转Bar基因甘蓝型油菜叶片蛋白质组变化的初步分析

采取SDS-PAGE与MALDI-TOF-MS联用的方法,对抗除草剂转Bar基因T1代甘蓝型油菜与普通栽培油菜的叶片蛋白质组进行比较性研究,获得差异蛋白质组的重要信息,并初步探讨差异蛋白的主要功能,以期找到与转Bar基因油菜抗除草剂有关的蛋白质,揭示其抗性机理.双向电泳表达图谱研究表明,Bar基因的转入使得转基因油菜中的差异蛋白表达质与量发生了显著变化,共得到16个发生差异表达的蛋白质点,其中11个经质谱分析功能得到鉴定,这些鉴定出的蛋白质涉及多个生理过程,如能量与代谢、信号转导、代谢相关蛋白离子转运和防御应答等.     

Efficient Gene Silencing Mediated by Tobacco Rattle Virus in an Emerging Model Plant Physalis

The fruit of Physalis has a berry and a novelty called inflated calyx syndrome (ICS, also named the ‘Chinese lantern’). Elucidation of the underlying developmental mechanisms of fruit diversity demands an efficient gene functional inference platform. Here, we tested the application of the tobacco rattle virus (TRV)-mediated gene-silencing system in Physalis floridana. First, we characterized the putative gene of a phytoene desaturase in P. floridana (PfPDS). Infecting the leaves of the Physalis seedlings with the PfPDS-TRV vector resulted in a bleached plant, including the developing leaves, floral organs, ICS, berry, and seed. These results indicated that a local VIGS treatment can efficiently induce a systemic mutated phenotype. qRT-PCR analyses revealed that the bleaching extent correlated to the mRNA reduction of the endogenous PfPDS. Detailed comparisons of multiple infiltration and growth protocols allowed us to determine the optimal methodologies for VIGS manipulation in Physalis. We subsequently utilized this optimized VIGS methodology to downregulate the expression of two MADS-box genes, MPF2 and MPF3, and compared the resulting effects with gene-downregulation mediated by RNA interference (RNAi) methods. The VIGS-mediated gene knockdown plants were found to resemble the mutated phenotypes of floral calyx, fruiting calyx and pollen maturation of the RNAi transgenic plants for both MPF2 and MPF3. Moreover, the two MADS-box genes were appeared to have a novel role in the pedicel development in P. floridana. The major advantage of VIGS-based gene knockdown lies in practical aspects of saving time and easy manipulation as compared to the RNAi. Despite the lack of heritability and mosaic mutation phenotypes observed in some organs, the TRV-mediated gene silencing system provides an alternative efficient way to infer gene function in various developmental processes in Physalis, thus facilitating understanding of the genetic basis of the evolution and development of the morphological diversities within the Solanaceae.     

β‐catenin regulates IRF3‐mediated innate immune signalling in colorectal cancer

Objective

β‐catenin is one of the most critical oncogenes associated with many kinds of human cancers, especially in the human CRC. Innate immunity recognizes tumour derived damage‐associated molecular patterns (DAMPs) and primes the anti‐tumour adaptive responses. While the function of β‐catenin in CRC tumourigenesis is well established, its impact on innate immune evasion is largely unknown. The aim of this study is to characterize the role of β‐catenin in inhibiting RIG‐I‐like receptor (RLR)‐mediated IFN‐β signalling in colorectal cancer.

Materials and Methods

Immunohistochemical staining and western blotting were conducted to study the expression of β‐catenin, IRF3 and phospho‐IRF3 (p‐IRF3) in CRC samples and cell lines. Plaque assay determining virus replication was performed to assess the regulation of β‐catenin on IFN‐β signalling. The inhibition of β‐catenin on RLR‐mediated IFN‐β signalling was further studied by real‐time analyses and reporter assays in the context of lentiviral‐mediated β‐catenin stably knocking down. Lastly, co‐immunoprecipitation and nuclear fractionation assay were conducted to monitor the interaction between β‐catenin and IRF3.

Results

We found that high expression of β‐catenin positively correlated with the expression of IRF3 in CRC cells. Overexpression of β‐catenin increased the viral replication. Conversely knocking down of β‐catenin inhibited viral replication. Furthermore, our data demonstrated that β‐catenin could inhibit the expression of IFN‐β and interferon‐stimulated gene 56 (ISG56). Mechanistically, we found that β‐catenin interacted with IRF3 and blocked its nuclear translocation.

Conclusion

Our study reveals an unprecedented role of β‐catenin in enabling innate immune evasion in CRC.

     

Novel dimeric bis(7)-tacrine proton-dependently inhibits NMDA-activated currents

Bis(7)-tacrine has been shown to prevent glutamate-induced neuronal apoptosis by blocking NMDA receptors. However, the characteristics of the inhibition have not been fully elucidated. In this study, we further characterize the features of bis(7)-tacrine inhibition of NMDA-activated current in cultured rat hippocampal neurons. The results show that with the increase of extracellular pH, the inhibitory effect decreases dramatically. At pH 8.0, the concentration-response curve of bis(7)-tacrine is shifted rightwards with the IC(50) value increased from 0.19+/-0.03 microM to 0.41+/-0.04 microM. In addition, bis(7)-tacrine shifts the proton inhibition curve rightwards. Furthermore, the inhibitory effect of bis(7)-tacrine is not altered by the presence of the NMDA receptor proton sensor shield spermidine. These results indicate that bis(7)-tacrine inhibits NMDA-activated current in a pH-dependent manner by sensitizing NMDA receptors to proton inhibition, rendering it potentially beneficial therapeutic effects under acidic conditions associated with stroke and ischemia.     

A loss-of-function mutant allele of a glycosyl hydrolase gene has been co-opted for seed weight control during soybean domestication

The resultant DNA from loss-of-function mutation can be recruited in biological evolution and development. Here, we present such a rare and potential case of “to gain by loss” as a neomorphic mutation during soybean domestication for increasing seed weight. Using a population derived from a chromosome segment substitution line of Glycine max(SN14) and Glycine soja(ZYD06), a quantitative trait locus(QTL) of 100-seed weight(q HSW) was mapped on chromosome 11, corresponding to a truncated β-1, 3-gl...     

Deciphering the Physalis floridana Double-Layered-Lantern1 Mutant Provides Insights into Functional Divergence of the GLOBOSA Duplicates within the Solanaceae

Glycosylphosphatidylinositol-anchor biosynthesis and glycosylphosphatidylinositol modification of proteins are central to coordinated plant development.Since their discovery (Low and Saltiel, 1988), glycosylphosphatidylinositol-anchored proteins (GPI-APs) have provoked intense interest as crucial regulators for growth, morphogenesis, reproduction, and disease pathogenesis in organisms ranging from yeast and trypanosomes to animals and plants. The lipid moiety, the glycosylphosphatidylinositol (GPI) anchor, is synthesized in the endoplasmic reticulum (ER); the protein component is cotranslationally inserted into the ER and posttranslationally modified by the addition of a GPI anchor (Kinoshita et al., 2013; Fig. 1). GPI-APs are then transported via the Golgi to the outer surface of the plasma membrane. The lipid anchor mediates stable attachment of these proteins to the cell surface, where some play important roles as signaling regulators from sphingolipid- and sterol-enriched membrane microdomains (Simons and Gerl, 2010). Some GPI-APs are released from the cell membrane by phosphatidylinositol-specific phospholipases to the extracellular matrix, where they might engage in processes such as cell adhesion and cell-cell communication. In Arabidopsis (Arabidopsis thaliana), there are about 250 predicted GPI-APs (Borner et al., 2003), a relatively large number compared with about 150 in mammals and 50 in the budding yeast (Saccharomyces cerevisiae). Important functions for plant GPI-APs have been elucidated through the study of individual proteins, such as the COBRA family in cell expansion and cell wall biosynthesis (Brady et al., 2007), ARABINOGALACTAN PROTEIN18 in megagametogenesis (Demesa-Arévalo and Vielle-Calzada, 2013), and LORELEI in the pollen tube-female gametophyte interaction (Capron et al., 2008; Tsukamoto et al., 2010; Duan et al., 2014). However, it is the studies of mutants defective in GPI biosynthesis that underscore the general importance of GPI-APs as a class: lacking the capacity to assemble the anchor is lethal.Open in a separate windowFigure 1.GPI anchor biosynthesis pathway. Ten or 11 stepwise modifications of phosphoinositide occur, starting from the synthesis of N-glucosamine-phosphoinositide on the cytoplasmic surface of the ER, followed by its flipping to the ER lumenal side for additional modifications, ending with the addition of the terminal ethanolamine phosphate. Proteins destined for GPI modification are synthesized with a C-terminal signature sequence recognized by the GPI transamidase (a five-protein-enzyme complex) that concomitantly cleaves the peptide at what is designated as the ω and ω+1 amino acids and attaches the GPI anchor in a transamination reaction (red arrows). The GPI-modified proteins are then sorted and transported via the Golgi apparatus to the cell membrane. The established biosynthetic proteins from Arabidopsis and their mammalian homologs are indicated; the galactosylation step appears to be plant specific. The diagram is modeled after figure 3 in Ellis et al. (2010), which also provided a complete list of potential plant orthologs of the human and yeast proteins in the pathway.The GPI anchor is synthesized by an elaborate biosynthetic pathway, starting on the cytoplasmic side of the ER and ending with a completely assembled core anchor on the lumenal surface of the ER (Fig. 1). Prior to their transport out of the ER, proteins destined for GPI modification are cleaved at a C-terminal signature sequence by a GPI transamidase complex that in two enzymatic steps concomitantly attaches a GPI anchor to the C terminus of processed proteins (Kinoshita, 2014). Most of the knowledge on GPI biosynthesis and GPI-AP modification is derived from studies in mammals and yeast, but the pathway is likely to be conserved in plants (Ellis et al., 2010). In a recent article in Plant Physiology, Dai et al. (2014) reported that a GPI anchor biosynthesis mutant, abnormal pollen tube guidance1 (atpg1), displays both embryo lethality and severely depressed male fertility. They determined that APTG1 is homologous to the yeast GPI10 and human PIG-B (for phosphatidylinositol glycan anchor biosynthesis) proteins, the last of three distinct mannosyltransferases that modify the precursor anchor (Fig. 1), and showed that APTG1 can functionally substitute for GPI10 in a conditionally lethal gpi10 yeast mutant. Previous studies have demonstrated that Arabidopsis SETH1 (a male fertility god in Egyptian mythology), SETH2, and PEANUT1 (PNT1), encoding homologs of mammalian PIG-C, PIG-A, and PIG-M (Fig. 1) and their corresponding yeast counterparts, respectively, are important for male fertility (Lalanne et al., 2004; Gillmor et al., 2005). In addition, loss of the first mannosyltransferase in the pathway in pnt1 results in early seedling lethality. pnt1 embryos are severely defective, displaying various cell division anomalies and exhibiting altered levels and ectopic deposition of cell wall polymers. The results reported by Dai et al. (2014), therefore, further demonstrate the conservation of the GPI biosynthesis pathway and the importance of GPI anchoring in plant development and reproduction.The aptg1 mutant was isolated in a search for mutants defective in pollen tube targeting of ovules (Fig. 2), an intriguing and crucial step in plant reproduction. A pollen tube is guided to an ovule by attractants, and upon reaching the target, the female gametophyte, the pollen tube ruptures, ejecting its cytoplasm and releasing sperm for fertilization (Dresselhaus and Franklin-Tong, 2013). aptg1 pollen tubes either fail to target ovules or undertake a more twisted pathway before entering an ovule. In an earlier study, Li et al. (2013) showed that a GPI-AP, COBRA-LIKE10 (COBL10), is required to maintain normal pollen tube growth rates and ovule targeting efficiency. In aptg1 pollen tubes, citrine fluorescent protein-COBL10 was absent from its normal apical membrane location while the citrine fluorescent signal in the cytoplasm was more intense, implying that the diminished presence of COBL10 on the apical membrane could be an underlying cause for the ovule-targeting phenotype. This observation also demonstrates that GPI anchoring is important for the subsequent sorting and transport of these proteins to their destined locations (Kinoshita et al., 2013) and consistent with a wholesale failure of GPI-APs to reach their functional locations as underlying lethality in GPI biosynthesis mutants.Open in a separate windowFigure 2.Pollen tube targeting of ovules in an Arabidopsis pistil. GUS-expressing pollen grains pollinated the pistil. Each blue dot represents discharged cytoplasm from a pollen tube that, in response to attractants, has successfully targeted the ovule and penetrated the female gametophyte and was induced to burst. The cytoplasmic discharge releases sperm for fertilization.While it is clear that major biological roles are played by GPI-APs, many questions remain. Most constituents of the plant GPI anchor biosynthetic pathway remain to be functionally established (Fig. 1). Much has been said about the membrane environments where GPI-APs are localized, but we are far from understanding the precise roles they play in assembling these domains and regulating their functional dynamics. Advances in high-resolution imaging at the cell surface and biochemical approaches to determine the constituents in these membrane microdomains (Simons and Gerl, 2010) should accelerate our understanding of the importance of GPI anchoring as a conserved strategy among eukaryotes to control a wide range of processes.     

Genetic variations in Tibetan populations and high-altitude adaptation at the Himalayas

Modern humans have occupied almost all possible environments globally since exiting Africa about 100,000 years ago. Both behavioral and biological adaptations have contributed to their success in surviving the rigors of climatic extremes, including cold, strong ultraviolet radiation, and high altitude. Among these environmental stresses, high-altitude hypoxia is the only condition in which traditional technology is incapable of mediating its effects. Inhabiting at >3,000-m high plateau, the Tibetan population provides a widely studied example of high-altitude adaptation. Yet, the genetic mechanisms underpinning long-term survival in this environmental extreme remain unknown. We performed an analysis of genome-wide sequence variations in Tibetans. In combination with the reported data, we identified strong signals of selective sweep in two hypoxia-related genes, EPAS1 and EGLN1. For these two genes, Tibetans show unusually high divergence from the non-Tibetan lowlanders (Han Chinese and Japanese) and possess high frequencies of many linked sequence variations as reflected by the Tibetan-specific haplotypes. Further analysis in seven Tibetan populations (1,334 individuals) indicates the prevalence of selective sweep across the Himalayan region. The observed indicators of natural selection on EPAS1 and EGLN1 suggest that during the long-term occupation of high-altitude areas, the functional sequence variations for acquiring biological adaptation to high-altitude hypoxia have been enriched in Tibetan populations.     

Low Genetic Diversity and Low Gene Flow Corresponded to a Weak Genetic Structure of Ruddy-Breasted Crake (<Emphasis Type="Italic">Porzana fusca</Emphasis>) in China

The Ruddy-breasted Crake (Porzana fusca) is an extremely poorly known species. Although it is not listed as globally endangered, in recent years, with the interference of climate change and human activities, its habitat is rapidly disappearing and its populations have been shrinking. There are two different life history traits for Ruddy-breasted Crake in China, i.e., non-migratory population in the south and migratory population in the north of China. In this study, mitochondrial control sequences and microsatellite datasets of 88 individuals sampled from 8 sites were applied to analyze their genetic diversity, genetic differentiation, and genetic structure. Our results indicated that low genetic diversity and genetic differentiation exit in most populations. The neutrality test suggested significantly negative Fu’s Fs value, which, in combination with detection of the mismatch distribution, indicated that population expansion occurred in the interglacier approximately 98,000 years ago, and the time of the most recent common ancestor (TMRCA) was estimated to about 202,705 years ago. Gene flow analysis implied that the gene flow was low, but gene exchange was frequent among adjacent populations. Both phylogenetic and STRUCTURE analyses implied weak genetic structure. In general, the genetic diversity, gene flow, and genetic structure of Ruddy-breasted Crake were low.