Expression and Application of a Female-Specific Protein in Cynoglossus semilaevis

Abstract

The present invention provides the use of the pitpβ_w protein in regulating the expression of growth-related genes and sex differentiation-related genes in male Cynoglossus semilaevis; the present invention successfully regulates growth-related genes and sex differentiation-related genes in male Cynoglossus semilaevis by using the pitpβ_w protein, providing a good research basis for studying the expression patterns and phenotypes of related genes, as well as for the growth and differentiation of male Cynoglossus semilaevis, and having broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an expression and application method of a female-specific protein of Cynoglossus semilaevis. Background Art

[0002] Cynoglossus semilaevis is a kind of flatfish, belonging to the family Cynoglossidae and the genus Cynoglossus, and is an important marine cultured fish in Northeast Asia. Cynoglossus semilaevis is deeply loved by consumers for its delicious meat and smooth taste, and has become a precious variety among marine cultured fish. There are obvious differences in growth between female and male Cynoglossus semilaevis. The growth of female fish can be 2-4 times larger than that of male fish. Therefore, increasing the proportion of female fish in aquaculture is of great significance for improving production. Cynoglossus semilaevis belongs to the ZW sex determination type, that is, theoretically, the ZW genotype will develop into female fish, and the ZZ type will develop into male fish. However, in actual production, a considerable number of ZW individuals will sex-reverse into "pseudo-male fish", and the growth of pseudo-male fish is similar to that of male fish, which greatly restricts the development of the tongue sole industry.

[0003] Through transcriptome analysis, we screened a gene module closely related to female differentiation. Its core network consists of 50 genes, most of which are located on the W chromosome. Further functional studies were carried out on a gene pitpβ_w located on the W chromosome, and it was found that it is not expressed in male fish, but specifically expressed only in female fish. It is mainly located in oocytes of stages I-III in the ovary. RNA interference in the Cynoglossus semilaevis ovarian cell line will cause a decrease in sox9a (Xu et al., 2021; Sun et al., 2022). The above results indicate that pitpβ_w plays an important role in female differentiation, but its role in vivo is not yet clear. Whether its expression in male fish has an impact on sex differentiation has important application significance. Expressing recombinant proteins and injecting them to study the expression patterns and phenotypes of related genes is an effective way to study the relationship between sex differentiation and growth. Summary of the Invention

[0004] The present invention provides the expression and application of a female-specific protein of Cynoglossus semilaevis. Specifically, the female-specific protein of Cynoglossus semilaevis is the pitpβ_w protein. Expressing it in male Cynoglossus semilaevis (male fish) can regulate the gene expression in male Cynoglossus semilaevis (male fish).

[0005] In the present invention, the pitpβ_w protein is phosphatidylinositol transfer protein; in one embodiment, the gene sequence of the pitpβ_w protein of the present invention is shown in SEQ ID No.1, and the amino acid sequence of the encoded protein is shown in SEQ ID No.2. The pitpβ_w protein is unique to female Cynoglossus semilaevis and is not found in male Cynoglossus semilaevis.

[0006] In one embodiment, the present invention provides the use of the pitpβ_w protein in regulating the expression levels of growth-related genes and / or sex differentiation-related genes in male Cynoglossus semilaevis.

[0007] In a preferred embodiment, introducing the pitpβ_w protein into male Cynoglossus semilaevis can reduce the expression levels of growth-related genes and / or sex differentiation-related genes.

[0008] The reduction in the expression levels of growth-related genes and sex differentiation-related genes means that, compared with the control without introducing the pitpβ_w protein, introducing the pitpβ_w protein can reduce the expression levels of growth-related genes and sex differentiation-related genes in male Cynoglossus semilaevis; for example, compared with the control, the gene expression level is reduced by at least 50%, 60%, 70% or 80%.

[0009] In one embodiment, the "introduction of the pitpβ_w protein" can be achieved by protein injection, injecting the pitpβ_w protein into male Cynoglossus semilaevis. In other embodiments, the "introduction of the pitpβ_w protein" can also be achieved by expressing or overexpressing the pitpβ_w protein in male Cynoglossus semilaevis.

[0010] In one embodiment, the growth-related gene is igf1, and the sex differentiation-related gene is cyp19a; preferably, the nucleic acid sequence of igf1 is shown in SEQ ID No.3, and the nucleic acid sequence of cyp19a is shown in SEQ ID No.4.

[0011] On the other hand, the present invention also provides a method for regulating the gene expression level in male Cynoglossus semilaevis (male fish), the method comprising the step of introducing the pitpβ_w protein into male Cynoglossus semilaevis.

[0012] In one embodiment, the "introduction of the pitpβ_w protein" can be achieved by protein injection, injecting the pitpβ_w protein into male Cynoglossus semilaevis. In other embodiments, the "introduction of the pitpβ_w protein" can also be achieved by expressing or overexpressing the pitpβ_w protein in male Cynoglossus semilaevis.

[0013] In one embodiment, the method for regulating gene expression in male half-smooth tongue sole (male fish) is to reduce the expression levels of growth-related genes and / or sex differentiation-related genes in male half-smooth tongue sole.

[0014] In one embodiment, the growth-related gene is igf1, and the sex differentiation-related gene is cyp19a; preferably, the nucleic acid sequence of igf1 is as shown in SEQ ID No.3, and the nucleic acid sequence of cyp19a is as shown in SEQ ID No.4.

[0015] The present invention uses the pitpβ_w protein to successfully regulate growth-related genes and sex differentiation-related genes in male half-smooth tongue sole, providing a good research basis for studying the expression patterns and phenotypes of related genes, as well as for the growth and differentiation of male half-smooth tongue sole, and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 .Electrophoresis diagram of the expression and purification of Pitpβ_w recombinant protein.

[0017] Figure 2 .Effect of Pitpβ_w recombinant protein on the expression of growth-related gene igf1 and sex differentiation-related gene cyp19a in male half-smooth tongue sole (* indicates significant difference p<0.05). Embodiment

[0018] The following further describes the present invention in conjunction with embodiments. The following descriptions are only preferred embodiments of the present invention, and do not limit the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make equivalent changes to equivalent embodiments. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention's solution falls within the protection scope of the present invention.

[0019] In this embodiment, the gene sequence of pitpβ_w (phosphatidylinositol transfer protein) is as shown in SEQ ID No.1, and the amino acid sequence of the encoded protein is as shown in SEQ ID No.2.

[0020] According to the gene sequence of pitpβ_w, expression primers for pitpβ_w were designed, and a pET28a and Escherichia coli expression system were used for recombinant protein expression. The primers are shown in Table 1.

[0021] Table 1 Primers for protein expression

[0022]

[0023]

[0024] The steps for vector construction, protein expression, and purification are as follows: Extract ovarian RNA and reverse-transcribe it to prepare cDNA. Use the cDNA as a template to amplify the pitp_w gene. After double digestion with NcoI and HindIII, ligate it to the pET-28a vector. After ligation and transformation into Escherichia coli Dh5a, screen for positive plasmids, and then transform them into Escherichia coli BL21(DE3) and culture overnight at 37°C. Pick monoclonal colonies and inoculate them into 5 ml of liquid LB medium and culture overnight at 37°C. Pipette 0.5 ml of the bacterial solution and transfer it to 50 ml of liquid LB medium, and culture at 37°C for 2 - 3 h. When the OD 600 is in the range of 0.6 - 0.8, add 0.5 mM IPTG and induce expression overnight in a shaker at 25°C. Centrifuge to collect the bacteria, resuspend them in 10 ml of Tris-HCL buffer at pH 7.2, break them by sonication and then centrifuge. Take the supernatant and precipitate for SDS-PAGE electrophoresis detection. Pass the supernatant through BeyoGold TM Hig-tag Purification Resin for affinity purification to obtain relatively pure protein (as Figure 1 shown), measure the concentration and then perform injection. (Note: Kanamycin is added to both the plate and liquid media at a final concentration of 50 μg / ml).

[0025] Inject the purified recombinant target protein - phosphatidylinositol transfer protein into male half-smooth tongue sole (male fish) in vivo. The specific steps are as follows: Select half-smooth tongue sole 120 days after hatching. The gonad position can be clearly distinguished by ventral irradiation with a flashlight. Use a microinjector to inject the protein into the testis position. The injection is divided into a control group (BSA) and an experimental group (Pitpβ_w recombinant protein). The dose of the protein (concentration 29.79 μg / ml, inject 100 μl), and a total of 2 injections are performed (with an interval of 24 h). After 48 h, take the gonads for RNA extraction, reverse-transcribe, and then detect the growth-related gene insulin-like growth factor (igf1, NM_001294198.1) and the sex differentiation-related gene cyp19a (NM_001294242.1) by quantitative PCR. The nucleic acid sequence of igf1 is as shown in SEQ ID No.3, and the nucleic acid sequence of cyp19a is as shown in SEQ ID No.4; the detection primers for quantitative PCR are shown in Table 2.

[0026] Table 2 Detection primers for quantitative PCR

[0027]

[0028] In actual operation, after sex identification, 3-5 male fish were selected from the control group and the experimental group respectively for qPCR verification to detect the growth-related gene igf1 and the sex differentiation-related gene cyp19a, and it was found that the expression levels of both igf1 and cyp19a decreased significantly (as Figure 2 shown).

[0029] References:

[0030] Sun et al., pitpβ_w Encoding Phosphatidylinositol Transfer Protein Is Involved in Female Differentiation of Chinese Tongue Sole, Cynoglossus semilaevis. Frontiers in Genetics, 2022, doi: 10.3389 / fgene.2022.861763.

[0031] Xu et al., Transcriptomic analysis revealed gene expression profiles during the sex differentiation of Chinese tongue sole (Cynoglossus semilaevis). Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics, 2021, 40: 100919.

[0032] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings that have been published, and these changes are within the protection scope of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. Use of the pitpβ_w protein in regulating the expression levels of growth-related genes and / or sex differentiation-related genes in male Cynoglossus semilaevis; The amino acid sequence of the pitpβ_w protein is shown in SEQ ID No. 2; The growth-related gene is igf1, and the sex differentiation-related gene is cyp19a; The use is to overexpress the pitpβ_w protein in male Cynoglossus semilaevis to thereby reduce the expression levels of growth-related genes and / or sex differentiation-related genes.

2. A method for reducing the expression levels of growth-related genes and / or sex differentiation-related genes in male Cynoglossus semilaevis, the method comprising the step of overexpressing the pitpβ_w protein in male Cynoglossus semilaevis; The amino acid sequence of the pitpβ_w protein is shown in SEQ ID No. 2; The growth-related gene is igf1, and the sex differentiation-related gene is cyp19a.

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