拟南芥RPP1A参与幼苗生长的蛋白质组学分析

核糖体蛋白不仅参与蛋白质合成,而且参与植物生长发育的调控.利用拟南芥核糖体磷酸蛋白P1(ribosomal phosphoprotein P1,RPP1)家族基因RPP1A缺失突变体rpp1a研究RPP1A缺失对幼苗蛋白质表达水平的影响,揭示其参与调控幼苗生长的作用机制.表型分析发现,与野生型WT相比,RPP1A缺失导...     

Molecular cloning of N-methylputrescine oxidase from tobacco

Nicotine biosynthesis in Nicotiana species requires an oxidative deamination of N-methylputrescine, catalyzed by N-methylputrescine oxidase (MPO). In a screen for tobacco genes that were down-regulated in a tobacco mutant with altered regulation of nicotine biosynthesis, we identified two homologous MPO cDNAs which encode diamine oxidases of a particular subclass. Tobacco MPO genes were expressed specifically in the root, and up-regulated by jasmonate treatment. Recombinant MPO protein expressed in Escherichia coli formed a homodimer and deaminated N-methylputrescine more efficiently than symmetrical diamines. These results indicate that MPO evolved from general diamine oxidases to function effectively in nicotine biosynthesis.     

AtMRD1 and AtMRU1, two novel genes with altered mRNA levels in the methionine over-accumulating mto1-1 mutant of Arabidopsis thaliana

The mto1-1 mutant of Arabidopsis thaliana over-accumulates soluble methionine (Met) up to 40-fold higher than that in its Col-0 wild type. In order to identify genes regulated by altered Met concentrations, microarray analysis of gene expression in young rosettes and developing siliques of the mto1-1 mutant were performed. Expression of selected genes was then examined in detail in three developmental stages of the mto1-1 mutant using a combination of Northern hybridisation analysis and real-time PCR. Eight genes were identified that had altered mRNA accumulation levels in the mto1-1 mutant compared to that in wild-type plants. Three of the genes have known roles in plant development unrelated to amino acid biosynthesis. One other gene up-regulated specifically in mto1-1 rosettes shared similarity with the embryo-specific protein 3 (ATS3). Two novel genes, referred to as AtMRD1 and AtMRU1, were also identified that were expressed in a developmental manner in wild-type Col-0 and do not share sequence similarity with genes of known function. AtMRD1 was strongly down-regulated in both rosette and young silique tissues of the mto1-1 mutant. AtMRU1 was up-regulated approximately 3-fold in young mto1-1 rosettes and exhibited a developmental response to the mto1-1 mutation.     

感染球孢白僵菌的家蚕免疫应答差异表达基因分析

【目的】筛选家蚕Bombyx mori应对白僵菌Beauveria bassiana侵染的应答基因, 以进一步研究家蚕抵御真菌侵染的分子机制。【方法】采用新一代Solexa高通量测序技术对感染白僵菌及未感染白僵菌的对照组家蚕进行了测序分析, 筛选差异表达基因; 结合生物信息学工具分析差异表达基因的功能注释、 分类及涉及的信号通路等; 应用荧光定量PCR技术验证10个基因的差异表达。【结果】通过测序和生物信息学分析共获得377个差异表达基因, 其中表达上调基因236个, 下调基因141个; KEGG通路分析表明, 各通路中既有表达上调的基因, 也有下调基因; 12个上调基因、 26个下调基因参与3个显著性富集的KEGG通路, 即核糖体、 氨酰tRNA生物合成和剪接体通路。定量PCR与测序结果显示, 溶菌酶、 热激蛋白、 谷胱甘肽S-转移酶、 肽聚糖识别蛋白等与免疫应激相关的蛋白基因均呈现表达上调。【结论】本研究筛选获得的差异表达基因, 特别是上调表达的基因可能与家蚕应对白僵菌侵染的应答机制有关, 其中与免疫应激相关的蛋白基因如溶菌酶、 热激蛋白、 谷胱甘肽S 转移酶、 肽聚糖识别蛋白基因等可能直接参与了家蚕对白僵菌的免疫识别和防御, 研究结果为从分子水平阐明家蚕抵御真菌侵染的防御机制和白僵菌对家蚕的致病机理提供新的依据。     

果生刺盘孢CfHAC1调控应答二硫苏糖醇胁迫的转录组分析

果生刺盘孢Colletotrichum fructicola是油茶炭疽病优势病原菌。前期研究发现bZIP转录因子CfHac1参与调控该菌的生长发育和致病性。为了揭示转录因子CfHac1调控果生刺盘孢响应内质网压力和致病机理,本研究测定了ΔCfhac1突变体对内质网压力胁迫剂的敏感性,发现突变体对二硫苏糖醇（dithiothreitol,DTT）的耐受性下降,说明CfHAC1基因可能参与调控果生刺盘孢响应内质网压力胁迫过程。进一步利用高通量RNA-seq技术对该病菌野生型菌株和CfHAC1敲除突变体菌株在DTT胁迫下的转录组进行了比较分析,结果表明差异表达基因共有2 680个,其中上调表达基因有1 181个,下调表达基因有1 499个。Gene Ontology 功能分析结果显示,差异表达基因主要参与催化活性、结合、代谢过程、细胞过程、细胞成分合成、生物过程调控和应激反应等生物学过程。KEGG功能富集分析表明,上调表达基因主要被富集到核糖体、真核细胞的核糖体生物合成、RNA转运和氰基氨基酸代谢通路中;下调表达基因显著富集在内质网蛋白质加工、N-聚糖生物合成、类固醇合成和蛋白质分泌等通路中。分析发现转录因子CfHac1调控内质网胁迫应答和致病相关基因的表达。本研究提供了在全基因组水平上对CfHAC1基因与内质网压力胁迫应答之间关联的新认识,为阐明果生刺盘孢响应内质网压力胁迫和致病机制奠定了基础。     

The genetic similarity of different generations of Neocallimastix frontalis SK

The genetic similarity of different generations of Neocallimastix frontalis SK was examined by random amplified polymorphic DNA (RAPD) profiling and internal transcribed spacer 1 (ITS1) sequence analysis. N. frontalis SK was subcultured every 2-4 days, and SK-1, SK-3M, and SK-1Y represented N. frontalis SK cultures after one subculture, 50 subcultures, and 150 subcultures. The DNA polymorphisms of the different N. frontalis SK generations were compared by RAPD profiling. The RAPD results gave the same patterns for SK-1, SK-3M and SK-1Y using 12 selected random primers. The partial 18S rDNA, 5.8S rDNA, and ITS1 regions of different generations of N. frontalis SK were amplified and sequenced. The results of alignment and pairwise similarity indicated that the analyzed rRNA sequences of SK-1, SK-3M and SK-1Y were totally identical. This study thus demonstrated genetically identical DNA polymorphisms by RAPD profiling and an unvaried ITS1 region for N. frontalis SK when the strain is subcultured frequently. This suggests that this strain is homokaryotic and grows via an asexual life cycle in vitro.     

代谢组与转录组联合解析赤皮青冈叶片黄化变异机制

为揭示赤皮青冈叶色黄化变异机制,该研究以赤皮青冈叶色变异植株和正常植株的叶片为试验材料,采用超高效液相色谱串联质谱法和高通量RNA测序技术分别进行代谢组和转录组分析。结果表明:(1)代谢组在正离子(POS)、负离子(NEG)模式下分别检测出正常植株和突变体之间存在257个和357个显著差异代谢物(SCMs),其中槲皮素、白矢车菊素、杨梅素等多种黄酮类化合物及其糖苷衍生物(吡喃酮啡肽A、异鼠李素3-葡糖苷酸等)在突变体中显著上调,而叶绿素a、叶绿素b、类胡萝卜素等色素含量则显著下降。(2)转录组测序检测出4 146个差异表达基因(DEGs),其中1 711个基因上调表达,2 435个基因下调表达。(3)KEGG富集分析表明,SCMs和DEGs显著富集到光合作用、卟啉与叶绿素代谢、类黄酮生物合成等途径。综上表明,突变体叶色黄化可能是受到叶绿素合成受阻、叶绿体发育异常及黄酮物质合成增加等因素的综合影响。此外,MYB和bHLH家族基因在突变体中显著上调,证实该两类转录因子参与调控类黄酮生物合成。该研究结果为植物黄化突变的分子机制研究提供了新的见解,也为叶色功能基因挖掘与园林植物育种工作提供了参考。     

白桦BpNAC012基因调控白桦木质部发育表达谱分析

NAC转录因子成员被认为是调控植物次生壁合成的开关。前期研究结果显示BpNAC012基因的表达能够调控白桦次生细胞壁的合成。为研究BpNAC012调控的下游靶基因,本研究分别以该基因的过表达,抑制表达株系茎为材料构建转录组,以野生型为对照分析差异表达基因。结果显示:与对照相比,过表达株系OE中上调的基因有627条,下调的基因有229条,抑制表达株系中上调的基因有299条,下调表达的基因有207条。过表达BpNAC012相对于抑制表达能够调控更多的基因表达变化。而抑制表达BpNAC012更多的是影响蛋白修饰和转运类基因的表达变化。在差异表达基因中,涉及受体信号通路,营养代谢,氨基酸合成,及苯丙烷生物合成相关代谢通路基因比较富集。BpNAC012能够调控纤维素、木质素合成及木质部发育相关基因的表达变化,同时能够调控多种转录因子的表达变化。该研究为深入分析BpNAC012在白桦次生细胞壁合成的分子调控机制奠定基础。     

Characterization of changes in the proteome in different regions of 3D multicell tumor spheroids

Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required.     

Proteome analysis of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> reveals proteins associated with the response to the antibiotic concanamycin A,produced by<Emphasis Type="Italic"> Streptomyces</Emphasis> species

Competition between microbes is common to all ecosystems, but the exact nature of the competition is in most cases unknown. We have previously studied the antagonism between Streptomyces halstedii and several fungi at both the organismal and gene expression levels. Here we analysed the effect of an antibiotic produced by Streptomyces, concanamycin A, on protein levels in the filamentous fungus Aspergillus nidulans. Two-dimensional gel electrophoresis revealed that 20 proteins either increased or decreased in abundance upon treatment of the fungus with the antibiotic. Five of the most prominent proteins which changed in abundance were identified based on peptide analysis by mass spectrometry. Two of these correspond to proteins previously described in A. nidulans, and three others are homologous to proteins found in other organisms. Of these, one down-regulated protein was identified as glyceraldehyde dehydrogenase, a protein involved in general metabolic pathways. A second down-regulated protein, CpcB, affects the initiation of sexual development. Among the proteins not previously described in A. nidulans, all of them up-regulated by concanamycin A, we found two proteins with described homologues in other fungal species. The first is homologous to a cadmium-induced protein in Candida sp. The second protein is homologous to LovC, an enoyl transferase involved in the biosynthesis of lovastatin, a secondary metabolite identified in A. terreus. A third protein has a homologue in A. niger, which is of unknown function. This study indicates that proteome analysis may be a useful method for studying effects on gene expression during competitive interactions between bacteria and filamentous fungi.     

Comparative Proteomics Analysis of Engineered Saccharomyces cerevisiae with Enhanced Biofuel Precursor Production

The yeast Saccharomyces cerevisiae was metabolically modified for enhanced biofuel precursor production by knocking out genes encoding mitochondrial isocitrate dehydrogenase and over-expression of a heterologous ATP-citrate lyase. A comparative iTRAQ-coupled 2D LC-MS/MS analysis was performed to obtain a global overview of ubiquitous protein expression changes in S. cerevisiae engineered strains. More than 300 proteins were identified. Among these proteins, 37 were found differentially expressed in engineered strains and they were classified into specific categories based on their enzyme functions. Most of the proteins involved in glycolytic and pyruvate branch-point pathways were found to be up-regulated and the proteins involved in respiration and glyoxylate pathway were however found to be down-regulated in engineered strains. Moreover, the metabolic modification of S. cerevisiae cells resulted in a number of up-regulated proteins involved in stress response and differentially expressed proteins involved in amino acid metabolism and protein biosynthesis pathways. These LC-MS/MS based proteomics analysis results not only offered extensive information in identifying potential protein-protein interactions, signal pathways and ubiquitous cellular changes elicited by the engineered pathways, but also provided a meaningful biological information platform serving further modification of yeast cells for enhanced biofuel production.     

Proteome analysis of programmed cell death and defense signaling using the rice lesion mimic mutant cdr2

We have previously identified three lesion-mimic mutants, cell death and resistance (cdr), in rice. These mutants induce a series of defense responses, including expression of defense-related genes and high accumulation of phytoalexins, indicating that the cdr mutants are useful materials to study programmed cell death and defense signaling in rice. Here, we carried out a proteome analysis of the cdr2 mutant. Total proteins prepared from the wild type and the cdr2 mutant at three different stages of lesion formation were compared using two-dimensional electrophoresis. We found a total of 37 proteins that were differentially expressed between cdr2 and wild type. Among them, 28 spots were up-regulated and nine were down-regulated in the cdr2 mutant. All the protein spots were identified by mass spectrometric analysis. These differentially regulated proteins included defense-related proteins. In addition, 27 proteins were classified as metabolic enzymes, suggesting that the programmed cell death that occurs in the cdr2 mutant is associated with active metabolic changes. Our study shows that proteome analysis is a useful approach to study programmed cell death and defense signaling in plants.