WO2024065376A1 - Utilisation d'un nouveau locus du gene callipyge dans l'elevage de moutons - Google Patents

Utilisation d'un nouveau locus du gene callipyge dans l'elevage de moutons Download PDF

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Publication number
WO2024065376A1
WO2024065376A1 PCT/CN2022/122505 CN2022122505W WO2024065376A1 WO 2024065376 A1 WO2024065376 A1 WO 2024065376A1 CN 2022122505 W CN2022122505 W CN 2022122505W WO 2024065376 A1 WO2024065376 A1 WO 2024065376A1
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sheep
body weight
gene
differentially methylated
breeding
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PCT/CN2022/122505
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Chinese (zh)
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邓学梅
王建魁
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中国农业大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

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  • the present invention belongs to the field of biotechnology, and relates to the application of a new site of a beautiful buttocks gene in sheep breeding, and specifically to the application of a differentially methylated site of a beautiful buttocks gene in breeding for meat traits.
  • Tan sheep have become smaller and their growth and development are slower. Therefore, solving the contradiction between lamb meat, fur and skin output is crucial to improving the economic benefits of fur-meat or wool-meat dual-purpose breeds such as Tan sheep.
  • the beautiful butt gene is located in the telomere of chromosome 18 of sheep, involving paternal and maternal imprinted genes in the Dlk1-Mirg region.
  • the beautiful butt phenotype of sheep is believed to be caused by a base mutation from A to G in this region, which leads to excessive hypertrophy of the buttocks muscles. This trait is currently considered to be inherited from the father.
  • the breeding application of the maternal beautiful butt gene has not yet been involved. Advanced breeding methods and means for the beautiful butt trait of the maternal will effectively supplement the traditional breeding methods for beautiful butt.
  • DMRs differentially methylated regions
  • the present invention claims a differentially methylated region (DMR).
  • DMR differentially methylated region
  • the differentially methylated region (DMR) claimed to be protected by the present invention comes from the sheep buttocks imprint region Dlk1-Mirg, and its nucleotide sequence is shown in SEQ ID No.1.
  • the DMR claimed in the present invention includes both its nucleotide sequence and its methylation modification status (level).
  • the present invention claims the use of the differentially methylated region (DMR) described in the first aspect as a methylation marker in any of the following:
  • the present invention claims the use of the sheep Gtl2 gene as a marker in any of the following:
  • the sheep Gtl2 gene is the gene associated with the DMR described in the first aspect above, the DMR is located at 66216001-66217000 of sheep chromosome 18, and the genome version is rambouillet_v1.0_genomic (the DMR is located in the promoter region of the sheep Gtl2 gene).
  • the sheep Gtl2 gene is specifically located at 66220214-66253349 of sheep chromosome 18, and the genome version is rambouillet_v1.0_genomic.
  • the present invention claims the use of a substance for detecting the methylation level of the differentially methylated region described in the first aspect above in identifying or assisting in identifying the body weight trait of sheep.
  • the present invention claims protection for the use of a substance for detecting the expression level of the sheep Gtl2 gene in identifying or assisting in identifying the body weight trait of sheep.
  • the sheep weight may be the sheep's early body weight, such as the body weight at 1 month of age or younger.
  • the early body weight of the sheep may specifically be the body weight of the sheep at one month of age.
  • the present invention claims protection for the use of the differentially methylated regions described in the first aspect as methylation markers in the breeding of sheep for meat traits.
  • the present invention claims protection for the use of the sheep Gtl2 gene as a marker in breeding sheep for meat traits.
  • the present invention claims the use of a substance for detecting the methylation level of the differentially methylated region described in the first aspect above in breeding for sheep meat traits.
  • the present invention claims protection for the use of a substance for detecting the expression level of the sheep Gtl2 gene in breeding for sheep meat traits.
  • the breeding of sheep for meat traits may be breeding of sheep for meat traits at an early stage (eg, 1 month old).
  • the substance used to detect the methylation level of the differentially methylated region of sheep described in the first aspect above can specifically be a reagent and/or instrument used for methylation sequencing of the DNA fragment shown in SEQ ID No. 1 in the sheep genome.
  • the substance used to detect the expression level of sheep Gtl2 gene can specifically be a primer pair consisting of two single-stranded DNAs shown in SEQ ID No.3 and SEQ ID No.4.
  • the present invention claims a method for identifying or assisting in identifying a body weight trait in sheep.
  • the method for identifying or assisting in identifying the weight trait of sheep claimed in the present invention may include the following steps (B1) or (B2):
  • the sheep weight may be the early weight of the sheep, such as the weight at 1 month of age or younger.
  • the early body weight of the sheep may be the body weight of the sheep at one month of age.
  • the present invention claims protection for the use of the method described in the tenth aspect above in breeding sheep for meat traits.
  • the breeding of sheep for meat traits may be breeding of sheep for early-stage (1 month old) meat traits.
  • the present invention claims a method for breeding a meat-type sheep breed.
  • the method for breeding a meat-type sheep breed claimed in the present invention may include the following steps (D1) or (D2):
  • the present invention claims the use of a differentially methylated region (DMR) of the mouse Dlk1-Mirg region as a methylation marker in regulating the body weight of mice;
  • DMR differentially methylated region
  • the nucleotide sequence of the differentially methylated region (DMR) of the mouse Dlk1-Mirg region is shown in SEQ ID No.2.
  • the present invention claims protection for the use of mouse Meg3 gene as a marker in regulating mouse body weight.
  • the mouse Meg3 gene is a gene associated with the differential methylation region (DMR) of the mouse Dlk1-Mirg region mentioned above, and the genebank accession number of the DMR is: AC107681.15.
  • the mouse Meg3 gene is specifically located on mouse chromosome 12: 109506879-109538163, and the reference genome version is: GRCm39.
  • the mouse MDR is located upstream of the Meg3 gene, far from the meg3 promoter, and belongs to the regulatory region (Dlk1-Mirg region) that regulates the expression of the meg3 gene.
  • the present invention claims a method for constructing a mouse model of reduced body weight.
  • the method for constructing a mouse model with reduced body weight claimed in the present invention may include the following steps: reducing the expression level of the mouse Meg3 gene to obtain a mouse model with reduced body weight.
  • the expression of mouse Meg3 gene is reduced by knocking out the differentially methylated region (DMR) regulating the expression of mouse Meg3 gene.
  • DMR differentially methylated region
  • it is carried out using CRISPR/Cas9 technology, wherein the gRNA sequence (targeting the differentially methylated region of the mouse Dlk1-Mirg region described above) is shown in Figure 5.
  • the body weight refers to the body weight of young mice (such as 2 days after birth).
  • the present invention claims the use of a differentially methylated region (DMR) of a mammalian Dlk1-Mirg region or a gene thereof in any of the following:
  • the sheep is Aohan fine-wool sheep.
  • the sample to be tested when detecting the methylation level of the differentially methylated region described in the first aspect and detecting the expression of the sheep Gtl2 gene can be a tissue sample, such as a skin tissue sample.
  • the sample to be tested is specifically sheep shoulder skin tissue.
  • Figure 1 shows the identification of DMRs in the sheep Gtl2 region.
  • Figure 2 shows the specific location information of DMR in the sheep Gtl2 region (genome version: Oar_rambouillet_v1.0).
  • Figure 3 shows the comparison and analysis of the body weight of lambs in the high and low methylation level groups. * indicates P ⁇ 0.05
  • FIG. 4 is a comparative analysis of the expression of the Gtl2 gene in the high body weight (low methylation level) group and the low body weight (high methylation level) group (** indicates extremely significant difference, P ⁇ 0.001).
  • Figure 5 shows the Guide RNA sequence
  • FIG6 shows the PCR positive identification results of Gtl2 (called Meg3 gene in mice) related DMR knockout mice, where the symbol “+/-” indicates the characteristics of positive individual bands, and the symbol “+/+” indicates the characteristics of negative control individual bands.
  • FIG. 7 is a schematic diagram of the selection of Gtl2 (called Meg3 gene in mice) related DMR knockout mice
  • Figure 8 shows the expression of maternal imprinted genes (Meg3, Mirg and Rian) such as Gtl2 (called Meg3 gene in mice) in knockout mice and wild type mice. *** indicates extremely significant difference (P ⁇ 0.0001).
  • Figure 9 shows the body weight analysis of young (2 days after birth) mice with Gtl2 (called Meg3 gene in mice) related DMR knockout mice. *** indicates extremely significant difference (P ⁇ 0.0001).
  • Example 1 Application of the expression of the maternal imprinted gene cluster Gtl2-Mirgs in the breeding of meat-type sheep
  • This embodiment involves a newly discovered DMR (SEQ ID No. 1) located in the sheep maternal imprinted gene cluster Gtl2-Mirgs, and the Gtl2 gene associated with the DMR.
  • the sheared DNA fragments were end-repaired, A-tailed, and connected to sequencing adapters in which all cytosines were methylated. Bisulfite treatment was then performed (EZ DNA Methylation Gold Kit, Zymo Research). The unmethylated C was converted to U (to T after PCR amplification), while the methylated C remained unchanged. Finally, PCR amplification was performed to obtain the final DNA library. The length of the insert fragment of the library was detected using Agilent 2100 (Agilent, USA). After the library inspection was qualified, different libraries were pooled according to the effective concentration and the target data volume requirements and then sequenced by Hiseq. The sequencing method was double-end sequencing.
  • the swDMR software http://122.228.158.106/swDMR/ was used to identify differentially methylated regions (DMR). Based on the methylation information of each site (set reads coverage ⁇ 5), the software used a sliding window method to scan the genome and identified the methylation degree of the promoter region of the Gtl2 gene in 6 individuals. The methylation levels were sorted from high to low, with the first three being the high methylation group and the last three being the low methylation group. Based on the important biological significance of DMR, the genomic location of DMR and the genomic structural annotation information were used to perform structural annotation on it.
  • the reaction system for reverse transcription into cDNA is 20 ⁇ L, including: primer, 3.0 ⁇ L; dNTP, 0.15 ⁇ L; Mautiscribe RT enzyme, 1.0 ⁇ L; 10 ⁇ RT Buffer, 1.5 ⁇ L; RNase inhibitor, 0.19 ⁇ L; RNA sample, 2.0 ⁇ L (1-10ng); DEPC water, 12.16 ⁇ L. Reaction conditions: 16°C, 15min; 42°C, 30min; 95°C, 5min; finally kept at 4°C.
  • Fluorescence quantitative PCR was used to detect the expression of Gtl2 gene.
  • the lambs were sorted according to the methylation level of the Gtl2 promoter region, and the methylation region shared by the Gtl2 promoter region of the six lambs was selected for further grouping. Specifically, for each methylation region in the region, the methylation level was sorted, and the first three lamb groups with high methylation and the last three lamb groups with low methylation levels formed differential methylation regions. The significance of the difference was calculated to obtain the regions shown in Figures 1 and 2. According to the methylation level of the region, they were divided into two groups (sorted from high to low according to the methylation level, the first three were high methylation groups, and the last three were low methylation groups).
  • the DMR in this application was obtained through whole genome methylation sequencing of skin tissues of 6 Aohan fine wool sheep.
  • Figure 1 shows the identification of DMR in the sheep Gtl2 region.
  • Figure 2 shows the specific location information of DMR in the sheep Gtl2 region (genomic version: Oar_rambouillet_v 1.0).
  • the DMR is located at 66216001-66217000 of chromosome 18 of sheep, and the genome version is rambouillet_v1.0_genomic (the DMR is located in the promoter region of the sheep Gtl2 gene).
  • FIG. 3 is a comparison and analysis of the body weight of lambs with high/low methylation levels.
  • Figure 4 is a comparative analysis of the expression of the Gtl2 gene in the high body weight (low methylation level) group and the low body weight (high methylation level) group.
  • CRISPR/Cas9 technology was used to perform gene editing on the Gtl2 (called Meg3 gene in mice) DMR sequence (SEQ ID No. 2), delete the sequence, construct C57/BL mouse MEG3 (Gtl2) DMR knockout mice, and identify their body weight traits.
  • the primer names and sequences are: Meg3_WT-F1: GGATTCAAGGAATGGAGTTTGGG; Meg3_WT-R1: GCCACTTGCATCAGAATGAAAGC; Meg3(DMR)_KO-F2: CTCTGCCATACATAGTGTTC TAGGCC; Meg3(DMR)_KO-R2: TGAGGAAACAGGCTGTTGAGTGG; Meg3(DMR)_KO-Seq: CTCTGCCATACATA GTG TTCTAGGCC.
  • Reaction system 0.5 ⁇ L of each primer F1/R1, F2/R2, (DMR)KO-seq, 10 ⁇ L of dNTP Mix, add DEPC water to make up to 20 ⁇ L system.
  • Reaction conditions first step, 95°C, 5 min; second step, 95°C, 30 s, 57°C, 30 s, 72°C, 30 s, 35 cycles; third step, 72°C, 5 min; fourth step, storage at 4°C.
  • Genotype determination criteria 1. WT/WT, Wild Type Wild-type genome: WT genome Meg3WT-F1/R1 primer pair PCR product obtained a single WT band of 435bp, WT genome Meg3(DMR)KO-F2/R2 primer pair PCR band (5167bp) exceeded the conventional PCR reaction capacity and no PCR product was obtained; 2.
  • (DMR)KO/(DMR)KO Homozygous homozygous genome: (DMR)KO genome Meg3WT-F1/R1 primer pair had no PCR product because the upstream and downstream primers could not anneal, (DMR)KO genome Meg3(DMR)(DMR)KO-F2/R2 primer pair PCR product A single (DMR) KO band of 492 bp was obtained from the WT/(DMR) KO, Heterozygous heterozygous genome: The WT genome Meg3WT-F1/R1 primer pair PCR product obtained a single WT band of 435 bp, the WT genome Meg3(DMR) KO-F2/R2 primer pair PCR band (5167 bp) exceeded the conventional PCR reaction capacity and no PCR product was obtained, the (DMR) KO genome Meg3WT-F1/R1 primer pair had no PCR product because the upstream and downstream primers could not anneal, and the (DMR) KO genome
  • each positive female mouse is mated with a wild-type C57BL/6J inbred male mouse for natural implantation and pregnancy, or male mice are taken for in vitro fertilization (IVF) and embryo implantation for pregnancy expansion, pregnancy and birth.
  • IVF in vitro fertilization
  • Meg3 fluorescence quantitative PCR the sample loading system (20 ⁇ L) included: 0.5 ⁇ L each of upstream and downstream primers (Meg3 upstream primer: TCTTCCTGTGCCATTTGCTGT; Meg3 downstream primer: TCTTCCTGTGCCATTTGCTGT.
  • Mirg upstream primer TTAGGAGCATTTCCAGGAGG; Mirg downstream primer: AAGCGAACTCATCACAGACAAC.
  • Rian upstream primer TGGAGGCCCTAATGTGAATG; Rian downstream primer: AAGCATCCACAGGACGCAAT), 10 ⁇ L of SYBGreen Mix, and 9 ⁇ L of DEPC water.
  • reaction conditions were as follows: the first step, 95°C, 30 s; the second step, 95°C, 5 s; annealing (Meg3 gene annealing temperature was 58°C, Mirg gene annealing temperature was 57°C, and Rian annealing temperature was 60°C), 20 s; repeated 42 times.
  • the positive heterozygous chimeric genotype F0 mice obtained in step 4 were used as the mother to mate with the wild-type father to obtain offspring heterozygous knockout mice (corresponding to the above steps 5 and 6).
  • the mating pattern is shown in FIG. 7 .
  • the present invention reveals the mechanism of action of a differential methylation site (DMR) in the imprinted region (Dlk1-Mirg) where the beautiful buttocks gene is located in regulating individual development, and discloses for the first time the application of a new DMR (SEQ ID No.1) in early breeding of sheep for meat traits.
  • DMR differential methylation site
  • SEQ ID No.1 a new DMR
  • the present invention provides a new molecular breeding method for selecting meat lambs.
  • knocking out the DMR (SEQ ID No.2) reported in mice can induce mice to lose weight and become smaller in size.
  • the present invention is of great significance for breeding young mammals for meat traits based on DMR in the Dlk1-Mirg region.

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Abstract

La présente invention concerne l'utilisation d'une région méthylée de manière différentielle du gène callipyge dans la sélection des caractéristiques de la viande. La présente invention concerne une région méthylée de manière différentielle à partir d'une région imprimée Dlk1-Mirg du gène callipyge de mouton, la séquence nucléotidique de la région méthylée de manière différentielle étant représentée par SEQ ID NO : 1. La région différentiellement méthylée et le gène Gtl2 qui lui est associé peuvent être utilisés pour la sélection précoce des caractéristiques de la viande de mouton. La présente invention concerne en outre une région méthylée de manière différentielle représentée par SEQ ID NO : 2 d'une région Dlk1-Mirg de souris et un gène associé Meg3 de celle-ci pouvant être utilisés pour réguler et contrôler le poids corporel des souris et construire un modèle de souris à perte de poids contrôlée.
PCT/CN2022/122505 2022-09-29 2022-09-29 Utilisation d'un nouveau locus du gene callipyge dans l'elevage de moutons WO2024065376A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN118374609A (zh) * 2024-06-21 2024-07-23 中国农业科学院北京畜牧兽医研究所 一种与绵羊生长性状相关的snp分子标记及其应用
CN118374609B (zh) * 2024-06-21 2024-08-30 中国农业科学院北京畜牧兽医研究所 一种与绵羊生长性状相关的snp分子标记及其应用

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