Method for observing dynamic changes of testis development of bama miniature pigs

Abstract

The invention discloses a bama miniature pig testis development dynamic change observation method which comprises the following steps: (1) collecting testis tissues, namely collecting the testis tissues of a bama miniature pig in 65-105 days of an embryonic period and 0-300 days after birth, determining various morphological indexes of the testis in different development stages, and observing the tissue characteristics of the testis through HE staining, measuring the reproductive hormone levels of different ages in days after birth; (2) deeply researching spermatogonium cells, supporting cells and interstitial cells in the bama miniature pig testis by adopting a molecular biological marker and combining immunofluorescence and RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) technologies; (3) performing transcriptome sequencing analysis on the testicular tissues at different development stages; and (4) constructing an eukaryotic expression vector through cDNA clone gene segments of the Bama miniature pig testis tissues, and performing bioinformatics analysis on the FRS2 gene sequence. The method has the advantages that testis development conditions of the bama miniature pigs in different development stages can be conveniently known, and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and in particular relates to a method for observing dynamic changes in testicular development of Bama miniature pigs. Background Art

[0002] Bama Xiang pig is a small pig breed in Guangxi, my country. It is produced in Bama Yao Autonomous County. Because of its black ends and white in the middle, locals also call it "two-headed black", "winter melon pig" and "banana pig". Bama Xiang pig has distinctive meat characteristics such as rich intramuscular fat, tender meat, fat but not greasy, and rich unsaturated fatty acids. It is sought after by consumers and has the reputation of mountain treasure civet cat. It is a superior raw material for preparing roast suckling pig and whole cured pig, and is famous for its rich meat flavor. Bama Xiang pig is small in size, with an average weight of 36.50kg for adult sows. Bama Xiang pig also has the characteristics of strong adaptability, tolerance to roughage, and strong disease resistance. However, due to the inconvenient transportation in its production area, local people mostly use the breeding method of keeping the son and mating the mother for breeding. Under the influence of long-term inbreeding and local feeding conditions, its gene homozygosity is high, invisible unfavorable genes are gradually eliminated, and genetic performance is stable, which strengthens the characteristics of early puberty, small body and fragrant meat.

[0003] Bama Xiang pigs reach sexual maturity early, with the average age of first mating for boars being 75.75±2.60 days. The average age of sexual maturity for sows is 120.08±2.31 days, and for boars is 67±8.40 days. The age of first mating for sows is mostly between 5 and 6 months old, with an average of 159.28±20.60 days, and the average estrus cycle for sows is 19.92±1.20 days[2]. Its reproductive characteristics of precocious puberty also make it a good material for studying the genetic mechanism of precocious puberty.

[0004] The current research content on the testicular development of Bama miniature pigs in Guangxi mainly focuses on: using second-generation sequencing, 10×Genomics and third-generation sequencing technology to sequence and assemble the testicular tissues of local pigs of different pig breeds, different estrus stages and different ages, and identify differentially expressed genes, RNA molecules, proteins and related pathways related to the testicular development of local pigs.

[0005] The above research plan mainly aims to identify and analyze the expression of different RNAs in the testicular tissues of foreign pig breeds and some local pigs, and to establish a library. The analysis found that these target factors are mainly enriched in the GO and KEGG signaling pathways related to testicular development and spermatogenesis, which provides a reference for the testicular development of local pigs. Summary of the invention

[0006] In view of the above problems, the present invention provides a method for observing the dynamic changes of testicular development in Guangxi Bama miniature pigs.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for observing the dynamic changes of testicular development in Bama miniature pigs, comprising the following steps:

[0009] (1) Testicular tissue was collected from Bama miniature pigs at embryonic stage 65-105 days and postnatal stage 0-300 days, and various morphological indicators of the testis at different developmental stages were measured. The tissue characteristics were observed by HE staining, and the reproductive hormone levels at different postnatal ages were measured.

[0010] (2) Using molecular biological markers, combined with immunofluorescence and RT-qPCR techniques, we conducted an in-depth study on spermatogonia, supporting cells, and interstitial cells in the testis of Bama miniature pigs;

[0011] (3) Transcriptome sequencing analysis of testicular tissue at different developmental stages;

[0012] (4) The cDNA fragment of the testicular tissue of Bama miniature pigs was cloned to construct a eukaryotic expression vector, and the FRS2 gene sequence was subjected to bioinformatics analysis.

[0013] Furthermore, the morphological indicators include the long diameter, short diameter, thick diameter and weight of the testicle.

[0014] Furthermore, the reproductive hormones include gonadotropin-releasing hormone, luteinizing hormone, follicle-stimulating hormone, and ketones.

[0015] Furthermore, in the step (1), the developmental period of the Bama miniature pig testicles is set to 15 periods, namely 65, 90, 105 days in the embryonic period and 0, 30, 35, 40, 45, 60, 90, 120, 150, 180, 300 days after birth and 6 years after birth; and the Bama miniature pig testicular tissue corresponding to each developmental period is collected.

[0016] Furthermore, the 15 developmental periods were divided into five developmental stages, and the core genes affecting testicular development in each stage were screened out; among them, the core genes in the mid-embryo period included COL1A1, COL1A2, COL3A1, BGN, and SPP1; the core genes in the late embryo period included SOX9, GATA4, AMH, CXCL12, and CXCL10; the core genes initiated in the first puberty period included CDK1, KIF4A, BUB1B, TOP2A, and DAZL; the core genes in the sexual maturity period included SPATA19, NME8, DNAH1, AKAP4, and SPA17; the core genes in the old age period included CYP11A1, CYB5A, FDX1, EGFR, and SREBF1.

[0017] Furthermore, in step (3), the gene expression FPKM value is obtained by standardizing the transcriptome sequencing data of testicular tissue of Bama miniature pigs at different developmental stages, and the weighted gene co-expression network analysis is completed with the help of the WGCNA shiny plug-in based on the R package in TBtools.

[0018] Furthermore, the weighted gene co-expression network analysis method comprises the following steps:

[0019] a. Upload the gene expression matrix and set the sample percentage threshold to 0.5 and the expression value threshold to 1 in the initial screening. Then use the median absolute deviation-based method for secondary screening, and finally retain 12,000 genes for subsequent analysis;

[0020] b. Use the sample outgroup value detection function to build a sample clustering tree to detect and remove outlier samples;

[0021] c. To construct a scale-free co-expression network, when selecting the topological threshold, β=20 was calculated as the optimal soft threshold under the premise of satisfying the scale-free property;

[0022] d. When constructing the gene module network, the minimum module size is set to 50 and the module merging and trimming height is set to 0.25, thereby generating a hierarchical clustering tree network topology. The gene modules are recombined according to the merging height to obtain a more stable gene cluster;

[0023] e. The correlation coefficient r between the characteristic genes of the module and different growth ages was obtained. The closer |r| is to 1 and the smaller the P-value is, the more significant the correlation between the genes in the module and the different growth ages of the testicles of Bama miniature pigs is.

[0024] Furthermore, the molecular biological markers include molecular markers of porcine undifferentiated spermatogonia, proliferative cell markers, molecular markers expressed in the cytoplasm of germ cells, molecular markers of testicular supporting cells, and molecular markers of testicular interstitial cells.

[0025] Furthermore, in step (4), the construction of the eukaryotic expression vector includes

[0026] (1) Total RNA: extract total RNA from testicular tissue;

[0027] (2) Obtaining the target fragment cDNA, reverse-transcribe the extracted total RNA from testicular tissue, and the cDNA obtained by this reverse transcription reaction can be directly used as a template for PCR reaction;

[0028] (3) Design of cloning primers, and connection of the target fragment with the empty plasmid by homologous recombination;

[0029] (4) Cloning technology: plasmids are obtained through cloning technology, and double enzyme digestion of plasmids is performed. The correct plasmids are identified and stored at -80℃.

[0030] Furthermore, the FRS2 gene regulates the development of interstitial cells in the Bama miniature pigs and the secretion of testosterone, affecting precocious puberty.

[0031] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0032] The present invention uses testicular tissues of Guangxi Bama miniature pigs at 15 developmental time points, based on histomorphological observation, to determine the serum reproductive hormone content of Bama miniature pigs after birth, and simultaneously uses immunofluorescence and RT-qPCR techniques to observe and quantify different types of cells in the testis, judge the developmental dynamics of different types of cells, and use transcriptome sequencing technology (RNA-seq) to reveal the expression characteristics of testicular development-related genes, and explore the developmental dynamics and gene expression characteristics of the testis from the levels of tissue, hormone, gene, etc. RT-qPCR, immunohistochemistry and other methods are further used to obtain that the FRS2 gene affects testicular development, interstitial cell secretion of testosterone and precocious puberty. It can lay a theoretical basis for analyzing the genetic basis of precocious puberty traits in local pigs in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic diagram of the technical route of the present invention.

[0034] Figure 2 The present invention summarizes the dynamic changes of testicular development of Bama miniature pigs.

[0035] Figure 3 Schematic diagram of the construction of the eukaryotic expression vector of FRS2 gene of Bama miniature pig. In the figure, A: gel electrophoresis of FRS2 gene fragment and linearized vector fragment; B: double restriction enzyme verification of recombinant plasmid pEGFP-N1-FRS2; M in figure A: DL5000Maeker; 1, 2, 3, 4: FRS2 CDS fragment; 5, 6, 7, 8: pEGFP-N1 linearized vector; M in figure B: DL5000Maeker; 1: different configuration fragments of empty plasmid pEGFP-N1; 2, 3, 4, 5, 6, 7, 8: double restriction enzyme fragments of recombinant plasmid pEGFP-N1-FRS2.

[0036] Figure 4 This is a schematic diagram of the cloning and sequence analysis of the CDS region of the FRS2 gene of the Bama miniature pig. In the figure, A: PCR amplification product of the FRS2 CDS region; B: nucleotide and amino acid sequences of the FRS2 CDS region of the Bama miniature pig; C: open reading frame (ORF) analysis; (D) prediction of the conserved domain M: DL2000Maeker; 1, 2: FRS2 RT-PCR products. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below by way of examples. These examples are only used to illustrate the present invention and do not limit the protection scope of the present invention.

[0038] like Figure 1-4 As shown, a method for observing the dynamic changes of testicular development in Bama miniature pigs comprises the following steps:

[0039] (1) Testicular tissues were collected from Bama miniature pigs at embryonic days 65 to 105 and days 0 to 300 after birth. The morphological indices of the testes at different developmental stages were measured. The tissue characteristics were observed by HE staining, and the reproductive hormone levels at different days after birth were measured.

[0040] Morphological parameters included the long diameter, short diameter, thick diameter and weight of the testis.

[0041] Reproductive hormones include gonadotropin-releasing hormone, luteinizing hormone, follicle-stimulating hormone, and ketones.

[0042] It should be noted that the Bama miniature pig is a miniature pig breed unique to Guangxi, with the characteristics of small size, early puberty, and genetic stability.

[0043] The developmental period of the testis of Bama miniature pigs was set to 15 periods, namely 65, 90, 105 days in the embryonic period and 0, 30, 35, 40, 45, 60, 90, 120, 150, 180, 300 days after birth and 6 years after birth; and the testicular tissues of Bama miniature pigs corresponding to each developmental period were collected.

[0044] It should be noted that the corresponding numbers of the 15 developmental times are:

[0045] Bama miniature pigs were at embryonic stages 65d (E65), 90d (E90), and 105d (E105).

[0046] 0 days after birth (D0), 30 days (D30), 35 days (D35), 40 days (D40), 45 days (D45), 60 days (D60), 90 days (D90), 120 days (D120), 150 days (D150), 180 days (D180), 300 days (D300), and 6 years (Y6).

[0047] (2) Molecular biological markers, combined with immunofluorescence and RT-qPCR techniques, were used to conduct in-depth research on spermatogonia, supporting cells and interstitial cells in the testis of Bama miniature pigs. Molecular biological markers include molecular markers of undifferentiated spermatogonia, proliferative cell markers, molecular markers expressed in the cytoplasm of germ cells, molecular markers of testicular supporting cells, and molecular markers of testicular interstitial cells.

[0048] It is understandable that the molecular markers of porcine undifferentiated spermatogonia include UCHL1; the proliferative cell markers include Ki-67 and PCNA; the molecular markers expressed in the cytoplasm of porcine germ cells include DDX4; the molecular markers of porcine testicular supporting cells include SOX9; and the molecular markers of testicular interstitial cells include CYP17A1.

[0049] (3) Transcriptome sequencing analysis of testicular tissue at different developmental stages. The FPKM values ​​of gene expression were obtained after normalization of transcriptome sequencing data of testicular tissue at different developmental stages of Bama miniature pigs, and weighted gene co-expression network analysis was completed using the WGCNA shiny plug-in based on the R package in TBtools.

[0050] Transcriptome sequencing analysis includes strand-specific transcriptome sequencing, fluorescence quantitative PCR verification, RNA-seq data quality control statistics, sample correlation and cluster analysis, differential analysis and differential gene enrichment analysis, time series analysis based on differential genes, weighted network co-expression analysis, and time series analysis and WGCNA combined screening of core genes in different developmental stages.

[0051] (4) The cDNA fragment of the testicular tissue of Bama miniature pigs was cloned to construct a eukaryotic expression vector, and the FRS2 gene sequence was subjected to bioinformatics analysis.

[0052] The FRS2 gene regulates the development of Leydig cells and testosterone secretion in Bama miniature pigs and affects precocious puberty.

[0053] In some embodiments of the present disclosure, a method for constructing a eukaryotic expression vector is provided, and the construction method comprises

[0054] (1) Total RNA: extract total RNA from testicular tissue;

[0055] (2) Obtaining the target fragment cDNA, reverse-transcribe the extracted total RNA from testicular tissue, and the cDNA obtained by this reverse transcription reaction can be directly used as a template for PCR reaction;

[0056] (3) Design of cloning primers, and connection of the target fragment with the empty plasmid by homologous recombination;

[0057] (4) Cloning technology: plasmids are obtained through cloning technology, and double enzyme digestion of plasmids is performed. The correct plasmids are identified and stored at -80℃.

[0058] The cloning techniques are as follows:

[0059] (1) Use cloning primers to perform ordinary PCR amplification of the target fragment. The reaction system and reaction procedure are as follows: PCR reaction system: Taq enzyme 25μL, FRS2_L1527_F 2.5μL, FRS2_L1527_R 2.5μL, cDNA 5μL, RNase FreedH2O 15μL.

[0060] The PCR cycle reaction program is as follows:

[0061]

[0062] (2) The amplified product was subjected to 1.5% agarose gel electrophoresis to detect whether it was the desired target fragment, and the PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing and identification. If the target fragment was identified to be correct, the TaKaRa Mini BEST Agarose Gel DNA Extraction Kit Ver.4.0 (Code No.9762) was used to recover and purify the target fragment.

[0063] (3) The pEGFP-N1 vector was double-digested using HindⅢ and KpnⅠ enzymes and buffer M in a PCR instrument at 37℃ for 1h30min to form a linear vector with sticky ends at both ends and the pEGFP-N1 linearized fragment (Cat. No. 9762) was recovered and purified.

[0064] (4) Prepare the recombination reaction system. The amount of vector fragment and insert fragment used is calculated according to the instructions for optimal ligation reaction. After reacting at 37°C for 30 minutes, immediately cool on ice.

[0065] The reaction system is as follows:

[0066]

[0067] (5) Thaw cloning competent cells (DH5α Competent cells, Vazyme#C502) on ice;

[0068] (6) Add 10 μL of the recombinant product to 100 μL of Trans 5α competent cells, mix well, place on ice for 30 min, incubate in a 42°C water bath for 45 s, cool on ice for 2 min, add 900 μL of SOC medium, shake the cells at 37°C (250 rpm) for 1 h, centrifuge at 5000 rpm for 5 min, discard 900 μL of supernatant, resuspend the cells, spread them evenly on an LA plate containing kanamycin using a sterile spreader, place the cells upright in a 37°C incubator for 0.5 h, and then invert and culture overnight.

[0069] (7) Pick several single colonies from the LA plate and add them to 50 μL of enzyme-free sterile water. Shake well to prepare several bacterial suspensions.

[0070] (8) Perform cross-primer PCR on bacterial liquid using FRS2_L1527_F as the upstream primer and EGFP-N (universal primer for vector) as the downstream primer or CMV-F (universal primer for vector) as the upstream primer and FRS2_L1527_R as the downstream primer, select the positive bacterial liquid for amplification and sequencing.

[0071] The primer sequences are shown below:

[0072]

[0073] (9) Add 50 μL of the correctly identified bacterial solution into a conical flask containing 250 mL of LB medium (with kanamycin added at a ratio of 1:1000 for antibiotics: medium) and shake overnight at 37°C at 200 rpm. Take 1 mL of the bacterial solution at a ratio of 1:1 for bacterial solution: 50% glycerol and store at -80°C.

[0074] (10) The endotoxin-free plasmid for transformation was extracted using the Endo-Free Plasmid Mega Kit D6228.

[0075] (11) The extracted plasmid is subjected to double enzyme digestion identification, and the enzyme digestion steps are the same as (3). The correctly identified plasmid is stored at -80°C.

[0076] The present invention successfully cloned the CDS sequence of the FRS2 gene from the testicular tissue of Bama miniature pigs and constructed its eukaryotic expression vector. The CDS is 1527 bp in length and encodes 508 amino acids in total. The gene was subjected to bioinformatics analysis, real-time fluorescence quantitative PCR, Western Blot protein detection and immunohistochemical analysis, and the expression trend and spatial expression characteristics of the gene at different testicular developmental times at the transcription and translation levels were obtained.

[0077] (1) The full length of the FRS2 gene CDS is 1527 bp, and it is predicted to encode an unstable extracellular hydrophilic protein. The amino acid sequence shows a high degree of evolutionary conservation among species, and it is predicted that the protein is highly expressed in testicular Leydig cells and ovarian granulosa cells.

[0078] (2) The results of FRS2 gene expression analysis showed that the gene was mainly expressed in the gonadal axis tissues; in the testis, the FRS2 gene was mainly expressed in the cytoplasm and cell membrane of interstitial cells, a small amount in spermatogonia, various spermatogenic cells and sperm, and not in spindle muscle cells and supporting cells.

[0079] (3) The expression trend of FRS2 gene transcription level was significantly positively correlated with the expression trend of steroid hormone synthesis-related genes, and there was a significantly positive correlation between FRS2 protein and CYP17A1 protein as well as testicular physical parameters.

[0080] It is understandable that the FRS2 gene plays a role in regulating the development of testicular interstitial cells and testosterone secretion in Bama miniature pigs, affecting precocious puberty.

[0081] In some embodiments of the present disclosure, a weighted gene co-expression network analysis method is provided, comprising the following steps:

[0082] a. Upload the gene expression matrix and set the sample percentage threshold to 0.5 and the expression value threshold to 1 in the initial screening. Then use the median absolute deviation-based method for secondary screening, and finally retain 12,000 genes for subsequent analysis;

[0083] b. Use the sample outgroup value detection function to build a sample clustering tree to detect and remove outlier samples;

[0084] c. To construct a scale-free co-expression network, when selecting the topological threshold, β=20 was calculated as the optimal soft threshold under the premise of satisfying the scale-free property;

[0085] d. When constructing the gene module network, the minimum module size is set to 50 and the module merging and trimming height is set to 0.25, thereby generating a hierarchical clustering tree network topology. The gene modules are recombined according to the merging height to obtain a more stable gene cluster;

[0086] e. The correlation coefficient r between the characteristic genes of the module and different growth ages was obtained. The closer |r| is to 1 and the smaller the P-value is, the more significant the correlation between the genes in the module and the different growth ages of the testicles of Bama miniature pigs is.

[0087] In the present invention, transcriptome chain-specific library sequencing was performed on testicular tissues of Bama miniature pigs at 15 different developmental stages, and core genes at each developmental stage were screened out through sample correlation analysis, principal component analysis, differential expression analysis, time series analysis, weighted gene co-expression network analysis (WGCNA) and enrichment analysis.

[0088] Specifically, the 15 developmental periods were divided into five developmental stages, and the core genes affecting testicular development in each stage were screened out; among them, the core genes in the mid-embryo period included COL1A1, COL1A2, COL3A1, BGN, and SPP1; the core genes in the late embryo period included SOX9, GATA4, AMH, CXCL12, and CXCL10; the core genes initiated in the puberty period included CDK1, KIF4A, BUB1B, TOP2A, and DAZL; the core genes in the sexual maturity period included SPATA19, NME8, DNAH1, AKAP4, and SPA17; the core genes in the old age period included CYP11A1, CYB5A, FDX1, EGFR, and SREBF1. It is understandable that the core genes of the five developmental stages are not limited to this.

[0089] The five developmental stages are: E65 (mid-embryo), D90-D0 (late-embryo), D30-D45 (onset of puberty), D60-D300 (sexual maturity), and Y6 (old age).

[0090] The core genes of the five developmental stages are shown in Table 1.

[0091] Table 1 Core genes of Bama miniature pig testis at different developmental stages

[0092]

[0093] The changes in FPKM expression levels of core genes in the mid-embryo (E65) during the five developmental stages are shown in Table 2 below.

[0094] Table 2 Changes in FPKM expression of core genes in mid-embryo (E65)

[0095]

[0096] Table 2 Changes in FPKM expression of core genes in mid-embryo (E65)

[0097]

[0098] The changes in FPKM expression levels of core genes in the late embryonic period (E90-D0) in the five developmental stages are shown in Table 3 below.

[0099] Table 3 Changes in FPKM expression of core genes in late embryonic period (E90-D0)

[0100]

[0101] Table 3 Changes in FPKM expression of core genes in late embryonic period (E90-D0)

[0102]

[0103] The changes in FPKM expression levels of the core genes at the onset of puberty (D30-D45) in the five developmental stages are shown in Table 4 below.

[0104] Table 4 Changes in FPKM expression of core genes during the onset of puberty (D30-D45)

[0105]

[0106] Table 6 Changes in FPKM expression of core genes during the onset of puberty (D30-D45)

[0107]

[0108] The changes in FPKM expression levels of core genes in the sexual maturity period (D60-D300) among the five developmental stages are shown in Table 6 below.

[0109] Table 6 Changes in FPKM expression of core genes during sexual maturity (D60-D300)

[0110]

[0111] Table 6 Changes in FPKM expression of core genes during sexual maturity (D60-D300)

[0112]

[0113] The changes in FPKM expression levels of core genes in the sexual maturity period (D60-D300) among the five developmental stages are shown in Table 7 below.

[0114] Table 7 Changes in FPKM expression of core genes in the elderly (Y6)

[0115]

[0116] Understandably, the core genes screened out are all related to testicular tissue structure development, sex differentiation and somatic cell development, cell cycle and meiotic process, spermatogenesis and sperm development, and steroid hormone synthesis and secretion.

[0117] Based on the above, we can know that at E65, a small amount of seminiferous tubules and undifferentiated spermatogonia and supporting cells can be seen in the testis; undifferentiated spermatogonia have proliferation characteristics before birth, differentiate at D30 after birth, and their number increases rapidly from D0 to D30; supporting cells have proliferation characteristics from E90 to D0, and their number continues to increase from E90 to D30, and they have differentiated and matured after D30 and their number tends to be stable; mature interstitial cells appear at E90, and the expression level of interstitial cell marker genes is the highest at Y6 stage, and no expression of various germ cell and supporting cell marker genes is observed in Y6 period; sperm marker genes begin to be expressed at D40, and meiosis and spermatogonia differentiation marker genes begin to be expressed at D30.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for observing the dynamic changes of testicular development in Bama miniature pigs, characterized in that: The developmental dynamic change observation comprises the following steps: (1) Testicular tissue was collected from Bama miniature pigs at embryonic stage 65-105 days and postnatal stage 0-300 days, and various morphological indicators of the testis at different developmental stages were measured. The tissue characteristics were observed by HE staining, and the reproductive hormone levels at different postnatal ages were measured. (2) Using molecular biological markers, combined with immunofluorescence and RT-qPCR techniques, we conducted an in-depth study on spermatogonia, supporting cells, and interstitial cells in the testis of Bama miniature pigs; (3) Transcriptome sequencing analysis of testicular tissue at different developmental stages; (4) The cDNA fragment of the testicular tissue of Bama miniature pigs was cloned to construct a eukaryotic expression vector, and the FRS2 gene sequence was subjected to bioinformatics analysis.

2. The method for observing the dynamic changes of testicular development of Bama miniature pigs according to claim 1, characterized in that: The morphological indicators include the long diameter, short diameter, thick diameter and weight of the testis.

3. The method for observing the dynamic changes of testicular development of Bama miniature pigs according to claim 1 is characterized in that: The reproductive hormones include gonadotropin-releasing hormone, luteinizing hormone, follicle-stimulating hormone, and ketones.

4. The method for observing the dynamic changes of testicular development of Bama miniature pigs according to claim 1, characterized in that: In the step (1), the developmental period of the testicles of the Bama miniature pig is set to 15 periods, namely 65, 90, 105 days in the embryonic period and 0, 30, 35, 40, 45, 60, 90, 120, 150, 180, 300 days after birth and 6 years after birth; and the testicular tissues of the Bama miniature pigs corresponding to each developmental period are collected.

5. The method for observing the dynamic changes of testicular development of Bama miniature pigs according to claim 4 is characterized in that: The 15 developmental periods were divided into five developmental stages, and the core genes affecting testicular development at each stage were screened; Among them, core genes in the mid-embryo include COL1A1, COL1A2, COL3A1, BGN, and SPP1; The core genes in the late embryonic period include SOX9, GATA4, AMH, CXCL12, and CXCL10; The core genes activated during puberty include CDK1, KIF4A, BUB1B, TOP2A, and DAZL; The core genes in sexual maturity include SPATA19, NME8, DNAH1, AKAP4, and SPA17; The core genes in old age include CYP11A1, CYB5A, FDX1, EGFR, and SREBF1.

6. The method for observing the dynamic changes of testicular development of Bama miniature pigs according to claim 1, characterized in that: In the step (3), the gene expression FPKM value is obtained by standardizing the transcriptome sequencing data of testicular tissue of Bama miniature pigs at different developmental stages, and the weighted gene co-expression network analysis is completed with the help of the WGCNA shiny plug-in based on the R package in TBtools.

7. The method for observing the dynamic changes of testicular development in Bama miniature pigs according to claim 6, characterized in that: The weighted gene co-expression network analysis method comprises the following steps: a. Upload the gene expression matrix and set the sample percentage threshold to 0.5 and the expression value threshold to 1 in the initial screening. Then use the median absolute deviation-based method for secondary screening, and finally retain 12,000 genes for subsequent analysis; b. Use the sample outgroup value detection function to build a sample clustering tree to detect and remove outlier samples; c. To construct a scale-free co-expression network, when selecting the topological threshold, β=20 was calculated as the optimal soft threshold under the premise of satisfying the scale-free property; d. When constructing the gene module network, the minimum module size is set to 50 and the module merging and trimming height is set to 0.25, thereby generating a hierarchical clustering tree network topology. The gene modules are recombined according to the merging height to obtain a more stable gene cluster; e. The correlation coefficient r between the characteristic genes of the module and different growth ages was obtained. The closer |r| is to 1 and the smaller the P-value is, the more significant the correlation between the genes in the module and the different growth ages of the testicles of Bama miniature pigs is.

8. The method for observing the dynamic changes of testicular development of Bama miniature pigs according to claim 1, characterized in that: The molecular biological markers include molecular markers of pig undifferentiated spermatogonia, proliferative cell markers, molecular markers expressed in the cytoplasm of germ cells, molecular markers of testicular supporting cells, and molecular markers of testicular interstitial cells.

9. The method for observing the dynamic changes of testicular development in Bama miniature pigs according to claim 1, characterized in that: In step (4), the construction of the eukaryotic expression vector includes (1) Total RNA: extract total RNA from testicular tissue; (2) Obtaining the target fragment cDNA, reverse-transcribe the extracted total RNA from testicular tissue, and the cDNA obtained by this reverse transcription reaction can be directly used as a template for PCR reaction; (3) Design of cloning primers, and connection of the target fragment with the empty plasmid by homologous recombination; (4) Cloning technology: plasmids are obtained through cloning technology, and double enzyme digestion of plasmids is performed. The correct plasmids are identified and stored at -80℃.

10. The method for observing the dynamic changes of testicular development in Bama miniature pigs according to claim 1, characterized in that: The FRS2 gene regulates the development of interstitial cells in the Bama miniature pig and the secretion of testosterone, affecting precocious puberty.

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