Method for regulating molting of shrimps and crabs

Abstract

The invention provides a method for regulating and controlling molting of shrimps and crabs based on interference of insulin receptor genes. By realizing effective silencing of a target gene in an animal body, the expression of an insulin receptor gene is obviously reduced, and the transcriptional level of ecdysis-related factors is changed, including but not limited to, the expression of blood lymphatic juvenile hormone binding protein is obviously increased, and the expression of crustacean hyperglycemia hormone and ligand gated ion channels is obviously reduced, so that the ecdysis-related factors are obviously improved. Therefore, the molting and growth processes of shrimp bodies are influenced. The target gene of the double-stranded molecular interference RNA based on the insulin receptor can obviously influence and regulate (block or inhibit) the ecdysis process. Normal expression of the insulin receptor plays an important role in regulating (maintaining or promoting) the ecdysis process, the ecdysis process of shrimp and crab animals can be regulated to a certain extent in a mode of artificially regulating the insulin receptor, and the insulin receptor has important significance on growth monitoring of aquaculture animals.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for regulating molting of shrimps and crabs, belonging to the field of biotechnology and aquaculture. BACKGROUND

[0002] Shrimp and crab molting refers to the process of updating the body surface by shedding the original shell and generating a new shell during the growth of crustaceans. Since the external shell of shrimps and crabs cannot stretch, as the shrimps and crabs grow, the original shell will limit the expansion of the shrimps and crabs' bodies. In order to adapt to the growth and development of the body, shrimps and crabs will regularly molt. Molting is very important for the growth and development of shrimps and crabs, as it not only allows the shrimps and crabs' bodies to be updated to adapt to the growth of the body, but also repairs body damage, enhances physiological functions, and promotes reproduction. Therefore, molting of shrimps and crabs is an essential process in the life cycle of shrimps and crabs.

[0003] Insulin receptor is a membrane protein located on the cell membrane, which mainly functions to transmit insulin signals after binding with insulin. Insulin receptor can regulate physiological processes such as metabolism, growth, and proliferation in cells by activating the insulin signaling pathway. The specific insulin-like receptor in the ovary of Macrobrachium rosenbergii has a conserved insulin receptor protein domain, which is speculated to be involved in the insulin-like signal cascade regulating the reproductive molting and growth of shrimps. In order to further clarify the function of this insulin receptor protein, the present application uses gene silencing method to inhibit the expression of insulin receptor gene in animals, so as to further analyze the function of insulin receptor and its application in reproductive molting and growth of shrimps and crabs. SUMMARY

[0004] The present application provides a method for regulating molting of animals based on interference of insulin receptor gene. The interference of insulin receptor gene can cause significant changes in the transcription level of blood lymph juvenile hormone binding protein, crustacean hyperglycemic hormone, and ligand-gated ion channel genes related to molting of animals, significantly affecting the molting process of shrimps, thereby being used for regulating the development process of reproductive molting and growth, and having potential economic benefits.

[0005] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions.

[0006] (1) According to the nucleic acid information corresponding to the insulin receptor sequence (WHM41614.1) published on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), primers are designed to clone the interference sequence of the insulin receptor gene using shrimp tissue cDNA as a template, and dsRNA samples for interfering with the insulin receptor gene are synthesized in vitro.

[0007] (2) using injection method to introduce dsRNA into the shrimp body to effectively interfere with the transcription product of the insulin receptor gene.

[0008] (3) detecting the transcription level change of the molting related gene or factor of the shrimp body after the insulin receptor gene is silenced.

[0009] The beneficial effects of the present application are that through animal experiments, the expression of the insulin receptor gene is obviously down-regulated, the effective silencing of the target gene in the animal body is realized, and the expression of the blood lymph juvenile hormone binding protein is obviously increased, the transcription level of the molting related factor crustacean hyperglycemic hormone and ligand-gated ion channel is obviously down-regulated, so as to affect the molting and growth process of the shrimp body. The target gene of the double-stranded molecular interference RNA based on the insulin receptor can significantly affect the development process of reproductive molting and growth, and can be used for molting regulation of animals. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 Detection of interference efficiency of the insulin receptor gene silencing.

[0011] Figure 2 Effect of the gene silencing of the insulin receptor on the transcription level of the molting related factor. DETAILED DESCRIPTION

[0012] The following examples are used to illustrate the present application, but are not used to limit the protection scope of the present application.

[0013] The experimental methods involved in the following examples are all conventional methods in the art if no special instructions are given.

[0014] The reagent consumables and instruments involved in the following examples are all commercially available products if no special instructions are given.

[0015] The contents described in the following examples are part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0016] Example 1: Gene silencing of the insulin receptor in the animal body.

[0017] According to the gene sequence of the insulin receptor, the dsRNA fragment of the insulin receptor synthesized in vitro is used for gene silencing in animal experiments. The experimental shrimps are taken from the Macrobrachium rosenbergii breeding farm in Ningbo, Zhejiang Province, and are bred in a water tank at 27±2℃, with feeding in the morning and evening, and natural light. Healthy mature female Macrobrachium rosenbergii in the ovary development period (body length 9-10 cm and body weight 18-20 g) are selected for interference. The experimental group is injected with 10 micrograms of double-stranded RNA per animal, and the GFP control group is injected with 10 micrograms of green fluorescent protein fragment GFP double-stranded RNA per animal. The interference experiment is set up with 10 animals in each group, and each group is injected with 2 interference samples, with an injection cycle of 10 days. After the animal experiment, the animal tissues are collected, and the RNA is extracted for interference efficiency detection. In the present application, when the mRNA level of the insulin receptor gene is significantly decreased, and the interference efficiency is more than 65%, the gene interference of the insulin receptor is considered to be effective.

[0018] The tissue samples are obtained from the animals with insulin receptor gene silencing, and 3 samples in each group are mixed. The total RNA is extracted by the column Trizol total RNA isolation kit, and the transcriptome sequencing is performed by the Illumina HiSeq method. The transcriptome data is removed by removing the adapter sequence, empty sequence and low-quality sequence, and assembled into non-redundant transcripts. The transcripts with p<0.05 and the absolute value of log2(fold change)>1 are selected, which are defined as the significantly differentially expressed genes of the transcriptome.

[0019] In the present application, after the insulin receptor gene interference, the data of the target insulin receptor gene sequence and its transcription level are obtained by screening and comparing in the transcriptome database. By analyzing the Mean TPM (Transcripts Per Million) of each million mapped reads, the log2(fold change) value, the p value and the q value data of the experimental group and the control group, the interference efficiency of the target gene is calculated.

[0020] The Mean TPM value of the insulin receptor in the experimental group is 4.43, and the Mean TPM value of the control group is 12.60, the log2(fold change) value is-1.51, and the interference efficiency of the insulin receptor gene is calculated to be 35.19%, which is effective (. Figure 1 ).

[0021] Example 2 Influence of gene silencing of the insulin receptor on the transcription level of important molting regulatory factors.

[0022] Silencing of insulin receptor and transcriptome sequencing: The effective silencing of insulin receptor gene and transcriptome sequencing analysis were carried out according to the conventional method in the art. Tissue samples were obtained from animals with insulin receptor gene silencing, and 3 samples from each group were mixed. Total RNA was extracted by column Trizol total RNA isolation kit, and transcriptome sequencing was performed by Illumina HiSeq method. The transcriptome data was removed by removing adapter sequences, empty sequences and low-quality sequences, and assembled into non-redundant transcripts. The transcripts with p<0.05 and the absolute value of log2(fold change)>1 were selected and defined as significantly differentially expressed genes of the transcriptome. In the present application, after the insulin receptor gene was interfered, the sequences and transcriptional level data of the molting related genes or factors were screened and compared and analyzed in the transcriptome database.

[0023] Effect of insulin receptor gene silencing on the transcriptional level of molting related genes or factors: The molting of shrimp and crab is a periodic physiological phenomenon, which is regulated and achieved through various pathways including ion channels. Ion channels are protein channels present on the cell membrane, which can regulate the transmembrane transport of specific ions (such as sodium ions, calcium ions, chloride ions, etc.) to maintain the ion balance inside and outside the cell and participate in physiological activities such as nerve transmission, muscle contraction, cell signal transduction, etc., and play a crucial role in cell function. Ion channels are usually divided into two categories: chemical-gated ion channels and voltage-gated ion channels. Analysis of transcriptome data after silencing of insulin receptor, screening and summarizing significantly differentially expressed genes, and selecting data enriched with molting related genes and factors will help to elucidate the mechanism of action of insulin receptor in the process of reproductive molting and growth.

[0024] In the present application, silencing of insulin receptor induces changes in the transcriptional level of important molting regulatory factors, including but not limited to significant increase in expression of blood lymph juvenile hormone binding protein (Juvenile hormone binding protein, JHBP), significant down-regulation of expression of crustacean hyperglycemic hormone (Crustacean hyperglycemic hormone, CHH) and ligand-gated ion channel (Ligand-gated ion hhannel, LGIC), indicating that insulin receptor regulates (maintains or promotes) the molting process through affecting various ways such as molting related hormones and ion channels.

[0025] Juvenile hormone is a class of hormones that maintain the larval characteristics of insects and promote the development of adult ovaries. In the larval stage, it can inhibit the appearance of adult characteristics, so that the larvae remain in the larval state after molting; in the adult stage, it has the functions of controlled development, production of sex attractants, and promotion of oocyte maturation. Juvenile hormone binding protein (JHBP) is a specific carrier protein present in the hemolymph and intracellularly. After binding with juvenile hormone, it protects juvenile hormone from enzymatic degradation and transports it to the site of action. Hemolymph juvenile hormone binding protein is mainly present on the surface of cells in the blood and lymphatic system, such as red blood cells, lymphocytes, and other immune cells. It forms a complex with juvenile hormone by transmitting the signal of juvenile hormone, and participates in the regulation of cell growth, proliferation, and differentiation processes in the body.

[0026] Crustacean hyperglycemic hormone (CHH) is an important hormone produced in crustaceans. CHH is involved in the regulation of blood glucose levels, molting, reproduction, and ion balance in crustaceans. CHH is found in the nervous system of crustaceans, mainly synthesized and released by the eyestalk neuroendocrine cells, and can be transmitted to target organs through the circulatory system to regulate blood glucose levels. CHH can coordinate the molting process of crustaceans, promoting the softening and dissolution of the exoskeleton, and promoting the synthesis and formation of new exoskeleton. CHH also participates in the regulation of reproduction and ion balance in crustaceans. CHH can regulate the development and function of sex glands, affecting the reproductive capacity of crustaceans; CHH can also regulate the balance of calcium, sodium, potassium and other ions in crustaceans, maintaining a stable environment in the body. In summary, crustacean CHH plays an important regulatory role in regulating blood glucose levels, molting, reproduction, and ion balance.

[0027] Ligand-gated ion channel (LGIC) is a class of transmembrane proteins located on the cell membrane, which regulates the opening and closing of ion channels by binding with specific ligands. When a specific ligand binds to the corresponding receptor, the ion channel undergoes a conformational change, transforming from a closed state to an open state, allowing specific ions to pass through the channel into or out of the cell. Ligand-gated ion channels play an important regulatory role in biological processes such as neurotransmission, sensory conduction, and muscle contraction.

[0028] During the molting process of shrimps and crabs, many growth and metabolism related hormones and neuroregulatory substances are involved, which usually affect the metabolism and growth and development of shrimps and crabs by acting on ligand-gated ion channels. In the nervous system of shrimps and crabs, many neurotransmitters also transmit nerve signals by acting on ligand-gated ion channels. Molting related hormones, as exogenous signal molecules, usually affect cell function and metabolism by acting on ligand-gated ion channels on the cell surface, thereby affecting the molting process of shrimps and crabs to some extent. In summary, the relationship between molting of shrimps and crabs and ligand-gated ion channels is realized through the regulation of the nervous and endocrine systems, which can affect the metabolism, growth, behavior and physiological state of shrimps and crabs, and is one of the important factors for shrimps and crabs to adapt to the environment and survive.

[0029] In the present application, the interference of insulin receptor can effectively silence the insulin receptor gene and induce changes in the transcription level of important molting related regulatory factors, including but not limited to the significant increase in the expression of blood lymph juvenile hormone binding protein, the significant down-regulation of the expression of crustacean hyperglycemic hormone and ligand-gated ion channels, indicating that insulin receptor regulates (blocks or inhibits) the molting process through various ways such as affecting molting related hormones and ion channels. Normal expression of insulin receptor plays an important role in regulating (maintaining or promoting) the molting process, and artificial regulation of insulin receptor can regulate the molting process of shrimps and crabs to some extent, which has important significance for the growth monitoring of aquaculture animals.

[0030] The above examples are only used to illustrate the technical solutions of the present application, and are not limited to the present application. Although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions described in the foregoing examples can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the present application. Therefore, the extended obvious changes or variations are still within the protection scope of the present application.

Claims

1. A method of regulating molting in a shrimp or crab animal, comprising, The method is to reduce or silence the expression of the insulin receptor or its derivative protein by reducing the expression of the insulin receptor gene.

2. The method of claim 1, wherein, The method is to reduce or silence the expression of the insulin receptor or its derivative protein by reducing the expression of the insulin receptor gene.

3. The method according to claims 1 and 2, characterized in that, The method is to reduce or silence the expression of the insulin receptor or its derivative protein by reducing the expression of the insulin receptor gene.

4. The transcript or translation product of the insulin receptor according to claims 1 to 3, characterized in that, The receptor gene molecule includes a sense strand and an antisense strand complementary to the sense strand; the translation product of the receptor includes the translation protein of the insulin receptor or its derivative protein.

5. The method of claim 1, wherein, The molting regulation includes regulating the transcription level of juvenile hormone binding protein in blood lymph, and then regulating the storage and transport of juvenile hormone and its analogues in the animal body, thereby affecting the molting and growth process of the animal.

6. The method of claim 1, wherein, The molting regulation includes regulating the transcription level of juvenile hormone binding protein in blood lymph, and then regulating the storage and transport of juvenile hormone and its analogues in the animal body, thereby affecting the molting and growth process of the animal.

7. The method of claim 1, wherein, The molting regulation includes regulating the transcription level of juvenile hormone binding protein in blood lymph, and then regulating the storage and transport of juvenile hormone and its analogues in the animal body, thereby affecting the molting and growth process of the animal.

8. The method according to claims 1 to 7, characterized in that, The regulation includes inhibiting or delaying the molting regulation, which in vivo includes double-stranded RNA or a carrier for inhibiting the expression of the gene by RNA interference, a chromosome with a functional defect of the gene, or an inhibitor against the translation product of the gene or its receptor.

9. The method according to claims 1 to 8, characterized in that, The animal includes Macrobrachium rosenbergii.

Images