阅读说明：本技术 一种Tris-HCl溶液的应用及其制备方法 (Application and preparation method of Tris-HCl solution ) 是由 田晓飞 张秀梅 封文萍 于 2020-12-22 设计创作，主要内容包括：本发明涉及生物技术领域,尤其涉及一种Tris-HCl溶液的应用及其制备方法。本发明所述的Tris-HCl溶液用于浮游动物的短期保存中,保存7天后检测浮游动物的蛋白酶活力时发现,保存7天的浮游动物的蛋白酶活力较活体样本并无显著差异。对大批量检测浮游生物具有重要的应用价值。(The invention relates to the technical field of biology, in particular to application of a Tris-HCl solution and a preparation method thereof. The Tris-HCl solution is used for short-term storage of zooplankton, and when the protease activity of the zooplankton is detected after the zooplankton is stored for 7 days, the protease activity of the zooplankton stored for 7 days is not obviously different from that of a living sample. Has important application value for detecting plankton in large batch.)

The application of Tris-HCl solution in the short-term storage of zooplankton.

2. A method of preparing a Tris-HCl solution according to claim 1, comprising the steps of:

(1) dissolving a Trizima base reagent in water to obtain a Tris solution with the molar concentration of 0.04-0.06 mol/L;

(2) and (3) regulating the pH value of the Tris solution in the step (1) by using hydrochloric acid to obtain a Tris-HCl solution.

3. The method of claim 1, wherein the Tris solution has a molarity of 0.05 mol/L.

4. The method according to claim 3, wherein the hydrochloric acid has a molar concentration of 0.5 to 1.5 mol/L.

5. The method according to claim 4, wherein the hydrochloric acid has a molar concentration of 1.0 mol/L.

6. The method according to any one of claims 2 to 5, wherein the pH is 7.9 to 8.1.

Technical Field

The invention relates to the technical field of biology, in particular to application of a Tris-HCl solution and a preparation method thereof.

Background

Zooplankton plays a very important role in the material and energy cycle of the water ecosystem. In nature, zooplankton populations are highly susceptible to external environmental factors. Among these, food conditions are one of the most important environmental factors. In poor food conditions, zooplankton increases survival by increasing the digestive absorption of key nutrients and energy. Meanwhile, researches show that the activity of the digestive enzyme of zooplankton plays an important role in adapting to the bad food environment. Therefore, the research on the digestive enzyme activity of zooplankton under different food conditions is especially necessary for exploring the adaptability of zooplankton populations to different food environments. However, due to the characteristic of easy decomposition of digestive enzymes, researchers need to take samples immediately for instant detection when performing digestive enzyme measurement, and the current fluorescent protein detection method commonly used for zooplankton has a long period (about 3 hours for each 12 samples), which limits the number of experimental samples, thereby restricting the development of large-sample experiments.

In addition, conventional zooplankton use 95% Ethanol (Ethanol) to preserve zooplankton for fluorescent protease detection, which results in egg release from oviparous adults due to high concentration of Ethanol (Ethanol) (> 70%) (Black and Dodson 2003), resulting in a decrease in total protein content of the sample. Therefore, the use of high Ethanol (Ethanol) to preserve the ovulated adults artificially increases the activity of the protease, and decreases the accuracy of the measurement. And the sample is removed and washed again before protease measurement by using high-concentration ethanol for storage, so that the opportunity of contacting the sample with the outside is increased in the process, and the decomposition probability of the protease is increased. Therefore, the development of a solution which has no influence on the activity of the zooplankton protease for short-term storage of plankton has important application value for large-scale detection of plankton.

Disclosure of Invention

The invention aims to provide a solution for short-term storage of plankton without influence on activity of zooplankton protease and a preparation method thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of a Tris-HCl solution in short-term storage of zooplankton.

The invention also provides a preparation method of the Tris-HCl solution, which comprises the following steps:

(1) dissolving a Trizima base reagent in water to obtain a Tris solution with the molar concentration of 0.04-0.06 mol/L;

(2) and (3) regulating the pH value of the Tris solution in the step (1) by using hydrochloric acid to obtain a Tris-HCl solution.

Preferably, the molar concentration of the Tris solution is 0.05 mol/L.

Preferably, the molar concentration of the hydrochloric acid is 0.5-1.5 mol/L.

Preferably, the molar concentration of the hydrochloric acid is 1.0 mol/L.

Preferably, the pH value is 7.9-8.1.

The invention provides an application of a Tris-HCl solution and a preparation method thereof, and the technical scheme of the invention has the following advantages:

the method not only improves the mode of taking and measuring when the traditional digestive enzyme is measured, improves the flexibility of experimental setting and sample amount, but also does not cause the release of eggs of an egg-carrying adult, and the preservation solution used by the method is the buffer solution for measuring the protease, can be used after being thawed, ensures that the sample does not need to contact the outside again, has the advantages of reducing the decomposition risk of the protease and the like, and can provide a convenient and practical sample preservation method for developing the research on the digestion and absorption capacity of zooplankton, so the method can be considered to have various advantages, is suitable for popularization and application in the field, and has great scientific research significance.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Taking a 100ml beaker, adding 60ml of distilled water, then weighing 0.484g of Trizima base, dissolving in water, fully stirring and dissolving, and adding distilled water to a constant volume of 100ml to obtain a Tris solution with a molar concentration of 0.04 mol/L.

Taking a 100ml beaker, adding 1ml of 37% concentrated hydrochloric acid into the beaker, then adding 23ml of distilled water, fully and uniformly mixing to prepare a 0.5N hydrochloric acid solution, and finally placing the solution in a reagent bottle for storage at 4 ℃.

The pH of the Tris solution with a molar concentration of 0.04mol/L was adjusted to 7.9 using a 0.5N hydrochloric acid solution, and finally stored in a reagent bottle at 4 ℃ to obtain the Tris-HCl storage solution of example 1.

Example 2

Taking a 100ml beaker, adding 60ml of distilled water, then weighing 0.605Trizima base, dissolving in water, fully stirring and dissolving, and adding distilled water to a constant volume of 100ml to obtain a Tris solution with a molar concentration of 0.05 mol/L.

Taking a 100ml beaker, adding 2ml of 37% concentrated hydrochloric acid into the beaker, then adding 22ml of distilled water, fully and uniformly mixing to prepare a 1N hydrochloric acid solution, and finally placing the solution in a reagent bottle for storage at 4 ℃.

The pH of the Tris solution with a molar concentration of 0.05mol/L was adjusted to 8.0 using a 1N hydrochloric acid solution, and finally stored in a reagent bottle at 4 ℃ to obtain a Tris-HCl storage solution of example 2.

Example 3

Taking a 100ml beaker, adding 60ml of distilled water, then weighing 0.726g of Trizima base, dissolving in water, fully stirring and dissolving, and adding distilled water to a constant volume of 100ml to obtain a Tris solution with a molar concentration of 0.06 mol/L.

Taking a 100ml beaker, adding 3ml of 37% concentrated hydrochloric acid into the beaker, then adding 22ml of distilled water, fully and uniformly mixing to prepare a 1.5N hydrochloric acid solution, and finally placing the solution in a reagent bottle for storage at 4 ℃.

The pH of the Tris solution with a molar concentration of 0.06mol/L was adjusted to 8.1 using a 1.5N hydrochloric acid solution, and finally stored in a reagent bottle at 4 ℃ to obtain the Tris-HCl storage solution of example 3.

Example 4

Healthy monoclonal daphnia magna (Daphniaapuex) born by the same female parent on the same day is cultured in a culture flask filled with aerated tap water, and Scenedesmus obliquus (Scenedesmus obliquus) with 1.0mg C/L is added into the culture flask. Culturing at 20 deg.C under 24h of light, changing water every two days, feeding Scenedesmus obliquus (Scenedesmus obliquus) 1.0mg C/L every day, and culturing for 5 days to obtain daphnia magna to be detected.

Application example 1

Randomly selecting 3 daphnia magna individuals to be detected, which are obtained in the embodiment 4; transferring the culture water from the culture water body to a 30ml culture dish by using a 10ml rubber head dropper, removing the culture water in the culture dish by using a 2ml rubber head dropper with a smaller suction port in order to prevent the zooplankton from being sucked away, then washing the surface of the zooplankton by using sufficient distilled water, removing the distilled water by using a 2ml rubber head dropper again, and repeating the washing for three times.

The alkaline phosphatase activity of 3 Daphnia magna was measured according to the method for measuring alkaline phosphatase reported in Tian et al 2019 in Evolution of advanced Daphnia pulex in Japan and variations of the digestive, morpholino and life history. The measurement results are shown in Table 1.

Application example 2

Randomly selecting 3 daphnia individuals to be detected obtained in the example 4, cleaning the daphnia individuals according to the cleaning method of the application example 1, storing the daphnia individuals to be detected in 0.6ml of centrifuge tubes filled with 200 mu L of the solution prepared in the example 1, closing the centrifuge tubes, and storing the daphnia individuals at-20 ℃. After 7 days, the centrifuge tubes were thawed at room temperature, and after sufficient thawing, the alkaline phosphatase activity in daphnia magna was measured after 7 days of storage per one according to the measurement method of alkaline phosphatase reported by Tian et al. The measurement results are shown in Table 1.

Application example 3

Randomly selecting 3 daphnia magna individuals to be detected, which are obtained in the embodiment 4; daphnia magna was washed according to the method of application example 1.

The cleaned 3 daphnia flea were stored in 0.6ml centrifuge tubes containing 200. mu.L of the solution prepared in example 2, respectively, and finally the centrifuge tubes were closed and stored at-20 ℃. After 7 days, the centrifuge tubes were thawed at room temperature, and after sufficient thawing, the alkaline phosphatase activity in daphnia magna was measured after 7 days of storage per one according to the measurement method of alkaline phosphatase reported by Tian et al. The measurement results are shown in Table 1.

Application example 4

Randomly selecting 3 daphnia magna individuals to be detected, which are obtained in the embodiment 4; daphnia magna was washed according to the method of application example 1.

The cleaned 3 daphnia flea were stored in 0.6ml centrifuge tubes containing 200. mu.L of the solution prepared in example 3, respectively, and finally the centrifuge tubes were closed and stored at-20 ℃. After 7 days, the centrifuge tubes were thawed at room temperature, and after sufficient thawing, the alkaline phosphatase activity in daphnia magna was measured after 7 days of storage per one according to the measurement method of alkaline phosphatase reported by Tian et al. The measurement results are shown in Table 1.

TABLE 1 determination of daphnia magna individual alkaline phosphatase activity

As can be seen from Table 1, the activity of alkaline phosphatase in an individual is detected after the daphnia pulex is stored for 7 days by using the storage liquid of the embodiment 1-3, and the result of directly detecting daphnia pulex living bodies is not different. The Tris-HCl preservation solution of the application can not influence the expression of the digestive enzyme activity of the zooplankton.

The embodiment shows that the method not only improves the mode of taking and measuring the traditional digestive enzyme when measuring, improves the flexibility of experimental setting and sample amount, but also can not cause the release of eggs of an oviparous adult, and the preservation solution used by the method is the buffer solution for measuring the protease, can be used after being thawed, ensures that the sample does not need to contact the outside again, has the advantages of reducing the decomposition risk of the protease and the like, and can provide a convenient and practical sample preservation method for developing the research on the digestion and absorption capacity of zooplankton, so the method can be considered to have various advantages, is suitable for popularization and application in the field, and has great scientific research significance.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.