Application of STC2 gene coding sequence mutant for improving growth speed and size of animals

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of an STC2 gene coding sequence mutant capable of improving the growth speed and size of animals, an SNP site is located at a cattle 20 # chromosome 4967596-5008911, and it is found that missense variation on STC2 can promote animal growth in the region; therefore, the SNP locus related to the animal growth traits in the region is determined, and the invention provides the application of the SNP locus of the STC2 gene in improvement of the animal growth traits.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an application of an STC2 gene coding sequence mutant for improving the growth rate and size of animals. Background Art

[0002] In the field of livestock breeding and farming, improving the size and growth rate of animals is one of the important goals. With the development of molecular genetics technology, more and more research focuses on identifying genetic variations associated with these traits, especially single nucleotide polymorphism (SNP) sites. SNP refers to the variation of a single nucleotide base in the genome. These variations may affect the expression and function of genes, thereby significantly affecting the growth, development, size and other traits of animals.

[0003] Animal size and growth rate are important indicators of their economic value. Animals with large size and fast growth usually have higher meat yield and better production performance. Therefore, improving these traits through genetic improvement is of great significance for the sustainable development of animal husbandry. At present, many studies have found that multiple genes are related to animal size and growth rate, but there are still a large number of gene loci that have not been fully explored.

[0004] The STC2 (Stanniocalcin 2) gene is a conserved gene that is widely present in many animals. The protein it encodes plays an important role in many physiological processes. The STC2 protein is mainly involved in the regulation of calcium and phosphorus metabolism inside and outside the cell, and has a significant effect on cell growth, differentiation and survival. In addition, STC2 is also related to inflammatory response, cell stress response and neural function, showing its functional complexity in many aspects. The variation of SNP sites may affect the structure and function of STC2 protein, but the SNP sites that improve animal growth traits are currently unclear, resulting in an unclear relationship between SNP sites and improving animal growth traits. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an application of a mutant of an STC2 gene coding sequence for improving the growth rate and size of an animal.

[0006] The invention discloses an application of a SNP site of an STC2 gene in improving animal growth traits, wherein the SNP site is located at positions 4967596-5008911 of chromosome 20 of cattle.

[0007] Preferably, the SNP site undergoes a mutation from C to G at the 9187 bp of the sequence shown in SEQ ID NO.2.

[0008] Preferably, the improved growth traits refer to increased body weight and increased body length.

[0009] Preferably, the animal is a cow or a mouse.

[0010]

[0011] CCTGATTTATCCATGCTGCCTGCCCACATGTCCTCTAGGAGATGACAAATT

[0012] TCTACAAATTTCATGTCTCTTTTATTCTTACCTATTAGGTGAAAATAGCTTTT

[0013] GGCATGTTTAATTGGAGTCCCCGAACTTTCCTGCCCTATCCCCAGCCTAAT

[0014] GTTATGGTCCTCACAGACTGGGTTCTGAGTAGCCAGACACCGTGTGTCCC

[0015] TTACAAGATGTTTATTATTTATTATGAGTTTTCTCTGCTAAGAATCCTCAAAT

[0016] AATTGTTCAGCTTGGTCTCCATCCCATTCTGTGCATGCAAACTTCCTCCGA

[0017] GTGGAGGAAGACTCGGCTGATATGACAGGTCCAGAAAGCTGAACCCCAG

[0018] GAGTGGAGTGGGGAGTCACTGGCACCCTGCTGCACACCTGGAAGGTCTC

[0019] TGGCAAGTACTAAAAGCCATCATGACGATCAGCTAGGAAACTCTGTTAAC

[0020] AGTTTTTTCCATTGAATGTTTCAGTCATGCTCTGCTAACCCAAAACATTCTT

[0021] GGTGCTCAATATGGTTTTTACCTGGGCATCAAATTTTCCAGCGTTGTGCAG

[0022] AAAAGTCATGCAAATTCCGTGTAAGCCCCGAATCTCACAAGAGTTGTTCT

[0023] CGAAACATTCAAACACGCCACACCCCACATCGCCAG(G)GTTGACCAAA

[0024] CAGTGCTGGATTTCCGCTAAAATGAGAATTACACACAATCATATACAAAAT

[0025] TCTCTGTCATGCTGGGGAATGCAGAGAGAGGGTGGTTGAAATAAAAGTAA

[0026] TGACTAAAAGGAAAATTAAGAAAAAGTGTTAGAAATGATTGTTTTGGGAT

[0027] CCAAAAACCAAGGAAAGAAAATTTCATCACTTAATCTACTTATAAAGATAA

[0028] CTAGTATTTGTAAATTTTTGTAAAAATTAGCCAGACCTACATTACAAAAGT

[0029] CACAAGAGAAGTTAGGTCAGAACACTCCATTTTTTCATCCTTCTCATCTTA

[0030] TTAAAGTATTTGTGGGAATACTTGGTTTGACAGCTGAAGATATTTCAGCTT

[0031] TCTAAACCCACTGACAAAAATAAAATTTCAACCCATTAGCTTGGTCTTTCC

[0032] CTTCTGTCCTTCATCAATGAAATACTCTTCTGTACTTGGATGAAGCTGCTG

[0033] CTATTAAATCACGGCCATTTCCTTCCTGACCCCCAAATGAGAGCGTCCAAA

[0034] AAGCAGTTTCAATTTAGCTTTATGATTCAACACCTAAAAATCAAAACAAAA

[0035] CTCCTATTAAGGCAGCGCCTCCAAACCATTCGCATTAAAAATACCAGGCTA

[0036] GGGAAACTGCGGTTTGCTTGCATGCAGGCTCAGGAGTCGAGTCAGATGG

[0037] GGGTTAGTCTGCAGAAACGCAACCTTTGCAGTTCGAGGTTCCTTTTTAATA

[0038] GAGATCTGGGGTGAGATGTCGCTTAATTCCCTAGACTTCTCGCGACCCGG

[0039] GTGTCCGCTGCCCTCCTTCGCAGAATGTGGCCCCAATTCGCCTCTCTGAA

[0040] AGGACAGGCTGAGAGGGAGTTAACTGTCAGGCTGCAAAGCCTCTCTCCC

[0041] TTCCTTCCCCCTACCCAATTAAACCAACCACAAAATTACATCAGAAGAGTA

[0042] TATTTTGGAAGAAGGGCGGGTCGGAGGAGATAATCCGCATTCAGGTTAAA

[0043] TCTGCCGAGAGAATGAGGGGCGTGTATTTGTTGAGGGTCACCAACTAGCG

[0044] AAAATCACGTTTGATTCTCAGGGAAAGCTCTGGGAGGGGAAAAGGTGGG

[0045] TGAGACGAGCGGGGCGCGAGCCGGAGGTGCTGGCATCTGCCTATGGAGG

[0046] TGGGAGACTCCCGGACGGTCCCATGCGCGGCCCCGGCGCGCCGGCCCGG

[0047] AGCGCGCAGGGCCGCGGTTCACTCGCTCCCGGCGCCCCTAATGGGCACTC

[0048] GTGCATTCCTGCTCGTGCGGTGAACGCACGCACGGCACTGCCGGGTTCGC

[0049] AGGCCCCCTTAATAAAGGGAGGGCCGTGAAGAGCGAAGAAGCGCTAGGC

[0050] AGATTTTCTTCCTCTCCCTGGAGCTGGGAAAAGGGCCAGCCCCGTGCCCT

[0051] GCACGCCTGGTTGTGCTTGGCAGCTCTCGGGTAAACGGAGAGCCGGTCG

[0052] AGCAGCTGCGGCGCACGGTTCGGGCCGTGTCACCATTTCTTCTCTTGCTG

[0053] ACCTGCTCTGGAGCTGGGGACGGAGGCAGCACCGCAGAGGGACGCTTCA

[0054] GTGCACCCGCCCCTGCCCCTTCCGAAAGGAATGCCCCACAAGGGGGAAA

[0055] GTTCACCATGCAATTTGTCTCGCCTACAAAGTTCTTGGATGTCGGGCATCT

[0056] TCTGAGGAGGGGGAGAAACAGTCCTGCCGCGGCCCGCCGGCTGGGAAG

[0057] AGAAGTTCTTCGGTTCAGAGCGCAGCCCGGTAGCGGGGACGCTCCGTGC

[0058] CTCCTGCAAGCCGGTTAGGATGGAGCAGGGAGGTTTGAACCGGTCCGGG

[0059] AACGCTGGAGCCGACCCTGGCCGCGTGATGCGGTGCTTCCTTTACCCTCG

[0060] ACCCCGGGAGGACAGCAACAAGTCCTGCCCCAGCCATCTCATCACCCGG

[0061] CCAGCACGTGCGGATTCCGGACGCGCGGAGCCCGGTGTGGACCTCGCTC

[0062] TGCCGCGCGCTTTCCTCTTCACGCCCCGCTCCACCCCAAGAGTTTGCGCG

[0063] GTCCAGGTTGGGCGCCCAGTTCTGGGCGAAGCGCCCGGGGCGCGCTGCG

[0064] TGGGTGCGGGATGTCTTCTCCCAGCAGCTCCCCGCCTCGGGGGCGCACGC

[0065] ACTTACCTGTGTTCTGCAAGGACAGACGGCCTTCTGCTGGGAGCCCCTG

[0066] TCTTGGGGACCTTCGGGCGTGTTGGTGGCGTCGGTCCCTCGAGCTGGATC

[0067] GAAGGTAACCAGCACCAAAGCCAGGGTCACGAACTGGCCCAGCCGCTCGGCACACAT, wherein the G in "()" is the mutated base. The STC2 protein encoded by the SNP nucleic acid molecule undergoes a mutation from A to P at position 60.

[0068] A mutant protein, the mutant protein is encoded by the nucleotide sequence of SEQ ID NO.2;

[0069] The amino acid sequence of the mutant protein is shown in SEQ ID NO.1, which is as follows: MCAERLGQFVTLALVLVTFDPARGTDATNTPEGPQDRGSQQKGRLSLQNTA EIQHCLVN(P)GDVGCGVFECFENNSCEIRGLHGICMTFLHNAGKFDAQGK SFIKDALKCKAHALRHRFSCISRKCPAIKEMVFQLQRECYLKHDLCSAAQENIRVMVEMIHFKDLLLHEPYVDLVNLLLTCGEEVKEAITHSVQAQCEQSWGSLCSILSFCTSAIQRPPTAPPEHQLQGDRAKLSRGHPAEMGHHLAEPSSRETGRGAKGERGSKSHPNAHARGRAAGPGAQGTSGSSEWEDEQSEYSDIRR, wherein the P in “()” is the mutated amino acid.

[0070] The application of the SNP nucleic acid molecule or the mutant protein in improving animal growth traits.

[0071] The use of the SNP nucleic acid molecule or the mutant protein in detecting animal growth traits.

[0072] Preferably, the primers used for detecting animal growth traits using the SNP nucleic acid molecule are as shown in SEQ ID NO.4-5.

[0073] Preferably, the animal is cattle.

[0074] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0075] The present invention uses population genetics to identify a gene locus STC2 that is selected in the process of domestic cattle breed improvement. Through local fine positioning, it is found that missense mutations on STC2 in the body length QTL region Chr20:4967596-5008911 promote animal growth, thereby determining the SNP sites related to animal growth traits in this region. Therefore, the present invention proposes the use of the SNP sites of the STC2 gene in improving animal growth traits.

[0076] In the body length QTL region Chr20:4967596-5008911, the present invention identified a missense mutation STC2 at 5008911 on STC2. A60P The SNP nucleic acid molecule corresponding to the SNP site was determined by driving selection clearance in the STC2 region of cattle, i.e., a mutation from A to P occurred at position 60 of the STC2 protein encoded by the SNP nucleic acid molecule, resulting in a rapid increase in the body size and growth rate of cattle.

[0077] STC2 constructed by CRISPR / Cas9 A60P Mutant mice, and monitoring their growth confirmed that STC2 A60P The mutation causes the mice to have a faster growth rate.

[0078] Therefore, the present invention provides a safe SNP site that can be gene-edited and can be used to produce animals with larger body size and faster growth rate.

[0079] The STC2 gene coding sequence mutant of the present invention is the SNP nucleic acid molecule and the mutant protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 Results of selection analysis on the STC2 locus in Hereford cattle.

[0081] Figure 2 For STC2 A60P Regional comparative genomic analysis.

[0082] Figure 3 For STC2 A60PThe location of the missense mutation site in the STC2·PAPP-A complex, where A is the three-dimensional structure of the STC2·PAPP-A complex and B is the STC2 A60P Effects of missense mutations on the local polarity of STC2.

[0083] Figure 4 For STC2 A60P Schematic diagram of the generation of gene-edited mice, where A is the gene-edited mouse strategy and B is STC2 + / + Mouse genotyping results, C is STC2 A60P / + Mouse genotyping results, D is STC2 A60P / A60P Genotyping results of mice.

[0084] Figure 5 For STC2 A60P Body length and growth curve of gene-edited mice, where A is a representative picture of three genotypes of mice, B is the body length data of male mice, C is the body length data of female mice, D is the growth curve of male mice, and E is the growth curve of female mice. DETAILED DESCRIPTION

[0085] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0086] (I) Identification of causal variation in body size and growth rate in cattle

[0087] First, 100 Hereford cattle were collected. The ancestral recombination map was constructed using the relate v1.1.9 software, and the selection analysis of each breed within the past 1000 years was performed using the CLUES software. The STC2 gene is located in the body length QTL region Chr20:4967596-5008911, and the reference genome is ARS-UCD1.2. In this region, a mutation occurred at 5008911 on chromosome 20, and the protein was expressed as a missense variant rs42661323*G. rs42661323*G showed a CLUES selection signal in Hereford cattle, such as Figure 1 , the C to G missense mutation at rs42661323 caused the amino acid at position 60 of the STC2 gene to change from alanine to proline, denoted as STC2 A60P Mutation. Alanine 60 of the STC2 gene is highly conserved in vertebrates, such as Figure 2 .

[0088] Structural analysis of the human PAPP-A·STC2 complex showed that the amino acids at positions 60-66 of STC2 interact with PAPP-A, e.g. Figure 3 , where the valine at position 63 of STC2 binds to the hydrophobic pocket in PAPP-A through van der Waals forces. A60P The mutation is located in this interaction region and is predicted to change the polarity of amino acids 60–66, e.g. Figure 3 .

[0089] Therefore, the present invention speculates that STC2 A60P The mutation reduced the binding of STC2 to PAPP-A and increased the biological activity of insulin-like growth factor (IGF), thereby promoting growth. This speculation was further verified by the BeAtMuSiC program, which predicted that the STC2 A60P mutation would reduce the binding affinity between STC2 and PAPP-A (ΔΔGBind = 0.65 kcal / mol).

[0090] (ii) STC2 A60P Preparation and functional verification of gene-edited mice

[0091] To verify STC2 A60P The mutation affects the body length in vivo. The present invention introduces this mutation into C57BL / 6J mice using the CRISPR-Cas9 system. Figure 4 . gRNA targeting the mouse STC2 gene contains a donor oligonucleotide Cas9 mRNA with a p.A60P (GCC to CCC) mutation. Injection of fertilized eggs: gRNA, donor oligonucleotide, and Cas9 mRNA were co-injected into fertilized eggs of C57BL / 6J mice to generate offspring carrying the targeted gene knock-in mutation. F0 founder animals were identified by PCR and sequencing analysis. The identified F0 founder animals were mated with wild-type mice to test gene transmission and generate F1 animals.

[0092] Among them, the gRNA targeting the mouse STC2 gene matches the forward chain of the gene, and the sequence of the gRNA is: CAGCACTGTTTGGTCAATGCCGG, recorded as SEQ IN NO.3, and the p.A60P mutation refers to the mutation from GCC to CCC.

[0093] The primers used in PCR are: F1: 5'-CGATAGAAGGAAGAAAAGAAAACGC-3', recorded as SEQ IN NO.4, R1: 5'-CAAACACCAGACTCTCTCAAGCAA-3', recorded as SEQ IN NO.5.

[0094] The present invention monitors the wild type (STC2 + / +), heterozygous (STC2 A60P / + ) and homozygous (STC2 A60P / A60P ) The body weight of mice from weaning to 9 weeks of age ( Figure 5 ). The results showed that STC2 A60P / A60P Mice were significantly heavier than STC2 mice starting at 3 weeks of age. + / + The effect of STC2A60P mutation on body weight showed a dose-dependent trend. A60P / + Male and female mice were compared with STC2 + / + mice weighed 6.5% (1.8 g) and 3.6% (0.7 g), while STC2 A60P / A60P Male and female mice were compared with STC2 + / + The mice weighed 10.5% (2.8 g) and 11.4% (2.3 g). Consistent with the expectations of the present invention, the STC2A60P mutation also showed an additive effect in terms of increased body length ( Figure 5 ). At 14 weeks of age, STC2 A60P / + Male and female mice were compared with STC2 + / + mice are 1.8% (1.8 mm) and 2.0% (1.8 mm) longer, while STC2 A60P / A60P Male and female mice were compared with STC2 + / + The mice were 4.6% (4.5 mm) and 4.0% (3.7 mm) longer.

[0095] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0096] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0097] 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 equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An application of a SNP site of an STC2 gene in improving animal growth traits, characterized in that: The SNP site is located at 4967596-5008911 on chromosome 20 of cattle.

2. The use according to claim 1, characterized in that: The SNP site undergoes a mutation from C to G at the 9187 bp of the sequence shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: The improved growth traits refer to increased body weight and increased body length.

4. The use according to claim 1, characterized in that: The animal is a cow or a mouse.

5. A SNP nucleic acid molecule for improving animal growth traits, characterized in that: The nucleotide sequence of the SNP nucleic acid molecule is shown in SEQ ID NO.

2.

6. A mutant protein, characterized in that The mutant protein is encoded by the nucleotide sequence of SEQ ID NO.2 described in claim 5; The amino acid sequence of the mutant protein is shown in SEQ ID NO.

1.

7. Use of the SNP nucleic acid molecule according to claim 5 or the mutant protein according to claim 6 in improving animal growth traits.

8. Use of the SNP nucleic acid molecule according to claim 5 or the mutant protein according to claim 6 in detecting animal growth traits.

9. The use according to claim 8, characterized in that: The primers used for detecting animal growth traits using the SNP nucleic acid molecules are shown in SEQ ID NO. 4-5.

10. The use according to claim 9, characterized in that: The animal is a domestic cattle.

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