Method for screening and identifying cattle and sheep embryo implantation key genes

Abstract

The invention relates to the technical field of gene expression and regulation, and discloses a method for screening and identifying cattle and sheep embryo implantation key genes, which comprises the following steps: bioinformatics identification, reverse transcription PCR (polymerase chain reaction), PCR amplification, fluorescent quantitative PCR, cell line construction and cell fusion test. According to the method for screening and identifying the cattle and sheep embryo implantation key genes, the expression difference between normal and cloned animal placentas is analyzed by analyzing env genes in cattle and sheep genomes and utilizing bulk transcriptome sequencing, candidate syncytin genes are screened, and the candidate syncytin genes are analyzed through the structure and expression, so that the cattle and sheep embryo implantation key genes are obtained. The typical functional characteristics of the cattle and sheep syncytial gene are identified through cell fusion and pseudotype virus tests, the key cattle and sheep syncytial gene is found in a cattle and sheep genome for the first time, the function of mediating placenta fusion promotion by the cattle and sheep syncytial gene is disclosed, receptors combined with the cattle and sheep syncytial gene are verified as ASCT1 and ASCT2, and the cattle and sheep syncytial gene can be used for identifying the cattle and sheep syncytial gene. And a new theoretical basis is provided for solving the problems of pregnancy failure and low somatic cell cloning efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of gene expression and regulation, and in particular to a method for screening and identifying key genes for embryo implantation in cattle and sheep. Background Art

[0002] Abnormal embryo implantation and placental development in cattle and sheep lead to pregnancy losses as high as 20%, which is also the main factor leading to the death of somatic cell cloned embryos. Pregnancy losses of normal and cloned embryos in cattle and sheep mainly occur during the embryo implantation period. Studies have shown that abnormal trophoblast cell differentiation is the main factor leading to pregnancy losses.

[0003] At present, the cellular and molecular mechanisms of trophoblast differentiation in cattle and sheep are still unclear. The syncytin gene evolved from the env gene of ERVs is a key gene that regulates the differentiation of mammalian placental trophoblast cells and the diversity of placental morphology. Therefore, only by identifying the key syncytialin genes in cattle and sheep and revealing their mechanism of regulating the differentiation of placental trophoblast cells can the problem of high pregnancy loss rate during the implantation period of normal embryos and somatic cell cloned embryos be solved.

[0004] Therefore, a method for screening and identifying key genes for embryo implantation in cattle and sheep was proposed. Summary of the invention

[0005] The invention aims to solve the problems existing in the prior art, and proposes a method for screening and identifying key genes for embryo implantation of cattle and sheep. The env gene in the cattle and sheep genome is analyzed, and the expression difference between the placenta of normal animals and cloned animals is analyzed by bulk transcriptome sequencing, and a candidate syncytin gene is screened. At the same time, the candidate syncytin gene is analyzed by structure and expression, and the typical functional characteristics of the cattle and sheep syncytin gene are identified by cell fusion and pseudotype virus tests. This scheme is the first to discover the key cattle and sheep syncytin gene in the cattle and sheep genome, reveal the function of the cattle and sheep syncytin gene mediating the placenta fusion promotion, and verify that the receptors bound by the cattle and sheep syncytial gene are ASCT1 and ASCT2, which provides a new theoretical basis for solving the problems of pregnancy failure and low somatic cell cloning efficiency, and solves the problem of high pregnancy loss rate during the implantation period of normal cattle and sheep embryos and somatic cell cloned embryos.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for screening and identifying key genes for embryo implantation in cattle and sheep comprises the following steps:

[0008] Step 1: Bioinformatics identification: Identify key syncytin genes in cattle and sheep through cattle and sheep reference genome data and cattle and sheep tissue and embryo transcriptome sequencing data;

[0009] Step 2: Reverse transcription PCR: collect cattle and sheep placentas, and process the placenta samples before performing 20 μL reverse transcription PCR;

[0010] Step 3: PCR amplification: Use the cDNA obtained by reverse transcription as a template and use a gene amplifier to perform PCR experiments;

[0011] Step 4: Fluorescence quantitative PCR: the cDNA obtained by reverse transcription is used as a template, and the expression levels of Sus1 and Sus2 are detected by fluorescence quantitative detection using a universal high-sensitivity dye-based quantitative PCR detection kit and StepOnePlus;

[0012] Step 5: Cell line construction: insert the CDS sequences of gASCT1 / 2 and bASCT1 / 2 into the pPB-Puro-EF1AEGFP plasmid (Addgene), revive and culture HEK293T cells, and co-transfect pPB-Puro-EF1AEGFP-gASCT1 / 2 or pPB-Puro-EF1AEGFP-bASCT1 / 2 with the pRP-CAGhyPBase plasmid to obtain cell lines;

[0013] Step 6: Cell fusion test: Identify the typical functional characteristics of cattle and sheep syncytin genes through cell fusion and pseudotype virus tests.

[0014] Preferably, the specific steps of the bioinformatics identification are:

[0015] The reference genome FASTA files of cattle and sheep GCF_002263795.3 (ARS-UCD2.0) and GCF_001704415.2 (ARS1.2) were downloaded from the Genome database of NCBI. The reference genome FATSA files were used to establish a nucleotide database using tBLASTNv2.5.0. The Accension numbers NP_041188.1, YP_004222726.1, NP_045939.1, NP_056899.1, NP_054717.1, NP_040334.1, NP_057856.1, NP_056883.1, and NP_043923.1 in the NCBI protein database were used as queries in the tBLASTn program, and the parameters were default;

[0016] Transcriptome sequencing data of cattle and sheep tissues and embryos were collected from the GEO database of NCBI. Fastq-dumpv2.9.6 was used to convert SRA objects into FASTQ files. HISAT2v2.2.1 was used to establish genome indexes for the cattle and sheep reference genomes GCF_002263795.3 (ARS-UCD2.0) and GCF_001704415.2 (ARS1.2). The FASTQ files were then aligned to the reference genomes to obtain the original bam files, and the bam files were sorted using Samtoolsv1.17 for the next step of TPM and Counts value calculation.

[0017] The hits obtained from the first tBLASTn were subjected to iterative tBLASTn with amino acids greater than or equal to 100 aa and without early stop codons, and the hits obtained from iterative tBLASTn were subjected to iterative tBLASTn with amino acids greater than or equal to 200 aa and without early stop codons to obtain 3285 and 2789 hits, respectively;

[0018] All hits obtained after de-redundancy were made into genome annotation files according to chromosome number, start and end position and direction, and added to GCF_002263795.3 (ARS-UCD2.0) and GCF_001704415.2 (ARS1.2) reference genome annotation files and reordered for transcriptome data calculation and analysis;

[0019] Stringtie2v2.2.1 was used to calculate the TPM and Counts of all hits after de-redundancy. The parameter "-rf" was set to specify the strand specificity of the transcript assembly of the bam file in (1). The obtained GTF files were merged using the "-merge" parameter in Stringtie2 to obtain a merged GTF file. The "-e-rf" parameter of Stringtie2 was used to calculate the TPM and Counts of the collected tissue and embryo samples. It was found that 13 and 11 hits in all the de-redundancy hits of cattle and sheep overlapped with the assembled transcripts obtained after merging, so they were named B1-B13 and G1-G11. It was also found that B3, B4, B5, B9, B11, B13, G1, G2, G5, G8 and G11 were differentially expressed in normal and cloned mature placentas, and not expressed in embryos.

[0020] Using NCBI's ConservedDomian database, it was found that B3, B4, B5, B9, B11, B13, G1, G2, G5, G8 and G11 all have the conserved domains TLVcoatsuperfamily and Ebola_HIV-1-like_HR1-HR2superfamily, and all have CXXC and RXXR motifs.

[0021] Preferably, the specific steps of the reverse transcription PCR are:

[0022] Placentas collected from slaughterhouses and cloning cattle and sheep bases were placed in a thermos containing 1× penicillin-streptomycin saline at a temperature of about 20°C and transported to the laboratory within 4 hours;

[0023] Cut off the excess tissue outside the ovary and wash it repeatedly with saline;

[0024] The placenta samples were washed three times with RNase-free PBS for 3 min each time, and soybean-sized tissues were cut with scissors and put into a mortar and ground with liquid nitrogen;

[0025] Add 1mL of pre-cooled TRIzol reagent and lyse at room temperature for 10min; add 200μL of chloroform, shake for 30s, and let stand for 3min; centrifuge at 12,000rpm for 15min at 4℃; aspirate the upper aqueous phase and mix it with an equal volume of isopropanol, and let stand for 30min; centrifuge at 12,000rpm for 10min at 4℃; wash the RNA pellets at the bottom of the tube with 1mL of 75% ethanol solution; centrifuge at 8,000rpm for 5min at 4℃; repeat the wash once and dry at room temperature for 5-10min; dissolve the RNA with an appropriate amount of RNase-free ddH2O, and finally measure the RNA concentration and OD value;

[0026] 20 μL reverse transcription PCR system: 10×RT Buffer 2 μL, dNTPMix 4 μL, Random Decamers 1 μL, Oligo (dT) Primers 1 μL, Reverse Transcriptase M-MLVRT 1 μL, Embryo Lysis Product 10 μL, RNase Inhibitor 1 μL;

[0027] The reaction procedure was 42°C, 30 min; 95°C, 10 min; cooling to 4°C, and the cDNA product was stored at –80°C or directly subjected to fluorescent quantitative PCR.

[0028] Preferably, the specific steps of the PCR amplification are:

[0029] The amplification primer sequences of eight bovine and sheep placenta-specific candidate syncytin genes were synthesized, and the cDNA obtained by reverse transcription was used as a template to perform PCR experiments using the gene amplification instrument QIQIAN-A100;

[0030] use HSDNA Polymerase kit for high-fidelity PCR test;

[0031] Reaction system (50 μL): 5× PrimeSTAR Buffer (Mg2+ Plus) 10 μL, dNTP Mixture (2.5 mM each) 4 μL, upstream and downstream primers (10-15 pmol) 1 μL each, Template 1 μL, PrimeSTAR HS DNA Polymerase (2.5 U / μl) 0.5 μL, ddH2O3 2.5 μL;

[0032] Reaction parameters: 98°C, 2 min pre-denaturation; 98°C, 20 s denaturation, 58-65°C, 34 s annealing, 72°C extension 2 min 30 s, 40 cycles; 72°C extension 5 min.

[0033] Preferably, the specific steps of the fluorescent quantitative PCR are:

[0034] The quantitative primer sequences of eight candidate syncytin genes specific to the placenta of cattle and sheep were sent for synthesis;

[0035] The cDNA obtained by reverse transcription was used as a template, and the expression levels of Sus1 and Sus2 were detected by fluorescence quantitative detection using a universal high-sensitivity dye-based quantitative PCR detection kit and StepOnePlus;

[0036] Reaction system (10 μL): 2×ChamQ Universal SYBR qPCR Master Mix 5 μL, upstream and downstream primers (10-15 pmol) 0.2 μL each, Template 1 μL, ddH2O 4.6 μL;

[0037] The reaction parameters were: 95°C, 30 s pre-denaturation; 95°C, 10 s denaturation, 60°C, 30 s annealing, according to the instrument default melting curve acquisition program;

[0038] The internal reference primer is the cattle and sheep histone H2A gene.

[0039] Preferably, the specific steps of constructing the cell line are:

[0040] The gASCT1 / 2 and bASCT1 / 2 eukaryotic vector cloning primer sequences were sent for synthesis;

[0041] The CDS sequences of gASCT1 / 2 and bASCT1 / 2 were inserted into the pPB-Puro-EF1AEGFP plasmid (Addgene) using the pEASY-Uni seamless cloning and assembly kit;

[0042] HEK293T cells were preserved by our laboratory. After revival, they were cultured in a pre-prepared culture medium (9% Gibco fetal bovine serum + 1% double antibody + 90% Gibco DF12 culture medium). The cells were allowed to migrate out slowly and were subcultured to 6-well plates after they were fully grown for subsequent experiments.

[0043] Co-transfect pPB-Puro-EF1AEGFP-gASCT1 / 2 or pPB-Puro-EF1AEGFP-bASCT1 / 2 with pRP-CAGhyPBase plasmid, add 5ng / ml puromycin for selection after 24-48h, change the culture medium every day, and obtain the cell line after 10-15 days;

[0044] Take appropriate amount of cells for immunofluorescence and western blot detection to obtain cell lines.

[0045] Preferably, the specific steps of the cell fusion assay are:

[0046] The CDS sequences of eight bovine and ovine placenta-specific candidate syncytin genes were inserted into the pcDNA3.1-3xFLAG-C plasmid and manipulated using the pEASY-Uni seamless cloning and assembly kit;

[0047] For the LacZ reporter plasmid, LacZ with an encephalomyocarditis virus internal ribosome entry site was expressed in the presence of T7 polymerase (pcDNA3.1-T7-EMCV-LacZ), the CMV promoter and enhancer sequences of pcDNA3.1(+) were removed as the backbone plasmid, and the EMCV, IRES, LacZ, and T7 terminator sequences were inserted, also using the pEASY-Uni Seamless Cloning and Assembly Kit;

[0048] The EMCV sequence was amplified by PCR and connected to the IRES sequence;

[0049] IRES and LacZ sequences were cloned from pMX-IRES-Puro and pUB6-V5-His-LacZ, respectively;

[0050] T7 terminator sequence was synthesized;

[0051] All the above PCRs were performed using PrimeSTARHS DNA polymerase;

[0052] HEK293T cells overexpressing gASCT1 / 2 and bASCT1 / 2 were transfected using PEI reagent, that is, cells were transferred into 24-well plates at a density of 2×105 or into 6-well plates at a density of 1×106 for 12 hours, and then the transfection premix was added. After transfection for 4-6 hours, the transfected cells were co-incubated.

[0053] Preferably, the transfection premix is ​​prepared as follows:

[0054] Prepare two EP tubes, add 300 μL Opti-MEM, pre-mix 2 μg plasmid (1 μg pcDNA3.1-3xFLAG-C-8 bovine and sheep placenta-specific candidate syncytin genes and 1 μg pcDNA3.1-T7-EMCV-LacZ) and 10 μL PEI reagent and 2 μg pCAG-T7-Pol in each tube, and let stand at room temperature for 5 min; gently mix and let stand again for 15 min, then add the transfection premix to one well of a 6-well plate;

[0055] After culturing for 24-48 hours, the culture medium was discarded, the cells were washed 5 times with PBS, the cells were fixed with 4% paraformaldehyde for 20 minutes, and then washed 5 times with PBS. Preheated Xgal staining solution was added, incubated in a 37 degree Celsius incubator for 24 hours, and then observed under a microscope.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] 1. The screening and identification method of key genes for embryo implantation in cattle and sheep is to analyze the env gene in the cattle and sheep genome and use bulk transcriptome sequencing to analyze the expression differences between normal and cloned animal placentas, screen out candidate syncytialin genes, and analyze the candidate syncytialin genes through structure and expression, and identify the typical functional characteristics of cattle and sheep syncytialin genes through cell fusion and pseudotype virus experiments. This scheme is the first to discover key cattle and sheep syncytialin genes in the cattle and sheep genome, revealing the function of cattle and sheep syncytialin genes in mediating placental fusion, and verifying that the receptors bound by cattle and sheep syncytialin genes are ASCT1 and ASCT2, providing a new theoretical basis for solving the problems of pregnancy failure and low efficiency of somatic cell cloning.

[0058] 2. The screening and identification method of key genes for cattle and sheep embryo implantation screened out 8 candidate syncytialin genes. Structural analysis found that the structures of the 8 candidate syncytialin genes were conserved. Expression analysis found that the 8 candidate syncytialin genes were highly expressed in the placenta, revealing that Sus1 and Sus2 may mediate the development of cattle and sheep embryos and play an important role. Cell fusion and pseudotype virus experiments verified that both Sus1 and Sus2 could promote cell fusion and mediate virus entry, which further indicated that the 8 cattle and sheep placenta-specific candidate syncytialin genes may be involved in early embryonic development and placenta formation. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a schematic diagram of the construction of the cell lines overexpressing gASCT1 / 2 and bASCT1 / 2 of the present invention;

[0060] Figure 2The figure is a schematic diagram of the cell fusion test of the bovine and sheep placenta-specific candidate syncytin gene of the present invention. DETAILED DESCRIPTION

[0061] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0062] Reference Figure 1-Figure 2 A method for screening and identifying key genes for embryo implantation in cattle and sheep, a method for screening and identifying key genes for embryo implantation in cattle and sheep, comprising the following steps:

[0063] Step 1: Bioinformatics identification;

[0064] The specific steps of bioinformatics identification are:

[0065] The reference genome FASTA files of cattle and sheep GCF_002263795.3 (ARS-UCD2.0) and GCF_001704415.2 (ARS1.2) were downloaded from the Genome database of NCBI. The reference genome FATSA files were used to establish a nucleotide database using tBLASTNv2.5.0. The Accension numbers NP_041188.1, YP_004222726.1, NP_045939.1, NP_056899.1, NP_054717.1, NP_040334.1, NP_057856.1, NP_056883.1, and NP_043923.1 in the NCBI protein database were used as queries in the tBLASTn program, and the parameters were default;

[0066] Transcriptome sequencing data of cattle and sheep tissues and embryos were collected from the GEO database of NCBI. Fastq-dumpv2.9.6 was used to convert SRA objects into FASTQ files. HISAT2v2.2.1 was used to establish genome indexes for the cattle and sheep reference genomes GCF_002263795.3 (ARS-UCD2.0) and GCF_001704415.2 (ARS1.2). The FASTQ files were then aligned to the reference genomes to obtain the original bam files, and the bam files were sorted using Samtoolsv1.17 for the next step of TPM and Counts value calculation.

[0067] The hits obtained from the first tBLASTn were subjected to iterative tBLASTn with amino acids greater than or equal to 100 aa and without early stop codons, and the hits obtained from iterative tBLASTn were subjected to iterative tBLASTn with amino acids greater than or equal to 200 aa and without early stop codons to obtain 3285 and 2789 hits, respectively;

[0068] All hits obtained after de-redundancy were made into genome annotation files according to chromosome number, start and end position and direction, and added to GCF_002263795.3 (ARS-UCD2.0) and GCF_001704415.2 (ARS1.2) reference genome annotation files and reordered for transcriptome data calculation and analysis;

[0069] Stringtie2v2.2.1 was used to calculate the TPM and Counts of all hits after de-redundancy. The parameter "-rf" was set to specify the strand specificity of the transcript assembly of the bam file in (1). The obtained GTF files were merged using the "-merge" parameter in Stringtie2 to obtain a merged GTF file. The "-e-rf" parameter of Stringtie2 was used to calculate the TPM and Counts of the collected tissue and embryo samples. It was found that 13 and 11 hits in all the de-redundancy hits of cattle and sheep overlapped with the assembled transcripts obtained after merging, so they were named B1-B13 and G1-G11. It was also found that B3, B4, B5, B9, B11, B13, G1, G2, G5, G8 and G11 were differentially expressed in normal and cloned mature placentas, and not expressed in embryos.

[0070] Using NCBI's ConservedDomian database, it was found that B3, B4, B5, B9, B11, B13, G1, G2, G5, G8 and G11 all have the conserved domains TLVcoatsuperfamily and Ebola_HIV-1-like_HR1-HR2superfamily, and all have CXXC and RXXR motifs.

[0071] Step 2: Reverse transcription PCR;

[0072] The specific steps of reverse transcription PCR are:

[0073] Placentas collected from slaughterhouses and cloning cattle and sheep bases were placed in a thermos containing 1× penicillin-streptomycin saline at a temperature of about 20°C and transported to the laboratory within 4 hours;

[0074] Cut off the excess tissue outside the ovary and wash it repeatedly with saline;

[0075] The placenta samples were washed three times with RNase-free PBS for 3 min each time, and soybean-sized tissues were cut with scissors and put into a mortar and ground with liquid nitrogen;

[0076] Add 1mL of pre-cooled TRIzol reagent and lyse at room temperature for 10min; add 200μL of chloroform, shake for 30s, and let stand for 3min; centrifuge at 12,000rpm for 15min at 4℃; aspirate the upper aqueous phase and mix it with an equal volume of isopropanol, and let stand for 30min; centrifuge at 12,000rpm for 10min at 4℃; wash the RNA pellets at the bottom of the tube with 1mL of 75% ethanol solution; centrifuge at 8,000rpm for 5min at 4℃; repeat the wash once and dry at room temperature for 5-10min; dissolve the RNA with an appropriate amount of RNase-free ddH2O, and finally measure the RNA concentration and OD value;

[0077] 20 μL reverse transcription PCR system: 10×RT Buffer 2 μL, dNTPMix 4 μL, Random Decamers 1 μL, Oligo (dT) Primers 1 μL, Reverse Transcriptase M-MLVRT 1 μL, Embryo Lysis Product 10 μL, RNase Inhibitor 1 μL;

[0078] The reaction procedure was 42°C, 30 min; 95°C, 10 min; cooling to 4°C, and the cDNA product was stored at –80°C or directly subjected to fluorescent quantitative PCR.

[0079] Step 3: PCR amplification;

[0080] The specific steps of PCR amplification are:

[0081] The amplification primer sequences of eight bovine and sheep placenta-specific candidate syncytin genes were synthesized, and the cDNA obtained by reverse transcription was used as a template to perform PCR experiments using the gene amplification instrument QIQIAN-A100;

[0082] use HSDNA Polymerase kit for high-fidelity PCR test;

[0083] Reaction system (50 μL): 5× PrimeSTAR Buffer (Mg2+ Plus) 10 μL, dNTP Mixture (2.5 mM each) 4 μL, upstream and downstream primers (10-15 pmol) 1 μL each, Template 1 μL, PrimeSTAR HS DNA Polymerase (2.5 U / μl) 0.5 μL, ddH2O3 2.5 μL;

[0084] Reaction parameters: 98°C, 2 min pre-denaturation; 98°C, 20 s denaturation, 58-65°C, 34 s annealing, 72°C extension 2 min 30 s, 40 cycles; 72°C extension 5 min.

[0085] Step 4: Fluorescence quantitative PCR;

[0086] The specific steps of fluorescence quantitative PCR are as follows:

[0087] The quantitative primer sequences of eight candidate syncytin genes specific to the placenta of cattle and sheep were sent for synthesis;

[0088] The cDNA obtained by reverse transcription was used as a template, and the expression levels of Sus1 and Sus2 were detected by fluorescence quantitative detection using a universal high-sensitivity dye-based quantitative PCR detection kit and StepOnePlus;

[0089] Reaction system (10 μL): 2×ChamQ Universal SYBR qPCR Master Mix 5 μL, upstream and downstream primers (10-15 pmol) 0.2 μL each, Template 1 μL, ddH2O 4.6 μL;

[0090] The reaction parameters were: 95°C, 30 s pre-denaturation; 95°C, 10 s denaturation, 60°C, 30 s annealing, according to the instrument default melting curve acquisition program;

[0091] The internal reference primer is the cattle and sheep histone H2A gene.

[0092] Step 5: Cell line construction;

[0093] The specific steps for cell line construction are:

[0094] The gASCT1 / 2 and bASCT1 / 2 eukaryotic vector cloning primer sequences were sent for synthesis;

[0095] The CDS sequences of gASCT1 / 2 and bASCT1 / 2 were inserted into the pPB-Puro-EF1AEGFP plasmid (Addgene) using the pEASY-Uni seamless cloning and assembly kit;

[0096] HEK293T cells were preserved by our laboratory. After revival, they were cultured in a pre-prepared culture medium (9% Gibco fetal bovine serum + 1% double antibody + 90% Gibco DF12 culture medium). The cells were allowed to migrate out slowly and were subcultured to 6-well plates after they were fully grown for subsequent experiments.

[0097] Co-transfect pPB-Puro-EF1AEGFP-gASCT1 / 2 or pPB-Puro-EF1AEGFP-bASCT1 / 2 with pRP-CAGhyPBase plasmid, add 5ng / ml puromycin for selection after 24-48h, change the culture medium every day, and obtain the cell line after 10-15 days;

[0098] Take appropriate amount of cells for immunofluorescence and western blot detection to obtain cell lines.

[0099] Step 6: Cell fusion test;

[0100] The specific steps of the cell fusion assay are:

[0101] The CDS sequences of eight bovine and ovine placenta-specific candidate syncytin genes were inserted into the pcDNA3.1-3xFLAG-C plasmid and manipulated using the pEASY-Uni seamless cloning and assembly kit;

[0102] For the LacZ reporter plasmid, LacZ with an encephalomyocarditis virus internal ribosome entry site was expressed in the presence of T7 polymerase (pcDNA3.1-T7-EMCV-LacZ), the CMV promoter and enhancer sequences of pcDNA3.1(+) were removed as the backbone plasmid, and the EMCV, IRES, LacZ, and T7 terminator sequences were inserted, also using the pEASY-Uni Seamless Cloning and Assembly Kit;

[0103] The EMCV sequence was amplified by PCR and connected to the IRES sequence;

[0104] IRES and LacZ sequences were cloned from pMX-IRES-Puro and pUB6-V5-His-LacZ, respectively;

[0105] T7 terminator sequence was synthesized;

[0106] All the above PCRs were performed using PrimeSTARHS DNA polymerase;

[0107] HEK293T cells overexpressing gASCT1 / 2 and bASCT1 / 2 were transfected using PEI reagent, i.e., cells were transfected into 24-well plates at a density of 2×105 or into 6-well plates at a density of 1×106 for 12 h, and then the transfection premix was added. After transfection for 4-6 h, the transfected cells were co-incubated.

[0108] Preparation of transfection premix:

[0109] Prepare two EP tubes, add 300 μL Opti-MEM, pre-mix 2 μg plasmid (1 μg pcDNA3.1-3xFLAG-C-8 bovine and sheep placenta-specific candidate syncytin genes and 1 μg pcDNA3.1-T7-EMCV-LacZ) and 10 μL PEI reagent and 2 μg pCAG-T7-Pol in each tube, and let stand at room temperature for 5 min; gently mix and let stand again for 15 min, then add the transfection premix to one well of a 6-well plate;

[0110] After culturing for 24-48 hours, the culture medium was discarded, the cells were washed 5 times with PBS, the cells were fixed with 4% paraformaldehyde for 20 minutes, and then washed 5 times with PBS. Preheated Xgal staining solution was added, incubated in a 37 degree Celsius incubator for 24 hours, and then observed under a microscope.

[0111] Transcriptome sequencing data of cattle and sheep tissues and embryos were collected from the GEO database of NCBI. Fastq-dumpv2.9.6 was used to convert SRA objects into FASTQ files. HISAT2v2.2.1 was used to establish genome indexes for the GCF_002263795.3 (ARS-UCD2.0) and GCF_001704415.2 (ARS1.2) cattle and sheep reference genomes. The FASTQ files were then aligned to the reference genomes to obtain the original bam files, and the bam files were sorted using Samtoolsv1.17 for the next step of TPM (transcripts per kilobase million) and Counts value calculations.

[0112] The GCF_002263795.3 (ARS-UCD2.0) and GCF_001704415.2 (ARS1.2) cattle and sheep reference genome FASTA files were downloaded from the Genome database of NCBI, and a nucleotide database was established using the reference genome FATSA files using tBLASTNv2.5.0. The Accension numbers NP_041188.1, YP_004222726.1, NP_045939.1, NP_056899.1, NP_054717.1, NP_040334.1, NP_057856.1, NP_056883.1, and NP_043923.1 in the NCBI protein library were used as queries in the tBLASTn program with default parameters.

[0113] The hits obtained from the first tBLASTn were iteratively tBLASTn through amino acids greater than or equal to 100 aa and without early stop codons, and the hits obtained from the iterative tBLASTn were iteratively tBLASTn through amino acids greater than or equal to 200 aa and without early stop codons to obtain 3285 and 2789 hits, respectively.

[0114] All hits obtained after de-redundancy were made into genome annotation files according to chromosome number, start and end position and direction, and added to GCF_002263795.3 (ARS-UCD2.0) and GCF_001704415.2 (ARS1.2) reference genome annotation files and reordered for transcriptome data calculation and analysis.

[0115] Stringtie2v2.2.1 was used to calculate the TPM and Counts of all hits after de-redundancy. The parameter "-rf" was set to specify the strand specificity of the transcript assembly of the bam file in (1). The obtained GTF files were merged using the "-merge" parameter in Stringtie2 to obtain a merged GTF file. The "-e-rf" parameter of Stringtie2 was used to calculate the TPM and Counts of the collected tissue and embryo samples. It was found that 13 and 11 hits in all the de-redundancy hits of cattle and sheep overlapped with the assembled transcripts obtained after merging, so they were named B1-B13 and G1-G11. It was also found that B3, B4, B5, B9, B11, B13, G1, G2, G5, G8 and G11 were differentially expressed in normal and cloned mature placentas, and not expressed in embryos.

[0116] Using the ConservedDomian database of NCBI, it was found that B3, B4, B5, B9, B11, B13, G1, G2, G5, G8 and G11 all have the conserved domains TLVcoatsuperfamily and Ebola_HIV-1-like_HR1-HR2superfamily, and all have CXXC and RXXR motifs;

[0117] Placentas collected from slaughterhouses and cloning cattle and sheep bases were placed in a thermos containing 1× penicillin-streptomycin saline at a temperature of about 20°C and transported to the laboratory within 4 hours. Excess tissue outside the ovary was cut off and repeatedly washed with saline.

[0118] The placenta samples were washed three times with RNase-free PBS for 3 min each time, and soybean-sized tissues were cut with scissors into a mortar and ground with liquid nitrogen.

[0119] Add 1 mL of pre-cooled TRIzol reagent and lyse at room temperature for 10 min; add 200 μL of chloroform, shake for 30 s, and let stand for 3 min; centrifuge at 12,000 rpm for 15 min at 4°C; aspirate the upper aqueous phase and mix it with an equal volume of isopropanol, and let stand for 30 min; centrifuge at 12,000 rpm for 10 min at 4°C; wash the small RNA pieces that precipitated at the bottom of the tube with 1 mL of 75% ethanol solution; centrifuge at 8,000 rpm for 5 min at 4°C; repeat the wash once and dry at room temperature for 5-10 min; dissolve the RNA with an appropriate amount of RNase-free ddH2O, and finally measure the RNA concentration and OD value.

[0120] 20μL reverse transcription PCR system: 10×RTBuffer 2μL, dNTPMix 4μL, RandomDecamers 1μL, Oligo(dT)Primers 1μL, Reverse Transcriptase M-MLVRT 1μL, Embryo lysate 10μL, RNase inhibitor 1μL.

[0121] The reaction procedure was 42°C, 30 min; 95°C, 10 min; cooling to 4°C, and the cDNA product was stored at –80°C or directly subjected to fluorescent quantitative PCR.

[0122] The amplification primer sequences of 8 bovine and sheep placenta-specific candidate syncytin genes were synthesized, and the cDNA obtained by reverse transcription was used as a template to perform PCR experiments using the gene amplification instrument QIQIAN-A100.

[0123] use HSDNA Polymerase Kit was used for high-fidelity PCR experiments.

[0124] Reaction system (50μL): 5×PrimeSTAR Buffer (Mg2+Plus) 10μL, dNTP Mixture (2.5mM each) 4μL, upstream and downstream primers (10-15pmol) 1μL each, Template 1μL, PrimeSTAR HS DNA Polymerase (2.5U / μl) 0.5μL, ddH2O3 2.5μL.

[0125] Reaction parameters: 98°C, 2 min pre-denaturation; 98°C, 20 s denaturation, 58-65°C, 34 s annealing, 72°C extension 2 min 30 s, 40 cycles; 72°C extension 5 min.

[0126] The quantitative primer sequences of 8 bovine and sheep placenta-specific candidate syncytin genes were sent to synthesis.

[0127] The cDNA obtained by reverse transcription was used as a template, and the expression levels of Sus1 and Sus2 were detected by fluorescence quantitative detection using a universal high-sensitivity dye-based quantitative PCR detection kit and StepOnePlus.

[0128] Reaction system (10 μL): 2×ChamQ Universal SYBR qPCR Master Mix 5 μL, upstream and downstream primers (10-15 pmol) 0.2 μL each, Template 1 μL, ddH2O 4.6 μL.

[0129] The reaction parameters were: 95°C, 30 s pre-denaturation; 95°C, 10 s denaturation, 60°C, 30 s annealing, according to the instrument default melting curve acquisition program.

[0130] The internal reference primer is the cattle and sheep histone H2A gene.

[0131] The gASCT1 / 2 and bASCT1 / 2 eukaryotic vector cloning primer sequences were sent to synthesis.

[0132] The CDS sequences of gASCT1 / 2 and bASCT1 / 2 were inserted into the pPB-Puro-EF1AEGFP plasmid (Addgene) using the pEASY-Uni seamless cloning and assembly kit.

[0133] HEK293T cells were preserved by our laboratory. After recovery, they were cultured in the prepared culture medium (9% Gibco fetal bovine serum + 1% double antibody + 90% Gibco DF12 culture medium). The cells were allowed to migrate out slowly and were subcultured to 6-well plates after they were fully grown for subsequent experiments.

[0134] Co-transfect pPB-Puro-EF1AEGFP-gASCT1 / 2 or pPB-Puro-EF1AEGFP-bASCT1 / 2 with pRP-CAGhyPBase plasmids, add 5 ng / ml puromycin for selection after 24-48 hours, change the culture medium every day, and obtain the cell line after 10-15 days. Take appropriate amount of cells for immunofluorescence and western blotting to detect the obtained cell line.

[0135] The CDS sequences of eight bovine and ovine placenta-specific candidate syncytin genes were inserted into the pcDNA3.1-3xFLAG-C plasmid and manipulated using the pEASY-Uni seamless cloning and assembly kit.

[0136] For the LacZ reporter plasmid, LacZ with an encephalomyocarditis virus internal ribosome entry site was expressed in the presence of T7 polymerase (pcDNA3.1-T7-EMCV-LacZ), the CMV promoter and enhancer sequences of pcDNA3.1(+) were removed as the backbone plasmid, and the EMCV, IRES, LacZ, and T7 terminator sequences were inserted, also using the pEASY-Uni seamless cloning and assembly kit.

[0137] The EMCV sequence was amplified by PCR and connected to the IRES sequence.

[0138] IRES and LacZ sequences were cloned from pMX-IRES-Puro and pUB6-V5-His-LacZ, respectively.

[0139] T7 terminator sequence synthesis.

[0140] All the above PCRs were performed using PrimeSTAR HS DNA polymerase.

[0141] HEK293T cells overexpressing gASCT1 / 2 and bASCT1 / 2 were transfected using PEI reagent, that is, cells were transferred into 24-well plates at a density of 2×105 or into 6-well plates at a density of 1×106 for 12 hours, and then the transfection premix was added. After transfection for 4-6 hours, the transfected cells were co-incubated.

[0142] Preparation of transfection premix: prepare two EP tubes, add 300 μL Opti-MEM, premix 2 μg plasmid (1 μg pcDNA3.1-3xFLAG-C-8 bovine and sheep placenta-specific candidate syncytin genes and 1 μg pcDNA3.1-T7-EMCV-LacZ) and 10 μL PEI reagent and 2 μg pCAG-T7-Pol in the two tubes respectively, and let them stand at room temperature for 5 minutes; gently mix and let it stand for 15 minutes again, then add the transfection premix to one well of a 6-well plate.

[0143] After culturing for 24-48 hours, the culture medium was discarded, the cells were washed 5 times with PBS, the cells were fixed with 4% paraformaldehyde for 20 minutes, and then washed 5 times with PBS. Preheated Xgal staining solution was added, incubated in a 37 degree Celsius incubator for 24 hours, and then observed under a microscope.

[0144] The beneficial effects of the present invention are:

[0145] The screening and identification method of key genes for embryo implantation of cattle and sheep analyzes the env gene in the cattle and sheep genome and uses bulk transcriptome sequencing to analyze the expression difference between normal and cloned animal placentas, screens out candidate syncytialin genes, and analyzes the candidate syncytialin genes through structure and expression, and identifies the typical functional characteristics of cattle and sheep syncytialin genes through cell fusion and pseudotype virus tests. This scheme is the first to discover key cattle and sheep syncytialin genes in the cattle and sheep genome, revealing the function of cattle and sheep syncytialin genes in mediating placental fusion, and verifying that the receptors bound by cattle and sheep syncytialin genes are ASCT1 and ASCT2. In order to provide a new theoretical basis for solving the problems of pregnancy failure and low efficiency of somatic cell cloning, a total of 8 candidate syncytialin genes were screened. Structural analysis showed that the structures of the 8 candidate syncytialin genes were conserved. Expression analysis found that the 8 candidate syncytialin genes were highly expressed in the placenta, revealing that Sus1 and Sus2 may mediate the development of cattle and sheep embryos and play an important role. Cell fusion and pseudotype virus experiments verified that both Sus1 and Sus2 can promote cell fusion and mediate virus entry, which further indicates that the 8 cattle and sheep placenta-specific candidate syncytialin genes may be involved in early embryonic development and placenta formation.

[0146] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for screening and identifying key genes for embryo implantation in cattle and sheep, characterized in that: The following steps are involved: Step 1: Bioinformatics identification: Identify key syncytin genes in cattle and sheep through cattle and sheep reference genome data and cattle and sheep tissue and embryo transcriptome sequencing data; Step 2: Reverse transcription PCR: collect cattle and sheep placentas, and process the placenta samples before performing 20 μL reverse transcription PCR; Step 3: PCR amplification: Use the cDNA obtained by reverse transcription as a template and use a gene amplifier to perform PCR experiments; Step 4: Fluorescence quantitative PCR: the cDNA obtained by reverse transcription is used as a template, and the expression levels of Sus1 and Sus2 are detected by fluorescence quantitative detection using a universal high-sensitivity dye-based quantitative PCR detection kit and StepOnePlus; Step 5: Cell line construction: insert the CDS sequences of gASCT1 / 2 and bASCT1 / 2 into the pPB-Puro-EF1AEGFP plasmid (Addgene), revive and culture HEK293T cells, and co-transfect pPB-Puro-EF1AEGFP-gASCT1 / 2 or pPB-Puro-EF1AEGFP-bASCT1 / 2 with the pRP-CAGhyPBase plasmid to obtain cell lines; Step 6: Cell fusion test: Identify the typical functional characteristics of cattle and sheep syncytin genes through cell fusion and pseudotype virus tests.

2. The method for screening and identifying key genes for embryo implantation in cattle and sheep according to claim 1, characterized in that: The specific steps of the bioinformatics identification are: The reference genome FASTA files of cattle and sheep GCF_002263795.3 (ARS-UCD2.0) and GCF_001704415.2 (ARS1.2) were downloaded from the Genome database of NCBI. The reference genome FATSA files were used to establish a nucleotide database using tBLASTNv2.5.

0. The Accension numbers NP_041188.1, YP_004222726.1, NP_045939.1, NP_056899.1, NP_054717.1, NP_040334.1, NP_057856.1, NP_056883.1, and NP_043923.1 in the NCBI protein database were used as queries in the tBLASTn program, and the parameters were default; Transcriptome sequencing data of cattle and sheep tissues and embryos were collected from the GEO database of NCBI. Fastq-dumpv2.9.6 was used to convert SRA objects into FASTQ files. HISAT2v2.2.1 was used to establish genome indexes for the cattle and sheep reference genomes GCF_002263795.3 (ARS-UCD2.0) and GCF_001704415.2 (ARS1.2). The FASTQ files were then aligned to the reference genomes to obtain the original bam files, and the bam files were sorted using Samtoolsv1.17 for the next step of TPM and Counts value calculation. The hits obtained from the first tBLASTn were subjected to iterative tBLASTn with amino acids greater than or equal to 100 aa and without early stop codons, and the hits obtained from iterative tBLASTn were subjected to iterative tBLASTn with amino acids greater than or equal to 200 aa and without early stop codons to obtain 3285 and 2789 hits, respectively; All hits obtained after de-redundancy were made into genome annotation files according to chromosome number, start and end position and direction, and added to GCF_002263795.3 (ARS-UCD2.0) and GCF_001704415.2 (ARS1.2) reference genome annotation files and reordered for transcriptome data calculation and analysis; Stringtie2v2.2.1 was used to calculate the TPM and Counts of all hits after de-redundancy. The parameter "-rf" was set to specify the strand specificity of the transcript assembly of the bam file in (1). The obtained GTF files were merged using the "-merge" parameter in Stringtie2 to obtain a merged GTF file. The "-e-rf" parameter of Stringtie2 was used to calculate the TPM and Counts of the collected tissue and embryo samples. It was found that 13 and 11 hits in all the de-redundancy hits of cattle and sheep overlapped with the assembled transcripts obtained after merging, so they were named B1-B13 and G1-G11. It was also found that B3, B4, B5, B9, B11, B13, G1, G2, G5, G8 and G11 were differentially expressed in normal and cloned mature placentas, and not expressed in embryos. Using NCBI's ConservedDomian database, it was found that B3, B4, B5, B9, B11, B13, G1, G2, G5, G8 and G11 all have the conserved domains TLVcoatsuperfamily and Ebola_HIV-1-like_HR1-HR2superfamily, and all have CXXC and RXXR motifs.

3. The method for screening and identifying key genes for embryo implantation in cattle and sheep according to claim 1, characterized in that: The specific steps of the reverse transcription PCR are: Placentas collected from slaughterhouses and cloning cattle and sheep bases were placed in a thermos containing 1× penicillin-streptomycin saline at a temperature of about 20°C and transported to the laboratory within 4 hours; Cut off the excess tissue outside the ovary and wash it repeatedly with saline; The placenta samples were washed three times with RNase-free PBS for 3 min each time, and soybean-sized tissues were cut with scissors and put into a mortar and ground with liquid nitrogen; Add 1mL of pre-cooled TRIzol reagent and lyse at room temperature for 10min; add 200μL of chloroform, shake for 30s, and let stand for 3min; centrifuge at 12,000rpm for 15min at 4℃; aspirate the upper aqueous phase and mix it with an equal volume of isopropanol, and let stand for 30min; centrifuge at 12,000rpm for 10min at 4℃; wash the RNA pellets at the bottom of the tube with 1mL of 75% ethanol solution; centrifuge at 8,000rpm for 5min at 4℃; repeat the wash once and dry at room temperature for 5-10min; dissolve the RNA with an appropriate amount of RNase-free ddH2O, and finally measure the RNA concentration and OD value; 20 μL reverse transcription PCR system: 10×RT Buffer 2 μL, dNTPMix 4 μL, Random Decamers 1 μL, Oligo (dT) Primers 1 μL, Reverse Transcriptase M-MLVRT 1 μL, Embryo Lysis Product 10 μL, RNase Inhibitor 1 μL; The reaction procedure was 42°C, 30 min; 95°C, 10 min; cooling to 4°C, and the cDNA product was stored at –80°C or directly subjected to fluorescent quantitative PCR.

4. The method for screening and identifying key genes for embryo implantation in cattle and sheep according to claim 1, characterized in that: The specific steps of the PCR amplification are: The amplification primer sequences of eight bovine and sheep placenta-specific candidate syncytin genes were synthesized, and the cDNA obtained by reverse transcription was used as a template to perform PCR experiments using the gene amplification instrument QIQIAN-A100; use HSDNA Polymerase kit for high-fidelity PCR test; Reaction system (50 μL): 5× PrimeSTAR Buffer (Mg2+ Plus) 10 μL, dNTP Mixture (2.5 mM each) 4 μL, upstream and downstream primers (10-15 pmol) 1 μL each, Template 1 μL, PrimeSTAR HS DNA Polymerase (2.5 U / μl) 0.5 μL, ddH2O3 2.5 μL; Reaction parameters: 98°C, 2 min pre-denaturation; 98°C, 20 s denaturation, 58-65°C, 34 s annealing, 72°C extension 2 min 30 s, 40 cycles; 72°C extension 5 min.

5. The method for screening and identifying key genes for embryo implantation in cattle and sheep according to claim 1, characterized in that: The specific steps of the fluorescent quantitative PCR are: The quantitative primer sequences of eight candidate syncytin genes specific to the placenta of cattle and sheep were sent for synthesis; The cDNA obtained by reverse transcription was used as a template, and the expression levels of Sus1 and Sus2 were detected by fluorescence quantitative detection using a universal high-sensitivity dye-based quantitative PCR detection kit and StepOnePlus; Reaction system (10 μL): 2×ChamQ Universal SYBR qPCR Master Mix 5 μL, upstream and downstream primers (10-15 pmol) 0.2 μL each, Template 1 μL, ddH2O 4.6 μL; The reaction parameters were: 95°C, 30 s pre-denaturation; 95°C, 10 s denaturation, 60°C, 30 s annealing, according to the instrument default melting curve acquisition program; The internal reference primer is the cattle and sheep histone H2A gene.

6. The method for screening and identifying key genes for embryo implantation in cattle and sheep according to claim 1, characterized in that: The specific steps of constructing the cell line are: The gASCT1 / 2 and bASCT1 / 2 eukaryotic vector cloning primer sequences were sent for synthesis; The CDS sequences of gASCT1 / 2 and bASCT1 / 2 were inserted into the pPB-Puro-EF1AEGFP plasmid (Addgene) using the pEASY-Uni seamless cloning and assembly kit; HEK293T cells were preserved by our laboratory. After revival, they were cultured in a pre-prepared culture medium (9% Gibco fetal bovine serum + 1% double antibody + 90% Gibco DF12 culture medium). The cells were allowed to migrate out slowly and were subcultured to 6-well plates after they were fully grown for subsequent experiments. Co-transfect pPB-Puro-EF1AEGFP-gASCT1 / 2 or pPB-Puro-EF1AEGFP-bASCT1 / 2 with pRP-CAGhyPBase plasmid, add 5ng / ml puromycin for selection after 24-48h, change the culture medium every day, and obtain the cell line after 10-15 days; Take appropriate amount of cells for immunofluorescence and western blot detection to obtain cell lines.

7. The method for screening and identifying key genes for embryo implantation in cattle and sheep according to claim 1, characterized in that: The specific steps of the cell fusion assay are: The CDS sequences of eight bovine and ovine placenta-specific candidate syncytin genes were inserted into the pcDNA3.1-3xFLAG-C plasmid and manipulated using the pEASY-Uni seamless cloning and assembly kit; For the LacZ reporter plasmid, LacZ with an encephalomyocarditis virus internal ribosome entry site was expressed in the presence of T7 polymerase (pcDNA3.1-T7-EMCV-LacZ), the CMV promoter and enhancer sequences of pcDNA3.1(+) were removed as the backbone plasmid, and the EMCV, IRES, LacZ, and T7 terminator sequences were inserted, also using the pEASY-Uni Seamless Cloning and Assembly Kit; The EMCV sequence was amplified by PCR and connected to the IRES sequence; IRES and LacZ sequences were cloned from pMX-IRES-Puro and pUB6-V5-His-LacZ, respectively; T7 terminator sequence was synthesized; All the above PCRs were performed using PrimeSTARHS DNA polymerase; HEK293T cells overexpressing gASCT1 / 2 and bASCT1 / 2 were transfected using PEI reagent, that is, cells were transferred into 24-well plates at a density of 2×105 or into 6-well plates at a density of 1×106 for 12 hours, and then the transfection premix was added. After transfection for 4-6 hours, the transfected cells were co-incubated.

8. The method for screening and identifying key genes for embryo implantation in cattle and sheep according to claim 7, characterized in that: Preparation of the transfection premix: Prepare two EP tubes, add 300 μL Opti-MEM, pre-mix 2 μg plasmid (1 μg pcDNA3.1-3xFLAG-C-8 bovine and sheep placenta-specific candidate syncytin genes and 1 μg pcDNA3.1-T7-EMCV-LacZ) and 10 μL PEI reagent and 2 μg pCAG-T7-Pol in each tube, and let stand at room temperature for 5 min; gently mix and let stand again for 15 min, then add the transfection premix to one well of a 6-well plate; After culturing for 24-48 hours, the culture medium was discarded, the cells were washed 5 times with PBS, the cells were fixed with 4% paraformaldehyde for 20 minutes, and then washed 5 times with PBS. Preheated Xgal staining solution was added, incubated in a 37 degree Celsius incubator for 24 hours, and then observed under a microscope.

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