应用DGGE研究微生物群落时的常见问题分析

变性梯度凝胶电泳(DGGE)是通过核酸片段对微生物群落进行研究,可以监测未培养细菌及其功能基因,被广泛地应用于微生物群落多样性和动态分析,并成为微生物分子生态学研究中的重要手段之一。文中论述了DGGE操作过程中遇到的常见问题,并提出了相应的解决方法。全面分析了样品预处理过程和PCR扩增效果对DGGE分析的影响,探讨了DGGE图谱的优化过程和图谱分析方法,并对DGGE的应用前景进行了综述。     

一株产乙酰酯酶细菌筛选、鉴定及酶学性质研究

【目的】利用筛选培养基,从肉牛瘤胃液中分离筛选产乙酰酯酶的细菌菌株,并研究菌株的产酶特征。【方法】利用厌氧培养技术,以木质素为唯一碳源,筛选并驯化所得菌株。根据菌株16S rDNA序列分析、革兰氏染色、伊红美蓝培养基培养、甲基红试验和柠檬酸盐利用试验,鉴定菌株。采用对-硝基苯乙酯测定酶活力。【结果】筛选得到产乙酰酯酶活力较高细菌菌株RB1,初步鉴定为Escherichia coli。菌株RB1的生长曲线表明,0 42 h为菌株的延迟期,42 60 h为菌株的对数期,60 66 h为菌株的稳定期,66 86 h为菌株的衰亡期。菌株所产乙酰酯酶最适温度为40°C,最适pH为8.0,在最适温度与pH条件下,培养基中添加玉米秸秆粉,乙酰酯酶最高酶活力达到0.52 U/mL。【结论】筛选获得产乙酰酯酶的细菌菌株RB1,其乙酰酯酶活力高于已报道的菌株,是一株具有研究和应用潜力的产乙酰酯酶的菌株。     

92份棉花资源遗传多样性的SSR分析

选用均匀分布于棉花全基因组的132个SSR标记,对精选的92个棉花品种(系)进行分析,结果表明,共扩增出494个多态性条带,每个引物平均3.69个.等位基因变异的多态性信息含量(PIC)在0.010 0～0.871 4之间,平均为0.546 6.采用NTSYS-pc软件计算品种对间的相似系数, 92个品种之间的成对相似系数平均为0.729 2±0.135 0,变幅在0.411 8～0.957 5之间,其中陆地棉种内48.94%的品种对相似系数≤0.800 0,海岛棉种内50.91%的品种对相似系数≤0.800 0,表明此自然群体的遗传基础较为宽泛.根据Jaccard's相似系数所得的UPGMA聚类图和系谱追溯的结果显示,此群体中品种的选育过程复杂,血统来源多样,存在较高程度的遗传多样性.     

Apple Sucrose Transporter SUT1 and Sorbitol Transporter SOT6 Interact with Cytochrome b5 to Regulate Their Affinity for Substrate Sugars

Sugar transporters are central machineries to mediate cross-membrane transport of sugars into the cells, and sugar availability may serve as a signal to regulate the sugar transporters. However, the mechanisms of sugar transport regulation by signal sugar availability remain unclear in plant and animal cells. Here, we report that a sucrose transporter, MdSUT1, and a sorbitol transporter, MdSOT6, both localized to plasma membrane, were identified from apple (Malus domestica) fruit. Using a combination of the split-ubiquitin yeast two-hybrid, immunocoprecipitation, and bimolecular fluorescence complementation assays, the two distinct sugar transporters were shown to interact physically with an apple endoplasmic reticulum-anchored cytochrome b5 MdCYB5 in vitro and in vivo. In the yeast systems, the two different interaction complexes function to up-regulate the affinity of the sugar transporters, allowing cells to adapt to sugar starvation. An Arabidopsis (Arabidopsis thaliana) homolog of MdCYB5, AtCYB5-A, also interacts with the two sugar transporters and functions similarly. The point mutations leucine-73 → proline in MdSUT1 and leucine-117 → proline in MdSOT6, disrupting the bimolecular interactions but without significantly affecting the transporter activities, abolish the stimulating effects of the sugar transporter-cytochrome b5 complex on the affinity of the sugar transporters. However, the yeast (Saccharomyces cerevisiae) cytochrome b5 ScCYB5, an additional interacting partner of the two plant sugar transporters, has no function in the regulation of the sugar transporters, indicating that the observed biological functions in the yeast systems are specific to plant cytochrome b5s. These findings suggest a novel mechanism by which the plant cells tailor sugar uptake to the surrounding sugar availability.     

Water‐use efficiency in response to climate change: from leaf to ecosystem in a temperate steppe

Water‐use efficiency (WUE) has been recognized as an important characteristic of ecosystem productivity, which links carbon (C) and water cycling. However, little is known about how WUE responds to climate change at different scales. Here, we investigated WUE at leaf, canopy, and ecosystem levels under increased precipitation and warming from 2005 to 2008 in a temperate steppe in Northern China. We measured gross ecosystem productivity (GEP), net ecosystem CO₂ exchange (NEE), evapotranspiration (ET), evaporation (E), canopy transpiration (T_c), as well as leaf photosynthesis (P_max) and transpiration (T_l) of a dominant species to calculate canopy WUE (WUE_c=GEP/T), ecosystem WUE (WUE_gep=GEP/ET or WUE_nee=NEE/ET) and leaf WUE (WUE_l=P_max/T_l). The results showed that increased precipitation stimulated WUE_c, WUE_gep and WUE_nee by 17.1%, 10.2% and 12.6%, respectively, but decreased WUE_l by 27.4%. Climate warming reduced canopy and ecosystem WUE over the 4 years but did not affect leaf level WUE. Across the 4 years and the measured plots, canopy and ecosystem WUE linearly increased, but leaf level WUE of the dominant species linearly decreased with increasing precipitation. The differential responses of canopy/ecosystem WUE and leaf WUE to climate change suggest that caution should be taken when upscaling WUE from leaf to larger scales. Our findings will also facilitate mechanistic understanding of the C–water relationships across different organism levels and in projecting the effects of climate warming and shifting precipitation regimes on productivity in arid and semiarid ecosystems.     

微波条件下LiCl/DMAc溶解稻秆纤维素制备还原糖

利用氯化锂/N,N-二甲基乙酰胺(LiCl/DMAc)溶剂体系在微波控制条件下对稻秆纤维素进行溶解预处理以提高纤维素酶解糖化效率。考察了微波时间和微波强度对产糖量及还原糖转化率的影响。通过扫描电镜(SEM)和热重分析仪(TG)对预处理前后稻秆纤维素的微观形貌及热稳定性进行表征,并利用高效液相色谱仪(HPLC)对酶解糖液进行糖成分鉴定和含量分析。结果表明,微波加热能够有效促进LiCl/DMAc对稻秆纤维素的溶解。与原生稻秆相比,经微波-LiCl/DMAc法溶解后再生纤维素出现明显解聚,热分解温度由290℃降至220℃。在微波功率为385 W、加热溶解时间为7 min时,所得稻秆纤维素还原糖转化率由30.90%上升至98.67%;HPLC谱图表明,糖液中主要成分为葡萄糖和木糖,分别占所得还原糖总量的43.74%和48.55%。     

俄罗斯大果沙棘无性繁殖技术的研究

无性繁殖技术是培育优良品系的极好手段,通过对俄罗斯大果沙棘进行多因子多水平正交试验,总结出沙棘嫩枝扦插育苗的最佳实施方案。     

非人灵长类个体的迁移与扩散

迁移现象在群居动物中普遍存在、在非人灵长类中尤为突出。在非人灵长类中,大多数的迁移表现出强烈的雄性偏向性和雌性不进行迁移的形式。在一些少数的物种中,也存在雌雄双方都进行迁移以及雌性偏向性迁移而雄性不迁移的形式。群居种类、一夫一妻制种类、独居种类的迁移模式上各有特色且不尽相同,这是动物社群结构多样性的体现。驱赶和异性的吸引是推动个体迁移的两大动力,驱赶多发生在一雄多雌的社群中,异性吸引多发生在无亲缘关系的个体之间。个体迁移过程,是个体付出与收益的平衡。迁移不仅是非人灵长类动物生活史中的一个重要环节,同时在不同种群间个体基因交流上也有明显的作用。     

家蚕微孢子虫孢壁蛋白与其发芽的相关性

为了研究孢壁蛋白与家蚕微孢子虫发芽（孢原质弹出）的相关性,我们采用碳酸钾诱导微孢子虫体外发芽结合密度梯度离心的方法（简称GDGC法）,收集纯化发芽后的孢子空壳（简称孢壳）,对发芽液、纯化的孢壳及成熟孢子的孢壁蛋白组分进行了分析。结果表明：GDGC法可以获得高纯度孢壳,计算出其密度为1．130g/cm^3;与发芽前成熟孢子提取的孢壁蛋白相比,空孢壳可以提取到主要孢壁蛋白SWP32、SWP30、SWP25,同时发现SWP32、SWP25丰度有所降低;结合碳酸钾发芽液的蛋白电泳分析,发现孢壳上丰度降低的SWP32在发芽液蛋白样品中存在,LC—MS／MS数据分析也发现SWP32、SWP30、SWP25在碳酸钾处理液中都有存在;而用碳酸钾溶液处理冷冻孢子时,未观察到发芽现象,电泳结果显示此时K2CO,溶液中只有SWP30条带,说明在碳酸钾溶液诱导的发芽过程中SWP32和SWP25从孢壳上脱落可能与发芽相关而不是被碱性的碳酸钾溶解下来的[动物学报54（6）：1068—1074,2008]。     

核酸技术在立枯丝核菌分类与生态学研究中的应用

立枯丝核菌是一种传播广、危害大的土传病原菌,对其展开的研究已涉及各个方面并逐渐深入,近几年借助于核酸技术对其所展开的研究更成为热点。对核酸技术在立枯丝核菌的分类学研究(主要包括G Cmol%含量测定、核酸杂交、各种DNA指纹技术、特异PCR扩增、测序DNA的序列分析)、监测群体动态变化的生态学研究(实时荧光PCR技术)中的应用作一简要的介绍。