Application of ACOT12 gene in regulating proliferation of bovine sertoli cells

Abstract

The application provides application of an ACOT12 gene in regulation of proliferation of bovine testis Sertoli cells and belongs to the technical field of biotechnology.The application observes the proliferation of bovine testis Sertoli cells by interfering with ACOT12 gene expression.The results show that interference with the ACOT12 gene can significantly inhibit the proliferation of bovine testis Sertoli cells, which indicates that the ACOT12 gene has a positive regulation effect on the proliferation of bovine testis Sertoli cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to application of ACOT12 gene in regulating proliferation of bovine testis Sertoli cells. BACKGROUND

[0002] In the beef and dairy cattle breeding industry, most of the cattle are single-birth animals (the twinning rate is usually less than 3%), and the gestation period is relatively long (about 280 days). Therefore, the reproductive performance of the cattle herd becomes one of the most important economic traits of beef and dairy cattle farms. With the wide application of artificial insemination technology, a single bull can provide breeding services for more than ten thousand cows per year, and an excellent breeding bull has a greater contribution to the genetic improvement of the cattle population, far more than that of a cow. Therefore, cultivating excellent high-breeding bulls is the core of improving the overall reproductive performance of the cattle population and is an important factor restricting the further development of the industry. However, to fully improve the reproductive capacity of the breeding bull, it is necessary to deeply understand the process of bovine spermatogenesis at the molecular level.

[0003] At present, some genes closely related to the fertility of cattle have been found. For example, five markers on the X chromosome are significantly associated with male fertility. These markers include the FAM9B, TBL1X and PIH1D3 genes, which are closely related to testosterone concentration, spermatogenesis and sperm motility. In addition, miR-34c in bovine testis Sertoli cells can target the AXL gene, which may regulate the proliferation and apoptosis of Sertoli cells, and then affect the transcription level of male reproductive genes, thereby participating in the process of spermatogenesis. Screening and identifying key genes related to bovine spermatogenesis are of great significance for improving the reproductive performance of bulls and reasonably guiding cattle breeding.

[0004] Spermatogenesis is a complex and strictly regulated biological process, mainly occurring in the seminiferous tubules of the testis. Sertoli cells are important somatic cells that directly contact with germ cells at all stages in the seminiferous tubules, and they play a crucial role in the initiation of spermatogenesis and maintenance in adulthood. Regulating the proliferation of bovine testis Sertoli cells can improve the reproductive capacity and reproductive health of cattle. SUMMARY

[0005] The application aims to provide the application of ACOT12 gene in regulating the proliferation of bovine testis Sertoli cells, and the two present a positive regulation mode.

[0006] The technical scheme of the application is as follows:

[0007] This invention provides the application of the ACOT12 gene as a target in regulating the proliferation of bovine testicular Sertoli cells. The ACOT12 gene includes the bovine ACOT12 gene with NCBI accession number NC_037334.1 or a homologous gene in bovine tissue. The regulation is positive regulation; that is, promoting ACOT12 gene expression promotes bovine testicular Sertoli cell proliferation, and inhibiting ACOT12 gene expression inhibits bovine testicular Sertoli cell proliferation. Through this method, the proliferation of bovine testicular Sertoli cells can be regulated, thereby improving bovine reproductive capacity and reproductive health.

[0008] This invention provides the application of a reagent that regulates ACOT12 gene expression in the preparation of products that affect the proliferation of bovine testicular supporting cells.

[0009] In one embodiment, the reagent includes a reagent that promotes ACOT12 gene expression and a reagent that inhibits ACOT12 gene expression. The reagent that promotes ACOT12 gene expression promotes the proliferation of bovine testicular Sertoli cells, and the reagent that inhibits ACOT12 gene expression inhibits the proliferation of bovine testicular Sertoli cells.

[0010] As one embodiment, the reagent includes a recombinant vector containing the ACOT12 gene or a recombinant vector that interferes with the expression of the ACOT12 gene.

[0011] This invention provides a reagent for regulating the proliferation of bovine testicular supporting cells, the reagent comprising at least one of the following RNAs or gene derivatives thereof targeting the ACOT12 gene: siRNA, shRNA, and sgRNA.

[0012] Furthermore, the gene-derived products include at least one of the following products: an expression vector containing siRNA, a CRISPER-Cas gene editing system containing sgRNA, and a recombinant expression vector containing shRNA.

[0013] Preferably, the nucleotide sequence of the siRNA is shown in SEQ ID NO:1.

[0014] The present invention also provides a method for promoting / inhibiting the proliferation of bovine testicular Sertoli cells by transfecting bovine testicular Sertoli cells with a reagent that promotes / inhibits ACOT12 gene expression.

[0015] Preferably, the reagent comprises at least one of the following RNAs or gene derivatives thereof targeting the ACOT12 gene: siRNA, shRNA, and sgRNA. The gene derivatives include at least one of the following products: an expression vector containing siRNA, a CRISPER-Cas gene editing system containing sgRNA, and a recombinant expression vector containing shRNA.

[0016] As an implementation, siRNA targeting the ACOT12 gene is transfected into bovine testicular Sertoli cells to inhibit the proliferation of bovine testicular Sertoli cells. Preferably, the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1. The present application evaluates the proliferation level of bovine testicular Sertoli cells by detecting the expression level of the proliferation marker genes CCNB1 and / or CDK1 of bovine testicular Sertoli cells, or detecting the cell proliferation condition by the Edu method.

[0017] Advantages:

[0018] The present application observes the proliferation condition of bovine testicular Sertoli cells by interfering with the expression of the ACOT12 gene. The mRNA level of the proliferation marker genes CCNB1 and CDK1 is detected by RT-qPCR, and the cell proliferation condition is detected by the Edu method to evaluate the cell proliferation level. The results show that interfering with the ACOT12 gene can significantly inhibit the proliferation of bovine testicular Sertoli cells, suggesting that the ACOT12 gene has a positive regulatory effect on the proliferation of bovine testicular Sertoli cells. The present application takes the ACOT12 gene as a target to regulate the proliferation of bovine testicular Sertoli cells, providing a new idea for studying and regulating the reproductive health of livestock. Through this technology, the reproductive capacity can be optimized in the process of animal breeding, and a new gene regulation tool is provided for improving the reproductive health of bovine. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 To isolate bovine primary testicular Sertoli cells;

[0020] Figure 2 To detect the expression of marker genes in bovine primary testicular Sertoli cells by RT-PCR;

[0021] Figure 3 To detect the interference effect of ACOT12 siRNA by RT-qPCR (ns P>0.05, **P<0.01, t-test);

[0022] Figure 4 To detect the mRNA level of the proliferation marker genes CCNB1 and CDK1 after interfering with ACOT12 by RT-qPCR (*P<0.05, t-test).

[0023] Figure 5 To detect the cell proliferation condition by the Edu method after interfering with ACOT12; in A, blue represents the cell nucleus and red represents the proliferating cells; in B, ****P<0.0001, t-test. DETAILED DESCRIPTION

[0024] In specific embodiments, the present application selects RNA interference (RNAi) technology, targets ACOT12 gene by small interfering RNA (siRNA), transfects the siRNA into bovine testicular Sertoli cells, specifically inhibits the transcription or translation of ACOT12 gene, thereby reducing the expression of ACOT12 in bovine testicular Sertoli cells, and observing the proliferation of bovine testicular Sertoli cells. The mRNA level of bovine testicular Sertoli cell proliferation marker gene CCNB1 and CDK1 is detected by RT-qPCR, and the cell proliferation is detected by Edu method, the proliferation level of bovine testicular Sertoli cells is evaluated, and the expression inhibition effect of ACOT12 gene is verified. The results show that the inhibition of ACOT12 gene expression inhibits the proliferation of bovine testicular Sertoli cells, indicating that ACOT12 gene has a positive regulatory effect on the proliferation of bovine testicular Sertoli cells.

[0025] In view of the positive correlation between the expression level of ACOT12 gene and the proliferation of bovine testicular Sertoli cells, the present application provides the application of the ACOT12 gene as a target in regulating the proliferation of bovine testicular Sertoli cells, which is positive regulation. Specifically, the application of ACOT12 gene as an overexpression target in promoting the proliferation of bovine testicular Sertoli cells, and the application of ACOT12 gene as a knockout or inhibition target in inhibiting the proliferation of bovine testicular Sertoli cells.

[0026] The present application provides the application of a reagent for regulating the expression of ACOT12 gene in preparing a product affecting the proliferation of bovine testicular Sertoli cells. As an embodiment, the reagent includes a reagent for promoting the expression of ACOT12 gene and a reagent for inhibiting the expression of ACOT12 gene, the reagent for promoting the expression of ACOT12 gene promotes the proliferation of bovine testicular Sertoli cells, and the reagent for inhibiting the expression of ACOT12 gene inhibits the proliferation of bovine testicular Sertoli cells.

[0027] In the present application, the reagent preferably includes a recombinant vector containing ACOT12 gene or a recombinant vector interfering with the expression of ACOT12 gene. The recombinant vector containing ACOT12 gene can promote the up-regulated expression of proliferation marker genes CCNB1 and / or CDK1 through positive regulation, promote the proliferation of bovine testicular Sertoli cells, and thus optimize the reproductive ability of breeding cattle. The recombinant vector interfering with the expression of ACOT12 gene inhibits the expression of ACOT12 gene through siRNA, inhibits the expression of proliferation marker genes CCNB1 and / or CDK1, and inhibits the proliferation of bovine testicular Sertoli cells, thereby reducing the reproductive ability of breeding cattle. As an embodiment, the recombinant vector interfering with the expression of ACOT12 gene contains siRNA with a nucleotide sequence as shown in SEQ ID NO: 1.

[0028] The present application provides an agent for regulating the proliferation of bovine testicular Sertoli cells, which comprises at least one of the following RNA or gene-derived products targeting the ACOT12 gene: siRNA, shRNA and sgRNA.

[0029] In the present application, the gene-derived products include an expression vector comprising siRNA, a CRISPER-Cas gene editing system comprising sgRNA, and a recombinant expression vector comprising shRNA. The present application does not have special restrictions on the preparation method of the gene-derived products, and the construction method of the recombinant expression vector or the CRISPER-Cas gene editing system known in the art can be used. In the embodiments of the present application, the effect of inhibiting the expression of the ACOT12 gene on the proliferation of bovine testicular Sertoli cells is illustrated by taking siRNA as an example. The siRNA is a nucleotide sequence as shown in SEQ ID NO: 1.

[0030] The present application provides a method for promoting / inhibiting the proliferation of bovine testicular Sertoli cells, which comprises transfecting bovine testicular Sertoli cells with an agent for promoting / inhibiting the expression of the ACOT12 gene.

[0031] In the present application, the agent for promoting / inhibiting the expression of the ACOT12 gene is the same as described above and will not be repeated here. The present application does not have special restrictions on the transfection method, and the conventional transfection method in the art can be used, such as liposome transfection. As an embodiment, the siRNA targeting the ACOT12 gene is transfected into bovine testicular Sertoli cells, the expression level of the proliferation marker gene CCNB1 and / or CDK1 of bovine testicular Sertoli cells is detected, or the cell proliferation is detected by the Edu method, the proliferation level of bovine testicular Sertoli cells is evaluated, and the proliferation of bovine testicular Sertoli cells is inhibited.

[0032] The technical solutions of the present application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0033] Example 1

[0034] Isolation and culture of bovine primary testicular Sertoli cells

[0035] Newborn calf testis tissue was collected and stored in PBS buffer (with penicillin and streptomycin). The surface of the tissue was sterilized by soaking in 75% ethanol for 30 seconds, and then washed with PBS three times. The tunica albuginea was cut open with scissors, and a number of testis tissues weighing about 1 g were cut into pieces in a 4 mL centrifuge tube using a cross-cut method. PBS was added and mixed well, and then centrifuged at 1500 rpm for 5 min. The PBS was discarded, and collagenase type IV was added at a concentration of 1.0 mg / mL for digestion. The tube was placed in a cell culture incubator for 30-40 min (shaken up and down every 10 min). After digestion, the tube was centrifuged at 1500 rpm for 5 min, and the collagenase was discarded. PBS was added and mixed well, and then centrifuged at 1500 rpm for 5 min. The PBS was discarded, and the cell pellet was digested with 2.5% trypsin in a cell culture incubator for 10 min. Cell culture medium containing 10% FBS was added to stop the digestion, and the filtrate was collected by filtering through a 120-mesh steel sieve. The filtrate was centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in DMEM / F12 cell culture medium containing 10% FBS, and inoculated in a 60 mm dish. After 6 h, the floating cells were gently aspirated and the culture medium was replaced, and the cultured cells were separated to obtain bovine primary testicular Sertoli cells, as shown in FIG. 1. Figure 1 .

[0036] Example 2

[0037] Identification of bovine primary testicular Sertoli cells

[0038] The total RNA of the bovine primary testicular Sertoli cells obtained by culture in Example 1 was extracted according to the operating instructions of the Omega total RNA extraction kit (R6834-01), and the RNA was reverse transcribed into first-strand cDNA using a reverse transcription kit (Thermo Scientific, 00238582).

[0039] An appropriate amount of cDNA was used as a template, and the bovine testicular Sertoli cell marker gene primers in Table 1 were used for detection, and PCR enzyme (NEB, E0555S) was used for amplification, and the PCR results were detected by gel electrophoresis.

[0040] The results showed that the bovine testicular Sertoli cell marker genes GDNF, SCF, and WT1 were all expressed Figure 2 , indicating that the bovine primary testicular Sertoli cells were successfully isolated and could be used for subsequent experiments.

[0041] Table 1 bovine testicular Sertoli cell marker gene primers

[0042]

[0043] Example 3

[0044] RT-qPCR detection of ACOT12 siRNA interference effect

[0045] (1) Design 3 siRNAs and a negative control (NC) using the mRNA sequence of the bovine ACOT12 gene in the NCBI website (Table 2).

[0046] Table 2 ACOT12 and NC siRNAs

[0047]

[0048] (2) When the bovine primary testicular Sertoli cells reached 60-70% confluence, transfect the siRNAs according to the operating instructions of the transfection reagent jetPRIME (Polyplus, 101000046).

[0049] (3) After 24 hours of transfection, extract the total RNA of the cells according to the operating instructions of the total RNA extraction kit of the Omega company (R6834-01), and reverse transcribe the RNA into first-strand cDNA using the reverse transcription kit (Thermo Scientific, 00238582).

[0050] (4) After 5-fold dilution of the cDNA with RNase Free H2O, perform qPCR using the Bio-Rad CFX96 fluorescence quantitative PCR instrument to detect the inhibitory effect of different siRNAs on the expression of the ACOT12 gene. At the same time, set the internal reference gene, and set 3 replicate wells for each sample. The amplification reaction conditions of the three-step method are as follows: pre-denaturation at 95°C for 4 min, cycle reaction at 95°C for 5 sec, annealing at 60°C for 20 sec, extension at 72°C for 20 sec, and collection of fluorescence signal, a total of 40 cycles, and finally extension at 72°C for 2 min. Increase the temperature from 65°C to 95°C at a rate of 0.5°C / 5 sec to detect the melting curve. The relative expression of the target gene is calculated according to The system and primers used are shown in Tables 3 and 4.

[0051] Table 3 SYBR amplification system

[0052]

[0053] Table 4 RT-qPCR primers

[0054]

[0055] The results show that siRNA-1 can significantly (P<0.01) inhibit the mRNA expression of ACOT12 in bovine primary testicular Sertoli cells Figure 3 , and can be used for subsequent experiments.

[0056] Example 4

[0057] Detection of bovine primary testicular Sertoli cell proliferation

[0058] The mRNA levels of proliferation marker genes CCNB1 and CDK1 were detected by RT-qPCR and the cell proliferation was detected by Edu experiment to evaluate the effect of interfering ACOT12 gene expression on the proliferation of bovine testicular Sertoli cells after transfection of siRNA-1 in bovine primary testicular Sertoli cells. The specific operation of RT-qPCR refers to Example 3, and the primers of proliferation marker genes are shown in Table 5. The Edu experiment was carried out according to the operating instructions of the EdU Cell Proliferation Imaging Analysis Kit (KTA2031) of Abbkine company.

[0059] Table 5 RT-qPCR primers of proliferation marker genes

[0060]

[0061]

[0062] Figure 4 The results show that interfering the expression of ACOT12 can significantly reduce the mRNA expression of proliferation marker genes CCNB1 and CDK1. Figure 5 In the NC group, a large number of blue nuclei can be seen in DAPI staining, and a few red fluorescent spots can be seen in Edu staining, indicating that some cells are undergoing DNA replication. In the siRNA-1 group, a large number of blue nuclei can also be seen, but the number of red positive cells in Edu staining is significantly reduced, indicating that cell proliferation is reduced after siRNA-1 treatment. It is proved that interfering the expression of ACOT12 gene can inhibit the proliferation of bovine testicular Sertoli cells.

[0063] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Application of the ACOT12 gene as a target point in regulating the proliferation of bovine testicular Sertoli cells for non-disease treatment purposes, characterized in that, Inhibition of ACOT12 gene expression can inhibit the proliferation of bovine testicular Sertoli cells.

2. Use of an agent for inhibiting the expression of ACOT12 gene for non-disease treatment purposes in the preparation of a product for inhibiting the proliferation of bovine testicular Sertoli cells.

3. Use according to claim 2, characterized in that, The agent comprises a recombinant vector interfering with the expression of the ACOT12 gene.

4. Use according to claim 2, characterized in that, The agent comprises at least one of the following RNA or gene-derived products targeting the ACOT12 gene: siRNA, shRNA and sgRNA.

5. Use according to claim 4, characterized in that, The gene-derived products comprise at least one of the following products: an expression vector comprising siRNA, a CRISPER-Cas gene editing system comprising sgRNA, and a recombinant expression vector comprising shRNA.

6. Use according to claim 4 or 5, characterized in that, The nucleotide sequence of the siRNA is shown in SEQ ID NO:

1.

7. A method of inhibiting proliferation of bovine Sertoli cells for non-disease therapeutic purposes, characterized in that, Transfect bovine testicular Sertoli cells with an agent for inhibiting the expression of ACOT12 gene.

8. The method of claim 7, wherein, Transfect bovine testicular Sertoli cells with siRNA targeting the ACOT12 gene to inhibit the proliferation of bovine testicular Sertoli cells.

9. The method of claim 7, wherein, Detect the expression level of the proliferation marker gene CCNB1 and / or CDK1 of bovine testicular Sertoli cells, or detect the cell proliferation by Edu method, to evaluate the proliferation level of bovine testicular Sertoli cells.

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