毕赤酵母发酵生产颗粒性乙肝表面抗原的条件优化

乙型肝炎病毒的流行对人们的生命健康造成了极大的威胁, 而有效准确的诊断和预防性疫苗是阻止其流行的主要手段, 乙肝表面抗原是诊断试剂和疫苗的主要成分。本试验在构建稳定表达HBsAg的毕赤酵母菌株后, 对其发酵条件进行了研究。采用摇瓶分批培养方法, 探讨了不同培养基、溶解氧、诱导物甲醇的浓度以及pH值等因素对菌体生长与重组蛋白表达的影响。在10 L发酵罐上采用分批补料培养的方法研究了进行扩大培养生产重组HBsAg。结果表明, FBS无机盐合成培养基是理想的工业发酵培养基, 溶解氧对菌体的生长与表达有显著的影响, 甲醇诱导最佳终浓度为1% (V/V), 发酵的最适pH值为5.4~6.0。发酵罐放大培养后, ELISA和 SDS-PAGE分析表明重组HBsAg获得了高效表达, 最终菌体生物量达到310 OD600, 表达量达到27 mg/L。电子显微镜观察表达重组乙肝抗原可以自组装为22 nm类病毒颗粒, 为HBV的新一代早期血清学诊断和疫苗的大规模生产提供了一定的参考。     

不同生境草鱼肠道微生物组成和群落特征分析

[目的]分析不同生境来源的草鱼前肠、中肠和后肠的微生物组成和群落特征.[方法]利用16S rRNA高通量测序技术比较河流、湖泊、高密度池塘养殖与水库低密度养殖4种不同生境来源的草鱼其前、中、后肠的微生物组成和群落特征.[结果]Venn图、稀释性曲线和Alpha指数分析结果显示,前肠微生物群落多样性以养殖生境草鱼更高,而...     

陕北黄土高原蒿属植物的分类与分析

陕北黄土高原有蒿属植物30种1变种,居该地产种子植物属中所含种数的首位。所产蒿属植物在不同的植被带中梯度变化明显,替代现象显著。在生态类型上,旱生类型从南向北递增,中生类型从南向北递减。在区系组成上可分为6种分布区类型,即:我国特有分布,3种;温带亚洲分布,14种1变种;北温带及中亚分布各4种;旧世界温带分布,3种;东亚分布的2种。可见陕北黄土高原蒿属植物种类丰富,梯度变化明显,旱化现象显著,地理成分复杂,但以温带亚洲分布类型为主,兼有其它成分,属典型的温带性质。     

Cutting edge: monovalency of CD28 maintains the antigen dependence of T cell costimulatory responses

CD28 and CTLA-4 are the major costimulatory receptors on naive T cells. But it is not clear why CD28 is monovalent whereas CTLA-4 is bivalent for their shared ligands CD80/86. We generated bivalent CD28 constructs by fusing the extracellular domains of CTLA-4 or CD80 with the intracellular domains of CD28. Bivalent or monovalent CD28 constructs were ligated with recombinant ligands with or without TCR coligation. Monovalent CD28 ligation did not induce responses unless the TCR was coligated. By contrast, bivalent CD28 ligation induced responses in the absence of TCR engagement. To extend these findings to primary cells, we used novel superagonistic and conventional CD28 Abs. Superagonistic Ab D665, but not conventional Ab E18, predominantly ligates CD28 bivalently at low CD28/Ab ratios and induces Ag-independent T cell proliferation. Monovalency of CD28 for its natural ligands is thus essential to provide costimulation without inducing responses in the absence of TCR engagement.     

厌氧氨氧化菌富集培养物对羟胺的转化研究

【目的】羟胺是厌氧氨氧化的重要中间产物,本研究旨在探明厌氧氨氧化菌对羟胺的转化特性。【方法】采用厌氧氨氧化菌富集培养物,以羟胺和亚硝酸盐为基质进行分批培养试验,检测反应液中基质和产物的消涨情况。【结果】不接种厌氧氨氧化富集培养物时,羟胺和亚硝酸盐具有化学稳定性,彼此不发生化学反应;接种厌氧氨氧化富集培养物后,羟胺和亚硝酸盐发生化学反应;反应过程中有中间产物氨的产生和转化,最大氨氮积累浓度为0.338mmol/L;液相中总氮浓度从起始的4.694mmol/L降至结束时的0.812mmol/L,转化率为82.7%。羟胺和亚硝氮浓度均为2.5mmol/L时,羟胺最大比污泥转化速率为0.535mmol/(gVSS.h),是厌氧氨氧化反应体系中氨氮最大比污泥转化速率的1.81倍。将羟胺浓度提高至5.0mmol/L时,羟胺和亚硝氮转化速率分别提高26.7%和120.7%,最大氨氮积累浓度为0.795mmol/L;将亚硝氮浓度提高至5.0mmol/L时,羟胺和亚硝氮转化速率分别提高6.9%和9.0%,最大氨氮积累浓度为1.810mmol/L。【结论】厌氧氨氧化富集培养物能够转化羟胺,其对羟胺的转化速率高于对氨的转化速率。羟胺相对过量可显著加快羟胺和亚硝酸盐的转化速率,亚硝酸盐相对过量对羟胺和亚硝氮转化速率影响不大,提高羟胺或亚硝氮浓度均会增大中间产物氨氮的积累。实验现象可用van de Graaf模型解释,对于进一步开发厌氧氨氧化工艺具有重要的理论意义。     

Imputation of missing genotypes from low‐ to high‐density SNP panel in different population designs

Imputation of missing genotypes, in particular from low density to high density, is an important issue in genomic selection and genome‐wide association studies. Given the marker densities, the most important factors affecting imputation accuracy are the size of the reference population and the relationship between individuals in the reference (genotyped with high‐density panel) and study (genotyped with low‐density panel) populations. In this study, we investigated the imputation accuracies when the reference population (genotyped with Illumina BovineSNP50 SNP panel) contained sires, halfsibs, or both sires and halfsibs of the individuals in the study population (genotyped with Illumina BovineLD SNP panel) using three imputation programs (fimpute v2.2, findhap v2, and beagle v3.3.2). Two criteria, correlation between true and imputed genotypes and missing rate after imputation, were used to evaluate the performance of the three programs in different scenarios. Our results showed that fimpute performed the best in all cases, with correlations from 0.921 to 0.978 when imputing from sires to their daughters or between halfsibs. In general, the accuracies of imputing between halfsibs or from sires to their daughters were higher than were those imputing between non‐halfsibs or from sires to non‐daughters. Including both sires and halfsibs in the reference population did not improve the imputation performance in comparison with when only including halfsibs in the reference population for all the three programs.     

Imaging Cell Wall Architecture in Single Zinnia elegans Tracheary Elements

The chemical and structural organization of the plant cell wall was examined in Zinnia elegans tracheary elements (TEs), which specialize by developing prominent secondary wall thickenings underlying the primary wall during xylogenesis in vitro. Three imaging platforms were used in conjunction with chemical extraction of wall components to investigate the composition and structure of single Zinnia TEs. Using fluorescence microscopy with a green fluorescent protein-tagged Clostridium thermocellum family 3 carbohydrate-binding module specific for crystalline cellulose, we found that cellulose accessibility and binding in TEs increased significantly following an acidified chlorite treatment. Examination of chemical composition by synchrotron radiation-based Fourier-transform infrared spectromicroscopy indicated a loss of lignin and a modest loss of other polysaccharides in treated TEs. Atomic force microscopy was used to extensively characterize the topography of cell wall surfaces in TEs, revealing an outer granular matrix covering the underlying meshwork of cellulose fibrils. The internal organization of TEs was determined using secondary wall fragments generated by sonication. Atomic force microscopy revealed that the resulting rings, spirals, and reticulate structures were composed of fibrils arranged in parallel. Based on these combined results, we generated an architectural model of Zinnia TEs composed of three layers: an outermost granular layer, a middle primary wall composed of a meshwork of cellulose fibrils, and inner secondary wall thickenings containing parallel cellulose fibrils. In addition to insights in plant biology, studies using Zinnia TEs could prove especially productive in assessing cell wall responses to enzymatic and microbial degradation, thus aiding current efforts in lignocellulosic biofuel production.The organization and molecular architecture of plant cell walls represent some of the most challenging problems in plant biology. Although much is known about general aspects of assembly and biosynthesis of the plant cell wall, the detailed three-dimensional molecular cell wall structure remains poorly understood. The highly complex and dynamic nature of the plant cell wall has perhaps limited the generation of such detailed structural models. This information is pivotal for the successful implementation of novel approaches for conversion of biomass to liquid biofuels, given that one of the critical processing steps in biomass conversion involves systematic deconstruction of cell walls. Therefore, a comprehensive understanding of the architecture and chemical composition of the plant cell wall will not only help develop molecular-scale models, but will also help improve the efficiency of biomass deconstruction.The composition and molecular organization of the cell wall is species and cell type dependent (Vorwerk et al., 2004). Thus, the development of a model plant system, which utilizes a single cell type, has enhanced our capacity to understand cell wall architecture. The ability to generate a population of single Zinnia elegans plant cells that were synchronized throughout cell wall deposition during xylogenesis was developed in the 1980s (Fukuda and Komamine, 1980). Mesophyll cells isolated from the leaves of Zinnia and cultured in the presence of phytohormones will transdifferentiate into tracheary elements (TEs), which are individual components of the xylem vascular tissue (Fukuda and Komamine, 1980). During this transdifferentiation process, TEs gradually develop patterned secondary wall thickenings, commonly achieving annular, spiral, reticulate, scalariform, and pitted patterns (Bierhorst, 1960; Falconer and Seagull, 1988; Roberts and Haigler, 1994). These secondary wall thickenings serve as structural reinforcements that add strength and rigidity to prevent the collapse of the xylem under the high pressure created by fluid transport. During the final stages of transdifferentiation, TEs accumulate lignin in their secondary walls and undergo programmed cell death, which results in the removal of all cell contents, leaving behind a “functional corpse” (Roberts and McCann, 2000; Fukuda, 2004).In broad terms, the primary cell wall of higher plants is mainly composed of three types of polysaccharides: cellulose, hemicelluloses, and pectins (Cosgrove, 2005). Cellulose is composed of unbranched β-1,4-Glc chains that are packed together into fibrils by intermolecular and intramolecular hydrogen bonding. Hemicelluloses and pectins are groups of complex polysaccharides that are primarily composed of xyloglucans/xylans and galacturonans, respectively. Hemicelluloses are involved in cross-linking and associating with cellulose microfibrils, while pectins control wall porosity and help bind neighboring cells together. The patterned deposits of secondary wall in Zinnia TEs primarily consist of cellulose microfibrils, along with hemicelluloses, and also lignin, a complex aromatic polymer that is characteristic of secondary walls and provides reinforcement (Turner et al., 2007). All the molecular components in the cell wall correspond to a multitude of different polysaccharides, phenolic compounds, and proteins that become arranged and modified in muro, yielding a structure of great strength and resistance to degradation.Currently, electron microscopy is the primary tool for structural studies of cell walls and has provided remarkable information regarding wall organization. Fast-freeze deep-etch electron microscopy in combination with chemical and enzymatic approaches have generated recent models of the architecture of the primary wall (McCann et al., 1990; Carpita and Gibeaut, 1993; Nakashima et al., 1997; Fujino et al., 2000; Somerville et al., 2004). Direct visualization of secondary wall organization has been focused toward the examination of multiple wall layers in wood cells (Fahlen and Salmen, 2005; Zimmermann et al., 2006). However, few studies have examined the secondary wall, so our knowledge regarding the higher order architecture of this type of wall is limited. Over the past few decades, atomic force microscopy (AFM) has provided new opportunities to probe biological systems with spatial resolution similar to electron microscopy techniques (Kuznetsov et al., 1997; Muller et al., 1999), with additional ease of sample preparation and the capability to probe living native structures. AFM has been successfully applied to studies of the high-resolution architecture, assembly, and structural dynamics of a wide range of biological systems (Hoh et al., 1991; Crawford et al., 2001; Malkin et al., 2003; Plomp et al., 2007), thus enabling the observation of the ultrastructure of the plant cell wall, which is of particular interest to us (Kirby et al., 1996; Morris et al., 1997; Davies and Harris, 2003; Yan et al., 2004; Ding and Himmel, 2006).To generate more detailed structural models, knowledge about the structural organization of the cell wall can be combined with spatial information about chemical composition. Instead of utilizing chromatography techniques to analyze cell wall composition by extracting material from bulk plant samples (Mellerowicz et al., 2001; Pauly and Keegstra, 2008), Fourier transform infrared (FTIR) spectromicroscopy can be used to directly probe for polysaccharide and aromatic molecules in native as well as treated plant material (Carpita et al., 2001; McCann et al., 2001). FTIR spectromicroscopy is not only able to identify chemical components in a specific system but also can determine their distribution and relative abundance. This technique also improves the sensitivity and spatial resolution of cellular components without the derivatization needed by chemical analysis using chromatography. Polysaccharide-specific probes, such as carbohydrate-binding modules (CBMs), can also be used to understand the chemical composition of the plant cell wall. CBMs are noncatalytic protein domains existing in many glycoside hydrolases. Based on their binding specificities, CBMs are generally categorized into three groups: surface-binding CBMs specific for insoluble cellulose surfaces, chain-binding CBMs specific for single chains of polysaccharides, and end-binding CBMs specific for the ends of polysaccharides or oligosaccharides. A surface-binding CBM with high affinity for the planar faces of crystalline cellulose (Tormo et al., 1996; Lehtio et al., 2003) has been fluorescently labeled and used to label crystals as well as plant tissue (Ding et al., 2006; Porter et al., 2007; Liu et al., 2009; Xu et al., 2009). The binding capacity of the CBM family has been further exploited for the detection of different polysaccharides, such as xylans and glucans, and can thus be used for the characterization of plant cell wall composition (McCartney et al., 2004, 2006).In this study, we used a combination of AFM, synchrotron radiation-based (SR)-FTIR spectromicroscopy, and fluorescence microscopy using a cellulose-specific CBM to probe the cell wall of Zinnia TEs. The Zinnia TE culture system proved ideal for observing the structure and chemical composition of the cell wall because it comprises a single homogeneous cell type, representing a simpler system compared with plant tissues, which may contain multiple cell types. Zinnia TEs were also advantageous because they were analyzed individually, and population statistics were generated based on specific conditions. Furthermore, cultured Zinnia TEs were used for the consistent production of cell wall fragments for analysis of the organization of internal secondary wall structures. In summary, we have physically and chemically dissected Zinnia TEs using a combination of imaging techniques that revealed primary and secondary wall structures and enabled the reconstruction of TE cell wall architecture.     

A new phytoplasma associated with little leaf disease in azalea: multilocus sequence characterization reveals a distinct lineage within the aster yellows phytoplasma group

An azalea little leaf (AzLL) disease characterised by abnormally small leaves, yellowing and witches'‐broom growth symptoms was observed in suburban Kunming, southwest China. Transmission electron microscopic observations of single‐membrane‐bound, ovoid to spherical bodies in phloem sieve elements of diseased plants and detection of phytoplasma‐characteristic 16S rRNA gene sequence in DNA samples from diseased plants provided evidence linking the disease to infection by a phytoplasma. Results from restriction fragment length polymorphism, phylogenetic and comparative structural analyses of multiple genetic loci containing 16S rRNA, rpsS, rplV, rpsC and secY genes indicated that the AzLL phytoplasma represented a distinct, new 16Sr subgroup lineage, designated as 16SrI‐T, in the aster yellows phytoplasma group. The genotyping also revealed that the AzLL phytoplasma represented new rp and secY gene lineages [rp(I)‐P and secY(I)‐O, respectively]. Phylogenetic analyses of secY and rp gene sequences allowed clearer distinctions between AzLL and closely related strains than did analysis of 16S rDNA.