土壤浸提液中硝酸盐氮氧同位素组成的反硝化细菌法测定

本研究优化了采用反硝化细菌法同时测定土壤浸提液中硝酸盐氮氧同位素组成的方法。在已有研究结果的基础上,通过采用5000~8000 r·min-1的转速离心、高纯氮气吹扫1 h、减少加样量及改造仪器自动进样器等措施对已发表方法进行了优化。对国际标准样品USGS34的分析表明,0.1~0.8μg NO-3-N样品量即可以得到较稳定、准确的测定值和校正值;同一时间内制备的硝酸盐δ15N的SD介于0.05‰~0.09‰之间,δ18O的SD介于0.28‰~0.48‰之间;在三个月之内δ15N和δ18O的测定值基本一致,表明该方法具有较好的准确度、精密度和稳定性。通过研究浸提剂、保存条件以及加热对测定土壤浸提液中硝酸盐氮氧同位素组成的影响,结果表明:常用的去离子水、KCl、Ca Cl2可能都含有微量的硝酸盐,随着加样量增大,浸提剂中含有的硝酸盐可能就会影响δ15N和δ18O的测定;对于土壤硝酸盐的浸提液,冷冻保存效果较好,保证了土壤硝酸盐氮氧同位素的准确性和稳定性;尽管加热对硝酸盐标准样品USGS34和IAEA-NO3的δ15N没有显著影响,但δ18O显著升高,说明加热易引起氧同位素分馏;而土壤硝酸盐浸提液样品加热前后的δ15N和δ18O的测定值没有显著变化,因此为避免产生氧同位素分馏和节省测试时间,建议同时测定土壤浸提液硝酸盐δ15N和δ18O时直接和反硝化细菌反应。应用本方法对不同肥料处理田间土壤浸提液硝酸盐的氮氧同位素组成进行了测定。     

松材线虫组DNA条形码筛选

本文基于Gen Bank中登录的14种松材线虫组线虫的28S、18S和ITS部分基因DNA序列,利用遗传距离法及分子生物学方法评价其各自作为松材线虫组DNA条形码序列的适用性。结果显示,28S(拟松材线虫不分亚种)、28S(拟松材线虫分亚种)、18S和ITS的基因序列种内成对遗传距离平均值分别为0.0071、0.0030、0.0007、0.0043,种间成对遗传距离平均值分别为0.0476、0.0454、0.0052、0.1556,其中28S、18S基因序列种内及种间距离存在一定程度的重叠,ITS具有一定程度的Barcoding gap。3个基因序列构建的NJ进化树显示,28S、ITS构建的系统进化树具有较高的节点支持率,可以有效将14个松材线虫组线虫分离成独立分支,而且28S可以有效鉴别出拟松材线虫的2个亚种;基于18S基因构建的进化树不能对吉拉尼伞滑刃线虫、日本冷杉伞滑刃线虫、拟松材线虫进行有效区分。因此,28S、ITS区具有一定的遗传距离间隔以及相对较高的物种识别率,可以作为松材线虫组线虫候选条形码基因。     

Exploring genetic diversity of rice cultivars for the presence of brown planthopper (BPH) resistance genes and development of SNP marker for Bph18

Brown planthopper (BPH) is the most devastating insect pest in rice‐growing areas. Information on availability of BPH resistance alleles and their sources enhances BPH‐resistant breeding programmes. In this study, 260 highly diversified rice cultivars or breeding lines were screened for the presence of five major BPH resistance genes (Bph10, Bph13, Bph18, Bph20 and Bph21) using gene‐specific markers. The analysis revealed that 137 of the 260 cultivars possess at least one BPH resistance gene. Bph10 was predominant while Bph20 was the least distributed. Moreover, two and three different resistance gene combinations were found in the cultivars. Molecular markers play an important role in molecular breeding programmes. A tightly linked PCR‐based co‐dominant Bph18 marker was developed, which is cost effective and time effective and simpler than available Bph18 CAPS marker (7312.T4A). We strongly believe that the identified BPH‐resistant cultivars can be used as alternative resistance gene sources and also as resource for novel BPH resistance genes. The developed Bph18 marker will be highly useful in molecular breeding applications of BPH‐resistant breeding programmes.     

宛麦18的生理特性及其与产量性状的关系

为了解小麦品种宛麦18高产的原因,以周麦22、内麦988和新麦19为对照,在高产麦田对宛麦18的生理特性及产量构成进行了研究.结果表明,宛麦18在灌浆期具有较高的叶面积指数、较稳定的净光合速率和灌浆速率,并且光合功能期长.宛麦18具有较多的次生根,根系吸收功能较强,营养物质积累量大.宛麦18较内麦988和新麦19相比分别增产21.3％和22.9％,差异达到显著水平.宛麦18的有效穗数和穗粒数与3个对照差异不显著,但千粒重显著高于内麦988和新麦19.说明生育后期较高的灌浆速率、光合性能、根系活力是宛麦18有较大千粒重,进而获得高产的重要原因.     

致猪水肿病大肠埃希菌多重PCR检测方法的建立及应用

本研究旨在建立一种快速鉴定致猪水肿病大肠埃希菌的多重PCR检测方法.分别针对大肠埃希菌16S rDNA、志贺毒素Stx2e A亚基和菌毛F18ab A亚基保守序列设计合成3对特异性引物,优化多重PCR反应条件,并进行特异性和敏感性检测.结果显示,阳性对照菌株扩增产物大小分别为1 062、733和313 bp.特异性和灵敏性检测结果表明,与肠炎沙门菌、多杀性巴氏杆菌、胸膜肺炎放线杆菌、副猪嗜血杆菌、支气管败血波氏杆菌和猪链球菌等猪常见致病菌均无交叉反应;菌体直接扩增法最低检出量为1 875 CFU.利用建立的多重PCR检测方法对分离收集的128株大肠埃希菌进行鉴定,得到36株致猪水肿病大肠埃希菌,其中30株既有菌毛F18ab又产志贺毒素Stx2e,另外6株仅产志贺毒素Stx2e.结果表明,本试验所建立的多重PCR检测方法对致猪水肿病大肠埃希菌的快速诊断和流行病学调查具有一定的应用价值.     

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.