Application of knock-down CDYL gene in improvement of cattle meat production performance

Abstract

The invention relates to the technical field of gene engineering, in particular to application of a knock-down CDYL gene in improvement of cattle meat production performance. The invention provides an application of knocking down a CDYL gene in improving the differentiation capacity of cattle muscle satellite cells and / or improving the meat production performance of cattle. The gene ID of the CDYL gene is 539013. The 404th basic group A on the upstream of the CDYL gene transcription start site is deleted through a gene editing method, so that the CDYL knock-down gene editing cattle is created, compared with wild cattle and gene editing cattle, the myotube forming ability of muscle satellite cells is obviously enhanced, the number of myotube marker intermuscular line protein positive cells is obviously increased, the number of multinuclear myotubes is obviously increased, and the CDYL knock-down gene editing cattle has the advantages that the CDYL knock-down gene editing cattle can be used for preparing the CDYL knock-down gene editing cattle. And the weight and body size data (body height, cross height, body oblique length, chest circumference and abdominal circumference) of the gene edited cattle are greater than those of the wild cattle. According to the invention, high-yield breeding genes of beef cattle are enriched, and technical support is provided for cultivation of novel gene editing animals.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, in particular to knocking down CDYL The application of genes in improving cattle meat production performance. Background Art

[0002] High efficiency, high benefits and high output in the beef cattle breeding process are the core goals of the industry. Traditional breeding is mainly carried out through four aspects: production performance measurement, hybrid breeding, pedigree records and body shape and appearance assessment. Compared with traditional breeding, gene editing breeding can greatly shorten the breeding time of new varieties (lines) and accurately obtain the expected phenotype. At present, the use of gene editing technology has made great contributions to high yield, high quality, disease resistance and stress resistance in cattle breeding.

[0003] Chromatin domain Y-like ( CDYL ) gene is a transcriptional corepressor that contains a typical amino-terminal chromatin domain and a carboxy-terminal enoyl-CoA hydratase / isomerase catalytic domain. CDYL It is reported to be involved in negative regulation of histone crotonylation modification, further affecting the transcription level of key functional genes. CDYL It was first reported as a regulatory factor for spermatogenesis. CDYL It plays a key role in controlling the intrinsic excitability of neurons. CDYL The relationship between genes and animal meat production performance has not been reported. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for knocking down CDYL The invention discloses the application of gene in improving the meat production performance of cattle. CDYL Genes can improve the meat production performance of cattle.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a knockdown CDYL Application of genes in improving the differentiation ability of cattle muscle satellite cells and / or improving cattle meat production performance, the CDYL The gene ID of the gene in the NCBI database is 539013.

[0006] Preferably, the improvement of the differentiation ability of bovine muscle satellite cells includes: improving the ability of bovine muscle satellite cells to form myotubes, increasing the expression of bovine desmin and increasing bovine multinucleated myotubes; the meat production performance includes: one or more of body weight, body height, cross height, body oblique length, chest circumference and abdominal circumference.

[0007] Preferably, the cattle are Simmental cattle.

[0008] Preferably, the knockdown CDYL Genetic methods include: deletion through gene editing CDYL The A base 404 upstream of the gene transcription start site.

[0009] The present invention provides a method for knocking down CDYL The biological material of the gene comprises: pegRNA, a recombinant plasmid containing the pegRNA, one or more of a gene-editing positive bovine fibroblast cell line and a gene-editing positive bovine blastocyst; the nucleotide sequence of the pegRNA is shown in SEQ ID NO: 1; the gene-editing positive bovine fibroblast cell line is CDYL Gene knockdown cell line; the gene editing positive bovine blastocyst is CDYL Gene knockdown bovine blastocyst; CDYL The gene ID of the gene in the NCBI database is 539013.

[0010] Preferably, the expression elements contained in the recombinant plasmid include: pegRNA, nCas9, M-MLV reverse transcriptase, GFP and EBNA1.

[0011] Preferably, the gene editing positive bovine fibroblast cell line is transfected with the recombinant plasmid and CDYL A bovine fibroblast cell line with gene knockdown; the bovine blastocyst is obtained by injecting the gene-editing positive bovine fibroblast cell line into an enucleated oocyte using a somatic cell nuclear transplantation method and culturing the bovine blastocyst.

[0012] The present invention provides the use of the biological material described in the above technical solution in the preparation of gene-edited cattle with improved meat production performance.

[0013] The present invention provides a method for preparing a gene-edited cattle with improved meat production performance using the biological material described in the above technical solution, comprising the following steps: The gene-edited bovine blastocyst is transplanted into the recipient cow to obtain gene-edited cows.

[0014] Preferably, the recipient cow is a Simmental cow.

[0015] Beneficial effects: The present invention provides a knockdown CDYL Application of genes in improving the differentiation ability of cattle muscle satellite cells and / or improving cattle meat production performance, the CDYL The gene ID of the gene in the NCBI database is 539013. CDYL The gene can improve the meat production performance of cattle. The results of the embodiment show that the present invention deletes the gene through the gene editing method. CDYLThe 404th base A upstream of the gene transcription start site was created CDYL Compared with wild-type cattle, the gene-edited cattle with knockdown have significantly enhanced myotube formation ability of muscle satellite cells, significantly increased the number of myotube marker desmin-positive cells, significantly increased the number of multinucleated myotubes, and the weight and body size data (body height, cross height, body oblique length, chest circumference and abdominal circumference) of gene-edited cattle are greater than those of wild-type cattle. The present invention enriches the breeding genes for high-yield beef cattle and provides technical support for the breeding of new gene-edited animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 Knockdown bovine CDYL Schematic diagram of editing sites of gene expression; Figure 2 To expand the bovine CDYL Electrophoresis of the gene core promoter sequence; Figure 3 For CDYL Genotype identification results of gene editing positive cell lines; Figure 4 For mutant cell lines CDYL Gene mRNA expression test results; Figure 5 The results of CDYL protein expression detection in mutant cell lines; Figure 6 for CDYL Image of gene-edited cloned embryos; Figure 7 for CDYL Gene-edited cow image; Figure 8 This is the result of CDYL protein expression test in gene-edited cattle; Figure 9 for CDYL Results of myogenic differentiation test of gene-edited bovine muscle satellite cells; Figure 10 for CDYL Results of weight and body size testing of gene-edited cattle. DETAILED DESCRIPTION

[0018] The present invention provides a knockdown CDYL The application of the gene in improving the differentiation ability of cattle muscle satellite cells and / or improving the meat production performance of cattle. CDYL The gene ID of the gene in the NCBI database is 539013.

[0019] As an embodiment, the method of improving the differentiation ability of bovine muscle satellite cells includes: improving the ability of bovine muscle satellite cells to form myotubes, increasing the expression of desmin and increasing bovine multinucleated myotubes; the meat production performance includes: one or more of body weight, body height, cross height, body length, chest circumference and abdominal circumference.

[0020] As an embodiment, the cattle are Simmental cattle.

[0021] As one embodiment, the knockdown CDYL Genetic methods include: deletion through gene editing CDYL The 404th base A (49745768th position of chromosome 23 of the cattle genome) upstream of the gene transcription start site (49745364th position of chromosome 23 of the cattle genome). CDYL The 404th base A upstream of the gene transcription start site is deleted. This editing site is located in the promoter region. By editing this editing site, the gene can be knocked down. CDYL Gene expression, thus creating CDYL Gene mutation bovine fibroblast cell line and CDYL Gene-edited cattle.

[0022] The present invention found that knockdown CDYL Genes can improve the meat production performance of cattle. Compared with wild-type cattle, knocking down CDYL The ability of muscle satellite cells to form myotubes in cattle with the gene was significantly enhanced, the number of positive cells for the myotube marker Desmin was significantly increased, the number of multinucleated myotubes was significantly increased, and the weight and body size data (body height, cross height, body oblique length, chest circumference and abdominal circumference) were greater than those of wild-type cattle. The present invention enriches the breeding genes for high-yield beef cattle and provides technical support for the cultivation of new gene-edited animals.

[0023] Based on the above advantages, the present invention provides a method for knocking down CDYL The biological material of the gene comprises: pegRNA, a recombinant plasmid containing the pegRNA, one or more of a gene-editing positive bovine fibroblast cell line and a gene-editing positive bovine blastocyst; the nucleotide sequence of the pegRNA is shown in SEQ ID NO: 1; the gene-editing positive bovine fibroblast cell line is CDYL Gene knockdown cell line; the gene editing positive bovine blastocyst is CDYL Gene knockdown bovine blastocyst; CDYL The gene ID of the gene in the NCBI database is 539013.

[0024] As an embodiment, the expression elements contained in the recombinant plasmid include: pegRNA, nCas9 (H840A), M-MLV reverse transcriptase, GFP and EBNA1; the gene editing positive bovine fibroblast cell line is transfected with the recombinant plasmid and CDYL Gene knockdown bovine fibroblast cell line. As an embodiment, the recombinant plasmid consists of an ePE plasmid and a pegRNA inserted into the ePE plasmid; the pegRNA is inserted between the Nhe I and Hind III restriction sites of the ePE plasmid; the ePE plasmid and its construction method are disclosed in Chinese patent CN119120578A.

[0025] As an embodiment, the bovine blastocyst is obtained by injecting the gene-editing-positive bovine fibroblast cell line into an enucleated oocyte using a somatic cell nuclear transplantation method and culturing the bovine blastocyst.

[0026] The biomaterial provided by the present invention uses the ePE plasmid as a skeleton and inserts bovine CDYL The pegRNA sequence of the gene editing site was obtained to obtain a recombinant plasmid, which was then transfected into bovine fibroblasts and successfully obtained CDYL Gene-edited fibroblast cell lines were further transformed with somatic cell nuclear transfer to obtain improved meat production performance CDYL Gene-edited cattle.

[0027] Based on the above advantages, the present invention provides the use of the biomaterial described in the above technical solution in preparing gene-edited cattle with improved meat production performance. As an embodiment, the recipient cow of the gene-edited cattle is Simmental cattle.

[0028] Based on the above advantages, the present invention provides a method for preparing gene-edited cattle with improved meat production performance using the biological material described in the above technical solution, comprising the following steps: The gene-edited bovine blastocyst is transplanted into the recipient cow to obtain gene-edited cows.

[0029] As an embodiment, the recipient cow is a Simmental cow.

[0030] In order to further illustrate the present invention, the knockdown provided by the present invention is described below in conjunction with the accompanying drawings and embodiments. CDYL The application of genes in improving the meat production performance of cattle is described in detail, but they should not be understood as limiting the scope of protection of the present invention.

[0031] The main reagents used in the following examples are: Collagenase IV used to isolate bovine fetal fibroblasts was purchased from Sigma; DMEM, FBS, PBS and Trypsase used for cell culture were all purchased from Gibco; primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.; fluorescent quantitative PCR kit was purchased from Novazon Biotech Co., Ltd.; antibodies used for immunoblotting: CDYL antibody was purchased from Santa Cruz, and α-Tubulin antibody was purchased from Proteintech.

[0032] The main instruments used in the following examples are: CO2 incubator (Thermo, USA); fluorescence quantitative PCR instrument (Roche, Switzerland); exposure instrument (Tanon, China); flow cytometer (SONY, Japan); micromanipulator (Nikon, Japan).

[0033] Example 1 CDYL Construction of gene editing plasmids 1.1. According to cattle CDYL Gene editing site ( Figure 1 ) designed and synthesized pegRNA-CDYL-1, the nucleotide sequence of which is shown in SEQ ID NO: 1, and is as follows: 5'-gctagcTATTTAGAGGTGAAGCTTTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTAAACACCCCAAAGCTTCACCTCTAAATAaagctt-3'; 5'-gctagc-3' is Nhe I restriction site, 5'-aagctt-3' Hind III restriction enzyme cutting site.

[0034] 1.2. Construction of recombinant plasmid Use restriction enzyme Nhe I and Hind Ⅲ The ePE plasmid was digested at 37°C for 1.5 h, and the linearized fragment was recovered by gel cutting; the ePE plasmid and the construction method are disclosed in Chinese patent CN119120578A.

[0035] The pegRNA-CDYL-1 double-stranded fragment synthesized in step 1.1 was mixed with the recovered linearized fragment and connected at 16°C overnight, then transformed into DH5α competent cells, spread on LB plates containing ampicillin for growth, and then single colonies were picked for expansion and sequencing. The sequencing primers are as follows: U6-promoter: 5'-CCGTAACTTGAAAGTATTTCG-3' (SEQ ID NO: 2); After culturing, the positive clones were extracted using a plasmid endotoxin removal kit (TIANGEN, China) to obtain the recombinant plasmid (named ePE-CDYL-Bos) for subsequent cell transfection.

[0036] Example 2 Construction of gene editing positive cell line 2.1. Isolation and culture of bovine fetal fibroblasts The bovine fibroblasts used in this example were obtained from wild Chinese Simmental fetuses (about three months old) at the grassland livestock germplasm innovation and breeding base. Primary fibroblasts were isolated and cultured using the tissue adhesion method. The fetal tissue was washed three times with PBS containing double antibodies, soaked in 75% alcohol for 5 seconds, and then quickly transferred to PBS. After washing three times, it was cut into 1-3 mm pieces. 3 , and evenly spread in a culture dish, inverted and cultured in a 38.5 ℃, 5% CO2 incubator for 1~2 hours, and add complete culture medium after the tissue block adheres to the culture dish. When the cell growth reaches 80%~90% confluence, subculture and cryopreservation.

[0037] 2.2 Cell transfection The primary bovine fibroblasts were seeded into six-well plates one day before transfection, and transfection was performed when the cells reached 70% to 80% confluence. The transfection steps were strictly performed in accordance with the instructions of the Lipofectamine 2000 (Invitrogen, USA) kit. Specifically, 6 μg of the recombinant plasmid ePE-CDYL-Bos in Example 1 was transfected into bovine fibroblasts.

[0038] 2.3. Flow cytometry sorting of gene-edited positive cells 36-48 h after transfection, the cells were inoculated into 96-well plates by flow cytometry to ensure that a single cell was inoculated into each well. The monoclonal cells were further amplified. When the density reached 80% in the 6-well plates, half of the cells were taken to extract genomic DNA for identification, and the remaining cells were frozen for somatic cell cloning.

[0039] 2.4. Identification of monoclonal cells According to the cattle CDYL The promoter region contains the DNA sequence of the editing site in Example 1, and PCR primers are designed, the specific sequences are as follows: Bos-CDYL-1F: 5'-ACAACTGAGGAAACTTAA-3' (SEQ ID NO: 3); Bos-CDYL-1R: 5'-GAAAAGTACAGGTACACG-3' (SEQ ID NO: 4); The DNA of edited cells and unedited cells were extracted and amplified separately. CDYL The sequence of the editing site is Figure 2 ) were purified and then sequenced. The results showed that the edited cell line was successfully obtained ( Figure 3 ).

[0040] Example 3 Mutation of cells CDYL Gene mRNA and protein level detection Real-time fluorescence quantitative PCR Total RNA from edited cells was extracted according to the instructions of HiPure total RNA Mini Kit (Magen, China), and the RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo, USA).

[0041] Using GoScript TM Total RNA was reverse transcribed into cDNA using Reverse Transcription Mix, Oligo(dT) (Promega, USA).

[0042] Primers were designed using Primer Premier 5.0 software. The specific sequences are as follows: Bos-CDYL-2F: 5'-GACCCGACTGGAACAAAGTGT-3' (SEQ ID NO: 5); Bos-CDYL-2R: 5'-CTTTCAACCTCGTACAGCTCCT-3' (SEQ ID NO: 6); PCR system: 80 ng cDNA, 10 μM upstream primer, 10 μM downstream primer, SYBR green master mix (TaKaRa, China) and 20 μL of RNase-free water.

[0043] The reaction system included: 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 34 s, for a total of 40 cycles.

[0044] GAPDH As internal reference, the primer sequences are as follows: Bos-GAPDH-F: 5'-TGGTGAAGGTCGGAGTGAAC-3' (SEQ ID NO: 7); Bos-GAPDH-R: 5'-ATGGCGACGATGTCCACTTT-3' (SEQ ID NO: 8); The results showed that compared with unedited cells, CDYL The mRNA expression level of Figure 4 ).

[0045] Immunoblotting The edited cells were lysed with RIPA Buffer (Beyotime, China), and the protein concentration was determined using BCA Protein Assy Kit (Beyotime, China). The protein samples were separated by 10% SDS-PAGE and transferred to a membrane.

[0046] The primary antibody was incubated at 4 °C overnight, and the secondary antibody was incubated at 37 °C for 1 h. After extensive washing with 1× TBST, the samples were exposed using an ECL chemiluminescence detection kit (Biosharp, China).

[0047] The results showed that compared with unedited cells, CDYL protein expression in mutant cells was significantly reduced ( Figure 5 ).

[0048] Example 4 Gene-edited cattle obtained by somatic cell nuclear transplantation 4.1. Oocyte collection and in vitro maturation Chinese Simmental ovaries were collected from local slaughterhouses and stored in saline containing penicillin and streptomycin at 20-25 °C. Cumulus-oocyte complexes (COCs) were collected from preantral follicles with a diameter of 3 to 8 mm using a needle. The oocytes were washed three times with a buffer (M199 medium containing 1% FBS) under a stereomicroscope and then placed in in vitro maturation medium for oocyte maturation. The culture conditions were 38.5 °C and 5% CO2 in a saturated humidity environment. After 22 h of in vitro maturation, cumulus cells were removed in 1 mg / mL hyaluronidase, and mature oocytes that expelled the first polar body were selected. The in vitro maturation medium was based on M199 medium and only contained 10% FBS, 20 ng / mL epidermal growth factor, 1 μg / mL β-estradiol, 1 μg / mL gonadotropin, and 0.1 IU / mL luteinizing hormone.

[0049] 4.2. Nuclear transfer, fusion, activation and embryo culture Before somatic cell nuclear transfer (SCNT), oocytes were exposed to 7.5 μg / mL Hoechst 33342 for 5 min and then placed in M199 medium containing 7.5 μg / mL cytochalasin B for somatic cell nuclear transfer. Under a brief (1-2 s) exposure to ultraviolet light, the position of the oocyte nucleus was located and removed with a glass needle (18-20 μm), and then CDYL Gene-edited positive cells were injected into enucleated oocytes and allowed to rest in the operating drop for 30 min to recover. The cloned embryos were then electrofused using a 1.8 kV / cm DC pulse. The electrofused cloned embryos were incubated in 5 μmol / L ionomycin for 5 min and then transferred to 10 μg / mL cycloheximide (CHX) for 5 h. Finally, the cloned embryos were transferred to SOFaa culture medium and cultured in a humidified environment at 38.5 °C and 5% CO2. Fresh SOFaa culture medium was replaced every 48 h until the cloned embryos developed to the blastocyst stage ( Figure 6 ).

[0050] Embryo transfer First, the recipient cows were treated for estrus synchronization. A progesterone vaginal plug (CIDR) was placed in the recipient cows, and after 7 days, prostaglandin (PG) was injected and the estrus status of the recipient cows was observed. CDYL The gene-edited cloned blastocysts were transplanted into 9 recipient cows in estrus, and pregnancy was determined by rectal ultrasound EVO scanner on the 35th day after transplantation.

[0051] Example 5 Detection of CDYL protein levels in edited cattle Immunoblotting The method is the same as step 3.2 in Example 3.

[0052] The results showed that compared with wild-type cattle, edited cattle ( Figure 7 ) in which CDYL protein expression was significantly decreased ( Figure 8 ).

[0053] Example 6 CDYL Detection of differentiation ability of muscle satellite cells from mutant cattle Muscle satellite cells of mutant and wild-type cattle were isolated and cultured for myogenic differentiation (see [LiShuang, Liu Dan, Fu Yuying et al. Podocan Promotes Differentiation of Bovine Skeletal Muscle Satellite Cells by Regulating the Wnt4-β-Catenin SignalingPathway. [J] . Front Physiol, 2019, 10: 1010.]), and myotube differentiation was observed on the third day of differentiation (D3). The results showed that CDYL After gene editing, the myotube formation ability of bovine muscle satellite cells was significantly enhanced, the number of myotube marker Desmin-positive cells was significantly increased, and the number of multinucleated myotubes was significantly increased ( Figure 9 ).

[0054] Example 7 Editing cattle weight and body size detection right CDYL The growth indicators of gene mutant cattle and wild-type cattle were tested, and the results are shown in Figure 10 , where * is P <0.05, ** P <0.01. The weight and body size data (body height, cross height, body oblique length, chest circumference and abdominal circumference) of mutant cattle from birth to 6 months of age were larger than those of wild-type cattle.

[0055] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Knockdown CDY Application of genes in improving the differentiation ability of cattle muscle satellite cells and / or improving cattle meat production performance, the CDY The gene ID of the gene in the NCBI database is 539013.

2. The use according to claim 1, characterized in that: The method for improving the differentiation ability of cattle muscle satellite cells includes: improving the ability of cattle muscle satellite cells to form myotubes, increasing the expression of cattle desmin and increasing cattle multinucleated myotubes; the meat production performance includes: one or more of body weight, body height, cross height, body oblique length, chest circumference and abdominal circumference.

3. The use according to claim 1 or 2, characterized in that: The cattle are Simmental cattle.

4. The use according to claim 1, characterized in that: The knockdown CDY Genetic methods include: deletion through gene editing CDY The A base 404 upstream of the gene transcription start site.

5. A Knockdown CDY Genetic biological material, characterized in that include: pegRNA, a recombinant plasmid containing the pegRNA, a gene-editing positive bovine fibroblast cell line, and a gene-editing positive bovine blastocyst; the nucleotide sequence of the pegRNA is shown in SEQ ID NO: 1; the gene-editing positive bovine fibroblast cell line is CDY Gene knockdown cell line; the gene editing positive bovine blastocyst is CDY Gene knockdown bovine blastocyst; CDY The gene ID of the gene in the NCBI database is 539013.

6. The biomaterial according to claim 5, characterized in that The expression elements contained in the recombinant plasmid include: pegRNA, nCas9, M-MLV reverse transcriptase, GFP and EBNA1.

7. The biomaterial according to claim 5 or 6, characterized in that: The gene editing positive bovine fibroblast cell line is transfected with the recombinant plasmid and CDY A bovine fibroblast cell line with gene knockdown; the bovine blastocyst is obtained by injecting the gene-editing positive bovine fibroblast cell line into an enucleated oocyte using a somatic cell nuclear transplantation method and culturing the bovine blastocyst.

8. Use of the biomaterial according to any one of claims 5 to 7 in the preparation of gene-edited cattle with improved meat production performance.

9. A method for preparing a gene-edited cattle with improved meat production performance using the biological material according to any one of claims 5 to 7, characterized in that: The following steps are involved: The gene-edited bovine blastocyst is transplanted into the recipient cow to obtain gene-edited cows.

10. The method according to claim 9, characterized in that The recipient cow is a Simmental cow.

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