Summary

通过RNA定位蝗虫天线中的气味受体基因<em>原位</em>杂交

Published: July 13, 2017
doi:

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

References

  1. Sato, K., et al. Insect olfactory receptors are heteromeric ligand-gated ion channels. Nature. 452 (7190), 1002-1006 (2008).
  2. Smart, R., et al. Drosophila odorant receptors are novel seven transmembrane domain proteins that can signal independently of heterotrimeric G proteins. Insect Biochem Mol Biol. 38 (8), 770-780 (2008).
  3. Wicher, D., et al. Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels. Nature. 452 (7190), 1007-1011 (2008).
  4. Carey, A. F., Wang, G., Su, C. Y., Zwiebel, L. J., Carlson, J. R. Odorant reception in the malaria mosquito Anopheles gambiae. Nature. 464 (7258), 66-71 (2010).
  5. Hallem, E. A., Carlson, J. R. Coding of odors by a receptor repertoire. Cell. 125 (1), 143-160 (2006).
  6. Wang, G., Carey, A. F., Carlson, J. R., Zwiebel, L. J. Molecular basis of odor coding in the malaria vector mosquito Anopheles gambiae. Proc Natl Acad Sci U S A. 107 (9), 4418-4423 (2010).
  7. Hassanali, A., Njagi, P. G., Bashir, M. O. Chemical ecology of locusts and related acridids. Annu Rev Entomol. 50, 223-245 (2005).
  8. Schaeren-Wiemers, N., Gerfin-Moser, A. A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labeled cRNA probes. Histochemistry. 100 (6), 431-440 (1993).
  9. Vosshall, L. B., Amrein, H., Morozov, P. S., Rzhetsky, A., Axel, R. A spatial map of olfactory receptor expression in the Drosophila antenna. Cell. 96 (5), 725-736 (1999).
  10. Yang, Y., Krieger, J., Zhang, L., Breer, H. The olfactory co-receptor Orco from the migratory locust (Locusta migratoria) and the desert locust (Schistocerca gregaria): identification and expression pattern. Int J Biol Sci. 8 (2), 159-170 (2012).
  11. Schultze, A., et al. The co-expression pattern of odorant binding proteins and olfactory receptors identify distinct trichoid sensilla on the antenna of the malaria mosquito Anopheles gambiae. PLoS One. 8 (7), e69412 (2013).
  12. Xu, H., Guo, M., Yang, Y., You, Y., Zhang, L. Differential expression of two novel odorant receptors in the locust (Locusta migratoria). BMC Neurosci. 14, 50 (2013).
  13. Saina, M., Benton, R. Visualizing olfactory receptor expression and localization in Drosophila. Methods Mol Biol. 1003, 211-228 (2013).
  14. Guo, M., Krieger, J., Große-Wilde, E., Mißbach, C., Zhang, L., Breer, H. Variant ionotropic receptors are expressed in olfactory sensory neurons of coeloconic sensilla on the antenna of the desert locust (Schistocerca gregaria). Int J Biol Sci. 10 (1), 1-14 (2013).
  15. You, Y., Smith, D. P., Lv, M., Zhang, L. A broadly tuned odorant receptor in neurons of trichoid sensilla in locust, Locusta migratoria. Insect Biochem Mol Biol. 79, 66-72 (2016).
  16. Jiang, X., Pregitzer, P., Grosse-Wilde, E., Breer, H., Krieger, J. Identification and characterization of two “Sensory Neuron Membrane Proteins” (SNMPs) of the desert locust, Schistocerca gregaria (Orthoptera: Acrididae). J Insect Sci. 16 (1), 33 (2016).
  17. Angerer, L. M., Angerer, R. C., Wilkinson, D. G. In situ. hybridization to cellular RNA with radiolabelled RNA probes. In situ hybridization. , 2 (1992).
  18. Komminoth, P., Merk, F. B., Leav, I., Wolfe, H. J., Roth, J. Comparison of 35S- and digoxigenin-labeled RNA and oligonucleotide probes for in situ hybridization. Expression of mRNA of the seminal vesicle secretion protein II and androgen receptor genes in the rat prostate. Histochemistry. 98 (4), 217-228 (1992).
  19. Leary, J. J., Brigati, D. J., Ward, D. C. Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc Natl Acad Sci U S A. 80 (13), 4045-4049 (1983).
  20. Hallem, E. A., Ho, M. G., Carlson, J. R. The molecular basis of odor coding in the Drosophila antenna. Cell. 117 (7), 965-979 (2004).
  21. Jones, W. D., Nguyen, T. A., Kloss, B., Lee, K. J., Vosshall, L. B. Functional conservation of an insect odorant receptor gene across 250 million years of evolution. Curr Biol. 15 (4), R119-R121 (2005).

Play Video

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).

View Video