Application of sheep ID2 gene in regulation and control of lambing traits of sheep

Abstract

The invention provides application of a sheep ID2 gene in regulation and control of lambing traits of sheep, and belongs to the technical field of gene functions. The invention finds that overexpression of the sheep ID2 gene can promote granulosa cell proliferation along with the increase of time, significantly down-regulate the expression of ID3, BMPR2, RBX1 and FSHR genes in cells, up-regulate the expression of ID1 gene, up-regulate the secretion of P4 progestational hormone and inhibit the secretion of E2 estradiol, and has a regulation effect on the lambing traits of sheep. The invention further provides a breeding method of the Hetian sheep with the multi-lambing character, a method for screening or breeding the Hetian sheep with the multi-lambing character and application of the Hetian sheep with the multi-lambing character, and economic benefits can be remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of gene function technology, and in particular to the application of sheep ID2 gene in regulating sheep lambing traits. Background Art

[0002] Fertility is one of the important economic indicators of sheep and directly affects the economic benefits of sheep. The number of lambs born by ewes is an important indicator to measure the fertility of sheep. Therefore, increasing the number of lambs born by sheep is very important for the development of sheep farming. Sheep lambing traits are affected by multiple factors such as genetics, hormones, environment, and nutrition. Ovulation rate and litter size are important reproductive traits of sheep and have high economic value. Reproductive traits usually have low to moderate heritability, so traditional breeding methods based only on phenotypic data will be a time-consuming process. Reproduction is a complex process. Traits such as ovulation rate and litter size are genetically affected by many genes and are the main internal factors affecting the number of lambs born in sheep. Related research on them continues to receive attention.

[0003] Hotan sheep is an excellent local sheep breed, with semi-coarse wool, famous for producing high-quality carpet wool and delicious meat. It is mainly distributed in Hotan area and is the main livestock breed in Hotan area. It is deeply loved by farmers, herdsmen and consumers. The exploration of genes related to the number of lambs born in Hotan sheep can improve the reproductive performance of Hotan sheep, promote the protection and utilization of Hotan sheep, and is of great significance to the breeding of Hotan sheep. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide the use of sheep ID2 gene and its encoded protein and related biological materials in regulating sheep lambing traits.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides application of sheep ID2 gene in regulating sheep lambing traits, and the nucleotide sequence of the sheep ID2 gene is shown as SEQ ID NO:1.

[0007] The present invention also provides the use of the protein encoded by the sheep ID2 gene in regulating the lambing traits of sheep. The amino acid sequence of the protein encoded by the sheep ID2 gene is shown in SEQ ID NO:2.

[0008] The present invention also provides the use of biological materials containing sheep ID2 gene in regulating sheep lambing traits, the nucleotide sequence of the sheep ID2 gene is shown in SEQ ID NO: 1, and the biological materials include lentivirus, recombinant expression vector or engineered cells.

[0009] Preferably, the sheep ID2 gene positively regulates the proliferation of ovarian granulosa cells.

[0010] Preferably, the sheep ID2 gene downregulates the expression of ID3, BMPR2, RBX1 and FSHR genes, and upregulates the expression of ID1 gene.

[0011] Preferably, the overexpression of the sheep ID2 gene promotes the secretion of P4 progesterone and inhibits the secretion of E2 estradiol.

[0012] Preferably, the sheep are Hetian sheep.

[0013] The present invention also provides a method for breeding Hetian sheep with multi-lamb trait, and the method comprises overexpressing the ID2 gene of Hetian sheep.

[0014] The present invention also provides a method for screening or breeding Hetian sheep with the trait of multiple lambs, the method comprising: detecting the relative expression amount of the ID2 gene of the Hetian sheep, and the Hetian sheep with relatively high expression of the ID2 gene exhibit the trait of multiple lambs; the relative expression amount of the ID2 gene uses β-actin or GAPDH as an internal reference gene.

[0015] The present invention also provides application of the method in Hetian sheep breeding.

[0016] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0017] The present invention finds that overexpression of the sheep ID2 gene can promote the proliferation of granulosa cells over time, significantly downregulate the expression of the ID3, BMPR2, RBX1 and FSHR genes in the cells, upregulate the expression of the ID1 gene, upregulate the secretion of P4 progesterone, inhibit the secretion of E2 estradiol, and has a regulatory effect on the lambing traits of sheep, especially Hotan sheep. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the distribution of whole genome variation in Example 1. In the figure, a: CG content; b: number of genes; c: number of SNPs; d: number of Insertions; e: number of Deletions.

[0019] Figure 2 This is the SNP distribution in each window when the step length is 100 kb in the whole genome in Example 1.

[0020] Figure 3 This is the distribution of INDELs in each window when the step length of the whole genome is 100 kb in Example 1.

[0021] Figure 4 This is the principal component analysis result diagram in Example 1.

[0022] Figure 5 This is a diagram of the structure analysis results in Example 1.

[0023] Figure 6 This is a graph of the PI_ratio significance analysis results in Example 1, wherein the X-axis represents the chromosome position and the Y-axis represents the P value.

[0024] Figure 7 This is a graph of the FST significance analysis results in Example 1, wherein the X-axis represents the chromosome position and the Y-axis represents the P value.

[0025] Figure 8 This is the GO enrichment analysis of excellent candidate genes of Hotan sheep in Example 1, where the X-axis represents the degree of enrichment and the Y-axis represents the signal pathway.

[0026] Fig. 9 This is the KEGG enrichment analysis of excellent candidate genes of Hotan sheep in Example 1, where the X-axis represents the number of enriched genes and the Y-axis represents the signal pathway.

[0027] Fig.10 This is a protein interaction diagram of lambing-related genes in Example 1.

[0028] Fig.11 These are pictures of the ID2 gene overexpression vector transfected into Hotan sheep ovarian granulosa cells in Example 2, wherein the left picture is a fluorescent picture of an empty vector transfection (500 μm), and the right picture is a fluorescent picture of a lentivirus transfection (200 μm).

[0029] Fig.12 This is a diagram showing the effect of ID2 overexpression on cell proliferation in Example 2.

[0030] Fig.13 This is the effect of overexpression of the ID2 gene on the expression of the ID1 gene in Example 2.

[0031] Fig.14 This is the effect of overexpression of the ID2 gene on the expression of the ID3 gene in Example 2.

[0032] Fig.15 This is the effect of overexpression of the ID2 gene on BMPR2 gene expression in Example 2.

[0033] Fig.16 This is the effect of ID2 gene overexpression on SMAD7 gene expression in Example 2.

[0034] Fig.17 This is the effect of overexpression of the ID2 gene on RBX1 gene expression in Example 2.

[0035] Fig.18 This is the detection result of progesterone hormone (P4) after transfection of ID2 gene overexpression vector in Example 2.

[0036] Fig.19This is the detection result of estrogen (E2) after transfection of the ID2 gene overexpression vector in Example 2. DETAILED DESCRIPTION

[0037] The invention provides an application of a sheep ID2 gene, a protein encoded by the sheep ID2 gene or a biological material containing the sheep ID2 gene in regulating sheep lambing traits. The nucleotide sequence of the sheep ID2 gene is: ATGAAAGCCTTCAGTCCAGTGAGGTCCGTTAGGAAAAACAGCCTTTCGGACCACGGCCTGGGCATCTCCCGGAGCAAAACCCCGGTGGACGACCCGATGAGCCTGCTGTACAACATGAACGACTGCTACTCCAAGCTCAAGGAGCTGGTCCCCAGCATCCCGCAAAACA AGAAGGTGAGCAAGATGGAAATCCTGCAGCACGTCATCGACTACATCTTGGACTTGCAGATCGCGCTAGACTCGCACCCCACCATCGTTAGCCTGCACCACCAGCGACCCGGGCAGAGCCAGGCGTCCAGGACGCCGCTCACCACCCTCAACACCGACATCAGCATCCTGTCCTTGCAGGCTTCTGAATTCCCTTCTGAATTAATGTCAAATGACAGCAAAGCGCTCTGTGGCTGA, as shown in SEQ ID NO: 1. The amino acid sequence of the protein encoded by the sheep ID2 gene is: MKAFSPVRSVRKNSLSDHGLGISRSKTPVDDPMS LLYNMNDCYSKLKELVPSIPQNKKVSKMEILQHVIDYILDLQIALDSHPTIVSLHHQRPGQS QASRTPLTTLNTDISILSLQASEFPSELMSNDSKALCG, as shown in SEQ ID NO: 2. The biological material of the present invention includes a lentivirus, a recombinant expression vector or an engineered cell.

[0038] The present invention obtained 801Gb of sequence data by analyzing the genome sequences of single-lamb and twin-lamb Hotan sheep flocks, identified 31,864,651 high-quality single nucleotide polymorphism sites, and scanned 290 selected regions and 332 genes in the whole genome. Functional annotation and enrichment analysis of genes identified 13 genes related to lambing rate, which were enriched in signaling pathways such as TGF-β signaling pathway (BMPR2, ID2, SMAD7, THBS1, RBX1), renal cell carcinoma (PAK1, ELOC, PLCZ), non-homologous end joining (RAD50), NETs pathway (H2B, H4, H2A) and ABC transporter (ABCC4). Based on the selected 13 genes related to lambing rate, the present invention further selected the ID2 gene, and verified by constructing an ID2 gene lentiviral vector and transfecting it into sheep ovarian granulosa cells: the sheep ID2 gene positively regulates the proliferation of ovarian granulosa cells, that is, the overexpression of the sheep ID2 gene can promote the proliferation of granulosa cells over time; the overexpression of the sheep ID2 gene can significantly downregulate the expression of the ID3, BMPR2, RBX1 and FSHR genes in the cells, upregulate the expression of the ID1 gene, upregulate the secretion of P4 progesterone, inhibit the secretion of E2 estradiol, and have a regulatory effect on the lambing traits of sheep. The sheep of the present invention is preferably a Hetian sheep.

[0039] The present invention also provides a method for breeding Hetian sheep with multi-lamb trait, the method being to overexpress the ID2 gene of Hetian sheep. The method for overexpressing the ID2 gene of Hetian sheep of the present invention can adopt conventional methods in the art.

[0040] The present invention also provides a method for screening or breeding Hetian sheep with the trait of multiple lambs, the method comprising: detecting the relative expression amount of the ID2 gene of the Hetian sheep, and the Hetian sheep with relatively high expression of the ID2 gene exhibit the trait of multiple lambs; the relative expression amount of the ID2 gene uses β-actin or GAPDH as an internal reference gene.

[0041] The present invention also provides an application of the above method in Hetian sheep breeding. Through the breeding method of Hetian sheep with multiple lambs traits and / or the method of screening and breeding Hetian sheep with multiple lambs traits, Hetian sheep with multiple lambs traits are bred to improve economic benefits.

[0042] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0044] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0045] Example 1

[0046] Screening of genes related to lambing traits in Hotan sheep:

[0047] 1. Sample Cohort and DNA Extraction

[0048] The reproductive phenotypes of Hotan sheep, such as the number of lambs born, were recorded in detail and statistically analyzed. According to the lambing records, multiparous ewes in the group without known major gene mutations were divided into a twin-lamb group and a single-lamb group. The relationship between SNP loci and lambing was verified using 30 single-lamb Hotan sheep (with litter number records; DGA group) and 30 twin-lamb Hotan sheep (with litter number records; DGB group). To ensure the quality of library construction, the genomic DNA was tested using the following methods, including sample concentration, integrity and purity. Library construction was performed after the samples were qualified. The specific contents include: (1) Whether the sample appearance contains foreign matter. (2) Genomic DNA was extracted using the standard phenol / chloroform extraction method. (3) 0.75% agarose gel electrophoresis was used to detect DNA fragment size and the degree of DNA degradation. (4) NanoDrop One spectrophotometer was used to detect DNA purity and OD 260 / 280 The ratio is between 1.8 and 2.2, and there is no protein or visible impurities contamination. (5) The DNA concentration is detected by Qubit 3.0 fluorescence instrument, and the detection concentration is greater than 50 ng / μL and the total amount is greater than 2 μg.

[0049] 2. Library construction, library testing and sequencing

[0050] After the DNA sample is qualified, it is sheared using a Covaris ultrasonic crusher, and the sheared sample magnetic beads are used for fragment selection, so that the sample band is concentrated in 200-400bp. Then the entire library preparation is completed through steps such as end repair, A tail addition, sequencing adapter addition, purification, PCR amplification, and PCR product circularization.

[0051] After the library is constructed, Qubit 2.0 is used for preliminary quantification to dilute the library, and then the insert fragment of the library is detected using Agilent2100. After the insert fragment size meets expectations, the effective concentration of the library is accurately quantified using the Q-PCR method to ensure the quality of the library.

[0052] Qualified libraries are arranged for sequencing (DNBSEQ): Single-stranded circular DNA molecules are replicated through rolling circles to form a DNA nanoball (DNB) containing more than 300 copies. The obtained DNBs are added to the mesh holes on the chip using high-density DNA nanochip technology and sequenced using combined probe anchor polymerization technology (cPAS).

[0053] 3. Quality control, comparison and identification of SNP sites

[0054] Fastp software was used for quality control and data filtering, and the filtering criteria were as follows: (1) polyG and polyX were removed at the end of the read (minimum length was 10 bp). (2) N reads greater than 5 were deleted. (3) Reads with a base-to-base ratio lower than 15 and higher than 40% were removed. (4) Filtered reads with a length less than 15 were deleted. The BWA software package was used for alignment analysis, and the filtered Cleanreads were aligned to the reference genome. Samtools was used to convert the alignment results from the sam (SequenceAlignment / MAP) file to the sorted bam (binaryAlignment / Map) file, and samtools was used to remove duplicates from the sorted alignment results. The python script was used to calculate the alignment rate and coverage. GATK was used to detect variant sites. All variant sites were annotated using ANNOVAR. SNP and insertion and deletion mutation sites were filtered out using GATK. The following criteria were used for SNP filtering: QualByDepth (QD) < 2.0; Quality (QUAL) < 30.0; StrandOddsRatio (SOR) > 3.0; FisherStrand (FS) > 60.0; RMSMappingQuality (MQ) < 40.0; MappingQualityRankSumTest (MQRankSu) < -12.5; ReadPosRankSumTest (ReadPosRankSum) < -8.0. The following criteria were used for INDEL filtering: QD < 2.0; QUAL < 30.0; FS > 200.0; MQ < 40.0; ReadPosRankSum < -20.0. For GATK-filtered sites, sites with a minor allele frequency (maf) less than 3% and a genotype missing rate greater than 20% were excluded using vcftools.

[0055] 4. Population genetic structure analysis

[0056] plink software was used to prepare input files (.bed, .bim, .fam) and perform simple filtering (-geno 0.1 -maf0.01). Neighbor-joining trees were constructed using PHYLIP, and data were visualized using MEGA. Principal component analysis (PCA) was performed using plink to obtain the principal component values ​​for each sample. The R package scatterplot was then used to analyze the PCA data. Figure 3 d The principal component scatter plot was drawn to further investigate the population genetic structure. The population structure was inferred using ADMIXTURE software, and the number of genetic clusters K ranged from 2 to 5.

[0057] 5. Selectivity feature analysis and protein interaction analysis

[0058] To identify selection signals, genome-wide differentiation index (FST) and nucleotide diversity (π) were calculated by vcftools using a sliding window method (parameters: --fst-window-size 100,000 --fst-window-step 50000) for each interval between the two populations (100KB as window and 10KB as step size). To reduce the number of false positives, only regions with significant FST and very low or high θπ ratios (5% left tail and 95% right tail) were selected as candidate genomic regions. Plots were drawn using the R package cmplot. Gene functions of genes in the selected regions were annotated by alignment with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Then, genes associated with sheep litter size were screened by reviewing the literature. Protein interaction analysis of the screened genes was performed using STRING.

[0059] The results showed that a total of 801G of clean reads were obtained, among which an average of 126,339,773 reads were retained for each sample. Q20>97.35%, Q30>91.50%, and the GC content was stable between 41.50% and 44.219%. The alignment rate of the 60 samples was between 99.81% and 99.98%, the average sequencing depth was between 4.657X and 9.9X, and the coverage of the reference genome was between 94.932% and 98.963%. After mapping using SAMtools, 42,782,967 high-quality SNPs and 5,705,707 high-quality InDels were obtained, as shown in the following table. Figure 1 shown.

[0060] According to the distribution in chromosomes, it can be found that the InDels results are consistent with the distribution trend of SNPs, and both are most distributed in the intergenic region. In order to more clearly understand the distribution of sheep chromosome SNPs and InDels, a 100Kb window was set, and the density map of SNPs and InDels in each window was counted. The results are shown in Figure 2. Figure 2 and Figure 3 As shown in Figure 2, it can be seen that SNPs and InDels have good homogeneity.

[0061] The principal component analysis based on SNPs in autosomes showed Figure 4 As shown, there is no obvious structure in these samples, probably because these individuals come from the same large population. Using ADMIXTURE software to infer population structure shows that the most likely value of genetic cluster K is 2. The results are shown in Figure 5 shown.

[0062] Based on selective elimination analysis ( Figure 6-7 ), 332 selected genes were identified. Based on GO annotation, KEGG enrichment analysis ( Figure 8-Figure 9 ), and consulted the relevant references of 332 genes, and obtained 13 genes (BMPR2, ID2, SMAD7, PLCZ, PAK1, ELOC, RAD50, H2A, H2B, THBS1, RBX1, H4, ABCC4) related to litter size, which were enriched in TGF-β signaling pathway (BMPR2, ID2, SMAD7, THBS1, RBX1), inositol phosphate metabolism, renal cell carcinoma (PAK1, ELOC, PLCZ), non-homologous end joining (RAD50), ABC transporter (ABCC4) and NETs pathway (H2B, H4, H2A) and other signaling pathways.

[0063] Protein interaction analysis revealed the associations among BMPR2, ID2, SMAD7, PAK1, ELOC, RAD50, THBS1, and RBX1, e.g. Fig.10 shown.

[0064] Example 2

[0065] Construct an overexpression vector of the ID2 gene and transfect it into sheep ovarian granulosa cells to analyze the function of the ID2 gene related to reproduction:

[0066] 1. Construction of lentiviral overexpression vector of ID2 gene

[0067] (1) Primers were designed as follows:

[0068] LV-s-ID2-E / BF: ACTAGAGGATCTATTTCCGGTGAATTCGCCACCATGAAAGCCTTCA GT (SEQID NO: 3);

[0069] LV-s-ID2-E / BR: AGTCACTTAAGCTTGGTACCGAGGATCCGCCACAGAGCGCTTTGCTGT (SEQ ID NO: 4).

[0070] (2) Using Hetian sheep cDNA as template DNA, PCR amplification was performed to obtain the target fragment of ID2 gene:

[0071] Prepare the system as shown in Table 1, mix gently, and place in a PCR instrument for reaction. The PCR program is shown in Table 2:

[0072] Table 1 Fragment PCR amplification system

[0073] Component name Volume (μL) 2×PCRBuffer 25 dNTPsmix (10 mM each) 1 F primer (10 μM) 2 R primer (10 μM) 2 Template DNA (200 ng / μL) 1 <![CDATA[ddH2O]]> 18 Total volume 50

[0074] Table 2 PCR program

[0075]

[0076] (3) Ligation of target fragment and vector

[0077] HB infusion TM one-step cloning and ligation system: Prepare the reaction system shown in Table 3 in an ice-water bath. After the ligation reaction solution reacts at 50°C for 30 minutes, it is placed on ice for 5 minutes and transformed immediately. The vector is pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO, purchased from Hanheng Biotechnology Co., Ltd.

[0078] Table 3 One-step cloning and ligation system

[0079]

[0080]

[0081] (4) Conversion

[0082] After taking out the DH5α competent cells from the -80℃ refrigerator, they should be immediately placed on ice to thaw. The competent cell packaging process should be gentle to reduce mechanical damage. After the competent cell is thawed, it should be packaged in a volume of 50μL per tube. After packaging, the ligation product is added in an amount not exceeding 1 / 10 of the competent cell volume (add 5μL ligation product), and placed on ice for 20-30min; heat shock at 42℃ for 90s, and immediately put it on ice for 2-3min after heat shock. In the clean bench, add 500μL of antibiotic-free LB culture medium and gently turn it upside down 4 times; shake and culture at 37℃ and 230rpm for 50min; apply the bacterial solution to the LB culture medium containing ampicillin, spread it evenly, and then invert the plate in a 37℃ constant temperature box for 15h.

[0083] (5) Plasmid extraction

[0084] After the positive clones screened out were sequenced successfully, plasmids were extracted using the Tiangen Plasmid Extraction Kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The concentration of the extracted plasmid QC was required to be greater than 200 ng / μL, and the A260 / 280 was between 1.8 and 2.0.

[0085] (6) Lentivirus packaging, concentration and purification

[0086] 293T cells were passaged in advance for transfection and placed in a 37°C, 5% CO2 incubator after the operation. Cell density was observed before transfection, and transfection could be performed when the confluence rate reached 70-80%; lipid transfection was performed, and the complex components for transfection of 100 mm dishes were shown in Table 4:

[0087] Table 4 Lipid transfer complex system

[0088] Plasmids and reagents Dosage pSPAX2 10 μg pMD2G 5μg Shuttle Plasmid 10 μg LipofiterTM Dosage 75μL

[0089] The shuttle plasmid in the table is a plasmid containing the target gene, which is constructed in step (3). After the lipid transfection complex is mixed and incubated at room temperature for 15 minutes, it is slowly added dropwise to the 293T cells and cultured in a cell culture incubator at 37°C and 5% CO2. 16 hours after transfection, fresh complete medium containing 10% fetal bovine serum (FBS) is replaced.

[0090] Virus collection: Virus supernatant was collected twice at 48h and 72h after transfection (fresh complete medium was replaced after 48h collection). When collecting virus at 48h, the medium in the 100mm culture dish was poured into a 50mL centrifuge tube, and attention was paid to not letting the wall of the culture dish touch the mouth of the centrifuge tube to prevent bacterial contamination. Then 10mL of fresh complete medium containing 10% fetal bovine serum FBS was added, and the culture was placed steadily in a constant temperature incubator at 37°C and 5% CO2 for continued cultivation. When collecting virus at 72h, the medium in the 100mm culture dish was directly poured into a 50mL centrifuge tube, and attention was paid to not letting the wall of the culture dish touch the mouth of the centrifuge tube to prevent bacterial contamination.

[0091] Ultracentrifugation: Centrifuge the virus supernatant in a 50mL centrifuge tube at 4°C, 2000×g for 10 min to remove cell debris; then collect the supernatant of the virus stock solution and place it in an ultracentrifuge tube, centrifuge it at 4°C, 82700×g for 120 min, resuspend the virus precipitate in complete culture medium, and finally dispense the ultracentrifuge resuspension into sterilized virus tubes and store them in a -80°C refrigerator.

[0092] 2. Cell preparation before transfection

[0093] (1) Recovery of frozen cells

[0094] Hotan sheep ovarian granulosa cells were collected from Hotan Huimin Co., Ltd. The frozen ovarian granulosa cells were taken out of liquid nitrogen and immediately placed in a 37°C water bath for rapid shaking to thaw. The thawed cells were placed in a centrifuge at 1000rpm and centrifuged for 5 minutes. Carefully take out the centrifuged cells, and completely discard the supernatant in the clean bench. White cell precipitation can be seen at the bottom. Add 1mL of DMEM containing 10% FBS to the precipitate, resuspend the precipitate, mix well and add it to the culture dish for culture.

[0095] (2) Cell subculture

[0096] After obtaining the primary cells, the culture medium was discarded first, and the adherent cells were washed with 2 mL of PBS buffer (shaking up and down and left and right), the floating impurities were washed away, the washing solution was discarded, and 7 mL of complete culture medium (containing 10% FBS) was added and placed in an incubator at 37° C. and 5% CO2 for 36 h until the cell adhesion rate reached 70-80%, and the culture medium was discarded; 3 mL of PBS buffer was added to wash the adherent cells twice, and the washing solution was discarded; 1 mL of 0.25% trypsin was added to each dish to completely cover the cells, and digested in an incubator at 37° C. for 3 min. When all the cells were wrinkled and rounded and began to flow in large quantities, FBS equal to that of trypsin was added to terminate the digestion, and the cells were completely peeled off from the bottom of the rinse dish with a pipette, and the mixed solution was transferred to a 15 mL centrifuge tube, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded; the cells were resuspended with 2 mL of complete culture medium (containing 10% FBS), and inoculated into 2 new 10 cm culture dishes (1 for 2) for subculture, and subcultured cells were obtained after culturing for 24 h.

[0097] 3. Lentiviral transfection of ovarian granulosa cells

[0098] (1) 20 h before lentiviral transfection, adherent cells were cultured at 1×10 5 / well in a 24-well plate. The number of cells during lentiviral transfection is 2×10 5 / hole left and right.

[0099] (2) Aspirate the culture medium, wash twice with PBS, and add 500 μL of complete culture medium to each well.

[0100] (3) The overexpression group was transfected with 45uL of lentiviral solution, and the empty vector group was transfected with 45μL of lentiviral empty vector (pSPAX2, pMD2G and shuttle plasmid (blank plasmid without target gene)). The blank group was not treated. After 4h, the culture medium was replenished and 500μL of complete culture medium was added.

[0101] (4) Continue culturing for 24 h and replace the virus-containing culture medium with fresh culture medium.

[0102] (5) Continue to cultivate.

[0103] 4. Analysis of Hotan sheep ovarian granulosa cells after lentivirus transfection

[0104] (1) The lentivirus contains fluorescent protein (GFP tag), and the transfected cells were observed under a fluorescence microscope. The picture of Hotan sheep ovarian granulosa cells 48 hours after transfection with the ID2 gene overexpression vector is as follows Fig.11 As shown, the left picture is the fluorescence picture of empty transfection (500μm), and the right picture is the fluorescence picture of lentiviral transfection (200μm). The results showed that the ID2 overexpression vector was successfully transfected into the ovarian granulosa cells of Hotan sheep.

[0105] (2) For transfected ovarian granulosa cells, cell proliferation was detected by CCK-8 kit (purchased from Shanghai Biotech Biotechnology Co., Ltd.) at 48h and 72h after lentiviral transfection. Fig.12 As shown, at 48h, the difference between the ID2 transfection group and the blank group was significant (P<0.05), and the difference between the ID2 transfection group and the empty group was extremely significant (P<0.01); at 72h, the cell proliferation rate of the ID2 transfection group was extremely significantly different from that of the blank group and the empty group (P<0.01). This shows that the overexpression of the ID2 gene has a certain promoting effect on the proliferation of ovarian granulosa cells, reaching an extremely significant promoting effect at 48h and 72h (P<0.01), preliminarily proving that the ID2 gene can promote the proliferation of ovarian granulosa cells.

[0106] (3) Cell RNA was extracted 48 h and 72 h after transfection for reverse transcription and synthesis of cDNA (using a reverse transcription kit purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.). The expression levels of the genes related to ID2 (ID1, ID3) predicted by STRING and the genes in the same signaling pathway as ID2 (BMPR2, RBX1, SMAD7) in the overexpression were detected. The primer sequences are shown in Table 5.

[0107] Table 5 Primer information

[0108]

[0109]

[0110] The results are as follows Figure 13 to Figure 17 As shown in the figure, HBLV-ZsGreen-PURO represents the empty group, and HBLV-s-ID2-3xflag-ZsGreen-PURO represents the ID2 transfection group. The results showed that the relative expression of ID1 in the ID2 transfection group was significantly higher than that in the empty group (P < 0.01). The relative expression of ID3 in the ID2 transfection group was significantly lower than that in the empty group 48 hours after transfection (P < 0.05), and was significantly lower than that in the empty group 72 hours after transfection (P < 0.01). The relative expression of BMPR2 in the ID2 transfection group was significantly lower than that in the empty group (P < 0.01). The relative expression of SMAD7 in the ID2 transfection group was significantly lower than that in the empty group (P < 0.01). The relative expression of RBX1 in the ID2 transfection group was significantly lower than that in the empty group (P < 0.01).

[0111] (4) The cell culture supernatant was collected 48h and 72h after transfection, and the concentrations of progesterone (P4) and estrogen (E2) were detected according to the test steps in the instructions of the Elisa kit (purchased from Shanghai Jining Biotechnology Co., Ltd.). Figure 18-19As shown in the figure, HBLV-ZsGreen-PURO represents the empty group, and HBLV-s-ID2-3xflag-ZsGreen-PURO represents the ID2 transfection group. The results showed that the P4 concentration of the ID2 transfection group was significantly higher than that of the empty group at 48h and 72h (P < 0.01); the E2 concentration of the ID2 transfection group was significantly lower than that of the empty group at 48h (P < 0.05), and was significantly lower than that of the empty group at 72h (P < 0.01).

[0112] The above results show that ID2 overexpression can promote the proliferation of granulosa cells over time. The ID2 gene can significantly downregulate the expression of ID3, BMPR2, RBX1 and FSHR genes in cells, upregulate the expression of ID1 gene, upregulate the secretion of P4 progesterone, and inhibit the secretion of E2 estradiol.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of sheep ID2 gene in regulating sheep lambing traits, characterized in that: The nucleotide sequence of the sheep ID2 gene is shown in SEQ ID NO:

1.

2. The use of the protein encoded by the sheep ID2 gene in regulating the lambing traits of sheep, characterized in that: The amino acid sequence of the protein encoded by the sheep ID2 gene is shown in SEQ ID NO:

2.

3. The use of biological materials containing sheep ID2 gene in regulating sheep lambing traits, characterized in that: The nucleotide sequence of the sheep ID2 gene is shown in SEQ ID NO: 1, and the biological material includes a lentivirus, a recombinant expression vector or an engineered cell.

4. The use according to any one of claims 1 to 3, characterized in that: The sheep ID2 gene positively regulates the proliferation of ovarian granulosa cells.

5. The use according to any one of claims 1 to 3, characterized in that: The sheep ID2 gene downregulates the expression of ID3, BMPR2, RBX1 and FSHR genes, and upregulates the expression of ID1 gene.

6. The use according to any one of claims 1 to 3, characterized in that: Overexpression of the sheep ID2 gene promotes the secretion of P4 progesterone and inhibits the secretion of E2 estradiol.

7. The use according to any one of claims 1 to 3, characterized in that: The sheep are Hetian sheep.

8. A method for breeding multi-lamb Hetian sheep, characterized in that: The method is to overexpress the ID2 gene of Hotan sheep.

9. A method for screening or breeding multi-lamb trait Hetian sheep, characterized in that: The method comprises the following steps: detecting the relative expression of the ID2 gene of Hetian sheep, wherein the Hetian sheep with relatively high expression of the ID2 gene exhibits the trait of having multiple lambs; and the relative expression of the ID2 gene uses β-actin or GAPDH as an internal reference gene.

10. Application of the method according to any one of claims 8 to 9 in Hetian sheep breeding.

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