云南腾冲热泉极端嗜酸热硫化叶菌多样性研究

硫化叶菌(Sulfolobus)是极端嗜热古菌遗传机制研究的重要模式菌株,广泛分布于酸性热泉中.对分离自云南腾冲酸性热泉的11株极端嗜酸热菌株进行16S rRNA基因序列测定,并通过菌体形态观察、16S rRNA基因可变区序列比较、菌株16S rRNA基因相似性比较及系统发育分析,研究腾冲热泉硫化叶菌的多样性.结果表明,分离自腾冲热泉的11株硫化叶菌的16S rRNA基因序列与分离自世界上其它地区的8个硫化叶菌的标准菌株的相似性为85.8%～94.9%,与同样分离自腾冲热泉的S.tengchongensis标准菌株的相似性为96.6%～97.5%,在分类学上具有形成新种的可能.在16S rRNA基因高可变区序列上,与分离自世界上其它地区的8个硫化叶菌的标准菌株存在明显的差异.系统发育分析表明,分离自腾冲热泉的硫化叶菌在系统发育树上形成两个亲缘关系较为紧密的簇群,可见腾冲热泉硫化叶菌不仅具有一定的多样性,同时具有较明显的地域性特征.     

硫化叶菌病毒dUTPase基因的表达及酶学活性分析

对硫化叶菌病毒(Sulfolobus virus)STSV2脱氧尿苷焦磷酸酶(dUTPase)基因进行克隆表达,并分析其酶学特征.根据STSV2的dUTPase基因序列设计特异性引物,以病毒STSV2 DNA为模板进行PCR扩增,将产物克隆至原核表达载体pET32a(+)中,然后,将其转化至Escherichia coli BL21(DE3)进行dUTPase的IPTG诱导表达及纯化,通过SDS-PAGE检测其大小,并测定其酶学活性.结果表明,诱导表达的重组dUTPase蛋白分子量(Mr)约为38×103(含pET32a(+)自带的助溶标签),重组dUTPase在Mg2+存在的情况下能特异性催化dUTP生成dUMP和PPi,其反应最适温度为60℃,在pH值3-8之间有较高的活性,其在提高PCR扩增效率和特异性方面具有一定的作用.本研究通过克隆表达获得了硫化叶菌病毒(Sulfolobus virus)STSV2脱氧尿苷焦磷酸酶,其在基因扩增领域有一定的应用价值.图5表1参18     

一个硫化叶菌病毒启动子的分离与鉴定

几乎所有古菌病毒基因组中无RNA聚合酶(RNA polymerase,RNAP)等组成基本转录装置的同源蛋白编码序列,而且启动子活性对病毒感染过程中病毒基因的转录上可能具有重要的影响.为进一步揭示古菌病毒基因启动子的序列结构特点和活性之间的关系,首先基于硫化叶菌质粒pSeSD,将β-半乳糖苷酶编码基因lacS克隆到阿拉伯糖启动子araS下游多克隆位点,构建重组表达载体pSeSD-lacS.将pSeSD-lacS转化冰岛硫化叶菌(Sulfolobus islandicus)E233S菌株后的功能分析结果表明,lacS基因成功表达.在此基础上,利用硫化叶菌病毒STSV2衣壳蛋白编码基因ORF37上游500bp的潜在启动子片段P37替换pSeSD-lacS中的araS启动子,构建出新的重组表达质粒pSeSD-P37-lacS,进一步将pSeSD-P37-lacS转化E233S菌株进行启动子活性分析.β-半乳糖苷酶酶活结果显示,诱导后araS启动子酶活为14 345.7±422.3 mU,P37酶活为13 723.1±370.9 mU,表明P37片段具有启动子功能,而且活性与araS启动子相当.序列分析也显示,P37具有与硫化叶菌基因启动子类似的基础序列元件initiator、TATA-box及BRE等.本研究表明pSeSD-lacS可作为一个硫化叶菌病毒基因启动子筛选载体,而且高活性的基因启动子可能在STSV2病毒生命过程具有重要的作用.(图4表1参27)     

一株产胞外多糖的山药内生细菌Lysinibacillus fusiformis S-1的分离和鉴定

微生物的胞外多糖是重要的生物资源,为获得新型的具有药用价值的胞外多糖产生菌,从山药、地瓜、马铃薯和胡萝卜的根茎组织中分离、筛选到11株能产胞外多糖的植物内生菌,利用苯酚–硫酸法对这11株菌的多糖产量进行了定量分析.对多糖产量最高的S-1菌株,检测其在发酵过程中菌体生长、胞外多糖生成以及发酵过程的pH变化,绘制其胞外多糖发酵代谢曲线.通过形态观察、培养特性观察、生理生化实验和16S rDNA序列分析对该菌株进行了鉴定.结果显示,S-1菌株在产糖培养基中可以产生1.50 g/L的胞外多糖,在11株菌中产量最高.16S rDNA序列分析显示该菌属于赖氨酸芽孢杆菌(Lysinibacillus),且与纺锤形赖氨酸芽孢杆菌(L.fusiformis)亲缘最近.综合其形态特征、培养特性和生理生化实验结果,将S-1菌株鉴定为纺锤形赖氨酸芽孢杆菌.该菌在胞外多糖产生菌中少有报道,为胞外多糖的进一步研究提供了菌种资源.     

不同宿主植物根瘤Frankia及其生物学特性

对从木麻黄、杨梅、桤木和胡颓子等宿主植物根瘤中获得的19株分离菌的离体培养形态特征、生理特性和交叉侵染特性等进行了比较分析.结果表明,各分离菌均有Frankia属所特有的分枝状菌丝、孢囊、泡囊或串珠状菌丝等形态结构;细胞壁类型多属胞壁Ⅲ型,生理类型多属B型,无氮诱导培养下都具有同氮酶活性,在BAP、JA或S培养液中菌体生长较好,以吐温-80和酪蛋白水解物为最佳碳、氮源.但不同宿主分离菌的形态和培养特征差异明显,木麻黄属分离菌菌丝较粗,孢囊数量较少,在BAP培养液中多旱荔肉白絮状颗粒沉淀;杨梅属菌丝较细,孢囊数量较多,在BAP培养液中多是浅红色颗粒沉淀;桤木属和胡颓子属的菌体形态特征在BAP培养液中与木麻黄属的相似,但桤木属菌丝较细,胡颓子属的菌丝较粗.根据回接及交叉侵染特性可将分离菌分为2个宿主特异类群:能侵染木麻黄苗木的木麻黄类群;只侵染原宿主并能在杨梅属、胡颓子属和桤木属间相互侵染,但不能使木麻黄属苗木结瘤的杨梅-桤木-胡颓子类群.图2表5参19     

一株嗜碱产甲烷八叠球菌的分离鉴定与系统发育分析

在15℃条件下用产甲烷菌培养基对采自四川省红原县的牦牛粪进行富集培养,采用Hungate厌氧操作技术从富集培养物中分离得到一株在8~45℃范围生长、最适生长pH为8.5的嗜碱产甲烷菌T13.该菌株革兰氏染色阳性,细胞聚集体,在液体培养基中为肉眼可见的颗粒状物,在固体培养基上菌落为淡黄色桑葚状;可利用甲醇、乙酸盐和甲胺作为唯一碳源生长;对氯霉素和庆大霉素敏感;生长pH范围为6.5~9.5;最适NaCl浓度为0~0.15 mol L-1;最适生长温度为30℃.形态和生理生化特征以及16S rDNA序列分析表明菌株T13为梅氏产甲烷八叠球菌(Methanosarcina mazei).由于该菌最适生长pH为8.5,所以初步认为菌株T13是一株梅氏产甲烷八叠球菌的新菌株.     

一株降解苄嘧磺隆光合细菌的分离鉴定及其降解特性

从农药厂工业废水和污泥中富集分离到一株能降解苄嘧磺隆(Bensulfuron methyl)的光合细菌PSB07-6,根据分离菌株的细胞形态结构、活细胞光吸收特征、生理生化特征以及系统发育分析将该菌初步鉴定为沼泽红假单胞菌(Rhodopseudomonas palustris).高效液相色谱法(HPLC)测定该菌降解光合细菌培养基中苄嘧磺隆的能力,在pH为6.5的光合细菌培养基中培养5 d,对350 mg·L-1苄嘧磺隆降解率达25.03%.添加回收率为105%～112%.降解特性研究结果表明,该菌能以苄嘧磺隆为唯一碳源和氮源,降解最佳条件为30℃、pH6.5.     

叶菌唑对斑马鱼的急性毒性及生物富集效应

为评价杀菌剂叶菌唑对水生生态系统的影响,以斑马鱼(Brachydanio rerio)为试验生物,采用半静态法研究了叶菌唑原药对斑马鱼的急性毒性以及生物富集效应。结果表明,95%叶菌唑原药对斑马鱼96 h的半数致死浓度(96 h-LC_(50))为3.89 mg·L~(-1),选用在3.90×10~(-1)和3. 90×10~(-2)mg·L~(-1)这2个处理浓度下连续暴露8 d,在3. 90×10~(-1)mg·L~(-1)浓度下生物富集系数(BCF)为53.3;在3.90×10~(-2)mg·L~(-1)浓度暴露下生物富集系数(BCF8d)为26.2。根据《化学农药环境安全评价试验准则》毒性划分标准,叶菌唑原药属于中等富集性农药。研究结果为叶菌唑田间安全使用提供理论依据。     

3株高温蛋白酶产生菌的分离与鉴定

从云南温泉的污泥样品中分离到3株产蛋白酶的高温菌SB11、SB31和SC5,通过其形态特征、生理生化特征和16S rDNA序列比对分析等,初步鉴定这3株菌都属于土芽孢杆菌属(Geobacillus),其最适生长温度为60℃,最适pH为6.三者的蛋白酶活力分别可达35.6、26.1和26.6UmL-1,最适酶反应温度都在70℃以上,其中菌株SB31的蛋白酶其最适酶反应温度高达80℃,远高于一般动植物来源的蛋白酶.图4表1参11     

柞蚕多角体病毒的纯化和鉴定

对柞蚕多角体病毒(ApNPV)的多角体颗粒进行了分离和纯化,并在显微镜下进行了形态观察．提取和纯化了柞蚕多角体病毒的基因组DNA,对DNA进行了部分限制性内切酶图谱鉴定．结果表明,在ApNPV基因组中只含少量的EcoRI位点,与家蚕核型多角体病毒差别很大,并提示这两种病毒的复制机制可能不同．图3参5。     

利用硫杆菌淋滤电镀污泥中的重金属(英文)

从广东云浮硫铁矿矿山酸性废水中分离得一株氧化亚铁硫杆菌(Thiobacillus ferrooxdans),从新兴县含硫化氢温泉中分离得一株氧化硫硫杆菌 (Thiobacillus thiooxidans), 并将其应用于电镀污泥的生物淋滤的研究,该污泥含铬、镍及铜分别为80.74 g/kg自然风干污泥、37.34g/kg自然风干污泥和13.42 g/kg自然风干污泥.利用未经驯化的氧化亚铁硫杆菌和氧化硫硫杆菌,以底物类型、菌液比、初始pH值和污泥浓度为四个因素,设置3个水平进行L9(34)正交实验,得出电镀污泥的最适淋滤条件:污泥浓度0.1%、硫酸亚铁和硫代硫酸钠作底物、菌液比T.f:T.t=:1:1、初始pH值2.0.为了将分离得的两种硫杆菌应用于更高浓度的电镀污泥,对所得菌进行驯化,以提高该菌种的重金属耐性,并应用于高浓度污泥的生物淋滤.经过驯化后的细菌,成功地适应了电镀污泥环境.结合上述最适条件,加入了合适的营养底物及还原剂之后,电镀污泥中的重金属,如铜、镍等,在30℃酸性好氧的环境中,能够在7天内被有效地淋滤出来,混合菌对污泥浓度为0.5%的污泥中铜的滤出率达90%以上,镍的滤出率40%以上;对1%的污泥的淋滤效果较差;对铬的淋滤效果不明显.本文还讨论了两株硫杆菌对高毒性电镀污泥的适应性.即在一定的污泥浓度范围内,氧化亚铁硫杆菌和氧化硫硫杆菌能够氧化底物,并产生硫酸将污泥中的重金属溶解,两株硫杆菌能够加快镍和铜的滤出效率.     

Dental fluorosis associated with drinking water from hot springs in Choma district in southern province,Zambia

This study was conducted to investigate the high incidence of mottled teeth among residents of an area with hot springs in the Choma District of the Southern Province of Zambia. A survey involving 128 pupils was conducted at a Basic School to collect data on pupil’s backgrounds and their main sources of drinking water between birth and age 7. A dental specialist examined the pupils’ teeth and samples of drinking water were collected from locations where the majority of the pupils lived. It was analysed for fluorides and other drinking water quality parameters. Results of the survey showed a highly significant (P < 0.001) association between pupils’ main sources of drinking water between birth and age 7 and the incidence of discoloured teeth. All (100%) pupils who drank water from hot springs before age 7 had moderate to severe fluorosis, while the majority (96.7%) of the pupils who drank water from other sources had no dental fluorosis. Fluoride concentrations ranged from 5.95 to 10.09 mg/l in water from hot springs, and from 0.03 to 0.6 mg/l in water from other sources. Fluoride levels in water from hot spring water samples exceeded the 1.5 mg/l WHO guideline value for drinking water, while those in water from other sources were significantly (P < 0.05) lower than this. We conclude that the high prevalence of mottled teeth among residents of the study area is a case of endemic dental fluorosis associated with drinking water from hot springs containing high concentrations of fluoride.     

Hydrodynamic study of the functional morphology of the benthic suspension feeder Phoronopsis viridis (Phoronida)

Ambient water currents were altered by the morphology of an active suspension feeder, Phoronopsis viridis Hilton (phylum Phoronida), to produce a flow around its ciliated crown of feeding tentacles (lophophore). To test the effects of specific morphological characteristics on patterns of water movement, the morphology of model phoronids was varied and the resultant paths of water movement were compared to those around living phoronids. Living individuals were collected from the intertidal sandflats at Bodega Bay, California/USA, in the springs of 1984 and 1985. Although P. viridis actively produce a feeding current, use of various models demonstrated that the gross pattern of flow around a living phoronid was created by the physical interaction of its morphology with ambient currents. The important aspects of that morphology were the presence of a wide, porous crown of tentacles atop a cylindrically-shaped body. A hydrodynamic consequence of this morphology was that dye eroded off the substratum from a circular area around the base of the body and entrained upwards into the lophophore. In addition, rates of water movement were slowed at the lophophore and near the substratum adjacent to a phoronid, particles were slowed and diverted from horizontal paths immediately downstream of the lophophore, and the number of visible suspended particles within the wake per unit time increased with ambient velocity. Paths of water movement around a phoronid were also influenced by its angle and height relative to the substratum, indicating that P. viridis could behaviorally modify their local flow environment.     

Cd2+胁迫对螺旋藻生长、光谱特性及藻胆蛋白质量浓度的影响

Cd2 是造成环境污染的重金属元素之一,如何治理Cd2 污染一直是一个难题。螺旋藻(Spirulina)是一种营光合自养的蓝藻,由于其突出的营养和保健价值而被誉为21世纪最优秀的食品。高浓度的某些重金属,如Cu2 ,Mn2 ,Zn2 等会抑制螺旋藻的生长并在藻细胞内富集,但有关Cd2 对螺旋藻的影响则未见相关报道。采用室内培养法,研究了不同质量浓度(0~25mg/L)Cd2 对钝顶螺旋藻突变株(SP-Dz)的生长、光谱特性及藻胆蛋白质量浓度的影响。结果表明,不同质量浓度的Cd2 处理均会抑制螺旋藻的生长,随着Cd2 质量浓度的增加,抑制作用增强。螺旋藻藻细胞有三个主要吸收峰,分别在440nm(叶绿素a在蓝紫光区的吸收峰),626nm(藻蓝蛋白的吸收峰)和676nm(别藻蓝蛋白和叶绿素a在红橙光区的吸收峰)。Cd2 处理会使藻细胞3个主要吸收峰中的两个发生红移,分别由440nm红移到448nm,676nm红移到684nm。Cd2 会使藻胆蛋白中藻蓝蛋白的吸收峰由626nm红移到630nm,而对别藻蓝蛋白在680nm的吸收峰没有明显影响。随着Cd2 质量浓度的增加,藻胆蛋白质量浓度下降。     

云南高黎贡山白颌大角蟾的核型,C—带及Ag—NORs的研究

比较了云南高黎贡山地区的贡山独龙江和腾冲大蒿坪白颌大角蟾（Megophryslateralis）两个地理种群的核型、C-带和Ag-NORs结果表明,两个地理种群在核型和带型上都有差异两个地理种群的核型均为2n＝26,NF＝52染色体形态差异不明显,而次缢痕的位置完全不同,贡山独龙江标本的次缢痕位于No．2的长臂上近着丝点处,腾冲标本的次缢痕位于No．5的短臂上近着丝点的部位在腾冲标本中发现一雄性个体中有一条额外的染色体,可能是B染色体两地标本的C-带差异不太显著,贡山独龙江标本的C-带相对较为显著．贡山独龙江标本的Ag-NORs位于No．2长臂近着丝点处,与次缢痕的部位对应,两条同源染色体上大小有显著差异．腾冲标本的Ag-NORs位于No．5短臂上近着丝点处,与次缢痕的部位对应依据核型和带型的比较,对白颌大角蟾的分类和进化问题进行了讨论。     

云南,西藏热泉中主要微生物类群及其金成矿作用的研究

从地质微生物生态学的角度研究和阐述了云南和西藏热泉中主要微生物类群及其金成矿作用．发现了热泉中存在的嗜热好氧菌优势菌属为并孢杆菌（Racillus）,放线菌中的优势菌属为链霉菌（Streptomyces）,真菌中优势菌属为曲霉（Aspergillus）,厌氧菌中优势菌为硫酸盐还原菌的脱硫肠状菌属（Desulfotomaculum）和核状芽孢杆菌属（Clostridium）,对吸附聚金、沉淀金的功能菌进行的研究发现．嗜热细菌对溶解金有明显的吸附聚金作用,表明原溶液金大多富集于细胞体;并以芽孢杆菌属K12－1菌株等进行了吸附聚金条件试验．     

Unique aluminosilicate-based natural nanoparticles in the volcanogenic Goshiki-numa pond

We report here the occurrence of uniquely shaped nanoparticles newly discovered in natural ponds. Nanoparticles originate from the Goshiki-numa pond community in Japan, where volcanic activity facilitated the formation of four specific ponds. We built a steric three-dimensional image of nanoparticles by integrating 120 transmission-electron-microscope image fragments obtained from various angle ranges. The thick-walled, cylindrically shaped particle has an outer diameter that measures approximately 40 nm and a length that measures 70 nm. A 10-nm-thick wall surrounds a 30 nm hole located in the particle centre. Particles are composed of an aluminium silicate-based material with an Al₂O₃:SiO₂ ratio of 2:1. They also exhibit an amorphous X-ray diffraction pattern. Although the water solubility characteristics and the infrared spectrum of these newly discovered particles resembles imogolite, these two materials do not have identical structural characteristics.     

Are meiofaunal species cosmopolitan? Morphological and molecular analysis of Xenotrichula intermedia (Gastrotricha: Chaetonotida)

Many meiofaunal species are reported to be cosmopolitan, but due to uncertainties of identification, the affiliation of specimens from geographically distant areas to the same species-taxon is problematic. In this study, we examined morphological and molecular variation in samples of Xenotrichula intermedia Remane (Gastrotricha: Chaetonotida) from the Mediterranean Sea, the northwestern Atlantic and the northern Gulf of Mexico. Univariate analysis of 16 morphological traits was unable to detect differences among populations, except for the length of the pharynx, which was significantly shorter in the Gulf of Mexico specimens. Canonical discriminant analysis separated the Gulf of Mexico specimens from the other two populations, with pharynx length contributing about half of the total discrimination. Molecular analysis based on restriction-fragment length polymorphisms (RFLPs) in a 710-base pair polymerase chain-reaction (PCR) produet representing roughly half of the mitochondrial cytochrome oxidase I (COI) gene detected four haplotypes: one each from the Mediterranean and the Gulf of Mexico populations and two coexisting within the Atlantic population. The estimated nucleotide-sequence divergence calculated for each pairwise combination of haplotypes (based on the proportion of shared fragments) ranged from 5.3 to 11.5%. The high genetic divergence and the inability to clearly separate populations based on morphology suggest that individuals characterized by different haplotypes are genetically isolated sibling species.     

不同粒径矿化垃圾对大麦生长发育和斑马鱼胚胎毒性效应研究

将太原东山新沟垃圾填埋场矿化垃圾筛分为900~300μm、300~150μm、150~105μm、105~90μm和90~0μm共5个不同粒径范围,制备浸出液。在分析不同粒径矿化垃圾浸出液的理化指标基础上,研究了矿化垃圾浸出液对大麦和斑马鱼胚胎生长发育的影响。结果显示,矿化垃圾浸出液p H值随粒径的减小而减小,电导率、CODCr、全盐量和总氮则随粒径的减小而增大。矿化垃圾浸出液对大麦的萌发、根长和芽长均表现出抑制作用,且抑制作用随粒径的减小而显著增加,90~0μm抑制作用最强,具体表现为染毒1 d、2 d和3 d后,大麦种子处理组萌发率为对照组的39.74%、56.38%和59.81%;染毒3 d、5 d和7 d后,根长分别为对照组的57.08%、48.33%和41.66%;芽长分别为对照组的66.60%、64.79%和61.65%。斑马鱼胚胎暴露于不同粒径的矿化垃圾浸出液中,胚胎生长发育的毒害作用与粒径大小呈现明显负相关关系,即在最小粒径90~0μm处理后,卵凝结、血循环异常、孵化率、心包水肿及脊柱畸形影响作用达到最大,这5个指标值分别为40.28%、50.00%、51.39%、31.94%和29.17%。以上研究表明,矿化垃圾浸出液对大麦和斑马鱼胚胎生长发育的毒性效应与矿化垃圾粒径分布显著相关,小粒径矿化垃圾(90~0μm)的生态毒性效应最大。