通过RNA定位蝗虫天线中的气味受体基因<em>原位</em>杂交
Summary
本文描述了一种详细和高效的RNA 原位杂交方案，特别是对于低水平表达的气味受体（OR）基因以及其他基因，在使用地高辛（DIG）标记或生物素标记的探针的昆虫天线中。
Abstract
昆虫已经发展了复杂的嗅觉接收系统来感测外源化学信号。这些化学信号由嗅觉受体神经元（ORNs）转导，其位于天线的头发状结构（称为化学感光体）中。在ORN的膜上，认为气味受体（ORs）参与气味编码。因此，能够识别定位于ORN的基因是识别OR基因所必需的，为进一步的功能性原位研究提供了基础。昆虫天线中特异性OR的RNA表达水平非常低，用于组织学的保存昆虫组织具有挑战性。因此，使用RNA 原位杂交难以将OR定位到特定类型的感觉器。在本文中，介绍了详细和高效的RNA 原位杂交方案，特别是对昆虫的低表达OR基因。另外还有一个具体的OR 基因哇通过使用共表达受体基因Orco进行双色荧光原位杂交实验鉴定 。
Introduction
作为最重要的化学感觉器官的昆虫天线被许多由嗅觉受体神经元（ORN）支配的头发状结构（称为感觉）覆盖。昆虫ORNs的膜，气味受体（ORS），一种类型的包含七个跨膜结构域的蛋白质，与共同受体（ORCO）以形成杂聚体表示其功能如同一个加臭剂门控离子通道1，2，3。不同的OR响应化学化合物4，5，6的不同组合。
蝗虫（ Locusta migratoria ）主要依靠嗅觉信号触发重要行为7 。蝗虫OR是理解分子嗅觉机制的关键因素。将特定的蝗虫OR基因定位到a的神经元通过RNA的形态特异性感觉类型原位杂交（RNA ISH）是探索ORs功能的第一步。
RNA ISH使用标记的互补RNA探针来测量和定位组织，细胞或整个原位部分的特异性RNA序列，从而提供对生理过程和疾病发病机理的见解。地高辛标记（DIG标记）和生物素标记的RNA探针已被广泛用于RNA杂交。用地高辛-11-UTP或生物素-16-UTP进行RNA标记可以通过SP6和T7 RNA聚合酶的体外转录来制备。 DIG-和生物素标记的RNA探针具有以下优点：非放射性;安全;稳定;高度敏感;高度特异性并且使用PCR和体外转录容易产生。 DIG-和生物素标记的RNA探针可以发色和荧光检测。 DIG标记的RNA探针可以用抗地高辛碱碱检测使用光学显微镜或2位可以用高度敏感的化学发光底物硝基蓝四唑氯化物/ 5-溴-4-氯-3-吲哚基 – 磷酸酯甲苯胺盐（NBT / BCIP）可视化的ne磷酸酶（AP）共轭抗体使用共聚焦显微镜与4-氯-2-甲基苯并噻唑半氯化锌盐（Fast Red）偶合的3-羟基-3-萘甲酸-2'-苯基苯胺磷酸盐（HNPP）。可以用抗生物素链霉亲和素马萝卜过氧化物酶（HRP） – 共轭抗体检测生物素标记的RNA探针，该抗体可以使用共聚焦显微镜用荧光素 – 酪酰胺显色。因此，可以进行双色荧光原位杂交以使用DIG-和生物素标记的RNA探针在一个切片中检测两个靶基因。
具有DIG和/或生物素标记的探针的RNA ISH已被成功地用于定位嗅相关基因，如OR ，离子型受体，加味剂结合蛋白和感觉神经元的膜蛋白，在昆虫天线，但不限于， 黑腹果蝇 ， 冈比亚按蚊 ，L.飞蝗和沙漠蝗虫Schistocera蝗 8，9，10，11，12，13，14，15，16。然而，为昆虫ORs进行RNA ISH时有两个实质性的挑战：（1）OR基因（ ORco除外）以低水平表达，仅在少数细胞中表达，使信号检测非常困难，（2）保存昆虫组织组织学，使得形态被保留并且背景噪声低，可能是具有挑战性的。在本文中，详细和有效的方案描述了用于在昆虫中定位OR基因的RNA ISH提出了天线，包括显色和Tyramide信号放大（TSA）检测。
Protocol
注意：为了限制RNA降解，请使用湿式高压灭菌蒸馏水（121℃，60分钟）和湿式高压釜材料制备溶液。 RNA ISH反义和检测探针的制备 靶基因扩增和纯化 首先，从含有LmigOR1全长cDNA的质粒与Taq DNA聚合酶一起产生A. migratoria OR1 （ LmigOR1 ，GenBank：JQ766965）的387bp双链片段，该Taq DNA聚合酶在片段的两端添加腺嘌呤…
Representative Results
通过显色检测，每个成年天线部分的小部分触角细胞由DIG标记的LmigOR1和LmigOR2反义探针表示（ 图3 ）。连续切片上的RNA ISH定位LmigOR1和LmigOR2显示表达两个基因的触角细胞位于表达LmigORco的 ORN簇中，表明推定的LmigOR1和LmigOR2实际上在ORN中表达（ 图4a -4d ）。有时，标记的?…
Discussion
这是很难执行RNA ISH本地化或昆虫触角基因，因为的表达水平或基因除了ORCO，是非常低的和保存的组织学切片的昆虫触角是非常困难的。另外TSA检测也很棘手。为解决这些问题，应采取以下措施。天线选自具有薄且软的触角表皮的新蜕皮成虫蝗虫，其保持其在幻灯片上的形态。将冷冻样品切成12μm厚的切片。使用高灵敏度和特异性的生物素和DIG标记的探针。洗涤剂如Tween-20和Triton X-100用于在?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到国家自然科学基金资助项目（No.1472037）的支持。本文中提及的商品名称或商业产品仅用于提供具体信息，并不意味着推荐。
Materials
| Materials | |||
| 2×TSINGKETM Master Mix | TSINGKE, China | TSE004 | |
| RNase-free H2O | TIANGEN, China | RT121-02 | |
| REGULAR AGAROSE G-10 | BIOWEST, SPAIN | 91622 | |
| Binding buffer | TIANgel Midi Purification Kit, TIANGEN, China | DP209-02 | |
| Balance buffer | TIANgel Midi Purification Kit, TIANGEN, China | DP209-02 | |
| Wash solution | TIANgel Midi Purification Kit, TIANGEN, China | DP209-02 | |
| T Vector | Promega, USA | A362A | |
| T4 DNA Ligase | Promega, USA | M180A | |
| Escherichia coli DH5α | TIANGEN, China | CB101 | |
| Ampicillin | Sigma, USA | A-6140 | |
| Isopropyl β-D-1-thiogalactopyranoside | Inalco, USA | 1758-1400 | |
| 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside | SBS Genetech, China | GX1-500 | |
| Nco I | BioLabs, New England | R0193S | |
| Spe I | BioLabs, New England | R0133M | |
| DIG RNA Labeling Kit | Roche, Switzerland | 11175025910 | |
| Biotin RNA Labeling Kit | Roche, Switzerland | 11685597910 | |
| DNase | DIG RNA Labeling Kit, Roche, Switzerland | 11175025910 | |
| LiCl | Sinopharm, China | 10012718 | |
| Ethanol | Sinopharm, China | 10009257 | |
| Acetic acid | BEIJING CHEMICAL REGENTS COMPANY, China | 10000292 | |
| Tissue-Tek O.C.T. Compound | Sakura Finetek Europe, Zoeterwoude, Netherlands | 4583 | |
| Slides | TINA JIN HAO YANG BIOLOGCAL MANUFACTURE CO., LTE, China | FISH0010 | |
| HCl | Sinopharm, China | 80070591 | |
| Millex | Millipore, USA | SLGP033RS | |
| Tween 20 | AMRESCO, USA | 0777-500ML | |
| Nitroblue tetrazolium chloride / 5-bromo-4-chloro-3-indolyl-phosphate toluidine salt | Roche, Switzerland | 11175041910 | |
| Glycerol | Sinopharm, China | 10010618 | |
| Name | Company | Catalog Number | Comments |
| Solutions | |||
| 1×Tris-acetate-EDTA | Sigma, USA | V900483-1KG | 0.04mol/L Tris-Base |
| 1×Tris-acetate-EDTA | BEIJING CHEMICAL REGENTS COMPANY, China | 10000292 | 0.12%acetic acid |
| 1×Tris-acetate-EDTA | Sigma, USA | 03677 | Ethylenediaminetetraacetic acid disodium salt (EDTA) |
| Luria-Bertani (LB) liquid medium | Sinopharm, China | 10019392 | 10g/L NaCl |
| Luria-Bertani (LB) liquid medium | MERCK, Germany | VM335231 | 10g/L Peptone from casein (Tryptone) |
| Luria-Bertani (LB) liquid medium | MERCK, Germany | VM361526 | 5g/L Yeast extract |
| LB solid substrate plate | Sinopharm, China | 10019392 | 10g/L NaCl |
| LB solid substrate plate | MERCK, Germany | VM335231 | 10g/L Peptone from casein (Tryptone) |
| LB solid substrate plate | MERCK, Germany | VM361526 | 5g/L Yeast extract |
| LB solid substrate plate | WISENT ING, Canada | 800-010-CG | 15g/L Agar Bacteriological Grade |
| 10×phosphate buffer saline (pH7.1) | Sinopharm, China | 10019392 | 8.5%NaCl |
| 10×phosphate buffer saline (pH7.1) | Sigma, USA | V900041-500G | 14mM KH2PO4 |
| 10×phosphate buffer saline (pH7.1) | Sigma, USA | V900268-500G | 80mM Na2HPO4 |
| 10×Tris buffered saline (pH7.5) | Sigma, USA | V900483-1KG | 1M Tris-Base |
| 10×Tris buffered saline (pH7.5) | Sinopharm, China | 10019392 | 1.5M NaCl |
| Detection Buffer (DAP) chromogenic detection pH9.5 TSA detection pH8.0 | Sigma, USA | V900483-1KG | 100mM Tris-Base |
| Detection Buffer (DAP) chromogenic detection pH9.5 TSA detection pH8.0 | Sinopharm, China | 10019392 | 100mM NaCl |
| Detection Buffer (DAP) chromogenic detection pH9.5 TSA detection pH8.0 | Sigma, USA | V900020-500G | 50mM MgCl2·6H2O |
| 20×saline-sodium citrate (pH7.0) | Sinopharm, China | 10019392 | 3M NaCl |
| 20×saline-sodium citrate (pH7.0) | Sigma, USA | V900095-500G | 0.3M Na-Citrate |
| 4% paraformaldehyde solution (pH9.5) | Sigma, USA | V900894-100G | 4% paraformaldehyde |
| 4% paraformaldehyde solution (pH9.5) | Sigma, USA | V900182-500G | 0.1M NaHCO3 |
| Sodium Carbonate Buffer (pH10.2) | Sigma, USA | V900182-500G | 80mM NaHCO3 |
| Sodium Carbonate Buffer (pH10.2) | Sigma, USA | S7795-500G | 120mM Na2CO3 |
| Formamide Solution (pH10.2) | MPBIO, USA | FORMD002 | 50% Deionized Formamide |
| Formamide Solution (pH10.2) | 5×saline-sodium citrate | ||
| Blocking Buffer in Tris buffered saline | Roche, Switzerland | 11175041910 | 1% Blot |
| Blocking Buffer in Tris buffered saline | AMRESCO, USA | 0694-500ML | 0.03% Triton X-100 |
| Blocking Buffer in Tris buffered saline | 1×Tris buffered saline | ||
| Alkaline phosphatase solution | Roche, Switzerland | 11175041910 | 1.5 U/ml anti-digoxigenin alkaline phosphatase conjugated antibody |
| Alkaline phosphatase solution | Blocking Buffer in Tris buffered saline | ||
| Alkaline phosphatase/ horse radish peroxidase solution | Roche, Switzerland | 11175041910 | 1.5 U/ml anti-digoxigenin alkaline phosphatase conjugated antibody |
| Alkaline phosphatase/ horse radish peroxidase solution | TSA kit, Perkin Elmer, USA | NEL701A001KT | 1% anti-biotin streptavidin horse radish peroxidase- conjugated antibody |
| Alkaline phosphatase/ horse radish peroxidase solution | Blocking Buffer in Tris buffered saline | ||
| Hybridization Buffer | MPBIO, USA | FORMD002 | 50% Deionized Formamide |
| Hybridization Buffer | 2×saline-sodium citrate | ||
| Hybridization Buffer | Sigma, USA | D8906-50G | 10% dextran sulphate |
| Hybridization Buffer | invitrogen, USA | AM7119 | 20 µg/ml yeast t-RNA |
| Hybridization Buffer | Sigma, USA | D3159-10G | 0.2 mg/ml herring sperm DNA |
| 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate | Roche, Switzerland | 11758888001 | 1% 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate (10mg/ml) |
| 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate | Roche, Switzerland | 11758888001 | 1% 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt (25mg/ml) |
| 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate | Detection Buffer | ||
| Tyramide signal amplification substrate | TSA kit, Perkin Elmer, USA | NEL701A001KT | 2% fluorescein-tyramides |
| Tyramide signal amplification substrate | TSA kit, Perkin Elmer, USA | NEL701A001KT | Amplification Diluent |
| Name | Company | Catalog Number | Comments |
| Instrument | |||
| Freezing microtome | Leica, Nussloch, Germany | Jung CM300 cryostat | |
| Spectrophotometer | Thermo SCIENTIFIC, USA | NANODROP 2000 | |
| Optical microscope | Olympus, Tokyo, Japan | Olympus IX71microscope | |
| Confocal microscope | Olympus, Tokyo, Japan | Olympus BX45 confocal microscope |
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Cite This Article
Xu, X., You, Y., Zhang, L. Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization. J. Vis. Exp. (125), e55924, doi:10.3791/55924 (2017).