CN111434779A - MAP2 mutant gene influencing pig hair follicle development and application thereof - Google Patents

Abstract

The invention provides a MAP2 mutant gene influencing pig hair follicle development, which has 10 mutant sites, the physical position of the mutant gene is between 112155861 and 112424202 of No. 15 chromosome of pig, and the nucleotide sequence is shown as SEQ ID NO. 1-4. The invention finds the pig hairless character key gene, the provided MAP2 mutant gene which influences the development of pig hair follicles can be applied to genotyping, screening, identifying and assisted breeding of pigs, the MAP2 mutant gene can be used for assisted breeding of pigs, sows with stable hereditary hairless characters can be effectively selected, disease model pigs with excellent varieties can be bred in a short time, at low cost and with high accuracy, and the pig can be used as a molecular marker for identifying the generation of hereditary hairless diseases and further applied to the hair development research of mammals.

Description

MAP2 mutant gene influencing pig hair follicle development and application thereof

Technical Field

The invention relates to the field of molecular biology medicine, in particular to a MAP2 mutant gene influencing pig hair follicle development and application thereof.

Background

Human beings are very concerned with hereditary alopecia and thinning hair, and the stability of the morphological structure and function of hair follicles is the key to the normal hair cycle. The morphological structure and function of hair follicles are congenital, and the genetic mechanism of the hair follicles is studied, and the problem of alopecia is expected to be solved from the source. The pig has important medical significance, the pig has very high genetic homology with human, the genome of the pig is similar to that of human, the complexity and the chromosome composition are relatively consistent, and the conservation of the human and pig homolinear region is 3 times that of the human and mouse. Thus, pigs are now an ideal model animal for medical research to study human medical problems.

The hairless pig has the characteristic of hair follicle hypoplasia, and the body hair is sparse, so the hairless pig can be used as an animal model for researching diseases in the field of human hair. In order to screen the hairless pig which can be used as an animal model for medical research, a simple, convenient and effective molecular marker for distinguishing the characters of the normal and hairless pigs is needed to realize the accurate identification of the hairless pig, the hair development starts in the embryonic period, and the molecular marker technology can be used for screening the sow and the matched boar during the pregnancy of the sow so as to ensure that all the piglets born after the fertilization pregnancy are hairless piglets. Therefore, related proteins and corresponding mutant genes influencing the pig hair follicle development need to be researched, and no related research report exists at present.

Microtubule-associated protein2 (MAP 2) is one of the major members of the MAP family, and in addition to playing an important role in microtubule assembly, synaptic growth, and information communication between microtubules and other components of nerve cells, abnormal expression of MAP2 is also closely related to the occurrence of neurodegenerative diseases. Research has shown that the reasonable administration of abnormal protein related to neuronal cytoskeleton can effectively inhibit the occurrence of neurodegenerative diseases or relieve symptoms thereof. In the preliminary experiments of the invention, some candidate genes of MAP2 and other proteins are found to be related to the hairless character and the development of hair follicles of animals through screening.

Disclosure of Invention

The invention aims to provide a MAP2 mutant gene influencing pig hair follicle development, which can be applied to pig genotyping, screening and assisted breeding.

The invention provides a MAP2 mutant gene influencing pig hair follicle development, which has 10 mutant sites, the physical position of the mutant gene is between 112155861 and 112424202 of No. 15 chromosome of pig, the nucleotide sequence is shown as SEQ ID NO. 1-4, and the 10 mutant sites are respectively:

1, G95 > C, the mutation site is positioned in a 5' untranslated region;

2, 129 th C > T, 173 th C > T, 274 th A > G and the mutation site is positioned in No. 12 exon;

3, 166 th C > G and 412 th A > G of SEQ ID NO, wherein the mutation site is positioned in a No. 13 exon;

4, C at position 89 > T, A at position 314 > G, G at position 550 > A, A at position 575 > G and the mutation site is positioned in the No. 16 exon.

The MAP2 mutant gene influencing the development of the pig hair follicles, which is provided by the invention, can be applied to genotyping, screening, identifying and assisted breeding of the pigs.

The invention is realized by the following technical means:

Step 1: the method comprises the following steps of (1) carrying out group expansion on a hairless individual of a large white pig through a previously discovered hairless large white pig to construct a hairless character experimental group of the large white pig;

Step 2: observing and identifying the hair-feather and hairless character types of the pig hair follicle and epidermal tissue sections, and excluding some hairy pigs with hair-feather but hair follicle tissues;

And step 3: mining a pig hairless character candidate gene region from a DNA level through whole genome association analysis (GWAS) and genome re-sequencing, searching a significant SNP locus of a hairless character through whole genome association analysis, searching related functions of candidate genes through GO analysis and KEGG analysis, mining a pig hairless character candidate gene region through genome re-sequencing, and performing multigroup mutual verification analysis;

And 4, step 4: the method comprises the steps of collecting hairless and hairless pig tissues, verifying the expression difference of MAP2 gene in different individuals and different periods on the level of RNA and protein through quantitative PCR, HE staining and immunohistochemistry, and verifying the function of target mutant genes.

The MAP2 mutant gene for influencing pig hair follicle development obtained by the invention has 10 mutation sites on the 15 th chromosome of a pig, and comprises two types of synonymous mutation and missense mutation, wherein the missense mutation occurs on 12 th exon and 13 th exon, the 274 th base of the 12 th exon is changed from A to G, the 166 th base of the 13 th exon is changed from C to G, the 412 th base of the 13 th exon is changed from A to G, and the corresponding amino acid is changed from N to S, Q to E, M to V respectively.

The invention has the beneficial effects that:

1. The mutant gene site is accurate, and an important animal model is provided: the invention provides a technical means for accurately screening candidate genes with hairless characters by using an integrated omics strategy, integrates and utilizes DNA horizontal multi-level biological information, accurately screens the candidate genes, detects the levels of RNA and protein at different periods, performs functional verification on the candidate genes, finds out the key genes with the hairless characters of pigs, and provides important reference and animal models for the research of human hereditary alopecia diseases by analyzing and researching the hairless characters. Provides a molecular marker of hairless character: the molecular marker provided by the invention can be used for breeding and purifying hairless pigs and also can be used for identifying hereditary hairless diseases.

2. A large white pig hairless test population is provided for research use: the experimental population is a small number of large white hairless pigs discovered in advance, and the genetic breeding theory is utilized to expand the population of the discovered large white hairless pigs, so that valuable experimental materials are provided for developing the research of hair follicle development and model animals.

3. The pig hairless character is genetically analyzed by integrating the multidisciplinary technology, and the result is more accurate: the invention integrates multiple groups of chemical ideas for cross validation analysis, relates to multiple disciplines, and comprises the genetic analysis of the pig hairless character in multiple fields of cytology, molecular biology, statistical genomics, bioinformatics, computer science and the like, obtains more accurate results, reduces the existence of some false positives, and provides a new idea for the research of complex characters while analyzing the genetic mechanism of the hairless character.

Drawings

FIG. 1 is a diagram of a mixed pool sequencing peak pattern of a MAP2 gene mutation site influencing pig hair follicle development.

Detailed Description

Step 1: a small number of hairless pigs are bred according to a breeding theory, and 12 boar families containing hairless individuals are established. A total of 120 big white pigs from the same field are collected, including 60 hairless individuals and 60 normal individuals; preparing for later phenotype verification and screening of hairless character candidate genes.

Step 2: at 19 days of age Piglets were the study subjects (hairless and normal pig hair shafts and hair follicles were most clearly compared) and the unit area (cm) was measured ²) The number of hair shafts. The number of hair shafts is detected at the same position on the back, and pigs with the number of hair shafts less than or equal to 3 (mean + standard deviation) per square centimeter area are judged as no-hair pigs, and pigs with the number of hair shafts more than or equal to 6 (mean-standard deviation) are judged as normal pigs. Meanwhile, the back tissues are transversely cut and longitudinally cut, the transverse cut HE is used for observing the number of hair follicles per unit area through HE staining observation, and the longitudinal cut is used for observing the hair follicle morphology and the development condition of the period. Thereby performing the auxiliary hairless property determination.

And step 3: collecting 200 families containing hairless and normal pig individuals, recording phenotype information and pedigree information of the selected individuals in detail, collecting later performance measurement information, collecting blood from ear vein, extracting genome DNA, and measuring genotype by adopting a column Porcine80K SNP chip. And (3) performing genome-wide association analysis (GWAS) on the traits affecting the pig hairless traits by adopting case-control family analysis, screening SNP sites obviously associated with the target traits, and further searching candidate genes related to the development of the pig hair follicles in the embryonic stage. The screened MAP2, UNC5D and other genes are related to the development of hair follicles of animals and are potential candidate genes for the hairless character of large white pigs. The GO functional analysis finds that the differential gene obviously enriches hair follicle morphological development, cell structure skeleton, fibroblast differentiation and other hair follicle development processes in the aspects of cell components, molecular functions, biological processes and the like. KEGG pathway analysis finds that partial differential genes are annotated in WNT, shh and other pathways, and important differential genes existing in the pathways can be used as candidate genes critical to hair follicle generation and development in porcine embryo stage.

After strict quality control is carried out on the typed original data, the analysis result is corrected by Bonferroni, 125 SNP sites which are obviously associated with the hairless character are screened, the genes of 1MB at the upstream and downstream of the obvious SNP sites are searched, and 23 genes are searched in total, wherein 13 genes contain associated SNP sites.

After 74 pig ear samples containing 7 boar families, including boar, sow and offspring pig family samples are collected for DNA extraction, the candidate gene is accurately analyzed and detected by a re-sequencing technology, and the candidate gene sequence information of the boar and the hairless pig is compared to find out the causative mutation generating character separation. And the association analysis is carried out on the mutation generating site and the hairless character. The mutation site is found to be significantly related to the hairless character. And screening related synonymous mutations and missense mutations from the mutation sites by using amino acid analysis software and protein alignment software. And comparing the amino acids of normal pigs, hairless pigs and other vertebrates, and observing whether the mutation sites are positioned in the high conservative region.

According to the experimental results, the MAP2 mutant gene influencing the pig hair follicle development is screened out, the mutant gene has 10 mutant sites, the physical position of the mutant gene is between 112155861 and 112424202 of No. 15 chromosome of the pig, and the nucleotide sequence of the mutant gene is shown as SEQ ID NO. 1-4.

And 4, step 4: based on the results of step 2, it was found that the hair follicle was completely developed in the skin tissue of the postnatal piglet and the period in which the hair follicle started to develop was in the embryonic period. Finding that the offspring of the pigs with the hair and the offspring of the pigs without the hair have two characters of the hair and the hairs, then carrying out abortion on the pregnant sows of which the parents are respectively the two characters of the hair and the hairs at different periods, taking embryos at different periods of hair follicle development, carrying out quantitative PCR, and verifying the expression difference condition of the MAP2 gene in the embryo periods of the pigs with the hair and the pigs without the hair and the expression difference in different tissues; HE staining is carried out on skin tissues of embryos at different development stages, and the hair-containing and hair-free properties and the development stages of hair follicles at different stages are judged by observing the staining results of transverse cutting and longitudinal cutting; the MAP2 gene of skin tissues of embryos at different development stages is immunohistochemically, and the expression condition and action part of MAP2 protein are observed.

TABLE 1 detection primers for MAP2 mutation site

The experimental results are as follows: the 10 mutation sites on the MAP2 mutant gene were identified.

10 mutation sites which influence the pig hair follicle development are co-found on a MAP2 mutant gene, the physical position of the mutation sites is between 112155861 and 112424202 of No. 15 chromosome of a pig, the nucleotide sequences of the mutation sites are shown as SEQ ID NO. 1-4, and the 10 mutation sites are respectively:

1, G95 > C, the mutation site is positioned in a 5' untranslated region;

2, 129 th C > T, 173 th C > T, 274 th A > G and the mutation site is positioned in No. 12 exon;

3, 166 th C > G and 412 th A > G of SEQ ID NO, wherein the mutation site is positioned in a No. 13 exon;

4, C at position 89 > T, A at position 314 > G, G at position 550 > A, A at position 575 > G and the mutation site is positioned in the No. 16 exon.

The above mutated bases include two types of synonymous mutations and missense mutations, wherein missense mutations occur on exons 12 and 13, and as shown in Table 2, the 274 th base of exon 12 is changed from A to G, the 166 th base of exon 13 is changed from C to G, the 412 th base of exon 13 is changed from A to G, and the corresponding amino acids are changed from N to S, Q to E, M to V, respectively.

TABLE 2 10 mutation sites of MAP2 mutant Gene affecting pig Hair follicle development

Through genome-wide association analysis, we found that all the 10 significant sites have a very significant relationship with the morphological development of hair follicles. The results show that the mutation conditions of the 10 sites have the possibility of influencing the hair follicle development process. Since the hairless trait is not a complete quality trait, there is usually a certain number of thin-haired pigs between hairless and normal pigs. The effect of these mutation sites on the phenotype was additive and the effect of each mutation was minimal. Mutations at these sites result in a greatly increased probability of hairless trait production. Through a quantitative PCR result, the remarkable difference of the MAP2 mutant gene between the hairless pig and the hairless pig in different gestation periods is further found, and the gestation period with high specificity expression of the MAP2 mutant gene is determined through HE staining observation. According to the results of immunohistochemistry, the specific site of expression and action of the MAP2 mutant gene is further determined. In conclusion, the MAP2 mutant gene disclosed by the invention is specifically and highly expressed in the hair follicle morphological development process and has obvious difference in hairy pigs and hairless pigs, so that the MAP2 mutant gene disclosed by the invention plays an important role in the hair follicle morphological development process.

Base changes can result in missense mutations and synonymous mutations in amino acids. Missense mutations result in amino acid changes that affect the normal functioning and expression of the spatial structure and function of the protein, which in turn affects the phenotype. Synonymous mutations do not result in changes in the corresponding amino acids, but many single nucleotide substitutions alter the efficiency or accuracy of cleavage of the mRNA. Exon Splicing Enhancer (ESE) and silencer (ESS) elements are present in most exons. Most of the ESE is far from the cleavage site, and some point mutations inactivating the ESE result in partial or complete exon walking, which affects the structure and number of the expressed protein product, and thus the phenotype.

According to the embodiment of the invention, the method is used for detecting the molecular marker of the pig to be detected so as to determine the hairless property of the pig to be detected, so that the MAP2 mutant gene disclosed by the invention is used for carrying out auxiliary breeding on the pig, the sow with stable hereditary hairless property can be effectively selected, and further the disease model pig with excellent breed can be bred in a short time, at low cost and with high accuracy, and the molecular marker can be used for identifying the genetic hairless disease, and further can be applied to the hair development research of mammals.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Sequence listing

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Claims (2)

1. A MAP2 mutant gene influencing pig hair follicle development is characterized in that the mutant gene has 10 mutant sites, the physical position of the mutant gene is between 112155861 and 112424202 of No. 15 chromosome of pig, the nucleotide sequence is shown as SEQ ID NO. 1-4, and the 10 mutant sites are respectively:

1, G95 > C, the mutation site is positioned in a 5' untranslated region;

2, 129 th C > T, 173 th C > T, 274 th A > G and the mutation site is positioned in No. 12 exon;

3, 166 th C > G and 412 th A > G of SEQ ID NO, wherein the mutation site is positioned in a No. 13 exon;

4, C at position 89 > T, A at position 314 > G, G at position 550 > A, A at position 575 > G and the mutation site is positioned in the No. 16 exon.

2. The use of the MAP2 mutant gene affecting pig hair follicle development of claim 1 in pig genotyping, screening, identification and assisted breeding.

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