抗吡虫啉单克隆抗体的制备及鉴定

为制备灵敏度高,特异性强的抗吡虫啉单克隆抗体,建立经济、快速、准确的吡虫啉残留免疫学分析方法,采用分子模拟技术分析吡虫啉及其类似农药的电荷分布后,选择1-[6-(2-羧乙硫基-3-吡啶)甲基]-N-硝基-2-咪唑啉亚胺 (H1) 作为免疫半抗原,1-(6-氯-3-吡啶甲基)-3-羧丙基-N-硝基-2-咪唑啉亚胺 (H2) 作为包被半抗原,利用NHS酯法将H1和H2分别与牛血清蛋白 (BSA) 和卵清蛋白 (OVA) 偶联合成免疫原与包被原。免疫BALB/c小鼠后,采用常规杂交瘤技术共获得2株稳定分泌抗吡虫     

Identification and molecular characterization of twin-arginine translocation system (Tat) in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> strain PXO99

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases in rice. This study identified and characterized the contribution of the twin-arginine translocation (Tat) pathway to motility, chemotaxis, extracellular polysaccharide (EPS) production and virulence in X. oryzae pv. oryzae strain PXO99. The tatC disruption mutant (strain TCM) of strain PXO99 were generated, and confirmed both by PCR and Southern blotting. Strain PXO99 cells were highly motile in NYGB 0.3% soft agar plate. In contrast, the tatC mutation impaired motility. Furthermore, strain TCM cells lacked detectable flagella and exhibited almost no chemotaxis toward glucose under aerobic conditions, indicating that the Tat secretion pathway contributed to flagellar biogenesis and chemotactic responses. It was also observed that strain TCM exhibited a reductive production of extracellular polysaccharide (EPS) and a significant reduction of virulence on rice plants when compared with the wild type PXO99. However, the tatC mutation in strain PXO99 did not affect growth rate and the ability to induce hypersensitive response (HR) in nonhost tobacco (Nicotiana tabacum L. cv. Samsun). Our findings indicated that the Tat system of X. oryzae pv. oryzae played an important role in the pathogen’s virulence. L. Chen, B. Hu, and G. Qian contributed equally to this research.     

The <Emphasis Type="Italic">luxS</Emphasis> Gene Is Involved in AI-2 Production,Pathogenicity, and Some Phenotypes in <Emphasis Type="Italic">Erwinia amylovora</Emphasis>

Erwinia amylovora causes fire blight of apple, pear, and other members of the Rosaceae family. The enzyme LuxS catalyzes the last step in the production of autoinducer-2 (AI-2), a molecule implicated with quorum sensing in many bacterial species. It is now well recognized that LuxS also plays a central role in sulfur metabolism and in the activated methyl cycle, which is responsible for the generation of S-adenosyl-l-methionine. A research paper has reported that luxS is not involved with quorum sensing in Er. amylovora, but in our study, Er. amylovora strain NCPPB1665 (Ea1665) produced luxS-dependent extracellular AI-2 activity. Additionally, the maximal AI-2 activity occurred during late-exponential and early-stationary growth phases and diminished during the stationary phase. The luxS mutant of Ea1665 was constructed, and the phenotypes of a defined luxS mutant have been characterized. Inactivation of luxS in Ea1665 impaired motility, extracellular polysaccharide (EPS) production, and tolerance for hydrogen peroxide, and reduced virulence on pear leaves. Yan Gao and Junxian Song contributed equally to this research.     

Efficient production of heat-stable antifungal factor through integrating statistical optimization with a two-stage temperature control strategy in <Emphasis Type="Italic">Lysobacter enzymogenes</Emphasis> OH11

Background

Heat-stable antifungal factor (HSAF) is a newly identified broad-spectrum antifungal antibiotic from the biocontrol agent Lysobacter enzymogenes and is regarded as a potential biological pesticide, due to its novel mode of action. However, the production level of HSAF is quite low, and little research has reported on the fermentation process involved, representing huge obstacles for large-scale industrial production.

Results

Medium capacity, culture temperature, and fermentation time were identified as the most significant factors affecting the production of HSAF and employed for further optimization through statistical methods. Based on the analysis of kinetic parameters at different temperatures, a novel two-stage temperature control strategy was developed to improve HSAF production, in which the temperature was increased to 32 °C during the first 12 h and then switched to 26 °C until the end of fermentation. Using this strategy, the maximum HSAF production reached 440.26?±?16.14 mg L^??1, increased by 9.93% than that of the best results from single-temperature fermentation. Moreover, the fermentation time was shortened from 58 h to 54 h, resulting in the enhancement of HSAF productivity (17.95%) and yield (9.93%).

Conclusions

This study provides a simple and efficient method for producing HSAF that could be feasibly applied to the industrial-scale production of HSAF.

     

Identification and Evolution of FYVE Domain-Containing Proteins and Their Expression Patterns in Response to Abiotic Stresses in Rice

The FYVE domain is a typical zinc finger motif containing four conserved CxxC pairs and has been shown to specifically binds to PtdIns(3)P on the surface of cell membrane. FYVE domain-containing proteins are commonly distributed in eukaryotic cells and have been implicated in diverse functions like signal transduction, membrane trafficking, exocytosis and endocytosis, phosphoinositides (PIs) metabolism, and cytoskeletal regulation. Analysis of the rice genome using comprehensive online databases and research tools resulted in the identification of 19 putative rice FYVE (OsFVYE) proteins. Based on domain architectural and phylogenetic analyses, these OsFYVEs were further classified into six groups. Groups I–V were conserved in Arabidopsis (dicots) and rice (monocot) at both the genetic and protein levels, while group VI was widely present in plants but truncated in Arabidopsis and species of Brassicaceae. Comprehensive and comparative investigation of their expression profiles showed that FYVE genes in plant exhibited a variety of expression patterns during different developmental stages and in response to phytohormones and abiotic stresses. These findings indicated that both OsFYVE and AtFYVE genes may played potential roles in normal plant growth, hormone signal transduction, and abiotic stress tolerance. Results from our study shed light on the potential roles of FYVE proteins in plant growth, development, and stress responses.     

水稻细菌性条斑病菌中受DSF 调控的鞭毛基因flgD、flgE 的功能分析

[目的]为了阐明可扩散性信号分子(diffusible signal factor,DSF)调控的鞭毛基因对水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)Rs105的致病性等方面的影响.[方法]采用PCR的方法克隆靶标基因flgDxoc和flgExoc,用同源重组法构建缺失突变体,测定突变体及其互补菌株的菌体形态、运动性、致病性及过敏性反应等表型,用反转录PCR(RT-PCR)的方法验证Rs105和ArpfFxoc(rpfFxoc基因的缺失突变体,不产生DSF)中flgDxoc、、flgExoc表达量的差异.[结果]从Rs105基因组中克隆到flgDxoc、flgExoc基因,并证实这两个基因在基因组中均为单拷贝.PCR和Southern杂交结果显示,flgDxoc、flgExoc基因被成功敲除.与野生型相比,突变体的鞭毛产生能力丧失,游动性和趋化性能力减弱,接种水稻叶片显示其致病性部分减弱,基因互补可使其恢复.生长能力和对烟草叶片的致敏性无明显改变.RT-PCR结果显示,flgDxoc、flgExoc基因在△rpfFxoc中的转录水平明显降低.[结论]本试验表明:FlgD、FlgE是水稻细菌性条斑病菌鞭毛形成所必需的因子;进一步证明了DSF通过调控flgDxoc、flgExoc基因表达,从而影响条斑病菌的致病性等表型.为深入认识DSF对细菌性条斑病菌鞭毛基因簇的调控提供了科学依据.     

Biocontrol of bacterial soft rot of calla lily by elicitor HarpinXoo and N-acyl homoserine lactonase (AttM)

Bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum is a serious plant disease in Zantedeschia spp. (also called calla lily). In this study, two independent genes (a N-acyl homoserine lactonase gene attM from Agrobacterium tumefaciens and a hypersensitive response and pathogenicity gene hrf1 from Xanthomonas oryzae pv. oryzae), transcribed by a strong and constitutive Escherichia coli promoter P _lpp , respectively, were cloned into plasmid pUC19, and was transformed into E. coli, creating strain JM109/pPHA. The result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) assay showed that both genes (hrf1 and attM) were successfully expressed in one plasmid system in strain JM109/pPHA. The expressed Harpin_Xoo (Hrf1) and AttM protein had the ability of inducing hypersensitive response (HR) in nonhost tobacco and degrading the N-acyl homoserine lactones (AHLs) produced by P. carotovorum subsp. carotovorum, respectively, whereas Harpin_Xoo and AttM protein did not seem to interfere with the normal growth of this pathogen. In planta, strain JM109/pPHA could significantly reduce the soft rot disease severity on dormant tubers (control efficiency: 92.8%) or potted plants (control efficiency: 92.4%) of calla lily. We have first demonstrated the both biocontrol effects of Harpin_Xoo and AttM proteins (also described as Quorum interference) on the bacterial soft rot disease of calla lily, caused by P. carotovorum subsp. carotovorum. This work provided a potential way to control this serious plant disease.