Identification of Coffee Genes Expressed During Systemic Acquired Resistance and Incompatible Interaction with Hemileia vastatrix

Coffee genes associated with systemic acquired resistance (SAR) and incompatible reaction against coffee leaf rust inoculation were identified by suppression subtractive hybridization. Analysis of 384 clones of each of the subtracted cDNA libraries identified genes involved in oxidative burst/apoptosis/hypersensitive response, synthesis of antimicrobial proteins, synthesis and transport of antimicrobial metabolites, signal perception and transduction, metabolism of lipids, regulated protein degradation and cell maintenance and development. Induction of distinct sets of genes in the two resistance responses was observed. A wide range of genes involved in defence responses described in other plant species was also found in coffee plants. Semi-quantitative and quantitative RT-PCR analysis of seven selected genes showed differences in their expression profile within 72 h after treatment. Full-length cDNA sequences of two β-1,3-glucanases, one induced during SAR and the other in the incompatible reaction, were obtained by 5' and 3' RACE and the sequence data suggest different properties and cellular localization of the encoded proteins.     

A phosphorylation code of the Aspergillus nidulans global regulator VelvetA (VeA) determines specific functions

The velvet protein VeA is a global fungal regulator for morphogenetic pathways as well as for the control of secondary metabolism. It is found exclusively in filamentous fungi, where it fulfills conserved, but also unique functions in different species. The involvement of VeA in various morphogenetic and metabolic pathways is probably due to spatially and timely controlled specific protein–protein interactions with other regulators such as phytochrome (FphA) or velvet‐like proteins (VelB). Here we present evidence that Aspergillus nidulans VeA is a multi‐phosphorylated protein and hypothesize that at least four specific amino acids (T167, T170, S183 and Y254) undergo reversible phosphorylation to trigger development and sterigmatocystin biosynthesis. Double mutation of T167 to valine and T170 to glutamic acid exerted the largest effects with regards to sexual development and veA gene expression. In comparison with wild‐type VeA, which shuttles out of the nuclei after illumination this VeA variant showed stronger nuclear accumulation than the wild type, independent of the light conditions. The interaction between VeA and VelB or FphA, respectively, was affected in the T167V‐T170E mutant. Our results suggest complex regulation of the phosphorylation status of the VeA protein.     

Metabolic alteration of the N-glycan structure of a protein from patients with a heterozygous protein deficiency

Glycosylation, an important post-translation modification, could alter biological activity or influence the clearance rates of glycoproteins. We report here the first example of a heterozygous protein deficiency leading to metabolic alteration of N-glycan structures in residual secreted protein. Analysis of C1 esterase inhibitor (C1INH) glycans from normal individuals and patients with hereditary deficiency of C1INH demonstrated identical O-glycan structures but the N-glycans of patients with a heterozygous genetic deficiency were small, highly charged and lacked sialidase releasable N-acetylneuraminic acid. Structural studies indicate that the charge character of these aberrant N-glycan structures may result from the presence of mannose-6-phosphate residues. These residues might facilitate secretion of C1INH through an alternate lysosomal pathway, possibly serving as a compensatory mechanism to enhance plasma levels of C1INH in these deficient patients.     

In toto differentiation of human amniotic membrane towards the Schwann cell lineage

Human amniotic membrane (hAM) is a tissue containing cells with proven stem cell properties. In its decellularized form it has been successfully applied as nerve conduit biomaterial to improve peripheral nerve regeneration in injury models. We hypothesize that viable hAM without prior cell isolation can be differentiated towards the Schwann cell lineage to generate a possible alternative to commonly applied tissue engineering materials for nerve regeneration. For in vitro Schwann cell differentiation, biopsies of hAM of 8 mm diameter were incubated with a sequential order of neuronal induction and growth factors for 21 days and characterized for cellular viability and the typical glial markers glial fibrillary acidic protein (GFAP), S100β, p75 and neurotrophic tyrosine kinase receptor (NTRK) using immunohistology. The secretion of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was quantified by ELISA. The hAM maintained high viability, especially under differentiation conditions (90.2 % ± 41.6 day 14; 80.0 % ± 44.5 day 21 compared to day 0). Both, BDNF and GDNF secretion was up-regulated upon differentiation. The fresh membrane stained positive for GFAP and p75 and NTRK, which was strongly increased after culture in differentiation conditions. Especially the epithelial layer within the membrane exhibited a change in morphology upon differentiation forming a multi-layered epithelium with intense accumulations of the marker proteins. However, S100β was expressed at equal levels and equal distribution in fresh and cultured hAM conditions. Viable hAM may be a promising alternative to present formulations used for peripheral nerve regeneration.     

2000-2019年长江流域植被覆盖时空演化及其驱动因素

长江流域作为中国重要的生态屏障,科学认识长江流域植被覆盖时空变化及其驱动因素,对有效开展长江流域生态工程建设具有重要的指导意义与应用价值。基于2000—2019年间MODIS-NDVI与相关气象等数据,采用Theil-Sen median趋势分析、Mann-Kendall检验、变异系数、偏相关分析、残差分析等方法,研究了近20年来长江流域植被覆盖的时空分布与变化特征,并探究了研究区植被覆盖的驱动因素。结果表明：(1)时间变化上,长江流域生长季NDVI呈现波动增长趋势,显著改善面积大于退化面积;(2)空间分布上,流域植被覆盖空间分布格局大致呈现为“中部高,东西低”,生长季NDVI多年均值为0.6164,呈较高植被覆盖状态;(3)变化趋势上,植被增长区域大于减少区域,具体表现为“中部强于东部、东部强于西部”;(4)变化稳定性上,流域植被变异系数介于0.0104—1.3199之间,呈现出“中间低,东西高,东西部局部区域高低波动并存,地域性差异明显”的空间变化稳定性特征;(5)影响因素上,流域植被覆盖变化受气温和降水的共同影响,大部分区域生长季NDVI变化以气候驱动为主,局部区域表现自然因素叠...     

‘凤丹’牡丹PoKAS基因的克隆及表达分析

为探究‘凤丹’牡丹(Paeonia ostii‘Feng Dan’)PoKAS基因在脂肪酸合成中的功能,从转录组数据中获得3个PoKAS基因,克隆基因全长并进行生物信息学分析,通过qRT-PCR检测它们在牡丹落花后第23、45、75、100和125天时的表达。结果显示：(1)克隆得到的3个基因序列全长分别为1 401、1 692和1 215 bp,分别编码466、563和404 aa;保守结构域分析发现,它们都含有KAS保守结构域,属于cond-enzymes超蛋白家族。(2)系统进化树将三者分为三大类,表明其在进化上相对独立,分别命名为PoKASⅠ、PoKASⅡ和PoKASⅢ(GenBank登录号分别为OP056413、OP056412和OP056414)。(3)qRT-PCR分析发现,在牡丹落花后种子发育的5个时期中,PoKASⅠ和PoKASⅡ基因在落花后75 d和45 d时的表达量分别显著高于其他发育时期;PoKASⅢ基因在落花后45～125 d时的表达量均显著高于落花后23 d,说明PoKASⅢ基因在牡丹种子脂肪酸合成的整个过程中发挥着重要作用,而PoKASⅡ基因主要在种子油脂...     

水稻品种次生物质与稻白叶枯病(Xanthomonas oryzae pv. oryzae)抗性的关系

应用高效液相色谱分析了对白叶枯病具有不同抗性水平的12个水稻品种中的19个(组)次生物质色谱峰(面积)的差异及其与白叶枯病抗性水平间关系.结果表明,水稻品种抗性水平与谱峰面积值之间相关极显著(R=0.992,p<0.01),被测的19个组分中,峰1、峰2、峰8、峰10、峰12、峰14、峰16和峰18是影响水稻对稻白叶枯病抗性水平的主要抗原次生物质.建立了水稻品种对白叶枯病抗性级别与以上次生物质含量谱峰面积之间的回归模型:Y=10.7603 0.1823X1-0.2287X2 0.2163X8-2.1975X10 0.0728X12 -0.7438X14 1.1484X16-0.7795X18.研究结果表明水稻品种中起抗病作用的抗原次生物质不止一种,而是几种的组合,而且它们对水稻抗病性的贡献作用是不完全相同的,这与它们的性质与含量密切相关.提出了以抗原次生物质为标记的快速分析、鉴定、预测水稻品种对稻白叶枯病抗性水平的新途径、新方法.     

体外重构STUB1介导α-1抗胰蛋白酶突变体Z蛋白泛素化修饰反应体系

α-1抗胰蛋白酶Z型突变体蛋白(α-1 antitrypsin Z-mutant protein, ATZ)是引发α-1抗胰蛋白酶缺陷症(α-1 antitrypsin deficiency, AATD)的主要原因,研究ATZ蛋白的泛素化修饰和降解对于治疗AATD具有重要意义。STUB1是一种重要的E3泛素连接酶,参与调节多种蛋白质的泛素化修饰。然而,STUB1是否参与ATZ的泛素化修饰尚未明确。本研究首先将ATZ和STUB1的编码基因克隆到pET28a质粒,构建了这2个蛋白的表达质粒。随后,将重组质粒转入大肠杆菌表达系统,在优化诱导条件实现了重组蛋白的异源表达。通过金属螯合亲和层析技术纯化得到目的蛋白,并通过蛋白质谱分析验证了其氨基酸序列的准确性。利用纯化的ATZ和STUB1重组蛋白,构建了一个体外泛素化修饰反应体系。实验结果显示,在ATP、E1泛素激活酶和E2泛素结合酶的协同作用下,STUB1成功催化了ATZ的泛素化修饰。本研究提供了一种体外获得Z型突变体ATZ纯化蛋白的方法,并确认了STUB1介导ATZ的泛素化修饰功能,推进了对α-1抗胰蛋白酶Z型突变体蛋白在细胞内降解过程的调控机制的理解。     

Elektronenmikroskopische Darstellung,Längenverteilung und Konfiguration einsträngiger Desoxyribonucleinsäure im Cytochrom-c-Film

Zusammenfassung Durch Alkali nachFreifelder undDavison einsträngig gemachte DNA aus Forellengonaden wird mittels Spreitung einer DNA/Protein-Lösung elektronenmikroskopisch dargestellt. Ihre Konfiguration, gemessen durch das mittlere Quadrat der Abstände der Molekülenden als Funktion der Konturenlänge, ist flexibler als bei zweisträngiger DNA unter gleichen Bedingungen, weicht aber wie diese infolge ihres Polyelektrolytcharakters vom statistischen Knäuel merklich ab. Die exponentielle Verteilung der Konturenlängen wird als statistische Verteilung der Bruchstellen gedeutet.

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Summary Single-stranded DNA is visualized by electron microscopy after the denaturation procedure by alkali (Freifelder andDavison) and by spreading to a mixed DNA-protein film. The configuration is measured by the mean squares of end-to-end distances as a function of contour lengths. Single-stranded DNA is more flexible than double-stranded DNA under the same conditions but like native DNA differs markedly from the random coil, probably because of electrostatic interactions along the polyelectrolyte molecule. The exponential distribution of the contour lengths is explained as a random distribution of breaks in single strands similar to these of native DNA.

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Wir danken Herrn Prof. Dr. K.Herzberg für die Unterstützung dieser Arbeit, Frau C.Plescher sowie Frl. I.Hortenbach für technische Mitarbeit und der Deutschen Forschungsgemeinschaft für Beihilfen.