微生物源农药申嗪霉素的研制与应用

申嗪霉素是中国自主研发的一种新型微生物源农药,具有高效、安全、广谱等特点,其主要成分是甜瓜根际促生菌M18产生的次级代谢产物吩嗪-1-羧酸。文章重点就申嗪霉素产生菌M18的分离及其代谢产物的鉴定、申嗪霉素生物合成及调控机理最新研究进展、申嗪霉素高产工程菌株的构建和产业化、以及申嗪霉素大田防病试验结果及推广应用情况等进行详细综述,并对其抗菌作用机理进行探讨,旨在为申嗪霉素的生物合成机理研究、遗传和代谢改造以及推广应用提供参考。     

小麦重要自噬相关基因ATG18的鉴定和表达分析

【目的】克隆小麦重要自噬相关基因ATG18,分析其编码产物的序列特征和高级结构,了解其在生物、非生物胁迫及激素处理条件下的表达模式,阐明其生物学功能。【方法】利用EST拼接和RT-PCR方法从白粉菌侵染的小麦叶片中克隆ATG18 cDNA序列,利用生物信息学方法进行基因的外显子-内含子结构分析、编码蛋白的结构域和保守氨基酸预测、高级结构分析和物种间同源蛋白的进化分析。采用实时荧光定量PCR方法研究基因表达对白粉菌侵染和外源激素处理的响应模式及对高盐、干旱、低温黑暗和缺氮培养等逆境胁迫处理的响应模式。【结果】获得了4个小麦ATG18家族成员（TaATG18a、TaATG18b、TaATG18c和TaATG18d）cDNA。4个基因高度相似,均含有1 158 bp开放阅读框（ORF）,编码385个氨基酸的蛋白质。4个基因的编码蛋白在一级结构上均含有典型的WD-40结构域、磷脂酰肌醇-3-磷酸（PI3P）结合基序和保守的ATG2结合位点氨基酸残基。4个基因均具有2种转录后的可变剪接方式,2种剪接产物mRNA分别编码完整的有功能蛋白和N端截短导致结构域和功能位点缺失的无功能蛋白。TaATG18a蛋白在高级结构上折叠成与其他WD-40蛋白类似的β-推进器样构象,PI3P结合基序位于推进器第五叶片的折叠4和第五、六叶片的连接部分,ATG2结合位点位于连接第二叶片和第三叶片的loop上。TaATG18s能够被白粉菌侵染诱导表达,但具体的诱导表达模式在抗、感白粉病反应之间存在明显差异。在广谱抗白粉病基因Pm21和小种专一性抗白粉病基因Pm3f介导的抗病反应中,TaATG18s均呈现接种白粉菌后0-36 h期间的2次诱导表达模式,2次诱导表达时间与白粉菌侵染进程密切相关。在中感材料扬麦158遗传背景上的感病反应中,TaATG18s尽管也呈现2次诱导表达,但第一次诱导表达持续时间短且强度低于含Pm21的近等基因系上抗病反应中的表现,相反第二次诱导表达强度高于抗病反应中的表现。高感材料Chancellor遗传背景上的TaATG18s响应白粉菌侵染的表达波动较小。外源乙烯或SA处理对TaATG18s表达的调控作用在抗、感白粉病材料上明显不同,在感病材料上表现激活作用,而在抗病材料上表现抑制作用。激素处理对TaATG18s表达的调控不仅作用于转录水平,还作用于转录后的mRNA剪接环节。高盐、干旱、低温黑暗和缺氮培养等逆境处理也能够上调TaATG18s的表达。【结论】推测鉴定的4个TaATG18s编码蛋白具有通过结合PI3P定位于自噬膜表面和通过形成ATG18-ATG2复合物参与小麦自噬过程的功能。TaATG18s及其参与的自噬过程与小麦对白粉菌侵染的免疫反应密切相关,也与小麦响应非生物逆境胁迫环境相关。抗、感材料TaATG18s对同种激素信号的不同响应模式可能是导致抗、感白粉病表型差异的原因之一。     

Surveying soil faunal communities using a direct molecular approach

Soil faunal communities are often phylogenetically diverse and the accurate assessment of the taxonomic structure of these communities is both time-consuming and requires a high level of taxonomic expertise. Here we describe a DNA sequence-based methodology for characterizing soil micro- and mesofaunal communities that is similar to the molecular approaches commonly used to survey soil microbial diversity. The technique involves the direct extraction of faunal DNA from soil, PCR amplification of the extracted DNA with metazoan-specific primers, followed by the construction of clone libraries and direct sequencing of individual PCR products. We used this technique to characterize micro- and mesofaunal community composition from six individual soils representing two land-use types. The technique captured the more abundant faunal groups in the soils (nematodes, Collembola, Acari, tardigrades, enchytraeids) and provided sufficient taxonomic resolution to describe the overall structure of the communities. We compared the results obtained using this molecular approach to results obtained using a traditional, microscopy-based approach and found that the results were broadly similar. However, since biases are inherent in both methods it remains unclear which method provides a more accurate assessment of soil faunal community composition. Although this molecular approach has some distinct disadvantages over the more widely-used direct extraction methods, one advantage is that the taxonomic identification it can provide will be more accurate and consistent across research groups, facilitating effective comparisons of mesofaunal surveys.     

基于PIC18F2580的畜禽舍温湿环境控制系统

温度和湿度是畜禽养殖环境中重要的环境因素,它们的高低是否合适直接影响到畜禽养殖中畜禽的健康和生长性能,对畜禽养殖有着重要的意义.根据畜禽舍温湿度控制的要求,应用单片机技术设计了畜禽舍温湿度控制系统,该系统采用PIC18F2580芯片,在控制软件的支持下,CPU对外围电路进行控制,实现了对温度的控制,有良好的适应性.实践表明,系统运行稳定,降温效果明显,平均降温幅度为4.15℃,温度测量精度可达到±0.4℃,湿度测量精度可达到±3.0%RH,有效地缓和了温度过高对于畜禽产生的影响,有较高的推广价值.     

Porcine CD18 mediates Actinobacillus pleuropneumoniae ApxIII species-specific toxicity

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, produces Apx toxins that are recognized as major virulence factors. Recently, we showed that ApxIIIA-cytotoxic activity specifically targets Sus scrofa leukocytes. Since both LtxA from Aggregatibacter actinomycetemcomitans (aggressive periodontitis in humans) and LktA from Mannheimia haemolytica (pneumonia in ruminants) share this characteristic, respectively towards human and ruminant leukocytes, and because both use the CD18 subunit to interact with their respective LFA-1, we hypothesized that ApxIIIA was likely to bind porcine CD18 to exercise its deleterious effects on pig leukocytes. A β ₂−integrin-deficient ApxIIIA-resistant human erythroleukemic cell line was transfected either with homologous or heterologous CD11a/CD18 heterodimers using a set of plasmids coding for human (ApxIIIA-resistant), bovine (-resistant) and porcine (-susceptible) CD11a and CD18 subunits. Cell preparations that switched from ApxIIIA-resistance to -susceptibility were then sought to identify the LFA-1 subunit involved. The results showed that the ApxIIIA-resistant recipient cell line was rendered susceptible only if the CD18 partner within the LFA-1 heterodimer was that of the pig. It is concluded that porcine CD18 is necessary to mediate A. pleuropneumoniae ApxIIIA toxin-induced leukolysis.     

羊白细胞介素-18成熟蛋白的酵母表达和生物学活性研究

为了研究重组羊白细胞介素(gIL)-18在酵母系统中的高效表达,进一步阐明该重组蛋白的生物学活性,以含有gIL-18基因的重组质粒为模板进行PCR扩增,构建重组表达质粒pPICZ-gIL-18,转化于毕赤酵母GSll5,以甲醇诱导表达,经sDS-PAGE和western blot分析证实了重组蛋白的表达,分泌的重组gIL-18表达量为100 mg/L.经纯化后用MTT法和MDBK-VSV法检测表达的重组IL-18体外生物活性,利用免疫试验检测了重组蛋白对羊痘疫苗的免疫增强作用.实验结果表明,该蛋白具有诱导MDBK细胞分泌IFN-γ和刺激PBMC增殖的生物学活性,比活性为1.5×105 u/mg.体外具有增强羊痘疫苗的活性.毕赤酵母分泌表达的重组gIL-18具有良好的生物学活性,为gIL-18作为免疫佐剂和免疫治疗剂的大规模应用奠定了基础.     

USE OF 3'-DEOXY-3'-[18F]FLUOROTHYMIDINE PET/CT FOR EVALUATING RESPONSE TO CYTOTOXIC CHEMOTHERAPY IN DOGS WITH NON-HODGKIN'S LYMPHOMA

Imaging and measurement of proliferation with computed tomography (CT) and positron emission tomography (PET) provide a noninvasive method for improved staging and monitoring of response to cancer treatment. We evaluated prospectively the proliferation marker 3'-deoxy-3'[¹⁸F] fluorothymidine (FLT) in the context of FLT-PET/CT for detection of early response, confirmation of posttreatment response, and prediction of relapse in dogs with non-Hodgkin's lymphoma. Nine dogs with non-Hodgkin's lymphoma who were scheduled to receive five cycles of an investigational cytotoxic chemotherapy agent were included. All dogs received baseline FLT-PET/CT imaging immediately before chemotherapy. Intent was to repeat imaging with FLT-PET/CT at various time points: group 1 ( n =4), 5 days after initiation of chemotherapy and 3 weeks following the last chemotherapy administration; group 2 ( n =5), before the fourth cycle of chemotherapy and 3 weeks following the last administration. Two dogs in group 2 did not undergo repeat PET/CT. Body mass standardized uptake values (SUV) for FLT were calculated for each dog. Eight dogs had initially increased FLT uptake (mean SUV_max=9.8 [2.6–22.3]). Mean SUV decreased significantly for the seven dogs that underwent follow-up PET/CT following chemotherapy (mean SUV_max=3.5 [1.1–7.9], P <0.016). Increased uptake preceded clinical and cytological evidence of relapse in two dogs. Ki-67 immunohistochemistry confirmed decreased proliferation corresponding to decreased SUV in three canine lymph node samples. FLT-PET/CT functional and anatomical imaging shows promise for the evaluation of response to cytotoxic chemotherapy in dogs with non-Hodgkin's lymphoma and for predicting relapse before standard clinical and clinicopathologic confirmation.     

Flow cytometric patterns in blood from dogs with non-neoplastic and neoplastic hematologic diseases using double labeling for CD18 and CD45

BACKGROUND: In dogs, flow cytometry is used in the phenotyping of immunologic cells and in the diagnosis of hemic neoplasia. However, the paucity of specific antibodies for myeloid cells and B lymphocytes and of labeled antibodies for multicolor techniques limits the ability to detect all leukocyte subpopulations. This is especially true for neoplastic and precursor cells. CD18 and CD45 are expressed on all leukocytes and are involved in cell activation, and together could be useful in helping determine cell lineage. OBJECTIVES: The purpose of this study was to double label canine blood for CD18 and CD45 and to use the differential expression of antigens to identify leukocyte populations in dogs with non-neoplastic and neoplastic hematologic diseases. METHODS: A template was developed using blood samples from 10 clinically healthy dogs and a back-gating technique. Differential leukocyte counts obtained with the template were compared with those obtained by manual and automated methods on blood samples from 17 additional healthy dogs. Blood samples obtained from 9 dogs with non-neoplastic (reactive) hematologic diseases and 27 dogs with hemic neoplasia were double stained for CD18 and CD45 using mouse anticanine CD18 monoclonal antibody (mAb) plus phycoerythrin-conjugated rat anticanine CD45 mAb and fluorescein isothiocyanate-conjugated rabbit antimouse IgG. Hemic neoplasms were diagnosed by cell morphology, and immunophenotypic and cytochemical markers. RESULTS: With the double label, neutrophils, eosinophils, monocytes, and T- and B-lymphocytes were identified. In reactive disorders, a population of activated neutrophils with high CD45 and CD18 expression was detected. In hemic neoplasia, cell lineage was easily determined, even in acute leukemia. CONCLUSIONS: Double labeling for CD18/CD45 may be useful as a screening method to evaluate hematologic diseases and help determine cell lineage, and to aid in the selection of a panel of antibodies that would be useful for further analysis.     

种植密度对新葵18号生长发育及产量品质的影响

[目的]通过研究不同密度下新葵18号的干物质积累、叶面积及叶面积指数、产量和含油率,分析种植密度对新葵18号生长发育及产量品质的影响.[方法]调查不同种植密度下新葵18号的生长数据,计算不同处理的干物质积累、叶面积、叶面积指数和产量,并测定含油率.[结果]种植密度对新葵18号的生长发育有着显著影响,低密度处理(4 000株/667 m2)的单株叶面积较高,高密度处理(5 500株/667 m2)的叶面积指数较高,产量、含油率和单位面积干物质积累方面以处理5 000株/667 m2的值最高.产量随密度的增大而增加,但是密度超过5 000株/667 m2后,产量则表现出减少的趋势.[结论]适宜的种植密度可以调节新葵18号个体发育,最大发挥群体效应,在新疆5 000株/667 m2是新葵18号的适宜种植密度,可以取得较高产量和品质.     

莱芜黑猪a1岩藻糖转移酶基因(FUT I)不同基因型对大肠杆菌F18抵抗力研究

为了研究不同猪种(山东省地方猪种莱芜黑猪和引进猪种大约克)和FUT I不同基因型仔猪对肠毒性大肠杆菌F18(ETEC F18)的抵抗能力和肠黏膜上皮细胞对ETEC F18 黏附能力的差异,在采用PCR-RFLP技术对莱芜黑猪和大约克FUT I基因型检测的基础上,选取易感性和抗性断奶仔猪,利用野生型ETEC F18标准株进行试验猪口服细菌攻毒试验和肠黏膜黏附试验.由于多方面原因,接受ETEC F18细菌攻毒的试验猪均未发病,无法判断试验猪对ETEC F18的抵抗能力.ETEC F18标准菌株能与所有FUT I敏感基因型仔猪的小肠黏膜上皮细胞黏附,而不能与抗性基因型仔猪小肠上皮黏膜细胞黏附,莱芜黑猪与大约克的肠黏膜上皮细胞与E.coli F18的黏附特性并无品种差异.但是养猪生产实践中,莱芜黑猪极少发生断奶仔猪水肿和腹泻病.所以除FUT I基因外,也许本地猪种有其他导致遗传抗性的突变或抗性基因.