Method for lossless evaluation of heat resistance of stichopus japonicus

Abstract

The invention relates to the technical field of stichopus japonicus breeding, in particular to a method for losslessly evaluating heat resistance of stichopus japonicus, which comprises the following steps: S1, collecting foot tissues from a living stichopus japonicus sample, and pretreating to obtain enzyme-containing tissue fluid; s2, determining the thermal stability of malate dehydrogenase of the enzyme-containing tissue fluid; and S3, performing covariance analysis on the measured data, and comparing the time change slopes of the hot residual enzyme activity of different stichopus japonicus samples, so that the smaller the slope is, the higher the heat resistance of the stichopus japonicus sample is. According to the technical scheme, the heat resistance of the stichopus japonicus individual can be accurately determined, the death of the living stichopus japonicus of the detected sample is avoided, and the method is suitable for screening the heat resistance in stichopus japonicus breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of sea cucumber breeding, and particularly relates to a method for non-destructively evaluating the heat resistance of sea cucumbers. Background Art

[0002] Sea cucumbers are an important species for aquaculture along the northern coast of my country, and are also one of the species with the highest output value in my country's marine aquaculture. The suitable growth temperature range for sea cucumbers is 10-20°C. When the water temperature exceeds 24°C, sea cucumbers will enter a state of summer hibernation. If the water temperature exceeds 32°C for a long time, sea cucumbers will die on a large scale. However, under the background of global climate change, the average temperature has gradually risen, and extreme high temperatures have frequently occurred in summer. In recent years, the continuous high temperature weather in my country in summer has caused the water temperature of land-based ponds to be high and last for a long time. In some areas, the bottom water temperature of ponds is as high as 34°C or above, which has had a serious impact on sea cucumber farming, and a large number of sea cucumbers have rotted and died. In addition, the "north sea cucumber and south sea cucumber farming" industry has put forward a demand for sea cucumber seedlings with high temperature resistance.

[0003] Since the tolerance of sea cucumbers to high temperatures is heritable, selecting sea cucumbers with high temperature tolerance is a key step in the breeding of sea cucumbers that are resistant to high temperatures. Indicators commonly used to reflect the heat resistance of sea cucumbers include initial lethal temperature and critical thermal extremes. Although the measurement methods of the two indicators are different, both will lead to a large number of deaths of the tested sea cucumbers, which is not conducive to the selection of individuals with high temperature tolerance. Therefore, the development of a non-destructive method for evaluating the heat resistance of sea cucumbers is crucial for the breeding of sea cucumbers. Summary of the invention

[0004] In view of the above technical problems, the present invention proposes a method for non-destructively evaluating the heat resistance of sea cucumbers, comprising the following steps:

[0005] S1 collected tube foot tissue from live sea cucumber samples and obtained enzyme-containing tissue fluid after pretreatment;

[0006] S2 determines the thermal stability of malate dehydrogenase in enzyme-containing tissue fluid;

[0007] S3 performed covariance analysis on the measured data to compare the time-varying slopes of thermal residual enzyme activity of different sea cucumber samples. The sea cucumber sample with a smaller slope had higher heat resistance.

[0008] Furthermore, the pretreatment in S1 specifically includes the following steps: cutting the tube foot tissue into pieces, grinding it in an ice bath, adding pre-cooled physiological saline to prepare a tissue homogenate, placing the tissue homogenate in a centrifuge tube, centrifuging it at 4°C, 4000 / min for 30 minutes, and the supernatant is the enzyme-containing tissue fluid, which is stored at 4°C.

[0009] Furthermore, S2 specifically includes the following steps: taking the enzyme-containing tissue fluid and heat-shock it at 32°C for 3 minutes, then dialyzing it at 4°C, heating the enzyme-containing tissue fluid after dialyzation, and when the temperature reaches 32°C, taking out a small amount of the enzyme-containing tissue fluid at multiple different time points, cooling it to 25°C to determine the malate dehydrogenase activity.

[0010] Furthermore, in S2, malate dehydrogenase activity was measured using a UV-1800 Shimadzu UV spectrophotometer.

[0011] Furthermore, the concentration of the reaction substrate oxaloacetate was 200 μmol / L, and the residual activity was defined as the percentage of the enzyme activity at time t to the enzyme activity at time 0.

[0012] Furthermore, the temperature of the cuvette in S2 is controlled by a circulating water bath, and the temperature is controlled within ±0.2°C of the set temperature.

[0013] Furthermore, the NADH concentration in S2 was 150 μmol / L.

[0014] Furthermore, the buffer used to measure the activity of malate dehydrogenase in S2 is Imidazole-HCl buffer.

[0015] Furthermore, the multiple different time points in S2 include time points at 0, 5, 10, 15, 20, 30, 45, 60 and 75 minutes respectively.

[0016] On the other hand, the present invention also provides a method for breeding heat-resistant sea cucumbers, which uses the above-mentioned method for non-destructively evaluating the heat resistance of sea cucumbers to breed heat-resistant sea cucumbers.

[0017] The technical solution of the present invention can accurately measure the heat resistance of individual sea cucumbers without causing the death of the living sea cucumbers of the tested samples, and is suitable for screening heat resistance in sea cucumber breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Changes in residual activity of sea cucumber samples collected from different populations after heat shock at 32°C; the illustration in the upper right corner is a plot of the logarithm of residual enzyme activity against time. DETAILED DESCRIPTION

[0019] The sea cucumber described in the present invention, also known as the imitation sea cucumber, is an animal of the genus Imitation Sea Cucumber of the family Stichopus in the order Stichopus. Its body wall is thick and soft, cylindrical; its back is raised, with 4-6 rows of irregularly arranged conical tube feet; its ventral surface is flat, with dense tube feet.

[0020] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0021] Live sea cucumber samples from four sea cucumber populations were collected, and the operating steps in the following examples and control examples were performed for the live sea cucumber samples from each population.

[0022] Example 1

[0023] The following steps are involved:

[0024] S1 collected tube foot tissue from live sea cucumber samples and obtained enzyme-containing tissue fluid after pretreatment;

[0025] The steps are as follows:

[0026] The podia tissue was cut into pieces, ground in an ice bath, and pre-cooled physiological saline was added to prepare a tissue homogenate. The tissue homogenate was placed in a centrifuge tube and centrifuged at 4°C, 4000 / min for 30 min. The supernatant was the enzyme-containing tissue fluid, which was stored at 4°C.

[0027] S2 determines the thermal stability of malate dehydrogenase in enzyme-containing tissue fluid;

[0028] This step specifically includes the following steps: taking the enzyme-containing tissue fluid, heat-shocking it at 32°C for 3 minutes, and then dialyzing it at 4°C; heating the enzyme-containing tissue fluid after dialyzation, and when the temperature reaches 32°C, taking out a small amount of the enzyme-containing tissue fluid at multiple different time points, cooling it to 25°C, and determining the malate dehydrogenase activity.

[0029] In this example, malate dehydrogenase activity was measured at time points of t=0, 5, 10, 15, 20, 30, 45, 60 and 75 min, respectively.

[0030] The steps for determining malate dehydrogenase activity are as follows:

[0031] The activity of malate dehydrogenase was determined by ultraviolet spectrophotometer. By comparing the absorbance changes before and after the reaction, the activity of the cytoplasmic malate dehydrogenase of the sea cucumber can be indirectly reflected. While MDH catalyzes the conversion of oxaloacetate into malate, it oxidizes NADH to NAD+, causing the absorbance of NADH at 340nm to decrease continuously. The enzyme activity can be calculated by continuously measuring the change of absorbance at 340nm during the enzyme reaction.

[0032] The assay buffer is Imidazole-HCl, pH 7.0, the reaction substrate oxaloacetic acid concentration is 200 μmol / L, and the NADH concentration is 150 μmol / L. The calculation formula is:

[0033]

[0034] Where: △A340nm / min: decrease in absorbance per minute

[0035] VT: total reaction volume, ml

[0036] Vs: sample volume, ml

[0037] df: dilution factor

[0038] e: molar absorptivity of NADH at 340 nm, 6.22×10 -3 mL / μmol.cm b: cuvette light path, cm

[0039] The concentration of the reaction substrate oxaloacetic acid (OAA) was 200 μmol l-1. The residual activity was defined as the percentage of the enzyme activity at time t to the activity at t=0.

[0040] S3 performed covariance analysis on the measured data to compare the time-varying slopes of thermal residual enzyme activity of different sea cucumber samples. The sea cucumber sample with a smaller slope had higher heat resistance.

[0041] The residual activity of live sea cucumber samples collected from different populations after heat shock at 32℃ is as follows Figure 1 .

[0042] Comparative Example 1

[0043] The living sea cucumber sample after the operation of Example 1 is heated at a certain heating rate. During the heating process, the body temperature of the living sea cucumber sample can be the same as the water temperature, until the sea cucumber loses its body balance and loses the temperature to escape from the dangerous environment. The specific operation method is as follows:

[0044] Before the experiment, 300 sea cucumbers of different species were taken and temporarily kept in an oxygenated water basin. The temperature was raised from 16℃ at a rate of 1℃ / h until all sea cucumbers died. 30℃ was found to be the mortality inflection point, and the death temperature of all groups was 36℃.

[0045] The formal heat resistance experiment was conducted in a water bath with healthy sea cucumbers that had been temporarily raised. Each type of imitation sea cucumber was divided into 3 groups, with 20 in each group. Starting from 16°C, the temperature was raised at a rate of 0.5°C / h to 30°C. After 30°C, the temperature was raised at a rate of 0.5°C / 2h until all sea cucumbers died. During the experiment, the survival status of the sea cucumbers was observed in time, and the survival time of each sea cucumber individual was recorded. The experiment ended when all sea cucumbers died. The time when all sea cucumbers died was the maximum critical temperature (CTMax).

[0046] The heat tolerance index CTMax results of the four sea cucumber populations are shown in Table 1.

[0047] Comparative Example 2

[0048] The live sea cucumber samples after the operation of Example 1 were suddenly exposed from a certain acclimation temperature to a series of higher experimental temperatures, and after 2 hours, they were transferred to the acclimation temperature for recovery, and then the mortality of the sea cucumbers at the experimental temperatures was observed to determine the initial lethal temperature. The specific operation method is as follows:

[0049] The experiment was conducted in a water bath with healthy imitation sea cucumbers that had been temporarily raised. Each type of sea cucumber was divided into 3 groups, with 20 in each group. Starting from 16°C, the temperature was raised to 30°C at a rate of 0.5°C / h. After 30°C, the temperature was raised at a rate of 0.5°C / 2h until all sea cucumbers died. During the experiment, the survival status of the sea cucumbers was observed in time, and the survival time of each sea cucumber individual was recorded. The experiment ended when all sea cucumbers died. The heat tolerance index (UTT) was calculated based on the death time. The heat tolerance index UTT results of the four sea cucumber groups are shown in Table 1.

[0050] Table 1. Heat resistance and critical high temperature values ​​of four sea cucumber populations

[0051]

[0052] Conclusion: According to the results of Example 1 and Control Examples 1 and 2, the results of Example 1 are consistent with the results of Control Examples 1 and 2, which can indicate that the technical solution of Example 1 can evaluate the heat resistance of sea cucumbers.

[0053] Comparative Example 3

[0054] The invention patent 202010704474.X discloses the determination of chronic high temperature tolerance of sea cucumbers. The specific method is as follows:

[0055] 30 sea cucumbers were selected for temporary culture and placed evenly in three water tanks. The initial temperature of the experimental water was the same as the temporary culture temperature. After the sea cucumbers stabilized, the temperature began to rise. The heating rate was 1°C / h, and the phenomenon was observed. When the experimental sea cucumbers lost their adhesion and floated or could be easily moved with a glass rod, the water temperature was measured and recorded. The experimental sea cucumbers were immediately fished out and placed at the corresponding starting temperature. If they could recover normal behavior, the data was valid. If they could not recover normal behavior, the data was discarded. It was ensured that each group obtained more than 5 valid data. The arithmetic mean of the water temperature was the CHT value. The larger the CHT value, the stronger the high temperature tolerance of the tested sea cucumbers. The thermal tolerance time (TTT) was determined, and the calculation formula was:

[0056]

[0057] In the formula, i is the number of hours, Ti is the temperature at the i-th hour, T0 is 25℃, the initial temperature of the experiment, and k is the number of hours of survival. 30 sea cucumbers were randomly selected and placed in three water tanks. The temperature was raised by 1℃ every 24 hours from 25℃. The temperature was stopped after reaching 32℃ and maintained until the end of the experiment. In the experiment, the temperature was measured once every hour, and the pool water was disturbed at the same time. Dead or dying individuals were fished out and the survival time was recorded. When the total number of dead individuals in each water tank reached 100%, the heat resistance experiment was terminated. The average TTT value of each group of sea cucumbers was obtained to determine the high temperature tolerance TTT value of the sea cucumber population.

[0058] Conclusion: The data measured by the control method are accurate and reliable, but all the tested sea cucumber samples died. Even if reliable data on the heat resistance of sea cucumber samples can be measured, living samples with good heat resistance cannot be obtained for breeding, and therefore it cannot be used in sea cucumber breeding.

[0059] Comparative Example 4

[0060] The invention patent 202010704474.X discloses the determination of the critical temperature of acute high temperature of sea cucumber, and the specific steps are as follows:

[0061] Record the stress temperature value of each tested sea cucumber. When the experimental sea cucumber loses its adhesion and floats or can be easily moved with a glass rod, immediately remove the experimental sea cucumber and place it at the corresponding starting temperature. If it can resume normal behavior, the data is valid; that is, the heat resistance CHT value of the sea cucumber is obtained.

[0062] Conclusion: The data measured by the method of the control example is accurate and reliable, but a large number of the tested sea cucumber samples died, and only a few samples survived, indicating that the cost of this embodiment is very high.

[0063] Although the present invention has been described in detail above by general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for non-destructive evaluation of the heat resistance of sea cucumbers, characterized in that: The following steps are involved: S1 collected tube foot tissue from live sea cucumber samples and obtained enzyme-containing tissue fluid after pretreatment; S2 determines the thermal stability of malate dehydrogenase in enzyme-containing tissue fluid; S3 performed covariance analysis on the measured data to compare the time-varying slopes of thermal residual enzyme activity of different sea cucumber samples. The sea cucumber sample with a smaller slope had higher heat resistance.

2. The method according to claim 1, characterized in that The pretreatment in S1 specifically includes the following steps: mincing the tube foot tissue, grinding it in an ice bath, adding pre-cooled physiological saline to prepare a tissue homogenate, placing the tissue homogenate in a centrifuge tube, centrifuging it at 4°C, 4000 / min for 30 minutes, and the supernatant is the enzyme-containing tissue fluid, which is stored at 4°C.

3. The method according to claim 1, characterized in that S2 specifically includes the following steps: taking the enzyme-containing tissue fluid, heat-shocking it at 32°C for 3 minutes, and then dialyzing it at 4°C; heating the enzyme-containing tissue fluid after dialyzation, and when the temperature reaches 32°C, taking out a small amount of the enzyme-containing tissue fluid at multiple different time points, cooling it to 25°C, and determining the malate dehydrogenase activity.

4. The method according to claim 3, characterized in that In S2, malate dehydrogenase activity was measured using a Shimadzu UV-1800 spectrophotometer.

5. The method according to claim 3, characterized in that: The concentration of the reaction substrate oxaloacetate in S2 was 200 μmol / L, and the residual activity was defined as the percentage of the enzyme activity at time t to the enzyme activity at time 0.

6. The method according to claim 3, characterized in that The temperature of the cuvette in S2 was controlled by a circulating water bath and the temperature was controlled within ±0.2°C of the set temperature.

7. The method according to claim 3, characterized in that The NADH concentration in S2 was 150 μmol / L.

8. The method according to claim 3, characterized in that The buffer used to measure the activity of malate dehydrogenase in S2 is Imidazole-HCl buffer.

9. The method according to claim 1, characterized in that: The multiple different time points in S2 include time points at 0, 5, 10, 15, 20, 30, 45, 60 and 75 minutes respectively.

10. A method for breeding heat-resistant sea cucumbers, characterized in that: Breeding heat-resistant sea cucumbers using any one of the methods for non-destructively evaluating the heat resistance of sea cucumbers as described in claims 1 to 9.

Images