Application of DIO1 gene in liver in controlling body color of scophthalmus maximus

Abstract

The invention relates to an application of a DIO1 gene in a liver in controlling the body color of a turbot, and belongs to the field of molecular biology, and the application method is as follows: the turbot with golden body color is obtained by overexpressing the DIO1 gene in the liver of the turbot; the amino acid sequence of the protein coded by the DIO1 gene is as shown in SEQ ID NO. 1. The invention discovers that the gene expression level change in the liver can cause the change of the blood hormone level by influencing the thyroid hormone pathway in the turbot for the first time, thereby influencing the color formation of the skin; therefore, the formation of the target body color of the scophthalmus maximus can be controlled by controlling the gene expression level, so that the market value of the scophthalmus maximus is improved.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically, it relates to the application of the DIO1 gene in the liver in controlling the body color of turbot. Background Technology

[0002] The diversity of fish body color has long been a classic model of adaptive evolution in vertebrates, with its regulatory mechanisms involving a multidimensional network of interactions among genetics, environment, and ecology. It plays a central role in species survival (such as camouflage and vigilance), reproduction (such as sexual selection), and population competition. In recent years, with consumption upgrades and market segmentation, the economic value of fish body color has significantly increased. Particularly in the ornamental fish sector, varieties such as koi and goldfish have achieved market premiums of hundreds of times due to variations in their body color. In the edible fish industry, the retail prices of mutant strains such as platinum-colored snakehead and red loach have also increased significantly. This indicates that targeted design of body color has become a new direction for value mining in the aquatic seed industry.

[0003] As a pillar species of marine aquaculture in northern my country, turbot exhibits unique adaptations in its body coloration. Its traditional grayish-brown coloration provides excellent camouflage for its benthic lifestyle; however, this coloration rarely commands a high premium in the market. Against this backdrop, the emergence of the golden-colored mutant offers a new breakthrough for industrialization. The phenotypic characteristics of the golden-colored turbot differ significantly from those of conventional individuals; its bright golden appearance not only visually attracts consumers but also brings new economic opportunities to the aquaculture industry.

[0004] With the deepening research into the genetic mechanisms of body color, scientists have successively discovered several key genes involved in the regulation of fish body color in recent years. For example, the Agouti gene, MC1R gene, and SLC24A5 gene directly affect changes in fish body color by regulating pigment synthesis and distribution. Specifically, the Agouti gene is related to the generation and distribution of pigment cells, and its variation may lead to different manifestations of fish coloration; the MC1R gene participates in the regulation of melanin synthesis and is one of the key genes causing color changes in many fish; while the SLC24A5 gene is closely related to pigment transport and stability, playing a crucial role, especially in the formation of gold and silver colors. These studies have not only helped us understand the genetic basis of fish body color but also provided theoretical support for body color improvement. However, although existing research has revealed some molecular mechanisms of body color changes, many mysteries remain. Changes in fish body color are not merely the result of a single gene, but involve complex gene interactions and the influence of environmental factors. Therefore, research on fish body color needs to focus more on the synergistic effects between genes and the regulatory role of environmental factors in the expression of these genes. With the development of genomics and functional genomics, scientists are gradually unraveling these mysteries.

[0005] Against this backdrop, systematically analyzing the molecular regulatory network of golden-colored turbot has become an important research direction. In-depth research on relevant key genes will help identify target genes that significantly influence body color changes, providing a theoretical basis for improving fish body color. In the future, based on these research findings, a controllable body color improvement technology system can be established, thereby promoting the industrial application of new golden-colored turbot strains. This will not only enhance the market premium of turbot but also promote the development of my country's aquaculture seed industry towards higher added value, bringing positive effects to the upgrading of the entire industry chain. Summary of the Invention

[0006] The technical problem this invention aims to solve is to provide a method for controlling the body color of turbot by regulating the expression level of the DIO1 gene in the liver. Our research found that elevated DIO1 expression in the turbot liver enhances the peripheral conversion of thyroxine (T4) to reactive triiodothyronine (T3), subsequently inhibiting thyroid-stimulating hormone (TSH) secretion through negative feedback. The resulting high T3 levels inhibit melanocyte proliferation and differentiation while promoting luteocyte differentiation and lutein accumulation, ultimately leading to a golden phenotype.

[0007] This invention is achieved through the following technical solution:

[0008] The application of the DIO1 gene in the liver in controlling the body color of turbot, wherein the method involves obtaining turbot with a golden body color by overexpressing the DIO1 gene in the liver of turbot; the amino acid sequence of the protein encoded by the DIO1 gene is (SEQ ID NO.1):

[0009] MLIQKLWVYFSTACMFCFMIGLNILLKVLHFISPSLARKQILKMGEKTTM

[0010] TQNQKFSYEDWGPTFLSFDFVKIAAQSMWLSLGQEAFEGGEAPDTPVVTM

[0011] DGERTTVCKFFKGNRPLVLSFGSCTUPPFMYKLDEFKQLVRDFSDVADFL

[0012] VVYVAEAHSTDGWAFTNNFNINQHQSLEDRLSAAQVLVEKEPLCPVVVDEMNDVTAVKYGALPERLYVLQVGKVVYKGGMGPWGYNPLEVRSFLEKMK.

[0013] The beneficial effects of this invention compared to the prior art are as follows: This invention is the first to discover that changes in gene expression levels in the liver of turbot can affect changes in blood hormone levels by influencing the thyroid hormone pathway, thereby affecting skin color formation; therefore, the formation of target body color in turbot can be controlled by controlling gene expression levels, thereby increasing the market value of turbot. Attached Figure Description

[0014] Figure 1 Bright yellow individuals and control individuals, where Y represents golden turbot and B represents black turbot;

[0015] Figure 2 Bar charts and volcano plots of differentially expressed genes in different tissues, where A is a bar chart of differentially expressed genes in the pituitary gland, B is a bar chart of differentially expressed genes in the liver, C is a bar chart of differentially expressed genes in the skin, D is a volcano plot of differentially expressed genes in the pituitary gland, E is a volcano plot of differentially expressed genes in the liver, and F is a volcano plot of differentially expressed genes in the skin.

[0016] Figure 3 Bubble plots of KEGG enrichment analysis in different tissues, where A is the bubble plot of KEGG enrichment analysis in the pituitary gland, B is the bubble plot of KEGG enrichment analysis in the liver, and C is the bubble plot of KEGG enrichment analysis in the skin.

[0017] Figure 4 The graphs show the serum total thyroid hormone, free thyroid hormone, and thyroid-stimulating hormone levels of golden turbot and common turbot. In the graphs, A shows a comparison of T3 levels in the serum of golden turbot and common turbot; B shows a comparison of fT3 levels in the serum of golden turbot and common turbot; C shows a comparison of TSH levels in the serum of golden turbot and common turbot; D shows a comparison of T4 levels in the serum of golden turbot and common turbot; and E shows a comparison of fT4 levels in the serum of golden turbot and common turbot.

[0018] Figure 5 Comparison of fT3 levels among different treatment groups;

[0019] Figure 6 Effect of T3 immersion on skin pigment cells of turbot. Detailed Implementation

[0020] The technical solution of the present invention will be further explained below through embodiments, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0021] Example 1: Transcriptomic analysis of skin, liver, and pituitary gland, and measurement of blood endocrine hormones.

[0022] The experimental fish were sourced from Liaoning Xingcheng Longyun Marine Aquaculture Co., Ltd. Three fish were randomly selected from 30 wild-type (normal body color) fish as the control group (Group B), and three fish were selected from 30 mutant (golden body color) fish as the experimental group (Group Y). A direct comparative observation between the two groups is shown below. Figure 1 As shown. All fish were anesthetized with MS-222, and after blood was drawn from the tail using a 1 ml syringe, they were dissected on ice to collect liver, pituitary, and skin tissue. All tissue samples were immediately rapidly frozen in liquid nitrogen and stored at -80°C. Total RNA was isolated from skin, liver, and brain tissue using TRIzol reagent. RNA purity and integrity were assessed using a NanoDrop 2000 spectrophotometer. Ultra TM RNA library preparation kits were used to construct RNA sequencing libraries, which were then sequenced on an Illumina NovaSeq 6000 platform (Illumina, USA). Serum concentrations of thyroid hormones, free thyroid hormones, and thyroid-stimulating hormone were detected using kits developed by Nanjing Jiancheng.

[0023] Compared to wild-type greyish-brown individuals, we detected relatively fewer differentially expressed genes (DEGs) in the skin of the golden turbot. Figure 2 The findings suggest that the pigmentation mutations may not be caused by widespread changes in the skin's gene network, but rather by targeted modifications to the pigment cell population, such as reducing the number of melanocytes and restricting dendritic development, accompanied by an increase in luteosomes. These findings are consistent with our previous histological observations, which showed fewer and morphologically shrunken melanocytes in golden individuals, along with an increase in the number and size of luteosomes (https: / / doi.org / 10.1016 / j.aqrep.2025.102848). In contrast, numerous differentially expressed genes identified in the liver and pituitary gland of golden individuals are primarily associated with metabolic regulation and hormone signaling pathways. Figure 2 This reflects the key role of endocrine mechanisms in the formation of the golden phenotype.

[0024] Differentially expressed genes in the liver are significantly enriched in metabolic pathways and thyroid hormone biosynthesis pathways. Figure 3The results indicate that the metabolic state and hormonal homeostasis of golden turbot have changed. These results suggest that the liver may indirectly but significantly affect pigmentation, as changes in the expression of hepatic metabolic enzymes and hormone-related genes may affect the availability of pigment precursors or hormone levels, thus affecting skin color. Specifically, we found high expression of the DIO1 gene in the liver of golden turbot, consistent with the liver's crucial role in thyroid hormone metabolism and supporting its potential contribution to pigment regulation. In fish, DIO1 converts T4 in peripheral tissues into circulating T3 and is highly expressed in peripheral organs such as the liver. Its hepatic activity is considered essential for regulating peripheral circulating T3 levels. Thyroid hormones, especially T3, are known to regulate growth, metabolism, and pigment cell differentiation in fish. Serum hormone detection results showed that both T3 and FT3 were elevated in golden individuals (…). Figure 4 Furthermore, immersion in T3-treated juvenile turbot also revealed an inhibition of skin melanocytes. In the pituitary gland, many differentially expressed genes were found to be enriched in hormone synthesis (including thyroid hormones), neuroendocrine signaling pathways, and metabolic regulatory pathways. Figure 3 This indicates that the regulation of the hypothalamus-pituitary-thyroid (HPT) axis plays a crucial role in pigmentation. In the gold standard individuals, pituitary TSHβ expression was significantly reduced, and plasma TSH levels were also lower. Figure 4 This aligns with the classic negative feedback regulation following elevated T3 levels. Therefore, endocrine changes in pituitary function are a crucial component of the golden turkey formation mechanism. Besides melanin, T3 also promotes luteinocyte differentiation and the accumulation of yellow pigment. In fish, lutein is primarily composed of pteridine pigment and carotenoids, with pteridine pigment synthesis dependent on the GCH pathway. We observed elevated GCH1 expression in the skin of golden turbot, suggesting that elevated T3 levels stimulate the pteridine biosynthesis pathway.

[0025] Integrating transcriptomic and endocrine data, we propose a potential mechanism for golden pigmentation: elevated DIO1 expression in the liver enhances the peripheral conversion of T4 to active T3, subsequently inhibiting TSH secretion through negative feedback. The resulting high T3 levels suppress melanocyte proliferation and differentiation while promoting luteocyte differentiation and lutein accumulation, ultimately leading to the golden phenotype. This study provides crucial insights into the molecular and endocrine mechanisms of pigmentation in fish and identifies promising molecular targets for selective breeding of golden turbot.

[0026] Example 2: Effect of DIO1 gene overexpression on T3 transformation

[0027] 1. Experimental Design:

[0028] Primer design: Based on the DIO1 gene ORF sequence (containing the start ATG and stop TGA), primers containing EcoRI and XhoI restriction sites were designed. The forward primer added the Kozak sequence and restriction site upstream of the start codon ATG; the reverse primer retained the stop codon and added the XhoI site. Additionally, 2–6 protective bases were added to the 5' end of the restriction sites to improve digestion efficiency. After primer design, the sequences were submitted to the company for synthesis.

[0029] PCR amplification and product purification: The coding sequence of the gene was amplified using designed primers. The amplified gene fragment was purified using a gel extraction kit.

[0030] Double digestion of plasmid and insert: The pcDNA3.1 vector plasmid and the purified PCR product were double-digested with EcoRI and XhoI, respectively. The digested vector and insert mixture were then subjected to agarose gel electrophoresis, and the target band was recovered.

[0031] T4 ligation reaction: The DIO1 gene fragment was inserted into the pcDNA3.1 vector using T4 DNA ligase. After ligation, it was used for E. coli transformation.

[0032] Primary hepatocyte transfection: The isolated turbot primary hepatocytes were cultured in a suitable medium. One day before transfection, cells were seeded into culture plates as needed, ensuring a cell confluence of approximately 70–90% at transfection. Transfection was performed according to Thermo Fisher Lipofectamine. TM Transfection was performed according to the instructions for reagent 3000.

[0033] Equal amounts of exogenous T4 were added to the culture medium of the target genome transfection group, the empty vector transfection group, and the control group. After culturing for 48 hours, cells and culture medium were collected, and the fT3 content was detected using the Nanjing Jiancheng reagent kit.

[0034] 2. Experimental Results: fT3 content as follows Figure 5 As shown, the transfection of the target genome was significantly higher than that of the empty vector group and the control group (P<0.05), indicating that DIO1 gene overexpression caused a higher level of T3 transformation.

[0035] Example 3: Effects of T3 Immersion on Skin Pigment Cells of Juvenile Turbot

[0036] 1. Experimental Design:

[0037] The experimental fish were obtained from the Xiaying Propagation Station of the Chinese Academy of Fishery Sciences, with a weight of 10±0.2g. All fish came from the same parent to reduce the influence of genetic background.

[0038] The experiment included a control group and a T3 immersion group, using T3 powder to prepare 10 -6mol / L solution. Three replicates per group, 15 fish per replicate, water temperature 16℃, fed once daily, water changed 1 hour after feeding, experimental period 7 days. After the experiment, the morphology of pigment cells was observed under a microscope.

[0039] 2. Experimental Results: Microscopic examination results are as follows Figure 6 As shown, in the control group, melanocytes were mostly radial with prominent dendrites; compared with the control group, melanocytes in the immersion group were round with significantly restricted dendrites, smaller in size, and the number of luteinocytes increased. The results indicate that T3 immersion can inhibit the development of melanocytes in turbot and promote the development of luteinocytes.

Claims

1. The application of the DIO1 gene in the liver in controlling body color in turbot, characterized in that, The application method described above involves obtaining turbot with a golden body color by overexpressing the DIO1 gene in the liver of turbot; the amino acid sequence of the protein encoded by the DIO1 gene is SEQ ID NO.1.

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