马克斯克鲁维酵母GX-UN120糖转运蛋白基因的克隆及表征

【背景】马克斯克鲁维酵母(Kluyveromyces marxianus)具有完整的木糖代谢途径,可以高效利用木质纤维素中的木糖,因此对其糖转运蛋白基因的研究或可有效解决酵母木糖转运的相关问题。【目的】根据马克斯克鲁维酵母DMKU3-1042中KLMA＿70145和KLMA＿80101基因位点的功能预测,获得马克斯克鲁维酵母GX-UN120相应的糖转运蛋白基因序列并探究其功能。【方法】将转运蛋白基因分别克隆表达至酿酒酵母EBY.VW4000中考察重组菌株生长特性,以此间接评价对应转运蛋白的转运能力。【结果】Km＿SUT2基因编码的糖转运蛋白可有效提高宿主细胞转运木糖、阿拉伯糖、山梨糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖、果糖、蔗糖和半乳糖。类似地,Km＿SUT3基因编码的糖转运蛋白可提高细胞转运木糖、阿拉伯糖、山梨糖、半乳糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖和果糖。然而在葡萄糖存在的条件下,重组菌株对各种碳源的利用均受抑制,但Km＿SUT3转运木糖和核糖过程中受葡萄糖的抑制作用较小。【结论】马克斯克鲁维酵母GX-UN120中转运蛋白Km＿SUT2和Km＿SUT3可...     

地衣芽胞杆菌FLP/FRT基因编辑系统的构建及验证

地衣芽胞杆菌有效的基因编辑工具有限,为了拓展和丰富其基因编辑手段,在地衣芽胞杆菌中构建一个抗性标记可重复使用的FLP/FRT基因编辑系统,并通过敲除α-淀粉酶基因amyL、蛋白酶基因aprE及敲入外源透明颤菌血红蛋白基因vgb对该系统进行初步验证。首先以温敏质粒pNZT1为载体分别构建amyL和aprE基因敲除质粒pNZTT-AFKF和pNZTT-EFKF,两个敲除质粒各自包含针对目标基因的同源臂、抗性基因及同向的FRT位点;将敲除质粒转化地衣芽胞杆菌并经过两次同源交换过程实现目标基因的敲除;最后导入一个FLP重组酶表达质粒通过FLP/FRT系统的重组作用介导抗性基因的回收。为进一步验证本系统的实用性及编辑效率,构建了包含透明颤菌血红蛋白编码基因vgb表达盒及基因组丙酮酸甲酸裂解酶编码基因pflB敲除盒的重组质粒pNZTK-PFTF-vgb,并以此进行外源基因vgb在基因组上的定向敲入。结果显示,成功敲除amyL及aprE并回收了抗性标记卡那霉素基因,敲除后淀粉酶活和蛋白酶活分别减少95.3%和80.4%;vgb基因成功整合入基因组pflB位点并回收了抗性标记四环素基因,并利用荧光定量PCR技术检测到vgb的整合表达。文中首次建立了一个适用于地衣芽胞杆菌的、抗性标记可重复使用的FLP/FRT基因编辑系统,并进行了基因敲除及基因敲入验证,为地衣芽胞杆菌遗传改造提供了良好的方法参考。     

一株产常温葡甘聚糖酶的芽孢杆菌的分离、鉴定及产酶发酵条件的研究

以魔芋生粉为唯一碳源的筛选培养基,从魔芋废渣中分离筛选出一株可以降解魔芋生粉的细菌菌株,编号为LS-6。根据16SrDNA序列和生理生化特性,将该菌株鉴定为枯草芽孢杆菌Bacillus subtilis。胞内外葡甘露聚糖酶酶活分析结果表明,LS-6产生的葡甘聚糖酶为胞外酶。LS-6产生葡甘聚糖酶的最佳培养基组分和条件是:1%魔芋生粉,0.5%酵母粉,0.5%NaCl,培养基初始pH为6.0,最佳发酵条件是25℃,摇床转速200r/min,培养48h。酶解产物的高效液相色谱分析结果表明,LS-6粗酶液的酶解产物主要为葡萄糖和甘露糖。LS-6的分离为进一步克隆常温葡甘聚糖酶基因和产酶工程菌的构建奠定了基础。     

几种控释氮肥减少氨挥发的效果及影响因素研究

采用“静态吸收法”和“土柱淋溶法”、室内模拟试验,研究几种控释氮肥施入土壤后的氨挥发损失情况、N溶出速率、土壤脲酶活性及pH值变化的关系．结果表明,施氮450mg·kg^-1土时,3种控释氮肥氨挥发损失氮总量分别比普通尿素减少49．7％、28．0％和71．2％;施氮600mg·kg^-1土时,3种控释氮肥氨挥发损失氮总量分别比普通尿素减少34．6％、12．3％和69．9％．控释氮肥能显著降低土壤氨挥发量,减少因施肥而引起的大气环境污染．控释氮肥氨挥发量与不同氮肥引起的土壤脲酶活性、pH值、土壤中氮溶出速率密切相关．土壤的氨挥发总量与肥料在土壤中溶出总量的相关系数达到0．9533,在肥料施入的前期土壤氨挥发量同土壤脲酶活性、pH值的相关系数达到0．9533和0．9908。     

广州地区晚稻田CH4和N2O的排放通量及其影响因素

用密闭箱法同时研究了广州地区晚稻田CH4和N2 O的排放通量。结果表明 ,连续淹水、常规连作和水旱轮作等 3种处理的CH4平均排放通量分别为 1 7 63、2 84和0 36mg·m- 2 ·h- 1 ,而N2 O的平均排放通量分别为 6 74、1 1 69和 55 0 7μgN2 O N·m- 2 ·h- 1 ,表明稻田连续淹水显著增加CH4的排放而降低N2 O的排放。水旱轮作降低CH4排放而提高N2 O的排放 ,说明稻田CH4和N2 O排放之间存在着消长关系。讨论了这 2种温室气体排放的影响因素 ,并初步分析了它们对温室效应的相对贡献。     

基于ITS和ndhF-rpl32序列的荞麦种间亲缘关系分析

荞麦起源于我国, 演化形成了丰富的物种和遗传多样性。为了有效研究和利用荞麦及其野生种资源, 以从四川、甘肃、贵州等地采集的荞麦属(Fagopyrum)10个种(含变种、亚种和复合体种)共71份材料为对象, 通过ITS和叶绿体ndhF-rpl32序列分析, 利用MEGA5.0构建系统进化树, 探讨了荞麦种内及种间亲缘关系。结果表明, 在ITS序列矩阵中, 序列长度为725 bp, 信息位点为150个, 占序列总长度的20.7%; 在ndhF-rpl32序列矩阵中, 序列长度为940 bp, 信息位点为158个, 占序列总长度的16.8%。由ITS序列和ndhF-rpl32序列构建的两个进化树都可以将71份材料分为大粒荞麦种组和小粒荞麦种组; 其中, 大粒荞麦种组包括栽培苦荞和米苦荞(F. tataricum)、金荞复合体(F. cymosum complex)、栽培甜荞(F. esculentum)和野生甜荞(F. esculentum ssp. ancestralis); 小粒荞麦种组包括齿翅野荞(F. gracilipes var. odontopterum)、疏穗小野荞(F. leptopodum var. grossii)、小野荞(F. leptopodum)、密毛野荞(F. densovillosum)、细柄野荞(F. gracilipes)和硬枝万年荞(F. urophyllum)。而ndhF-rpl32序列构建的系统发育树还能区分栽培甜荞和野生甜荞, 具有更好的聚类效果。另外, 与栽培甜荞相比, 金荞复合体与栽培苦荞的亲缘关系更近。该研究为荞麦属种的分类和条形码研究提供了一定的科学依据。     

Magnetophoretic position detection for multiplexed immunoassay using colored microspheres in a microchannel

This paper demonstrates a new magnetophoretic position detection method for multiplexed immunoassay using colored microspheres as an encoding tool in a microchannel. Colored microspheres conjugated with respective capture molecules are incubated with a mixture of target analytes, followed by reaction with the probe molecules which had been conjugated with superparamagnetic nanoparticles (SMNPs). Under the magnetic field gradient, the resulting microspheres are deflected from their focused streamlines in a microchannel, and respective colored microspheres are detected using color charge-coupled device (CCD) in a specific detection region of the microchannel. The color and position of respective colored microspheres are automatically decoded and analyzed by MATLAB program, and the position was correlated with the concentration of corresponding target analytes. As a proof-of-concept, we attempted to assay simultaneously three types of biotinylated immunoglobuline Gs (IgGs), such as goat, rabbit and mouse IgGs, using colored microspheres (red, yellow and blue, respectively). As the capture molecules, corresponding anti-IgGs were employed and target analytes were probed using streptavidin-modified superparamagnetic nanoparticles. As a result, three analytes were simultaneously assayed using colored microspheres with high accuracy, and detection limits of goat IgG, rabbit IgG and mouse IgG were estimated to be 10.9, 30.6 and 12.1fM, respectively. In addition, with adjustment of the flow rate and detection zone, the dynamic range could be controlled by more than one order of magnitude.     

不同土壤水盐条件下多枝柽柳(Tamarix ramosissima)对胡杨(Populus euphratica)幼苗的影响

目前胡杨更新困难,种群处于退化阶段,而与其生态位高度重叠的多枝柽柳却在扩张。为探讨多枝柽柳对胡杨生长的影响,本研究设置了3个水平的水分、盐分梯度,对胡杨进行了单种和混种的盆栽控制实验,通过测定胡杨幼苗的生长和存活状况,分析不同水、盐梯度下多枝柽柳如何影响胡杨生长。结果表明：（1）多枝柽柳伴生降低当年生胡杨幼苗的存活率,随着水分条件改善,胡杨存活率提高,而盐分对存活率没有显著的影响。（2）水分、盐分和伴生模式几个因子对胡杨的生长特征的影响存在显著的交互作用（P<0.05）。各水盐条件下,多枝柽柳会不同程度降低胡杨幼苗的株高、主根长和地上地下生物量,而根冠比增大。混种条件下,胡杨幼苗对水盐的响应更为敏感。（3）相对竞争强度对水盐环境有明显的响应（P<0.05）,随水分条件改善,地上、地下相对竞争强度降低。多枝柽柳对胡杨的生长产生不良影响的机制是通过快速消耗土壤的水分,从而导致胡杨幼苗水分亏缺。土壤水分条件是胡杨和多枝柽柳幼苗共存的关键因素,在水分供给不足的情况下,对水分偏好的差异导致胡杨无法通过实生苗进行有效的更新从而加剧胡杨种群的衰退,而多枝柽柳更为耐旱的特性使其逐步占据河岸的生境。我们的研究结果强调了两个树种对环境因子的适应性差异决定了河岸带植被发育过程中植物-土壤的相互作用导致胡杨在演替过程中表现出的衰退现象。     

Glycyrrhizin protects against sodium iodate‐induced RPE and retinal injury though activation of AKT and Nrf2/HO‐1 pathway

Glycyrrhizin is a bioactive triterpenoid saponin extracted from a traditional Chinese medicinal herb, glycyrrhiza, and has been reported to protect the organs such as liver and heart from injuries. However, there is no report about the effects of glycyrrhizin on atrophic age‐related macular degeneration (AMD). This study investigated the effects of glycyrrhizin on retinal pigment epithelium (RPE) in vitro and retina of mice in vivo treated with sodium iodate (SI). Glycyrrhizin significantly inhibited SI‐induced reactive oxygen species (ROS), and decreased apoptosis of RPE in vitro. The underlying mechanisms included increased phosphorylation of Akt, and increased expression of nuclear factor erythroid 2‐related factor2 (Nrf‐2) and HO‐1, thereby protecting RPE from SI‐induced ROS and apoptosis. Furthermore, glycyrrhizin significantly decreased the apoptosis of retinal cells in vivo, resulting in the inhibition of thinning of retina, decreasing the number of drusen and improving the function of retina. These findings suggested that glycyrrhizin may be a potential candidate for the treatment of atrophic AMD in clinical practice.     

Recombinant microsatellite amplification: a rapid method for developing simple sequence repeat markers

Recombinant microsatellite (simple sequence repeat; SSR) amplification is a technique by which DNA sequences flanking microsatellites can be isolated rapidly on a large scale. The approach selectively amplifies microsatellite-containing sequences and recombines the amplicons by redigestion and ligation, in order to increase the yield of microsatellite flanks per clone two-fold and to further increase selectivity of amplification. Since this method does not require prior knowledge of the genomic sequence, it is especially useful for species for which abundant genomic sequences are not available. The feasibility of this approach was demonstrated by developing SSR markers in cultivated oats.