Application of circ-CREBBP in identifying or regulating porcine semen quality

Abstract

The present invention provides the application of circ-CREBBP in identifying or regulating pig semen quality. The present invention has discovered an important circRNA molecular marker that affects pig semen quality. By detecting the expression level of circ-CREBBP, assisted breeding can be performed for boar semen quality identification and early screening of high-semen-quality boars, selecting individuals with superior semen quality and eliminating individuals with inferior quality, thereby achieving a short-term, low-cost, and high-accuracy breeding of a boar population with excellent semen quality; by regulating the expression of circ-CREBBP, pig sperm motility, ATP content in sperm cells, and sperm survival rate can also be regulated, thereby achieving improved pig semen quality. At the same time, as pigs are a good model for human disease research, circ-CREBBP can also be used as an important molecular target for male reproductive diseases, and further applied to rapid and non-invasive diagnosis of male reproductive diseases and research on improving male sperm motility.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of circ-CREBBP in identifying or regulating pig semen quality. Background Art

[0002] High-quality boars are one of the most important factors in the development of the pig industry. The quality of boar semen directly impacts the economic profitability of pig farms. In many pig-raising countries around the world, artificial insemination has become the primary method of breeding commercial sows. Using artificial insemination not only accelerates genetic improvement and distributes superior genes to more farms, but also significantly improves labor efficiency and reduces breeding costs. Selecting boars with excellent semen quality is a prerequisite and guarantee for implementing artificial insemination. Traditional methods struggle to quickly and accurately select for semen quality. However, marker-assisted selection (MAS) using molecular markers can effectively accelerate the breeding process, select superior individuals, eliminate inferior boars, and improve boar semen quality traits. Furthermore, pigs share a high degree of genetic homology with humans. Their genomes are highly similar, with similar chromosome composition and organ size. Furthermore, the conservation of syntenic regions between humans and pigs is three times that between humans and mice. As an ideal model animal for modern medical research, pigs are widely used in the study of human diseases. Therefore, boars with poor semen quality can serve as animal models for studying human male reproductive disorders.

[0003] Mammalian seminal plasma extracellular vesicles (SPEVs) are 50-500nm in diameter and are primarily produced by the epididymis and prostate. They are characterized by high cholesterol and sphingomyelin content and a complex protein composition. Extracellular vesicles contain a large number of molecules, including nucleic acids (DNA and RNA), proteins, and lipids. Since SPEVs were first discovered, increasing evidence has shown that they play an important role in sperm function. SPEVs can promote sperm motility, participate in the acrosome reaction, and help sperm to become capacitated at the right time. Studies have found that SPEVs can protect sperm from the immune response of the female reproductive tract, which is very important for sperm to successfully pass through the zona pellucida and combine with the egg. Studies have shown that some small non-coding RNAs in SPEVs can bind to target cells in the female reproductive tract and affect the expression of related genes. The most extensive research on the contents of SPEVs currently includes proteins and non-coding RNA. Most studies focus on "comparative omics" with the aim of exploring certain substances in SPEVs as biomarkers for certain diseases such as general infertility, obstructive and non-obstructive azoospermia, asthenozoospermia, prostate cancer, etc., making the diagnosis of diseases faster and non-invasive.

[0004] Circular RNA (circRNA) is a type of non-coding RNA produced through a unique alternative backsplicing mechanism: the 3' end of an exon is linked to its own 5' end or to an upstream exon via a 3', 5'-phosphodiester bond, forming a closed loop with a backsplicing junction site. Unlike most linear RNAs, circRNAs lack a 5'-3' polarity or an adenosine tail, making them resistant to exonuclease degradation and, therefore, more stable than linear RNAs. Studies have shown that circRNAs are not only structurally stable but also possess a rich diversity, sequence conservation, and specific expression in cells and tissues. They possess numerous potential functions and play a vital role in regulating gene expression and various biological functions. In recent years, numerous studies using circRNA sequencing have revealed abundant circRNA expression profiles in human, mouse, and pig testes. These circRNAs are derived from genes involved in spermatogenesis, sexual reproduction, germ cell development, and the meiotic cell cycle, suggesting that circRNAs may also play a significant role in spermatogenesis. A research team compared circRNA expression profiles in the testes of Duroc and Large White boars, finding significant differences in expression abundance. In 2017, researchers conducted circRNA sequencing in testicular tissue from 30- and 180-day-old Shaziling pigs. A total of 14,108 circRNAs were identified in immature and mature testes, with 2,819 and 8,069 circRNAs specifically expressed in immature and mature testes, respectively. Of these, 12,274 were differentially expressed. Analysis revealed that the genes from these differentially expressed circRNAs were primarily enriched in signaling pathways involved in testicular development and spermatogenesis. Studies have shown that circRNAs are abundant in various types of human spermatocytes, suggesting a potential differential regulatory role for circRNAs in sperm differentiation. International researchers have reported that circNAPEPLDiso1 and circNAPEPLDiso2 are expressed in both human and mouse sperm, with significant changes in expression levels before and after fertilization. These studies suggest that circRNAs could serve as biomarkers for predicting male fertility.

[0005] At present, there are no reports on the research of circRNA in porcine SPEVs. How circRNA regulates the motility of mature sperm and whether it can serve as a marker of semen quality needs further study. Summary of the Invention

[0006] The purpose of the present invention is to provide a circ-CREBBP marker for identifying or regulating pig semen quality, and a method for identifying or regulating pig semen quality based on circ-CREBBP.

[0007] To achieve the above objectives, the present invention first provides circ-CREBBP or a new use of circ-CREBBP as a marker.

[0008] The present invention provides circ-CREBBP or the use of circ-CREBBP as a marker in any of the following A1)-A8):

[0009] A1) Identify or assist in identifying the quality of pig semen;

[0010] A2) Screening or assisting in screening pigs with high or low semen quality;

[0011] A3) Screening or assisting in screening high-quality or low-quality pig semen;

[0012] A4) Regulating pig semen quality;

[0013] A5) Regulates pig sperm motility;

[0014] A6) Regulates ATP content in pig sperm cells;

[0015] A7) regulating the survival rate or apoptosis rate of pig sperm;

[0016] A8) Pig breeding.

[0017] In the above application, the regulation of pig semen quality is reflected in that the higher the expression level of circ-CREBBP in pig seminal plasma extracellular vesicles or sperm, the higher the semen quality produced by the pig; the lower the expression level of circ-CREBBP in pig seminal plasma extracellular vesicles or sperm, the lower the semen quality produced by the pig. Specifically, the higher the expression level of circ-CREBBP in pig seminal plasma extracellular vesicles or sperm, the higher the sperm motility, the stronger the ability of sperm to move rapidly, the higher the ATP content in sperm cells, and the higher the sperm survival rate produced by the pig; the lower the expression level of circ-CREBBP in pig seminal plasma extracellular vesicles or sperm, the lower the sperm motility, the weaker the ability of sperm to move rapidly, the lower the ATP content in sperm cells, and the higher the sperm apoptosis rate produced by the pig.

[0018] In a specific embodiment of the present invention, the regulation is reduction.

[0019] The purpose of pig breeding is to select pigs with high semen quality.

[0020] In order to achieve the above objectives, the present invention further provides a new use of a substance for detecting the expression level of circ-CREBBP.

[0021] The present invention provides the use of a substance for detecting the expression level of circ-CREBBP in any of the following B1)-B6):

[0022] B1) Identify or assist in identifying the quality of pig semen;

[0023] B2) preparing products for identifying or assisting in identifying the quality of pig semen;

[0024] B3) Screening or assisting in screening pigs with high or low semen quality;

[0025] B4) preparing products for screening or assisting in screening pigs with high or low semen quality;

[0026] B5) Screening or assisting in screening high-quality or low-quality pig semen;

[0027] B6) Prepare products for screening or assisting in screening high-quality or low-quality pig semen.

[0028] In order to achieve the above objectives, the present invention also provides a product for identifying or assisting in identifying the quality of pig semen.

[0029] The product provided by the present invention for identifying or assisting in identifying the quality of pig semen includes a substance for detecting the expression level of circ-CREBBP.

[0030] In the above applications or products, the substance for detecting the expression level of circ-CREBBP is a reagent and / or instrument for detecting the expression level of circ-CREBBP.

[0031] Furthermore, the reagent and / or instrument for detecting the expression level of circ-CREBBP may be a reagent and / or instrument for detecting the expression level of circ-CREBBP using a sequencing method or a reagent and / or instrument for detecting the expression level of circ-CREBBP using a fluorescence quantitative PCR method.

[0032] Furthermore, the reagents and / or instruments for detecting the expression level of circ-CREBBP by sequencing include reagents and / or instruments used for library construction and sequencing.

[0033] The reagents and / or instruments for detecting the expression of circ-CREBBP using the fluorescent quantitative PCR method include reagents and / or instruments used for reverse transcription and fluorescent quantitative PCR. Specifically, the reagents used for the fluorescent quantitative PCR include a primer pair consisting of the single-stranded DNA molecule shown in SEQ ID No. 1 and the single-stranded DNA molecule shown in SEQ ID No. 2.

[0034] In order to achieve the above objectives, the present invention also provides a new use of a substance for inhibiting the expression of circ-CREBBP.

[0035] The present invention provides use of a substance that inhibits circ-CREBBP expression in any of the following C1)-C10):

[0036] C1) Reduce the quality of pig semen;

[0037] C2) preparing products that reduce the quality of pig semen;

[0038] C3) Reduced sperm motility in pigs;

[0039] C4) preparing a product for reducing sperm motility in pigs;

[0040] C5) Reduce the ATP content in pig sperm cells;

[0041] C6) preparing a product for reducing ATP content in pig sperm cells;

[0042] C7) reducing the sperm survival rate or increasing the sperm apoptosis rate of pigs;

[0043] C8) preparing a product for reducing the survival rate of pig sperm or increasing the apoptosis rate of pig sperm;

[0044] C9) Breeding pigs with low semen quality;

[0045] C10) Preparation of products for breeding pigs with low semen quality.

[0046] In order to achieve the above objectives, the present invention also provides a product for regulating the quality of pig semen.

[0047] The product for regulating pig semen quality provided by the present invention includes a substance for regulating the expression amount of circ-CREBBP.

[0048] Furthermore, the substance that regulates the expression of circ-CREBBP is a substance that inhibits the expression of circ-CREBBP.

[0049] In the above-mentioned application or product, the substance that inhibits the expression of circ-CREBBP can specifically be ssc-circ-CREBBP, the nucleotide sequence of the ssc-circ-CREBBP positive chain is shown in SEQ ID No. 3, and the nucleotide sequence of the ssc-circ-CREBBP antisense chain is shown in SEQ ID No. 4.

[0050] In order to achieve the above object, the present invention finally provides any one of the following methods D1) to D6):

[0051] D1) A method for identifying or assisting in identifying pig semen quality, comprising the following steps: detecting the expression level of circ-CREBBP in seminal plasma extracellular vesicles or sperm of a pig to be tested, and judging the semen quality of the pig to be tested based on the circ-CREBBP expression level: the semen quality of pigs with high circ-CREBBP expression levels is higher than that of pigs with low circ-CREBBP expression levels;

[0052] D2) A method for screening or assisting in screening pigs with high semen quality, comprising the following steps: detecting the expression level of circ-CREBBP in seminal plasma extracellular vesicles or sperm of the pigs to be tested, and selecting pigs with high circ-CREBBP expression levels;

[0053] D3) A method for screening or assisting in screening pigs with low semen quality, comprising the steps of: detecting the expression level of circ-CREBBP in seminal plasma extracellular vesicles or sperm of the pigs to be tested, and selecting pigs with low circ-CREBBP expression;

[0054] D4) A method for screening or assisting in screening high-quality porcine semen, comprising the following steps: obtaining pigs with high semen quality according to the method described in D2), and obtaining high-quality porcine semen from the pigs with high semen quality;

[0055] D5) A method for screening or assisting in screening low-quality porcine semen, comprising the following steps: obtaining pigs with low semen quality according to the method described in D3), and obtaining low-quality porcine semen from the pigs with low semen quality;

[0056] D6) A method for breeding pigs with low semen quality, or reducing pig semen quality, or reducing pig sperm motility, or reducing ATP content in pig sperm cells, or reducing pig sperm survival rate, or increasing pig sperm apoptosis rate, comprising the step of reducing the expression level of circ-CREBBP in pig sperm.

[0057] In any of the methods D1) to D5) above, the method for detecting the expression level of circ-CREBBP in the seminal plasma extracellular vesicles or sperm of the pig to be tested can be method 1 or method 2:

[0058] Method 1: Transcriptome sequencing was performed using RNA from seminal plasma extracellular vesicles or sperm of the pigs to be tested as templates to obtain the expression level of circ-CREBBP;

[0059] Method 2: Fluorescence quantitative PCR was performed using the cDNA obtained by reverse transcription of RNA in the seminal plasma extracellular vesicles or sperm of the pig to be tested as a template to obtain the expression level of circ-CREBBP (relative expression level or absolute expression level).

[0060] Furthermore, the transcriptome sequencing method may include the steps of preparing a sequencing library and sequencing. The preparation of the sequencing library may be performed using a QIAseq miRNA Library Kit. The sequencing may be performed using an Illumina Hiseq2500.

[0061] The primer pair used for the fluorescent quantitative PCR detection of the relative expression level of circ-CREBBP can specifically be a primer pair consisting of a single-stranded DNA molecule shown in SEQ ID No. 1 and a single-stranded DNA molecule shown in SEQ ID No. 2.

[0062] Furthermore, the pigs with high circ-CREBBP expression levels are pigs with higher circ-CREBBP expression levels than control pigs, and the pigs with low circ-CREBBP expression levels are pigs with lower circ-CREBBP expression levels than control pigs. The control pigs are pigs with total sperm motility greater than 88%.

[0063] In the method described in D6) above, the method for reducing the expression level of circ-CREBBP in pig sperm is to introduce a substance that inhibits the expression of circ-CREBBP into pig sperm.

[0064] Furthermore, the substance that inhibits circ-CREBBP expression may be ssc-circ-CREBBP, the nucleotide sequence of the ssc-circ-CREBBP positive chain is shown in SEQ ID No. 3, and the nucleotide sequence of the ssc-circ-CREBBP antisense chain is shown in SEQ ID No. 4.

[0065] Furthermore, the introduction method includes the step of transferring the substance that inhibits circ-CREBBP expression into seminal plasma extracellular vesicles and then incubating sperm, which can be carried out specifically as follows: 20 μL of seminal plasma extracellular vesicles, 20 μL of trehalose solution (PBS solution containing 5% trehalose) and 2 μL of the above-mentioned ssc-circ-CREBBP solution (concentration of 20 μM) are mixed and added to an electroporation cup for electroporation. After electroporation, the electroporation cup is first placed in an ice box for 30 minutes, and then incubated at 17°C for 30 minutes to obtain a mixed system after electroporation; the mixed system after electroporation is mixed and incubated with pig sperm, and incubated at 17°C for 4 days.

[0066] In any of the above-mentioned uses, products, or methods, the pigs with high semen quality are pigs that produce high-quality semen; the pigs that produce high-quality semen are pigs with a total sperm motility of >88%. The pigs with low semen quality are pigs that produce low-quality semen; the pigs that produce low-quality semen are pigs with a total sperm motility of <73%.

[0067] The high-quality semen is semen with a total sperm motility of >88%; the low-quality semen is semen with a total sperm motility of <73%.

[0068] In any of the above applications, products, or methods, sperm motility refers to the ability of sperm to move rapidly. The higher the sperm motility, the stronger the ability to move rapidly, and the higher the semen quality.

[0069] In any of the above-mentioned applications, products or methods, the seminal plasma extracellular vesicles and sperm may be the seminal plasma extracellular vesicles and sperm of pigs at or after sexual maturity.

[0070] In any of the above applications, products or methods, the pig is a Large White boar, specifically a Large White boar aged 14 to 36 months.

[0071] In any of the above-mentioned applications, products or methods, the circ-CREBBP is located on porcine chromosome 3, and the nucleotide sequence is shown as SEQ ID No. 5.

[0072] The quality of boar reproductive performance directly impacts the reproductive performance of the sow herd, including key indicators such as conception rate and litter size. Therefore, boar semen quality has a significant economic impact on the pig production system. The greater the amount of semen produced by each boar and the higher the semen quality, the lower the cost of using high-quality boars and the higher the economic benefits. When conducting artificial insemination, priority should be given to individual boars with superior breeds and excellent performance across all indicators, while those with poorer performance should be promptly eliminated.

[0073] The method provided by the present invention can be used to screen pig individuals with high sperm motility and with high accuracy. It can be used for early screening of boars, and can even be accurately screened when the boars are rigidly mature, greatly accelerating the breeding process of large white pigs, selecting groups with superior semen quality, and eliminating inferior boars, thereby achieving a short-term, low-cost, and highly accurate selection of boar groups with excellent semen quality. The method provided by the present invention can also regulate pig sperm motility, ATP content in sperm cells, and sperm survival rate or apoptosis rate, thereby improving pig semen quality. The present invention has significant application value in establishing and cultivating boar groups with high-quality semen. At the same time, pigs are a good model for human disease research, and circ-CREBBP can also be used as an important molecular target for male reproductive diseases, and further applied to the rapid and non-invasive diagnosis of male reproductive diseases and the study of improving male sperm motility. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 This is the normalized expression result of circ-CREBBP identified in the transcriptome sequencing results in Example 1.

[0075] Figure 2 The expression results of circ-CREBBP in Large White pig seminal plasma extracellular vesicles (SPEVs) detected by fluorescence quantitative PCR in Example 2.

[0076] Figure 3 The expression level of circ-CREBBP in Large White pig sperm detected by fluorescence quantitative PCR in Example 3.

[0077] Figure 4 This is the expression level of circ-CREBBP in Large White pig sperm after incubation with si-circCREBBP in Example 4.

[0078] Figure 5 This is the test result of Large White pig sperm motility after incubation with si-circCREBBP in Example 5.

[0079] Figure 6 This is the detection result of ATP content in Large White pig sperm cells after incubation with si-circCREBBP in Example 6.

[0080] Figure 7 This is the detection result of the apoptosis rate of Large White pig sperm after incubation with si-circCREBBP in Example 7. DETAILED DESCRIPTION

[0081] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0082] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0083] The pigs in the following examples are all Large White boars.

[0084] Example 1: Transcriptome sequencing revealed that circ-CREBBP is differentially expressed in extracellular vesicles of seminal plasma of Large White pigs with high and low semen quality

[0085] 1. Experimental animals and grouping

[0086] All semen samples used in this study were obtained from Henan Jingwang Breeding Pig Improvement Co., Ltd. They were Large White pigs, aged 14 to 36 months, raised to sexual maturity under normal nutritional conditions. Boars with high and low semen quality were selected based on semen collection records from boar stations over the past six months. Twenty-four boars were selected and divided into two groups: a high-quality group (H group, n = 12) and a low-quality group (L group, n = 12). Total sperm motility was >88% in the high-quality group and <73% in the low-quality group, with a highly significant difference in sperm motility between the two groups (P < 0.001). Semen samples were collected from all boars at the same time by a semen collector. The total sperm motility (%) of the 24 boars, by group, age, and semen samples, is shown in Table 1.

[0087] Table 1

[0088] Grouping Farm number Age in months Total sperm motility (%) H Y913447 16 90.86 H Y908883 18 93.73 H Y911751 17 85.09 H Y808188 31 93.31 H Y902687 21 88.82 H Y907074 19 90.16 H Y822641 14 98.50 H Y803771 15 98.70 H Y700486 29 99.60 H Y700486 35 96.80 H Y607647 30 97.70 H Y703589 27 99.60 L Y808525 31 59.46 L Y915873 15 20.67 L Y901507 22 13.59 L Y823963 24 47.34 L Y919289 14 13.06 L Y822354 22 28.10 L Y804123 31 10.08 L Y822395 14 63.20 L Y97910 36 73.20 L Y702447 17 65.80 L Y608742 29 55.30 L Y702624 28 31.30

[0089] Note: H represents high semen quality group; L represents low semen quality group.

[0090] 2. Isolation of porcine seminal plasma extracellular vesicles (SPEVs)

[0091] (1) Pretreatment

[0092] Fresh semen from 24 boars was inspected for quality and equilibrated at 17°C for 2 hours before undergoing pretreatment. This pretreatment process consisted of three steps. First, the semen was centrifuged at 800g for 15 minutes at 17°C. The supernatant was removed and the sperm pellet discarded. Next, the semen was centrifuged at 10,000g for 30 minutes at 4°C. The supernatant was removed and the pellet discarded. Finally, the semen was centrifuged at 12,000g for 60 minutes at 4°C. The supernatant was removed and the pellet discarded.

[0093] (2) Ultracentrifugation

[0094] Transfer the supernatant from the previous step to an ultracentrifuge tube and equalize with DPBS. Then, centrifuge at 120,000 g at 4°C for 1.5 hours, discard the supernatant, and resuspend the pellet in DPBS. Centrifuge again at 12,000 g at 4°C for 1.5 hours, discard the supernatant, and resuspend the pellet in an appropriate amount of DPBS. Finally, sterilize the SPEV sample by filtration using a 0.22 μm filter membrane and store at -80°C until further use.

[0095] 3. RNA extraction and concentration determination in porcine SPEVs

[0096] (1) Add 700 μL of QIAzol lysis buffer to 200 μL of SPEVs sample, vortex to mix, and let stand at room temperature (15-25°C) for 5 min.

[0097] (2) Add 140 μL of chloroform to each tube, cap tightly, shake vigorously for 15 seconds, and let stand at room temperature for 2-3 minutes. Centrifuge at 12,000 g for 15 minutes at 4°C.

[0098] (3) Transfer the supernatant to a new EP tube, taking care not to mix it with the lower liquid phase, add 1.5 times the volume of anhydrous ethanol, and then mix well.

[0099] (4) Transfer 700 μL of the sample to the RNeasy Mini column and place it in a 2 mL collection tube. Centrifuge at 8000 g for 15 seconds at room temperature and discard the liquid in the collection tube.

[0100] (5) Repeat step (4) to collect the remaining sample. Add 700 μL of Buffer RWT to the centrifuge column, centrifuge at 8000 g for 15 seconds, and discard the filtrate.

[0101] (6) Pipette 500 μL of Buffer RPE into the centrifuge column, centrifuge at 8000 g for 15 s, and discard the filtrate. Pipette 500 μL of Buffer RPE into the centrifuge column, centrifuge at 8000 g for 15 s, and discard the filtrate.

[0102] (7) Transfer the RNeasy Mini column to a new 2 mL collection tube and centrifuge at 12000 g for 2 min.

[0103] (8) Transfer the RNeasy Mini column to a new 1.5 mL EP tube and leave it at room temperature with the lid open for 3 min.

[0104] (9) Add 35 μL of RNA-free water to the RNeasy Mini column, cover, and let stand at room temperature for 2 min. Centrifuge at 12,000 g for 1 min at room temperature and discard the column.

[0105] (10) The purity and concentration of total RNA in extracellular vesicles of boar seminal plasma were detected using Nanodrop nucleic acid analyzer (Thermo Scientific, USA), and the integrity of total RNA in extracellular vesicles of boar seminal plasma was detected using Agilent2100 (Agilent, USA).

[0106] 4. RNA library construction and sequencing

[0107] (1) After total RNA extraction, RNA library construction and sequencing were performed by Beijing Enze Kangtai Biological Co., Ltd. The sequencing library was prepared using the QIAseq miRNA Library Kit. The simplified library construction steps are as follows: ① 3' and 5' end adapter ligation; ② Biotinylated Random Primers and mRNA mixed reverse transcription; ③ mRNA capture by magnetic beads, elution and cDNA purification; ④ PCR amplification. After the library construction is completed, the constructed library is quality checked using the Agilent Bioanalyzer 2100. After passing the quality check, it is sequenced using the Illumina Hiseq2500, and the sequencing read length is 10×150bp per end.

[0108] (2) 24 cDNA libraries were obtained and paired-end sequenced using the Illumina HiSeq 2500 sequencing platform with a single-end read length of 10×150 bp. The raw reads were filtered using CutAdapt software to remove the adapter sequences and low-quality reads added during library construction to form clean reads. The clean reads were aligned with the Silva, GtRNAdb, Rfam, and Repbase databases using Bowtie software to filter out ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and repetitive sequences. Unannotated reads were obtained after filtering for subsequent analysis. Clean reads were then aligned to the pig reference genome sequence sscrofa11.1 and the corresponding gene annotation file (sscrofa11.1) using BWA software.

[0109] 5. Screening and expression of differentially expressed circRNAs in transcriptome sequencing

[0110] (1) Based on the sequencing data, reads mapped to the reference genome were aligned with circRNA sequences in the known circRNAs database using find-circ and CIRI2 software. The intersection of the two softwares was used to identify candidate circRNAs. The candidate circRNAs were further compared with the positions of the reference genome genes. The screened circRNAs were classified into three categories: exonic regions, intronic regions, and intergenic regions. The expression levels of circRNAs in each sample were statistically analyzed and normalized using the RPM (Read per million mapped reads, RPM) algorithm: the normalization formula is: RPM = (mapped reads × 1,000,000) / total reads.

[0111] (2) The edgeR package was used to analyze the differential circRNAs between the high and low groups, and the L / H ratio was used for analysis. The significant difference thresholds in this study were set at |logFC|>2 and P-value<0.05.

[0112] The results are shown in Table 2 and Figure 1 The results showed that circ-CREBBP was a significantly different circRNA in the extracellular vesicles of pigs with high and low semen quality. The RPM value of circ-CREBBP in the extracellular vesicles of the high semen quality group was significantly higher than that of the low semen quality group, that is, the expression level of circ-CREBBP in the extracellular vesicles of the high semen quality group was significantly higher than that of the low semen quality group.

[0113] Table 2

[0114] circRNA_ID Source gene Gene name logFC(L vs.H) P Value 3:38470924|38472523 ENSSSCG00000007951 CREBBP -8.992870075 0.018040593

[0115] Example 2: Detection of differential expression of circ-CREBBP in Large White boar SPEVs with high and low semen quality by quantitative PCR

[0116] 1. Preparation of primer pairs

[0117] Primers for qPCR were designed based on the ssc-circ-CREBBP (3_38470924_38472523) sequence in the circNet:circRNA in pigs database. The nucleotide sequences of the upstream and downstream primers are shown in Table 3.

[0118] Table 3

[0119]

[0120] 2. Extraction of total RNA from extracellular vesicles of seminal plasma of experimental animals

[0121] Three Large White boars were randomly selected from the high semen quality group and the low semen quality group in Example 1 as experimental animals, and seminal plasma extracellular vesicles were isolated and total RNA was extracted according to the method in steps 2-3 of Example 1.

[0122] 3. Reverse transcription of total RNA from seminal plasma extracellular vesicles

[0123] PrimeScript reverse transcription kit TM The total RNA of the sample was reverse transcribed according to the instructions of RT reagent Kit (Perfect Real Time) (RR037, TAKARA, Dalian) to obtain cDNA. The entire reaction was carried out on ice.

[0124] The reverse transcription reaction system (total volume of 10 μL) is as follows: 5 μL of template total RNA; 2 μL of 5X PrimeScript Buffer; 0.5 μL of PrimeScript RT Enzyme Mix I; 0.5 μL of Oligo dT Primer (50 μM); 0.5 μL of Random6mers; and 1.5 μL of Nuclease-free H2O.

[0125] The reverse transcription reaction conditions were 37°C for 60 min and 85°C for 5 s.

[0126] After the reaction, store at -20°C.

[0127] 4. Real-time fluorescence quantitative PCR detection of circ-CREBBP expression

[0128] Using the cDNA obtained in step 3 as a template, according to TB Premix Ex Taq TM II (Tli RNaseHPlus) (RR820, Takara, Dalian) kit instructions, and real-time fluorescence quantitative PCR was performed using the primer pairs in step 1 to detect the expression level of circ-CREBBP.

[0129] The PCR reaction system (total volume 20 μL) was as follows: 5.0 μL of template cDNA; 0.8 μL each of primers F and R; 0.4 μL of ROX Reference Dye II; 10.0 μL of TB Green Premix Ex TaqII (Tli RNaseH Plus); and 3.0 μL of ddH2O. The final concentration of primers F and R in the reaction system was 0.4 μM.

[0130] PCR reaction conditions are shown in Table 4.

[0131] Table 4

[0132]

[0133]

[0134] See the results Figure 2 The results showed that in Large White pig samples, the expression level of circ-CREBBP in "H group SPEVs" was greater than that in "L group SPEVs", and reached a significant level (P<0.001).

[0135] Example 3: Detection of differential expression of circ-CREBBP in spermatozoa of Large White boars with high and low semen quality by quantitative PCR

[0136] 1. Preparation of primer pairs

[0137] The primer pair is the primer pair in step 1 of Example 2.

[0138] 2. Extraction of total RNA from sperm of experimental animals

[0139] Three Large White boars were randomly selected from the high-quality semen group and the low-quality semen group in Example 1 as experimental animals for sperm RNA extraction and concentration determination. The specific steps are as follows:

[0140] (1) Take 1×10 8 Sperm was added with 1 mL of Trizol and mixed by vortexing; the sample was ground with a homogenizer for 30 seconds per sample.

[0141] (2) Place on ice for 15 min, centrifuge at 12,000 rpm and 4°C for 5 min, collect the supernatant, and transfer the supernatant to a new 2 mL centrifuge tube.

[0142] (3) Add 200 μL of pre-cooled chloroform, shake vigorously for 15 seconds, and let it stand on ice for 5 minutes.

[0143] (4) Centrifuge at 12000 rpm and 4°C for 15 minutes, collect the supernatant, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0144] (5) Add 500 μL of isopropanol and place in a -20°C refrigerator overnight.

[0145] (6) Centrifuge at 12000 rpm and 4°C for 10 min, discard the supernatant, add 1 mL of pre-cooled 75% alcohol to the precipitate, and flick the bottom of the tube to allow the precipitate to be fully washed with alcohol.

[0146] (7) Add 10 μL of nucleic acid precipitation aid to each tube and let it stand at room temperature for 10 minutes.

[0147] (8) Centrifuge at 7500 rpm and 4°C for 5 minutes, discard the supernatant, let it air dry for 5 minutes, and then place it on ice to air dry for 5 minutes.

[0148] (9) Add 10 μL of ddH 2 O and vortex briefly to fully dissolve the RNA precipitate. Use Nanodrop Nucleic Acid Analyzer (Thermo Scientific, USA) to detect the purity and concentration of boar sperm total RNA and store at −80°C.

[0149] 3. Reverse transcription of pig sperm total RNA

[0150] The total RNA of the sample was reverse transcribed using a reverse transcription kit to obtain cDNA. The entire reaction was performed on ice.

[0151] The reverse transcription reaction system and reaction conditions were the same as those in step 3 of Example 2.

[0152] 4. Real-time fluorescence quantitative PCR detection of circ-CREBBP expression

[0153] Using the cDNA obtained in step 3 as a template, real-time fluorescence quantitative PCR was performed using the primer pair.

[0154] The PCR reaction system and reaction conditions were the same as those in step 4 of Example 2.

[0155] See the results Figure 3 The results showed that the expression level of circ-CREBBP in "H group sperm" was greater than that in "L group sperm" and reached a significant level (P<0.001).

[0156] Example 4: Fluorescence quantitative PCR detection of the effect of si-circ-CREBBP on the expression of circ-CREBBP in pig sperm

[0157] 1. Preparation of si-circ-CREBBP sequences

[0158] An interfering sequence binding to the circulated site was designed based on the sequence of ssc-circ-CREBBP (3_38470924_38472523) in the circNet:circRNA in pigs database and named si-circ-CREBBP. The nucleotide sequence of the positive chain of si-circ-CREBBP is shown in SEQ ID No. 3, and the nucleotide sequence of the antisense chain is shown in SEQ ID No. 4. They were synthesized by Shanghai Jima Biotechnology Co., Ltd. Specific sequence information is shown in Table 5.

[0159] Table 5

[0160]

[0161] 2. Sperm incubation with si-circ-CREBBP

[0162] According to the method in step 2 of Example 1, seminal plasma extracellular vesicles were extracted from the seminal plasma of healthy Large White boar individuals, and then si-circ-CREBBP was loaded into the seminal plasma extracellular vesicles using electroporation technology using an electroporator (Gene Pulser Xcell electroporator). The experiment was divided into an interference group and a blank group. The specific operations of each group were as follows:

[0163] Interference group: 20 μL of seminal plasma extracellular vesicles, 20 μL of trehalose PBS solution (PBS solution containing 5% trehalose), and 2 μL of si-circ-CREBBP solution (solvent: DEPC water, concentration: 20 μM) were mixed and added to the electroporation cup;

[0164] Blank group: 20 μL of seminal plasma extracellular vesicles, 20 μL of trehalose solution (PBS containing 5% trehalose) and 2 μL of PBS solution were mixed and added to the electroporation cup.

[0165] The electroporation conditions for each group were 250 V, 100 μF, R = ∞, and cuvette size = 4 mm. Immediately after electroporation, the electroporation cuvette was placed in an ice box and allowed to stand for 30 min, and then transferred to a 17°C incubation chamber for 30 min.

[0166] 3. Sperm Co-incubation and RNA Extraction

[0167] Sperm co-incubation: 42 μL of the blank group and interference group electroporated mixed system were mixed with 1×10 8 The spermatozoa from healthy Large White boars were mixed and incubated at a constant temperature of 17℃ for 4 days.

[0168] RNA extraction: Spermatozoa were collected on the second and fourth days of co-incubation for RNA extraction. The RNA extraction method was the same as step 2 in Example 3.

[0169] 4. Reverse transcription of pig sperm total RNA

[0170] The total RNA of the sample was reverse transcribed using a reverse transcription kit to obtain cDNA. The entire reaction was performed on ice.

[0171] The reverse transcription reaction system and reaction conditions were the same as those in step 3 of Example 2.

[0172] 5. Real-time fluorescence quantitative PCR detection of circ-CREBBP expression

[0173] Using the cDNA obtained in step 4 as a template, real-time fluorescence quantitative PCR was performed using the primer pair.

[0174] The PCR reaction system and reaction conditions were the same as those in step 4 of Example 2.

[0175] See the results Figure 4 The results showed that on days 2 and 4 of incubation with the interference group and blank group, the expression of circ-CREBBP in sperm from the blank group was significantly higher than that in sperm from the interference group (P < 0.001). This suggests that si-circ-CREBBP can significantly reduce the expression of circ-CREBBP in sperm.

[0176] Example 5: CASA system detection of the effect of si-circ-CREBBP on pig sperm motility

[0177] Take 42 μL of the mixed system of blank group and interference group obtained in step 2 of Example 4 after electroporation and mix with 1×10 8 Sperm from healthy Large White boars were mixed and incubated at 17°C. The motility of sperm in the blank and interference groups was measured and analyzed using the CASA system after 12, 24, 48, 60, 72, 84, and 96 hours of incubation.

[0178] See the results Figure 5 The results showed that compared with the blank group, the si-circ-CREBBP interference group significantly reduced sperm motility from 12h to 96h (P<0.05).

[0179] Example 6: Detection of the effect of si-circ-CREBBP on pig sperm ATP by microplate reader

[0180] 1. Take 42 μL of the mixed system of blank group and interference group obtained in step 2 of Example 4 after electroporation and mix with 1×10 8 The spermatozoa from healthy Large White boars were mixed and incubated at a constant temperature of 17℃ for 4 days.

[0181] 2. According to the operation steps of A luciferase-based enhanced ATP assay kit, take 1×10 8 The sperm cells were lysed, and 100 μL was taken after lysis to detect the ATP content of the sperm cells using a microplate reader; at the same time, 20 μL was taken and the protein content was detected using a microplate reader according to the operating procedures of the BCA assay kit for ATP content correction.

[0182] See the results Figure 6 The results showed that on the second day of incubation of Large White pig sperm in the interference group and the blank group, the "sperm ATP content" in the blank group was greater than that in the interference group, and reached a significant level P < 0.001; on the fourth day of incubation of Large White pig sperm in the interference group and the blank group, the "sperm ATP content" in the blank group was greater than that in the interference group, and reached a significant level (P < 0.05).

[0183] Example 7: Flow cytometry detection of the effect of si-circ-CREBBP on the apoptosis rate of pig sperm

[0184] 1. Take 42 μL of the mixed system of blank group and interference group obtained in step 2 of Example 4 after electroporation and mix with 1×10 8 The spermatozoa from healthy Large White boars were mixed and incubated at 17℃ for 4 days.

[0185] 2. According to the operating procedures of the Annexin V-FITC Apoptosis Detection Kit, 100,000 sperm cells were collected for Annexin V and PI staining, and the sperm cell apoptosis rate was detected by flow cytometry. Since sperm motility is a more intuitive and good indicator of semen quality, the sperm survival rate was calculated as the phenotype according to the following formula: Sperm survival rate = 1 - sperm cell apoptosis rate.

[0186] See the results Figure 7 The results showed that on the second and fourth days of incubation of Large White pig sperm in the interference group and blank group, the "sperm survival rate" of the blank group was greater than that of the interference group, and reached a significant level (P<0.05).

[0187] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims. Sequence Listing <110> China Agricultural University <120> Application of circ-CREBBP in identifying or regulating porcine semen quality <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 ctgtcctgtt tgcctccctt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 cctctgacac ttgtgagcgt 20 <210> 3 <211> 21 <212> RNA <213> Artificial Sequence <400> 3 gcgaaaccaa caaaucucat t 21 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <400> 4 ugagauuugu ugguuucgct t 21 <210> 5 <211> 355 <212> DNA <213> Artificial Sequence <400> 5 tctcagatgc aaacgtcggt gggaattgtg cccacgcagg cgattgccac gggcccgaca 60 gcagacccag agaagcgcaa actgattcag cagcagctgg tcctcctgct tcacgctcac 120 aagtgtcaga ggcgggaaca agccaatgga gaggttcgag cctgctccct cccacactgt 180 cgaaccatga aaaatgtcct gaatcacatg acacattgtc aggctgggaa agcctgtcag 240 gttgcccatt gtgcatcttc acgacaaatc atctctcatt ggaagaactg cacgcgacat 300 gactgtcctg tttgcctccc tttgaaaaat gccagtgaca agcgaaacca acaaa 355

Claims

1. Use of circ-CREBBP or circ-CREBBP as a marker in any of the following A1)-A8): A1) Preparation of products for identifying or assisting in identifying the quality of pig semen; A2) preparing products for selecting or assisting in selecting pigs with high or low semen quality; A3) Prepare products for screening or assisting in screening high-quality or low-quality pig semen; A4) Preparation of products for regulating pig semen quality; A5) Preparation of products for regulating porcine sperm motility; A6) Preparation of products for regulating ATP content in porcine sperm cells; A7) Preparation of products for regulating porcine sperm survival or apoptosis rate; A8) Preparation of pig breeding products; The nucleotide sequence of the circ-CREBBP is shown in SEQ ID No.

5.

2. Use of a substance for detecting circ-CREBBP expression in any of the following B1)-B3): B1) Preparation of products for identifying or assisting in identifying the quality of pig semen; B2) preparing products for selecting or assisting in selecting pigs with high or low semen quality; B3) preparing products for screening or assisting in screening high-quality or low-quality pig semen; The nucleotide sequence of the circ-CREBBP is shown in SEQ ID No.

5.

3. The use according to claim 2, characterized in that: The substance for detecting the expression level of circ-CREBBP is a reagent and / or instrument for detecting the expression level of circ-CREBBP.

4. The use according to claim 3, characterized in that: The reagent for detecting the expression level of circ-CREBBP includes a primer pair consisting of a single-stranded DNA molecule shown in SEQ ID No. 1 and a single-stranded DNA molecule shown in SEQ ID No.

2.

5. The use according to any one of claims 1 to 4, characterized in that: The pig is a Large White boar.

6. Use of a substance that inhibits circ-CREBBP expression in any of the following C1)-C5): C1) Preparation of products that reduce the quality of pig semen; C2) Preparation of products for reducing sperm motility in pigs; C3) preparing a product for reducing the ATP content in pig sperm cells; C4) preparing products for reducing the survival rate of pig sperm or increasing the apoptosis rate of pig sperm; C5) Preparation of products for breeding pigs with low semen quality; The nucleotide sequence of the circ-CREBBP is shown in SEQ ID No. 5; The substance that inhibits circ-CREBBP expression is ssc-circ-CREBBP, the nucleotide sequence of the ssc-circ-CREBBP positive chain is shown in SEQ ID No. 3, and the nucleotide sequence of the ssc-circ-CREBBP antisense chain is shown in SEQ ID No.

4.

7. The use according to claim 6, characterized in that: The pig is a Large White boar.

8. Use any of the following methods D1)-D6): D1) A method for identifying or assisting in identifying pig semen quality, comprising the following steps: detecting the expression level of circ-CREBBP in seminal plasma extracellular vesicles or sperm of a pig to be tested, and determining the semen quality of the pig to be tested based on the circ-CREBBP expression level: the semen quality of pigs with high circ-CREBBP expression levels is higher than that of pigs with low circ-CREBBP expression levels; D2) A method for screening or assisting in screening pigs with high semen quality, comprising the following steps: detecting the expression level of circ-CREBBP in seminal plasma extracellular vesicles or sperm of the pigs to be tested, and selecting pigs with high circ-CREBBP expression levels; D3) A method for screening or assisting in screening pigs with low semen quality, comprising the following steps: detecting the expression level of circ-CREBBP in seminal plasma extracellular vesicles or sperm of the pigs to be tested, and selecting pigs with low circ-CREBBP expression; D4) A method for screening or assisting in screening high-quality porcine semen, comprising the following steps: obtaining pigs with high semen quality according to the method described in D2), and obtaining high-quality porcine semen from the pigs with high semen quality; D5) A method for screening or assisting in screening low-quality porcine semen, comprising the following steps: obtaining pigs with low semen quality according to the method described in D3), and obtaining low-quality porcine semen from the pigs with low semen quality; D6) A method for breeding pigs with low semen quality, reducing pig semen quality, reducing pig sperm motility, reducing pig sperm ATP content, reducing pig sperm survival rate, or increasing pig sperm apoptosis rate, comprising the step of reducing the expression of circ-CREBBP in pig sperm; The method is for non-disease diagnosis and treatment purposes.

9. The method according to claim 8, characterized in that: The pig is a Large White boar.

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