我们感谢他们的技术贡献Servane Contal和鲍里斯Untereiner。我们承认FNR的“FUTOX”项目的支持。

该协议要求16天完成。详细的时间表是在图1。 缓冲液配制的详细信息<ul><li>制备细胞裂解液（每个样品3ML）。 </li><li>制备的洗涤液（50毫升每个样品）。这个缓冲区应预先冷冻和储存在-20 ° C在冰箱，直到进一步的分析，并在整个过程中保持在冰上。 </li><li>制备沉淀的缓冲液（每个样品20ML）。这个缓冲区应预先冷冻和储存在冰箱，直到需要在整个过程中，冰在-20 ° C。 </li><li>应当优先进行的工作，进入冷室在4 ° C。 </li></ul>真菌种类用于蛋白质组学分析三株被列为其形态特征和翻译延伸因子-α- 1分析的基础上禾谷镰刀菌（奥唐奈等， 1998） 这些菌株属于CRP -加布里埃尔李普曼收集，维护和存放在 - 80℃在15％甘油。 菌丝体的制备真菌培养4天，在25 ° C交替光明与黑暗的时期每12小时在V8上。文化支离破碎，用消毒的刀片和这些文化被用来接种250毫升锥形瓶含有100毫升的毒素诱导媒体件。收获后10天菌丝体，并从中分离，用​​无菌过滤器的过滤文化。菌丝体均用无菌水冲洗3次，以去除介质的残留物和其他化合物。我们然后立即加入液氮冻结的细胞，保持样品在-80 ° C。 蛋白提取的一般描述使用在图3a和3b中所描述的方法提取蛋白样品。 镰刀菌的样品在液氮研磨的粉末收集在10毫升聚四氟乙烯管。样品与裂解液孵育30分钟，煮沸10分钟，离心2次，在室温（15分钟12000克）。收集上清和沉淀缓冲液孵育过夜在-20 ° C。后离心4 ° C30000克45分钟，沉淀蛋白质的沉淀物洗净，用冷丙酮含数码地面电视的3倍。最后，沉淀空气干燥和蛋白质resolubilized标签缓冲区中，调整pH值至8。 30毫升的子样本被删除总蛋白定量和蛋白电泳在-20 ° C保存，直到剩余的上清液。 <img src="/files/ftp_upload/1690/1690fig1.jpg" alt="图1" /> 图1。时间轴的过程。 <img src="/files/ftp_upload/1690/1690fig2.jpg" alt="图2" /> 图2样品处理多个运营商的计划。 <img src="/files/ftp_upload/1690/1690fig3atmb.jpg" alt="图3a" border="0" /> 图3a。 <img src="/files/ftp_upload/1690/1690fig3btmb.jpg" alt="图3b" border="0" /> 图3b，请<a href="/files/ftp_upload/1690/1690fig3a.jpg">点击这里</a>看到的图3A更大的版本，或<a href="/files/ftp_upload/1690/1690fig3b.jpg">在</a>这里为图3b更大的版本。 图3a - B蛋白的提取过程的流程图（A：第一天，B：第二天） 。 <img src="/files/ftp_upload/1690/1690fig4tmb.jpg" alt="图4" border="0" /> 图4。蛋白提取物的一维图像。蛋白的运行，直到蓝色熔岩紫染色的凝胶年底完成迁移。图4看到一个更大的版本， <a href="/files/ftp_upload/1690/1690fig4.jpg">请点击</a>这里。

<ol>
	<li>Bridge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Extraction+from+Fungi.">Protein Extraction from Fungi.</a> Protein Purification Protocols. , 39-48 (1996).</li><li>Jiao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+different+carbon+sources+on+trichothecene+production+and+Tri+gene+expression+by+Fusarium+graminearum+in+liquid+culture.">Effects of different carbon sources on trichothecene production and Tri gene expression by Fusarium graminearum in liquid culture.</a> FEMS Microbiol Lett. 285, 212-219 (2008).</li><li>Kim, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+of+filamentous+fungi.">Proteomics of filamentous fungi.</a> Trends in Biotechnology. 25, 395-400 (2007).</li><li>Milles, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+proteomic+approach+to+monitor+protein+synthesis+in+mycotoxin+producing+moulds.">Development of a proteomic approach to monitor protein synthesis in mycotoxin producing moulds.</a> Mycotoxin Res. 23, 161-165 (2007).</li><li>Morrison, N., Field, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concept+of+sample+in+OMICS+technology.">Concept of sample in OMICS technology.</a> OMICS. 10, 127-137 (2006).</li><li>O'Donnell, K., Kistler, H. C., Cigelnik, E., Ploetz, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+evolutionary+origins+of+the+fungus+causing+Panama+disease+of+banana:+Concordant+evidence+from+nuclear+and+mitochondrial+gene+genealogies.">Multiple evolutionary origins of the fungus causing Panama disease of banana: Concordant evidence from nuclear and mitochondrial gene genealogies.</a> PNAS. 95, 2044-2049 (1998).</li><li>Paper, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+proteomics+of+extracellular+proteins+in+vitro+and+in+planta+from+the+pathogenic+fungus+Fusarium+graminearum.">Comparative proteomics of extracellular proteins in vitro and in planta from the pathogenic fungus Fusarium graminearum.</a> Proteomics. 7, 3171-3183 (2007).</li><li>Pasquali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video+in+science.+Protocol+videos:+the+implications+for+research+and+society.">Video in science. Protocol videos: the implications for research and society.</a> EMBO Rep. 8, 712-716 (2007).</li><li>Taylor, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+minimum+information+about+a+proteomics+experiment+(MIAPE).">The minimum information about a proteomics experiment (MIAPE).</a> Nat Biotechnol. 25, 887-893 (2007).</li><li>Taylor, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analyses+of+Fusarium+graminearum+grown+under+mycotoxin-inducing+conditions.">Proteomic analyses of Fusarium graminearum grown under mycotoxin-inducing conditions.</a> Proteomics. 8, 2256-2265 (2008).</li></ol>

使用这种技术（Kim 等人 ，2007年审查）的出版物的数量增加导致真菌生物学领域内的蛋白质组学方法的近期利益。依靠蛋白的提取方法提取程序的组合，他们往往是很难描述在方法论部分和需要的专业知识，在处理潜在的故障排除方法。 至于其他“组学”的办法，设置样品和数据处理的标准是必要的，才能产生可​​靠的科学信息。此外，样品和操作程序应得到充分的描述，以允许共享不同的“组学”实验结果的解释。跨数据挖掘在基因组学，蛋白质组学，代谢组学，充分利用潜力，这将是必不可少的... （莫里森等人 ，2006年）。约蛋白质组学实验的最低限度的信息已经起草工作组已成立，以解决一个实验的所有方面（凝胶电泳，质谱，分子间的相互作用，蛋白质修饰，蛋白质组信息，来样加工）。目前没有明确的信息，来样加工程序进行。由于蛋白的提取程序，在确定蛋白质组研究的最终质量，以实施程序，可以增加MIAPE指导方针的框架内的标准化（Taylor等，2007）的重要性，我们提出了使用视频协议详述样品处理。影片描述的实验可能会作出重大贡献的来样加工的信息数量增加，可能会导致更好的“组学”实验（Pasquali，2007年）的可重复性。 事实上，整个实验室的可重复性是一项基本要求，为了保证蛋白质组学研究结果（http://www.fixingproteomics.org）的有效性。跨实验室的可重复性是由两个因素：不同的仪器和不同的运营商操纵样品的影响。 在描述的协议，我们建议执行生物复制涉及多个运营商增加（图2和视频）数据（即技术变化的结果是考虑到）的可靠性。 蛋白提取描述的过程是基于SDS和暖气。这是以前，以保证良好的纯度和蛋白质的数量（1996年桥）。 SDS的耦合，用沸水溶解细胞壁，疏水蛋白，防止形成的低聚物，中止蛋白质沉淀。沸点还允许蛋白酶失活。 EDTA，PMSF和完整的迷你蛋白酶抑制剂也产生同样的效果。数码地面电视的消除和促进蛋白增溶蛋白质之间的二硫键。 为了消除前IEF SDS，蛋白质沉淀丙酮/ TCA /数码地面电视。此外，丙酮/ TCA /数码地面电视的沉淀可以去除一些污染物，如脂类，核酸，盐类或/和酚类化合物，这些化合物存在时。 SDS的一样，这些分子可以防止在国际盛事基金的一个很好的迁移。这种沉淀，这是必要的丙酮/数码地面电视，洗沉淀的蛋白质，以去除TCA的，因为它可以干扰IEF。 一个良好的样品制备是好的结果的关键。对于这一点，它也是必不可少的，以避免污染环境工作无粉手套和尊重良好实验室规范的蛋白质。从技术角度来看，在所描述的协议，获得足够数量和纯度蛋白质最关键的步骤是分两个阶段。首先，研磨阶段，完整的细胞破坏是根本释放的蛋白质;和第二，总蛋白的纯化，一个阶段，包括取消大多数脂类，DNA和其它污染物，可能会干扰蛋白质的迁移。

媒体和解决方案 掌上电脑 ：39克马铃薯葡萄糖琼脂（Difco公司）; 1升蒸馏水。 V8琼脂 ：200毫升V8（坎贝尔，美国），2G碳酸钙（Sigma公司），16G琼脂（DIFCO），800毫升蒸馏水 毒素诱导媒体 ：1G K 2 HPO 4，0.5克氯化钾，0.5克硫酸镁4 7H 2 O，10毫克FeEDTA，2G大号谷氨酸，蒸馏水1L 10G蔗糖。 裂解缓冲液 ： <table border="1"><tr><td></td><td> 100毫升</td><td>终浓度</td><td></td></tr><tr><td>三，盐酸PH8 </td><td> 5ML </td><td> 50MM </td><td> 1M </td></tr><tr><td> SDS </td><td> 2克或10ml </td><td> 2％ </td><td> 20％ </td></tr><tr><td>数码地面电视</td><td> 500毫升</td><td> 10MM </td><td> 2M </td></tr><tr><td> EDTA </td><td> 20ML </td><td> 0.1MM </td><td> 0.5M </td></tr><tr><td> PMSF </td><td> 200毫升</td><td></td><td></td></tr><tr><td>完整的迷你蛋白酶抑制剂</td><td> 1片</td><td></td><td></td></tr><tr><td>水</td><td> QSP </td><td></td><td></td></tr></table> 沉淀缓冲液 ： <table border="1"><tr><td></td><td> 100毫升</td><td>终浓度</td></tr><tr><td>三氯乙酸</td><td> 20ML </td><td> 20％ </td></tr><tr><td>数码地面电视</td><td> 0.1ml的</td><td> 0.1％ </td></tr><tr><td>丙酮</td><td> QSP </td><td></td></tr></table> 洗涤缓冲液： <table border="1"><tr><td></td><td> 100毫升</td><td>终浓度</td></tr><tr><td>数码地面电视</td><td> 0.1ml的</td><td> 0.1％ </td></tr><tr><td>丙酮</td><td> 100毫升</td><td></td></tr></table> 标签缓冲区（储存在-18 ° C的小等分）： <table border="1"><tr><td></td><td> 1ML </td><td>终浓度</td></tr><tr><td>尿素</td><td> 140毫克</td><td> 7M </td></tr><tr><td>硫脲</td><td> 50毫克</td><td> 2M </td></tr><tr><td> CHAPS </td><td> 40毫克</td><td> 4％ </td></tr><tr><td>三</td><td> 30μL </td><td> 30毫米</td></tr><tr><td>水</td><td> 1毫升</td><td></td></tr></table>

toxin induction and protein extraction from fusarium spp. cultures for proteomic studies

Fusaria是丝状真菌能够产生不同的毒素。镰刀霉菌毒素，如呕吐毒素，nivalenol，T2，zearelenone，镰刀菌酸moniliformin，等..对人类和动物健康有不良影响，有些是致病因素考虑。蛋白质组学研究表明，要有效地破译毒素生产机制（Taylor等，2008），以及确定潜在的致病因素（纸张等人，2007 年，Houterman等，2007） ，在Fusaria。因此，它成为根本，以建立可靠的方法之间的比较蛋白质组学研究，以依靠蛋白表达实验，菌种和实验室之间的真正差异。将要描述的过程应该通过两种方式蛋白质组学程序的标准化水平的提高作出贡献。拍过的协议是用来增加可以精确地描述的细节水平。此外，标准化的程序来处理生物可用性，复制，应保证较高的鲁棒性的数据，同时也考虑到内的提取过程中的技术可重复性的人为因素。 描述该协议要求16天完成14天的文化和两天蛋白的提取（图1）。 简单地说， 镰刀菌菌株4天的固体培养基上生长，然后手动割裂为10天，并转移到一个修改后的毒素诱导媒体（ 焦等，2008）。。通过Miracloth层过滤收集菌丝。在冷室中进行研磨。不同的运营商进行提取，复制（N = 3），以便考虑到由于技术的变化（图2）偏置。提取是基于Taylor等人所描述的SDS /数码地面电视的缓冲区。 （2008年），稍作修改。总蛋白的提取所需的使用丙酮/ TCA / DTT缓冲区隔夜和丙酮/数码地面电视洗衣机（图3A，3B）的蛋白质的降水过程。终于resolubilized蛋白在蛋白标签缓冲区和量化。提取的结果是一维凝胶（图4 SDS - PAGE），然后再进行二维凝胶电泳（IEF / SDS - PAGE）。同样的过程可以应用于其他越来越多的媒体和其他丝状真菌（ 万里等人，2007年）的蛋白质组分析。

Watch this Scientific Journal Video about Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies at JoVE.com

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

在真菌的蛋白质组分析蛋白质的提取要求高的水平要完成标准化，按照有关一个蛋白质组学实验（MIAPE）指引的最低限度的信息。我们目前的视频协议，其中包括一个程序，最大限度地减少在毒素诱导和蛋白提取实验偏见<em&gt;镰刀菌。</em

毒素诱导和蛋白质提取镰刀 SPP。</em蛋白质组学研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

toxin-induction-protein-extraction-from-fusarium-spp-cultures-for

Research

JoVE Journal

Biology

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

16.9K 次观看.  Centre de Recherche Public-Gabriel Lippmann. 在真菌的蛋白质组分析蛋白质的提取要求高的水平要完成标准化，按照有关一个蛋白质组学实验（MIAPE）指引的最低限度的信息。我们目前的视频协议，其中包括一个程序，最大限度地减少在毒素诱导和蛋白提取实验偏见镰刀菌。

视频: Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Gibberella zeae子囊孢子生产和收集微阵列实验。

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

科研

生物学

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p&#8804;0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng.g-1 of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng.g-1. This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other mycotoxins in major food crops.

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

We demonstrate a method for the analysis of aflatoxins and transgene expression in peanut seeds that contain RNA-interference signals for silencing aflatoxin-synthesis genes in the fungus Aspergillus flavus. RNAi-mediated control of mycotoxins in plants has not been reported previously.

黄曲霉毒素在花生RNAi介导的控制:方法分析霉菌毒素的产生和转基因表达的花生/<em&gt;黑</em&gt;病害系统

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That ...

环境科学

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

真菌 DNA 分析用职业空气标本的收集与提取

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

使用内核生物测定真菌孢子发生和霉菌毒素生产的定量

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

免疫与感染

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are potent carcinogens that substantially reduce crop value in maize and other oil rich crops like peanut besides posing serious threat to human and animal health. Different approaches, including traditional breeding, transgenic expression of resistance associated proteins, and RNA interference (RNAi)-based host-induced gene silencing of critical A. flavus gene targets, are being evaluated to increase aflatoxin resistance in susceptible crops. Past studies have shown an important role of &#945;-amylase in A. flavus pathogenesis and aflatoxin production, suggesting this gene/enzyme is a potential target to reduce both A. flavus growth and aflatoxin production. In this regard, the current study was undertaken to evaluate heterologous expression (under control of the constitutive CaMV 35S promoter) of a Lablab purpureus L. &#945;-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is a competitive inhibitor of A. flavus &#945;-amylase enzyme and belongs to the lectin&#8211;arcelin&#8211;&#945;-amylase inhibitor protein family in common bean. In vitro studies prior to the current work had demonstrated the role of AILP in inhibition of A. flavus &#945;-amylase activity and fungal growth. Fungal growth and aflatoxin production in mature kernels were monitored in real time using a GFP-expressing A. flavus strain. This kernel screening assay (KSA) is very simple to set up and provides reliable and reproducible data on infection and the extent of spread that could be quantified for evaluation of germplasm and transgenic lines. The fluorescence from the GFP strain is closely correlated to fungal growth and, by extension, it is well-correlated to aflatoxin values.&#160; The goal of the current work was to implement this previous knowledge in a commercially important crop like maize to increase aflatoxin resistance. Our results show a 35%&#8211;72% reduction in A. flavus growth in AILP-expressing transgenic maize kernels which, in turn, translated into a 62%&#8211;88% reduction in aflatoxin levels.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Here we present a protocol to analyze Aspergillus flavus growth and aflatoxin production in maize kernels expressing an antifungal protein.&#160; Using a GFP-expressing A. flavus strain we monitored the infection and spread of the fungus in mature kernels in real time. The assay is rapid, reliable, and reproducible.

转基因玉米中黄曲霉生长和黄曲霉毒素的产生表达表达.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

生物化学

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

无标签定量蛋白经济学工作流程，用于发现驱动的宿主-病原体交互

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species. 
We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis. 
The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

从土壤中分离可培养酵母和霉菌以研究真菌种群结构

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

分离、表征和总 DNA 提取以鉴定真菌异养植物中的内生真菌

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...

毒素诱导和蛋白质提取镰刀 SPP。文化

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