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Environment

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

Published: May 04, 2018
doi:
10.3791/57390
, , ,
1Invasive Insect Biocontrol and Behavior Laboratory, Agricultural Research Service,United States Department of Agriculture, 2Horticultural Research Laboratory, Agricultural Research Service,United States Department of Agriculture

Summary

本文介绍了通过植物的血管组织在韧皮部 sap 喂养昆虫中进行 rna 干扰 (rna 干涉) 来口服双链 rna (dsRNA) 的新方法。

Abstract

韧皮部和植物 sap 喂养昆虫侵入作物和水果的完整性, 以检索养分, 在过程中破坏粮食作物。Hemipteran 昆虫占了许多经济上大量的植物害虫, 它们在韧皮部汁液上喂食, 对农作物造成了损害。褐大理石纹蝽臭味虫 (BMSB) 、 Halyomorpha 蝮蛇 (翅: 蝽) 和亚洲柑橘木虱 (非加太)、 Diaphorina 桔 Kuwayama (半翅目: Liviidae) 是北美洲引进的害虫, 在那里他们是一种具有高价值特产、排菜、主食作物和柑橘类水果的侵入性农业害虫, 在室内聚集时会造成害虫的危害。杀虫剂抗药性在许多种类导致了其他方法的虫害管理战略的发展。双链 rna (dsRNA) 介导的 rna 干涉 (rna 干扰) 是一种基因沉默机制的功能基因组研究, 具有潜在的应用工具, 以管理害虫。外部合成的 dsRNA 或小干扰 rna (siRNA) 可以通过降解内源 rna 而触发高效的基因沉默, 这与所提出的是同源的。利用 rna 干扰作为 hemipteran 昆虫生物防治的分子生物农药的有效和环境应用需要通过喂养来进行 dsRNAs 的体内传递. 在这里, 我们演示了向昆虫运送 dsRNA 的方法: 通过浸泡将 dsRNA 装入绿豆中, 通过摄取来吸收基因特异的 dsRNA 和口服分娩。我们还概述了非转基因植物提供方法使用叶面喷雾剂, 根淋, 树干注射以及粘土颗粒, 这一切可能是必不可少的持续释放 dsRNA。口服 dsRNA 有效的分娩是一种有效的剂量, 可以显著降低靶向基因的表达, 如青少年激素酸 O 型甲基 (JHAMT) 和卵黄蛋白原 (Vg)。这些创新方法代表了在作物保护中使用 dsRNA 的战略, 并克服了虫害管理方面的环境挑战。

Introduction

Hemipteran 昆虫包括一些最经济上重要的害虫, agriculturebecause 他们的能力, 以达到较高的人口增长和传播疾病的植物。BMSB, H. 蝮蛇Stål, 是一种侵入性害虫, 是在西半球的西伦敦, 从亚洲 (中国, 台湾, 韩国和日本) 意外引入的, 第一次目击报告在 1996年1。自介绍以来, 已在43个国家发现了 BMSB, 其中大西洋中部 (DE、马里兰州、PA、新泽西州、弗吉尼亚州和 WV) 以及加拿大和欧洲的人口最多, 对农业的潜在威胁是2。作为一种多食性害虫, BMSB 可能会对大约300种植物寄主造成伤害, 包括诸如苹果、葡萄、观赏植物、种子作物、大豆和玉米等高价值作物。损害主要是由于喂养方式被称为撕裂和冲洗, 动物刺穿宿主作物与它的针状鞘, 以获得从血管组织的营养物2,3。BMSB 也是一个室内害虫, 因为他们可能会发现居住地区, 如学校和房屋在秋季通过冬季2。据报道, BMSB 释放的化学物质和 aeroallergens 对水果作物工人的非法过敏反应。BMSB 也可能导致过敏性疾病导致接触皮炎, 结膜炎和鼻炎在敏感的个人4,5。另一 hemipteran 昆虫, 非加太,桔Kuwayama (半翅目: Liviidae), 是一种严重的柑橘类水果害虫, 并传送韧皮部有限的细菌 (Candidatus Liberibacter 胭脂) 导致黄龙 (HLB), 更好地知道作为柑橘绿化病害6,7。HLB 最初是从中国南方报告, 并已蔓延到40不同的亚洲, 非洲, 大洋洲, 南美和北美洲国家7。柑橘的绿色化是一个世界性的问题, 因柑橘果实的流失而造成经济和金融损失;因此, 对非加太的管理被认为是预防和控制 HLB 的最重要的。

有效控制这些害虫的措施通常需要使用相对短命的化学杀虫剂。化学杀虫剂控制战略往往缺乏安全的环境管理战略, 或降低了虫害的敏感性, 由于抗药性在害虫人口8,9。因此, 用分子生物农药对害虫进行生物控制是一种潜在的替代方法, 但它在全球的使用仍然是适度的, 而各种寄生昆虫 (例如、Trisolcus) 也可能作为天然生物而有效. 控制。rna 干扰是一种潜在的新兴技术, 用于管理侵入性害虫的分子生物农药 10.rna 干扰是一种很好描述的基因调控机制, 它促进了有效的转录后水平基因沉默和入侵 dsRNAs 以序列特定的方式, 最终导致调控基因表达在 mRNA级别11,12。简单地说, 当外源 dsRNA 被内化成细胞时, 它被二齿核酸酶 RNase III 超家族的成员加工成 siRNAs, 被称为 Dicer, 在蠕虫、苍蝇、植物、真菌和哺乳动物中进化保存13,14,15. 这21-25 核苷酸 siRNA 复式然后被解除和集成在 RNA 诱导的沉默综合体 (RISC) 作为指南 rna。这个 RISC-RNA 复合体允许沃森-克里克基配对的互补靶 mRNA;这最终导致分裂的 Argonaute 蛋白, 一个多域蛋白, 含有一个 RNase 的 H 样域, 降低相应的 mRNA 和减少蛋白质的翻译, 从而导致转录后水平基因沉默16,17,18。

害虫管理的 rna 干扰需要引入 dsRNA体内来保持兴趣基因的沉默, 从而激活 siRNA 通路。各种方法, 已用于 dsRNA 提供给昆虫和昆虫细胞诱导系统的干扰包括喂养10,19, 浸泡20,21, 显微注射22, 载体, 如脂质体23和其他技术24。在秀丽线虫中首次显示了 rna 干扰, 以保持unc-22基因表达的火和梅洛25, 其次是在卷曲基因表达的果蝇的26。初步功能研究利用显微注射在昆虫中提供 dsRNA, 如api 蜜蜂22,27, Acyrthosiphon 豌豆28,德国小蠊29,H. 蝮蛇30和鳞翅目昆虫 (由 Terenius et 等人审阅)31). 显微注射有利于对昆虫感兴趣的部位提供准确精确的剂量。尽管此类脓毒穿刺可能引起损伤的免疫相关基因的表达32, 因此, 排除其在农业生物农药发展的实用性。

另一种提供 dsRNA在体内的方法是浸泡, 它包括通常在含有 dsRNA 的胞外介质中悬浮动物或细胞来摄取或吸收 dsRNA。浸泡已被用来有效地诱导在果蝇S2 组织培养细胞中的 rna 干扰抑制 Downstream-of-Raf1 (DSOR1) 丝裂原活化蛋白激酶激酶 (MAPKK)20, 以及在C. 线虫沉默pos-1基因33。然而, 使用浸泡提供的 dsRNA 比微注射20更有效地诱导 rna 干扰。咀嚼昆虫中的干扰介导的沉默首先显示在西部玉米 rootworm (西铁) (Diabrotica virgifera virgifera) 通过注入 dsRNA 到一个人工琼脂饮食10。早先的报告总结了将 dsRNA 注入到节肢动物34特有的自然饮食中的方法。这些交付方法进一步被确定对人为交付手段是相对地有效的;如采舌蝇 (舌蝇 morsitansmorsitans) 的情况, 当 dsRNA 通过血餐或杏仁35传递时, 观察到与免疫相关的基因的同等击倒.同样, 通过在淡褐色苹果蛾 (Epiphyas postvittana)36, 小菜蛾 (小菜蛾) 幼虫37, 以及蜂蜜蜂38,39 dsRNA 通过水滴传递诱导有效的 rna 干扰。hemipteran 的大多数有效的 rna 干扰实验都利用了 dsRNA40的注入, 因为 hemipteran 昆虫 dsRNA 的口服分娩是艰巨的, 因为它必须通过宿主植物的血管组织来传递。在非加太和玻翅射手叶蝉 (GWSS)、 Homalodisca 金小蜂中也观察到有效的 rna 干扰: dsRNA 是通过通过根系淋湿吸收 dsRNA 到血管组织中的柑橘和葡萄藤提供的, 叶面喷淋, 树干注射, 或吸收通过扦插41,42,43,44,45,46。这也导致了第一个专利为 dsRNA 反对非加太 (2016, 美国 20170211082 A1)。使用载体, 如纳米粒子和脂质体传递 siRNA 和 dsRNA 提供稳定性, 并增加交付的 dsRNA 功效正在迅速涌现23,47,48,49 ,50。一种新型的基于纳米微粒的用于核酸的用于体外和体内的载体, 它是专门用于治疗应用的, 可以作为合适的传递载体51提供巨大的潜能。纳米微粒作为 dsRNA 的运载工具可能有缺点, 包括溶解度, 疏水性, 或有限的生物积累52, 但适当的聚合物协助交付可能补偿这些缺点。开发和使用自我交付核苷酸也出现称为 “反义寡核苷酸”, 这是单链 RNA/DNA 复式46。

节肢动物卵黄是控制生殖的关键过程, 是由幼激素和昆虫蜕皮激素调节的, 是人体脂肪合成 Vg 的关键诱导剂;vg 最终被开发的卵母细胞通过 Vg 受体介导的吞53。Vg 是一组多肽合成的 extraovarially, 这是关键的发展的主要蛋黄蛋白, 卵黄蛋白 54, 55, 因此, 它是重要的繁殖和老化 56.vg 已成功地在线虫57以及蜂蜜蜂 (api 蜜蜂) 中被静音, 在成人和卵子22中观察到了 vg 的 rna 介导的损耗。rna 介导的转录后水平基因沉默是测试, 因为它被认为它的耗尽将导致一个可观察的表型效应, 如降低生育率和生育力, 以潜在的帮助 BMSB 控制。JHAMT 基因编码的 S-s-甘基-l-蛋氨酸 (SAM) 依赖的 jh 酸 O-甲基, 催化的最后一步, jh 生物合成途径 58.在这一途径中, 法尼基焦磷酸盐 (FPP) 依次由金合欢转化为 farnesoic 酸, 随后由 farnesoate 转化成甲基 JHAMT 为 JH。这种通路是保存在昆虫和节肢动物专门为蜕变, 这一进程是由荷尔蒙调节59,60,61。在B. 森中, JHAMT 基因表达和咽侧体的生物合成活动表明, 对JHAMT基因的转录抑制是对合成58终止的关键.因此, 使用 rna 干扰为目标损耗选择了JHAMT和Vg基因。在柑橘树上也进行了 rna 干扰试验, 以控制非加太和 GWSS。柑橘树通过根淋、茎龙头 (树干注射) 以及叶面喷剂与 dsRNAs 对昆虫特异精氨酸激酶 (AK) 转录的 dsRNA 处理42,44。dsRNA 的局部应用被发现在柑橘树的树冠上, 表明通过植物血管组织有效的传递, 并导致增加的死亡率在非加太和 GWSS41,42,45。

在目前的研究中, 我们已经确定了一种自然的饮食提供方法, 如 dsRNA 的治疗。这种新开发的技术后来被用于沉默的 JHAMT 和 Vg mRNA 使用的基因特异 dsRNAs 在 BMSB 若虫如前所示的62。这里演示的这些新的交付协议取代了使用局部喷雾或 microinjections 的常规 RNA 传递系统。在 5月, 蔬菜和水果、茎秆、土壤淋湿和粘土吸收剂用于 dsRNA 的输送, 对生物农药害虫和病原体管理的持续发展至关重要。

Protocol

1. BMSB 饲养 后 BMSB 昆虫按标准实验室实践和以前描述的63。 在温室 (22 摄氏度) 和自然光下, 在柑橘秦艽上加注非加太 (D. 桔) 昆虫。使用成人非加太, 在大约5-7 天后羽化。 2. 基因区域的选择和体外合成 dsRNA 从以前发布的转录配置文件32中选择特定于 BMSB 的基因。 确保所选感兴趣的区域?…

Representative Results

以 BMSB 4th龄若虫为载体, 通过对侵袭性害虫进行 rna 干扰的分子生物农药的开发, 对蔬菜介导的 dsRNA 进行了试验。BMSBs 饲料使用他们的针状 stylets 被称为撕裂和冲洗的机制, 这对农作物造成相当大的损害。细长的有机青豆,寻常的L, 被用来测试如果营养素或 dsRNA 可以提供体内到 BMSB 通过喂养3。在 BMSB (图 1A) 中, 青…

Discussion

rna 干扰已被证明是探索基因生物学功能和调控的重要工具, 具有很大的潜力, 可用于害虫的管理19,68,69,70, 71. 在特定昆虫种类中进行沉默的适当基因的设计和选择以及向昆虫提供相应 dsRNA 的方法都是极其重要的。将 dsRNA 到昆虫中的最佳方法必须经过经验主义的确定, 连同相对…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感激地承认唐纳德-韦伯和梅根 Herlihy (美国农业部, 贝尔茨维尔, MD) 为实验和维护殖民地提供 BMSB 和 HB;和玛丽亚 t. 冈萨雷斯, 萨尔瓦多 p. 洛佩兹, (美国农业部, ARS, 皮尔斯, 佛罗里达州) 和杰姬·梅茨 (佛罗里达大学, 皮尔斯堡, 佛罗里达州) 为殖民地维护, 样品准备和分析。

Materials

BMSB (H. halys) insects  USDA
ACP (D. citri) insects  USDA
organic green beans N/A
Citrus plants USDA
sodium hypochlorite solution J.T. Baker
green food coloring  McCormick & Co., Inc
Thermo Forma chambers  Thermo Fisher Scientific
Magenta vessel (Culture) Sigma
Primers  IDT DNA
SensiMix SYBR Bioline
qPCR ABI 7500 Applied Biosystems 
Spray bottle N/A
Parafilm American Can Company
TaKaRa Ex Taq Clontech
QIAquick Qiagen
DOWNLOAD MATERIALS LIST

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RNA InterferenceDsRNA DeliveryPhloemPlant-sap-feeding InsectsHemipteran PestsBrown Marmorated Stink BugOral DeliveryGene SilencingMolecular BiopesticidesCapillary ActionGreen Beans4th Instar Nymphs

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Ghosh, S. K. B., Hunter, W. B., Park, A. L., Gundersen-Rindal, D. E. Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects. J. Vis. Exp. (135), e57390, doi:10.3791/57390 (2018).
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