Application of crassostrea gigas miR-182-5p in negative regulation of melanin synthesis

Abstract

The invention discloses application of crassostrea gigas miR-182-5p in negative regulation of melanin synthesis, and belongs to the technical field of aquatic genetic breeding and biology. The miR-182-5p in the crassostrea gigas is interfered by the miR-182-5p inhibitor, synthesis of melanin of the crassostrea gigas is remarkably enhanced after the miR-182-5p is interfered, and the color of the crassostrea gigas is black. In addition, the invention also synthesizes an exclusive miR-182-5p stimulant to treat the crassostrea gigas. Results show that compared with wild crassostrea gigas, the content of melanin in the crassostrea gigas injected with the simulant is lower, and the shell color is white. Therefore, the miR-182-5p is in negative correlation with the synthesis of the melanin of the crassostrea gigas, so that the miR-182-5p not only can be used for selecting or predicting the shell color character of the crassostrea gigas, but also can be used for assisting in screening or culturing varieties with high melanin content by silencing or knocking out the miR-182-5p in the crassostrea gigas. The important effect of miR-182-5p in oyster melanin synthesis is clarified for the first time, the molecular mechanism of mollusk melanin formation can be deeply understood, and the miR-182-5p has important significance in promoting practical application of crassostrea gigas quality breeding.

Description

Technical Field

[0001] This invention belongs to the field of aquatic genetics and breeding and biotechnology, specifically relating to the application of miR-182-5p in the regulation of melanin synthesis and improvement of shell color in Pacific oyster. Background Technology

[0002] Oyster ( Crassostrea gigas Oysters are an important marine aquaculture species in my country. Their shell color and mantle color are important economic traits that directly affect their commercial value. Melanin not only affects the appearance of oysters but also effectively enhances their immunity and may ensure the healthy and sustainable development of oyster farming.

[0003] Currently, breeding work on the Pacific oyster mainly focuses on growth rate and stress resistance, while research on the genetic improvement of pigment traits lags behind. Shell color is a key element in the selection of distinctive varieties of Pacific oysters, primarily derived from the presence of biological pigments, with melanin being a crucial component and playing a major role in shell color development. Shell color breeding can enhance the economic value of shellfish, and optimizing shell color breeding techniques for oysters can make it possible to simultaneously improve the health benefits and aesthetics of oysters. Therefore, strengthening research on the melanin synthesis mechanism of Pacific oysters, in conjunction with current research in pigments, can improve the economic value of shellfish in oyster farming through shell color breeding, which has significant application value for the development of aquaculture.

[0004] MicroRNAs (miRNAs), as an important class of gene expression regulators, play a crucial role in the melanin synthesis pathway by targeting and binding to the mRNA of target genes. Studies have found that specific miRNAs can influence melanin synthesis and deposition by regulating the expression of key genes involved in melanin synthesis, such as tyrosinase and tyrosinase-related proteins. However, the effects and applications of miRNAs on melanin formation and genetic shell color traits in oysters have not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to provide the application of miR-182-5p in regulating melanin synthesis and improving shell color in Pacific oysters.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: The present invention discovered miR-182-5p in the oyster *Crassostrea gigas*. The gene sequence of this miRNA is shown in SEQ ID NO.1, with a coding sequence length of 22 nt, and its sequence is 3'-AGUCACUUAAGAUGUUCACGGUUC-5', which can also be represented as 3'-AGTCACTTAAGATGTTCACGGTTC-5'. The target gene of the oyster miR-182-5p is predicted through an RNAhybrid database. It is found that the long oyster miR-182-5p has partial binding sites in the 3'UTR region of the microphthalmia-associated transcription factor (MITF) gene. The present application verifies the MITF gene through a double luciferase reporter experiment by injecting the miR-182-5p mimic and the MITF gene into the oyster. It is found that when the miR-182-5p is overexpressed, the luciferase activity of the MITF gene is significantly reduced, proving the targeted inhibition of the MITF by the miR-182-5p.

[0007] The present application detects the expression amount of the mantle miR-182-5p of the long oyster larvae through real-time fluorescent quantitative PCR (qRT-PCR). The expression amount of the miR-182-5p is the highest in the white mantle of the long oyster, the melanin content of the mantle is low, the shell color is light (white or light gray) after adulthood, the expression amount is the lowest in the black mantle, and the shell color is dark (black or dark brown).

[0008] The miR-182-5p targets the MITF to affect the expression of the melanin generation related genes, and then regulates the melanin synthesis. It is found through in-depth research that the miR-182-5p mimic and the inhibitor are prepared and microinjected into the long oyster larvae. The melanin content is detected after the injection. After the miR-182-5p is inhibited, the melanin synthesis of the long oyster is significantly enhanced, that is, the miR-182-5p is negatively correlated with the melanin synthesis of the long oyster, and the oyster shell color is significantly darkened. The present application can lay a certain genetic resource foundation for improving the shell color of the long oyster.

[0009] Advantages of the present application: (1) The present application first finds the miR-182-5p, which can be targeted to combine the target gene MITF, affect the expression of the MITF, and then regulate the melanin synthesis, which is helpful for deeply understanding the molecular mechanism of the melanin formation of the mollusk, and has important significance for promoting the practical application of the quality breeding of the long oyster.

[0010] (2) The present application can screen the oyster with the required shell color at the larva stage through the qRT-PCR detection without waiting for the oyster shell color to be completely formed (more than 6 months are required for the traditional phenotype selection). The expression level of the miR-182-5p directly reflects the genetic background, is not affected by the environmental factors such as water temperature and bait, and avoids the "false positive" (such as the light shell color induced by the environment) of the traditional phenotype selection.

[0011] (3) The present application can artificially intervene in the melanin synthesis process of the oyster by improving or inhibiting the expression of the miR-182-5p, is used for repairing the abnormal shell color caused by the environmental stress (such as high temperature and pollution), or is used for batch preparing the oyster product with the black shell color, and improves the commodity value.

[0012] (4) The detection of miR-182-5p only needs a conventional qRT-PCR device, without the need of a high-throughput sequencing platform, and is simple to operate, suitable for large-scale application in breeding farms and breeding enterprises. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 : A. Column chart of relative expression of miR-182-5p in mantle of Crassostrea gigas; Error bar: mean ± SD, n = 12; *P < 0.05, **P < 0.01.

[0014] Figure 2 : Schematic diagram of binding of miR-182-5p to 3'-UTR of MITF gene; In the diagram, upper: sequence of miR-182-5p (SEQ ID NO. 1); lower: sequence of binding site of 3'-UTR of MITF gene (SEQ ID NO. 2, sequence: 3'-AGAGCCGUCUAAGGAAAGCGGUAACGUGAA-5', or also expressed as 3'-AGAGCCGTCTAAGGAAAGCGGTAACGTGAA-5'); red dotted line: base complementary pairing relationship.

[0015] Figure 3 : Luciferase activity of MITF gene inhibited by miR-182-5p mimic; In the diagram, abscissa: empty plasmid pmirGLO group, MITF sequence inserted in empty plasmid pmirGLO (pmirGLO-MITF-WT group), plasmid with MITF binding site mutated (pmirGLO-MITF-MUT group); ordinate: relative luciferase activity; Error bar: mean ± SD, n = 12; *P < 0.05, **P < 0.01.

[0016] Figure 4 : Expression of MITF after injection of miR-182-5p; In the diagram, abscissa: negative control, injection of miR-182-5p mimic, injection of miR-182-5p inhibitor; ordinate: relative expression of MITF; Error bar: mean ± SD, n = 12; *P < 0.05, **P < 0.01.

[0017] Figure 5 : Contrast diagram of melanin granules in mantle of Crassostrea gigas after injection of miR-182-5p; In the diagram, from left to right, there are blank control group, miR-182-5p mimic group, and miR-182-5p inhibitor group; Staining method: ferrous sulfate melanin staining, scale = 100 μm. DETAILED DESCRIPTION

[0018] The advantages and features of the present application will be more clearly presented with the description of specific embodiments. It should be noted that, in the following examples, the specific experimental operations involved, if not specifically mentioned, are in accordance with the conventional experimental methods in the art, or are performed according to the recommended conditions provided in the instructions of the relevant reagents and instruments manufacturers; the technical means used are conventional techniques familiar to those skilled in the art; and the reagents and materials used in the experimental process, if not specifically mentioned, are conventional products that can be purchased through commercial channels.

[0019] In addition, all the professional terms and scientific expressions in the present application are consistent with the meanings generally recognized by those skilled in the art; any technical solution with similar or equivalent effects to the methods and materials described in the present application can be used in the present application; the preferred embodiments and materials mentioned in the present application are only examples and do not limit the scope of protection of the present application.

[0020] The black and white mantle membranes of long oyster were used for RNA extraction. The kit used was SYBR Green Premix Pro TaqHS qPCR (Accurate Biology). The synthesized cDNA was used as a template, U6 was used as a reference gene for quantification of miR-182-5p, and pigment-related genes were standardized with β-actin internal reference genes. The PCR reaction conditions were as follows: 95°C pre-denaturation for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 55°C for 30 seconds, and finally melting curve analysis (95°C for 15 seconds, 60°C for 1 minute, 95°C for 30 seconds, and 60°C for 15 seconds). The 2-ΔΔCT method was used to calculate the relative change in gene expression, and each sample was detected three times to ensure accuracy and repeatability.

[0021] The primer sequence of the reference gene U6 is as follows: U6-F: 5'-ATTGGAACGATACAGAGAAGATT-3' U6-R: 5'ATTTGCGTGTCATCCTTGC-3' The present application detects the expression amount of miR-182-5p in the mantle membrane of long oyster larvae by real-time fluorescence quantitative PCR (qRT-PCR). The results show that the expression amount of miR-182-5p is the highest in the white mantle membrane of long oyster, the content of melanin in the mantle membrane is low, the shell color is light (white or light gray) after adulthood, the expression amount is the lowest in the black mantle membrane, and the shell color is dark (black or dark brown) Figure 1 ).

[0022] HEK-293T cells used were cultured in an incubator at a temperature of 37°C and containing 5% CO2. The empty plasmid pmirGLO, the empty plasmid pmirGLO inserted with MITF sequence (pmirGLO-MITF-WT), and the plasmid with MITF binding site mutated (pmirGLO-MITF-MUT) were constructed. After the HEK-293T cells were cultured in a culture plate to a density of 70%-80%, the HEK-293T cells were inoculated into a 24-well culture plate, and when the cell density was about 80%-90%, transfection was performed. The plasmid was transfected using Lipofectamine 2000 reagent (Milipore, China) according to the manufacturer's instructions.

[0023] 200 ng of pmirGLO-MITF-WT plasmid / pmirGLO-MITF-MUT plasmid and 400 ng of miR-182-5p mimic were co-transfected into cells. 24 h after transfection, luciferase activity was detected using the Dual-Glo Luciferase Detection System kit from Promega, USA. Each group of experiments was repeated 3 times to ensure the reproducibility of the results.

[0024] In this experiment, a luciferase reporter vector containing MITF 3'UTR was used for dual-luciferase reporter gene experiment. Figure 2 The results showed that when miR-182-5p was overexpressed, the luciferase activity of pmirGLO-MITF-WT decreased, while the luciferase activity of pmirGLO-MITF-MUT did not change significantly. Figure 3 In summary, the results showed that there was a target regulation relationship between MITF and miR-182-5p.

[0025] Experimental grouping: Before the experiment started, all Crassostrea gigas were acclimated in the laboratory circulating seawater for 7 days. The experiment was set up in 3 groups (10 in each group): miR-182-5p mimic group, miR-182-5p inhibitor group and negative control group. 10 μg of the corresponding reagent was injected into the adductor muscle of each Crassostrea gigas in the miR-182-5p mimic group and the inhibitor group, and the negative control group was not treated. Injection was performed every 4 days, and a total of 8 injections were performed.

[0026] The miR-182-5p mimic and inhibitor were constructed according to the nucleotide sequence of miR-182-5p as follows.

[0027] The single-stranded sequence of the mimic is as follows: miR-182-5p single strand mimic (5'- CTTGGCACTTGTAGAATTCACTGA-3') Inhibitor single strand sequence is shown as follows: miR-182-5p single strand inhibitor (5'- TCAGTGAATTCTACAAGTGCCAAG-3') The reaction system of real-time fluorescent quantitative PCR (qRT-PCR) is shown in Table 1.

[0028] Table 1 qRT-PCR reaction system

[0029] Condition setting: pre-denaturation 95℃ 30s, then 95℃ 5s, 55℃ 30s for 40 cycles, and then perform melting curve analysis 95℃ 15s, 60℃ 1min, 95℃ 30s, 60℃ 15s.

[0030] II. qRT-PCR technology verifies the relationship between miR-182-5p and melanin synthesis of Crassostrea According to the nucleotide sequence of the specific binding site of the target gene MITF, the primers are designed as follows: Forward primer: MITF-F (5'- CTGACACTCACCCAGCAACC-3') Reverse primer: MITF-R (5'- GGCTCTGACGACAGCATAGG-3') Reference primer sequence: β-actin-F: 5'- GTGCTACGTTGCCCTGGACTT-3' β-actin-R: 5'- TCGCTCGTTGCCAATGGTGAT-3' In the experiment, the SYBR® Green Premix Pro Taq HS qPCR reagent kit of Accurate Biology Company was used. The synthesized cDNA was used as a template, and the pigment-related genes were standardized with β-actin (β-actin) as an internal reference gene. After all the injections of the oysters were completed, the mantle tissue of the Crassostrea was sampled and total RNA was extracted for subsequent experiments. The results show that compared with the negative control group, after injection of miR-182-5p mimic, the expression of MITF in the mantle of the Crassostrea increases; while after injection of miR-182-5p inhibitor, its expression decreases (Fig. 3). Figure 4 It is proved that the miR-182-5p mimic and the inhibitor are successfully constructed, and the results also show that the relationship between miR-182-5p and melanin synthesis is negative regulation.

[0031] The treated mantle tissue pieces were fixed in 4% paraformaldehyde, routinely dehydrated, immersed in wax, embedded, and sectioned. After deparaffination and rehydration, the sections were washed with distilled water. According to the manufacturer's instructions, melanin staining was performed using a ferrous sulfate method melanin staining kit (Solarbio, China). The sample sections were immersed in FeSO4 solution for 1 h, washed with distilled water several times, then placed in an acidic potassium ferric oxide solution for 3 min, followed by washing with distilled water for 3-4 times, and then immersed in nuclear fast red for 2 min. After washing with distilled water twice, the tissue samples were observed under a microscope (Nikon Corporation, CI-L, Japan).

[0032] The results show that a small amount of brown particles were observed in the negative control group; compared with the negative control group, there were fewer brown particles in the miR-182-5p mimic group, and more brown particles in the miR-182-5p inhibitor group Figure 5 . This further indicates that miR-182-5p is involved in the regulation of the formation of long oyster mantle melanin.

[0033] II. Shell color observation Thirty long oysters with a shell length of 6 cm (all with a light brown shell color) were selected and randomly divided into 3 groups (10 oysters per group). The long oysters were injected according to the above experimental grouping, and after all the injections were completed, they were placed in a circulating seawater culture system (water temperature 20℃, salinity 30‰) for further culture for 10 days. The shell color was observed, and the results showed that the shell color of the control group did not change significantly, 7 oysters in the injection mimic group were observed to be slightly lighter, and 3 oysters did not change significantly, while 8 oysters in the injection inhibitor group were slightly darker, and 2 oysters did not change significantly.

[0034] In summary, the present application verifies that miR-182-5p specifically targets and inhibits the expression of MITF gene (binding site: SEQ ID NO. 2) by using a dual luciferase reporter gene detection technology, which significantly reduces the synthesis of melanin in the mantle of long oysters. In addition, the present application interferes with miR-182-5p in long oysters through overexpression or inhibition experiments, and significantly enhances the synthesis of melanin in long oysters after inhibiting oyster miR-182-5p, and affects its specific shell color traits. It can be seen that miR-182-5p is negatively correlated with the melanin synthesis of long oysters, and therefore, the content of adult melanin can be artificially regulated by overexpression, inhibition or knockout of miR-182-5p in juvenile long oysters, and the shell color traits of adult long oysters can be changed, thereby providing a new method for long oyster shell color breeding.

[0035] The above-mentioned embodiments are only preferred embodiments of the present application, and their role is only to explain and describe the technical solutions of the present application, but not to limit the scope of the present application. Based on the technical content disclosed in the specification, it is obvious for those skilled in the art to derive other implementation forms through replacement, adjustment, etc. Therefore, any improvement, equivalent change or adjustment made according to the core principle of the present application should be included in the patent protection scope of the present application.

Claims

1. The application of miR-182-5p in the negative regulation of melanin synthesis in Pacific oyster, characterized in that, The nucleotide sequence of miR-182-5p is shown in SEQ ID NO.1, and the negative regulation is manifested in that miR-182-5p inhibits melanin synthesis in oysters.

2. The application according to claim 1, characterized in that, The miR-182-5p affects the formation of melanin in the oyster by targeting and regulating the expression of melanin synthesis-related genes; the melanin synthesis-related gene is MITF.

3. A method for regulating melanin synthesis in the Pacific oyster, characterized in that, When it is necessary to increase melanin content, the expression of miR-182-5p is inhibited; when it is necessary to decrease melanin content, the expression of miR-182-5p is increased; the nucleotide sequence of miR-182-5p is shown in SEQ ID NO.

1.

4. The method for regulating melanin synthesis in the Pacific oyster according to claim 3, characterized in that, The method to inhibit miR-182-5p expression is as follows: construct a miR-182-5p inhibitor and introduce it into the adductor muscle of Ostrea gigas via microinjection to interfere with miR-182-5p in Ostrea gigas.

5. The method for regulating melanin synthesis in the Pacific oyster according to claim 3, characterized in that, The method for increasing the expression of miR-182-5p is as follows: a miR-182-5p mimic is constructed and introduced into the adductor muscle of oyster by microinjection to increase the expression level of miR-182-5p in oysters.

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