Method for regulating female reproduction based on insulin receptor

Abstract

The invention discloses a method for regulating female reproduction based on interference of insulin receptor genes. The expression quantity of the insulin receptor gene in the shrimp body can be efficiently reduced in an insulin receptor gene silencing mode, and the production process of ovary yolk of the shrimp body and the metabolism process of lipid and protein can be obviously influenced, so that the development and maturation of female gonads are regulated and controlled. The method for regulating female reproduction by the insulin receptor can obviously influence ovary development and maturation of the shrimp body, and has potential economic benefits. The invention provides a means for regulating (promoting or inhibiting) ovaries of full-eye crustaceans such as shrimps, crabs and the like, including regulating the functions of insulin receptor genes or the functions of transcription products or translation products of the insulin receptor genes.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for regulating female reproduction based on the interference of insulin receptor gene, belonging to the field of biotechnology. BACKGROUND

[0002] Insulin signaling pathway is a key intracellular signaling pathway involved in the regulation of blood glucose levels and energy metabolism. Follicle cells in the ovary express insulin receptors, which act as transmembrane proteins and are important members of the insulin signaling pathway. Insulin directly acts on follicle cells through its signaling pathway to promote maturation and ovulation. Abnormal insulin signaling pathway can lead to insulin resistance and the occurrence of polycystic ovary syndrome, interfering with follicular development and ovulation process, resulting in infertility and irregular menstruation.

[0003] Aquaculture of shrimps and crabs is an important artificial breeding strain in the field of aquaculture. China is the world's largest shrimp and crab farming country, accounting for about 70% of the total global shrimp and crab farming output value. The ovarian development process of shrimps and crabs is a complex biological process, including oocyte development, oocyte maturation and follicle formation, and is influenced by environmental factors, seasonal changes, nutrient intake and growth status. Therefore, artificially regulating the ovarian development and maturation of shrimps and crabs has important guiding significance for the breeding and breeding of economic shrimps and crabs. In addition, the precocious puberty phenomenon of shrimps and crabs also occurs in aquaculture. Under normal circumstances, animals need to go through a certain growth cycle to reach the stage of sexual maturity, but some shrimps and crabs begin to reproduce at a young age, or shrimps and crabs reach sexual maturity at a young age, affecting the economic benefits and sustainable development of aquaculture.

[0004] The insulin receptor of Macrobrachium rosenbergii has a conserved insulin receptor protein domain composition, is specifically expressed in the ovary, and its expression gradually increases with the development and maturation of the ovary, suggesting that it is involved in the insulin signaling cascade to regulate the reproduction and maturation of the ovary of shrimps. In order to further clarify the function of this insulin receptor protein, the present application uses the method of gene silencing to inhibit the expression of insulin receptor gene in the animal body, so as to further analyze the function of insulin receptor and its application in female reproduction. SUMMARY

[0005] The present application aims to provide a method for regulating female reproduction based on the interference of insulin receptor gene. The dsRNA interference of the insulin receptor can slow down the ovarian development of shrimps, significantly affect the maturation of the ovary of shrimps, and thus regulate the development and maturation process of female gonads, with potential economic benefits.

[0006] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions.

[0007] According to the insulin receptor gene sequence information published on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), the primers are designed around the insulin receptor gene, and the interference sequence of the insulin receptor gene is cloned by taking the ovarian tissue cDNA of the shrimp as the template, and the dsRNA sample for interfering the insulin receptor gene is synthesized in vitro.

[0008] The dsRNA is introduced into the shrimp body by injection method, and the transcription product of the insulin receptor gene is subjected to RNA interference.

[0009] The change of the ovarian maturation process and the vitellogenesis of the shrimp body after the silencing of the insulin receptor gene is observed.

[0010] The change of the expression of the vitellogenesis related genes in the ovary of the shrimp body after the gene silencing is detected.

[0011] The beneficial effects of the present application are that the animal experiments show that the expression of the insulin receptor gene can be obviously down-regulated, the effective silencing of the target gene in the animal body is realized, and the expression of multiple key genes and factors in the vitellogenesis and lipid transport of the female shrimp can be obviously affected, so as to affect the development and maturation process of the ovary. The target gene of the double-stranded molecular interference RNA based on the insulin receptor can obviously affect the development and maturation of the ovary of the animal. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Analysis of the interference efficiency of the insulin receptor.

[0013] Figure 2 Effect of the silencing of the insulin receptor on the ovarian development and vitellogenesis.

[0014] Figure 3 Effect of the silencing of the insulin receptor on the expression of the vitellogenesis related genes.

[0015] Figure 4 Effect of the silencing of the insulin receptor on the expression of the key genes of lipid transport. DETAILED DESCRIPTION

[0016] The following examples are used to illustrate the present application, but are not used to limit the protection scope of the present application.

[0017] The experimental methods involved in the following examples are all conventional methods in the art if no special instructions are given.

[0018] The reagent consumables and instruments involved in the following examples are all commercially available products if no special instructions are given.

[0019] The contents described in the following examples are part of the embodiments of the present application, but not all the embodiments.

[0020] All other embodiments obtained by those of ordinary skill in the art with benefit of the embodiments of the present application without creative effort shall fall within the scope of the present application.

[0021] Example 1 dsRNA synthesis of insulin receptor gene fragment and gene silencing

[0022] (1) dsRNA synthesis of insulin receptor gene: total RNA was extracted from ovary tissue using TRIzol method, dissolved in appropriate DEPC water, and stored in a -20 degree refrigerator. The first strand of insulin receptor gene cDNA was synthesized using a reverse transcription kit. According to the sequence of insulin hormone receptor, primers with enzyme cutting sites were designed in the open reading frame. The upstream primer sequence dsRNA-F is shown in SEQ ID No. 1: 5'-GC TCTAGAG ATTATAAGTACTTAGGGT-3' (underlined represents Xbal enzyme cutting site) and the downstream primer sequence is shown in SEQ ID No. 2 dsRNA-R: 5'-CG GGATCC TCATCTCCAGGTTTTCGTT-3' (underlined represents BamHI enzyme cutting site). dsRNA fragment preparation was performed by PCR amplification method, enzyme cutting, ligation, and transformation into double-stranded prepared plasmid. Then, the pET-T7 vector was used to prepare the target double-stranded RNA, and the concentration of the purified dsRNA was detected. The nucleic acid concentration was determined by OD260, and the double-stranded RNA concentration was calculated. The RNA was stored at -20 degrees for use. Green fluorescent protein double-stranded samples were prepared as a control group.

[0023] (2) RNA interference of insulin receptor gene: the shrimps used in the experiment were taken from the Rongshimeng shrimp farm in Ningbo, Zhejiang Province, and were raised in a 27±2℃ aquaculture tank, with feeding in the morning and evening, and natural light. Healthy mature shrimps (body length 9-10 cm and body weight 18-20 g) were selected for interference. The experimental group was injected with 10 micrograms of target double-stranded RNA per shrimp, and the GFP control group was injected with 10 micrograms of GFP double-stranded RNA per shrimp. The interference experiment was set up with 10 shrimps in each group, and each group was injected twice, with a 10-day injection cycle. After the experiment, the reproductive system was collected and stored for use.

[0024] (3) Detection of interference efficiency of insulin receptor silencing: ovarian RNA samples of experimental animals were extracted for quantitative PCR to detect the interference efficiency. The expression of the target gene was detected using a fluorescent quantitative kit (SYBR Green RT PCR assay), and the GAPDH gene was used as an internal reference gene. The data of fluorescent quantitative PCR were analyzed by 2-ΔΔCt method, and plotted using Graph-Pad software.

[0025] In the animal experiment, double-stranded RNA was delivered to the female shrimp by injection, and the interference efficiency was detected by extracting RNA from the ovary tissue after 10 days of gene silencing. The results showed that compared with the gene expression level of insulin receptor in the control group, the mRNA level of insulin receptor gene in the experimental group decreased significantly, and the interference efficiency could reach about 70% (Figure 1), indicating that the insulin receptor successfully realized gene silencing.

[0026] Example 2 Insulin receptor expression silencing affects ovary development and yolk formation.

[0027] The ovary development process of shrimp and crab is a complex biological process, including oocyte development, oocyte maturation and follicle formation, etc. The ovary development cycle of Macrobrachium rosenbergii can be divided into juvenile shrimp period, oocyte proliferation period, development period, maturation period and ovulation period, etc. Among them, during the ovary development stage, the oocytes in the primary follicle gradually mature, and the follicle cells also increase. During the process of oocyte maturation, oocytes continue to synthesize and accumulate a large amount of nutrients, and form one or more follicles. Therefore, the shrimps in the ovary development period were selected to carry out animal experiments of dsRNA interference injection of insulin receptor, and the function of insulin receptor and its application in female reproduction were analyzed.

[0028] The ovary and control group ovary tissue of insulin receptor gene silenced animals were dissected to observe the appearance of the ovary tissue, the histological section and hematoxylin-eosin (HE) staining of the ovary tissue. First, as shown in Figure Figure 2 -A, the ovary of the experimental shrimp was full and showed obvious swelling and wide strip shape. The ovary was orange yellow, and the dorsal part had brown pigment deposited in flaky shape, which was in the ovary development period. Subsequently, each sample was fixed with 4% paraformaldehyde, dehydrated with ethanol gradient, transparent in xylene, embedded with paraffin, and tissue sections of 5 microns were made with a paraffin sectioning machine. Hematoxylin-eosin reagent was used for staining of the tissue cells. Generally, various oocytes at different stages are distributed in the peripheral region of the ovary, and are called oogenic zone (OZ), previtellogenic zone (PZ), vitellogenic zone (VZ) and maturation zone (MZ) from the center to the outside. From the histological point of view, the oocytes in the rapid development period of the ovary migrate radially to the periphery of the ovary.

[0029] As shown in Figure Figure 2In the GFP control group, the ovaries were in the rapid development stage. The results of tissue section showed that the ovaries were subdivided into multiple ring-like regions, with the core region in the center, the OZ region and the PV region in the outer peripheral region were relatively narrow, and the VZ region rapidly expanded, with a large number of primary late vitellogenic oocytes (Oc3) in the outer peripheral region. In the control group, the Oc3 cell volume increased, the nucleus contained darkly colored nuclear chromatin, and the cytoplasm was full of yolk and secretory granules, as well as a small amount of lipid droplets. Compared with the control group, in the ovary of the female shrimp with insulin receptor silenced, the number of oogonia, previtellogenic oocytes (Oc1) and early vitellogenic oocytes (Oc2) near the central core was less; and there was a higher proportion of abnormal Oc3 cells with unclear cell boundaries in the VZ region (Figure 2-B, experimental group). These abnormal Oc3 oocytes in the insulin receptor gene knockdown group showed broken cell boundaries and acidophilic cytoplasm overflow. This indicates that the silencing of the insulin receptor gene leads to the destruction of the cell structure and the disorder of the yolk granule distribution in Oc3. That is, in vivo, the knockdown of the insulin receptor will affect the proliferation of oogonia and various oocytes in the ovary, especially the abnormal development of Oc3 cells. In summary, the normal expression of the insulin receptor is involved in the maintenance or promotion of oogonia proliferation and the accumulation of yolk substances during the Oc3 stage of the ovarian development process. The ovarian development process of shrimp and crab animals is affected by many factors, and understanding the mechanism of ovarian development and maturation has important guiding significance for healthy breeding and breeding.

[0030] Example 3 Effect of silencing of the insulin receptor on the expression of yolk formation-related genes

[0031] The ovarian development of animals includes oogonia stage, previtellogenic stage and vitellogenic stage. During the development of oocytes, vitellogenesis is a key step for the accumulation of proteins, fats and other nutrients. In terms of protein transport and accumulation, vitellogenin (Vg) is the precursor of yolk protein, and exogenous Vg binds to the vitellogenin receptor (VgR) on the plasma membrane of oocytes to form a Vg / VgR complex. After endocytosis into the cytoplasm, Vg is processed into mature yolk protein, and VgR is recruited to the cell membrane through tubules / bubbles. During oocyte maturation, oocytes synthesize and accumulate a large amount of nutrients, and form one or more follicles.

[0032] In the present application, the ovaries are obtained from the female prawns with insulin receptor gene silencing, and each group of 3 samples is mixed. Total RNA is extracted by column Trizol total RNA isolation kit, and transcriptome sequencing is performed by Illumina HiSeq method. The transcriptome data is removed by linker sequence, empty load 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 and defined as the significantly differentially expressed genes of the transcriptome. In the ovary transcriptome, the expression changes of genes and regulatory factors related to fat and protein transport and accumulation in the yolk formation process are focused on. The results show that after the insulin receptor gene silencing, several regulatory factors in the process of ovary maturation and yolk formation show a significant upward trend, including but not limited to low-density lipoprotein receptor (Low-density lipoprotein receptor, LDLR), lipocalin, fatty acid hydroxylase (Fatty acid hydroxylases, FAH), peptidase (Peptidase) and significantly down-regulated trypsin (Trypsin) and the like.

[0033] Low-density lipoprotein receptor LDLR is a protein receptor located on the cell membrane, mainly involved in the uptake and metabolism of low-density lipoprotein (LDL) in the body. LDLR is combined with the protein on the LDL particle through its domain on the cell membrane surface, so as to realize the uptake of LDL. Once LDL is combined with LDLR, LDLR will be further internalized into the cell, and LDL particles will be transported to the endoplasmic reticulum through endocytosis. In the endoplasmic reticulum, cholesterol will be released for intracellular cholesterol utilization and metabolism. The function of LDLR is regulated by various factors, including the regulation of gene expression level, the activation of transcription factor and the influence of waste decomposition pathway.

[0034] Lipocalin is a kind of protein involved in the formation and stability of liposome. Lipocalin exists on the surface of liposome, and can regulate the structure and function of liposome by interacting with the lipids in the liposome. Lipocalin plays an important physiological function in the organism, including regulating lipid metabolism, carrying and delivering bioactive substances, etc.

[0035] Fatty acid hydroxylase FAH is a kind of enzyme involved in fatty acid metabolism, which is involved in cholesterol synthesis pathway, fatty acid beta oxidation, lipid regulation of cell membrane and signal transduction process.

[0036] Peptidase is a kind of enzyme that can hydrolyze peptide bond, which is involved in the degradation and metabolism of intracellular and extracellular polypeptides. Peptidase is involved in protein degradation and synthesis, and maintains the balance of amino acids in cells.

[0037] Trypsin is an enzyme that cleaves peptide bonds and primarily hydrolyzes proteins. Trypsin breaks down proteins into smaller peptides and amino acids for absorption and utilization by the body. The activity of trypsin is influenced by various regulatory factors, including neural regulation, hormonal regulation, and endogenous inhibitors.

[0038] In summary, after insulin receptor gene silencing, several regulatory factors involved in ovarian maturation and vitellogenesis, such as low-density lipoprotein receptor, liposome protein, and fatty acid hydroxylase related to lipid metabolism, showed a significant upregulation trend. Protein metabolism-related enzymes exhibited diverse expression changes; for example, peptidase transcript expression was significantly upregulated, while trypsin transcript expression was significantly downregulated. These results indicate that insulin gene silencing can inhibit or delay lipid metabolism in the ovary and interfere with protein metabolism, suggesting that the insulin receptor gene plays an important role in regulating (maintaining or promoting) ovarian maturation and vitellogenesis.

[0039] Example 4: The effect of insulin receptor silencing on the expression of key lipid transport genes In oviparous organisms, oocyte maturation depends on the massive production of vitellogenin, a precursor protein. Vitellogenin (Vg) is absorbed by the developing oocyte through receptor-mediated endocytosis, a necessary process for successful reproduction. This invention investigates insulin receptor gene silencing in mature animals. Reproductive system tissues from female shrimp after gene silencing were collected to examine changes in the gene expression levels of key lipid transport genes during ovarian maturation and vitellogenesis, including vitellogenin receptor (VgR), lipoprotein (LSD), and apolipoprotein D (ApD), aiming to elucidate the role of insulin receptor in lipid transport during ovarian development. The expression of target genes was detected using a quantitative real-time PCR assay (SYBR Green RT-PCR), with the GAPDH gene used as an internal control. The data from the quantitative real-time PCR were analyzed using the 2-ΔΔCt method and plotted using GraphPad software.

[0040] In 2012, scholars found that the specific localization of the vitellogenin receptor VgR protein in the oocyte membrane of Macrobrachium rosenbergii, and the expression of VgR in oocytes increased during vitellogenesis. Therefore, the study of Vg and VgR is helpful to understand the maturation process of oocytes in crustaceans, especially to clarify the absorption mechanism of Vg through VgR in RME. Using the method of fluorescent quantitative PCR, the expression level of vitellogenin receptor gene in the experimental group and the control group after insulin receptor gene silencing was detected under the amplification of forward primer VgR-qF: 5'-CAACGGGGATCTCGACTG-3' and reverse primer VgR-qR: 5'-CATCTTGAGGTCTCGCAGTG-3' of vitellogenin receptor. The results showed that after the insulin receptor gene was silenced, the transcription level of vitellogenin receptor VgR slightly increased Figure 4 ).

[0041] Lipid droplet protein LSD regulates the storage and utilization of intracellular lipids, and the utilization of oocyte lipids will affect embryonic development. As early as 2010, scholars have studied the size and localization of a lipid droplet protein perilipin-2 in mouse oocytes during the periovulatory period, and determined it as a potential biomarker for the lipid content of oocytes, and showed that there were significant changes in lipid utilization during oocyte maturation. Using the method of fluorescent quantitative PCR, the expression level of lipid droplet protein gene in the experimental group and the control group after insulin receptor gene silencing was detected under the amplification of forward primer LSD-qF: 5'-TCGCCAGAGCAGTTCGT-3' and reverse primer LSD-qR: 5'-CGGTTGTCAGGTCTTCCAC-3' of lipid droplet protein transcript. The results showed that after the insulin receptor gene was silenced, the transcription level of lipid droplet protein increased to 149% of the control group, and its expression level increased significantly Figure 4 ).

[0042] Apolipoprotein is an important component of plasma lipoprotein, which is a kind of protein combined with lipids in blood and lymph. Apolipoprotein can combine with lipids such as cholesterol and triglyceride to form a lipid protein complex, thereby helping the transport and metabolism of lipids in the body. Apolipoprotein endows lipids with a soluble form, and plays a role in promoting lipid transport, regulating enzyme activity, and guiding the combination of plasma lipoprotein with cell surface receptors in plasma lipoprotein metabolism. It can combine and transport blood lipids to all tissues of the body for metabolism and utilization, and is a group of extremely active plasma proteins in function. Apolipoprotein D is a 25-30 kDa glycosylated protein belonging to the lipocalin superfamily, which can be used as a transporter for several small hydrophobic molecules and plays an important role in parent ovary lipid accumulation and offspring lipid balance. The expression level of apolipoprotein D gene in the experimental group and the control group after insulin receptor gene silencing is detected by amplification of the forward primer of apolipoprotein D transcript, APD-qF: 5'-GGAGCCTAAACTCAGCAATACG-3' as shown in SEQ ID No. 5, and the reverse primer APD-qR: 5'-CTTAGCCTTCTTGGGGTCC-3' as shown in SEQ ID No. 6. The results show that after the insulin receptor gene is silenced, the transcription level of apolipoprotein D increases to 615% of the control group, and the expression amount significantly increases Figure 4 ).

[0043] The insulin receptor in the application is an ovary-specific receptor gene, and its expression amount significantly increases with the development and maturation of the ovary, so it is speculated to be closely related to yolk production and lipid transport in the development of the ovary. The results of the application show that after the insulin receptor gene is silenced, the key factors of lipid transport in the process of ovary maturation and yolk production show a significant upward trend, including but not limited to the transcript expression levels of vitellogenin receptor VgR gene, lipid droplet protein LSD and apolipoprotein APD Figure 4 ). The above results show that the silencing of the insulin gene accelerates the expression of lipid metabolism-related genes and regulatory factors, but disturbs the process of lipid metabolism and the transport and accumulation of vitellogenin in the ovary, and it is speculated that the insulin receptor gene plays an important role in maintaining the balance of lipid transport and metabolism in the process of ovary maturation and yolk production.

[0044] The above examples show that the insulin receptor can interfere with the development of oocytes and the yolk production process by affecting the lipid storage and transport and protein metabolism process in the ovary, thereby affecting the development and maturation of the ovary in the shrimp body, which is essential for reproductive development, and has application potential in regulating the development and maturation of female gonads.

[0045] The above examples are only used for illustrating the technical solutions of the present application, and are not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some or all of the technical features thereof can be replaced equivalently, and these modifications or replacements do not cause the nature of the corresponding technical solutions to deviate from the scope of the technical solutions of the present application. Therefore, the extended obvious changes or modifications are still within the protection scope of the present application.

Claims

1. A method of modulating female reproduction based on insulin receptor, characterized by, 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.

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.

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.

4. The transcript or translation product of the insulin receptor according to claims 1 to 4, characterized in that, The receptor gene molecule includes a sense strand and an antisense strand complementary to the sense strand; and 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 insulin receptor regulates female reproduction by affecting the expression of genes and factors related to lipid metabolism and protein metabolism, thereby regulating the process of yolk formation in the ovary, lipid and protein metabolism, and thus regulating the development and maturation of the female gonad.

6. The method of claim 1, wherein, The insulin receptor regulates female reproduction by affecting the development of multiple oocytes in the ovary, especially the development of Oc3 oocytes, thereby regulating tissue morphology and cell proliferation in the ovary, and thus regulating the development and maturation of the female gonad.

7. The method of claim 1, wherein, The insulin receptor regulates female reproduction by affecting the expression of genes and factors related to yolk formation and lipid transport, thereby regulating the process of yolk formation in the ovary and lipid metabolism, and thus regulating the development and maturation of the female gonad.

8. The method according to claims 1 to 7, characterized in that, The female reproduction regulation includes the inhibition of the function of the gene or its transcription product or translation product, which in vivo includes double-stranded RNA or vectors for inhibiting the expression of the gene using RNA interference, chromosomes with functional defects of the gene, or inhibitors 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.

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