Fluorescence labeling method for rapidly detecting free calcium ion distribution in rice anther

Abstract

The invention discloses a fluorescence labeling method for rapidly detecting the free calcium ion distribution in rice anther. The method comprises: (1) carrying out embedding freezing and slicing on rice anther, and pasting the slice containing the rice anther tissue onto a slide glass; (2) adding a Fluo-3AM fluorescence probe incubation liquid in a dropwise manner, carrying out dark incubation for 1-120 min in an incubator with a temperature of 4-37 DEG C, adding a fluorescence quenching slicing sealing liquid in a dropwise manner, and covering with a cover glass; and (3) observing the slice loading the Fluo-3AM fluorescence probe by using a fluorescence microscope. According to the present invention, the method is rapid and accurate, is suitable for the works requiring large batch detection and research, and can be applied to the detection of the free calcium ion distribution in other plant tissues.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fluorescent labeling method for rapidly detecting the distribution of free calcium ions in rice anthers. Background technique [0002] Calcium ion is an important element in plants and animals. At the same time, as a second messenger in cells, it activates a variety of protein kinases by binding to calmodulin, thus playing an important role in many life activities, such as cell division, apoptosis, cell It is an indispensable element for various biological activities such as extracellular stimuli and environmental ecological adaptation. Therefore, the detection of calcium ion activity and distribution in crop organs is of great significance for revealing the physiological activity and apoptosis changes of crop organs. [0003] Floral organs are the most sensitive and vulnerable organs for crop growth and development in response to adverse climates such as drought, high temperature...

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Problems solved by technology

[0003] Floral organs are the most sensitive and vulnerable organs for crop growth and development in response to adverse climates such as drought, high temperature, and cold damage, especially for traditional self-pollinating crops (such as rice, wheat, etc.), the development of floral organs The adversity damage is usually not easily detected by the sense organs, and the complex physiological and biochemical changes in the pollen development process (including the meiosis of microspore mother cells, the development of microspores, and the formation of male gametes) are difficult to be detected by conventional physiological methods. Exploring Ca during anther development of crops such as rice and wheat 2+ The activity and its distribution changes have become an extremely important means to study the physiological process of floral organ stress damage and the formation of sterile lines and their identification

[0004] At present, widely used in wheat and rice to detect its floral organ Ca 2+ The method of distribution is paraffin section or semi-thin section potassium pyroantimonate precipitation method. The advantages of these two methods are that after the section is stained, the shape and structure of the biological tissue structure are relatively clear, and the cell boundary is clear, but the disadvantages of these two methods are: Yes: (1) The film production needs to go through the steps of material collection, fixation, gradient dehydration, infiltration, embedding, sectioning, and staining, especially for paraffin sections. The time is long, the workload is heavy, and it is difficult to detect a large number of samples; (2) During the process of making paraffin sections and semi-thin sections, they must be fixed to kill cells, so that the section tissue loses enzyme activity, and cannot be used to detect living cells

[0008] However, the application of loading Fluo-3AM probes to observe the distribution of calcium ions in crop anthers, especially rice anthers, by making frozen sections has not been reported.

The main reasons are as follows: firstly, Liriodendron is a large deciduous tree,

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