Application of LCORL gene Indel mutant for improving growth speed and size of animals
By identifying the INDEL loci of the NCAPG-LCORL region in the home cattle, especially the CT deletion mutation at 37401770, the unclear relationship between INDEL mutations in the LCORL gene fragment INDEL mutations and animal growth traits in existing studies, and the effect of improving animal growth speed and size through gene editing is achieved.
Patent Information
- Application Number
- CN202411951967.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-06-27
AI Technical Summary
The existing research mainly focuses on single nucleotide polymorphism variants, and the relationship between fragment INDEL variants in the LCORL gene and animal growth traits is still unclear.
Through population genetics, NCAPG-LCORL, the most selected locus, was identified in domestic cattle, and the INDEL locus at 37349373-37438248 of domestic cattle chromosome 6, was found, especially the CT deletion mutation at 37401770, which was inferred that it was related to animal growth traits.
The introduction of mutations similar to home-box rs384548488*A in mice through CRISPR/Cas9 technology confirmed that the loss of the PIP domain in the LCORL gene caused an increase in growth rate in mice, providing a gene editing method to improve the growth rate and size of animals.
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Figure CN120210291A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of genetic engineering, and particularly relates to the application of an LCORL gene Indel mutant for improving the growth rate and size of animals. Background Art
[0002] The body size of European cattle has increased significantly in the past 1000 years. This change not only reflects the evolutionary history of domestic cattle but also reveals the continuous selection for body size and growth rate in human breeding activities. Body size and growth rate are important selection criteria in domestic animals. Especially for meat livestock, these traits directly affect their economic value and production efficiency. Therefore, studying the genetic variations of these traits helps to improve the yield and quality of beef cattle. Various livestock breeds exhibit significant phenotypic diversity, which provides a rich resource for exploring genetic variations that have a great impact on body size differences. Mammals have a set of conserved genes regulating body size, indicating that studying these genes is not only important for the breeding of domestic cattle but also related to human health and animal body size regulation.
[0003] Domestic common cattle (Bos taurus) were domesticated from ancient cattle (Bos primigenius) in the Fertile Crescent about 10,500 years ago and introduced into Europe about 8,500 years ago. Archaeological evidence shows that after domestication, the body size of cattle gradually decreased before the Middle Ages and then gradually increased. This trend of change indicates that the selection for larger body size may have left traces of selective sweeps in the genome. In recent years, with the development of genome sequencing technology, more and more genetic variations related to body size and growth rate have been discovered. The LCORL gene (ligand dependent nuclear receptor corepressor like) is an important candidate gene, which is known to be related to body size and growth rate in various mammals. Although some studies have reported the relationship between the LCORL gene and the body size and growth rate of domestic cattle, these studies mainly focus on single nucleotide polymorphism variations. Single nucleotide polymorphism variations only involve the variation of a single base, and the relationship between the INDEL variation sites of fragments and animal growth traits is still unclear. Therefore, it is necessary to provide the use of INDEL sites in animal growth traits. Summary of the Invention
[0004] To solve the above problems, the present invention provides the application of an LCORL gene Indel mutant for improving the growth rate and size of animals.
[0005] Application of an Indel locus of the LCORL gene in improving animal growth traits, wherein the Indel locus is located at positions 37349373 - 37438248 on chromosome 6 of cattle.
[0006] Preferably, the Indel locus is located at position 37401770 on chromosome 6 of cattle, and a base CT deletion occurs at the first and second positions of 37401770 on chromosome 6 of cattle.
[0007] Preferably, the reference genome of the Indel locus is ARS - UCD1.2.
[0008] Preferably, the improvement of growth traits refers to an increase in body weight.
[0009] Preferably, the animal is cattle or mice.
[0010] An Indel nucleic acid molecule for improving animal growth traits, wherein the Indel nucleic acid molecule has a mutation from base ACT to base A;
[0011] The nucleotide sequence of the Indel molecular marker is shown in SEQ ID NO.2, and SEQ ID NO.2 is as Figures 9 to 23 , wherein, Figure 10 The yellow - marked and underlined bases in the figure are the bases after the deletion of CT. When no bases are deleted, the bases at the yellow - marked and underlined positions are ACTCTGGG.
[0012] A mutant protein encoded by the nucleotide sequence of SEQ ID NO.2 as described above;
[0013]
[0014] Use of the Indel nucleic acid molecule or the mutant protein in improving animal growth traits.
[0015] Use of the Indel nucleic acid molecule or the mutant protein in detecting animal growth traits.
[0016] Preferably, the primers for detecting animal growth traits using the Indel nucleic acid molecule are as shown in SEQ ID NO. 6-7, and the animal is Bos taurus.
[0017] Compared with the prior art, the beneficial effects of the present invention are reflected in:
[0018] Through population genetics, the present invention identified a locus NCAPG-LCORL that has been under the strongest selection during the improvement of Bos taurus breeds in the past 1000 years. Through local fine mapping, the INDEL with the largest frequency difference was found within Chr6: 37349373-37438248, thereby determining the INDEL locus related to animal growth traits in this region. Therefore, the present invention proposes the application of the INDEL locus of the LCORL gene in improving animal growth traits.
[0019] The present invention identified a frameshift mutation located on LCORL within Chr6: 37349373-37438248, that is, a deletion of the bases CT occurred at the first and second positions of 37401770, forming the frameshift mutation rs384548488 (LCORL.p.Glu1159ValfsTer28), which most drives the selective sweep in the Bos taurus NCAPG-LCORL region, resulting in a rapid increase in the body size and growth rate of Bos taurus in the past 1000 years.
[0020] The LCORL gene Indel mutant of the present invention is the Indel nucleic acid molecule and the mutant protein.
[0021] By constructing mutant mice that mimic the rs384548488 mutation in cattle using CRISPR / Cas9 and monitoring the growth of the mice, it was confirmed that the loss of the PIP domain of LCORL results in faster growth of the mice.
[0022] Therefore, the present invention provides a safe INDEL locus for gene editing to produce Bos taurus with a larger body size and a faster growth rate. Description of the Drawings
[0023] Figure 1Results of the selection analysis of the whole genome of domestic cattle. Among them, A is the result of the selection analysis of Charolais cattle, B is the result of the selection analysis of Simmental cattle, C is the result of the selection analysis of Limousin cattle, D is the result of the selection analysis of Hereford cattle, and E is the result of the selection analysis of Angus cattle.
[0024] Figure 2 Results of the local CLUES analysis of NCAPG-LCORL. Among them, A is the result of the local CLUES analysis of NCAPG-LCORL in Charolais cattle, B is the result of the local CLUES analysis of NCAPG-LCORL in Simmental cattle, C is the result of the local CLUES analysis of NCAPG-LCORL in Limousin cattle, D is the result of the local CLUES analysis of NCAPG-LCORL in Hereford cattle, E is the result of the local CLUES analysis of NCAPG-LCORL in Angus cattle, F is the frequency change trajectory of 5 shared CLUES selected SNVs in Charolais cattle, G is the frequency change trajectory of 5 shared CLUES selected SNVs in Simmental cattle, H is the frequency change trajectory of 5 shared CLUES selected SNVs in Limousin cattle, I is the frequency change trajectory of 5 shared CLUES selected SNVs in Hereford cattle, and J is the frequency change trajectory of 5 shared CLUES selected SNVs in Angus cattle.
[0025] Figure 3 Distribution of haplotype A and haplotype D in domestic cattle. Among them, A is the distribution of haplotype D, B is the distribution of haplotype A, C is the composition of different haplotypes in Charolais cattle, D is the composition of different haplotypes in Simmental cattle, E is the composition of different haplotypes in Limousin cattle, F is the composition of different haplotypes in Hereford cattle, and G is the composition of different haplotypes in Angus cattle.
[0026] Figure 4 NCAPG-LCORL comparative haplotype analysis.
[0027] Figure 5 Effect of rs384548488 on LCORL.
[0028] Figure 6For the correlation analysis of rs384548488 with the growth traits of domestic cattle, where A is the GWAS analysis result of the NCAPG-LCORL region on the daily weight gain of domestic cattle, B is the correlation analysis between the GWAS analysis result of the NCAPG-LCORL region on the daily weight gain of domestic cattle and linkage disequilibrium, C is the GWAS analysis result of the NCAPG-LCORL region on the birth weight of domestic cattle, D is the correlation analysis between the GWAS analysis result of the NCAPG-LCORL region on the birth weight of domestic cattle and linkage disequilibrium, E is the GWAS analysis result of the NCAPG-LCORL region on the weaning weight of domestic cattle, F is the correlation analysis between the GWAS analysis result of the NCAPG-LCORL region on the weaning weight of domestic cattle and linkage disequilibrium, G is the GWAS analysis result of the NCAPG-LCORL region on the one-year-old weight of domestic cattle, and H is the correlation analysis between the GWAS analysis result of the NCAPG-LCORL region on the one-year-old weight of domestic cattle and linkage disequilibrium.
[0029] Figure 7 For the gene editing strategy of mice, where A is the comparison schematic diagram of the LCORL genes of domestic cattle and mice, and B is the schematic diagram of the gene structure of the mouse LCORL gene and the gene editing strategy.
[0030] Figure 8 Frameshift mutations similar to rs384548488*A can increase the growth rate of mice, where A is the growth curve of male mice with different genotypes, and B is the growth curve of female mice with different genotypes.
[0031] Figures 9 - 23 For the nucleotide sequence of the Indel molecular marker, where Figure 10 The bases marked in yellow and underlined are the bases after deleting AT. When no bases are deleted, the bases marked in yellow and underlined are ACTCTGGG. Specific implementation manners
[0032] The following describes the specific implementation manners of the present invention in detail, but it should be understood that the protection scope of the present invention is not limited by the specific implementation manners. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.
[0033] (1) Identification of causal variations in the growth traits of domestic cattle
[0034] First, collect beef cattle breeds originating from the UK region: Angus cattle, Hereford cattle, and beef cattle breeds originating from the European continent region: Simmental cattle, Limousin cattle, and Charolais cattle, with 100 individuals in each breed.
[0035] Construct an ancestral recombination graph using the relate v1.1.9 software, and perform selection analysis on each breed within the last 1000 years using the CLUES software. The NCAPG-LCORL region located at bovine body size quantitative trait locus Chr6: 37,180,233 - 37,768,454 was identified as the most significant region in the CLUES analysis of Charolais and Simmental cattle, as Figure 1 shown. In this region, the frequency of the selected mutations is relatively high, with a frequency ≥ 0.69. However, in Angus and Hereford cattle in the UK, either there is no significant selection signal in this region, or the frequency of the selected mutations is very low, with a frequency ≤ 0.1, as Figure 2 shown. Within the region Chr6: 37,180,233 - 37,768,454, 85 SNVs exceeded the genome-wide Fst threshold (top 0.05%), and 65 of these SNVs showed selection signals identified by CLUES, becoming the focus of further analysis.
[0036] The present invention hypothesizes that the alleles driving selection within this region have a single common origin in different breeds of domestic cattle. The spread of this allele in various breeds has led to a heterogeneous linkage disequilibrium pattern with the driving selection allele. Therefore, this allele should show a selection signal in all four breeds (Charolais, Simmental, Limousin, and Hereford) that display a selection signal in the NCAPG-LCORL region. Alleles that are only selected in certain breeds may be hitchhiking due to genetic drift. Therefore, performing a cross-ancestry analysis by combining the CLUES results of different breeds can assist in fine mapping by leveraging the LD patterns of ancestral differences.
[0037] Among the selected SNVs identified by CLUES, five SNVs span 89 kb, Chr6 being 37349373 - 37438248, and share selection signals in four breeds of domestic cattle that display a selection signal in the NCAPG-LCORL region, as Figure 2 shown. These five mutations sharing selection signals define a narrower selection target haplotype.
[0038] Among the four breeds that display a selection signal at the NCAPG-LCORL locus, 315 haplotypes carry the derived alleles of these five shared mutations, while 425 haplotypes carry the ancestral alleles. 315 haplotypes are D haplotypes and 425 haplotypes are A haplotypes, as Figure 3 shown. The D haplotype mainly exists in Charolais and Simmental cattle, but is hardly found in Angus cattle, as Figure 3 shown.
[0039] In addition to these five SNVs sharing selection signals, there are three INDELs showing the highest frequency differences between the A haplotype and the D haplotype, Δ frequency = 0.98 - 0.99, as Figure 4 . Two of the SNVs of all five shared selection signals and two of the three INDELs with the largest frequency differences are located in non-coding regions, while the other locus is at 37401770 on chromosome 6 of Bos taurus, the reference genome is ARS-UCD1.2, and 37401770 is a frameshift INDEL, rs384548488, ACT to A. The 2bp mutation on rs384548488 is located in "PRC2-related LCORL isoform 2", resulting in the complete loss of the PIP domain in PALI2, as Figure 5 . This may interfere with the PRC2 methyltransferase activity. Among them, PALI2 is encoded by an alternative transcript of the LCORL gene, PIP represents the interaction between PALI and PRC2, and PRC2 is Polycomb Repressive Complex 2.
[0040] Using the GWAS results of the whole genome association analysis of the body weight of Red Angus cattle and the GWAS results of the average daily gain (ADG) of the mixed Bos taurus population, the present invention infers that rs384548488 is highly correlated with the LD(r 2 ) value of the -log(GWAS p-value) level of each variant (r > 0.76, p < 1×10^-25), as Figure 6 . This indicates that rs384548488 is highly correlated with the growth trait variations in multiple beef cattle populations.
[0041] Therefore, rs384548488*A, a predicted mutation leading to the loss of the PIP domain, is very likely to be the allele driving selection and is the causal variant affecting the body size and growth rate of the NCAPG-LCORL locus in Bos taurus. The amino acid sequence of the PIP domain is shown in SEQ.ID.NO.3, and SEQ.ID.NO.3 is as follows: ITNAWVPVSGDEAENCVHKKRDHIENDNFKIASPLETCLLELEVSPVK MLFQKKYDLNELCTWFMQTTETQSLSLVRKANARNPLEVINTRGIKLGTKYSDFNTSPFRKHFKKFALSSPSK.
[0042] (II) The loss of the PIP domain in LCORL leads to an increase in the growth rate of mice
[0043] The present invention uses the CRISPR-Cas9 technology to introduce a pLoPD mutation similar to rs384548488*A in Bos taurus into C57BL / 6J mice, asFigure 7 。The MUSCLE algorithm in the MEGA 11 software was used to perform sequence alignment on the exons containing the PIP domain in the LCORL genes of domestic cattle and mice. Through this analysis, the homologous position of the bovine rs384548488 variant in mice was determined to be Chr5:45,882,519 - 45,882,520, as Figure 7 。To introduce a frameshift mutation in mice, the present invention selected the region Chr5:45,882,459 - 45,882,558 for CRISPR / Cas9-mediated deletion. Two gRNAs were designed to target the mouse LCORL gene, and these two gRNAs were co-injected with Cas9 mRNA into fertilized C57BL / 6J mouse oocytes to generate offspring carrying targeted gene deletions. Through PCR and sequencing analysis, the present invention identified F0 founder animals. Subsequently, these founder animals were mated with wild-type mice to test gene transmission and produce F1 animals.
[0044] The two gRNAs are reverse-strand matching gRNA-A1 and forward-strand matching gRNA-A2. The nucleotide sequence of gRNA-A1 is: AGGGTTAAAAGATTCATTTTGGG, denoted as SEQ ID NO.4, and the nucleotide sequence of gRNA-A2 is: TTGTCACTGTTGTTTATGGAAGG, denoted as SEQ ID NO.5.
[0045] Primers used in PCR:
[0046] F1: 5’-CAAGAAGACCCTAAGGAAAAGTCA-3’, denoted as SEQ ID NO.6, R1: 5’-TCTGAGGTATCATAGACTTGCTCT-3’, denoted as SEQ ID NO.7.
[0047] Wild-type, heterozygous, and homozygous LCORL PIP domain knockout mice were weighed from weaning to 9 weeks of age, as Figure 8Male: Starting from 7 weeks of age, p = 1 x 10^-7; Female: Starting from 25 days of age, p = 0.0. Repeated measures two-way ANOVA and Tukey's multiple comparison test were performed, and it was found that the homozygous LCORL PIP domain knockout mice were significantly heavier than the wild-type mice. The effect of the LCORL loss of PIP domain mutation on body weight showed a dose-dependent trend. At 9 weeks of age, the heterozygous and homozygous LCORL PIP domain knockout male mice were 5.9% (1.5 g) and 9.4% (2.4 g) heavier than the wild-type male mice, respectively. Similarly, the heterozygous and homozygous LCORL PIP domain knockout female mice were 4.8% (1.0 g) and 12.4% (2.6 g) heavier than the wild-type female mice, respectively.
[0048] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To prevent redundancy, the present invention describes preferred embodiments.
[0049] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments and all changes and modifications falling within the scope of the present invention.
[0050] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.
Claims
1. An application of an Indel site of an LCORL gene in improving animal growth traits, characterized in that: The Indel site is located at 37349373-37438248 of chromosome 6 of cattle.
2. The use according to claim 1, characterized in that: The Indel site is located at 37401770 of chromosome 6 of cattle, and the base CT is deleted at the first and second positions of 37401770 of chromosome 6 of cattle.
3. The use according to claim 1, characterized in that: The reference genome of the Indel site is ARS-UCD1.
2.
4. The use according to claim 1, characterized in that: The improved growth trait refers to weight gain.
5. The use according to claim 1, characterized in that: The animal is a cow or a mouse.
6. An Indel nucleic acid molecule for improving animal growth traits, characterized in that: The nucleotide sequence of the Indel nucleic acid molecule is shown in SEQ ID NO.
2.
7. A mutant protein, characterized in that The mutant protein is encoded by the nucleotide sequence of SEQ ID NO.2 described in claim 6; The amino acid sequence of the mutant protein is shown in SEQ ID NO.
1.
8. Use of the Indel nucleic acid molecule according to claim 6 or the mutant protein according to claim 7 in improving animal growth traits.
9. Use of the Indel nucleic acid molecule according to claim 6 or the mutant protein according to claim 7 in detecting animal growth traits.
10. The use according to claim 9, characterized in that: The primers for detecting animal growth traits using the Indel nucleic acid molecule are shown in SEQ ID NO.6-7, and the animal is cattle.