Application of vdr gene in improving high calcium tolerance of fish

Abstract

This invention relates to vdr The application of genes in improving the high calcium tolerance of fish belongs to the fields of aquaculture technology and molecular biology technology. This invention provides... vdr Application of genes in improving calcium tolerance in fish. This invention, through the determination of calcium ion content in water bodies in Jiuzhaigou and Longnan regions, and by comparing the differences in gene expression in the same species of Jialing fish, screens for... vdr The gene was differentially expressed in Jialing fish from different regions, and this was further verified by gene knockout experiments. vdr The influence of genes on the calcium tolerance of fish was eventually determined. vdr Knockout or low expression of genes can improve the tolerance of fish to high calcium levels.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and more particularly to... vdr Application of genes in improving calcium tolerance in fish. Background Technology

[0002] Calcium is a crucial component of fish bones, scales, and shells, playing a vital role in environmental remediation and physiological regulation in aquaculture. However, unlike terrestrial vertebrates, fish live their entire lives in aquatic environments, and their gill epithelium is the primary pathway for calcium ions to enter their bodies. When the calcium ion concentration in aquaculture water is excessively high, fish face continuous high-calcium stress. Studies have shown that high-calcium environments trigger a series of physiological disorders in fish: Nile tilapia exposed to high-calcium water exhibit significantly decreased growth performance, serum antioxidant levels, and protein profiles, while ALT, AST, urea, creatinine, and malondialdehyde levels significantly increased. High-calcium exposure also causes pathological changes in multiple organs and tissues, including the gills, liver, kidneys, spleen, and brain, with liver and kidney damage being particularly severe. In commercial Atlantic salmon farming, nephrocalcinosis, characterized by excessive mineral deposition in the kidneys, has become an increasingly serious production problem, affecting up to 41% of the tested fish populations. Therefore, high-calcium stress has become one of the important factors restricting the healthy development of aquaculture.

[0003] In the study of calcium metabolism regulation in fish, the vitamin D endocrine system has received considerable attention. Vitamin D exerts its biological functions through its nuclear receptor—the vitamin D receptor (VDR). Studies in mammals have shown that vitamin D-VDR signaling stimulates active calcium absorption by inducing the expression of various calcium-binding proteins and calcium transporters in the gut. In fish, VDR is mainly expressed in the kidneys, liver, stomach, and intestines. Studies using zebrafish as a model have found that vitamin D-VDRa signaling stimulates calcium absorption by upregulating the expression of epithelial calcium channels (ECaC). 2+ Absorption; a low-calcium environment can induce calcium absorption in zebrafish. 2+ Increased inflow and ecac , vdra The expression of genes such as [list of genes] is upregulated. In Atlantic salmon, the vitamin D system plays a crucial role in calcium and phosphorus processing. Furthermore, exogenous vitamin D treatment can inhibit [the processing of these genes]. cyp27b1 The expression and stimulation at the same time cyp24a1 The expression of these substances forms a homeostatic feedback regulation of vitamin D levels.

[0004] However, existing research on the regulation of calcium metabolism in fish mainly focuses on two aspects: first, studying the mechanism by which vitamin D-VDR signaling promotes calcium absorption under low calcium conditions; and second, exploring the optimal amount of vitamin D added to feed from the perspective of nutritional requirements. To date, there are no reports on enhancing fish tolerance to high calcium environments through active regulation of the vitamin D receptor signaling pathway. Current technologies for addressing high calcium stress mainly rely on passive measures such as changing the aquaculture water source (e.g., water exchange and dilution) or adjusting feed formulations, lacking active intervention methods based on the fish's own physiological regulation. The regulatory role of the vitamin D-VDR signaling pathway in the adaptation process of fish to high calcium stress and its feasibility as a target for enhancing high calcium tolerance remain unexplored.

[0005] Based on this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide vdr The application of genes in improving the tolerance of fish to high calcium levels aims to address the lack of existing technologies for improving the adaptability of fish to high calcium stress.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides vdr The application of genes in improving calcium tolerance in fish, the vdr Genes include vdra Genes and / or vdrb Gene.

[0008] Preferred, by knockout vdr Genes or downregulation vdr Gene expression enhances the fish's tolerance to high calcium levels.

[0009] The present invention has the following technical effects and advantages: This invention, by measuring the calcium ion content in water bodies in Jiuzhaigou and Longnan regions and comparing differences in gene expression within the same species of Jialing fish, screens for... vdr The gene was differentially expressed in Jialing fish from different regions, and this was further verified by gene knockout experiments. vdr The influence of genes on the calcium tolerance of fish was eventually determined. vdr Knockout or low expression of genes can improve the tolerance of fish to high calcium levels. Attached Figure Description

[0010] Figure 1 The results of calcium ion content determination in water bodies in Jiuzhaigou and Longnan areas; Figure 2 Results of calcium ion content determination in Jialing fish from different regions; Figure 3The results show the survival rate of Longnan Jialing fish in high-calcium and low-calcium environments. Figure 4 The results of calcium ion content determination in Longnan Jialing fish of different treatment groups; Figure 5 Jialing fish from different regions vdr Gene expression status; Figure 6 zebrafish vdr Results of gene expression level measurement; Figure 7 The results show the survival rate of zebrafish in different treatment groups in high-calcium ion water. Figure 8 Alizarin Red staining results for zebrafish from different treatment groups; Figure 9 The results of morphological observations of different zebrafish in different aquatic environments. Detailed Implementation

[0011] This invention provides vdr The application of genes in improving calcium tolerance in fish, the vdr Genes include vdra Genes and / or vdrb Gene.

[0012] In this invention, by knocking out vdr Genes or downregulation vdr Gene expression enhances the fish's tolerance to high calcium levels.

[0013] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0014] Example 1

[0015] Jiuzhaigou is located in Zhangzha Town, Jiuzhaigou County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan Province, while Longnan is located in southeastern Gansu Province, at the junction of Gansu, Shaanxi, and Sichuan provinces. Due to their different geographical locations, the calcium ion content in their water bodies also differs. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the calcium ion content in the water bodies of Jiuzhaigou and Longnan. The results of the calcium ion content determination in the water bodies of Jiuzhaigou and Longnan are as follows: Figure 1 As shown in the figure, HJ represents Jiuzhaigou and LL represents Longnan.

[0016] according to Figure 1 It can be seen that the calcium ion content in the water bodies of Jiuzhaigou and Longnan is extremely different, with the calcium ion content in the water bodies of Jiuzhaigou exceeding that of Longnan by more than 3 times.

[0017] Meanwhile, the inventors discovered the same fish (Jialing naked carp, scientific name: *Gymnocypris jialingensis*) in Jiuzhaigou, Sichuan and Longnan, Gansu. Schizopygopsis kialingensis (Hereinafter referred to as Jialing fish), serum was collected from the same fish species in two different regions. According to the instructions of the colorimetric assay kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number: S1063S), the calcium content was determined using the colorimetric-catechol-nitrite complex method. The specific steps are as follows: Prepare the detection working solution by mixing the detection buffer and the chromogenic solution at a 1:1 ratio. Add 50 μL of standard or serum sample to a 96-well plate, then add 150 μL of the detection working solution. Incubate at room temperature in the dark for 5 min. Measure the absorbance at 575 nm using an ELISA reader within 30 min. Subtract the blank absorbance and calculate the serum calcium ion content based on the standard curve.

[0018] Results of calcium ion content determination in Jialing fish from different regions are as follows: Figure 2 As shown in the figure, HJ represents the Jialing fish from Jiuzhaigou area, and LL represents the Jialing fish from Longnan area.

[0019] according to Figure 2 It can be seen that there is no significant difference in calcium ion content between the Jialing fish in Jiuzhaigou and the Jialing fish in Longnan.

[0020] Example 2

[0021] Forty Jialing fish from Longnan were selected and divided into a high-calcium group and a low-calcium group (calcium ion concentration in the water of the high-calcium group was 10 mM, and calcium ion concentration in the water of the low-calcium group was 0.3 mM), with 20 fish in each group. The fish were placed in the high-calcium and low-calcium water respectively for 10 days. Mortality was recorded daily, and 50% of the water was replaced daily. The survival rate of the Jialing fish in the high-calcium and low-calcium environments was calculated. After the rearing period, serum samples were collected from the high-calcium and low-calcium groups to determine the calcium ion content (the determination method was the same as in Example 1). The survival rate results of the Jialing fish in the high-calcium and low-calcium environments are as follows: Figure 3 As shown in the figure, the calcium ion content in the Longnan Jialing fish of different treatment groups was measured as follows: Figure 4 As shown.

[0022] according to Figure 3 and Figure 4 It was found that the survival rate of Jialing fish decreased significantly within 10 days in a high-calcium environment, and its blood calcium concentration increased significantly.

[0023] Further transcriptome sequencing of Jialing fish from different regions yielded two differentially expressed gene sequences. These sequences were then submitted to NCBI BLAST for homology comparison, and the results showed that they were similar to those of zebrafish. vdra Genes (NCBI Registry Number: 30076) and vdrbThe two gene sequences (NCBI accession number: 564511) are homologous, thus determining that they are differentially expressed. vdr Genes, respectively vdra Genes and vdrb Genes, sequencing revealed vdra vdrb

[0024] RT-PCR was used to determine the composition of Jialing fish from different regions. vdr Gene expression status, results as follows Figure 5 As shown in the figure, A represents vdra Gene expression level, B represents vdrb Gene expression levels.

[0025] according to Figure 5 It can be seen that the Jialing fish in the Jiuzhaigou area vdr The gene expression level was significantly lower than that of the Jialing fish from Longnan region, therefore it is speculated that... vdr Low expression or knockout of genes may increase calcium tolerance in fish.

[0026] Example 3

[0027] 1. vdr Construction of gene knockout zebrafish

[0028] Based on the method described in the article "Enhanced insulin activity achieved in VDRa / bablation zebrafish" published on February 13, 2023, a construction was made... vdr Gene knockout zebrafish.

[0029] Using RT-PCR vdr Gene knockout zebrafish and wild-type zebrafish vdr Gene amplification and measurement vdr The gene expression level was measured using the following methods: Total RNA was extracted from zebrafish juveniles using an RNA extraction kit (Ciscojet), and cDNA was synthesized using PrimeScript™ RTMaster Mix (Takara) via reverse transcription. Using the cDNA as a template, amplification and detection were performed on a real-time quantitative PCR instrument using THUNDERBIRD™ SYBR® qPCR Mix (Toyobo). β-actin was used as an internal control gene, and a 2... - The relative expression level of the target gene was calculated using the ΔΔCt method.

[0030] According to wild zebrafish vdra Genes (NCBI Registry Number: 30076) and vdrb Primers were designed based on the gene sequence (NCBI accession number: 564511). The primer sequences are as follows: vdra -F: 5'-GCACCTCTGCCACCGGG-3' (SEQ ID NO.3); vdra-R: 5'-GGTAAAACTGGCCTTACGCT-3' (SEQ ID NO.4); vdrb -F: 5'-ACACAGCGTGGAGTGGAGT-3' (SEQ ID NO.5); vdrb -R: 5'-ACACTCCATGGCAAGAACA-3' (SEQ ID NO. 6); β-actin-F: 5'-ATGAGGAAATCGCTGCCCTG-3' (SEQ ID NO.7), β-actin-R: 5'-CGTCCAACAATGGAGGGGAA-3' (SEQ ID NO. 8).

[0031] Reaction system (20 μL): 10 μL SYBR Premix, 1 μL each of forward and reverse primers (10 μM), 1 μL cDNA template, and ddH2O to bring the total to 20 μL.

[0032] Reaction procedure: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing extension for 45 s, for a total of 40 cycles; after the cycles, melting curve analysis (95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s) was performed to verify amplification specificity. Each sample was set to 3 replicates, and the experiment was independently repeated 3 times.

[0033] vdr Gene expression level measurement results as follows Figure 6 As shown in the figure, A represents vdra Gene expression level, B represents vdrb Gene expression levels.

[0034] according to Figure 6 It can be seen that the system was successfully constructed. vdr Gene knockout zebrafish.

[0035] 2. High calcium tolerance test

[0036] Set up a control group (WT) and a treatment group ( vdrb - / - The control group consisted of wild-type zebrafish, while the treatment group consisted of... vdrb Gene-knockout zebrafish were used in a 6-well plate containing high-calcium ion (10 mM) water, with 20 zebrafish per well and 6 replicates. The zebrafish were fed for 9 days, with 50% of the water changed daily. Mortality was monitored daily, and the survival rate of zebrafish in the high-calcium ion water was determined. The results of the survival rate determination for different treatment groups of zebrafish in the high-calcium ion water are shown below. Figure 7 As shown.

[0037] according to Figure 7 It can be seen that, vdrb The survival rate of gene-knockout zebrafish was significantly higher than that of wild-type zebrafish, indicating that... vdrb Gene knockout can improve the tolerance of fish to high calcium levels.

[0038] 3. Alizarin Red staining

[0039] The principle of alizarin red staining is through the interaction of its hydroxyl and carbonyl groups with calcium ions (Ca). 2+ Specific chelation forms a stable orange-red complex, thus visualizing calcium salt deposition. After 8 days of feeding, [the samples were]... vdrb Gene knockout zebrafish and wild-type zebrafish were fixed with 4% paraformaldehyde, bleached with 2% potassium hydroxide and 3% H2O2, and then stained overnight in 0.05% alizarin red solution. After washing, the staining area and total staining density were measured under a stereomicroscope. The alizarin red staining results of zebrafish from different treatment groups are shown below. Figure 8 As shown, A represents the alizarin red staining pattern of zebrafish in different treatment groups, B represents the staining area of ​​zebrafish in different treatment groups, and C represents the overall staining density of zebrafish in different treatment groups.

[0040] according to Figure 8 It can be seen that, vdrb Compared to wild-type zebrafish, gene knockout zebrafish also show a significantly reduced ability to absorb calcium ions.

[0041] 4. vdr Effects of gene knockout on zebrafish morphology

[0042] Observe wild-type zebrafish and vdr Morphology of gene-knockout zebrafish in different aquatic environments; morphological observation results of different zebrafish in different aquatic environments, as shown below. Figure 9 As shown.

[0043] according to Figure 9 It can be seen that, vdr The knockout of the gene does not affect the morphology of the zebrafish.

[0044] In summary, by knocking out vdr Inhibiting the vitamin D receptor signaling pathway can enhance the tolerance of fish to high-calcium environments, and vdr The gene knockout did not have a significant impact on the morphology of zebrafish.

[0045] As can be seen from the above embodiments, the present invention provides vdr Application of genes in improving calcium tolerance in fish. This invention, through the determination of calcium ion content in water bodies in Jiuzhaigou and Longnan regions, and by comparing the differences in gene expression in the same species of Jialing fish, screens for... vdr The gene was differentially expressed in Jialing fish from different regions, and this was further verified by gene knockout experiments. vdr The influence of genes on the calcium tolerance of fish was eventually determined. vdr Knockout or low expression of genes can improve the tolerance of fish to high calcium levels.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of the vdr gene in improving the high calcium tolerance of fish, wherein the vdr gene includes the vdra gene and / or the vdrb gene.

2. The application according to claim 1, characterized in that, Fish can improve their tolerance to high calcium levels by knocking out or downregulating the expression of the vdr gene.

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