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1.
获得功能基因是对植物次生代谢产物代谢途径进行遗传操作的必要条件。通过引入脱氧次黄嘌呤碱基和分解引物,大幅度降低高简并度引物的简并程度,并优化PCR扩增体系和扩增条件,从栀子果实中成功扩增出藏红花素生物合成途径的两个关键酶——玉米黄素剪切加双氧酶（zeaxanthin cleavage dioxygenase,ZCD）和糖基转移酶（glucosyltransferase, GTase）基因的保守区段,分别长170bp和250bp。研究结果为今后获得全长基因,并应用于植物藏红花素生物合成途径的调控奠定了基础。 相似文献
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云南3种野生稻中抗病基因同源序列的克隆及序列分析 总被引：18，自引：4，他引：18
根据已报道的NBS LRR类和STK类抗病基因结构中的氨基酸保守区域 ,设计简并引物 ,通过PCR扩增及克隆 ,从普通野生稻 (OryzarufipogonGriff.)、药用野生稻 (OryzaofficinalisWall.)、疣粒野生稻 (Oryzameye rianaBaill.)中共获得 14类NBS LRR类抗病基因同源序列 ,其中普通野生稻中的有 7类 ,药用野生稻中的有 2类 ,疣粒野生稻中的有 6类。药用野生稻中TO12代表序列与普通野生稻中TR19代表序列 ,同属一类 ,且具有 10 0 %的同源性 ,说明不同种野生稻中的同一类 (聚类 )抗病基因同源序列是完全一致的。同时 ,还获得 5类STK类抗病基因同源序列 ,其中普通野生稻中的有 4类 ;药用野生稻中的有 1类。通过氨基酸同源性比较分析 ,发现笔者克隆到的抗病基因同源序列与已克隆的抗病基因L6、N、Bs2、Prf、Pto、Lr10和Xa2 1等的氨基酸同源性都相当低 (均低于 2 5 % ) ,暗示了这些抗病基因同源序列可能是目前尚未报道的抗病基因的同源序列。 相似文献
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辣椒RGA的分离将为进一步从辣椒中分离功能性抗病基因和抗病机理分析打下基础,为辣椒相关抗病基因的克隆提供依据。根据已知抗病基因保守氨基酸结构域设计简并引物,以高抗辣椒品种88为试材,通过PCR扩增抗病基因同源序列。结果表明:从辣椒基因组DNA中分离出1条辣椒抗病基因同源序列WD1。对其编码的氨基酸序列进行分析表明:该序列同时含有P-环(GGVGKTT)、kinase-2(VLDD)、kinase-3(GSRII)及HD(即GLPLAL)保守域结构,分离的辣椒抗病基因同源序列(RGA)与已报道的辣椒抗病基因同源序列A5和A13都有99%的同源性。 相似文献
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Phototropins are light-activated kinases important for plant responses to blue light. Light initiates signaling in these proteins by generating a covalent protein-flavin mononucleotide (FMN) adduct within sensory Per-ARNT-Sim (PAS) domains. We characterized the light-dependent changes of a phototropin PAS domain by solution nuclear magnetic resonance spectroscopy and found that an alpha helix located outside the canonical domain plays a key role in this activation process. Although this helix associates with the PAS core in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent bond formation to kinase activation and identifies a signaling pathway conserved among PAS domains. 相似文献
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The Wnt-Wingless (Wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulates many aspects of metazoan development. Aberrant Wnt signaling has been linked to human disease. In the present study, we used a genomewide RNA interference (RNAi) screen in Drosophila cells to screen for regulators of the Wnt pathway. We identified 238 potential regulators, which include known pathway components, genes with functions not previously linked to this pathway, and genes with no previously assigned functions. Reciprocal-Best-Blast analyses reveal that 50% of the genes identified in the screen have human orthologs, of which approximately 18% are associated with human disease. Functional assays of selected genes from the cell-based screen in Drosophila, mammalian cells, and zebrafish embryos demonstrated that these genes have evolutionarily conserved functions in Wnt signaling. High-throughput RNAi screens in cultured cells, followed by functional analyses in model organisms, prove to be a rapid means of identifying regulators of signaling pathways implicated in development and disease. 相似文献
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W Liu E Chun AA Thompson P Chubukov F Xu V Katritch GW Han CB Roth LH Heitman AP IJzerman V Cherezov RC Stevens 《Science (New York, N.Y.)》2012,337(6091):232-236
Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We reengineered the human A(2A) adenosine receptor by replacing its third intracellular loop with apocytochrome b(562)RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp(2.50). Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function. 相似文献
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GID1作为赤霉素(GA)受体蛋白,是GA信号通路的重要组成部分,其编码基因GID1在被子植物中已经被广泛克隆,但在针叶树种中的研究十分滞后。为了分离针叶树GA受体GID1基因并推测其功能,本研究以拟南芥GID1s序列为探针,在油松高质量参考转录组内筛选并鉴别出了油松GID1直系同源基因;基于该基因序列同源克隆了樟子松、白皮松、赤松GID1基因,通过BLAST获得了日本落叶松、火炬松、白云杉与挪威云杉的GID1-like基因;对针叶树GID1基因进行序列保守性、蛋白结构和组织表达活性分析。结果表明:针叶树种很可能只含有一个GID1基因,该基因在针叶树中具有很高的保守性;虽然与被子植物GID1之间的序列一致性较低,但其保持GA亲和活性所必需的氨基酸残基十分保守,与其下游DELLA蛋白相互作用的功能域与结构同样十分保守,推测其在针叶树GA信号转导中具有受体功能;表达分析显示GID1在挪威云杉不同组织和油松雌雄球花不同发育阶段间表达较为稳定,表明GID1可能广泛参与这些组织的发育过程,针叶树GA信号调控通路中GA受体的转录调控可能并不是核心调控机制。研究结果为GID1基因在针叶树生长发育过程中的分子调控机制研究奠定了基础。 相似文献
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The evolutionarily conserved actin-related protein (Arp2/3) complex is a key component of actin filament networks that is dynamically regulated by nucleation-promoting and inhibitory factors. Although much is known about actin assembly, the physiologic functions of inhibitory proteins are unclear. We generated coronin 1-/- mice and found that coronin 1 exerted an inhibitory effect on cellular steady-state F-actin formation via an Arp2/3-dependent mechanism. Whereas coronin 1 was required for chemokine-mediated migration, it was dispensable for T cell antigen receptor functions in T cells. Moreover, actin dynamics, through a mitochondrial pathway, was linked to lymphocyte homeostasis. 相似文献
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Sulfonamide-resistant strains of Staphylococcus aureus produce greater amounts of p-aminobenzoic acid than do their parent strains. This synthesis occurs both in the absence and in the presence of sulfonamides. The quantity of p-aminobenzoic acid synthesized by resistant strains appears sufficient to account for their resistance to sulfonamide drugs. On the basis of this evidence, it is suggested that the development of ability to synthesize p-aminobenzoic acid in excess of the normal metabolic requirements, as a result of continued exposure to sulfonamides, explains the phenomenon of sulfonamide fastness in Staphylococcus aureus. 相似文献
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OsNHO1是一种甘油激酶,受多种因素的诱导,在植物先天免疫反应中起着重要的作用。笔者采用同源序列法,根据已报道的拟南芥NHO1基因序列结合水稻基因组测序结果,筛选水稻OsNHO1基因,采用RT-PCR获得水稻OsNHO1基因全长cDNA(1 590 bp,529AA),并通过半定量RT-PCR方法分析OsNHO1基因在SA,PXO99刺激下的表达模式,结果显示OsNHO1基因的表达受SA,PXO99的诱导,为进一步研究OsNHO1的具体功能和作用机理奠定了基础。 相似文献
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Weller GR Kysela B Roy R Tonkin LM Scanlan E Della M Devine SK Day JP Wilkinson A d'Adda di Fagagna F Devine KM Bowater RP Jeggo PA Jackson SP Doherty AJ 《Science (New York, N.Y.)》2002,297(5587):1686-1689
In eukaryotic cells, double-strand breaks (DSBs) in DNA are generally repaired by the pathway of homologous recombination or by DNA nonhomologous end joining (NHEJ). Both pathways have been highly conserved throughout eukaryotic evolution, but no equivalent NHEJ system has been identified in prokaryotes. The NHEJ pathway requires a DNA end-binding component called Ku. We have identified bacterial Ku homologs and show that these proteins retain the biochemical characteristics of the eukaryotic Ku heterodimer. Furthermore, we show that bacterial Ku specifically recruits DNA ligase to DNA ends and stimulates DNA ligation. Loss of these proteins leads to hypersensitivity to ionizing radiation in Bacillus subtilis. These data provide evidence that many bacteria possess a DNA DSB repair apparatus that shares many features with the NHEJ system of eukarya and suggest that this DNA repair pathway arose before the prokaryotic and eukaryotic lineages diverged. 相似文献
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转几丁质酶和 β-1,3-葡聚糖酶基因提高棉花对枯萎病和黄萎病的抗性 总被引：8，自引：0，他引：8
枯、黄萎病是世界棉花生产中的两大重要病害。传统育种缺乏抗源，几丁质酶和 -1,3-葡聚糖酶是植物防御体系中的两种防卫因子，两者之间存在协同增效作用。据此构建了4个单价和2个双价基因(分别定位于细胞内或细胞外)的植物表达载体，通过花粉管通道法转化棉花，经PCR和Southern杂交检测以及1996 2000年温室及病圃多代筛选鉴定，已培育出对枯、黄萎病抗性提高的转基因棉花株系。将抗病基因导入国产抗虫棉品种GK19中，还获得了兼抗病、虫的转基因优系。 相似文献
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为探索水环境因子对磺胺类抗性基因传播机制的影响,以磺胺类抗性基因Sul1为目的基因,采用绝对定量PCR技术,考察了pH、盐度、腐植酸、总氨氮和活性磷酸盐对Sul1含量的影响。研究结果表明,Sul1含量在pH 6~8时最高,而在酸性(pH 3~5)或碱性(pH 9~10)条件下均显著降低,表现出明显的抑制作用(P0.05)。Sul1含量随着盐度或腐植酸浓度升高而降低,呈显著的抑制作用(P0.05);总氨氮或活性磷酸盐对Sul1含量的影响表现为高浓度抑制、低浓度促进作用。当总氨氮或活性磷酸盐的浓度低于0.05 mg·L~(-1)时,Sul1含量显著增加(P0.05);而当浓度升高到1 mg·L~(-1),Sul1的含量显著降低(P0.05)。上述结果表明,水环境因子会影响磺胺类抗性基因Sul1的存在及分布。 相似文献
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Plant development: regulation by protein degradation 总被引：2，自引：0，他引：2
Many aspects of eukaryotic development depend on regulated protein degradation by the ubiquitin-proteasome pathway. This highly conserved pathway promotes covalent attachment of ubiquitin to protein substrates through the sequential action of three enzymes called a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3). Most ubiquitinated proteins are then targeted for degradation by the 26S proteasome. Recent studies have also shown that the ubiquitin-related protein RUB/Nedd8 and the proteasome-related COP9 signalosome complex cooperate with the ubiquitin-proteasome pathway to promote protein degradation. Most of these components are conserved in all three eukaryotic kingdoms. However, the known targets of the pathway in plants, and the developmental processes they regulate, are specific to the plant kingdom. 相似文献
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【目的】已有研究表明稻瘟菌Cdc42(MgCdc42)与酵母Cdc42(ScCdc42)高度同源，参与调控其形态分化和侵染过程，通过分析可能的MgCdc42互作蛋白，以明确这些蛋白其结构和功能。【方法】用ScCdc42互作蛋白经BLAST搜索获得了稻瘟菌基因组中的相应同源物，分析了这些同源物的结构，并通过半定量RT-PCR分析MgCdc42不同突变情况下这些可能的调控蛋白及效应蛋白的表达情况。【结果】MgCdc42正显性突变后，导致所有推测互作蛋白表达量均有所提高；MgCdc42负显性突变，除MgBem1、Chm1、MgGic1表达量未见明显变化外，其余表达量均有所降低；当MgCdc42失活后，所有可能的MgCdc42调控蛋白及效应蛋白之表达量均有所降低。【结论】稻瘟菌可能存在酵母Cdc42相似的信号途径，MgCdc42在其中起着重要的调控作用。 相似文献
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The ABC transporters are ubiquitous membrane proteins that couple adenosine triphosphate (ATP) hydrolysis to the translocation of diverse substrates across cell membranes. Clinically relevant examples are associated with cystic fibrosis and with multidrug resistance of pathogenic bacteria and cancer cells. Here, we report the crystal structure at 3.2 angstrom resolution of the Escherichia coli BtuCD protein, an ABC transporter mediating vitamin B12 uptake. The two ATP-binding cassettes (BtuD) are in close contact with each other, as are the two membrane-spanning subunits (BtuC); this arrangement is distinct from that observed for the E. coli lipid flippase MsbA. The BtuC subunits provide 20 transmembrane helices grouped around a translocation pathway that is closed to the cytoplasm by a gate region whereas the dimer arrangement of the BtuD subunits resembles the ATP-bound form of the Rad50 DNA repair enzyme. A prominent cytoplasmic loop of BtuC forms the contact region with the ATP-binding cassette and appears to represent a conserved motif among the ABC transporters. 相似文献
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根据丝氨酸/苏氨酸蛋白激酶类(STK)结构域Ⅰ和Ⅷ氨基酸保守序列设计了1对简并引物,以细菌性萎蔫病抗/感木薯种质E1340和GR911基因组DNA为模板,通过PCR扩增,均获得大小约0.5 kb的扩增片段。两个片段纯化、克隆、测序后获得完全一致的505 nt序列,比对发现,木薯种质AM560带有和该序列高度同源的核苷酸片段。对包括同源片段上下游各约2.0 kb的大片段序列进行基因预测,获得1个有4个外显子、ORF全长1866 nt的预测基因,命名SSK1。序列分析表明,该基因编码621 aa,有细胞壁受体激酶结合、偶联位点和8个跨膜结构域,具有典型的STK类抗病基因的保守结构,是一个候选的抗病基因,可能在木薯抗病反应中发挥重要作用。 相似文献