Optimization of RNA In Situ Hybridization for mRNA Localization Detection in Mature Tissue of Cucumber Seedlings.

Zixi Liu,Jing Nie,Shunli Gao,Wenqian Liu,Tao Wang,Lihong Gao,Qing Wang,Xiaohong Lu,Xi Hu,Xiaojun Li,Wenna Zhang

doi:10.3390/plants9111461

Abstract

Cucumber (Cucumis sativus L.) is one of the main vegetable crops in China. The physiological cultivation mechanism and gene function characteristics of cucumber are of great significance to the construction of modern agriculture. Due to the low genetic transformation rate of cucumber, only in situ hybridization, which does not involve the progress of gene modified transformation, is convenient to study mRNA localization, so it is more suitable for determination on mRNA localization in the mature tissue of cucumber. At present, the existing in situ hybridization technology system is more suitable for cucumber meristem than for the mature tissue of cucumber seedlings. Therefore, we optimized the traditional plant in situ hybridization protocol. Taking a known gene CsNPF7.2 (Nitrate Transporter Families protein) as an example, we then optimized the steps of plant tissue culture, gene probe preparation, plant material sampling and fixation, preparation of cross section, hybridization pretreatment, hybridization incubation, chromogenic reaction, microscopy examination, and treatment after reaction termination in order to obtain a new RNA in situ hybridization technique suitable for identification on mRNA localization in mature tissues of cucumber seedlings. This optimized technique will ensure the yield of probes, the integrity of RNA molecules, and the clarity and integrity of plant tissue structure, which is conducive to the study of gene function and screening of key genes in cucumber.

Highlights

Cucumber (Cucumis sativus L.) is a main vegetable crop in China, which is the country with the largest cucumber production in the world
In situ hybridization is more suitable for mRNA localization at the tissue level in cucumber
The existing in situ hybridization technology system applied to plants is suitable for meristems of plants as well as most mature tissues of plants

Summary

Introduction

Cucumber (Cucumis sativus L.) is a main vegetable crop in China, which is the country with the largest cucumber production in the world. Organization of the United Nations (FAO), the proportion of cucumber annual yield in China accounted for 76.5% of the world in 2017, compared with 48.3% in 1961. With the progress of genomics and bioinformatics, the whole-genome information of cucumber was deciphered [1]. Most of these nucleotide sequences that were deciphered are new genes with missing annotation information. At this point, it is an urgent challenge for us to figure out the function of these new genes

Methods

Results

Conclusion