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| An optimized protocol using Steedman’s wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber |
WANG Cui1*, SUN Jin-jing2*, YANG Xue-yong2, WAN Li2, ZHANG Zhong-hua1, ZHANG Hui-min1
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1 College of Horticulture, Qingdao Agricultural University/Engineering Laboratory of Genetic Improvement of Horticultural Crops of Shandong Province/Laboratory of Quality & Safety Risk Assessment for Fruit (Qingdao), Ministry of Agriculture and Rural Affairs, Qingdao 266109, P.R.China
2 Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Horticultural Crops of the Ministry of Agriculture and Rural Affairs/Sino-Dutch Joint Laboratory of Horticultural Genomics, Beijing 100081, P.R.China.
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摘要
黄瓜是研究花发育的重要模式作物，控制花发育相关的基因通常具有特异的时空表达模式，原位杂交技术（ISH）被广泛应用在分析黄瓜茎端分生组织（SAM）和花芽组织（FM）的基因时空表达模式上。原位杂交的常用方法是石蜡原位杂交，然而石蜡原位杂交中组织包埋周期长，RNA容易降解，导致信号减弱。本研究基于石蜡原位杂交进行了优化建立了碳蜡原位杂交方法，具有操作简便，灵敏度高的优点。该方法一是使用低熔点的碳蜡，RNA保存好；二是结合了整体原位杂交的方法，在溶液中处理组织切片，解决了碳蜡不易粘片的问题。在文章中，利用碳蜡原位杂交方法，我们在黄瓜的茎端分生组织（SAM）和花芽组织（FM）中检测了CLV3和WUS1基因的表达模式，实验结果与前期研究结果一致，而且该方法比石蜡原位杂交周期缩短4-5天，信号强。同样地，该方法也成功用于拟南芥中CLV3和WUS1的表达模式分析，表明碳蜡原位杂交方法在大多数物种中具有普遍适用性。
Abstract
In situ mRNA hybridization (ISH) is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber. The most common ISH protocol uses paraffin wax; however, embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals. Here, we developed an optimized protocol to simplify the process and improve RNA sensitivity. We combined embedding tissue in low melting-point Steedman’s wax with processing tissue sections in solution, as in the whole-mount ISH method in the optimized protocol. Using the optimized protocol, we examined the expression patterns of the CLAVATA3 (CLV3) and WUSCHEL (WUS) genes in shoot apical meristems and floral meristems of Cucumis sativus (cucumber) and Arabidopsis thaliana (Arabidopsis). The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin wax. Moreover, the optimized protocol achieved high signal sensitivity. The optimized protocol was successful for both cucumber and Arabidopsis, which indicates it might have general applicability to most plants
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Received: 23 March 2022
Accepted: 09 May 2022
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Fund: We thank Dr. Hu Bowen (Hunan Agricultural University, China) for valuable suggestions on this program. This work was supported by the National Natural Science Foundation of China (32002036).
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| About author: WANG Cui, E-mail: wangcui280@163.com; SUN Jin-jing, E-mail: sunjinjing@caas.cn; Correspondence ZHANG Hui-min, Tel/Fax: +86-532-58957760, E-mail: Zhanghm@qau.edu.cn
* These authors contributed equally to this study. |
Cite this article:
WANG Cui, SUN Jin-jing, YANG Xue-yong, WAN Li, ZHANG Zhong-hua, ZHANG Hui-min.
2023.
An optimized protocol using Steedman’s wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber. Journal of Integrative Agriculture, 22(2): 464-470.
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