CN117070521A - Goat MC4R defective mutant and application thereof - Google Patents
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Abstract
The invention discloses a goat MC4R defective mutant and application thereof, belonging to the field of molecular marker assisted breeding. The invention discloses an MC4R defect type mutant related to goat body size characters, wherein the nucleotide sequence of a coding gene is shown as SEQ ID NO:3, there is an A/C base mutation at 906 bp. The invention discovers that a defective mutant exists in the goat MC4R, a mutation site C906A is positioned on a seventh transmembrane region, and a base C is mutated into A; the mutant obviously reduces the level of induced cAMP and influences the growth and development of goats; the invention uses MC4R defective mutant as genetic marker to detect whether the MC4R receptor of the goat has defects, wherein the designed primer group can be applied to the detection of the genetic polymorphism of the MC4R receptor of the goat, and further can be applied to the identification and breeding of the body size character of the goat, can accelerate the breeding process, and has important economic application value.
Description
Technical Field
The invention relates to the field of molecular marker assisted breeding, in particular to a goat MC4R defective mutant and application thereof.
Background
Weight is one of the main conditions for identifying the variety of livestock and poultry. Body weight depends on appetite control and energy metabolism. MC4R is a member of the superfamily of G protein-coupled receptors, a peptide, and is secreted at the medial hypothalamic nucleus of mammals. MC4R is present not only in the hypothalamus but also in the central nervous system such as the cerebral cortex, thalamus, brain stem and spinal cord. MC4R is involved in leptin signaling in the appetite regulating region of the brain and is an important signaling molecule for regulating energy homeostasis. alpha-MSH is a decomposition product of opium-melanocyte pro-corticotropin (POMC), and is an endogenous agonist of MC4R. MC4R controls appetite and maintains posture through interaction with agonists, antagonists. Mice with MC4R gene knocked out show the characteristics of increased feed intake, faster fat deposition, excessive insulin secretion and the like. Normally, activation of MC4R by alpha-MSH produces the second messenger cAMP and phosphorylates extracellular regulatory protein kinase (extracellular regulated protein kinases, ERK). ASIP (agouti-signaling peptide) and related proteinsAGRPs (agouti-relatedproteins) bind MC4R and inhibit cAMP production. MC4R controls appetite and maintains posture by combination with agonists, antagonists [5] . The upper effects of ASIP are represented by: excessive antagonist/inhibitor expression, reduced cAMP content, increased body weight, and obesity; or the expression of the antagonist/inhibitor is lost, the cAMP content is increased, the body weight is reduced, and the occurrence of obesity is inhibited.
Research shows that the MC4R gene is candidate gene of important economic character of pig, cow, chicken and other animal. Correlation analysis of pig MC4R gene and fat character shows that MC4R gene is obviously related to pig chest and lumbar interfat thickness, buttock fat thickness, average backfat, eye muscle width, eye muscle area and skin rate. Correlation analysis of cattle MC4R gene and carcass traits shows that MC4R gene is obviously related to Qinchuan cattle backfat thickness. The different genotypes of the chicken MC4R are also obviously or extremely obviously related to the existence of weight, full-breech-free weight, leg meat weight and the like.
The MC4R gene is rich in polypeptides and has species specificity. More than one hundred of MC4R high constitutive active mutation sites are found by shape association analysis in various mammals, D90N, D298A, T150I, D146A, M161A, T A, G1392A of pigs. These sites share the common feature of being able to generate cAMP signals. Functional studies on mutation sites of goat MC4R genes have not been found, so that functional identification of the found natural mutants is urgently needed in the field, and a theoretical basis is provided for marker-assisted selection of genetic breeding.
According to the invention, through mutation site analysis of MC4R genes of the black goats and the Boer goats in the hemp city, the C906A natural mutant of MC4R is found. Based on the important influence of MC4R mutant on energy metabolism, a natural mutation carrier is constructed to perform function identification of the receptor.
Disclosure of Invention
The invention aims to provide a goat MC4R defective mutant and application thereof, so as to solve the problems in the prior art, and the invention proves that the MC4R defective mutant obviously reduces the level of induced cAMP and influences the growth and development of goats, and the mutant can be applied to the identification and breeding of the goat body size characters, can accelerate the breeding process and has important economic application value.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides an MC4R defect type mutant related to goat body size traits, wherein the nucleotide sequence of a coding gene of the MC4R defect type mutant is shown as SEQ ID NO:3, there is an A/C base mutation at 906 bp.
Further, the amino acid sequence of the MC4R defective mutant is shown as SEQ ID NO: 2.
The invention also provides a primer group for detecting the MC4R defect type mutant, and the nucleotide sequence of the primer group is shown as SEQ ID NO: 4-5.
The invention also provides a kit for identifying the goat body ruler character, which comprises the primer group.
The invention also provides a method for identifying the body size character of the goat, which comprises the steps of carrying out PCR amplification by using the primer group, detecting the mutation site of the MC4R defective mutant of the goat to be detected, and if the base mutation of the mutation site is A, obtaining the body size character of the goat to be detected; if the base mutation of the mutation site is C, the size of the goat body to be detected is poor.
The invention also provides application of the MC4R defect type mutant or the primer group or the kit in goat body size character breeding.
The invention discloses the following technical effects:
the invention discovers that a defective mutant exists in the goat MC4R, a mutation site C906A is positioned on a seventh transmembrane region, and a base C is mutated into A. The defective mutant of MC4R significantly reduces the level of induced cAMP, which results in reduced cAMP-PKA signaling pathway, affecting the growth and development of goats. The invention uses MC4R defective mutant as genetic marker to detect whether the MC4R receptor of the goat has defects, wherein the designed primer group can be applied to the detection of the genetic polymorphism of the MC4R receptor of the goat, and further can be applied to the identification and breeding of the body size character of the goat, can accelerate the breeding process, and has important economic application value.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a natural mutation site sequencing chromatogram detected by PCR amplified goat MC4R gene sequence sequencing;
FIG. 2 is an agarose gel electrophoresis of a goat MC4R gene sequence obtained by PCR amplification, wherein lane 1 is a MC4R gene band and lane M is a DL2000 Marker;
FIG. 3 shows the levels of cAMP generated by wild type receptor and natural mutant of MC4R in goat under induction of alpha-MSH;
FIG. 4 shows the total expression of wild-type receptor and natural mutant of MC4R in goat on the cell surface.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
In the early stage of the invention, mutation sites of MC4R genes of the black goats and the Boer goats in the Macheng are analyzed, so that the MC4R is found to have a C906A natural mutant. Based on the important influence of MC4R mutant on energy metabolism, a natural mutation carrier is constructed to perform function identification of the receptor.
The invention selects commercial plasmids pcDNA3.1-EGFP and pcDNA3.1-myc-HisA as expression vectors, and constructs an expression vector of a Wild Type (WT) goat melanocortin receptor 4 (MC 4R): pcDNA3.1-EGFP-MC4R and pcDNA3.1-myc-His A-MC4R. That is, the expression sequence of the coding region of MC4R gene was inserted into the multiple cloning sites of pcDNA3.1-EGFP and pcDNA3.1-myc-HisA.
The invention uses the constructed wild pcDNA3.1-EGFP-MC4R and pcDNA3.1-myc-HisA-MC4R as templates, and utilizes the point mutation technology to construct the natural mutation vector.
The invention uses human embryo kidney cell 293 (HEK 293) which does not express MC4R receptor as a transient expression system of MC4R and natural mutant thereof, and uses 5% CO at 37 DEG C 2 Cells were cultured in an incubator in DMEM medium containing 10% fetal bovine serum. The day before transfection, cells were plated at 0.14X10 6 Cells were inoculated at a density of one/mL into 6-well plates, after 30h (after cell attachment spread), MC4R mutant and wild type positive control plasmid were transfected with calcium phosphate, and after 48h, samples were collected.
The present invention relates to the ability to produce the inducible signaling molecule cAMP, and quantitative analysis of cell surface expression.
Example 1
Construction of MC4R wild and natural mutant eukaryotic expression vector
The bioinformatics analysis result of MC4R shows that MC4R has no intron; the open reading frame of the gene (GenBank accession number: NM_ 001285591.1) contains 999bp and codes 332 amino acids; the receptor protein has 7 transmembrane regions, 3 outer membrane loops and 3 inner membrane loops.
In this example, MC4R wild-type plasmids and natural mutant plasmids were constructed using eukaryotic expression vectors pcDNA3.1-EGFP and pcDNA3.1-myc-His A (both purchased from Wuhanaoke Biotechnology Co., ltd.) and were specifically prepared by: the coding region of wild MC4R gene is amplified by PCR method using specific primer (forward primer sequence is 5'-CCCGCTAGCGCCACCATGAACTCTACCCAGCCCCA-3'; SEQ ID NO: 4; reverse primer is 5'-CGCAAGCTTATATCTGCTAGACAAATCACAGAGGC-3' (SEQ ID NO: 5) wherein the shaded part sequence is complementary to the target gene), the cleavage site of NheI/HindIII is filled into pcDNA3.1-EGFP vector and pcDNA3.1-myc-HisA vector to construct wild expression vector pcDNA3.1-EGFP-MC4R and pcDNA3.1-myc-HisA-MC4R, and then pcDNA3.1-EGFP-MC4R and pcDNA3.1-myc-HisA-MC4R are used as template to construct expression vector of natural mutant of MC4R by enzyme digestion and sequencing verification.
Sequencing to obtain the amino acid sequence of the goat MC4R wild receptor as shown in SEQ ID NO:1 is shown in the specification; the amino acid sequence of the goat MC4R natural mutant (named C906A) is shown in SEQ ID NO:2 is shown in the figure; the full-length sequence of the cloned gene MC4R related to goat body weight is shown as SEQ ID NO:3, wherein the length of the sequence is 999bp, and an A/C substitution exists at 906bp of the sequence, a wild receptor is a C base, and a C906A mutant is mutated into an A base (allelic mutation, and the mutation site is written as m); as set forth in SEQ ID NO:4-5 are the nucleotide sequences of the primer pairs designed according to the invention, wherein the shadow tag sequence is located in SEQ ID NO: positions 1-20 and 971-996 of the 3 sequence.
SEQ ID NO：1
SEQ ID NO：2
Sequencing results show that the mutation site (named as Y302) of the goat MC4R natural mutant is positioned in a seventh transmembrane region, and the wild receptor is shown as SEQ ID NO:1, tyrosine (Y) at position 302 is replaced by a stop codon (x).
SEQ ID NO：3
FIG. 1 is a sequence chromatogram of a natural mutation site detected by sequencing a goat MC4R gene sequence amplified by PCR, which shows that the sequence is shown in SEQ ID NO:3, there is an A/C substitution at 906bp of the full-length sequence of the goat MC4R gene.
FIG. 2 is an agarose gel electrophoresis of a goat MC4R gene sequence obtained by PCR amplification, wherein lane 1 is a MC4R gene band and lane M is a DL2000 Marker.
Example 2
Comparison of MC4R wild type and Natural mutant production of inducible cAMP Activity
pcDNA3.1-EGFP-MC4R wild-type and natural mutant plasmids were transfected into HEK293 cells using calcium phosphate transfection and, after 24h, were isolated at different concentrations (10 -11 -10 -5 M) of alpha-MSH stimulation treatment cells, 37 ℃ temperature 6h incubation. Then, the culture solution was removed, and the cells were washed with PBS for 2 times, and the firefly fluorescence value and the Renilla fluorescence value of the sample were measured using a double luciferase kit from Shanghai Biyun biotechnology Co. By firefly fluorescence value/Renilla fluorescence valueRatios to represent cAMP activity of MC4R wild type and mutant. And the zero concentration value (Basal) is calculated by using GraphPadprism 9 software, and the alpha-MSH concentration value (EC 50) corresponding to 50% of the maximum biological effect and the maximum response value (Rmax) after alpha-MSH stimulation are reached.
The results of the α -MSH treatment showed that the C906A mutant had significantly reduced ability to produce cAMP activity compared to the wild type receptor (see FIG. 3). Studies have demonstrated that reduced cAMP levels, increased body weight, and obesity; increase in cAMP content, decrease in body weight, and inhibit obesity. This example reveals that the C906A mutant can increase body weight by decreasing the ability to produce cAMP activity, i.e., the C906A mutant can be used as a molecular marker in the selection of goat body weight traits.
Example 3
Quantitative analysis of MC4R wild type and natural mutant expression on cell surface
pcDNA3.1-myc-MC4R wild type and natural mutant plasmids were transfected into HEK293 cells using calcium phosphate transfection, after 24h, the medium was discarded, 2mLPBS was added to each well, and the cells were collected from the culture plates by pipetting into 1.5mL centrifuge tubes, 1500rpm, and centrifuged for 5min. 4% paraformaldehyde solution was added and the mixture was fixed at room temperature for 15min, after which it was washed with PBS 3 times for 5min each. Primary antibody was added and incubated for 1h at room temperature, washed 3 times with pbs for 5min each. Secondary antibody was added and incubated at room temperature for 1h in the dark, washed 3 times with pbs for 5min each. Finally, the detection is carried out by using a flow cytometer.
The results are shown in fig. 4, and the results indicate that the expression of Y302 natural mutant (i.e., C906A mutant) on the cell surface is significantly reduced compared to the wild type, belonging to the defective mutation.
Example 4
Accuracy verification of C906A mutant serving as molecular marker in goat population body size character breeding
In order to determine whether the goat MC4R gene C906A is related to the growth characteristics of the goat MC4R gene C906A, dongbao black head sheep is selected as a test material, and sample collection and related data information are all from a sheep farm of a research institute of livestock and veterinary science of the national academy of agricultural sciences of Hubei province. The traits analyzed are mainly growth traits. The growth traits include body weight, height, body diagonal length, chest circumference, tube circumference, etc. of the goat individual measured at 3, 6, 12 months of age, respectively. Polymorphism testing was performed using one-generation sequencing and correlation between individuals of corresponding different genotypes and their growth traits was analyzed. Performing association analysis between individual traits of different genotypes of molecular markers by adopting a GLM program in SAS statistical analysis software, wherein an analysis model is as follows:
model 1: y=global mean + genotype + sheep field environmental effect + residual.
The statistical analysis results are shown in table 1:
TABLE 1 analysis of correlation of MC4R Gene C906A and growth traits thereof
Note that: the values are least square values plus or minus standard error. In the same row comparison, the different lower case letters represent significant differences (P < 0.05).
The result shows that the AA genotype has higher three-month age body height, three-month age chest circumference and six-month age body height, and the Y302 natural mutant (namely the C906A mutant) can be used as a molecular marker for goat body size character breeding.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (6)
1. The MC4R defect type mutant related to goat body size characters is characterized in that the nucleotide sequence of a coding gene of the MC4R defect type mutant is shown as SEQ ID NO:3, there is an A/C base mutation at 906 bp.
2. The MC 4R-deficient mutant according to claim 1, characterized in that the amino acid sequence of the MC 4R-deficient mutant is as set forth in SEQ ID NO: 2.
3. A primer set for detecting the MC 4R-deficient mutant according to claim 1, wherein the primer set has a nucleotide sequence set forth in SEQ ID NO: 4-5.
4. A kit for identifying a goat body size trait, comprising the primer set of claim 3.
5. A method for identifying the body size character of a goat, which is characterized in that the primer set of claim 3 is used for PCR amplification, the mutation site of the MC4R defective mutant of the goat to be detected in claim 1 is detected, and if the base mutation of the mutation site is A, the body size character of the goat to be detected is good; if the base mutation of the mutation site is C, the size of the goat body to be detected is poor.
6. Use of the MC 4R-deficient mutant according to claim 1 or the primer set according to claim 3 or the kit according to claim 4 in goat body-scale trait breeding.
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| CN106498052A (en) * | 2016-11-01 | 2017-03-15 | 江苏省农业科学院 | The molecular labeling of one impact goat early growth, detection method and its application |
| CN107022022A (en) * | 2016-03-02 | 2017-08-08 | 湖北省农业科学院畜牧兽医研究所 | Three kinds of goat MC1R defects mutant and its application |
| CN114164284A (en) * | 2021-12-07 | 2022-03-11 | 华中农业大学 | Application of MC4R gene coding region mutation in early selection of number of lambs born by Hu sheep |
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| US20150150224A1 (en) * | 2012-06-14 | 2015-06-04 | Universite De Montreal | Transgenic mouse models for mc4r |
| CN107022022A (en) * | 2016-03-02 | 2017-08-08 | 湖北省农业科学院畜牧兽医研究所 | Three kinds of goat MC1R defects mutant and its application |
| CN106498052A (en) * | 2016-11-01 | 2017-03-15 | 江苏省农业科学院 | The molecular labeling of one impact goat early growth, detection method and its application |
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