Application of insulin in regulation and control of LC-PUFA biosynthesis of eriocheir sinensis

Abstract

The invention discloses application of insulin in regulation and control of LC-PUFA (liquid chromatography-polyunsaturated fatty acid) biosynthesis of eriocheir sinensis, and belongs to the technical field of bioengineering. The invention provides an application of insulin in preparation of a preparation for regulating and controlling eriocheir sinensis LC-PUFA biosynthesis, insulin is taken as an active component, and the content of LC-PUFA is regulated and controlled in a targeted manner by improving the PIK3Ralpha gene expression of eriocheir sinensis. The invention further provides application of the combination of the insulin and the PI3K agonist 740Y-P in preparation of a preparation for regulating and controlling the LC-PUFA biosynthesis of the eriocheir sinensis. The invention also provides a preparation for regulating and controlling the LC-PUFA biosynthesis of the eriocheir sinensis, which comprises an effective amount of insulin as an active component and also comprises a PI3K agonist 740Y-P. Experiments prove that the insulin regulates and controls the LC-PUFA biosynthesis of the eriocheir sinensis in a targeted manner through a PIK3 signal channel, and a general strategy is provided for nutrition regulation and control of crustaceans.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the application of insulin in regulating the LC-PUFA biosynthesis of Chinese mitten crab. Background Technology

[0002] Fatty acids are an important source of nutrition for humans. Among them, polyunsaturated fatty acids (PUFAs) are important physiologically active substances in the human body. PUFAs are not only basic components of phospholipids and triglycerides, but also metabolic products of biological activities. Long-chain polyunsaturated fatty acids (LC-PUFAs) refer to polyunsaturated fatty acids with ≥20 carbon atoms and ≥3 double bonds, and they play an important role in biological growth and development and disease prevention.

[0003] Insulin is a polypeptide hormone that lowers blood glucose and promotes the synthesis of fat, glycogen, and protein. Studies have found that insulin can increase the mRNA expression levels of Δ6FAD, Δ6Δ5FAD2, ELOVL5, ELOVL2, and SREBP1. The synthesis of LC-PUFA is regulated by these enzyme genes and transcription factors. Therefore, investigating the effects of insulin on LC-PUFA synthesis in the Chinese mitten crab is of great significance for studying the biosynthesis of LC-PUFA in aquatic organisms and reducing the dependence of aquaculture on fish oil. In recent years, some studies have been conducted on the regulatory mechanisms of insulin on key enzyme genes and transcription factors in LC-PUFA biosynthesis in fish, but no relevant reports have been found in crustaceans.

[0004] The Chinese mitten crab (Eriocheir sinensis), also known as the river crab or mitten crab, belongs to the phylum Arthropoda, class Crustacea, order Decapoda. It is widely distributed in the Yellow Sea and eastern coastal areas of my country and has high nutritional and economic value. Lipids play a crucial regulatory role in the growth and gonadal development of the Chinese mitten crab. Meanwhile, EPA, DHA, and other n-3 fatty acids are essential fatty acids for crab growth and development, playing important regulatory roles in growth, reproduction, and metabolism. However, current technologies still suffer from insufficient species applicability and a lack of systematic analysis. For example, the insulin signaling pathway and LC-PUFA synthesis mechanism in the Chinese mitten crab, and especially in other crustaceans, remain unclear. Transcriptome sequencing technology has not been used to comprehensively analyze the global impact of insulin on lipid metabolism pathways, making it difficult to screen key regulatory targets. Therefore, researching the LC-PUFA biosynthesis of the Chinese mitten crab will provide reference data for optimizing nutritional regulation strategies such as the lipid ratio in formulated feeds, which is beneficial for the cultivation of the Chinese mitten crab. Summary of the Invention

[0005] The purpose of this invention is to provide the application of insulin in regulating the biosynthesis of LC-PUFA in Chinese mitten crab, utilizing the endogenous hormone insulin to target and regulate the biosynthesis of LC-PUFA in Chinese mitten crab through the PIK3 signaling pathway.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the application of insulin in regulating the LC-PUFA biosynthesis of Chinese mitten crab.

[0008] Preferably, the insulin targets and regulates the LC-PUFA content of the Chinese mitten crab by increasing the expression of the PIK3Rα gene.

[0009] Preferably, the concentration of the insulin is 0.5-5 ug / uL.

[0010] Preferably, the concentration of the insulin is 1.1 ug / uL.

[0011] A second aspect of the invention provides the use of insulin in the preparation of formulations that regulate the biosynthesis of LC-PUFA in the Chinese mitten crab.

[0012] Preferably, the formulation uses insulin as the active ingredient, with an insulin concentration of 0.5-5 ug / uL. Insulin enhances the expression of the PIK3Rα gene in Chinese mitten crab, thereby targeting and regulating the LC-PUFA content of Chinese mitten crab.

[0013] More preferably, the concentration of the insulin is 1.1 ug / uL.

[0014] Preferably, the dosage form of the preparation includes at least one of the following: tablets, powders, granules, capsules, oral liquids, and injections.

[0015] A third aspect of the present invention provides the use of a combination of insulin and PI3K agonist 740Y-P in the preparation of a formulation that regulates the biosynthesis of LC-PUFA in the Chinese mitten crab.

[0016] In a fourth aspect, the present invention provides a formulation for regulating the LC-PUFA biosynthesis of Chinese mitten crab, comprising an effective amount of insulin as an active ingredient, wherein the concentration of insulin is 0.5-5 ug / uL.

[0017] Preferably, the formulation further includes the PI3K agonist 740Y-P, and the concentration of 740Y-P is 1-10 ug / uL.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. Improved regulatory efficiency: After optimization, the duration of insulin action was shortened to 1.5 h, the injection concentration was reduced to 1.1 μg / μL, and the expression levels of Δ6FAD and SREBP1 were significantly increased.

[0020] 2. Molecular mechanism clarified: The PIK3Rα gene of Chinese mitten crab was cloned for the first time, confirming that it regulates LC-PUFA synthesis through the PIK3 signaling pathway.

[0021] 3. Enhanced technical universality: Transcriptome data reveals lipid metabolism-related pathways, providing a more universal strategy for the nutritional regulation of crustaceans. Attached Figure Description

[0022] Figure 1 The figures represent the relative expression levels of the Δ6FAD and SREBP1 genes under different treatment times with the same concentration of insulin in the examples.

[0023] Figure 2 The figures represent the relative expression levels of the Δ6FAD and SREBP1 genes after treatment with different concentrations of insulin at the same time point in the examples.

[0024] Figure 3 The bar chart shows the statistical results of DEGs in the example. The vertical axis represents the number of differentially regulated genes in the comparison group, where pink represents the number of differentially regulated genes that are significantly upregulated and blue represents the number of differentially regulated genes that are significantly downregulated.

[0025] Figure 4 The KEGG enrichment bubble plot in this example shows the enrichment ratio on the X-axis, the specific pathway on the Y-axis, the size of the color dot representing the number of different expressed genes in each pathway, and the color of the bubble changing from purple to blue to green to red. The smaller the P-value, the stronger the significance.

[0026] Figure 5 For verification of differentially expressed genes in the transcriptome in the examples, * indicates significant differences (P < 0.05).

[0027] Figure 6 The effects of each treatment group on the TG content and LPS activity of Chinese mitten crab are shown in the examples.

[0028] Figure 7 The example shows a phylogenetic tree constructed based on the amino acid PI3KRα of the Chinese mitten crab. Detailed Implementation

[0029] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.

[0030] The following examples demonstrate the regulation of LC-PUFA content in Chinese mitten crab by insulin, mainly including:

[0031] (1) Determination of the optimal conditions for insulin action

[0032] Six healthy and vigorous Chinese mitten crabs were selected for each group. 1 μg / g (the crab's dry body weight) was injected into the base of the third appendage using a sterilized microsyringe. The control group was injected with DMSO. The effects of different insulin concentrations (0 μg / uL, 0.3 μg / uL, 0.6 μg / uL, 1.2 μg / uL, 2.4 μg / uL) at different time points (1 h, 1.5 h, 3 h) on the expression levels of Δ6FAD and SREBP1 genes in the Chinese mitten crabs were investigated. The optimal insulin treatment time and concentration were determined to be 1.5 h and 1.1 μg / uL, respectively.

[0033] Step 2: Transcriptome sequencing

[0034] Injection experiments were conducted using insulin treatment groups (1.5 h and 1.1 ug / uL) and a control group (DMSO), with three replicates per group and six crabs per replicate. Hepatopancreatic samples were collected, RNA was extracted using the Trizol method, purified into cDNA, amplified by PCR, and sequenced after passing quality control.

[0035] To obtain reliable reads and assess the quality of the raw data, Trimmomatic software was first used for quality control and adapter removal. Then, clean reads were aligned to the *Eriocheir sinensis* reference genome using hisat2 and assembled using String Tie software. Subsequently, Cuffcompare was used to compare the annotation information of the new transcripts with the reference sequence. For differentially expressed genes, the number of transcript reads was calculated using eXpress, the data was processed using the estimate size factors function, and the P-value and fold change value were calculated using the Nbinom Test. Transcripts with a fold change greater than 2 were screened, and finally, GO and KEGG enrichment analyses were performed. RNA-Seq technology was used to analyze differentially expressed genes between the insulin-treated group and the control group, screening for potential regulatory pathways and elucidating the regulatory role of insulin on lipid metabolism pathways in *Eriocheir sinensis* at the transcriptional level.

[0036] Step 3: Cloning of PIK3Rα

[0037] In the insulin signaling pathway, an important gene, PIK3Rα, was screened out. By cloning the full-length cDNA of the PIK3Rα gene, the structural features, evolutionary relationships, and tissue-specific expression of its encoded protein were analyzed. This provides a molecular basis for revealing the regulatory mechanism of PIK3Rα in the crustacean insulin-PI3K signaling pathway, and also provides a reference for exploring the biologically specific role of PI3K and the physiological regulatory mechanisms of lipid metabolism and immunity in Chinese mitten crab.

[0038] Step 4: PIK3 signal path function verification

[0039] Chinese mitten crabs were treated individually with insulin, the PI3K agonist 740Y-P, and the inhibitor LY294002, as well as in combination with insulin and the agonist, and with insulin and the inhibitor. The expression of key enzyme genes and transcription factors in LC-PUFA synthesis was detected, along with triglyceride (TG) levels and lipase (LPS) activity. Changes in fatty acid composition were also analyzed. This study aimed to explore whether insulin regulates LC-PUFA synthesis in Chinese mitten crabs from a physiological and biochemical perspective, and to clarify the role of the PI3K signaling pathway in insulin regulation.

[0040] Example 1

[0041] The insulin injection experiment is as follows:

[0042] (1) Determining the duration of insulin action: Six healthy and vigorous Chinese mitten crabs were selected for each group. 1 μg / g (the crab's body weight after drying) was injected into the base of the third appendage of the crab using a sterilized microsyringe. The control group was injected with DMSO. Samples were taken at 1 h, 1.5 h, and 3 h after injection to detect the expression levels of Δ6FAD and SREBP1 genes.

[0043] Experimental results showed that insulin had the best effect on upregulating the expression of Δ6FAD and SREBP1 genes at 1.5 h. Figure 1 The study determined that the optimal time for insulin to regulate LC-PUFA synthesis is 1.5 h. Compared with other time points, it can more efficiently activate key genes in LC-PUFA synthesis (Δ6FAD is the key enzyme gene and SREBP1 is the core transcription factor), improve regulatory efficiency, and achieve rapid nutritional regulation.

[0044] (2) Determining the optimal concentration of insulin: Several concentrations of 0.3 ug / uL, 0.6 ug / uL, 1.2 ug / uL, and 2.4 ug / uL were set for injection experiments, with an injection volume of 1 uL / g. The control group was injected with DMSO. Samples were taken 1.5 h after injection to detect the expression levels of Δ6FAD and SREBP1 genes.

[0045] like Figure 2 As shown, the effects of different insulin concentrations on the expression of Δ6FAD and SREBP1 genes were compared. Finally, the insulin injection concentration that most significantly upregulated the expression of Δ6FAD and SREBP1 genes was determined to be 1.1 ug / uL. This concentration can maximize the activation of key gene expression while avoiding the cost waste that may be caused by excessive concentration.

[0046] (3) The injection experiment determined that the optimal insulin action time and concentration were 1.5 h and 1.1 ug / uL. Chinese mitten crabs were treated with insulin, injected with 1.1 ug / uL of insulin. Hepatopancreatic samples were collected 1.5 h after treatment, while the control group was injected with DMSO. Transcriptome sequencing was used to analyze differentially expressed genes and metabolic pathways among the different treatment groups, such as… Figure 3 As shown, transcriptome sequencing results reveal the number of differentially expressed genes between the insulin-treated group and the control group. Significantly upregulated (pink) and downregulated (blue) genes are clearly visible, indicating that insulin treatment leads to differential expression of numerous lipid metabolism-related genes. To analyze the pathways affected by insulin treatment, differentially expressed genes from the insulin-treated and control groups were mapped to the KEGG database. The KEGG enrichment bubble plot results show the KEGG pathways enriched by differentially expressed genes in the insulin-treated group, including the PI3K signaling pathway and fatty acid metabolism pathway. Figure 4The results of qPCR validation of these transcriptome data are as follows: Figure 5 As shown, the expression changes of key differentially expressed genes (such as PIK3Rα, SREBP1, and Δ6FAD) in the transcriptome were verified. The results showed that the expression of these genes was significantly upregulated in the insulin-treated group (P<0.05), confirming the reliability of the transcriptome data and directly demonstrating that insulin targets and regulates LC-PUFA synthesis by upregulating genes such as PIK3Rα, providing a molecular basis for formulation development.

[0047] Example 2

[0048] Agonist and inhibitor injection experiments:

[0049] To investigate the regulatory role of insulin in the synthesis of LC-PUFA in Chinese mitten crabs and whether this process is mediated by the PI3K signaling pathway, healthy, vigorous female Chinese mitten crabs with intact limbs were selected for injection experiments. Six groups were set up as follows: (1) Blank control group: injected with DMSO; (2) Insulin group: injected with insulin at a concentration of 1.1 ug / uL; (3) 740Y-P group: injected with 740Y-P at a concentration of 5 ug / uL; (4) LY294002 group: injected with LY294002 at a concentration of 5 ug / uL; (5) Insulin+740Y-P group: injected with 1.1 ug / uL Insulin and 5 ug / uL 740Y-P; (6) Insulin+LY294002 group: injected with 1.1 ug / uL Insulin and 5 ug / uL LY294002. The injection dose was 1 uL / g. Hepatocellular and pancreatic samples were collected 7 days after injection. The expression of PIK3Rα, Δ6FAD, Δ9FAD, ELOVL6, SREBP and HNF4 genes was detected by qPCR. TG content and LPS activity were detected, and fatty acid analysis was performed.

[0050] Weigh the collected liver and pancreatic tissue samples, add physiological saline for homogenization, centrifuge at 2500 rpm for 10 min at 4℃, and collect the supernatant. Follow the instructions of the triglyceride (TG) assay kit (A110-1) and lipase (LPS) assay kit (A054-2) to calculate the TG content and LPS activity, and determine the fatty acid composition using the direct methyl esterification method.

[0051] The results showed that insulin treatment alone significantly upregulated the expression of PIK3Rα, SREBP1, Δ6FAD, and ELOVL6 genes; the agonist 740Y-P further enhanced the expression of HNF4 and Δ9FAD, while the inhibitor LY294002 significantly inhibited these genes. Combined treatment showed that gene expression was synergistically enhanced in the INS+740Y-P group (e.g., PIK3Rα and Δ9FAD were significantly higher than in the insulin-only group), while gene expression was significantly inhibited in the INS+LY294002 group, indicating that the PI3K signaling pathway plays a central role in insulin-mediated LC-PUFA synthesis, with agonists enhancing the insulin effect and inhibitors antagonizing it. Figure 6 As shown, both insulin and agonist treatments significantly increased TG levels, while the inhibitor group showed a decrease in TG levels. Regarding LPS activity, insulin significantly inhibited LPS activity, while the inhibitor significantly upregulated it, and the agonist had no significant effect. The insulin + agonist group had significantly higher ΣSFA than the control group, while there was no difference between the inhibitor and control groups. Insulin significantly increased ΣMUFA, while the inhibitor group significantly decreased it. Insulin significantly increased ΣPUFA (such as DHA and LA), and the combined agonist treatment enhanced the effect, while the inhibitor group showed a significant decrease in ΣPUFA (especially DHA). This physiologically and biochemically validates that insulin promotes lipid accumulation and inhibits lipolysis through the PI3K pathway, thereby enhancing LC-PUFA synthesis. The combined use of insulin and agonists can synergistically improve the effect, providing an optimized solution for formulation development.

[0052] Example 3

[0053] Cloning of the PIK3Rα gene:

[0054] (1) Based on the predicted PIK3Rα cDNA sequence in the whole genome database of Chinese mitten crab, primers were designed using PrimerPremier 5.0 software. Total RNA was extracted from the hepatopancreas of juvenile Chinese mitten crabs using the Trizol method and reverse transcribed into the first strand of cDNA. This was then used as a template for PCR reaction.

[0055] PCR loading system: 1 µL cDNA template, 10 µL 2×Taq Master Mix, 0.5 µL each of forward and reverse primers, 3 µL ddH2O, total volume 15 µL.

[0056] PCR reaction conditions: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 15 s; 60℃ annealing for 30 s; 72℃ extension for 60 s; 34 cycles, 72℃ extension for 7 min, and storage at 4℃.

[0057] The PIK3Rα core fragment was amplified, cloned into the pMD-19T vector, and sequenced.

[0058] (2) Based on the core fragment of PIK3Rα obtained above, a 3'PIK3Rα-RACE upstream primer and a 5'PIK3Rα-RACE downstream primer were designed. The extracted total RNA of Chinese mitten crab was reverse transcribed into the first strand of 3'-cDNA and 5'-cDNA, respectively. Using these as templates, 3' and 5' end fragments were RACEed:

[0059] First-strand cDNA was synthesized using the SMARTer RACE 5' / 3' Kit as a template for rapid gene amplification. The contents of the RACE cDNA template were as follows: 2.5 µL; 5 µL of 10× Universal Primer A Mix; 1 µL of specific primers (GSP, 10 µM); 1 µL of dNTP Mixture (10 mM each); 5 µL of 10×Advantage 2 PCR Buffer; 1 µL of 50×Advantage 2 Polymerase Mix; and 34.5 µL of PCR-Grade Water, for a total volume of 50 µL.

[0060] 5'RACE and 3'RACE PCR reaction programs: 94℃ for 30s, 72℃ for 2min, 5 cycles; 94℃ for 30s, 70℃ for 30s, 72℃ for 2min, 40 cycles; 94℃ for 30s, 68℃ for 30s, 72℃ for 2min, 25 cycles.

[0061] The obtained 3' and 5' end fragments were ligated into pMD-19T, cloned, and sequenced.

[0062] (3) The obtained 3' and 5' end fragments were spliced ​​with the core fragment to obtain the full-length PIK3Rα cDNA sequence of Chinese mitten crab, as shown in SEQ ID NO.1, as follows:

[0063]

[0064]

[0065] Analysis of the amino acid sequence encoding PIK3Rα revealed a molecular weight of 112.19 kDa and a theoretical isoelectric point of 6.78. BLASTp alignment showed that the PIK3Rα amino acid sequence of *Eriocheir sinensis* was homologous to that of *Scylla serrata*, *Portunus trituberculatus*, *Snow Crab*, *Procambarus clarkii*, zebrafish, and humans. Figure 7 As shown, the PIK3Rα of the Chinese mitten crab has high homology with the PIK3Rα of other shrimp and crab species such as the mud crab, swimming crab, snow crab, and red swamp crayfish. Among them, the homology with the mud crab is the highest at 83.05%, which confirms the conservation of PIK3Rα in crustaceans. The first cloning of the PIK3Rα gene of the Chinese mitten crab provides a molecular tool for the study of the insulin PI3K signaling pathway in crustaceans.

[0066] The above studies validated the effectiveness of insulin in targeting and regulating LC-PUFA biosynthesis in *Eriocheir sinensis* via the PI3K signaling pathway, optimizing the insulin action time (1.5 h) and concentration (1.1 ug / uL) to rapidly activate key gene expression. Transcriptome and KEGG analyses revealed the core role of the PI3K pathway, and cloning the PIK3Rα gene provided a molecular basis. Phylogenetic analysis showed that PIK3Rα is conserved in crustaceans, providing a general strategy for crustacean nutrient regulation. These conclusions provide solid data support for the application of this invention in the field of bioengineering, especially in reducing dependence on fish oil in aquaculture and optimizing feed formulation.

[0067] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of insulin in regulating LC-PUFA biosynthesis in Chinese mitten crab.

2. The application according to claim 1, characterized in that, The insulin targets and regulates the LC-PUFA content of Chinese mitten crab by increasing the expression of the PIK3Rα gene.

3. The application according to claim 1, characterized in that, The concentration of insulin is 0.5-5 ug / uL.

4. The application according to claim 3, characterized in that, The concentration of insulin was 1.1 ug / uL.

5. Application of insulin in the preparation of formulations that regulate the biosynthesis of LC-PUFA in Chinese mitten crab.

6. The application according to claim 5, characterized in that, The formulation uses insulin as the active ingredient, with an insulin concentration of 0.5-5 ug / uL. It enhances the expression of the PIK3Rα gene in Chinese mitten crabs through insulin, thereby targeting and regulating the LC-PUFA content in Chinese mitten crabs.

7. The application according to claim 5, characterized in that, The concentration of insulin was 1.1 ug / uL.

8. The application according to claim 5, characterized in that, The dosage form of the preparation includes at least one of the following: tablets, powders, granules, capsules, oral liquids, and injections.

9. Application of the combination of insulin and PI3K agonist 740Y-P in the preparation of formulations that regulate the biosynthesis of LC-PUFA in Chinese mitten crab.

10. A formulation for regulating LC-PUFA biosynthesis in the Chinese mitten crab, characterized in that, It contains an effective amount of insulin as the active ingredient, and the concentration of insulin is 0.5-5 ug / uL; It also includes the PI3K agonist 740Y-P, and the concentration of 740Y-P is 1-10 ug / uL.

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