番茄RBX1基因的功能初探

E3泛素连接酶是泛素蛋白酶体途径中非常重要的蛋白复合体,而大部分的E3连接酶都以Cullin蛋白作为支架,所有基于Cullin蛋白的 E3 连接酶都含有一个催化亚基RBX1。RBX1对于E3复合体结合E2和Cullin蛋白的活化具有重要作用。利用酵母双杂交技术鉴定出番茄RBX1蛋白与番茄CUL4蛋白有直接的相互作用。通过荧光定量PCR（Real-time PCR）技术分析野生型番茄中RBX1基因的组织特异性,结果显示RBX1基因在番茄各组织中均有表达,但在花与果实中表达量较高,茎和叶中相对较少。同时构建植物过量表达载体 PBI121-35S-RBX1并转化番茄,获得 3 株过量表达和 3 株RBX1共抑制转基因植株。研究发现共抑制的转基因植株表现出叶片变大、顶端优势变弱和不育等表型,说明RBX1基因在番茄的生长发育中发挥重要作用,推测植物RBX1基因可能与生长素和油菜素内酯等植物激素应答以及细胞增殖有关,为进一步研究该基因在植物中的生物学功能提供了线索。     

细菌铜抗性机制研究进展

硫化铜矿生物冶金过程中,由于高浓度铜离子对浸矿细菌具有毒性,抑制了细菌的浸矿活性.一些细菌,如丁香假单胞菌、大肠杆菌、肠球菌存在铜抗性机制.在丁香假单胞菌中.铜抗性是由4个结构基因copABCD和2个调节基因copRS控制的.在大肠杆菌中与之相似的基因被称为pcoABCD和pcoRS.pcoABCDRS的基因产物与copABCDRS的基因产物的氨基酸相似性分别达到76%,55%,60%,38%,61%,和30%.在大肠杆菌pcoABCDRS的末端还有一个pcoE基因,其功能目前还不清楚.另外,大肠杆菌的铜抗性还受染色体基因的调控.G+肠球菌的铜抗性机制研究的最清楚,其操纵子基因包括copYZAB,copYZ为调节基因.肠球菌copAB编码的蛋白CopA和CopB为P型ATP酶,分别负责铜的摄入和排出.对细菌铜抗性机制的研究进展进行了论述.对于浸矿细菌铜抗性基因工程改造具有理论指导意义.     

Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato

The resistance in tomato plants to bacterial speck caused by Pseudomonas syringae pv. tomato is triggered by the interactions between the plant resistance protein Pto and the pathogen avirulence proteins AvrPto or AvrPtoB. Fen is a gene encoding closely related functional protein kinases as the Pto gene. To investigate the status of resistance to the pathogen and natural variation of Pto and Fen genes in tomato, 67 lines including 29 growing in China were subject to disease resistance evaluation and fenthion-sensitivity test. Alleles of Pto and Fen were amplified from genomic DNA of 25 tomato lines using polymerase chain reaction (PCR) and sequences were determined by sequencing the PCR products. The results indicated that none of the 29 cultivars/hybrids growing in China were resistant to bacterial speck race 0 strain DC3000. Seven of eight tomato lines resistant to DC3000 were also fenthion-sensitive. Analysis of deduced amino acid sequences identified three novel residue substitutions between Pto and pto, and one new substitution identified between Fen and fen. A PCR-based marker was developed and successfully used to select plants with resistance to DC3000.     

Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase

Ubiquitination controls a broad range of cellular functions. The last step of the ubiquitination pathway is regulated by enzyme type 3 (E3) ubiquitin ligases. E3 enzymes are responsible for substrate specificity and catalyze the formation of an isopeptide bond between a lysine residue of the substrate (or the N terminus of the substrate) and ubiquitin. MIR1 and MIR2 are two E3 ubiquitin ligases encoded by Kaposi's sarcoma-associated herpesvirus that mediate the ubiquitination of major histocompatibility complex class I (MHC I) molecules and subsequent internalization. Here, we found that MIR1, but not MIR2, promoted down-regulation of MHC I molecules lacking lysine residues in their intracytoplasmic domain. In the presence of MIR1, these MHC I molecules were ubiquitinated, and their association with ubiquitin was sensitive to beta2-mercaptoethanol, unlike lysine-ubiquitin bonds. This form of ubiquitination required a cysteine residue in the intracytoplasmic tail of MHC I molecules. An MHC I molecule containing a single cysteine residue in an artificial glycine and alanine intracytoplasmic domain was endocytosed and degraded in the presence of MIR1. Thus, ubiquitination can occur on proteins lacking accessible lysines or an accessible N terminus.     

S-nitrosylation of parkin regulates ubiquitination and compromises parkin's protective function

Parkin is an E3 ubiquitin ligase involved in the ubiquitination of proteins that are important in the survival of dopamine neurons in Parkinson's disease (PD). We show that parkin is S-nitrosylated in vitro, as well as in vivo in a mouse model of PD and in brains of patients with PD and diffuse Lewy body disease. Moreover, S-nitrosylation inhibits parkin's ubiquitin E3 ligase activity and its protective function. The inhibition of parkin's ubiquitin E3 ligase activity by S-nitrosylation could contribute to the degenerative process in these disorders by impairing the ubiquitination of parkin substrates.     

A bacterial virulence protein suppresses host innate immunity to cause plant disease

Plants have evolved a powerful immune system to defend against infection by most microbial organisms. However, successful pathogens, such as Pseudomonas syringae, have developed countermeasures and inject virulence proteins into the host plant cell to suppress immunity and cause devastating diseases. Despite intensive research efforts, the molecular targets of bacterial virulence proteins that are important for plant disease development have remained obscure. Here, we show that a conserved P. syringae virulence protein, HopM1, targets an immunity-associated protein, AtMIN7, in Arabidopsis thaliana. HopM1 mediates the destruction of AtMIN7 via the host proteasome. Our results illustrate a strategy by which a bacterial pathogen exploits the host proteasome to subvert host immunity and causes infection in plants.     

A plant miRNA contributes to antibacterial resistance by repressing auxin signaling

Plants and animals activate defenses after perceiving pathogen-associated molecular patterns (PAMPs) such as bacterial flagellin. In Arabidopsis, perception of flagellin increases resistance to the bacterium Pseudomonas syringae, although the molecular mechanisms involved remain elusive. Here, we show that a flagellin-derived peptide induces a plant microRNA (miRNA) that negatively regulates messenger RNAs for the F-box auxin receptors TIR1, AFB2, and AFB3. Repression of auxin signaling restricts P. syringae growth, implicating auxin in disease susceptibility and miRNA-mediated suppression of auxin signaling in resistance.     

Molecular Basis of Gene-for-Gene Specificity in Bacterial Speck Disease of Tomato

Transient expression of the Pseudomonas syringae avirulence gene avrPto in plant cells resulted in a Pto-dependent necrosis. The AvrPto avirulence protein was observed to interact directly with the Pto resistance protein in the yeast two-hybrid system. Mutations in the Pto and avrPto genes which reduce in vivo activity had parallel effects on association in the two-hybrid assay. These data suggest that during infection the pathogen delivers AvrPto into the plant host cell and that resistance is specified by direct interaction of Pto with AvrPto.     

Activation of a phytopathogenic bacterial effector protein by a eukaryotic cyclophilin

Innate immunity in higher plants invokes a sophisticated surveillance system capable of recognizing bacterial effector proteins. In Arabidopsis, resistance to infection by strains of Pseudomonas syringae expressing the effector AvrRpt2 requires the plant resistance protein RPS2. AvrRpt2 was identified as a putative cysteine protease that results in the elimination of the Arabidopsis protein RIN4. RIN4 cleavage serves as a signal to activate RPS2-mediated resistance. AvrRpt2 is delivered into the plant cell, where it is activated by a eukaryotic factor that we identify as cyclophilin. This activation of AvrRpt2 is necessary for protease activity. Active AvrRpt2 can then directly cleave RIN4.     

Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity

Innate immune responses are triggered by the activation of pattern-recognition receptors (PRRs). The Arabidopsis PRR FLAGELLIN-SENSING 2 (FLS2) senses bacterial flagellin and initiates immune signaling through association with BAK1. The molecular mechanisms underlying the attenuation of FLS2 activation are largely unknown. We report that flagellin induces recruitment of two closely related U-box E3 ubiquitin ligases, PUB12 and PUB13, to FLS2 receptor complex in Arabidopsis. BAK1 phosphorylates PUB12 and PUB13 and is required for FLS2-PUB12/13 association. PUB12 and PUB13 polyubiquitinate FLS2 and promote flagellin-induced FLS2 degradation, and the pub12 and pub13 mutants displayed elevated immune responses to flagellin treatment. Our study has revealed a unique regulatory circuit of direct ubiquitination and turnover of FLS2 by BAK1-mediated phosphorylation and recruitment of specific E3 ligases for attenuation of immune signaling.     

PoprL启动子对丁香假单胞菌DC3000合成冠菌素的影响

冠菌素（coronatine, COR）是由多种丁香假单胞菌致病变种产生的次生代谢产物，是一种新型植物生长调节剂。为了提高菌株冠菌素产量并优化发酵培养条件，将来源于假单胞菌肽聚糖脂蛋白编码基因oprL高效表达组成型启动子PoprL，通过质粒pUCP24/recTE同源重组到丁香假单胞菌DC3000冠菌素合成基因cfl中，得到启动子重组菌株CO，在18和28 ℃培养条件下发酵培养，结果表明，CO菌株在18 ℃培养条件下冠菌素产量是出发菌株DC3000的1.8倍，在28 ℃培养条件下冠菌素产量是出发菌株DC3000的4.1倍。利用启动子替换的方法有效提高了冠菌素的产量，并且改善了高温发酵条件对菌株冠菌素产量的制约影响，为冠菌素的菌种改造和工业化生产应用提供有力支持与借鉴。     

Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.     

Phosphatidylinositol as a Component of the Ice Nucleating Site of Pseudomonas syringae and Erwinia herbiola

Phosphatidylinositol has been identified as a major component of the ice nucleating site on the outer surface of two bacteria, Pseudomonas syringae and Erwinia herbicola. Plant lectins binding to inositol and a highly purified phosphatidylinositol-specific hydrolase (a C(II) lipase) inhibited or decreased the efficiency of the ice nucleating activity (INA) of both bacteria. Extracts of these two INA(+) bacteria had phosphatidylinositol synthase activity while extracts from related INA(-) Pseudomonas or Erwinia strains had no detectable synthase activity. An Escherichia coli strain acquired phosphatidylinositol synthase activity when transformed to the INA(+) phenotype with recombinant plasmids containing fragments of P. syringae DNA.     

Species of Ice Nucleation Active Bacteria on the Apricot and the Relationship Between Their Activity and Flower Frost

During 1996- 1997, sixty samples were collected from apricot in Hebei Province, from which nineteen ice nucleation active bacterial strains were isolated. Nine stains were identified as Pseudomonas syringae pv. syringae by bacteriological determination, while the others were Erwinia uredovora. Assay of ice nucleation activity (INA) showed the activity of the Pseudomonas strains was higher than that of the Erwinia.According to the number of bacterial cells required to produce one ice nucleus active at - 3℃, four stains was classified as strong ones, three as medium-strong, the remainder as weak. In general, the INA of these strains were regarded as medium-strong. Under stress of Iow temperatures, treatment of INA bacteria can greatly raised relative electric conductivity of petals and permeability of cell membrane. The treated petals showed symptoms of serious frost at - 3 - - 4℃ and had supercooling points of 2 - 3℃ higher than controls. Our results demon strated that INA bacteria are one major factor to incite frost damage to apricot flowers. We may reduce frost injury to apricot during flowering phage through control of INA bacteria.     

黄瓜细菌性角斑病菌的分离与鉴定

黄瓜细菌性角斑病是黄瓜生产中的重要病害之一,在陕西的危害严重,发病率在50%以上。本研究采用常规组织分离法和柯赫氏证病律从杨凌地区黄瓜细菌性角斑病标样中分离到1株病原菌,结合形态学、生理生化指标及16S rDNA序列分析,鉴定为丁香假单胞杆菌(Pseudomonas syringestrain)CP001。初步确定适合丁香假单胞杆菌CP001菌株生长的最佳pH为6~8,最适温度为22~29℃。     

丁香假单胞菌中六个致病变种的全细胞蛋白质电泳分析

 利用聚丙稀酰胺电泳技术首次对丁香假单胞菌中6个致病变种全细胞蛋白电泳,发现同一致病变种内菌株蛋白条带一致,不同致病变种之间存在差异.以往对丁香假单胞菌中致病变种的鉴定方法主要是根据致病性的差异,结合营养筛选和血清学方法来进行.对丁香假单胞菌的6个致病变种的全细胞蛋白分析表明:全细胞蛋白质电泳能成功地鉴定到致病变种.     

两个高度同源的RING结构域蛋白功能初步研究

泛素化途径在植物应答生物和非生物胁迫中起着重要作用,其中E3连接酶在泛素化途径中起着决定性作用。室通过在线基因芯片预测发现2个与非生物胁迫相关的拟南芥RING结构域蛋白基因At2g24480（HHR1）、At5g43200(HHR2)通过体外泛素化实验证明了二者具有E3连接酶活性。对这两个E3连接酶进行了亚细胞定位,表达谱和组织特异性分析,发现二者受诱导后的表达情况、组织特异性、亚细胞定位均不同。HHR1定位于细胞膜上,HHR2定位于细胞核;表达谱分析表明HHR1响应热和H2O2胁迫,而HHR2响应盐和H2O2胁迫;HHR1在根和花中的表达量较高,在茎和叶中的表达量较低,而HHR2在根、茎、叶中的表达量都较高,在花中表达量较低。该研究结果证实了HHR1和HHR2的一些性质和亚细胞定位,为深入研究其功能奠定了基础。     

Road to ruin: targeting proteins for degradation in the endoplasmic reticulum

Some nascent proteins that fold within the endoplasmic reticulum (ER) never reach their native state. Misfolded proteins are removed from the folding machinery, dislocated from the ER into the cytosol, and degraded in a series of pathways collectively referred to as ER-associated degradation (ERAD). Distinct ERAD pathways centered on different E3 ubiquitin ligases survey the range of potential substrates. We now know many of the components of the ERAD machinery and pathways used to detect substrates and target them for degradation. Much less is known about the features used to identify terminally misfolded conformations and the broader role of these pathways in regulating protein half-lives.