rna 干扰的实际应用: 口服双链 rna 在脂质体载体为蟑螂
Summary
本手稿通过口服双链 RNA 包裹在脂质体中, 证明了德国蟑螂中肠内基因表达的耗竭。
Abstract
rna 干扰在揭示众多基因的生物学功能方面得到了广泛的应用, 并被设想为一种害虫控制工具, 其主要基因表达被打乱。虽然报告了不同的方法, 如注射, 喂养和浸泡, 成功地交付双链 RNA (dsRNA), 通过口服 dsRNA 的 rna 干扰的效率在不同的昆虫群中是高度可变的。德国蟑螂, 德国的大蠊, 对 dsRNA 的注射高度敏感, 正如此前发表的许多研究显示的那样。本研究描述了用脂质体载体包裹 dsRNA 的方法, 足以延缓中肠果汁对 dsRNA 的降解。值得注意的是, 脂质体包覆的 dsRNA 的连续喂养大大降低了中肠蛋白的表达, 导致了蟑螂的死亡。总之, dsRNA lipoplexes 的配制和利用, 保护 dsRNA 免受核酸的危害, 是今后害虫防治的一种实用方法。
Introduction
rna 干扰已被证明是一种有效的方法来击倒基因表达通过一个机制的转录后沉默途径触发的 dsRNA 分子在许多真核生物1。在过去的十年中, rna 干扰已经成为一个有用的工具来研究基因从发展到行为的功能, 通过消耗特定基因的表达, 通过注射和/或喂养 dsRNA 在昆虫的各种分类器2,3。由于损耗效应的特殊性和鲁棒性, 目前正在将 rna 干扰的应用视为害虫控制管理的潜在策略4,5。然而, 在昆虫种类之间, rna 干扰的效率差别很大, 这取决于不同基因的靶向和传递方法。越来越多的证据表明, 由 ribonucleases 降解的 dsRNA 的不稳定性是 rna 干扰5、6的有限功效的一个关键因素。例如, Manduca 天蛾中的低干扰敏感度被解释为: 与血淋巴混合的 dsRNA 在1小时的7中迅速退化。同样, 中肠碱性核酸的存在, 有效地降低了摄取 dsRNA, 在不同的昆虫顺序中与低 rna 干扰效率有很强的相关性8,9,10。
dsRNA 的口头交付对于在害虫防治战略中应用 rna 干扰特别有趣, 但尚未开发出一种延缓中肠核酸降解 dsRNA 的方法, 这将有可能确保有效通过喂食进行 rna 干扰。然而, dsRNA 的口服的对停止响应是通过喂养大量的 dsRNA ,例如50µg在家蚕, 或连续喂养8天 (总共8µg dsRNA) 的蝗虫物种。德国蟑螂,德国 germinica, 对通过 dsRNA11,12,13,14的注入而对 rna 的干扰非常敏感, 但对 dsRNA 通过喂食没有响应。最近, 林等。(2017) 证明了脂质体载体封装的 dsRNA 成功地进行了 rna 干扰, 以击倒中肠中的α蛋白基因表达, 并触发德国蟑螂15的显著死亡率。由于中肠 dsRNA 的降解是口服 rna 干扰的限制因素, 脂质体载体作为一种保护 dsRNA 免受降解的载体, 在肠道内具有较强核酸酶活性的其他昆虫中很容易被应用。值得注意的是, 选择特定转染试剂的原因 (参见材料表) 我们作为脂质体载体在目前的协议是, 它已被测试的昆虫细胞线转染较少的毒性, 根据制造商的说明。通过对不同脂质体转染系统的比较 Gharavi et 。(2013)16中, 转染小干扰 RNA (siRNA) 的效率大约与其他昆虫17、18中用于 dsRNA 传送系统的其他商用系统之间的效果大致相同..此外, 我们的喂养方法是足够小心, 以确保每只蟑螂摄取适量的 dsRNA, 结果是稳健的和确认的。总之, 本协议和结果表明, 使用 dsRNA lipoplexes 提高了 dsRNA 的稳定性, 为 rna 干扰的策略设计开辟了大门, 这是今后害虫防治的一个有前途的途径。
Protocol
Representative Results
Discussion
本协议提出了一种有效的 rna 干扰方法, 通过口服 dsRNA lipoplexes, 包括防止核糖核酸消化在中肠果汁的德国蟑螂。正如在其他昆虫种类的研究中所显示的, 通过口服 dsRNA 的干扰效应很差, 主要是由于 dsRNA8,9,10的退化。该协议产生脂质体, 作为保护性的工具, 在口头交付 dsRNA 抗降解的肠道。此外, dsRNA lipoplexes 的制备方法简单, 易于应…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究得到了台湾的赠款 (科学技术部, 多数 100-2923-002-002-MY3 和 106-2313-b-002-011-MY3 H.J.L.)、捷克共和国 (南波希米亚大学赠款机构、GAJU 赠款 065/2017/P y.h. L) 和西班牙 (西班牙经济和竞争力部, 赠款 CGL2012-36251 和 CGL2015-64727-P 王啸波和加泰罗尼亚政府, 赠款 2014 SGR 619 王啸波);它还得到欧洲经济和区域发展基金 (菲德基金王啸波) 的财政支助。
Materials
| GenJe Plus DNA in vitro Transfection reagent | SignaGen | SL100499 | for lipoplexes preparation |
| Blend Taq plus | TOYOBO | BTQ-201 | for PCR |
| Fast SYBR Green Master Mix | ABI | 4385612 | for qPCR |
| FirstChoice RLM-RACE Kit | Invitrogen | AM1700 | for 3' UTR identification |
| MEGAscript T7 Transcription Kit | Invitrogen | AMB13345 | for dsRNA synthesis |
| TURBO DNase | Invitrogen | AM2239 | remove DNA template from dsRNA |
| TRIzol | Invitrogen | 15596018 | for dsRNA or total RNA extraction |
| RQ1 RNase-Free Dnase | Promega | M6101 | remove DNA template from total RNA |
| chloroform | Sigma-Aldrich | C2432 | for dsRNA or total RNA extraction |
| 2-Propanol | Sigma-Aldrich | I9516 | for dsRNA or total RNA extraction |
| ethanol | Sigma-Aldrich | 24102 | for dsRNA or total RNA extraction |
| Diethyl pyrocarbonate, DEPC | Sigma-Aldrich | D5758 | for RNase free water preparation |
| glucose solution | Sigma-Aldrich | G3285 | for lipoplexes preparation |
| Sodium chloride, NaCl | Sigma-Aldrich | S7653 | insect saline buffer formula |
| Potassium chloride, KCl | Sigma-Aldrich | P9333 | insect saline buffer formula |
| Calcium chloride, CaCl2 | Sigma-Aldrich | C1016 | insect saline buffer formula |
| Magnesium chloride hexahydrate, MgCl2.6H2O | Sigma-Aldrich | M2670 | insect saline buffer formula |
| EGTA | Sigma-Aldrich | E3889 | enzyme inhibitor |
| dissecting scissor | F.S.T. | cockroach dissection | |
| fine tweezers | F.S.T. | cockroach dissection | |
| flexible tweezer | F.S.T. | cockroach holding | |
| pipetman | RAININ | P10 | sample preparation |
| microcentrifuge tube | Axygen | MCT175C, PCR02C | sample preparation |
| pipette tip | Axygen | sample preparation | |
| vortexter | Digisystem | vm1000 | sample preparation |
| Minispin centrifuge | The Gruffin Group | GMC 206 | for liquid spin down |
| Centrifuge | ALC | PK121R | sample preparation |
| pH meter | JENCO | 6071 | for pH adjust |
| micro-volume spectrophotometer | Quawell | Q3000 | nucleic acid quantitative |
| PCR Thermal cycler | ABI | 2720 | for template PCR or dsRNA synthesis incubation |
| quantitative real-time PCR | ABI | StepOne plus | gene expression quantitative |
| Centrifugal Vacuum Concentrators | eppendorf | 5301 | for dsRNA or total RNA extraction |
| Multipette | eppendorf | xstream | for real-time PCR sample loading |
| Agarose I | amresco | 0710 | for nucleic acid electrophoresis |
| tub gene specfifc forward preimer | tri-I biotech | GGG ACA AGC CGG AGT GCA GA | |
| tub gene specfifc reverse preimer | tri-I biotech | TCC TGC TCC TGT CTC GCT GA | |
| dsTub template forward primer | tri-I biotech | TAA TAC GAC TCA CTA TAG GGA CAA GCC GGA GTG CAG | |
| dsTub template reverse primer | tri-I biotech | TAA TAC GAC TCA CTA TAG GGT CCT GCT CCT GTC TCG CTG | |
| dsEGFP template forward preimer | tri-I biotech | TAA TAC GAC TCA CTA TAG GGT ATG GTG AGC AAG GGC GAG GAG | |
| dsEGFP template reverse preimer | tri-I biotech | TAA TAC GAC TCA CTA TAG GGT GGC GGA TCT TGA AGT TCA CC | |
| tub qPCR forward primer | tri-I biotech | GGA CCG CAT CAG GAA ACT GGC | |
| tub qPCR reverse preimer | tri-I biotech | CCA CAG ACA GCC TCT CCA TGA GC | |
| ef1 qPCR forward primer | tri-I biotech | CGC TTG AGG AAA TCA AGA AGG A | |
| ef1 qPCRreverse preimer | tri-I biotech | CCT GCA GAG GAA GAC GAA G |
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