Method for evaluating high temperature resistance of abalones by using mitochondrial quantity

Abstract

The invention discloses a method for evaluating high-temperature resistance of abalones by using mitochondrial quantity. The method comprises the steps of sample collection, DNA extraction, mitochondrial DNA copy number determination, standard establishment, high-temperature resistance evaluation and the like. The method has the advantages of rapidness, accuracy, no damage, low cost and the like, and the evaluation of the high-temperature resistance of the abalone can be completed in a short time by measuring the copy number of the mitochondrial DNA; only a small amount of abalone tissue samples need to be collected, and the influence on abalone growth and development is small; required equipment and reagents are relatively common, and the cost is low; the method can be applied to early screening and evaluation of abalone seedlings, can also be applied to high-temperature resistance evaluation of adult abalones, and is wide in application range; the mitochondrial DNA copy number has significant correlation with the high temperature resistance of abalones, and can be used as a reliable biological marker; and the method can also be combined with other physiological indexes or molecular markers to establish a multi-index comprehensive evaluation model, so that the accuracy and reliability of evaluation are improved, and the expandability is high.

Description

Technical Field

[0001] This invention relates to a method for assessing the heat resistance of abalone using mitochondrial count, belonging to the field of marine bioengineering technology. Background Technology

[0002] Shellfish are cold-blooded marine animals, highly sensitive to changes in environmental temperature. With the intensification of global warming and the increasing frequency of extreme heat waves, the resulting extreme summer temperatures will significantly impact shellfish growth and reproduction, even threatening their survival. Marine aquaculture, especially shellfish farming relying on raft culture, is therefore facing severe damage and losses. Abalone is an important marine aquaculture species in my country. After years of large-scale production, farmed abalone faces problems such as frequent disease outbreaks and slowed growth rates, which have become major factors restricting the industry's development. Currently, the main production area of ​​abalone, one of my country's major marine aquaculture shellfish, is located in the south. Against the backdrop of global warming, summer temperatures in the southern sea areas are constantly rising, with extreme seawater temperatures reaching above 30°C. The sustained high temperatures in summer have significantly exceeded the suitable temperature range for abalone, resulting in a summer mortality rate of over 30%, severely restricting the sustainable development of the abalone farming industry.

[0003] Abalone is a high-value aquatic product, but its cultivation also carries high costs and risks. Currently, global warming is causing seawater temperatures to rise, potentially exceeding the heat tolerance limits of some abalone habitats (such as shallow seas and intertidal zones). High summer temperatures can lead to excessively high water temperatures in aquaculture ponds, causing stress, slowed growth, or death in abalone. Human intervention is difficult to implement, or its costs are high, or timely responses are limited, resulting in significant economic losses. Therefore, assessing the heat tolerance of abalone is crucial for preventing heat-related problems and selecting suitable species to address the challenges of climate change. Temperature control in aquaculture environments and the selection of appropriate species are also vital for ensuring the development of aquaculture, promoting genetic breeding innovation, and maintaining ecological balance. These factors also hold significant importance for future sustainable fisheries and marine conservation. Summary of the Invention

[0004] This invention provides a method for assessing the heat resistance of abalone using mitochondrial count, which can effectively solve the above-mentioned problems.

[0005] This invention is implemented as follows: A method for assessing the heat tolerance of abalone using mitochondrial count includes the following steps: (1) Collect tissue samples from abalone and extract DNA; (2) Mitochondrial DNA copy number determination: The relative concentrations of mitochondrial and nuclear genomes were detected by using the COX1 gene on mitochondria and the single-copy gene RPL7 in the nucleus, respectively. The Ct values ​​of mitochondrial genes and the Ct values ​​of nuclear single-copy gene amplification were obtained, and the mtDNA copy number in each cell was calculated. (3) Establish standards and evaluate heat resistance: By measuring the mitochondrial DNA copy number of abalone individuals with different heat resistance, establish a correlation standard between mitochondrial DNA copy number and heat resistance of abalone; then establish an evaluation relationship based on the mitochondrial DNA copy number measured in step 2) and the standard to obtain the heat resistance of the abalone to be evaluated.

[0006] In some embodiments, the abalone is selected from healthy abalone individuals that are over 1 year old or have a shell length of over 4 cm.

[0007] In some embodiments, the tissue sample collected in step (1) is gill tissue or mantle tissue. In some embodiments, the gill tissue is the apical region of the gill tissue of the target organism as the sampling site.

[0008] During the sample preparation stage, in order to meet the requirements of transmission electron microscopy observation, the apical region of the gill tissue of the target organism was selected as the sampling site (because this region is directly involved in material exchange and environmental response, its ultrastructural characteristics have important research value). The specific procedure is as follows: Using sterile dissecting instruments (such as scalpels and forceps), precisely cut a tissue block of approximately 1 mm³ from the gill apex. Immediately immerse it in pre-cooled 2.5% glutaraldehyde fixative (prepared with 0.1 mol / L phosphate buffer, pH 7.2-7.4) for primary fixation to rapidly stabilize the cell ultrastructure (avoiding organelle autolysis or morphological distortion). After fixation for at least 2 hours (or overnight at 4°C), wash three times with the same concentration of buffer (15 minutes each time), then transfer to 1% osmium tetroxide fixative for secondary fixation (1-2 hours) to enhance the electron density of the cell membrane and organelles. Subsequently, dehydrate using a gradient of ethanol (30%, 50%, 70%, 80%, 90%, 95%, 100%) (15-20 minutes each step), transition with acetone, and embed in epoxy resin (such as Epon 812). Polymerize in a polymerization oven at 60°C for 48 hours to form an embedded block. Finally, use an ultramicrotome (such as Leica EM) for embedding. UC7) Sections were prepared along the functional axis of the gill apex tissue to obtain ultrathin sections with a thickness of 60-80 nm. After double staining with uranium acetate and lead citrate, the sections were used for observation and analysis of ultrastructure (such as mitochondrial morphology, cell membrane integrity, organelle distribution, etc.) under transmission electron microscopy.

[0009] In some embodiments, the DNA extraction method in step (1) is as follows: DNA is extracted from the collected tissue sample using the EasyPure Genomic DNA Kit.

[0010] In some embodiments, the method for determining the mitochondrial DNA copy number in step (2) is as follows: the mitochondrial DNA copy number is determined by real-time quantitative PCR (qPCR) using the SYBR Green II dye method, and the reaction is performed on a Bio-Rad CFX96™ Real-Time PCR Systems detection system; 2-4 technical replicates are performed for each sample.

[0011] In some implementations, the formula for calculating the mitochondrial DNA copy number in step (2) is as follows: N = 2 -Ctmt ÷2 -Ctn Where N: mtDNA copy number per cell; Ctmt: mitochondrial gene Ct value; Ctn: nuclear single-copy gene amplification Ct value.

[0012] In some implementations, the correlation criteria between mitochondrial DNA copy number and abalone's heat resistance in step (3) are established through the following steps: 1) Select abalone populations from different sources, including abalone varieties known to be heat-resistant and common abalone varieties; 2) Determine the copy number of mitochondrial DNA in each abalone individual; 3) Determine the high-temperature tolerance indicators of each abalone individual; 4) Analyze the correlation between mitochondrial DNA copy number and thermodynamic tolerance indicators to establish evaluation criteria. In some embodiments, the thermodynamic tolerance indicators include at least the high initial lethal temperature and the critical temperature.

[0013] In some implementations, step (3) further includes combining other physiological indicators or molecular markers with mitochondrial DNA copy number to establish a multi-indicator comprehensive evaluation model and obtain the heat resistance of the abalone to be evaluated.

[0014] The beneficial effects of this invention are: This method is based on the correlation between mitochondrial copy number and the heat resistance of abalone. It assesses heat resistance by measuring the mitochondrial DNA copy number in abalone tissue. It has the following advantages: Rapid and accurate: The heat resistance of abalone can be assessed in a short time by measuring the mitochondrial DNA copy number; Non-invasive: Only a small amount of abalone tissue sample needs to be collected, which has little impact on the growth and development of abalone; Lower cost: Compared to methods such as gene expression analysis and genome-wide association analysis, the equipment and reagents required by the method of this invention are relatively common; Wide range of applications: It can be used for early screening and evaluation of abalone seedlings, and also for evaluating the high-temperature resistance of adult abalone; High reliability: Mitochondrial DNA copy number is significantly correlated with the heat resistance of abalone, and can be used as a reliable biological marker; Highly scalable: It can be combined with other physiological indicators or molecular markers to establish a multi-indicator comprehensive evaluation model.

[0015] This invention utilizes changes in the mitochondrial DNA copy number of abalone to assess its heat tolerance. It offers a rapid, accurate, non-destructive, and low-cost method for selecting heat-tolerant strains, significantly improving selection efficiency. It can be applied to the early screening and evaluation of abalone seedlings, providing a scientific basis for breeding heat-tolerant abalone varieties. Furthermore, this method can be combined with other physiological indicators and molecular markers to establish a multi-indicator comprehensive evaluation model, further improving the accuracy and reliability of the assessment. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0017] Figure 1 This is a graph showing the difference in gill mitochondrial DNA copy number between the heat-training group and the control group provided in Example 1 of the present invention.

[0018] Figure 2 The ABT high-temperature heart rate evaluation charts for the heat training group (HA) and the control group (C) provided in Embodiment 1 of the present invention.

[0019] Figure 3 This is a transmission electron microscope (TEM) image of gill epithelial cells provided in Embodiment 1 of the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] A method for assessing the heat tolerance of abalone using mitochondrial count includes the following steps: (1) Sample collection: Collect tissue samples from abalone, preferably gill tissue or mantle tissue; (2) DNA extraction: Total DNA was extracted from the collected tissue samples using the EasyPure Genomic DNA Kit. (3) Mitochondrial DNA copy number determination: The relative concentrations of the mitochondrial and nuclear genomes were detected using the COX1 gene in mitochondria and the single-copy gene RPL7 in the nucleus, respectively. Mitochondrial DNA copy number was determined using real-time quantitative PCR (qPCR). This application used the SYBR Green II dye method, and the reaction was performed on a Bio-Rad CFX96™ Real-Time PCR Systems detection system. Three technical replicates were performed for each sample. The formula for calculating mitochondrial DNA copy number is as follows: N = 2 -Ctmt ÷2 -Ctn Where, N: mtDNA copy number per cell; Ctmt: mitochondrial gene Ct value; Ctn: nuclear single-copy gene amplification Ct value; q-PCR primer information table

[0022] (4) Establishment of standards: By measuring the mitochondrial DNA copy number of abalone individuals with different heat resistance, a correlation standard between mitochondrial DNA copy number and heat resistance of abalone was established; The correlation criteria between mitochondrial DNA copy number and abalone's heat tolerance were established through the following steps: 1) Select abalone populations from different sources, including abalone varieties known to be heat-resistant and common abalone varieties; 2) Determine the copy number of mitochondrial DNA in each abalone individual; 3) Determine the high-temperature tolerance indicators of each abalone individual, including the highest initial lethal temperature and critical temperature; 4) Analyze the correlation between mitochondrial DNA copy number and thermostable performance indicators, and establish evaluation criteria.

[0023] (5) Evaluation of heat resistance: The heat resistance of abalone was evaluated based on the mitochondrial DNA copy number determined in step (3) and the evaluation criteria established in step (4).

[0024] Furthermore, by combining mitochondrial DNA copy number with other physiological indicators or molecular markers, a multi-indicator comprehensive evaluation model can be established to improve the accuracy and reliability of the evaluation.

[0025] Compared with traditional methods, the method of this invention is characterized by its speed, accuracy, non-destructive nature, and low cost. It can be applied to the early screening and evaluation of abalone seedlings, providing a scientific basis for the breeding of heat-resistant abalone varieties. Furthermore, the method of this invention can be combined with other physiological indicators and molecular markers to establish a multi-indicator comprehensive evaluation model, further improving the accuracy and reliability of the evaluation.

[0026] Example 1 Abalone mitochondrial DNA copy number determination Sample collection: 30 abalone were selected from both the control group and the heat-trained group, and their origin and environment were recorded. A small amount of gill tissue (approximately 50 mg) was collected from each abalone using sterile scissors and immediately placed in liquid nitrogen for cryopreservation.

[0027] DNA extraction: Total DNA was extracted from abalone gill tissue using the EasyPure Genomic DNA Kit. The specific steps included tissue grinding, digestion, and purification. DNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer. The DNA concentration was adjusted to 50 ng / μL and stored at -20℃ for later use.

[0028] Mitochondrial DNA copy number determination: Mitochondrial DNA copy number was determined using the two-color fluorescent TaqMan probe quantitative PCR technique. Specific primers and probes were designed, standards were prepared, a standard curve was established, and the fluorescent quantitative PCR reaction was performed and the data were analyzed.

[0029] Results and Analysis: The results show ( Figure 1 Compared to the control group, the number of mitochondrial DNA copies in the gill tissue of wrinkled abalone was significantly reduced after heat training.

[0030] Example 2 Correlation analysis of abalone mitochondrial DNA copy number and heat resistance Sample selection: A full-sib family of 18-month-old wrinkled abalone was used. The ABT (Average Temperature Spectrum) method was used to compare the heat-treated (HA) group with the untreated (C) group. After 14 days of recovery following heat treatment, the ABT values ​​ranged from 32.02 to 33.43℃, while the ABT values ​​of the control group ranged from 31.34 to 31.59℃. The ABT results showed that after 14 days of recovery, the ABT value of the heat-treated group (32.81 ± 0.63℃) was significantly higher than that of the control group (31.47 ± 0.12℃) by 1.34℃ (p<0.01). Figure 2 ) This study used transmission electron microscopy to observe the number and morphology of mitochondria in cells of the control group and the heat-training group. Figure 3 In both groups of cells, most mitochondria exhibited normal morphology, uniform size, and a clearly defined double membrane structure with distinct cristae. In the control group, a few mitochondria showed localized vacuoles, but most remained intact. Within the field of view, the number of mitochondria in the control group was slightly higher than that in the heat-trained group, suggesting that the number of mitochondria did not significantly increase under heat-training conditions, but a trend towards improved mitochondrial morphology was observed in localized areas. Mitochondrial DNA copy number was detected using quantitative real-time fluorescence, thus indirectly reflecting the relative changes in the number of mitochondria in the cells.

[0031] Figure 3 In the middle, A~B are gill epithelial cells of the control group at magnifications of 8000 and 2000, respectively; C~D are gill epithelial cells of the heat-training group at magnifications of 8000 and 2000, respectively; MT: mitochondria; NC: cell nucleus.

[0032] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A method for evaluating the high temperature tolerance of abalone using the number of mitochondria, characterized by, The method comprises the following steps: (1) collecting tissue samples of abalones and extracting DNA; (2) determining the copy number of mitochondrial DNA: the relative concentrations of mitochondria and nuclear genome are detected by using COX1 gene on mitochondria and single copy gene RPL7 in nucleus respectively, the Ct value of mitochondrial gene and the Ct value of single copy gene amplification in nucleus are obtained, and the copy number of mtDNA in each cell is calculated; (3) establishing a standard and evaluating the high-temperature resistance: by determining the copy number of mitochondrial DNA of abalones with different high-temperature resistance, the correlation standard of the copy number of mitochondrial DNA and the high-temperature resistance of abalones is established; then, the evaluation relationship is established according to the copy number of mitochondrial DNA determined in step 2) and the standard, and the high-temperature resistance of the abalones to be evaluated is obtained.

2. The method for evaluating the high-temperature tolerance of abalone using the number of mitochondria according to claim 1, characterized in that, The abalones are selected from healthy abalones with more than 1 year or more than 4 cm in shell length.

3. The method for evaluating the heat tolerance of abalone using the number of mitochondria according to claim 1, characterized in that, The tissue sample collected in step (1) is gill tissue or mantle tissue.

4. The method for evaluating the high-temperature tolerance of abalone using the number of mitochondria according to claim 3, characterized in that, The gill tissue is the top region of the gill tissue of the target organism as the sampling site.

5. The method for evaluating the heat tolerance of abalone using the number of mitochondria according to claim 1 or 3, characterized in that, The DNA extraction method in step (1) is to extract DNA in the tissue from the collected tissue sample by using the whole EasyPure Genomic DNA Kit reagent box.

6. The method for evaluating heat tolerance of abalone using mitochondrial number according to claim 1, wherein, The determination method of the copy number of mitochondrial DNA in step (2) is to determine the copy number of mitochondrial DNA by using the real-time fluorescent quantitative PCR method, to use the SYBR Green II dye method, and to perform the reaction on the Bio-Rad CFX96TM Real-Time PCR Systems detection system; 2-4 technical repeats are performed for each sample.

7. The method for evaluating heat tolerance of abalone using mitochondrial number according to claim 1 or 6, characterized in that, The calculation formula of the copy number of mitochondrial DNA in step (2) is: N =2 -Ctmt ÷2 -Ctn Wherein, N: the copy number of mtDNA in each cell; Ctmt: the Ct value of mitochondrial gene; Ctn: the Ct value of single copy gene amplification in nucleus.

8. The method for evaluating heat tolerance of abalone using mitochondrial number according to claim 1, wherein, The correlation standard of the copy number of mitochondrial DNA and the high-temperature resistance of abalones in step (3) is established by the following steps: 1) selecting abalones of different sources, including abalones of known high-temperature resistance and ordinary abalones; 2) determining the copy number of mitochondrial DNA of each abalone; 3) determining the high-temperature resistance index of each abalone; 4) analyzing the correlation between the copy number of mitochondrial DNA and the high-temperature resistance index, and establishing the evaluation standard.

9. The method for evaluating the high-temperature tolerance of abalone using the number of mitochondria according to claim 8, characterized in that, The high-temperature resistance index at least includes high initial lethal temperature and critical temperature.

10. The method for evaluating heat tolerance of abalone using mitochondrial number according to claim 1 or 8, characterized in that, Step (3) further comprises combining other physiological indexes or molecular markers with the copy number of mitochondrial DNA, establishing a multi-index comprehensive evaluation model, and obtaining the high-temperature resistance of the abalones to be evaluated.

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