬以灰冰为主,间有初生冰和白冰;2月下旬到3月上旬的海冰类型以灰冰和初生冰为主。辽东湾内部结冰概率存在差异,北部沿岸结冰概率高于南部,东部结冰概率高于西部。辽东湾海冰冰情受海水深度、海温和气温影响明显,受风速影响较小。     

Oxygen isotopic composition and its application to the study of tracing oceanographical process in Bering Sea and Chukchi Sea

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Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

The wild Porphyra yezoensis collected from the Qingdao coast was used to prepare protoplasts by enzyme digestion. The pure line was constructed by cultivating the protoplasts. The 18S rDNA of the P. yezoensis pure line was cloned and sequenced. Sequence analysis was executed for this sequence and other 22 sequences retrieved from GenBank. A phylogenetic tree was constructed using the neighbor-joining method. The results revealed a high diversity of 18S rDNA sequences in genus Porphyra and the considerable variation of 18S rDNA sequences in different strains of the same species P. yezoensis and P. tenera. Significant difference of 18S rDNA sequence was observed between P. yezoensis from Qingdao, China, and the two strains of P. yezoensis from Japan, but the three strains of P. yezoensis formed a stable clade in the phylogenetic tree. These results indicate the possibility of interspecies and intraspecies discrimination of Porphyra using the 18S rDNA sequences.     

珠母贝人工苗养殖研究

在不同海区、不同水层养殖珠母贝人工苗,及用不同方法进行养殖小贝。结果表明:在开放性、半开放性、内湾性海区养殖11个月后,珠母贝壳高分别为64.5、51.4、69.8 mm,成活率分别为6.4%、0.8%、0.5%,其生长和成活率具有显著差异(P<0.05);同海区中3m、6m、9m、12m、15m水层养殖11个月后,珠母贝壳高分别为64.5、67.4、62.8、56.9、51.9mm,成活率分别为6.4%、6.8%、8.1%、11.7%、13.9%,随着水层的加深,珠母贝的生长变慢,成活率提高且差异显著(P<0.05);6个月龄小贝用穿耳法、片笼法、锥型笼法养殖5个月后,壳高分别长成56.1、55.7、51.7mm,成活率分别为72.6%、59.9%、51.7%,其生长和成活率具有显著差异(P<0.05)。     

Phylogenetic analysis of cultivable bacteria isolated from Arctic sea-ice

Phylogenetic analysis based on 16S rDNA of 8 strains of cultivable bacteria isolated from Arctic sea-ice was studied.The results showed that strain BJ1 belonged to genus Planococcus,which was a genus of low mole percent G C gram-positive bacteria;strain BJ6 belonged to genus Burkholderia of β-proteobacteria and the rest 6 strain all belonged to γ-proteobacteria,of which strain BJ8 was a species of Pseudoalteromonas,strain BJ2-BJ5 and BJ7 were members of genus Psychrobacter.Phylogenetic analysis also indicated that bacteria of genus Psychrobacter of the isolates formed a relatively independent phylogenetic cluster in comparison with other bacteria belonged to genus Psychrobacter.     

Extracellular enzymatic activities of cold-adapted bacteria from polar oceans and effect of temperature and salinity on cell growth

The potential of 324 bacteria isolated from different habitats in polar oceans to produce a variety of extracellular enzymatic activities at low temperature was investigated. By plate assay, lipase, protease, amylase, gelatinase, agarase, chitinase or cellulase were detected. Lipases were generally present by bacteria living in polar oceans. Protease-producing bacteria held the second highest proportion in culturable isolates. Strains producing amylase kept a relative stable proportion of around 30% in different polar marine habitats. All 50 Arctic sea-ice bacteria producing proteases were cold-adapted strains, however, only 20% were psychrophilic. 98% of them could grow at 3% NaCl, and 56% could grow without NaCl. On the other hand, 98% of these sea-ice bacteria produced extracellular proteases with optimum temperature at or higher than 35℃, well above the upper temperature limit of cell growth. Extracellular enzymes including amylase, agarase, cellulase and lipase released by bacteria from seawater or sediment in polar oceans, most expressed maximum activities between 25 and 35℃. Among extracellular enzymes released by bacterial strain BSw20308, protease expressed maximum activity at 40℃, higher than 35℃ of polysaccharide hydrolases and 25℃ of lipase.     

北极熊栖息地稳定性的遥感评估方法

北极熊是北极最重要的哺乳动物之一,近年来数量却在减少。海冰作为北极熊狩猎、活动和繁殖的平台,是其栖息地的重要组成部分。因此其种群栖息地变化主要依赖于海冰变化。本文基于美国雪冰中心的海冰密集度和NOAA提供的ETOPO1基岩数据,分析了北极海冰密集度、开阔水域面积、海冰消退时间、海冰出现时间、开阔水域季节长度的年际变化,进而评价北极熊栖息地的稳定性。结果表明,海冰密集度呈现降低的趋势,开阔水域面积增大,多年冰数量减少,大多变为一年冰。海冰消退时间提前,海冰出现时间延后,开阔水域季节长度大幅增加,与1992年相比增加了72 d。19个栖息地中,巴伦支海是开阔水域面积和季节长度变化贡献最大的海域,增加速度分别为9.71×10³ km²/a和71.69 d/10a。以开阔水域季节长度变化率为依据,将北极熊栖息地划分为稳定、次稳定和不稳定3个等级。总共有3个稳定栖息地,包括分布在相对其他栖息地而言纬度较低的楚科奇海、西哈得孙湾和南哈得孙湾。13个次稳定栖息地,包括拉普捷夫海、喀拉海、东格陵兰、巴芬湾、戴维斯海峡、福克斯湾、布西亚湾、麦克林托克海峡、梅尔维尔子爵海峡、挪威湾、北波弗特、南波弗特和兰开斯特海峡。3个不稳定栖息地,均位于70°N以北,包括北极盆地、巴伦支海和凯恩盆地。稳定区主要位于低纬度,不稳定区全部位于高纬度。该分级结果表明高纬度地区虽然海冰覆盖多,但是年际变化十分显著,不稳定的3个区域内北极熊对海冰变化适应时间更少,年际迁移变化大,对北极熊的生存发展更为不利。     

一株H_5亚型禽流感病毒血凝素基因的克隆与序列分析

根据GenBank中发表的H5亚型禽流感病毒HA基因序列设计2对引物,用RT-PCR方法从禽流感病毒广东分离病毒株(A/Chicken/Guangdong/1997)中扩增HA基因cDNA片段,并将其克隆至pMD-18T载体进行核苷酸序列测定。结果表明:用2对引物所扩增的片段大小分别约为1300 bp和800 bp,经序列拼接获得的HA基因cDNA长度约为1601 bp,编码533个氨基酸,与国内己发表的11个代表株的核苷酸和氨基酸序列同源性为分别为96.9%~99.9%和86.5%~93.0%;HA基因编码的氨基酸序列的系统进化树也表明A/Chicken/Guang-dong/1997、A/Goose/Huadong/01/2000、A/Ck/Hk/37.4/2002、A/Chicken/Zhoukou/2/02、A/Duck/Guangxi/53/2002、A/Duck/Fujian/01/2002等毒株处于同一进化枝,亲缘关系较近;而与A/Silly/Chicken/Hongkong/SF189/01株处于不同进化枝,亲缘关系较远。     

Isolation and characterization of a fucoidan-degrading bacterium from Laminaria japonica

Fucoidan, a polysaccharide containing abundant fucose and sulfate ester group, was prepared from Laminaria japonica. In order to obtain fucoidan-degrading enzyme, bacteria capable of degrading fucoidan were screened from kelp. A bacterial strain named RC2-3 was obtained, which degraded fucoidan by the maximum extent of 54% ± 1.3%, the highest among all bacterial isolates. High-performance size exclusion chromatography(HPSEC) showed that the molecular weight of fucoidan was gradually reduced by RC2-3 with culturing time, suggesting the production of fucoidan-degrading enzyme by RC2-3. Phylogenetic analysis of partial 16S ribosomal RNA gene(16S rDNA) sequence showed that RC2-3 belonged to the family Flavobacteriaceae. However, it showed different physiological and biochemical characteristics from the known Flavobacteriaceae members producing fucoidan-degrading enzyme, thus RC2-3 was proposed to be a new member of this family.     

一株拮抗芒果炭疽菌海洋细菌的鉴定和发酵培养基优化

从湛江近海海水和海泥分离的229株细菌中,选出对芒果炭疽菌(Colletotrichum gloeosporioides)具有较强拮抗作用的海洋细菌BSW03,抑菌圈直径为14.0 mm。根据菌体形态、生理生化特性和16s rDNA序列分析,BSW03鉴定为枯草芽孢杆菌(Bacillus subtilis)。从9种培养基中筛选出BDPB作为BSW03的发酵培养基,通过单因子碳源、氮源试验和L9(34)正交试验对发酵培养基成分进行优化。结果表明:发酵培养基最佳配方为乳糖20 g/L,牛肉膏15 g/L,K2HPO3.3H2O 0.75 g/L,MgSO4 0.75 g/L,pH自然。转速170 r/min、温度28℃的恒温摇床培养3 d后的无菌发酵液抑菌效果最佳,抑菌圈直径为27.3 mm。     

Response of the Dominant Modes of Atmospheric Circulation in the Northern Hemisphere to a Projected Arctic Sea Ice Loss in 2007

This study revisits the Arctic sea ice extent(SIE) for the extended period of 1979-2015 based on satellite measurements and finds that the Arctic SIE experienced three different periods: a moderate sea ice decline period for 1979-1996, an accelerated sea ice decline period from 1997 to 2006, and large interannual variation period after 2007, when Arctic sea ice reached its tipping point reported by Livina and Lenton(2013). To address the response of atmospheric circulation to the lowest sea ice conditions with a large interannual variation, we investigated the dominant modes for large atmospheric circulation responses to the projected 2007 Arctic sea ice loss using an atmospheric general circulation model(ECHAM5). The response was obtained from two 50-yr simulations: one with a repeating seasonal cycle of specified sea ice concentration for the period of 1979-1996 and one with that of sea ice conditions in 2007. The results suggest more occurrences of a negative Arctic Oscillation(AO) response to the 2007 Arctic sea ice conditions, accompanied by an North Atlantic Oscillation(NAO)-type atmospheric circulation response under the largest sea ice loss, and more occurrences of the positive Arctic Dipole(AD) mode under the 2007 sea ice conditions, with an across-Arctic wave train pattern response to the largest sea ice loss in the Arctic. This study offers a new perspective for addressing the response of atmospheric circulation to sea ice changes after the Arctic reached the tipping point in 2007.