转基因玉米中黄曲霉生长和黄曲霉毒素的产生表达表达.
Summary
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。
Abstract
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%。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Agar | Caisson | ||
| Amazing Marine Goop | Eclectic Products | ||
| C1000 Touch CFX96 Real-Time System | Bio-Rad | ||
| Corning Falcon Tissue Culture Dishes, 60 mm | Fisher Scientific | 08-772F | |
| Eppendorf 5424 Microcentrifuge | Fisher Scientific | ||
| Erlenmeyer flask with stopper, 50 mL | Ace Glass | 6999-10 | |
| Ethanol | |||
| FluoroQuant Afla | Romer Labs | COKFA1010 | |
| Fluted Qualitative Filter Paper Circles, 15 cm | Fisher Scientific | 09-790-14E | |
| Force Air Oven | VWR | ||
| FQ-Reader | Romer Labs | EQFFM3010 | |
| Geno/Grinder 2010 | OPS Diagnostics | SP 2010-115 | |
| Innova 44 Incubator Shaker | Brunswick Scientific | ||
| iScript cDNA Synthesis Kit | Bio-Rad | 1708890 | |
| liquid Nitrogen | |||
| Low Form Griffin Beakers, 100 mL | DKW Life Sciences | 14000-100 | |
| Methanol | |||
| Methylene Chloride | |||
| Nexttec 1-step DNA Isolation Kit for Plants | Nexttec | 47N | |
| Nikon Eclipse E600 microscope with Nikon DS-Qi1 camera | Nikon | ||
| Nikon SMZ25 stereomicroscope with C-HGFI Episcopic Illuminator and Andor Zyla 4.2 sCMOS camera | Nikon | ||
| Nunc Square BioAssay Dishes | ThermoFisher Scientific | 240835 | |
| Phire Plant Direct PCR Kit | ThermoFisher Scientific | F130WH | |
| Polycarbonate Vials, 15 ml | OPS Diagnostics | PCRV 15-100-23 | |
| Potato Dextrose Broth | |||
| Snap Cap, 22 mm | DKW Life Sciences | 242612 | |
| Sodium Phosphate dibasic heptahydrate | Sigma-Aldrich | ||
| Sodium Phosphate monobasic | Sigma-Aldrich | ||
| Spectrum Plant Total RNA Kit | Sigma-Aldrich | STRN50 | |
| Stainless Steel Grinding Balls, 3/8'' | OPS Diagnostics | GBSS 375-1000-02 | |
| Stir Plate | |||
| Synergy 4 Fluorometer | Biotek | ||
| T100 Thermal Cycler | Bio-Rad | ||
| Triton X-100 | Sigma-Aldrich | T-9284 | |
| V8 juice | Campbell's | ||
| Whatman Qualitative Grade Plain Sheets, Grade 3 | Fisher Scientific | 09-820P | |
| Wrist-Action Shaker | Burrell Scientific |
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