阅读说明：本技术 一种被子植物花部器官的线粒体荧光标记方法 (Mitochondrial fluorescence labeling method for angiosperm flower organs ) 是由 康萨如拉 赵坤 哈斯敖其尔 宝海风 于 2019-09-27 设计创作，主要内容包括：本发明公开了生物研究技术领域的一种被子植物花部器官的线粒体荧光标记方法,该标记方法如下：步骤一：将用于标记的荧光剂加入稀释液进行稀释；步骤二：将稀释后的荧光剂升温溶解,然后加入活性剂；步骤三：将植物花部器官制作成标本；步骤四：将荧光剂滴入在标本上；步骤五：通过显微镜观察状态,所述步骤一中稀释液为生理盐水,所述生理盐水与荧光剂的比例为100:1,所述荧光剂为健那绿染液,通过在对荧光剂中添加活性剂,提高荧光剂的表面活性,使其在滴入到实验标本上时可以快速的与线粒体中的细胞色素氧化酶进行氧化作用,提高荧光标记速度。(The invention discloses a fluorescence labeling method for mitochondria of angiosperm floral organs, belonging to the technical field of biological research, which comprises the following steps: the method comprises the following steps: adding a fluorescent agent for marking into a diluent for dilution; step two: heating and dissolving the diluted fluorescent agent, and then adding an active agent; step three: making plant flower organs into specimens; step four: dripping the fluorescent agent on the specimen; step five: observing the state through a microscope, wherein the diluent in the first step is physiological saline, the ratio of the physiological saline to the fluorescent agent is 100:1, the fluorescent agent is Jianna green dye solution, and the surface activity of the fluorescent agent is improved by adding an active agent into the fluorescent agent, so that the fluorescent agent can be quickly oxidized with cytochrome oxidase in mitochondria when being dripped onto an experimental specimen, and the fluorescence labeling speed is improved.)

1. A fluorescence labeling method for mitochondria of angiosperm floral organs is characterized in that: the marking method comprises the following steps:

the method comprises the following steps: adding a fluorescent agent for marking into a diluent for dilution;

step two: heating and dissolving the diluted fluorescent agent, and then adding an active agent;

step three: making plant flower organs into specimens;

step four: dripping the fluorescent agent on the specimen;

step five: the state was observed by a microscope.

2. The method of claim 1, wherein the method comprises the steps of: in the first step, the diluent is physiological saline, the ratio of the physiological saline to the fluorescent agent is 100:1, and the fluorescent agent is the Jiannalv dye solution.

3. The method of claim 1, wherein the method comprises the steps of: and the temperature for raising the temperature of the fluorescent agent in the second step is 30-40 ℃.

4. The method of claim 1, wherein the method comprises the steps of: the preparation method of the specimen in the third step comprises the steps of obliquely downwards cutting the separated flower part of the plant into 3-5mm thin slices by using a blade, placing the bottom of the thin slices in an experimental dish, adding cell culture solution, and standing for 24 hours.

5. The method of claim 1, wherein the method comprises the steps of: the fourth specific operation step is to drop the diluted fluorescent agent onto a thin sheet in a laboratory dish through a dropper, and standing for 10-15 min.

6. The method of claim 1, wherein the method comprises the steps of: and the observation method in the fifth step is to record the fluorescence label of the mitochondria at the same position after the microscope is adjusted to the state that the mitochondria can be clearly observed.

Technical Field

The invention relates to the technical field of biological research, in particular to a mitochondrial fluorescence labeling method for angiosperm flower organs.

Background

Angiosperms are the most evolved, most diverse, most widely distributed and most adaptable group in the world plant kingdom today. It is known that the angiosperms account for more than 20 ten thousand species in total and account for more than half of the total number of plant kingdoms all over the world. The known angiosperm in China belong to more than 2700 genera and more than 3 ten thousand species. The angiosperm and human being have close relationship, for example, the angiosperm in China can provide more than 2000 kinds of food; more than 300 fruit trees are available; the number of the flower plants is not enough; the medicinal angiosperm plants comprise 10027 (including the following classification units) which account for 90 percent of the total number of medicinal plants in China and are the group with the most medicinal types, and most of the traditional Chinese medicines are from the angiosperm plants.

Mitochondria, a two-layer membrane-coated organelle present in most cells, are structures that produce energy in cells, are the main sites for aerobic respiration of cells, and are called "powerhouse". The diameter is about 0.5 to 1.0 micron.

Most eukaryotic cells, except entamoeba histolytica, giardia lamblia and several microsporidia, possess more or less mitochondria, but their individual mitochondria differ in size, number and appearance.

Mitochondria possess their own genetic material and genetic system, but have a limited genome size and are a semi-autonomous organelle. In addition to powering cells, mitochondria are involved in processes such as cell differentiation, cell information transmission, and apoptosis, and possess the ability to regulate cell growth and cell cycle.

At present, when mitochondria are researched, the positions of the mitochondria need to be marked through fluorescence, so that the moving track of the mitochondria can be observed through a microscope, but the fluorescence is between the mitochondria marking, a user needs to inject a fluorescent agent into flower organs of plants, then waits for the fluorescent agent to be contacted with the mitochondria, and the fluorescent agent cannot be attracted by the mitochondria due to the characteristics of the fluorescent agent and needs to freely fall onto the mitochondria, so that the waiting time of the step is longer, and the whole experiment time is increased.

Disclosure of Invention

The invention aims to provide a mitochondrial fluorescence labeling method for angiosperm floral organs, which aims to solve the problem that the experiment process is too long due to the slow reaction time of a fluorescent agent and mitochondria in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a mitochondrial fluorescence labeling method of angiosperm floral organs comprises the following steps:

the method comprises the following steps: adding a fluorescent agent for marking into a diluent for dilution;

step two: heating and dissolving the diluted fluorescent agent, and then adding an active agent;

step three: making plant flower organs into specimens;

step four: dripping the fluorescent agent on the specimen;

step five: the state was observed by a microscope.

Preferably, the diluent in the first step is physiological saline, the ratio of the physiological saline to the fluorescent agent is 100:1, and the fluorescent agent is the Jianna green dye solution.

Preferably, the temperature for raising the temperature of the fluorescent agent in the second step is 30-40 ℃.

Preferably, the preparation method of the specimen in the third step is that the position of the separated plant flower part is obliquely cut downwards into a 3-5mm thin slice by using a blade, the bottom of the thin slice is placed in a laboratory dish, then the cell culture solution is added, and the thin slice is stood for 24 hours.

Preferably, the fourth specific operation step is to drop the diluted fluorescent agent onto a sheet in a laboratory dish through a dropper, and standing for 10-15 min.

Preferably, the observation method in the fifth step is to record the fluorescence label of the mitochondria at the same position after the microscope is adjusted to a state in which the mitochondria can be clearly observed.

Compared with the prior art, the invention has the beneficial effects that: by the fluorescent marking method for the mitochondria of the flower organs of angiosperm, at present, when the mitochondria are researched, the position of the mitochondria needs to be marked by fluorescence, so that the moving track of the mitochondria can be observed through a microscope, but the fluorescence is between the mitochondria markers, a user needs to inject the fluorescent agent into the flower organ of the plant and then wait for the fluorescent agent to contact the mitochondria, because the fluorescent agent is not attracted by mitochondria due to the characteristics of the fluorescent agent, the fluorescent agent is required to fall onto the mitochondria freely, the waiting time of the step is longer, the whole experiment time is increased, in the application document, the surface activity of the fluorescent agent is improved by adding the active agent into the fluorescent agent, so that the fluorescent agent can be quickly oxidized with cytochrome oxidase in mitochondria when being dripped onto an experimental specimen, and the fluorescence labeling speed is improved.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides the following technical scheme: a fluorescence labeling method for mitochondria of angiosperm floral organs is used for increasing fluorescence labeling speed and reducing experiment time.