Fluorescent labeling method for rapid detection of free calcium ion distribution in rice anthers

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 of plants and animals. At the same time, it acts as a second messenger in the cell to activate a variety of protein kinases by binding to calmodulin, thus playing an important role in many life activities, such as cell division, cell apoptosis, and cell Feel the indispensable elements of various biological activities such as extracellular stimulation and environmental and ecological adaptation. Therefore, the detection of calcium ion activity and distribution in crop organs is of great significance for revealing the physiological vitality and cell apoptosis changes of crop organs. [0003] Floral organs are the most sensitive and susceptible organs for crop growth and development to drought, high temperature and cold da...

AI Technical Summary

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, and its anthers are large in volume, which is easy to handle when making frozen slices. The volume of rice anthers is much smaller than that of Liriodendron, which is difficult to handle; secondly, Liriodendron belongs to cold-resistant plants. Growth at -15°C is completely unharmed. When making frozen sections, ice crystals are not easy to form during low-temperature freezing, which can better maintain the fine structure of plant cells and maintain the integrity of plant tissues. However, the water content of rice anthers High, not cold-resistant, poor transparency, its large amount of water makes plant samples easy to form ice crystals during low-temperature freezing, damages the fine structure of plant cells, and makes it difficult to maintain the integrity of plant tissues; again, the loading effect of Fluo-3AM is affected by incubation Objects, incubation temperature, time and concentration, etc.

[0009] The embedding methods currently applied to plant frozen sections can be summarized into three types: ① direct embedding method, which is the most convenient method, but the solidification process of embedding agent is slow, and it is easy to form ice crystal damage, resulting in slice damage and incomplete structure; ② liquid embedding method. Nitrogen quick-freezing method, that is, put the tissue into a soft plastic bottle cap, add an appropriate amount of OCT embedding agent to immerse, and then put it above the liquid nitrogen liquid surface, so that the embedded tissue can be quickly frozen into a block, this method will cause tissue and embedding Bubbles are very easy to appear on the contact surface of the agent, and it is difficult to fix the direction for irregular tissues, which affects the angle of sectioning, and the effect of forming slices is poor; The freezing point, but during the fixed osmotic process, it will affect the osmotic potential of the cells and cause changes in the distribution of calcium ions in the cells

The rice anthers are small and the water content is high, which increases the difficulty of frozen sectioning. These three methods cannot obtain the best results

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