Application of AMH gene in regulation and control of spermatogenesis of plateau zokor

Abstract

The invention discloses application of an AMH gene in regulation and control of spermatogenesis of plateau zokor, and relates to the field of molecular biology. According to the research basis that AMH gene expression is up-regulated in individual testis of Platealax baileyi reproduction inhibition, the spermatogenesis condition of overexpression of the gene in testis of model animals is analyzed. Results show that AMH gene expression is closely related to spermatogenesis of animals. A recombinant 9-type adeno-associated virus vector carrying and expressing an AMH gene is injected into testis of a rat before adolescence (four weeks old), and it is observed that first wave sperm is delayed. The APP-CD74 axis is found to be an important signal channel for the AMH gene to regulate and control the abnormal sperm by using methods of single cell sequencing, immunofluorescence staining, in-vitro cell culture and the like. The function of the AMH gene in spermatogenesis is determined, and a new molecular target is provided for maintaining the population density of the plateau zokor based on a breeding regulation strategy.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and discloses a... RAW Application of genes in regulating spermatogenesis in highland mole rats. Specifically, this involves the testes. RAW Application of gene overexpression in delayed spermatogenesis. Background Technology

[0002] plateau zokor ( Eospalax baileyi As a small rodent endemic to the Qinghai-Tibet Plateau, the plateau mole rat constructs its underground burrow system through digging and piling. Under natural conditions, its activity accelerates soil nutrient cycling and improves soil aeration and permeability. However, against the backdrop of grassland degradation, its population density continues to increase, further exacerbating grassland degradation and posing a serious threat to the local ecological environment. Previous studies have found that some individuals do not participate in reproduction during the plateau mole rat's breeding season, a phenomenon known as reproductive suppression. Therefore, elucidating the regulatory mechanisms of reproductive suppression is crucial for maintaining the plateau mole rat population density based on reproductive regulation strategies.

[0003] Reproductive suppression refers to the active or passive delay or inhibition of reproductive activity by animals to improve their survival fitness; it is a natural phenomenon widely present in mammals. This reproductive strategy involves both social and non-social species and plays an important role in maintaining population stability and promoting adaptive evolution. Reproductive suppression includes behavioral and physiological suppression, with dysfunction of the hypothalamus-pituitary-gonadal (HPG) axis constituting the regulatory basis for physiological suppression. Hormone secretion disorders of the HPG axis leading to abnormal germ cell differentiation are a direct manifestation of reproductive suppression in animals. However, the regulatory mechanisms by which endocrine signaling modulates abnormal germ cell differentiation under reproductive suppression remain unclear.

[0004] The continuous production of sperm is crucial for maintaining male fertility. Spermatogenesis is a complex biological process involving transcriptional regulation of genes, behavioral changes in chromosomes, and precise coordination among various functional cell populations. Although significant progress has been made in understanding the regulatory mechanisms of spermatogenesis, many causes of abnormal spermatogenesis remain unclear. In this invention, the abnormal spermatogenesis in individuals under reproductive inhibition provides an ideal model for addressing this critical issue. Our existing results show that... RAW Gene expression was upregulated in the testes of reproductively suppressed individuals of the plateau mole rat and was associated with hormonal imbalances along the HPG axis and spermatogenesis arrest, suggesting... RAW This gene can serve as a key functional gene for regulating abnormal spermatogenesis under reproductive repression.

[0005] RAWThis gene is specifically expressed in Sertoli cells of the testes and granulosa cells of the ovary. Its protein product belongs to the TGF-β superfamily and plays a key role in male sex differentiation. Clinical studies have found that AMH levels significantly decrease after the onset of puberty and remain at low levels in adulthood. However, in patients with delayed puberty, significantly increased AMH levels may affect spermatogenesis by altering the testicular microenvironment. Therefore, [further research is needed]. RAW Using genes as key targets, this study reveals the functional role and regulatory mechanism of their overexpression in spermatogenesis, providing new molecular targets for maintaining the population density of plateau mole rats based on reproductive regulation strategies. Summary of the Invention

[0006] This invention provides RAW The application of this gene in regulating spermatogenesis in plateau mole rats, clarifying its functional role and regulatory mechanism in spermatogenesis, is of great significance for understanding the molecular mechanism of abnormal spermatogenesis under reproductive inhibition.

[0007] The technical solution adopted in this invention is as follows: One of the objectives of this invention is to provide RAW The application of genes in regulating spermatogenesis in plateau mole rats provides molecular targets for maintaining mole rat population density based on reproductive regulation strategies.

[0008] Further: for the amplification of the plateau mole rat RAW The primer pair for the gene is: upstream primer sequence: SEQ ID No. 2, downstream primer sequence: SEQ ID No. 3.

[0009] The second objective of this invention is to provide a testis. RAW Animal models of gene overexpression provide experimental tools for revealing the regulatory mechanism of this gene in spermatogenesis.

[0010] Furthermore: the method for constructing the animal model is as follows: 4-week-old rats are anesthetized with isoflurane, and rats carrying the expression... RAW Recombinant adeno-associated virus type 9 (AAV9-) RAW The vector was injected into both testes of animals at a dose of 3E10 μg / tube / testis. It was detected in the testes at 6 weeks of age. RAW Upregulated gene expression indicates the successful construction of an animal model of testicular overexpression of the target gene; this model was used to amplify the expression of the target gene in the rats. RAW The primer pair for the gene is: upstream primer sequence: SEQ ID No.4, downstream primer sequence: SEQ ID No.5.

[0011] The AAV9- RAWThe vector was constructed as follows: the target fragment was obtained by PCR amplification and ligated into the AAV9 cis plasmid. The plasmid, along with helper and packaging plasmids, was co-transfected into HEK293T cells using Lipofiter liposome transfection reagent. The resulting vector was then amplified and purified to obtain the vector carrying the expression. RAW AAV9 stock solution for gene amplification in rats; RAW The primer pair for the gene fragment is: upstream primer sequence: SEQ ID No. 6, downstream primer sequence: SEQ ID No. 7.

[0012] The third objective of this invention is to provide a RAW A testicular support cell model with gene overexpression provides an experimental tool for verifying that this gene regulates the expression of downstream target genes.

[0013] Furthermore: the method for constructing the cell model is as follows: [The text abruptly shifts to a seemingly unrelated topic about cell models.] RAW Lv-CMV- gene lentivirus RAW -T2A-Puro, abbreviated as Lv- RAW The vector was used to infect cultured Support cells at 2.5E8 TU / mL, MOI=20. After 48 h, the infected cells were treated with puromycin for 24 h, and uninfected cells were discarded. The virus was detected in the cells... RAW Upregulation of gene expression indicates that a cell model of target gene overexpression has been successfully constructed.

[0014] The Lv- RAW The vector was constructed as follows: the target fragment was obtained by PCR amplification and ligated into a lentiviral cis plasmid. The plasmid, along with helper and packaging plasmids, was co-transfected into HEK293T cells using Lipofiter liposome transfection reagent. The resulting vector was then amplified, purified, and used to express the target fragment. RAW Lentiviral stock solution for gene amplification in rats; RAW The primer pair for the gene fragment is: upstream primer sequence: SEQ ID No. 8, downstream primer sequence: SEQ ID No. 9.

[0015] The advantages of this invention are as follows: This invention discovers RAW Gene expression is closely related to spermatogenesis. Specifically, upregulation of this gene expression is associated with spermatogenesis arrest in plateau mole rats, while downregulation of this gene expression is associated with the initiation of spermatogenesis in rats. In the testes... RAW In animal models of gene overexpression, the occurrence of the first wave of sperm was delayed, and the APP-CD74 axis was found to be... RAW A crucial signaling pathway regulating abnormal spermatogenesis. Clarifying the testes... RAWThe functional role and regulatory mechanism of gene overexpression in spermatogenesis provide a new molecular target for maintaining the population density stability of plateau mole rats based on reproductive regulation strategies. Attached Figure Description

[0016] Figure 1 for RAW Analysis of the relationship between genes and spermatogenesis in plateau mole rats; (A) Testicular spermatogenesis in plateau mole rats at different developmental stages RAW (A) Gene expression; (B) Sperm count in the epididymal tail of plateau mole rat at different developmental stages; (C) HE staining of the testis and epididymis of plateau mole rat at different developmental stages; Figure 2 In this invention RAW Gene expression analysis in rat testes at different developmental stages; (A) Gene expression in rat testes at different developmental stages RAW (a) Gene expression of AMH receptor in the testis; (b) Immunohistochemical results of AMH receptor in the testis; (c) HE staining of the testis and epididymis at different developmental stages of rats; Figure 3 The overexpression in the testes of this invention RAW First wave of spermatogenesis in genetically delayed animals; (A) Schematic diagram of experimental animal treatment; (B) HE staining of the testes and epididymis of 6-week-old rats; (C) Analysis of the proportion of different spermatogenic cell types; (D) Fertility testing of 6-week-old rats; AAV9: Empty vector of adeno-associated virus type 9; AAV9- RAW : Carrying expression RAW Adeno-associated virus vector type 9 of the gene; Figure 4 The overexpression in the testes of this invention RAW Gene suppression supports cell proliferation; (A) Count of supporting cells; (B) Immunofluorescence staining of SOX9; (C) Cell cycle analysis; (D) Schematic diagram of experimental design for in vitro cell culture; (E) Cell cycle detection; (F) Detection of supporting cell proliferation. ISC: Immature supporting cells; MSC: Mature supporting cells; AAV9: Empty vector of adeno-associated virus type 9; AAV9- RAW : Carrying expression RAW Adeno-associated virus vector type 9 of the gene; Figure 5 The overexpression in the testes of this invention RAW Genes cause abnormal differentiation and increased apoptosis of spermatogonia. (A) Spermatogonia cell clustering; (B) Spermatogonia cell frequency analysis; (C) Pseudo-temporal analysis of spermatogonia; (D) Immunofluorescence staining with STRA8 and TUNEL; (E) Statistical analysis of apoptotic spermatogonia. AAV9: Empty vector of adeno-associated virus type 9; AAV9- RAW : Carrying expression RAWAdeno-associated virus vector of type 9 gene; SPG_1: type I spermatogonia; SPG_2: type II spermatogonia; SPG_3: type III spermatogonia; SPG_4: type IV spermatogonia; Figure 6 The APP-CD74 axis in this invention is RAW By supporting important signaling pathways for regulating abnormal spermatogonial differentiation: (A) Signal input in different cell types; (B) Signal output in different cell types; (C) Common probability analysis of receptor-ligand pairs in the APP signaling pathway; (D) CD74 Gene expression levels in the pseudo-chronic differentiation trajectory of spermatogonia; (E) Immunofluorescence staining of APP and SOX9; (F) Immunofluorescence staining of CD74 and STRA8; AAV9: empty vector of adeno-associated virus type 9; AAV9- RAW : Carrying expression RAW Adeno-associated virus vector type 9 of the gene; Figure 7 The in vitro validation results of APP and CD74 expression in this invention are as follows: (A) RAW Relative gene expression levels; (B) CD74 (C) Relative gene expression levels; (D) Protein expression levels of p-smad5 and APP; (E) Schematic diagram of experimental design for in vitro cell culture; (V) Protein expression level of CD74; Lv-control: Empty lentiviral vector; Lv- RAW : Carrying expression RAW Lentiviral vectors of genes. Detailed Implementation

[0017] To clearly illustrate the technical features of the present invention, the present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods reported in the literature. Unless otherwise specified, the instruments, reagents, and materials in the following embodiments can be obtained commercially.

[0018] Due to the imperfections in indoor breeding techniques for plateau mole rats, this invention selects the rat as a model animal for further research. RAW Gene function verification. This was achieved by constructing a testis. RAW Animal models of gene overexpression, immunofluorescence staining, single-cell sequencing, and in vitro cell culture were used to clarify the functional role and regulatory mechanism of this gene in spermatogenesis, which is of great significance for understanding the molecular mechanism of abnormal spermatogenesis under reproductive inhibition.

[0019] Example 1, RAW Analysis of the relationship between gene expression and spermatogenesis at different developmental stages of plateau mole rats 1.1 Preparation of Experimental Materials Based on the capture time and testicular coefficient of the plateau mole rat, the reproductive status of the mole rat was divided into four periods: peak reproductive period, degenerative period, end of reproductive period and non-reproductive period, with each stage ensuring sample replication of 6 individuals.

[0020] 1.2, Test Methods 1) Based on the plateau mole rat RAW Primer pairs were designed based on the gene's nucleotide sequence: upstream primer sequence SEQ ID No. 2, downstream primer sequence SEQ ID No. 3, as shown in Table 1. The plateau mole rat... RAW The nucleotide sequence of the gene is shown in SEQ ID No. 1, obtained from transcriptome sequencing data, provided through the GenBank database https: / / www.ncbi.nlm.nih.gov / nuccore / , Bio Project sequence number: PRJNA1178356.

[0021] Detected using real-time quantitative PCR technology RAW Gene expression in the testes at different developmental stages.

[0022] Table 1 2) Transfer 6 mL of physiological saline into a 10 mL centrifuge tube and preheat to 37°C. Anesthetize the plateau mole rat with isoflurane, and quickly surgically remove one epididymis. Place the tail in the centrifuge tube and thoroughly mince it. Let it stand at 37°C for 15 minutes to allow complete sperm release. Place one drop of the diluted suspension on a cell counting plate for counting. Use a standard optical microscope to count the total number of sperm (N) in 5 squares. The sperm count per milliliter of suspension is calculated as follows: Sperm count (per milliliter) = N × 5 × dilution factor × 10 4 .

[0023] 3) HE staining was used to observe the histological structure of the testes and epididymis of plateau mole rats at different developmental stages.

[0024] 4) Results are shown Figure 1 During the developmental process of the plateau mole rat from its breeding season to its non-breeding season, RAW Upregulated gene expression is associated with spermatogenesis arrest. HE staining revealed abnormalities in the degenerative phase of spermatogenesis in the testes, and detached spermatogenic cells were observed in the epididymal tail. RAW Gene expression begins to be upregulated in vestigial testes.

[0025] Example 2, RAW Analysis of the relationship between gene expression and rat spermatogenesis 2.1 Preparation of Experimental Materials Rats aged 0 days, 2 weeks, 4 weeks, 6 weeks, 8 weeks, and 10 weeks were selected as experimental subjects. Testicular and epididymal tissues were collected from animals of each age group, with 6 individuals in each age group being replicated.

[0026] 2.2, Test Methods 1) Designing rats RAW Gene (rat) RAW The nucleic acid sequence of gene NM_012902.2 was obtained from the GenBank database (https: / / www.ncbi.nlm.nih.gov / nuccore / ). The primer pair was as follows: upstream primer sequence SEQ ID No. 4, downstream primer sequence SEQ ID No. 5, as shown in Table 2. Detection was performed using real-time quantitative PCR technology. RAW The relative expression levels of the gene in the testes of rats at 0 d, 2 weeks, 4 weeks, 6 weeks, 8 weeks and 10 weeks of age.

[0027] Table 2 2) HE staining was used to observe the histological structure of the testes and epididymis of 0-day, 2-week-old, 4-week-old and 6-week-old rats.

[0028] 3) Results are shown in Figure 2 In rat testes RAW Gene expression is downregulated at 4 weeks of age. Histological observation of the testes and epididymis reveals that spermatogenesis begins in the testes during this period.

[0029] Example 3, Overexpression in the testes RAW The Influence of Genes on Spermatogenesis and Individual Fertility 3.1 Preparation of Experimental Materials Based on the histological observations of the testes and epididymis in Example 2, 4-week-old rats were selected as experimental subjects. This is because animals at this age are pre-pubertal and spermatogenesis has begun, making them an ideal age for constructing testes. RAW The optimal period for gene overexpression.

[0030] 3.2, Test Methods 1) Based on rats RAW The gene NM_012902.2 is shown RAW The primer pair designed to amplify the target fragment using the gene nucleic acid sequence is as follows: upstream primer sequence SEQ ID No. 6, downstream primer sequence SEQ ID No. 7, as shown in Table 3. The target fragment was obtained by PCR amplification and ligated into an adeno-associated virus type 9 (AAV9) cis plasmid. Using Lipofiter liposome transfection reagent, the above plasmid, helper plasmid, and packaging plasmid were co-transfected into HEK293T cells. The fragment was then amplified, purified, and used to express the target fragment. RAWAAV9 mother liquor of the gene.

[0031] Table 3 2) After anesthetizing 4-week-old rats with isoflurane, the experimental group was treated with AAV9- RAW Bilateral testicular injections of AAV9 (3E10 μg / tube / testis) were performed, while the control group received bilateral testicular injections of empty AAV9 (3E10 μg / tube / testis). All animals were raised to 6 weeks of age and then anesthetized with isoflurane. Testicular tissue was rapidly surgically removed from each individual, and the levels of AAV9 in the testes of 6-week-old rats were detected. RAW The upregulation of gene expression indicates the successful construction of an animal model of testicular gene overexpression. Specifically, the rats used for amplification... RAW The primer pair for the gene is: upstream primer sequence: SEQ ID No.4, downstream primer sequence: SEQ ID No.5.

[0032] 3) Fertility testing was performed on 4-week-old animals that had completed the injection. Specifically, the empty AAV9 group (2 animals) and the AAV9- RAW Two male rats were housed together with eight healthy adult female rats, with each male paired with two females, for two weeks. During the experiment, the rats were provided with ample water and food. The number of offspring born to each female was recorded and compared with a control group at the same developmental stage to assess their reproductive capacity. RAW The impact of genes on fertility at the individual level.

[0033] 4) Testicular tissue from 6-week-old rats was fixed with 10% formaldehyde solution. Histological changes in the testes and epididymis were observed under a microscope using HE staining, and spermatogenesis within the seminiferous tubules was analyzed. The percentage of different types of seminiferous tubules in the testes of the experimental and control groups was statistically analyzed.

[0034] 5) Results are shown Figure 3 Seminiferous tubules were classified into three types—complete spermatogenesis, incomplete spermatogenesis, and azoospermia—using HE staining. The percentage of each type in the testes was then calculated, revealing AAV9- RAW Vector injection resulted in a small percentage of seminiferous tubules undergoing complete spermatogenesis, and most of these were concentrated in the incomplete spermatogenesis type, thus determining the testis. RAW Gene overexpression delays spermatogenesis and testes RAW Gene overexpression resulted in infertility in 6-week-old rats.

[0035] Example 4, Overexpression in the testes RAW Analysis of the mechanism by which genes cause delayed spermatogenesis 4.1 Preparation of Experimental Materials Overexpression in 6-week-old rats RAW Single-cell sequencing was performed on testes from both the gene-producing and control groups, with three replicates per group. Sequencing data was provided by Guangzhou GeneDio Biotechnology Co., Ltd. The single-cell sequencing results were then validated in vitro using testicular cells.

[0036] 4.2, Test Methods 1) Use marker genes for testicular cell types to identify different cell clusters in a single-cell atlas and screen out Sertoli cells and spermatogonia clusters.

[0037] 2) Based on typical marker factors, Sertoli cells and spermatogonia are classified into subcellular populations. Sertoli cells are divided into immature Sertoli cells and mature Sertoli cells, and spermatogonia are divided into type I spermatogonia, type II spermatogonia, type III spermatogonia and type IV spermatogonia.

[0038] 3) Utilize cell frequency analysis, immunofluorescence, and cell cycle detection to explore... RAW The effect of gene overexpression on supporting cell proliferation.

[0039] 4) Monocle2 was used to construct the pseudo-chronological differentiation trajectory of spermatogonia, and immunofluorescence staining was used to determine the apoptosis status of the cells.

[0040] 5) Screening using cell communication analysis RAW Genes support potential signaling pathways that regulate abnormal spermatogonial differentiation.

[0041] 6) According to NM_012902.2, the rat RAW Primer pairs for amplifying the target fragment were designed based on the gene nucleic acid sequence. The upstream primer sequence is SEQ ID No. 8, and the downstream primer sequence is SEQ ID No. 9, as shown in Table 4. The target fragment was obtained by PCR amplification and ligated into a lentiviral cis plasmid. Using Lipofiter liposome transfection reagent, the above plasmid, helper plasmid, and packaging plasmid were co-transfected into HEK293T cells. The fragment was then amplified, purified, and expressed. RAW Lentiviral mother liquor of the gene, hereinafter referred to as Lv- RAW Carrier.

[0042] Table 4 7) Mouse testicular Sertoli cells and spermatogonia, which are well-suited for culture, were selected for in vitro validation experiments. The cell culture conditions are shown in Table 5. Specific methods included... RAW The vector was used to infect cultured Support cells at 2.5E8 TU / mL, MOI=20. After 48 h, the infected cells were treated with puromycin for 24 h, and uninfected cells were discarded. The virus was detected in the cells... RAW Upregulated gene expression indicates the successful construction of a cell model for overexpression of the target gene. Among these, the gene used to amplify the target gene... RAW The primer pair for the gene is: upstream primer sequence: SEQ ID No. 4, downstream primer sequence: SEQ ID No. 5. For spermatogonial cell culture, the gene expressing... RAW The supernatant of supporting cells and complete culture medium for spermatogonia were mixed at a 1:1 ratio, and the cells were cultured for 24 h before the experiment. Cell samples were collected after the above experiment, and the expression of APP in supporting cells and CD74 in spermatogonia was detected, respectively.

[0043] Table 5 GC-1 spg CL-0600 DMEM high glucose+10%FBS+1%P / S 8) Results are shown Figure 4-7 . RAW Gene overexpression inhibited the proliferation of Sertoli cells in mice, while also causing abnormal spermatogonial differentiation and increased apoptosis. RAW The gene upregulates CD74 expression in spermatogonia by activating APP signaling in supporting cells. Therefore, the APP-CD74 axis has been found to serve as a mechanism for... RAW An important signaling pathway regulating spermatogenesis. APP The primer pair for (NM_001198823.1) is: upstream primer sequence SEQ ID No. 10, downstream primer sequence SEQ ID No. 11, as shown in Table 6; Design CD74 The primer pair for (NM_001042605.1) is: upstream primer sequence SEQ ID No.12 and downstream primer sequence SEQ ID No.13, as shown in Table 6.

[0044] Table 6 The above embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.

Claims

1. AMH Application of genes in regulating spermatogenesis of plateau zokor (Eolagurus floccidus) 2. Use according to claim 1, characterized in that, primers for amplifying the gene of the plateau zokor AMH The primer pair for amplifying the gene of the plateau zokor is: the upstream primer sequence: SEQ ID No. 2, and the downstream primer sequence: SEQ ID No.

3.

3. A testis AMH Animal models with overexpression of a gene are used to analyze the role of the gene in regulating spermatogenesis.

4. Use according to claim 3, characterized in that, The animal model was constructed as follows: 4-week-old rats were anesthetized with isoflurane, and rats carrying the expression... AMH Recombinant adeno-associated virus type 9 (AAV9-) AMH The vector was injected into both testes of animals at a dose of 3E10 μg / tube / testis. It was detected in the testes at 6 weeks of age. AMH Upregulated gene expression indicates the successful construction of an animal model of testicular overexpression of the target gene; this model was used to amplify the expression of the target gene in the rats. AMH The primer pair for the gene is: upstream primer sequence: SEQ ID No.4, downstream primer sequence: SEQ ID No.

5.

5. The method of claim 4, wherein, The AAV9- AMH The AAV9 mother liquor carrying the expression of the rat AMH gene is constructed by the method of obtaining the target fragment by PCR amplification and connecting to the AAV9 cis plasmid, co-transfecting the above-mentioned plasmid, auxiliary plasmid and packaging plasmid into the tool cell HEK293T by using Lipofiter liposome transfection reagent, and obtaining the AAV9 mother liquor carrying the expression of the rat AMH The primer pair for amplifying the rat gene fragment is: the upstream primer sequence is SEQ ID No. 6, and the downstream primer sequence is SEQ ID No.

7.

6. A kind AMH The testicular Sertoli cell model with gene overexpression was used to validate the application of this gene in regulating the expression of downstream target genes in vitro.

7. Use according to claim 6, characterized in that, The method for constructing the cell model is as follows: [The text abruptly shifts to a seemingly unrelated topic about cell models.] AMH Lv-CMV- gene lentivirus AMH -T2A-Puro, abbreviated as Lv- AMH The vector was used to infect cultured Support cells at 2.5E8 TU / mL, MOI=20. After 48 h, the infected cells were treated with puromycin for 24 h, and uninfected cells were discarded. The virus was detected in the cells... AMH Upregulation of gene expression indicates that a cell model of target gene overexpression has been successfully constructed.

8. Use according to claim 7, characterized in that, The Lv- AMH The construction method of the vector is: obtaining the target fragment by PCR amplification and connecting to the lentivirus cis-plasmid, co-transfecting the above-mentioned plasmid, auxiliary plasmid and packaging plasmid into the tool cell HEK293T by using Lipofiter liposome transfection reagent, and obtaining the lentivirus mother liquor carrying the expression AMH gene of the rat by amplification and purification; the lentivirus mother liquor is used for amplifying the rat AMH The primer pair for amplifying the gene fragment is: the upstream primer sequence is SEQ ID No. 8, and the downstream primer sequence is SEQ ID No. 9.

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