Name: inhibition of aspergillus flavus growth and aflatoxin production in transgenic maize expressing the &#945;-amylase inhibitor from lablab purpureus l.
Uploaded: 2019-02-15T15:00:00
Description: Watch this Scientific Journal Video about Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the Î±-amylase Inhibitor from Lablab purpureus L. at JoVE.co

我们感谢阿肯色州大学的大卫·梅林特在早期开发和分析转基因玉米方面提供的协助。这项工作得到了 usda-ars cris 项目6054-4000-025-00d 的财政支助。在本文中提及商品名称或商业产品仅用于提供特定信息, 并不意味着美国农业部的推荐或认可。usda-ars 的平等就业机会政策规定人人机会平等, 并禁止在该机构人事政策、做法和业务的所有方面的歧视。

1. 质粒结构和玉米转化
<ol><li>pcr 利用引物 5 '-ttatatgatgatgatgcctccc-3 ' 和 5 '-atactggggggggggggggggtactagtagtagtagtagtagtagtagg-3 ' ' 对 lablab ailp进行扩增。pcr 条件包括在98°c 下的初始变性步骤 30秒 (步骤 1), 然后是98°c 下的变性 10秒 (步骤 2), 55°c 退火 30秒 (步骤 3), 72°c 的伸长率为 20秒 (步骤 4), 步骤2至步骤4的31个周期, 最后的伸长率步骤在 72°c 5分钟, 克隆 pcr 产品到一个修改后的 pcambia 1, 300 矢量使用xba...

<ol>
	<li>Ismaiel, A., Papenbrock, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycotoxins:+Producing+fungi+and+mechanisms+of+phytotoxicity.">Mycotoxins: Producing fungi and mechanisms of phytotoxicity.</a> Agriculture. 5 (3), 492-537 (2015).</li><li>Mitchell, N., Bowers, E., Hurburgh, C., Wu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+economic+losses+to+the+USA+corn+industry+from+aflatoxin+contamination.">Potential economic losses to the USA corn industry from aflatoxin contamination.</a> Food Additives &amp; Contaminants: Part A. 33 (3), 540-550 (2016).</li><li>Umesha, S., Manukumar, H. M., Chandrasekhar, B., Shivakumara, P., Shiva Kumar, J., Raghava, S., Avinash, P., Shirin, M., Bharathi, T. R., Rajini, S. B., Nandhini, M., Vinaya Rani, G., Shobha, M., Prakash, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aflatoxins+and+food+pathogens:+Impact+of+biologically+active+aflatoxins+and+their+control+strategies.">Aflatoxins and food pathogens: Impact of biologically active aflatoxins and their control strategies.</a> Journal of the Science of Food and Agriculture. , (2016).</li><li>Brown, R. L., Menkir, A., Chen, Z. Y., Bhatnagar, D., Yu, J., Yao, H., Cleveland, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+aflatoxin-resistant+maize+lines+using+recent+advances+in+technologies+-+a+review.">Breeding aflatoxin-resistant maize lines using recent advances in technologies - a review.</a> Food Additives &amp; Contaminants - Part A Chemistry, Analysis, Control, Exposure &amp; Risk Assessment. 30 (8), 1382-1391 (2013).</li><li>Abbas, H., Accinelli, C., Shier, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+control+of+aflatoxin+contamination+in+U.S.+crops+and+the+use+of+bioplastic+formulations+of+Aspergillus+flavus+biocontrol+strains+to+optimize+application+strategies.">Biological control of aflatoxin contamination in U.S. crops and the use of bioplastic formulations of Aspergillus flavus biocontrol strains to optimize application strategies.</a> Journal of Agricultural and Food Chemistry. 65, 7081-7087 (2017).</li><li>Udomkun, P., Wiredu, A. N., Nagle, M., M&#252;ller, J., Vanlauwe, B., Bandyopadhyay, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative+technologies+to+manage+aflatoxins+in+foods+and+feeds+and+the+profitability+of+application+&#8211;+A+review.">Innovative technologies to manage aflatoxins in foods and feeds and the profitability of application &#8211; A review.</a> Food Control. 76, 127-138 (2017).</li><li>Dohroo, N. P., Bhardwaj, S. S., Shyram, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amylase+and+invertase+activity+as+influenced+by+Pythium+pleroticum+causing+rhizome+rot+of+ginger.">Amylase and invertase activity as influenced by Pythium pleroticum causing rhizome rot of ginger.</a> Plant Disease Research. 2, 106-107 (1987).</li><li>Singh, R., Saxena, V. S., Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pectinolytic,+cellulolytic,+amylase+and+protease+production+by+three+isolates+of+Fusarium+solani+variable+in+their+virulence.">Pectinolytic, cellulolytic, amylase and protease production by three isolates of Fusarium solani variable in their virulence.</a> Indian Journal of Mycology and Plant Pathology. 19, 22-29 (1989).</li><li>Fakhoury, A. M., Woloshuk, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amy1,+the+&#945;-amylase+gene+of+Aspergillus+flavus:+Involvement+in+aflatoxin+biosynthesis+in+maize+kernels.">Amy1, the &#945;-amylase gene of Aspergillus flavus: Involvement in aflatoxin biosynthesis in maize kernels.</a> Phytopathology. 89 (10), 908-914 (1999).</li><li>Bluhm, B. H., Woloshuk, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amylopectin+induces+Fumonisin+B1+production+by+Fusarium+verticillioides+during+colonization+of+maize+kernels.">Amylopectin induces Fumonisin B1 production by Fusarium verticillioides during colonization of maize kernels.</a> Molecular Plant-Microbe Interactions. 18 (12), 1333-1339 (2005).</li><li>Gilbert, M. K., Majumdar, R., Rajasekaran, K., Chen, Z. Y., Wei, Q., Sickler, C. M., Lebar, M. D., Cary, J. W., Frame, B. R., Wang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference-based+silencing+of+the+a-amylase+(amy1)+gene+in+Aspergillus+flavus+decreases+fungal+growth+and+aflatoxin+production+in+maize+kernels.">RNA interference-based silencing of the a-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels.</a> Planta. 247 (6), 1465-1473 (2018).</li><li>Chen, Z. Y., Brown, R. L., Russin, J. S., Lax, A. R., Cleveland, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+corn+trypsin+inhibitor+with+antifungal+activity+inhibits+Aspergillus+flavus+&#945;-amylase.">A corn trypsin inhibitor with antifungal activity inhibits Aspergillus flavus &#945;-amylase.</a> Phytopathology. 89 (18944733), 902-907 (1999).</li><li>Fakhoury, A. M., Woloshuk, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+growth+of+Aspergillus+flavus+and+fungal+&#945;-amylases+by+a+lectin-like+protein+from+Lablab+purpureus.">Inhibition of growth of Aspergillus flavus and fungal &#945;-amylases by a lectin-like protein from Lablab purpureus.</a> Molecular Plant-Microbe Interactions. 14 (8), 955-961 (2001).</li><li>Mirkov, T. E., Wahlstrom, J. M., Hagiwara, K., Finardi-Filho, F., Kjemtrup, S., Chrispeels, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+relationships+among+proteins+in+the+phytohemagglutinin-arcelin-a-amylase+inhibitor+family+of+the+common+bean+and+its+relatives.">Evolutionary relationships among proteins in the phytohemagglutinin-arcelin-a-amylase inhibitor family of the common bean and its relatives.</a> Plant Molecular Biology. 26 (4), 1103-1113 (1994).</li><li>Kim, Y. H., Woloshuk, C. P., Cho, E. H., Bae, J. M., Song, Y. S., Huh, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+functional+expression+of+the+gene+encoding+an+inhibitor+against+Aspergillus+flavus+a-amylase,+a+novel+seed+lectin+from+Lablab+purpureus+(Dolichos+lablab).">Cloning and functional expression of the gene encoding an inhibitor against Aspergillus flavus a-amylase, a novel seed lectin from Lablab purpureus (Dolichos lablab).</a> Plant Cell Reports. 26 (4), 395-405 (2007).</li><li>Frame, B., Main, M., Schick, R., Wang, K., Thorpe, T. A., Yeung, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+22.">Ch. 22.</a> Plant Embryo Culture. 710, 327-341 (2011).</li><li>Rajasekaran, K., Sickler, C. M., Brown, R. L., Cary, J. W., Bhatnagar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+resistance+to+aflatoxin+contamination+in+kernels+of+maize+genotypes+using+a+GFP-expressing+Aspergillus+flavus+strain.">Evaluation of resistance to aflatoxin contamination in kernels of maize genotypes using a GFP-expressing Aspergillus flavus strain.</a> World Mycotoxin Journal. 6 (2), 151-158 (2013).</li><li>Rajasekaran, K., Sayler, R. J., Sickler, C. M., Majumdar, R., Jaynes, J. M., Cary, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Aspergillus+flavus+growth+and+aflatoxin+production+in+transgenic+maize+kernels+expressing+a+tachyplesin-derived+synthetic+peptide,+AGM182.">Control of Aspergillus flavus growth and aflatoxin production in transgenic maize kernels expressing a tachyplesin-derived synthetic peptide, AGM182.</a> Plant Science. , 150-156 (2018).</li><li>Shu, X., Livingston, D. P., Franks, R. G., Boston, R. S., Woloshuk, C. P., Payne, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+gene+expression+in+maize+seeds+during+colonization+by+Aspergillus+flavus+and+Fusarium+verticillioides.">Tissue-specific gene expression in maize seeds during colonization by Aspergillus flavus and Fusarium verticillioides.</a> Molecular Plant Pathology. 16 (4), 662-674 (2015).</li><li>Savary, S., Ficke, A., Aubertot, J. -. N., Hollier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crop+losses+due+to+diseases+and+their+implications+for+global+food+production+losses+and+food+security.">Crop losses due to diseases and their implications for global food production losses and food security.</a> Food Security. 4, 519-537 (2012).</li><li>Damalas, C. A., Eleftherohorinos, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pesticide+exposure,+safety+issues,+and+risk+assessment+indicators.">Pesticide exposure, safety issues, and risk assessment indicators.</a> International Journal of Environmental Research and Public Health. 8 (5), 1402-1419 (2011).</li><li>Kowalska, A., Walkiewicz, K., Kozie&#322;, P., Muc-Wierzgo&#324;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aflatoxins:+Characterisitcs+and+impact+on+human+health.">Aflatoxins: Characterisitcs and impact on human health.</a> Post&#281;py Higieny i Medycyny Do&#347;wiadczalnej (Online). 71, 315-327 (2017).</li><li>Rajasekaran, K., Cary, J. W., Cotty, P. J., Cleveland, T. E. <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+GFP-expressing+Aspergillus+flavus+strain+to+study+fungal+invasion,+colonization,+and+resistance+in+cottonseed.">Development of a GFP-expressing Aspergillus flavus strain to study fungal invasion, colonization, and resistance in cottonseed.</a> Mycopathologia. 165 (2), 89-97 (2008).</li><li>Punt, P., Dingemanse, M. A., Kuyvenhoven, A., Soede, R. D., Pouwels, P. H., van den Hondel, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+elements+in+the+promoter+region+of+the+Aspergillus+nidulans+gpdA+gene+encoding+glyceraldehyde-3-phosphate+dehydrogenase.">Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase.</a> Gene. 93 (1), 101-109 (1990).</li><li>Lee, L. W., Chiou, C. H., Klomparens, K. L., Cary, J. W., Linz, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+aflatoxin+biosynthetic+enzymes+Nor-1,+Ver-1,+and+OmtA+in+time-dependent+fractionated+colonies+of+Aspergillus+parasiticus.">Subcellular localization of aflatoxin biosynthetic enzymes Nor-1, Ver-1, and OmtA in time-dependent fractionated colonies of Aspergillus parasiticus.</a> Archives of Microbiology. 181 (3), 204-214 (2004).</li><li>Bhatnagar, D., Cary, J. W., Ehrlich, K., Yu, J., Cleveland, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+genetics+of+regulation+of+aflatoxin+production+and+Aspergillus+flavus+development.">Understanding the genetics of regulation of aflatoxin production and Aspergillus flavus development.</a> Mycopathologia. 162, 155-166 (2006).</li><li>Williams, W. P., Krakowsky, M. D., Scully, B. T., Brown, R. L., Menkir, A., Warburton, M. L., Windham, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+and+developing+maize+germplasm+with+resistance+to+accumulation+of+aflatoxins.">Identifying and developing maize germplasm with resistance to accumulation of aflatoxins.</a> World Mycotoxin Journal. 8 (2), 193-209 (2015).</li><li>Broekaert, W. F., van Parijs, J., Leyns, F., Joos, H., Peumans, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chitin-binding+lectin+from+stinging+nettle+rhizomes+with+antifungal+properties.">A chitin-binding lectin from stinging nettle rhizomes with antifungal properties.</a> Science. 245 (4922), 1100-1102 (1989).</li><li>Vanparijs, J., Broekaert, W. F., Goldstein, I. J., Peumans, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hevein-an+antifungal+protein+from+rubber-tree+(Hevea+brasiliensis)+latex.">Hevein-an antifungal protein from rubber-tree (Hevea brasiliensis) latex.</a> Planta. 183, 258-264 (1991).</li><li>Gozia, O., Ciopraga, J., Bentia, T., Lungu, M., Zamfirescu, I., Tudor, R., Roseanu, A., Nitu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+properties+of+lectin+and+new+chitinases+from+potato+tubers.">Antifungal properties of lectin and new chitinases from potato tubers.</a> Comptes Rendus de l'Academie des Sciences - Series III. 316 (8), 788-792 (1993).</li><li>Wisessing, A., Choowongkomon, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amylase+inhibitors+in+plants:+Structures,+Functions+and+Applications.">Amylase inhibitors in plants: Structures, Functions and Applications.</a> Functional Plant Science and Biotechnology. 6 (1), 31-41 (2012).</li><li>Tyagi, B., Trivedi, N., Dubey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a-amylase+inhibitor:+A+compelling+plant+defense+mechanism+against+insect/pests.">a-amylase inhibitor: A compelling plant defense mechanism against insect/pests.</a> Environment &amp; Ecology. 32 (3), 995-999 (2014).</li><li>Powers, J. R., Culbertson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+effect+of+bean+amylase+inhibitor+on+insect+amylases.">In vitro effect of bean amylase inhibitor on insect amylases.</a> Journal of Food Protection. 45, 655-657 (1982).</li><li>Gatehouse, A. M. R., Fenton, K. A., Jepson, I., Pavey, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+a-amylase+inhibitors+on+insect+storage+pests:+Inhibition+of+a-amylase+in+vitro+and+effects+on+development+in+vivo.">The effects of a-amylase inhibitors on insect storage pests: Inhibition of a-amylase in vitro and effects on development in vivo.</a> Journal of the Science of Food and Agriculture. 37, 727-734 (1986).</li><li>Blanco-Labra, A., Chagolla-Lopez, A., Martinez-Gallardo, N., Valdes-Rodriguez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+characterization+of+the+12-kDa+protease+a-amylase+inhibitor+present+in+maize+seeds.">Further characterization of the 12-kDa protease a-amylase inhibitor present in maize seeds.</a> Journal of Food Biochemistry. 19, 27-41 (1995).</li><li>Abdollahi, A., Buchanan, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+aflatoxin+biosynthesis:+Induction+of+aflatoxin+production+by+various+carbohydrates.">Regulation of aflatoxin biosynthesis: Induction of aflatoxin production by various carbohydrates.</a> Journal of Food Science. 46, 633-635 (1981).</li><li>Liu, J., Sun, L., Zhang, N., Zhang, J., Guo, J., Li, C., Rajput, S. A., Qi, D. <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+nutrients+in+substrates+of+different+grains+on+aflatoxin+B1+production+by+Aspergillus+flavus.">Effects of nutrients in substrates of different grains on aflatoxin B1 production by Aspergillus flavus.</a> BioMed Research International. 2016, (2016).</li><li>Uppala, S. S., Bowen, K. L., Woods, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-harvest+aflatoxin+contamination+and+soluble+sugars+of+peanut.">Pre-harvest aflatoxin contamination and soluble sugars of peanut.</a> Peanut Science. 40 (1), 40-51 (2013).</li></ol>

由于病原体和害虫造成的农作物产量损失是一个全球性问题20。目前, 合成杀菌剂和农药的应用是控制植物病原体和害虫的主要手段, 但这些生化物质在食品和饲料中的残留毒性会对人类和动物健康构成严重威胁21。考虑到玉米作为食品和饲料作物的经济重要性, 减少或消除黄曲霉毒素污染至关重要.<sup class="xref...

真菌属、曲霉菌、镰刀菌、青霉素、黄曲霉等霉菌毒素污染是世界范围内种植的 1,2,3种食品和饲料作物的主要问题. 在这些植物致病性真菌中,曲霉菌对作物价值和人类及动物健康的不良影响最大。黄曲霉(黄曲霉) 是一种机会性植物病原体, 感染石油丰富的作物, 如玉米, 棉籽和花生, 并产生强效致癌物质, 黄曲霉毒素, 以及许多有毒的次生代谢物 (sm)。玉米是世界上种植的一种重要食品和饲料作物, 极易受到黄曲霉的污染。黄曲霉毒素污染对玉米损失和贬值的经济影响在美国可能高达 $686.6/年2随着全球气候的预测, 黄曲霉毒素的影响可能会给玉米带来更大的经济损失。估计在不久的将来为1.68 亿美元.鉴于黄曲霉毒素对人类和牲畜的不利经济和健康影响, 收获前控制玉米黄曲霉毒素可能是防止食品和饲料产品中黄曲霉毒素污染的最有效方法。
玉米黄曲霉毒素耐药性的主要收获前控制方法在过去几十年中得到了广泛的应用, 主要是通过育种, 这需要大量的时间4。近年来, 生物控制在大规模野外应用 5、6 中的黄曲霉毒素还原取得了一定的成功。除了生物控制外, 通过 rnai 应用尖端分子工具, 如 "宿主诱导基因沉默" (higs), 以及抗性相关蛋白的转基因表达, 在减少黄曲霉生长和黄曲霉毒素方面取得了一定的成功。在小型实验室和实地研究中生产。这些方法目前正在优化, 除了确定新的潜在的a. 黄酮基因目标, 为未来的操作。
除了直接参与霉菌毒素生产的基因作为转基因控制策略的潜在靶点外, 真菌淀粉酶在维持早期成功的发病机制和霉菌毒素的产生方面发挥着至关重要的作用宿主植物感染。几个例子包括百合 (生姜根茎腐病的因果剂)、香菇(菜花枯萎病的因果剂), 在那里观察到致病性与α-淀粉酶表达和活性之间的正相关7,8。通过基因敲除或敲除方法抑制α-淀粉酶活性对真菌生长和毒素产生有负面影响。一种α-淀粉酶敲除突变体在淀粉基板或脱胶玉米果仁9上生长时, 无法产生黄曲霉毒素.同样, 在黄萎病中, α-淀粉酶敲除菌株在玉米果仁感染 10期间未能产生富莫尼辛 b1 (霉菌毒素).在最近的一项研究中, gilbert 等人 (2018年) 表明, 通过 higs 对黄曲霉α-淀粉酶的 rnai 表达进行的 rnai 敲除, 可显著降低玉米籽粒感染期间黄曲霉的生长和黄曲霉毒素的产生 11.
α-淀粉酶活性的特定抑制剂也产生了类似于α-淀粉酶表达的下调结果。关于α-淀粉酶抑制剂在真菌抗真菌作用的第一份报告来自于对抗黄酮类化合物的玉米系14-kda 胰蛋白酶α-淀粉酶抑制剂的分离和表征。fakhoury 和 woloshuk 进一步筛选了数百种植物, 从 lablab pureus l. 13 种子中鉴定出一种 36 kda α-淀粉酶抑制剂样蛋白 (ailp). ailp 的肽序列类似于普通豆 14,15 中报道的属于凝集素-arcelin-α-淀粉酶抑制剂家族的凝集素。纯化的 ail 对哺乳动物胰蛋白酶没有任何抑制作用, 进一步的体外表征显示出显著抑制黄曲霉的生长和圆锥形萌发 13.这里提供的报告清楚地表明, α-淀粉酶可以作为控制方法的目标, 以限制依赖淀粉动员 (通过α-淀粉酶活性) 和获取可溶性糖作为能源在与宿主植物的致病性相互作用。
众所周知, α-淀粉酶在a.黄酮致病性9、10、11中至关重要, 并考虑到 ilp 作为一种有效的抗 a.黄酮类药物 (α-淀粉酶抑制剂/抗生长)的重要性,在本构 camv 35s 启动子下, 我们产生了表达 lablab ailp 基因的转基因玉米植株。目的探讨这种α-淀粉酶抑制剂在玉米籽粒感染过程中是否能有效对抗黄曲霉的发病机制和黄曲霉毒素的产生。结果表明, 转基因玉米籽粒在籽粒感染过程中, 其含量呈 ilp, 显著降低了黄曲霉的生长和黄曲霉毒素的产生。

<table>
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Caisson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amazing Marine Goop</td>
			<td>Eclectic Products</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C1000 Touch CFX96 Real-Time System</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;Falcon Tissue Culture Dishes, 60 mm</td>
			<td>Fisher Scientific</td>
			<td>08-772F</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf&#160;5424 Microcentrifuge</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flask with stopper, 50 mL</td>
			<td>Ace Glass</td>
			<td>6999-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FluoroQuant Afla</td>
			<td>Romer Labs</td>
			<td>COKFA1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluted Qualitative Filter Paper Circles, 15 cm</td>
			<td>Fisher Scientific</td>
			<td>09-790-14E</td>
			<td></td>
		</tr>
		<tr>
			<td>Force Air Oven</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FQ-Reader</td>
			<td>Romer Labs</td>
			<td>EQFFM3010</td>
			<td></td>
		</tr>
		<tr>
			<td>Geno/Grinder 2010</td>
			<td>OPS Diagnostics</td>
			<td>SP 2010-115</td>
			<td></td>
		</tr>
		<tr>
			<td>Innova 44 Incubator Shaker</td>
			<td>Brunswick Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad</td>
			<td>1708890</td>
			<td></td>
		</tr>
		<tr>
			<td>liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low Form Griffin Beakers, 100 mL</td>
			<td>DKW Life Sciences</td>
			<td>14000-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene Chloride</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nexttec 1-step DNA Isolation Kit for Plants</td>
			<td>Nexttec</td>
			<td>47N</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse E600 microscope with Nikon DS-Qi1 camera</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon SMZ25 stereomicroscope with C-HGFI Episcopic Illuminator and Andor Zyla 4.2 sCMOS camera</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Square BioAssay Dishes</td>
			<td>ThermoFisher Scientific</td>
			<td>240835</td>
			<td></td>
		</tr>
		<tr>
			<td>Phire Plant Direct PCR Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>F130WH</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate Vials, 15 ml</td>
			<td>OPS Diagnostics</td>
			<td>PCRV 15-100-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Potato Dextrose Broth</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Snap Cap, 22 mm</td>
			<td>DKW Life Sciences</td>
			<td>242612</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate dibasic heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrum Plant Total RNA Kit</td>
			<td>Sigma-Aldrich</td>
			<td>STRN50</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Grinding Balls, 3/8&#39;&#39;</td>
			<td>OPS Diagnostics</td>
			<td>GBSS 375-1000-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir Plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy 4 Fluorometer</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T-9284</td>
			<td></td>
		</tr>
		<tr>
			<td>V8 juice</td>
			<td>Campbell&#39;s</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Qualitative Grade Plain Sheets, Grade 3</td>
			<td>Fisher Scientific</td>
			<td>09-820P</td>
			<td></td>
		</tr>
		<tr>
			<td>Wrist-Action Shaker</td>
			<td>Burrell Scientific</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

inhibition of aspergillus flavus growth and aflatoxin production in transgenic maize expressing the &#945;-amylase inhibitor from lablab purpureus l.

食品和饲料作物中的黄曲霉毒素污染是全世界面临的主要挑战。黄曲霉( 黄曲霉)真菌产生的黄曲霉是一种强效致癌物质, 除了对人类和动物健康构成严重威胁外, 还能大幅降低玉米和花生等其他盛产石油作物的作物价值。不同的方法, 包括传统育种, 抗性相关蛋白的转基因表达, 以及 rna 干扰 (rna) 为基础的宿主诱导基因沉默的关键a. 黄酮基因靶材, 正在评估增加易感作物中的黄曲霉毒素耐药性。过去的研究表明α-淀粉酶在黄曲霉发病机制和黄曲霉毒素生产中的重要作用, 这表明这种基因酶是减少黄曲霉生长和黄曲霉毒素生产的潜在目标。在这方面, 目前的研究是为了评估玉米中一种乳突状蛋白 (ail) 对黄曲霉的异源表达 (在本构的 camv 35s启动子的控制下)。ailp 是一种-kDa 蛋白, 是黄曲霉α-淀粉酶的竞争抑制剂, 属于普通豆中的凝集素-arcelin-α-淀粉酶抑制剂蛋白家族。在目前的研究之前的体外研究已经证明了自吧升在抑制黄曲霉α-淀粉酶活性和真菌生长方面的作用。采用 gfp 表达的黄曲霉菌株对成熟籽粒的真菌生长和黄曲霉毒素的产生进行了实时监测。这种核筛选试验 (ksa) 是非常简单的建立, 并提供可靠和可重复的数据感染和传播的程度, 可以量化的种质和转基因线的评估。gfp 菌株的荧光与真菌生长密切相关, 进而与黄曲霉毒素值密切相关。 目前工作的目标是在玉米等具有商业重要性的作物上落实这一先前的知识, 以提高黄曲霉毒素的抗性。我们的结果显示, 在 ailp 表达转基因玉米籽粒中, 黄曲霉的生长减少了 35%--72%, 进而转化为黄曲霉毒素水平下降 62%-88%。

转基因植物的玉米转化与分子筛选
利用农杆菌eha101 菌株, 在 camv 35s 的控制下, 利用含有表达 lablab purpureus aip 基因的最终植物目的载体的农杆菌 eha101 菌株转化玉米 hi-ii 系的未成熟胚胎促进。五条独立转化的玉米系被推进到 t6 一代, 以便随后进行研究。转基因玉米植株较小, 活力较低, 但?...

Watch this Scientific Journal Video about Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the Î±-amylase Inhibitor from Lablab purpureus L. at JoVE.co

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

本文提出了一种分析具有抗真菌蛋白的玉米籽粒黄曲霉生长和黄曲霉毒素生产的方案。 利用 gfp 表达的黄曲霉菌株, 实时监测了成熟籽粒中真菌的感染和传播情况。该检测快速、可靠且重现性好。

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 ...

在这里，我们提出一个简单的内核筛选分析，以分析在玉米核表达抗真菌蛋白的阿斯佩吉卢斯飞松生长和黄曲霉素生产。使用表达阿斯佩吉卢斯黄花菌株的绿色荧光蛋白，我们实时监测成熟内核中真菌的感染和传播。这种简单的测定为评估玉米仁抵抗阿斯佩吉卢斯黄曲霉菌感染和黄曲霉素生产的能力提供了可靠的方法。在成熟内核的受控条件下进行的实验室检测结果与现场条件下的感染结果相关。演示该程序将是来自我们实验室的博士后拉杰蒂拉克·马朱姆达尔博士，以及来自我们实验室的生物科学技术人员克里斯汀·西克勒和大卫·安布罗西奥。首先，在培养皿中粘合 20 个两毫米的扣帽，以创建 KSA 盖。在使用瓶盖之前，让胶水干燥 48 小时。在生物安全柜中喷洒一个方形生物测定托盘，水中有70%乙醇。让托盘空气干燥。将无菌色谱纸添加到干纸盒中。用 70% 乙醇喷洒 9 个 KSA 瓶盖，让它们风干。然后把帽子放在生物测定托盘上。对于正在测试的每个转基因玉米生产线，选择20个未损坏的玉米仁，并把它们放入50ml离心管中。使用台式立体显微镜仅选择未损坏的内核进行检测。受损的内核更容易受到阿斯佩吉卢斯攻击，并产生更多的黄曲霉毒素，从而扭曲结果，不代表品种的真正抗真菌或抗菌特性。在每个管中加入70%乙醇。让内核消毒四分钟，偶尔轻轻摇动它们。在此轻轻倒出乙醇后，轻轻冲洗的内核在无菌去ion化水三次。获得为期六天的阿斯佩吉卢斯火焰染色70表达GGFP生长在V8介质上的六天文化。在去化水中加入20毫升无菌0.02%Triton X-100。并使用无菌循环刮掉孢子。从幼崽上移出，将其转移到300毫升无菌烧杯中。用 0.02% Triton X-100 准备五倍稀释，并使用血细胞计执行孢子计数。如有必要，再次稀释，以获得100毫升，浓度为每毫升400万孢子。将烧瓶倒入空烧杯中。将内核放入无菌的 300 ml 烧杯中，搅拌杆，搅拌三分钟。使用钳子将内核转移到生物测定盘中，确保每个盖中只放置一个内核。在每个生物测定托盘的底部加入30毫升的去维化水。并在31摄氏度下孵育7天。使用 A.flavus 接种七天后，拍摄四个内核的照片，这些内核被指定为显微分析和摄影。然后使用软组织和去化水来清洁内核的外部。执行内核的纵向部分，并立即在荧光显微镜下拍照。要准备内核进行分析，请用软组织和去维水清洁剩余的内核外部。将四个内核（组成一个代表）放入包含两个不锈钢球的 15 ml 螺帽聚碳酸酯小瓶中。立即将小瓶冷冻在液氮中，并储存在80摄氏度，直到准备好进行进一步处理和分析。准备就绪后，从冰柜中取出内核，并使用均质器以 1500 rpm 转速研磨三分钟。使用相同的地面材料进行GP分析、黄曲霉毒素测定和分子分析，使所有值都代表样品。虽然建议至少进行三次复制，但更多复制总是更好的，具体取决于高质量内核的可用性。在这项研究中，利用含有最终植物目标载体的农业细菌纪元EHA101菌株转化了玉米HI-II线的未成熟胚胎，该菌株在花椰菜马赛克病毒的35S促进剂的控制之下表达Lablab purpureus AILP基因。目标AILP基因的PCR扩增显示，仅在转基因玉米线中观察到548bp安培。AILP基因在转基因线中表现出表达，而在对照植物中未观察到AILP表达。早期发芽孢子暴露在来自年轻转基因玉米植物的粗叶提取物中20小时。与负对比相比，暴露于转基因叶提取物的孢子的全长比负控制减少了58%至80%。使用的 A.flavus 菌株包含一个 GFP 报告器，能够实时监测和定量真菌生长。与等源负对比，转基因AILP内核中GGFP荧光显著减少。AILP线4和5、69%和72%的真菌生长显著下降。与控制内核相比，其他行显示减少了 35 到 60%。与控件相比，在 AILP 内核中观察到 Aflatoxin 含量减少了 62% 到 88%。第四行的Aflatoxin含量最低，为每克486纳克，其次是内核中每克640至1498纳克的其他行。按照此过程，可以执行其他方法，例如使用荧光计来量化 GFP 及其真菌生长。免疫性商用试剂盒或 HPLC 或 EPLC 可用于执行黄曲霉毒素定量。KSA 的可用性提供了一种快速而简单的方法，用于确定对黄曲霉毒素污染的耐药性。使用这种技术可以快速评估不同玉米育种线。虽然内核筛选检测不危险，但操作者应保护自己免受接触 Aspergillus 飞毛孢菌，使用适当的防护设备。

Research

JoVE Journal

Environment

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

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

巴西田间放生转基因 伊 蚊的大规模生产

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the ...

科研

环境科学

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

在使用高通量微孔板测定水，土壤，沉积物和微生物测定胞外酶活性的

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

优化烟气设置通过电脑模拟来促进微藻生长的光生物反应器中

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

方法对纸浆厂污水流和培养微生物的生物降解能力的表征促进微生物生长

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

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

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

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 ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

提取及土壤微生物磷脂脂肪酸分析

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

一个插角Thermogradient表来控制土壤温度为评估植物生长和监测土壤过程

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber

哈佛环境室内有机颗粒物的生产与测定

The production and the evolution of atmospheric organic particulate matter (PM) are insufficiently understood for accurate simulations of atmospheric ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

植物感染试验: 以植物病原菌为载体的喷雾和伤口介导接种

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L.

玉米加工参数和营养品质分析的高通量、微尺度协议 (玉蜀黍)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole ...