Name: double-stranded rna oral delivery methods to induce rna interference in phloem and plant-sap-feeding hemipteran insects
Uploaded: 2018-05-04T14:00:00.000+00:00
Duration: 10 min 14 s
Description: Watch this Scientific Journal Video about Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects at JoVE.com

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。

1. BMSB 饲养
<ol><li>后 BMSB 昆虫按标准实验室实践和以前描述的63。</li>
	<li>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。</li>
</ol>2. 基因区域的选择和体外合成 dsRNA
<ol><li>从以前发布的转录配置文件32中选择特定于 BMSB 的基因。</li>
	<li>确保所选感兴趣的区域在200到500基对之间有所不同。</li>
	<li>使用以下描述的条件进行聚合酶链反应 (PCR), 生成与基因组 DNA 感兴趣的基因片段。有关特定于基因的寡核苷酸, 请参见表 1 。<ol>
<li>pcr 反应: 在0.25 毫升的 pcr 管中, 结合5µL 10X PCR 缓冲器, 4 µL dNTP 混合物 (2.5 毫米每), 2 µL dna 模板 (50 ng/µL), 2.5 µL 每个引物1和 2 (10 µM), 0.25 µL 的 dna 聚合酶 (5 U/µL), DNase/RNase 免费水可达50µL。</li>
		<li>pcr 条件: 循环 pcr 反应放大感兴趣的区域在95°c 为 3 min 跟随30个周期98°c 十年代, 55 °c 为三十年代, 72°C 为1分钟. 孵育反应在72°c 为另外的10分钟. 纯化 PCR 反应用净化套件。</li>
	</ol>
</li>
	<li>T7 RNA 聚合酶启动子序列 (5 '-GAA TTA ata GGG AGA-3 ') 的基因特异引物进一步放大获得的 PCR 片段, 如前所述62。</li>
	<li>使用 LacZ 基因作为阴性对照 (模拟) 的 rna 干扰。 注: LacZ 是一种基因, 编码β乳糖酶从 Escherichiacoli 基因组 DNA 中扩增 (使用的引物在 表1 中列出)。</li>
	<li>执行体外转录以产生 dsRNA, 如前面所描述的62。</li>
	<li>溶解和并用重悬产生的 dsRNA 在150µL DNase/RNase 自由水, 测量浓度, 并存储在-80 °c, 以供将来使用。</li>
</ol>3. 使用青豆运送 dsRNA
<ol><li>选择早 4th龄 BMSB 若虫孵化从相同的卵质量和饿死24小时之前, dsRNA 喂养。</li>
	<li>选择细长认证的有机青豆 (菜豆), 用0.2% 次氯酸钠溶液冲洗5分钟。 注: 选用细长青豆, 可方便地在2毫升离心管中容纳豆类。</li>
	<li>清洗3次与 ddH2O 和允许空气干燥。</li>
	<li>用干净的刀片修剪从花萼末端到总长度为7.5 厘米的青豆。</li>
	<li>将洗净和修剪过的青豆浸泡在含300µL 控制溶液的无盖2毫升离心管中 (1:10 的绿色食品着色 (配料: 水、丙烯乙二醇、Fd & C 黄5、Fd & C 蓝1和 Propylparaben 作为防腐剂))。</li>
	<li>使稀释的体外合成 LacZ、JHAMT 或 Vg dsRNAs, 稀释5µg 或20µg 300 µL RNase/DNase 游离水, 分别产生0.017 µg/µL 或0.067 µg/µL 的最终浓度。</li>
	<li>将洗净和修剪过的青豆浸入2毫升的离心管中, 其中含有300µL 的 dsRNA 溶液 (从步骤 3.6)。</li>
	<li>包裹和密封的离心管包围浸泡豆的边缘, 以避免蒸发的 dsRNA 溶液和防止动物进入离心管。</li>
	<li>将这些管子以直立的方式放置在室温下3小时, 使 dsRNA 溶液通过毛细管作用在整个绿豆中加载。</li>
	<li>把这些管子放在干净的培养容器里 (聚丙烯)。将三饥饿的 4th龄 BMSB 若虫放在养殖容器中。</li>
	<li>每种养殖容器治疗三只动物, 每只含有三个青豆和绿色食物着色或 dsRNA 溶液。保持昆虫在25°c 和72% 相对湿度, 在16升: 8 d 光周期在孵化器。</li>
	<li>允许昆虫在青豆 (浸泡和吸收 dsRNA) 5 天, 但补充新鲜的饮食 dsRNA 处理绿色珠后3天。</li>
</ol>4. BMSB 干扰介导沉默后基因表达的实时定量 (qPCR) 分析
<ol><li>测量 qPCR 对转录表达水平的影响。</li>
	<li>从 dsRNA 处理的动物中分离出总 RNA 并合成 cDNA62。</li>
	<li>使用实时 PCR 系统和在表 1中列出的引物设置 qPCR 反应。使用以下 qPCR 循环条件:95 °c 为10分钟跟随40个周期95°c 为十五年代, 60 °c 为1分钟, 连同离解步包括95°c 为十五年代, 60 °c 为1分钟, 95 °c 为十五年代和60°c 十五年代。</li>
	<li>确定 qPCR 标准: 使用从正常动物中分离出来的总 RNA 所制备的 cDNA 的序列稀释作为定量的参考标准。</li>
	<li>使用 BMSB 18s rna 作为内部标准, 以纠正从组织中 RNA 恢复的差异32。</li>
</ol>5. 叶面喷施在大型盆栽柑桔树和幼苗中的应用
注: 柑橘品种卡里佐枳橙 (中华柑桔 XPoncirus枳芸香) 的植物, 在自然光照和温度下, 在1.2 升容器中生长。这些植物不断修剪以促进新的叶面芽的生长, 称为 "冲洗"。非加太更喜欢在柑橘的新增长中喂养和产卵64。
<ol><li>选择植物或幼苗, 不要用水2-3 天之前, 让土壤干燥, 以潮湿, 但不完全干燥。</li>
	<li>使用手泵喷雾瓶将200毫升的 dsRNA 溶液 (0.5 毫克/毫升) 应用于下部树冠 (图 4A)。 注: 在 DNase/RNase 自由水中准备上述 dsRNA 溶液。</li>
	<li>喷后应用, 允许应用 dsRNA 溶液完全吸收叶子。 注: 柑橘树吸收了应用的 dsRNA, 然后从新的生长或在应用之前覆盖的树枝上提取出来;dsRNA 是可检测的使用 qPCR 和显示系统的移动到树顶篷叶在 3-4 h44,45,46。</li>
	<li>从四个分支的小贴士中收集大约10叶, 以25-40 天后的新增长为例。提取总 RNA 并通过反向转录 pcr (rt-pcr) 和 qPCR 来分析应用的 dsRNA 触发器是否存在, 使用表1中列出的引物和以前描述的方法46。 注意: 示例收集、总 RNA 隔离、RT PCR 和 qPCR 都是按照前面描述的46进行的。</li>
	<li>同样, 局部喷洒10毫升 dsRNA 到下部的幼苗或小盆栽树叶。 注: Hemipteran 昆虫 (非加太和 GWSS) 通常在24小时喂食, 在新的生长 (叶子) 的治疗后, 没有直接喷洒, 或生长几周后, 或整个植物。这产生的昆虫, 测试阳性的 dsRNA 在 3, 6, 10 天, 产后喂养。</li>
</ol>6. 土壤/根系淋湿在大、小盆栽柑桔树和幼苗中的应用
<ol><li>选择植物或幼苗, 不要用水2-3 天之前, 让土壤干燥, 以潮湿, 但不完全干燥 (这创造空气空间, 以保持液体溶液的应用)。</li>
	<li>将1升的 dsRNA 溶液 (0.2 毫克/毫升) 添加到大型盆栽植物 (约2.5 米) 的土壤中, 并在 1 h 之后添加1升水 (追赶)。</li>
	<li>在部分干土中应用100毫升的 dsRNA 溶液 (1.33 毫克/毫升) 到1米高的盆栽树木的土壤中。</li>
	<li>对于小苗, 应用10毫升的 dsRNA 溶液 (1 毫克/毫升) 到土壤中的锥或裸根 (图 4B, C)。</li>
	<li>允许接收 dsRNA 溶液的植物作为土壤淋湿浸泡30分钟。然后应用纯水处理, 以辅助吸收根 (20 毫升的植物在黄色容器, 或100毫升, 如果较大的盆栽是 > 1 加仑)。 注: 局部应用 dsRNA, 在3-6 小时后的治疗中发现了分支机构的远端尖端, 显示系统的运动通过树木。新的生长分支检测阳性 dsRNA 60-90 天后治疗。在 dsRNA 喂养生物测定44中, 向昆虫 (ACP 和 GWSS) 提供扦插。</li>
</ol>7. 茎龙头 (树干注射) 在大盆栽柑桔树和幼苗中的应用
<ol><li>选择柑橘幼苗, 新的, 或大约3.5 年的老植物注射 dsRNA 使用茎水龙头 (树干注射) 方法。</li>
	<li>在柑橘类植物钻孔中使用钻头和10毫米钻头, 注意不要超过2厘米, 或大约一半直径的茎。</li>
	<li>用0.6 厘米 (¼英寸) 宽条密封膜将每个喷油器的铜尖包裹4-6 次, 以防止靠近尖端的渗漏。</li>
	<li>填充树干喷油器与6毫升 (1.7 毫克/毫升) 的 dsRNA 溶液稀释在 DNase/RNase 自由水 (这里表示为有色溶液)。</li>
	<li>将溶液注入树的树干中, 将注射器留在树干中6-10 小时, 以允许吸收 dsRNA 溶液。允许昆虫在3、10和30天后处理后, 从处理过的树木的扦插中觅食。 注意: 使用主干注入方法注射的 dsRNA 在树中持续了30-60 天41,42,44。qPCR 使用表 1中列出的引物执行了对 rna 干扰的验证。</li>
</ol>8. dsRNA 处理的粘土颗粒通过土壤输送到昆虫
<ol><li>将粘土吸收剂倒入50毫升锥形管中, 管上35毫升 (约30克粘土吸收剂)。</li>
	<li>倒入20毫升的 dsRNA 溶液稀释在 DNase/RNase 自由水 (100 µg/毫升) 入管, 以湿所有的吸收剂。将锥形管盖上, 尖管以帮助去除空气, 并盖上管。</li>
	<li>把管子直立, 让它代表1-2 分钟的粘土颗粒吸收溶液。</li>
	<li>添加足够的 dsRNA 浸泡粘土到土壤混合, 以填补1加仑锅。</li>
	<li>用手把泥土拌匀, 把 dsRNA 浸泡的粘土彻底拌入泥土中。</li>
	<li>用此土壤 repot 苗选择 dsRNA 处理。</li>
	<li>用水200毫升的平原水, 没有 dsRNA 的土壤。30分钟到1小时后, 跟随100毫升的清水。24小时后, 把植物放在正常的浇水时间表上。</li>
	<li>用粘土吸收剂和 dsRNA 对经过处理的盆栽植物的4-6 叶进行每月的 dsRNA 后处理, 收集新植物生长的最顶端叶。 注: 在24小时后处理后, 这些被处理植物的植物或扦插会被喂食到昆虫身上, 并且能够对昆虫进行长达一年的 rna 干扰 (未发表的数据)。</li>
</ol>

作者没有什么可透露的。

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...

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。

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

double-stranded rna oral delivery methods to induce rna interference in phloem and plant-sap-feeding hemipteran insects

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。

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...

Watch this Scientific Journal Video about Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects at JoVE.com

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

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

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。

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

double-stranded-rna-oral-delivery-methods-to-induce-rna-interference

Universidad San Sebastian

Research

JoVE Journal

Environment

13.8K 次观看.  United States Department of Agriculture. 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。

视频: Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

科研

遗传学

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

水生甲壳虫中的RNA干扰是特定发育时间点操纵基因表达的有力工具

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

环境科学

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

RNAi介导的双基因敲除和味觉感知测量蜜蜂（<em&gt;蜜蜂</em&gt;）

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

神经科学

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi的干扰双链RNA注射到果蝇胚胎

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

生物学

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAi的触发传送到<em&gt;冈比亚按蚊</em&gt;蛹

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

rna 干扰的实际应用: 口服双链 rna 在脂质体载体为蟑螂

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

对成年 冈比亚按蚊 的有效口服RNA干扰（RNAi）给药

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) ...

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. 
This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5&#8242;-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 &#181;g/mL) in the soaking buffer for 24 h in the dark at 25 &#176;C.
The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

Here, we introduce a detailed soaking method of RNA interference in Bursaphelenchus xylophilus to facilitate the study of gene functions.

RNA干扰在松林线虫、木犀牛线虫中的应用

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of ...

推进树 突毛 虫 RNAi 技术用于基因功能分析

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura

The egg parasitoids, Trichogramma spp, are recognized as efficient biological control agents against various lepidopteran pests in agriculture and forests. The immature stages of Trichogramma offspring develop within the host egg, exhibiting remarkable diminutiveness (approximately 0.5 mm in adult length). RNA-interference (RNAi) methodology has emerged as a crucial tool for elucidating gene functions in numerous organisms. However, manipulating RNAi in certain small parasitoid species, such as Trichogramma, has generally posed significant challenges. In this study, we present an efficient RNAi method in Trichogramma denrolimi. The outlined procedure encompasses the acquisition and isolation of individual T. dendrolimi specimens from host eggs, the design and synthesis of double-stranded RNA (dsRNA), the in vitro transplantation and cultivation of T. dendrolimi pupae, the micro-injection of dsRNA, and the subsequent assessment of target gene knockdown through RT-qPCR analysis. This study furnishes a comprehensive, visually detailed procedure for conducting RNAi experiments in T. dendrolimi, thereby enabling researchers to investigate the gene regulation in this species. Furthermore, this methodology is adaptable for RNAi studies or micro-injections in other Trichogramma species with minor adjustments, rendering it a valuable reference for conducting RNAi experiments in other endoparasitic species.

作者聚焦:研究 Wolbachia 诱导的 Thelytokous 孤雌生殖和通过 RNA 干扰的遗传工具包开发

The manipulation of RNA interference (RNAi) presents a formidable challenge in many parasitoid species with diminutive size, such as Trichogramma wasps. This study delineated an efficient RNAi method in Trichogramma denrolimi. The present methodology provides a robust model for investigating gene regulation in Trichogramma wasps.

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

摘要

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此视频中的章节

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，

水生甲壳虫中的RNA干扰是特定发育时间点操纵基因表达的有力工具

RNAi介导的双基因敲除和味觉感知测量蜜蜂（

RNAi的干扰双链RNA注射到果蝇胚胎

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

RNAi的触发传送到

rna 干扰的实际应用: 口服双链 rna 在脂质体载体为蟑螂

对成年冈比亚按蚊的有效口服RNA干扰（RNAi）给药

RNA干扰在松林线虫、木犀牛线虫中的应用

卵寄生虫中的RNA干扰， Trichogramma dendrolimi Matsumura

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

摘要

探索更多视频

此视频中的章节

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，

水生甲壳虫中的RNA干扰是特定发育时间点操纵基因表达的有力工具

RNAi介导的双基因敲除和味觉感知测量蜜蜂（

RNAi的干扰双链RNA注射到果蝇胚胎

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

RNAi的触发传送到

rna 干扰的实际应用: 口服双链 rna 在脂质体载体为蟑螂

对成年 冈比亚按蚊 的有效口服RNA干扰（RNAi）给药

RNA干扰在松林线虫、木犀牛线虫中的应用

卵寄生虫中的RNA干扰， Trichogramma dendrolimi Matsumura

对成年冈比亚按蚊的有效口服RNA干扰（RNAi）给药