Differential genes related to black goat reproduction and detection method and application
By screening and verifying the expression differences of miR-novel-535 and GAB2, their regulatory relationship in the reproductive traits of Yunshang black goats was revealed, and detection and regulation methods were provided, thereby improving the reproductive capacity of high-yielding black goats.
Patent Information
- Application Number
- CN202310056012.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-01-17
- Publication Date
- 2025-10-10
- Estimated Expiration
- 2043-01-17
AI Technical Summary
In Yunshang black goats, the gene regulatory mechanism affecting fertility is still unclear, especially the functional relationship between miR-novel-535 and GAB2 has not been fully elucidated, resulting in no obvious differences in reproductive traits between high- and low-yielding goats.
The differentially expressed genes miR-novel-535 and GAB2 were screened through transcriptome sequencing, and specific primer pairs were designed to detect their expression levels. The qPCR method was used to verify their differential expression in high-yielding and low-yielding black goats. The software was further used to predict that miR-novel-535 targets the 3′-UTR of GAB2 and regulates its expression level.
It was confirmed that miR-novel-535 was significantly lowly expressed in high-yielding black goats, while GAB2 was significantly highly expressed in high-yielding black goats. Regulating GAB2 expression by miR-novel-535 mimics or inhibitors can theoretically improve the reproductive performance of Yunshang black goats.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biological breeding, and in particular relates to differential genes related to black goat reproduction, a detection method and an application thereof. Background Art
[0002] Many factors influence goat litter size and ovulation, but the genes that significantly influence goat fertility remain largely unknown. High- and low-yielding breeds are characterized by different estrous cycles. Therefore, identifying the molecular mechanisms underlying follicular development has been a key focus of goat genetics and breeding research. In recent years, research on goat fertility mechanisms has primarily focused on candidate genes and their regulatory factors, such as miRNAs, transcription factors, and various regulatory pathways. Furthermore, numerous factors influence goat reproductive capacity, including follicle recruitment, development, selection, and ovulation. Signaling pathways, such as the Wnt / β-catenin and bone morphogenetic protein (BMP) / Smad pathways, are involved in follicular development. The Yunshang Black Goat is a premium meat-producing goat breed native to Yunnan Province, my country, characterized by tender meat and a unique flavor. Despite its high fertility, the molecular mechanisms regulating its reproductive traits remain largely unknown.
[0003] Growth factor receptor-binding protein 2 (GAB2) is a conjugate protein belonging to the Gab family. This protein contains binding sites for signaling molecules, such as SH2 domain protein tyrosine phosphatase-2 (SHP2) and phosphatidylinositol 3-kinase (PI3K). These sites act downstream of membrane receptors for several cytokines, antigens, hormones, and growth factors, regulating multiple signaling pathways. Studies have reported that the interaction between GAB2 and PI3K regulates the PI3K-AKT signaling pathway, participating in multiple regulatory pathways such as cell proliferation, differentiation, apoptosis, and glucose transport. However, the regulatory elements and regulatory relationships of the GAB2 gene in the ovaries of Yunshang black goats remain unclear. Summary of the Invention
[0004] The purpose of the present invention is to provide differentially expressed genes related to black goat reproduction, as well as a detection method and application thereof, and to confirm that miR-novel-535 and GAB2 have a functional relationship and can affect the reproductive traits of black goats through mutual regulation.
[0005] The present invention provides differential genes related to black goat reproduction, wherein the differential genes include miR-novel-535 and GAB2;
[0006] The nucleotide sequence of miR-novel-535 is shown in SEQ ID NO.1.
[0007] The application also provides a set of primer pairs for detecting the expression amount of the difference gene, including primer pairs miR-novel-535-F and miR-novel-535-R for miR-novel-535, wherein the nucleotide sequence of the miR-novel-535-F is shown as SEQ ID NO. 2, and the nucleotide sequence of the miR-novel-535-R is shown as SEQ ID NO. 3.
[0008] The primer pairs GAB2-F and GAB2-R for GAB2, wherein the nucleotide sequence of the GAB2-F is shown as SEQ ID NO. 4, and the nucleotide sequence of the GAB2-R is shown as SEQ ID NO. 5.
[0009] The application also provides a method for evaluating the reproductive traits of black goats, including the following steps: detecting the expression amount of the difference gene by using the primer pairs, and the expression amount of miR-novel-535 in the genome of the high-yield black goat is significantly lower than that of the low-yield black goat; and the expression amount of GAB2 in the genome of the high-yield black goat is significantly higher than that of the low-yield black goat.
[0010] Preferably, the expression amount of the difference gene is detected by using the method of qPCR, and the procedure of the qPCR includes: 95℃ pre-denaturation for 5s; 95℃ denaturation for 5s, 60℃ annealing for 30s, and 40 cycles.
[0011] Preferably, the goat RPL19 is used as a correction gene, and the primer pairs designed according to the correction gene include RPL19-F and RPL19-R, wherein the nucleotide sequence of the RPL19-F is shown as SEQ ID NO. 6, and the nucleotide sequence of the RPL19-R is shown as SEQ ID NO. 7.
[0012] The application also provides the use of a reagent for regulating the expression of the GAB2 gene in the preparation of a product for affecting the reproductive traits of black goats.
[0013] Preferably, the reagent for regulating the expression of the GAB2 gene includes one or more of miR-novel-535, a mimic of miR-novel-535 and / or an inhibitor of miR-novel-535.
[0014] The nucleotide sequence of the miR-novel-535 is shown as SEQ ID NO. 1.
[0015] The application also provides the use of miR-novel-535 or a mimic of miR-novel-535 in the preparation of a preparation for reducing the expression of the GAB2 gene, wherein the nucleotide sequence of the miR-novel-535 is shown as SEQ ID NO. 1.
[0016] The present invention also provides use of an inhibitor of miR-novel-535 in preparing a preparation for enhancing GAB2 gene expression. The nucleotide sequence of the miR-novel-535 is shown in SEQ ID NO.1.
[0017] The present invention also provides the use of miR-novel-535 and / or GAB2 in breeding high-yielding black goats. The nucleotide sequence of the miR-novel-535 is shown in SEQ ID NO.1.
[0018] Beneficial effects: The present invention screened out the differentially expressed gene GAB2 and miR-novel-535 (referred to as miR-535) related to the reproduction of Yunshang black goats through transcriptome sequencing. The present invention also designed a detection primer pair for the above-mentioned differentially expressed genes, and confirmed that the expression level of miR-535 in high-yield Yunshang black goats was significantly lower than that in low-yield Yunshang black goats (P<0.05); the expression level of the GAB2 gene in high-yield Yunshang black goats was significantly higher than that in low-yield Yunshang black goats (P<0.05). In the embodiment of the present invention, the software also predicted the binding site with the target gene GAB2, that is, miR-535 targets the 3′-UTR of the GAB2 gene and inhibits its expression level. The present invention determines the regulatory relationship between miR-novel-535 and GAB2, and elaborates and explains the discovery and regulatory relationship of the candidate gene GAB2 related to the reproductive traits of Yunshang black goats and its key regulatory element miR-novel-535. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0020] Figure 1 The expression results of miR-novel-535 in the ovaries of high- and low-yielding Yunshang black goats;
[0021] Figure 2 This is the expression result of GAB2 gene in the ovaries of high- and low-yielding Yunshang black goats;
[0022] Figure 3 This is the result of dual fluorescence activity detection of miR-novel-535 and GAB2 genes. DETAILED DESCRIPTION
[0023] The present invention provides differential genes related to black goat reproduction, wherein the differential genes include miR-novel-535 and GAB2;
[0024] The nucleotide sequence of miR-novel-535 is shown in SEQ ID NO.1: ucccgggcgggacgagacg.
[0025] The present invention screened the differentially expressed gene GAB2 (XM_018043071.1) and miR-novel-535 related to the reproduction of Yunshang black goats through transcriptome sequencing. The expression level of miR-novel-535 in high-yield Yunshang black goats was significantly lower than that in low-yield Yunshang black goats (P<0.05); the expression level of the GAB2 gene in high-yield Yunshang black goats was significantly higher than that in low-yield Yunshang black goats (P<0.05), and software predicted and confirmed that miR-novel-535 targeted the 3′-UTR of the GAB2 gene and inhibited the expression level of the GAB2 gene.
[0026] The present invention also provides a set of primer pairs for detecting the differential gene expression, including a primer pair miR-novel-535-F and miR-novel-535-R designed for miR-novel-535, wherein the nucleotide sequence of miR-novel-535-F is shown in SEQ ID NO. 2, and the nucleotide sequence of miR-novel-535-R is shown in SEQ ID NO. 3;
[0027] The primer pair GAB2-F and GAB2-R were designed for GAB2, the nucleotide sequence of GAB2-F is shown in SEQ ID NO.4, and the nucleotide sequence of GAB2-R is shown in SEQ ID NO.5.
[0028] Table 1 Gene names and primer sequences
[0029]
[0030]
[0031] The present invention also provides a method for evaluating the reproductive traits of black goats, comprising the following steps: using the above-mentioned primer pairs to detect the expression levels of the above-mentioned differential genes, the expression level of miR-novel-535 in the genome of high-yield black goats is significantly lower than that of low-yield black goats; and the expression level of GAB2 in the genome of high-yield black goats is significantly higher than that of low-yield black goats.
[0032] The present invention preferably uses the qPCR method to detect the expression level of the differentially expressed genes. The qPCR system is constructed in 20 μL, preferably including: 0.8 μL each of 10 μmol / μL upstream and downstream primers, 10 mL SYBR Green qPCR, 50 ng / μL cDNA2 mL, and 6.4 μL ddH2O. The cDNA of the present invention is preferably obtained by reverse transcription of RNA extracted from fresh goat ovarian tissue. When performing the qPCR detection of the present invention, goat RPL19 is preferably used as the correction gene. The primer pair designed based on the correction gene includes RPL19-F and RPL19-R. The nucleotide sequence of RPL19-F is shown in SEQ ID NO.6, and the nucleotide sequence of RPL19-R is shown in SEQ ID NO.7. The qPCR program of the present invention preferably includes: pre-denaturation at 95°C for 5s; denaturation at 95°C for 5s, annealing at 60°C for 30s, and 40 cycles.
[0033] The present invention also provides an application of a reagent for regulating the expression of the GAB2 gene in the preparation of a product affecting the reproductive traits of black goats.
[0034] The reagent for regulating GAB2 gene expression of the present invention preferably comprises one or more of miR-novel-535 and mimics of miR-novel-535; the nucleotide sequence of miR-novel-535 is shown in SEQ ID NO. 1. The nucleotide sequence of the mimics of the present invention is preferably shown in SEQ ID NO. 9: UCCCCGGGCGGGACGAGACG.
[0035] The present invention also provides use of miR-novel-535 and miR-novel-535 mimics in preparing a preparation for reducing GAB2 gene expression. The nucleotide sequence of miR-novel-535 is shown in SEQ ID NO.1.
[0036] In the examples of the present invention, experiments confirmed that miR-novel-535 targets the 3′-UTR of the GAB2 gene and inhibits the expression level of the GAB2 gene. The GAB2 gene is a candidate gene related to the reproductive traits of Yunshang black goats and is significantly highly expressed in high-yielding Yunshang black goats. Therefore, by inhibiting the expression of the GAB2 gene through miR-novel-535, the breeding performance of the Yunshang black goats can theoretically be inhibited.
[0037] The present invention also provides use of an inhibitor of miR-novel-535 in preparing a preparation for enhancing GAB2 gene expression. The nucleotide sequence of the miR-novel-535 is shown in SEQ ID NO.1.
[0038] The present invention also provides the use of miR-novel-535 and / or GAB2 in breeding high-yielding black goats. The nucleotide sequence of the miR-novel-535 is shown in SEQ ID NO.1.
[0039] In the present invention, it is preferred to reduce the expression of miR-novel-535 to achieve the purpose of breeding high-yielding black goats.
[0040] To further illustrate the present invention, the differential genes related to black goat reproduction, the detection method and the application provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.
[0041] Example 1
[0042] 1.1 Sample collection
[0043] Tissue samples for this study were obtained from farms in Honghe Hani and Yi Autonomous Prefecture, Yunnan Province. All Yunshang Black goats were of similar age and weight, bred using random mating methods, and maintained under identical housing and nutritional conditions. Ovarian tissue was collected from five high- and five low-yielding goats (mean litter size was 3.00 ± 0.38 in the high-yielding group and 1.32 ± 0.19 in the low-yielding group), placed in liquid nitrogen, and then stored at -80°C for long-term storage.
[0044] 1.2 RNA extraction and RT-qPCR
[0045] RNA was extracted using the Animal Tissue / Cell RNA Extraction Kit from Beijing Tiangen Biochemical Technology Co., Ltd. according to the instructions for the reagent. After extraction, the concentration and quality of the RNA sample were detected by NanoDrop 2000 and 1.2% agarose gel. Then cDNA was obtained according to the instructions of the Reversal Kit (Takara, Japan). RT-qPCR was performed using cDNA as a template. The RT-qPCR system was 20 μL: 0.8 μL each of 10 μmol / μL upstream and downstream primers, 10 mL SYBR Green qPCR, 50 ng / μL cDNA 2 mL, 6.4 μL ddH2O. The qPCR program was: 95°C pre-denaturation for 5s, 95°C for 5s, 60°C for 30s, and 40 cycles. Goat RPL19 was used as the correction gene. The relative template amount algorithm (2 -△△CT ) The relative expression levels of each gene were calculated. RT-qPCR primers were designed using Primer 5 software and synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequences are shown in Table 1.
[0046] 1.3 Vector construction and cell transfection
[0047] The mimic (SEQ ID NO. 9) and mimic blank control (mimic NC, nonsense sequence) of miR-novel-535 were designed and synthesized based on the sequence of miR-novel-535. The mimic and mimic NC were synthesized at Beijing Xinnuo Jinda Biotechnology Co., Ltd.
[0048] 293T cells were cultured at 1×10 6 Cells were seeded at a density of 100 cells / well in a 6-well plate and 2 mL of complete DMEM (DMEM) was added to each well. When the cell density reached 70% to 80%, transfection was performed. The culture medium was discarded and the cells were washed twice with 2 mL of PBS (Gibco, USA).
[0049] For transfection of a 12-well plate, the following system was used: 121 μL of Opti-MEM and 4 μL of Lipofectamine 2000 reagent were thoroughly mixed to create Tube A. 115 μL of Opti-MEM and 10 μL of 200 nmol / μL vector (miR-novel-535mimic, miR-novel-535mimic NC) were thoroughly mixed to create Tube B, and the mixture was allowed to stand for 5 minutes. Tube B was added to Tube A, mixed thoroughly, and allowed to stand for 20 minutes before being added to a 6-well plate. 1.5 mL of Opti-MEM was then added. Three replicates were set up for each group, and the culture medium was switched to complete DMEM 6 hours after transfection.
[0050] 1.5 Dual luciferase activity assay
[0051] According to the sequence provided by NCBI, the GAB2 gene 3′-UTR-WT (SEQ ID NO.10: atccctaggacagaagcacaacggtTCTCaGCCTGcCCCGGGGctaaggcccccagctgtggccacccc a) and 3′-UTR-MUT (SEQ ID NO.11: atccctaggacagaagcacaacggtAGAGaCGGACcGGGCCCCctaaggcccccagctgtggccacccca) were synthesized (Shanghai Jima Pharmaceutical Technology Co., Ltd.) and connected to the psiCHECK2 vector (linker site: / Xhol). After screening and sequencing confirmation, positive clones were co-transfected with miR-novel-535 mimic and mimic NC into 293T cells. After 48 hours of culture, the cells were collected into a 1.5 mL centrifuge tube, centrifuged at 1500 rpm for 5 minutes, resuspended in 75 μL PBS per well, and transferred to a 96-well white cell culture plate. 75 μL of firefly luciferase was added and the fluorescence value was measured using a multifunctional microplate reader; after adding 75 μL of Renilla salina, the fluorescence value was measured.
[0052] 2.1 Expression of miR-novel-535 and GAB2 genes in ovarian tissue of Yunshang black goats
[0053] To clarify the expression levels of miR-novel-535 and GAB2 genes in the ovarian tissues of high- and low-yielding Yunshang black goats, RT-qPCR technology was used to detect the relative expression levels of miR-novel-535 and GAB2 genes. The results showed that the expression level of miR-novel-535 in high-yielding Yunshang black goats was significantly lower than that in low-yielding Yunshang black goats (P<0.05). Figure 1 ); The expression level of GAB2 gene in high-yield Yunshang black goats was significantly higher than that in low-yield Yunshang black goats (P<0.05) ( Figure 2 ).
[0054] 2.2 miR-novel-535 targets the 3′-UTR of the GAB2 gene
[0055] In this study, Target Scan and miRnda online software were used to predict the binding sites of miR-novel-535, and GAB2 was initially screened as a candidate target gene. To further confirm the targeting relationship between miR-novel-535 and GAB2, psiCHECKTM Vector-GAB2-WT and psiCHECKTM Vector-GAB2-MUT dual-luciferase reporter gene vectors were constructed and co-transfected with miR-novel-535 mimics and mimics NC into 293T cells. The dual-luciferase activity test results are shown in Figure 2. Figure 3 As shown in the figure, the dual luciferase activity of the GAB2-WT+miR-novel-535 mimic co-transfection group was significantly lower than that of the other three groups (P<0.05). In summary, miR-novel-535 targets the 3′-UTR of the GAB2 gene and inhibits its expression level.
[0056] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.
Claims
1. Differential genes related to black goat reproduction, characterized in that: The differential genes include miR-novel-535 and GAB2 ; The nucleotide sequence of miR-novel-535 is shown in SEQ ID NO.1, and the GAB2 The Genbank sequence number is XM_018043071.
1.
2. A set of primer pairs for detecting the differential gene expression of claim 1, characterized in that: The invention comprises a primer pair miR-novel-535-F and miR-novel-535-R designed for miR-novel-535, wherein the nucleotide sequence of miR-novel-535-F is shown in SEQ ID NO.2, and the nucleotide sequence of miR-novel-535-R is shown in SEQ ID NO.3; against GAB2 The designed primer pair is GAB2-F and GAB2-R, the nucleotide sequence of GAB2-F is shown in SEQ ID NO.4, and the nucleotide sequence of GAB2-R is shown in SEQ ID NO.
5.
3. A method for evaluating the reproductive traits of black goats, characterized in that: The following steps are involved: The primer pair described in claim 2 was used to detect the expression level of the differential gene described in claim 1. The expression level of miR-novel-535 in the genome of high-yield black goats was significantly higher than that in low-yield black goats; GAB2 The expression level of β-catenin was significantly lower than that of low-yielding black goats.
4. The method according to claim 3, characterized in that The expression levels of the differentially expressed genes were detected using a qPCR method, wherein the qPCR procedure included: pre-denaturation at 95°C for 5 s; denaturation at 95°C for 5 s, annealing at 60°C for 30 s, and 40 cycles.
5. The method according to claim 4, characterized in that: Goat RPL19 To correct the gene, the primer pair designed according to the correction gene includes RPL19-F and RPL19-R, the nucleotide sequence of RPL19-F is shown in SEQ ID NO.6, and the nucleotide sequence of RPL19-R is shown in SEQ ID NO.
7.
6. A regulation GAB2 The invention relates to a method for preparing a product for influencing the reproductive traits of black goats by using a gene expression reagent, wherein: The regulation GAB2 The gene expression agent is miR-novel-535 and / or a mimic of miR-novel-535; The nucleotide sequence of miR-novel-535 is shown in SEQ ID NO.1, the nucleotide sequence of the mimic is shown in SEQ ID NO.9, and the GAB2 The Genbank sequence number is XM_018043071.
1.
7. Preparation of miR-novel-535 and miR-novel-535 mimics to reduce GAB2 Use of a gene expression preparation, characterized in that The nucleotide sequence of miR-novel-535 is shown in SEQ ID NO.1, the nucleotide sequence of the mimic is shown in SEQ ID NO.9, and the GAB2 The Genbank sequence number is XM_018043071.
1.
8. Utilization of miR-novel-535 and / or GAB2 The application of the method in breeding high-yield black goats is characterized in that: The nucleotide sequence of miR-novel-535 is shown in SEQ ID NO.1, and the GAB2 The Genbank sequence number is XM_018043071.1.