Optimization of RNA In Situ Hybridization for mRNA Localization Detection in Mature Tissue of Cucumber Seedlings.
Optimization of RNA In Situ Hybridization for mRNA Localization Detection in Mature Tissue of Cucumber Seedlings.
复制标题
黄瓜幼苗成熟组织中 mRNA 定位检测的 RNA 原位杂交优化
DOI：
10.3390/plants9111461
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发表时间：
2020-10-29
期刊：
影响因子：
--
通讯作者：
Zhang W
中科院分区：
文献类型：
--
作者：
Liu Z;Hu X;Nie J;Li X;Wang Q;Liu W;Wang T;Lu X;Gao S;Gao L;Zhang W
黄瓜（Cucumis sativus L.）是我国主要蔬菜作物之一。黄瓜的生理栽培机理和基因功能特性对现代农业建设具有重要意义。由于黄瓜的遗传转化率较低，只有原位杂交才能方便地研究mRNA定位，而原位杂交不涉及基因修饰转化的进展，因此更适合于黄瓜成熟组织中mRNA定位的测定。目前，现有的原位杂交技术体系更适用于黄瓜分生组织，而不适用于黄瓜幼苗的成熟组织。因此，我们对传统的植物原位杂交方法进行了优化。取已知基因CsNPF7.2（硝酸盐转运蛋白家族）为例，对植物组织培养、基因探针制备、植物材料取样固定、横切面制备、杂交预处理、杂交孵育、显色反应、显微镜检查等步骤进行了优化，并在反应终止后进行处理，以获得一种新的RNA原位杂交技术，幼苗这种优化的技术将保证探针的产量、RNA分子的完整性以及植物组织结构的清晰和完整性，有利于黄瓜基因功能的研究和关键基因的筛选。
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.
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DOI：
10.1073/pnas.83.22.8447
发表时间：
1986-11-01
影响因子：
11.1
作者：
JEFFERSON, RA;BURGESS, SM;HIRSH, D
通讯作者：
HIRSH, D
影响因子：
9.2
作者：
Chiu, WL;Niwa, Y;Sheen, J
通讯作者：
Sheen, J
影响因子：
7.2
作者：
SHEEN, J;HWANG, SB;GALBRAITH, DW
通讯作者：
GALBRAITH, DW
影响因子：
3
作者：
ORIDATE, T;ATSUMI, H;ARAKI, H
通讯作者：
ARAKI, H
转录抑制因子 HANABA TARANU 通过整合拟南芥中多种激素、花器官规格基因和 GATA3 家族基因的作用来控制花发育
影响因子：
11.6
作者：
Zhang, Xiaolan;Zhou, Yun;Meyerowitz, Elliot M.
通讯作者：
Meyerowitz, Elliot M.