Application of ACOT12 gene in regulation and control of bovine sertoli cell proliferation

Abstract

The invention provides application of an ACOT12 gene in regulation and control of bovine sertoli cell proliferation, and belongs to the technical field of biology. According to the invention, the proliferation condition of the bovine sertoli cells is observed by interfering the expression of the ACOT12 gene. Results show that the ACOT12 interference gene can significantly inhibit the proliferation of the bovine sertoli cells, which prompts that the ACOT12 gene has a positive regulation effect in the proliferation of the bovine sertoli cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of ACOT12 gene in regulating the proliferation of bovine testicular supporting cells. Background Art

[0002] In the beef and dairy cattle farming industry, most cattle are single-calf animals (twin calf rate is usually less than 3%), and the gestation period is long (about 280 days). Therefore, the reproductive performance of cattle has become one of the most important economic traits in beef and dairy farms. With the widespread application of artificial insemination technology, a single bull can provide breeding services for more than 10,000 cows each year, and excellent bulls have a greater contribution to the genetic improvement of cattle populations, far exceeding the role of cows. Therefore, breeding excellent high-breeding bulls is the core of improving the overall reproductive performance of cattle populations, and it is also an important factor restricting the further development of the industry. However, to fully improve the reproductive capacity of bulls, it is necessary to have a deep understanding of the spermatogenesis process of cattle at the molecular level.

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

[0004] Spermatogenesis is a complex and strictly regulated biological process that mainly occurs in the seminiferous tubules of the testis. Sertoli cells are important somatic cells in the seminiferous tubules that are in direct contact with germ cells at all levels. They play a vital role in the initial stage of spermatogenesis and its maintenance in adulthood. Regulating the proliferation of bovine testicular Sertoli cells can improve the reproductive capacity and reproductive health of cattle. Summary of the invention

[0005] The purpose of the present invention is to provide the application of ACOT12 gene in regulating the proliferation of bovine testicular supporting cells, and both of them show a positive regulatory action mode.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention provides an application of the ACOT12 gene as a target in regulating the proliferation of bovine testicular supporting cells. The ACOT12 gene includes the ACOT12 gene with NCBI accession number NC_037334.1 in cattle or a gene homologous to the gene in cattle. The regulation is positive regulation, that is, promoting the expression of the ACOT12 gene can promote the proliferation of bovine testicular supporting cells, and inhibiting the expression of the ACOT12 gene can inhibit the proliferation of bovine testicular supporting cells. Through this method, the proliferation of bovine testicular supporting cells can be regulated, thereby improving the reproductive capacity and reproductive health of cattle.

[0008] The present invention provides an application of a reagent for regulating the expression of ACOT12 gene in the preparation of a product for influencing the proliferation of bovine testicular supporting cells.

[0009] As an embodiment, the reagent includes a reagent that promotes ACOT12 gene expression and a reagent that inhibits ACOT12 gene expression, wherein the reagent that promotes ACOT12 gene expression promotes the proliferation of bovine testicular supporting cells, and the reagent that inhibits ACOT12 gene expression inhibits the proliferation of bovine testicular supporting cells.

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

[0011] The present invention provides a reagent for regulating the proliferation of bovine testicular supporting cells, wherein the reagent comprises at least one of the following RNAs or gene-derived products thereof targeting the ACOT12 gene: siRNA, shRNA and sgRNA.

[0012] Furthermore, the gene-derived product includes 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 as shown in SEQ ID NO:1.

[0014] The present invention also provides a method for promoting / inhibiting the proliferation of bovine testicular supporting cells, wherein a reagent for promoting / inhibiting the expression of ACOT12 gene is transfected into the bovine testicular supporting cells.

[0015] Preferably, the reagent comprises at least one of the following RNAs or gene-derived products thereof targeting the ACOT12 gene: siRNA, shRNA and sgRNA. The gene-derived product comprises 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.

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

[0017] Beneficial effects:

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

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

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

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

[0022] Figure 4 After interfering with ACOT12, RT-qPCR was used to detect the mRNA levels of proliferation marker genes CCNB1 and CDK1 (*P<0.05, t-test).

[0023] Figure 5 After interfering with ACOT12, the Edu method was used to detect cell proliferation; in A, blue indicates cell nuclei, and red indicates proliferating cells; in B, ****P<0.0001, t-test. DETAILED DESCRIPTION

[0024] The present invention uses RNA interference (RNAi) technology in a specific embodiment, targets the ACOT12 gene by small interfering RNA (siRNA), and transfects the siRNA into bovine testicular supporting cells to specifically inhibit the transcription or translation of the ACOT12 gene, thereby reducing the expression of ACOT12 in bovine testicular supporting cells, and observing the proliferation of bovine testicular supporting cells. The mRNA levels of bovine testicular supporting cell proliferation marker genes CCNB1 and CDK1 were detected by RT-qPCR, and the cell proliferation was detected by the Edu method to evaluate the proliferation level of bovine testicular supporting cells and verify the expression inhibition effect of the ACOT12 gene. The results show that interfering with the expression of the ACOT12 gene inhibits the proliferation of bovine testicular supporting cells, suggesting that the ACOT12 gene has a positive regulatory effect in the proliferation of bovine testicular supporting cells.

[0025] In view of the fact that the expression level of the ACOT12 gene is positively correlated with the proliferation of bovine testicular supporting cells, the present invention provides the use of the ACOT12 gene as a target in regulating the proliferation of bovine testicular supporting cells, wherein the regulation is positive regulation. Specifically, the use of the ACOT12 gene as an overexpression target in promoting the proliferation of bovine testicular supporting cells, and the use of the ACOT12 gene as a knockout or inhibition target in inhibiting the proliferation of bovine testicular supporting cells.

[0026] The present invention provides an application of an agent for regulating the expression of the ACOT12 gene in the preparation of a product that affects the proliferation of bovine testicular supporting cells. As an embodiment, the agent includes an agent for promoting the expression of the ACOT12 gene and an agent for inhibiting the expression of the ACOT12 gene, wherein the agent for promoting the expression of the ACOT12 gene promotes the proliferation of bovine testicular supporting cells, and the agent for inhibiting the expression of the ACOT12 gene inhibits the proliferation of bovine testicular supporting cells.

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

[0028] The present invention provides an agent for regulating the proliferation of bovine testicular supporting cells, wherein the agent comprises at least one of the following RNAs or gene-derived products thereof targeting the ACOT12 gene: siRNA, shRNA and sgRNA.

[0029] In the present invention, the gene-derived product includes an expression vector comprising siRNA, a CRISPER-Cas gene editing system comprising sgRNA, and a recombinant expression vector comprising shRNA. The present invention has no particular restrictions on the preparation method of the gene-derived product, and the construction method of the recombinant expression vector or CRISPER-Cas gene editing system known in the art can be used. In the embodiment of the present invention, siRNA is used as an example to illustrate the effect of the present invention on the proliferation of bovine testicular supporting cells by inhibiting the expression of ACOT12 gene. The siRNA is a nucleotide sequence as shown in SEQ ID NO: 1.

[0030] The invention provides a method for promoting / inhibiting the proliferation of bovine testicular supporting cells, and a reagent for promoting / inhibiting the expression of ACOT12 gene is transfected into the bovine testicular supporting cells.

[0031] In the present invention, the reagents for promoting / inhibiting the expression of the ACOT12 gene are the same as the above-mentioned technical scheme and are not described in detail here. The present invention does not particularly limit the transfection method, and conventional transfection methods in the art can be used, such as liposome transfection. As an embodiment, the present invention transfects bovine Sertoli cells with siRNA targeting the ACOT12 gene, detects the expression of bovine Sertoli cell proliferation marker genes CCNB1 and / or CDK1, or uses the Edu method to detect cell proliferation, evaluates the proliferation level of bovine Sertoli cells, and inhibits the proliferation of bovine Sertoli cells.

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

[0033] Example 1

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

[0035] Testicular tissue of newborn calves was collected and stored in PBS buffer (with penicillin and streptomycin added). The tissue surface was disinfected by soaking in 75% ethanol for 30 seconds, and then washed with PBS three times. The testicular tunica albuginea was cut open with scissors, and several testicular tissues weighing about 1g were taken from the inside by the cross method and cut into pieces in a 4mL centrifuge tube. PBS was added, blown and evenly mixed, and centrifuged at 1500rpm for 5min. PBS was discarded, and type IV collagenase with a concentration of 1.0mg / mL was added for digestion. Placed in a cell culture incubator for digestion for 30-40min (shaking up and down every 10min). After digestion, centrifuged at 1500rpm for 5min, and the collagenase was discarded. PBS was added and blown and evenly mixed repeatedly, and centrifuged at 1500rpm for 5min. PBS was discarded, and the cell pellet was digested with 2.5% trypsin in the incubator for 10min. Cell culture medium was added to terminate digestion, and the filtrate was collected by filtering with a 120-mesh steel sieve. Centrifuged at 1500rpm for 5min, and the supernatant was discarded. Add DMEM / F12 cell culture medium containing 10% FBS to resuspend the cells and inoculate them in a 60 mm dish. After 6 hours, gently aspirate and discard the floating cells, replace the culture medium, and separate the cultured cells to obtain bovine primary testicular Sertoli cells. Figure 1 .

[0036] Example 2

[0037] Identification of primary bovine testicular Sertoli cells

[0038] The total RNA of the bovine primary Sertoli cells cultured in Example 1 was extracted according to the operating instructions of the total RNA extraction kit (R6834-01) of Omega Company, and the RNA was reverse transcribed into the 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 supporting cell marker gene primers in Table 1 were used for detection. PCR enzyme (NEB, E0555S) was used for amplification, and the PCR results were detected by gel electrophoresis.

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

[0041] Table 1 Primers for marker genes of bovine testicular supporting cells

[0042]

[0043] Example 3

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

[0045] (1) Three siRNAs and a negative control (NC) were designed using the mRNA sequence of the bovine ACOT12 gene from the NCBI website (Table 2).

[0046] Table 2 ACOT12 and NC siRNA

[0047]

[0048] (2) When the confluence of bovine primary testicular supporting cells reached 60% to 70%, siRNA transfection was performed according to the operating instructions of the transfection reagent jetPRIME (Polyplus, 101000046).

[0049] (3) 24 hours after transfection, total cellular RNA was extracted according to the instruction manual of the total RNA extraction kit (R6834-01) of Omega Company, and the RNA was reverse transcribed into the first-strand cDNA using a reverse transcription kit (Thermo Scientific, 00238582).

[0050] (4) After the cDNA was diluted 5 times with RNase Free H2O, qPCR was performed using a 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, an internal reference gene was set, and 3 replicate wells were set for each sample. The amplification reaction conditions of the three-step method were: pre-denaturation at 95°C for 4 minutes, cycle reaction was denaturation at 95°C for 5 seconds, annealing at 60°C for 20 seconds, extension at 72°C for 20 seconds, and collection of fluorescence signals, a total of 40 cycles, and finally extension at 72°C for 2 minutes. The temperature was raised from 65°C to 95°C at a rate of 0.5°C / 5 seconds to detect the melting curve. The relative expression of the target gene was calculated according to The systems and primers used for calculation are shown in Tables 3 and 4.

[0051] Table 3 SYBR amplification system

[0052]

[0053] Table 4 RT-qPCR primers

[0054]

[0055] The results showed that siRNA-1 could 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] Proliferation assay of primary bovine testicular Sertoli cells

[0058] After transfection of siRNA-1 in primary bovine testicular supporting cells, 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 with ACOT12 gene expression on the proliferation of bovine testicular supporting 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 Abbkine's Ed method U cell proliferation imaging analysis kit (KTA2031).

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

[0060]

[0061]

[0062] Figure 4 The results showed that interfering with the expression of ACOT12 could 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 some red fluorescent spots can be seen in Edu staining, indicating that some cells are undergoing DNA replication. A large number of blue nuclei can also be seen in the siRNA-1 group, 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 with the expression of ACOT12 gene can inhibit the proliferation of bovine testicular supporting cells.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of ACOT12 gene as a target in regulating the proliferation of bovine testicular Sertoli cells.

2. Use of a reagent for regulating ACOT12 gene expression in the preparation of a product that affects the proliferation of bovine testicular supporting cells.

3. The use according to claim 2, characterized in that: The reagents include reagents for promoting the expression of ACOT12 gene and reagents for inhibiting the expression of ACOT12 gene. The reagents for promoting the expression of ACOT12 gene promote the proliferation of bovine testicular supporting cells, and the reagents for inhibiting the expression of ACOT12 gene inhibit the proliferation of bovine testicular supporting cells.

4. The use according to claim 2 or 3, characterized in that: The reagent includes a recombinant vector containing the ACOT12 gene or a recombinant vector interfering with the expression of the ACOT12 gene.

5. A reagent for regulating the proliferation of bovine testicular supporting cells, characterized in that: The reagent includes at least one of the following RNAs or gene-derived products thereof targeting the ACOT12 gene: siRNA, shRNA, and sgRNA.

6. The reagent according to claim 5, characterized in that 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.

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

1.

8. A method for promoting / inhibiting the proliferation of bovine testicular Sertoli cells, characterized in that: Bovine Sertoli cells were transfected with agents that promote / inhibit ACOT12 gene expression.

9. The method according to claim 9, characterized in that: The siRNA targeting ACOT12 gene was transfected into bovine Sertoli cells to inhibit the proliferation of bovine Sertoli cells.

10. The method according to claim 9, characterized in that: The expression levels of bovine Sertoli cell proliferation marker genes CCNB1 and / or CDK1 were detected, or the cell proliferation was detected using the Edu method to evaluate the proliferation level of bovine Sertoli cells.

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