Barley aleurone layer cell protoplasts as a transient expression system

Protoplasts were prepared from barley aleurone layers using Onozuka cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of -amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley -amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormoneresponsive elements in hydrolase genes.     

Influence of plant development and environment on transgene expression in potato and consequences for insect resistance

Clonal replicates of different transformed potato plants expressing transgene constructs containing the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter, and sequences encoding the plant defensive proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA), and bean chitinase (BCH) were propagated in tissue culture. Plants were grown to maturity, at first under controlled environmental conditions, and later in the glasshouse. For a given transgene product, protein accumulation was found to vary between the different lines of clonal replicates (where each line was derived from a single primary transformant plant), as expected. However, variability was also found to exist within each line of clonal replicates, comparable to the variation of mean expression levels observed between the different clonal lines. Levels of GNA, accumulated in different parts of a transgenic potato plant, also showed variation but to a lesser extent than plant–plant variation in expression. With the majority of the clonal lines investigated, accumulation of the transgene product was found to increase as the potato plant developed, with maximum levels found in mature plants. The variation in accumulation of GNA among transgenic plants within a line of clonal replicates was exploited to demonstrate that the enhanced resistance towards larvae of the tomato moth, Lacanobia oleracea L., caused by expression of this protein in potato, was directly correlated with the level of GNA present in the plants, and that conditions under which the plants were grown affect the levels of GNA expression and subsequent levels of insect resistance.     

盐穗木金属硫蛋白HcMT的体外自由基清除活性及抗氧化能力*

目的: 原核表达盐穗木(Halostachys caspica C. A. Mey.)金属硫蛋白HcMT并探究其抗氧化活性。方法: 构建原核表达载体pET-32a-HcMT,转化至大肠杆菌Escherichia coli BL21,加入Zn²⁺胁迫培养(终浓度为200 μmol/L),分离纯化得到Zn-HcMT,测定Zn-HcMT自由基清除活性和总抗氧化能力,制备复合物Zn-HcMT/TiO₂并做FTIR表征。结果: 通过原核表达获得融合蛋白Zn-HcMT,对·OH、O₂·^-、DPPH自由基具有较强的清除活性,对·OH、O₂·^-的IC₅₀分别为0.386 mg/mL、0.038 mg/mL。融合蛋白浓度为0.01 mg/mL时,对DPPH清除率达(37.43 ± 0.006 8)%,浓度为0.3mg/mL时TEAC(trolox-equivalent antioxidant capacity)值为(1.023 ± 0.01)mmol/L,融合蛋白还原力A₇₀₀为0.142 ± 0.055,FTIR图谱同时表现了Zn-HcMT和TiO₂吸收特性。结论: Zn-HcMT具有良好的清除ROS活性及较强的抗氧化能力,在化妆品领域有潜在应用前景。     

The emerging role of mass spectrometry-based proteomics in molecular pharming practices

Molecular pharming relies on the integration of foreign genes into a plant system for production of the desired recombinant protein. The speed, scalability, and lack of contaminating human pathogens highlights plants as an enticing and feasible system to produce diverse protein-based products, including vaccines, antibodies, and enzymes. However, limitations of expression levels, host defense responses, and production irregularities underscore distinct areas for improvement within the molecular pharming pipeline. Within the past five years, mass spectrometry-based proteomics has begun to address these critical areas and show promise in advancing our understanding of the complex biological systems driving molecular pharming. Further, opportunities to leverage comprehensive proteome profiling have surfaced to meet good manufacturing practice regulations and move biopharmaceuticals derived from plants into mainstream production.     

Genotype‐dependent regulation of vitamin E biosynthesis in olive fruits as revealed through metabolic and transcriptional profiles

• Vitamin E is a general term used to describe a group of eight lipophilic compounds known as tocochromanols. These vitamin E variants are chemically categorised into two classes formed by α‐, β‐, γ‐ and δ‐ tocopherols and tocotrienols isoforms, respectively.
• The present study describes the concurrent regulation of genes and metabolites orchestrating vitamin E biosynthesis in olive drupes of five distinctive Greek olive cultivars. A combination of analytical, biochemical and molecular approaches was employed in order to carry out comparative analyses, including real‐time RT‐qPCR for gene expression levels and HPLC analysis of metabolite content.
• Findings indicated that tocochromanol levels and composition, oil content, gene expression levels as well as total antioxidant activity were highly dependent on cultivar and, to a lesser extent, on fruit developmental stage. Specifically, cultivars ‘Kalokairida’ and ‘Lianolia Kerkyras’ demonstrated the highest vitamin E content. The latter possessed high tocochromanol content combined with highest overall antioxidant activity in all developmental stages, concomitant with the up‐regulation expression profile of HPPD.
• The genotypic imprint versus the temporal contribution to vitamin E levels, as well as the potential link to lipid peroxidation amelioration, are discussed.

     

Complex patterns of dopamine‐related gene expression in the ventral tegmental area of male zebra finches relate to dyadic interactions with long‐term female partners

Dopaminergic projections from the ventral tegmental area (VTA) to multiple efferent targets are implicated in pair bonding, yet the role of the VTA in the maintenance of long‐term pair bonds is not well characterized. Complex interactions between numerous neuromodulators modify activity in the VTA, suggesting that individual differences in patterns of gene expression in this region may explain individual differences in long‐term social interactions in bonded pairs. To test this hypothesis we used RNA‐seq to measure expression of over 8000 annotated genes in male zebra finches in established male‐female pairs. Weighted gene co‐expression network analysis identified a gene module that contained numerous dopamine‐related genes with TH found to be the most connected gene of the module. Genes in this module related to male agonistic behaviors as well as bonding‐related behaviors produced by female partners. Unsupervised learning approaches identified two groups of males that differed with respect to expression of numerous genes. Enrichment analyses showed that many dopamine‐related genes and modulators differed between these groups, including dopamine receptors, synthetic and degradative enzymes, the avian dopamine transporter and several GABA‐ and glutamate‐related genes. Many of the bonding‐related behaviors closely associated with VTA gene expression in the two male groups were produced by the male's partner, rather than the male himself. Collectively, results highlight numerous candidate genes in the VTA that can be explored in future studies and raise the possibility that the molecular/genetic organization of the VTA may be strongly shaped by a social partner and/or the strength of the pair bond.     

用杆状病毒-昆虫细胞系统表达、纯化重组的人源化氨基末端LBP

根据已有的资料显示LBP与LPS的结合位点位于其氨基末端的第 91～ 1 0 0个氨基酸残基。在体外构建含有人LBP与LPS结合活性部分的穿梭质粒pBacmidtLBP ,并且带上 6×his的标签 ,用此穿梭质粒转染sf2 1昆虫细胞 ,获得重组病毒。然后用重组病毒液感染对数生长期的sf2 1昆虫细胞 ,72h后收获含有这种截断型人源化氨基末端LBP-(NH-LBP)的培养上清。用金属亲和树脂纯化后 ,采用SDS-PAGE电泳以及Western-Blot鉴定所得到的纯化物。成功获得相对分子质量约为 30 0 0 0的NH-LBP。为进一步研究LBP在介导LPS活化靶细胞中的作用机制奠定了基础。