利用rep-PCR研究云南尼泊尔桤木根瘤内Frankia基因多样性

利用rep-PCR指纹技术对云南5个地区的尼泊尔桤木根瘤内Frankia菌基因多样性进行研究,实验发现Frankia菌呈现丰富的基因多样性.所有71个样品被分为11种不同的基因类型.除高黎贡山外,不同地域都有某种基因型占优势,显示Frankia菌基因型与地域有紧密关系.所观察的5个地区中,高黎贡山Frankia菌基因型种类最多,多样性指数最高,基因多样性最为丰富.结合高黎贡山地理资料及其它生物多样性相关研究,推测与高黎贡山尼泊尔桤木共生的Frankia可能作为种储备库,为其它地区的寄主植物提供了祖先菌株.图8表1参13     

真菌核酸的一种快速提取方法

在总结多种真菌DNA和RNA提取方法的基础上，发展了一种提取真菌核酸新方法，与其它方法相比，该方法具有快速、操作简便等特点，而且提取DNA和RNA步骤基本一致，用该方法提取的DNA完整、纯度高，可用于PR、限制性内切酶酶切和Southern杂交等分子生物学操作；提取RNA产量高，RNA无降解，纯度高，适用Northern和cDNA合成等分子生物学操作。     

活性污泥总DNA不同提取方法的比较

为了快速、简便和经济地获得活性污泥的总DNA,以用于分子生物学研究,采用5种不同的DNA提取方法提取活性污泥的总DNA,并通过提取的核酸总量、纯度、是否含有聚合酶链反应抑制剂等指标综合分析不同的提取方法对活性污泥总DNA提取效果的影响.结果表明:蛋白酶K-SDS-酚氯仿法和柱提取DNA试剂盒法提取活性污泥总DNA效果最好,提取DNA总量多,纯度高,可不经纯化直接进行PCR扩增反应。     

一种经济、简单的微生物基因组DNA的提取方法

获得一定浓度和纯度的DNA是进行分子生物学研究的基础。破解细胞壁与细胞膜是获得基因组DNA的前提，而蛋白质和核酸物质的分离是获得高质量DNA产物的关键。目前，主要采用的破壁方法有：冷冻研磨法、溶菌酶法、EDTA测等，这些方法一般采用复杂的裂解液体系，并借助蛋白酶K和RNA酶的帮助来获得高质量的抽提产物。由于细胞裂解体系不仅配制十分麻烦，而日部分药品有毒操作危险性大，此外部分药品及相关酶试剂价格昂贵。本文充分利用DNA在不同温度下自身可变性与复性的特性和在高盐与高温条件下蛋白质能够变性并沉淀。以无菌的SDS（c／c=20％）和NaCl（c/c=8％）的混合液作裂解体系，在沸水浴中破壁膜并使得部分的蛋白质变性和DNA变性并得到初步分离；随后在60℃和72℃水浴中使变性的DNA复性和重新凝聚，同时让RNA、蛋白质和细胞壁碎片等杂质降解或沉淀，从而获得高质量的DNA产物。     

底泥水体中适用于PCR的不同形态DNA的提取方法（Methods of Extraction Different Gene Types of Sediments and Water for PCR Amplification）

提出直接从环境河流底泥和表层水体中同时提取不同形态DNA(胞内、胞外、染色体和质粒DNA)的有效方法.利用该方法提取了海河典型区域底泥的胞外DNA和胞内DNA及水体中细菌染色体DNA和质粒DNA.结果表明,NaH2PO4提取的胞外DNA分子量大于1kb,提取率40%～72%,没有共提取胞内DNA.胞内DNA提取的分子量均大于23.1kb,细菌质粒DNA与染色体DNA同时分离.16SrDNA与sul2扩增验证提取方法可靠性.16SrDNA扩增结果显示所有胞外DNA呈阴性,胞内DNA呈阳性.质粒DNA和染色体DNA的16SrDNA扩增显示,染色体DNA结果显阳性,质粒DNA呈阴性,sul2的结果相反.     

活性污泥宏基因组芯片DNA的制备方法

宏基因组芯片的探针是通过构建环境DNA的宏基因组文库而获得的,为了获得理想的活性污泥宏基因组文库和宏基因组芯片探针,必须获得高质量的活性污泥DNA.本文作者采用8种不同的DNA提取方法提取活性污泥的总DNA,并通过比较DNA总量、纯度、完整性、是否含有PCR酶抑制剂、能否进行限制性内切酶酶切等指标来评价不同提取方法对活性污泥总DNA提取效果的影响.结果表明,溶菌酶-SDS-蛋白酶K-酚氯仿抽提处理活性污泥所提取的总DNA效果最好,提取的DNA总量多、纯度高,DNA片段大于23 kb,无需进一步纯化就可直接进行PCR扩增反应或SauA Ⅰ限制性内切酶酶切,但DNA如果需要EcoRⅠ、HindⅢ等限制性内切酶酶切,则需采用Roche公司琼脂糖凝胶回收试剂盒将DNA进一步回收纯化.图7表3参14     

硝酸盐对毛萼田菁茎瘤固氮的影响

ＫＮＯ３对毛萼田菁（Ｓｅｓｂａｎｉａｒｏｓｔｒａｔａ）茎瘤和根瘤固氮的抑制作用与瘤中积累，碳水化合物减少（特别是蔗糖）以及豆血红蛋白（ｌｇｈｅｍｏｇｌｏｂｉｎ，Ｌｂ）浓度减少和功能受阻相关．经ＫＮＯ３处理的植株的茎瘤类菌体的呼吸作用和固氮活性明显下降．从ＫＮＯ３处理的植株茎瘤中提取的类菌体悬液加上未经ＫＮＯ３处理的正常Ｌｂ可提高固氮活性和耐ｐＯ２；加５ｍｍｏｌ／ＬＫＮＯ２于正常的茎瘤类菌体悬液和Ｌｂ反应体系，引起固氮活性和耐ｐＯ２都降低，证明是Ｌｂ功能受阻的重要因素．施ＫＮＯ３可能还导致茎瘤Ｏ２扩散的阻力增加．     

应用全细胞蛋白SDS-PAGE分子标记技术验证含羞草根瘤菌的结瘤能力

对云南省热带及亚热带地区的含羞草根瘤菌进行了分离,选择其中40株菌为接种菌株,通过结瘤试验并采用全细胞蛋白SDS-PAGE分子标记方法研究了其结瘤能力.经过结瘤试验,发现除菌株SWF66075和SWF66093没有结瘤外,其它38株菌株均与含羞草植物结瘤.结瘤率为95%.从结瘤试验所获根瘤中,分离得到结瘤菌株,采用全细胞蛋白SDS-PAGE分子标记对结瘤菌株与接种菌株进行了比较研究.蛋白图谱及聚类分析显示,26株接种菌株与其结瘤菌株的全细胞蛋白分子图谱完全相同,在100%的相似水平上与其结瘤菌株聚在一起,说明宿主植物所形成的根瘤确系接种菌株侵入所致,因而可将这些菌株确认为根瘤菌菌株;而SWF66012、SWF66029、SWF66044和SWF66058等12株菌株的结瘤菌株与其各自接种菌株的全细胞蛋白图谱存在较大差异,推测这12株接种菌株与其结瘤菌株可能不是同一菌株,尚不能确定它们与含羞草植物的结瘤能力,这些菌株是否为根瘤菌菌株仍需进一步验证.研究结果表明,全细胞蛋向SDS-PAGE分子标记技术是一种快速、准确地验证根瘤菌结瘤能力的方法.该方法进一步完善了结瘤试验,并初步揭示了根瘤菌的竞争结瘤能力,适用于对大量根瘤内分离菌株进行根瘤菌的证实研究.图2参28     

水稻总DNA的快速制备

采用两种不同的简单方法,均不接触有毒的有机试剂,且不需液氮速冻研磨,快速地制备水稻总DNA,用于SSR、STS等检测,效果稳定可靠.其中TritonX-100法提取的DNA具有很好的完整性,可用于后续其他分子生物学操作;NaOH法提取的DNA虽然降解严重,但是及时用于PCR及相关分析同样可行.两种方法均可在短时间内处理大批量的样品,操作简单,效果可靠,适于对杂种后代进行遗传连锁分析和分子标记辅助选育时的单株检测.图2表1参8     

鹤望兰基因组DNA的提取方法

由于鹤望兰组织内含有大量多糖类及多酚类物质，严重干预DNA的抽提，利用常规的SDS法，CTAB法和高盐低pH值法都难以抽提出高质量的鹤望兰基因组DNA，本文报道了在进行若干预处理之后，再用常规的SDS，CTAB，SDS裂解的高盐低pH值法和CTAB裂解的高盐低pH值法提取基因组DNA的改进方法，根据外观，琼脂糖凝胶电泳检测，D260nm/D280nm比值的测定。限制性酶切反应，PCR扩增的结果表明，用改进方法提取的基因组DNA无论在纯度上还是在完整性上都比常规方法要好，其中改进SDS裂解的高盐低pH值法是提取鹤望兰基因组DNAS的最好方法。     

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     

固氮放线菌Frankia与放线菌根植物共生进化的研究进展

固氮放线菌Frankia是能够诱导大范围的放线菌根植物(actinorhizal plants)产生根瘤放线菌.放线菌根植物是植物受Frankia侵染后能够形成根瘤的植物[1].目前,它们之间的进化研究虽然取得了很大进展,但是仍然存在许多不清楚的东西.本文根据最近对植物和Frankia系统进化研究成果、根瘤中放线菌Frankia的多样性研究情况,综述放线菌根植物和共生Frankia进化的研究进展.     

电感耦合等离子体质谱法分析锰结核中的多种元素含量

本文使用岛津ICPMS-2030电感耦合等离子体质谱仪,采用微波消解法对锰结核样品进行前处理,测定了锰结核样品中32种元素含量.实验结果表明,各元素在1.0—100μg·L^-1范围内线性关系良好,相关系数均大于0.9998,检出限低,各元素检出限都在0.00004—0.73μg·g^-1之间.该方法操作简便、快速,测试结果准确,使用锰结核标准物质GBW07296进行验证,测定值与标准值基本吻合,该方法可适用于锰结核样品中多元素含量的测定.     

Fish Disease Monitoring in the Dutch Part of the North Sea in Relation to the Dumping of Waste From Titanium Dioxide Production

Gross pathologies were recorded in a total of 5942 individual common dab (Limanda limanda L.) at 5 sites in and around Dutch coastal waters, in spring, from 1986 to 1988. Two of these sites (an offshore dumping ground and an inshore site influenced by direct river discharge) received large quantities of diluted acids of titanium dioxide waste (TDW); the other three were selected as reference sites for comparison.

The main diseases recorded were epidermal hyperplasia/papilloma, lymphocystis, liver nodules (pre-neoplastic and neoplastic lesions), and infections caused by the protozoan Glugea sp. Frequencies of disease were analysed using a logit model. There was a consistently high prevalence of epidermal hyperplasia/papilloma in dab from the two sites that received TDW when compared to the other sites. However, no dear relationships were found between the prevalence of epidermal hyperplasia/papilloma and dumping-associated heavy metals or other relevant environmental and biological factors. No significant spatial trend was revealed for liver nodules, although there was a statistically significant association between the occurrence of epidermal hyperplasia/papilloma or of lymphocystis and that of liver nodules in individual fish. Prevalences of lymphocystis were usually higher at offshore sites than in inshore areas, while prevalences of Glugea showed the opposite trend.

Although at first sight the pattern of disease prevalence would appear to furnish a strong case for a cause-and-effect relationship between TDW and epidermal hyperplasia/papilloma, interpretation of the data is complicated by interference from riverine inputs, long-distance dispersion of discharged wastes, local hydrographic conditions, and possible local migration of dab. On the basis of present results, therefore, the possibility that discharges of TDW contributed to the occurrence of this disease cannot be proven or discounted.     

Microscopic structure of soil Fe-Mn nodules: environmental implication

The internal structure, iron and manganese distributions of Fe-Mn nodules from imperfectly drained soils of central Greece were studied by scanning electron microscopy (SEM) and elemental analysis. Nodules exhibited a differentiated structure with discontinuous concentric rings as expression of their redox history. Fe and Mn have shown distinct repartition and were distributed mainly in concentric bands. The microstructure and the arrangement of iron—manganese compounds reflect the periodical development and the relative slow accretion rates of the studied nodules. These properties provide ample opportunity for adsorption and isolation of heavy metals from the soil system.     

Stromatolitoid microbial nodules from Bermuda —a special micro habitat for meiofauna

In Bermuda, stromatolitoid microbial nodules are found in dense groups in seagrass beds and on subtidal sandy or rocky bottoms. They develop between January and the beginning of August and may reach 15 cm in diameter and 6 cm in height. Nodules grow on top of the sediment surface and consist of convex interwoven mats of the cyanobacterium Phormidium corium and fine sediment particles trapped in these mats. Nodules were studied in the field and laboratory (in 1989–1992) with respect to their structure, microbial community, sediment chemistry and associated meiofauna. Vertical profiles taken with microelectrodes showed steep gradients of oxygen and sulfide. While free oxygen was only detected in the upper 2 mm, the sulfide concentrations increased with depth and reached maximal 1250 moll^-1 inside the nodules. On the basis of microscopic observations and sediment chemistry, vertical sections through a nodule reveal four distinct layers: (A) an oxic green surface layer of growing cyanobacteria mats, (B) an anoxic yellow-white laminated layer of sediment particles, mucus and unpigmented cyanobacteria sheaths, (C) an anoxic sulfidic (sulfide <50 moll^-1) interface where colour changes from yellow to gray and (D) an anoxic sulfidic (sulfide can increase to 1250 moll^-1) gray core composed of sediment and decomposing cyanobacteria. The following meiofauna taxa were found in the nodules: Ciliata, Turbellaria, Gnathostomulida, Gastrotricha, Nematoda, Kinorhyncha, Polychaeta, Ostracoda and Harpacticoida. The distribution of the meiofauna was significantly different (p<0.05) within the four layers. The highest density of individuals was found in the sulfidic interface and core. Nematodes represented the dominant group in general. Thiobiotic organisms, such as the Gnathostomulida, Solenofilomorphidae, and Stilbonematinae were primarily found in the anoxic sulfidic layers of the nodules.     

White locoweed toxicity is facilitated by a fungal endophyte and nitrogen-fixing bacteria

Mutualistic interactions with fungal endophytes and dinitrogen-fixing bacteria are known to exert key biological influences on the host plant. The influence of a fungal endophyte alkaloid on the toxicity of a plant has been documented in Oxytropis sericea. Oxytropis sericea is a perennial legume responsible for livestock poisoning in western North America. Livestock poisoning is attributed to the alkaloid swainsonine, which is synthesized inside the plant by the fungal endophyte Embellisia sp. In this study, the ability of Oxytropis sericea to form a dinitrogen-fixing symbiosis with Rhizobium and the effects of this symbiosis on the production of swainsonine by Embellisia sp. were evaluated in a greenhouse environment. Seeds of O. sericea were grown in plastic containers. Twenty-week-old O. sericea seedlings were inoculated with four strains of Rhizobium. Twenty weeks after inoculation, plant growth and root nodulation by Rhizobium were measured. Dinitrogen fixation was confirmed using an acetylene reduction assay (ARA) on excised root nodules. Dry leaves were analyzed for swainsonine content. A second set of plants was treated with fungicide to evaluate the effect of reduced fungal endophyte infection on plant growth and swainsonine production. All inoculated plants produced indeterminate nodules. The ARA indicated that 98% of the excised nodules were fixing dinitrogen. Rhizobium-treated plants had greater swainsonine concentrations than the non-inoculated controls. Plants that established from seeds treated with fungicide had lower biomass than non-fungicide-treated controls and plants treated with foliar fungicide. Plants treated with foliar fungicide and the controls had greater swainsonine concentrations than the plants that received seed fungicide. This greenhouse study is the first report of nodulation and dinitrogen fixation in O. sericea. It also demonstrates that dinitrogen fixation increases the production of swainsonine in O. sericea plants infected with Embellisia sp. Results from this study suggest that dinitrogen fixation affects swainsonine production and has the potential to support the symbiosis between Embellisia sp. and O. sericea when soil nitrogen is limited. Oxytropis sericea competitiveness appears to be facilitated by an ability to simultaneously associate with Rhizobium and a fungal symbiont.