MiRn35 inhibitor for improving high temperature stress resistance of crassostrea gigas and application of miRn35 inhibitor
By designing miR_n35 inhibitors to inhibit the expression of miR_n35, the problem of dysfunction of oysters in high temperature stress was solved, and their antioxidant ability and resistance to high temperature were significantly improved.
Patent Information
- Application Number
- CN202510165564.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-14
- Publication Date
- 2025-06-10
AI Technical Summary
Long oysters have dysfunctional antioxidant capacity under high temperature stress, resulting in large-scale deaths, which are difficult to effectively prevent and control in the existing technology.
A miR_n35 inhibitor is designed with a nucleotide sequence of 5’-CATACAGCCAAGCAACCAAAGA-3’, which is used to inhibit the expression of miR_n35, thereby enhancing the antioxidant capacity of long oysters.
By inhibiting miR_n35, the accumulation of lipid peroxides in the body of long oysters is significantly reduced, the antioxidant enzyme activity and total antioxidant ability are improved, and the resistance of long oysters to high temperatures is enhanced.
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Figure CN120118902A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of molecular biology, and particularly relates to a non-coding small RNA (miRNA) inhibitor of the Pacific oyster (Crassostrea gigas) and its application in enhancing the high-temperature stress resistance of the Pacific oyster. Background Art
[0002] As an aquaculture variety widely cultured globally, in recent years, large-scale deaths of the Pacific oyster caused by high summer temperatures have caused serious economic losses to the aquaculture industry. High-temperature stress can affect the energy metabolism mode of the Pacific oyster, leading to the excessive production of reactive oxygen species and causing oxidative stress responses. Under high-temperature stress, the ability of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) to scavenge reactive oxygen species decreases, the function of the intracellular antioxidant defense system is blocked, and ultimately tissue damage and the death of oysters occur. Therefore, the imbalance of antioxidant capacity under high-temperature stress is considered to be one of the key factors leading to the massive death of oysters. Currently, effective prevention and control means for high-temperature stress are still insufficient.
[0003] miRNA is an endogenous small non-coding RNA that regulates gene expression by binding to target mRNAs and plays an indispensable role in the post-transcriptional regulation of gene expression. A number of studies have shown that miRNAs can respond to abiotic stresses such as temperature changes and heavy metal pollution and participate in post-transcriptional regulation. In the study of Caenorhabditis elegans, it was found that 19 miRNAs were significantly expressed under heat stress. Among them, C. elegans lacking mir-71, mir-80, mir-229, and mir-64-66 showed obvious heat sensitivity, while C. elegans lacking miR-239 showed stronger heat tolerance. In Apostichopus japonicus, multiple miRNAs were differentially expressed during the summer aestivation induced by high temperature. These miRNAs may enhance the high-temperature resistance of A. japonicus by adjusting metabolic pathways and antioxidant capacity. Under high-temperature stress, maintaining antioxidant capacity helps organisms reduce oxidative stress pressure and thus enhance the high-temperature resistance of organisms. More and more studies have shown that miRNAs play an important role in regulating the antioxidant capacity of organisms. In a mouse model, miR-93 alleviated ischemic injury through the Nrf2 / HO-1 antioxidant pathway, and an inhibitor of miR-93 could reduce hydrogen peroxide-induced cell damage.
[0004] In H9C2 cardiomyocytes, an inhibitor of miRNA-29a promoted the expression of PGC-1a / Nrf2 and eNOS, and at the same time reduced the levels of iNOS and MDA during I / R. Therefore, under high-temperature stress, miRNAs may affect their adaptability to high temperature by negatively regulating antioxidant capacity. Summary of the Invention
[0005] The object of the present invention is to provide an miRNA inhibitor for enhancing the ability of the Pacific oyster (Crassostrea gigas) to resist high-temperature stress, which can significantly inhibit the accumulation of lipid peroxides in the Pacific oyster and effectively resist the damage caused by high temperature to the Pacific oyster during the cultivation process.
[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:
[0007] An miR_n35 inhibitor for the Pacific oyster to resist high-temperature stress, and its nucleotide sequence is 5’-CATACAGCCAAGCAACCAAAGA-3’.
[0008] Another object of the present invention is to provide the application of the above miR_n35 inhibitor.
[0009] Specifically, it relates to the application of the above miR_n35 inhibitor in the preparation of a preparation for the Pacific oyster to resist high-temperature stress.
[0010] Another object of the present invention is to provide a preparation for the Pacific oyster to resist high-temperature stress.
[0011] Specifically, for the preparation for the Pacific oyster to resist high-temperature stress, its main component includes the miR_n35 inhibitor, and its nucleotide sequence is 5’-CATACAGCCAAGCAACCAAAGA-3’.
[0012] Another object of the present invention is to provide a method for improving the ability of the Pacific oyster to resist high-temperature stress.
[0013] Specifically, for the method for improving the ability of the Pacific oyster to resist high-temperature stress, during the cultivation process, the Pacific oyster is given the miR_n35 inhibitor, and the nucleotide sequence of the miR_n35 inhibitor is 5’-CATACAGCCAAGCAACCAAAGA-3’.
[0014] The concentration of the miR_n35 inhibitor for the Pacific oyster is 20 μM, and the dosage is 100 μL / individual.
[0015] The water temperature of the cultivation seawater used for cultivation is 28 °C.
[0016] This invention studies the expression of miRNAs in Crassostrea gigas during high-temperature stress, and finds that the expression level of miR_n35 in the exosomes of Crassostrea gigas significantly increases after high-temperature stress. miR_n35 is a novel miRNA found in tissues and exosomes. By analyzing the differential expression heat map and hierarchical clustering of miRNAs under different stress conditions, it is predicted that miR_n35 may be involved in the response to high-temperature stress, indicating that miR_n35 may be involved in the high-temperature stress process of Crassostrea gigas. This invention designs an antisense miRNA of miR_n35 as an inhibitor to inhibit the expression level of miR_n35. After injecting the miR_n35 inhibitor and performing high-temperature stress, the lipid peroxide in the hemolymph of Crassostrea gigas significantly decreases compared with the negative control NC group injected with the miR_n35 inhibitor. The results show that the miR_n35 inhibitor can play an oxidative stress role by promoting the increase of antioxidant capacity in Crassostrea gigas under high-temperature stress.
[0017] The miR_n35 inhibitor involved in this invention can be used as an antioxidant capacity regulator to effectively improve the ability of Crassostrea gigas to resist high-temperature stress. Brief Description of the Drawings
[0018] Figure 1 It is a result diagram (*p<0.05; ***p<0.001) of the inhibition of SOD and CAT enzyme activities, T-AOC and MDA content changes by the miR_n35 inhibitor provided in the embodiment of this invention under high-temperature stress.
[0019] The Crassostrea gigas without any treatment is the blank control group (BLANK). The negative control NC group (antagomir-NC) or the miR_n35 inhibitor group (CgmiR_n35 antagomir) is subjected to high-temperature stress after injection, and the activities of SOD and CAT, the changes of T-AOC and MDA content in the gill tissues of Crassostrea gigas are detected at 12 h. The values are the averages of three experiments and are expressed as mean ± standard deviation.
[0020] Figure 2 It is the hematoxylin-eosin staining of the gill tissue pathological section provided in the embodiment of this invention.
[0021] A: Morphological changes of the oyster gill tissue in the antagomir-NC group after high-temperature stress. B: Morphological changes of the gill after high-temperature stress in the oysters with inhibition of exosomal CgmiR_n35. The red arrows indicate widened cell spacing and gill filament swelling. The triangular arrows point to loose cytoplasm. Detailed Embodiments
[0022] The following further illustrates the specific embodiments of the present invention with examples. It should be noted that the specific embodiments described herein are only for the purpose of illustrating and explaining the present invention and are not limited to the present invention.
[0023] Example 1: Experiment on miR_n35 inhibitor against high temperature stress
[0024] miR_n35 is a non-coding small RNA expressed in the Pacific oyster Crassostrea gigas. Its sequence was obtained by nucleic acid sequencing and is shown as SEQ ID NO:1: 5’-CTTGGCACTGGCGGAATAGTCAC-3’.
[0025] 1. Preparation of miR_n35 inhibitor and miR_n35 inhibitor NC (negative control):
[0026] Design the miR_n35 inhibitor according to the sequence in Example 1 above, and at the same time design the control sequence of the miR_n35 inhibitor (named antagomir-nc); dissolve the miR_n35 inhibitor and antagomir-nc in DEPC water (purchased from GenePharma Co., Ltd.) to 10 μM respectively for standby.
[0027] Among them, the miR_n35 inhibitor sequence is: 5’-CATACAGCCAAGCAACCAAAGA-3’.
[0028] The antagomir-nc sequence is: 5’-CAGTACTTTTGTGTAGTACAA-3’.
[0029] 2. Injection into the Pacific oyster Crassostrea gigas
[0030] Take 27 Pacific oysters Crassostrea gigas, each weighing about 90 - 110 g, and randomly divide them into 3 groups, named Blank, antagomir-nc, and CgmiR_n35 antagomir respectively, with 9 in each group, and culture them in seawater at 15°C. The Blank group is the blank control group without treatment. Inject 100 μL of miR_n35 inhibitor NC (negative control, 20 μM) and miR_n35 inhibitor (20 μM) into the Pacific oysters Crassostrea gigas in the antagomir-nc group and CgmiR_n35 antagomir group respectively. After 12 h, place the Pacific oysters Crassostrea gigas in the antagomir-nc group and CgmiR_n35 antagomir group in the culture environment at 28°C.
[0031] 3. Detection of antioxidant indexes
[0032] After culturing under high temperature stress for 12 h in Step 2 above, gill tissues of oysters in the Blank, antagomir-nc, and CgmiR_n35 antagomir groups were taken for tissue damage detection. Kits produced by Nanjing Jiancheng Bioengineering Institute were used to measure the content of total protein (A045-2-2), the activities of SOD (A001-3-2), CAT (A001-3-2), T-AOC (A015-1-2), and the content of MDA (A003-1-2). Three parallels were set for the measurement of all indicators.
[0033] Method for measuring protein concentration: Accurately weigh 0.1 g of tissue, add 9 times the volume of PBS buffer solution according to the ratio of weight (g): volume (mL) = 1:9, grind using a grinder to obtain 10% tissue homogenate, centrifuge at 2500 rpm for 10 min, and take 0.1 ml of the supernatant for detection according to the operation manual.
[0034] Method for measuring SOD activity: Accurately weigh 0.1 g of tissue, add 9 times the volume of PBS buffer solution according to the ratio of weight (g): volume (mL) = 1:9, grind using a grinder to obtain 10% tissue homogenate, centrifuge the 10% tissue homogenate at 3000 rpm for 10 min, and take the supernatant for testing.
[0035] Method for measuring CAT activity: Accurately weigh 0.1 g of tissue, add 9 times the volume of PBS buffer solution according to the ratio of weight (g): volume (mL) = 1:9, grind using a grinder to obtain 10% tissue homogenate, centrifuge the 10% tissue homogenate at 4000 rpm for 10 min, and take the supernatant for testing.
[0036] Method for measuring T-AOC: Accurately weigh 0.1 g of tissue, add 9 times the volume of PBS buffer solution according to the ratio of weight (g): volume (mL) = 1:9, grind using a grinder to obtain 10% tissue homogenate, centrifuge at 2500 rpm for 10 min, and take 0.1 ml of the supernatant for detection according to the operation manual.
[0037] Method for measuring MDA content: Accurately weigh 0.1 g of tissue, add 9 times the volume of PBS buffer solution according to the ratio of weight (g): volume (mL) = 1:9, grind using a grinder to obtain 10% tissue homogenate, centrifuge at 2500 rpm for 10 min, and take 0.1 ml of the supernatant for detection according to the operation manual.
[0038] It was found that Figure 1In the gill tissues of the Pacific oysters in the CgmiR_n35 antagomir group injected with miR_n35 inhibitor, the activities of SOD, CAT, and T-AOC increased significantly, being 1.27 times (p<0.05), 2.30 times (p<0.05), and 1.27 times (p<0.05) that of the antagomir-nc group, respectively. The content of MDA decreased significantly, being 0.79 times that of the antagomir-nc group.
[0039] 4. Histological examination and pathological observation of the Pacific oyster
[0040] The gill tissues of the Blank, antagomir-nc, and CgmiR_n35 antagomir groups were taken for histological and pathological observation, and the specific operations were as follows:
[0041] (1) Cut each tissue sample into small pieces about 3 mm square, soak them in Bouin's fluid at room temperature for 24 h for fixation;
[0042] (2) Soak them in 70% ethanol for decolorization, change the liquid every 2 h until the samples return to their original color;
[0043] (3) Dehydration and infiltration with paraffin: The samples were successively placed in 80% ethanol for 60 min, 95% ethanol at room temperature for 60 min, absolute ethanol at room temperature for 30 min, absolute ethanol at room temperature for 30 min, absolute ethanol:xylene (1:1) at room temperature for 30 min, xylene at room temperature for 20 min, xylene at room temperature for 10 min, xylene:paraffin (1:1) at room temperature for 30 min, and paraffin at 70 °C overnight, 70 °C;
[0044] (4) Embed them in a paraffin embedding machine and make paraffin sections;
[0045] (5) Dewax the paraffin sections to water: The sections were successively placed in xylene I for 20 min, xylene II at room temperature for 20 min, absolute ethanol I at room temperature for 5 min, absolute ethanol II at room temperature for 5 min, 75% alcohol at room temperature for 5 min, room temperature, and pure water wash;
[0046] (6) Hematoxylin staining: Place the sections in hematoxylin stain for 3 min, wash with pure water, differentiate with differentiating solution, wash with pure water, blue with bluing solution, and rinse with running water;
[0047] (7) Eosin staining: The sections were successively placed in 85% and 95% gradient ethanol for dehydration for 5 min each, and then placed in eosin stain for 5 min;
[0048] (8) Dehydration and mounting: The sections were successively placed in absolute ethanol I for 5 min - absolute ethanol II for 5 min - absolute ethanol III for 5 min - xylene I for 5 min - xylene II for 5 min for clearing, and then mounted with neutral gum;
[0049] (9) Microscopic examination and image sorting and analysis. As Figure 2 shown, the cell spacing in the antagomir-NC group became wider, the gill filaments swelled, and the cytoplasm was loose. In the CgmiR_n35 antagomir group, the cell spacing did not become wider, the gill filaments did not swell, and the cytoplasm was dense.
[0050] The above results indicate that the miR_n35 inhibitor can effectively improve the ability of the Pacific oyster to resist high-temperature stress.
Claims
1. A miR_n35 inhibitor for oysters to resist high temperature stress, characterized in that: The nucleotide sequence is 5'-CATACAGCCAAGCAACCAAAGA-3'.
2. Use of the miR_n35 inhibitor described in claim 1 in the preparation of a long oyster anti-high temperature stress preparation.
3. A preparation for oysters to resist high temperature stress, characterized in that: Its main components include miR_n35 inhibitor, and its nucleotide sequence is 5'-CATACAGCCAAGCAACCAAAGA-3'.
4. A method for improving the ability of Crassostrea gigas to resist high temperature stress, characterized in that During the culture process, the long oyster miR_n35 inhibitor is given, and the nucleotide sequence of the miR_n35 inhibitor is 5'-CATACAGCCAAGCAACCAAAGA-3'.
5. The method for improving the high temperature stress resistance of Crassostrea gigas according to claim 4, characterized in that: The long oyster miR_n35 inhibitor has a concentration of 20 μM and a dosage of 100 μL / oyster.
6. The method for improving the high temperature stress resistance of Crassostrea gigas according to claim 4 or 5, characterized in that: The temperature of the aquaculture seawater used is 28℃.