CN103966231B - Pig flesh characters is correlated with the molecular cloning of GLP2R gene fragment and application - Google Patents
Pig flesh characters is correlated with the molecular cloning of GLP2R gene fragment and application Download PDFInfo
- Publication number
- CN103966231B CN103966231B CN201410237979.4A CN201410237979A CN103966231B CN 103966231 B CN103966231 B CN 103966231B CN 201410237979 A CN201410237979 A CN 201410237979A CN 103966231 B CN103966231 B CN 103966231B
- Authority
- CN
- China
- Prior art keywords
- glp2r
- pig
- gene
- primer
- gene fragment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to domestic animal technical field of molecular biology, be specifically related to a kind of pig flesh characters to be correlated with the molecular cloning of GLP2R gene fragment and application, for Seed Sequences with the GLP2R gene order of people, BLASTN is utilized in GenBank, special primer is designed with reference to the pig EST of its homology more than 80%, RACE is utilized to clone pig GLP2R gene cDNA total length, is the DNA sequence dna of this pig GLP2R gene fragment as SEQ? ID? shown in NO:1.GLP2R gene polynorphisms utilizes comparative genomics method according to the GLP2R gene order design primer of people, with the genomic dna of pig for template amplification, amplified fragments utilizes order-checking screening SNP, and utilize PCR-RFLP to carry out gene type, find that there is the base mutation of an A/G at 244bp place, cause PCR-RFLP-BstE II polymorphism, for pig marker assisted selection provides new molecule marker, the situation of this marker detection China and foreign countries pig kind can be utilized.
Description
Technical field
The invention belongs to domestic animal technical field of molecular biology, be specifically related to a kind of nucleic acid molecule comprising pig gene nucleotide as shown in SEQIDNO:1.The invention still further relates to the mononucleotide polymorphism site in the molecular cloning method of the GLP2R gene as shown in SEQIDNO:1 and polynucleotide sequence and the method for mononucleotide polymorphism site as described in detecting.
Background technology
Pork is the main source of animal protein, along with people are to the increase of meat requirement, to the requirement also corresponding raising of meat.Affecting pork qualitative factor has hereditary and extragenetic, and pig intramuscular fat content has a great impact Meat Quality, and in general, intramuscular fat content is higher, and Meat is tenderer, thus meat is better.Meat Quality belongs to quantitative character mostly, is controlled by minor-polygene.Many Meat Qualities could can only measure after animal slaughtering, so conventional herd breeding can only carry out compatriot or half-sibs test, must strengthen seed selection cost like this, and genetic progress are slow.The structure of pig draft genome, makes to find the key-gene affecting Meat Quality or the molecule marker being close to lock with it becomes possibility, and these molecule markers, once confirmation, just can carry out marker assisted selection breeding.Utilizing molecule marker to carry out assisted Selection (MAS), to carry out Meat Quality improvement be a kind of effective approach.
Glucagon-like peptide 2 (Glucagon-likepeptide-2, GLP2) be a kind of enteric epithelium specific growth factor of discovered in recent years, contribute to enteron aisle to grow, the pathology damage of repairing intestinal mucosa can be promoted, improve intestinal barrier function, suppress enterocyte apoptosis, accelerate the transhipment of enteric cavity nutritive substance, strengthen enteron aisle to the absorption of nutritive substance.In addition, have report to point out, in serum, GLP-2 content is relevant to 2 type glycosurias.
Glucagon-like peptide 2 acceptor (Glucagon-likepeptide-2recepter, GLP2R) g protein coupled receptor family is belonged to, with glucagon-like-peptide-1 (Glucagon-likepeptide-1, GLP1), hyperglycemic-glycogenolytic factor (Glucagon, and the high receptor homology of glucose-dependent-insulinotropic polypeptide (Glucose-dependentinsulinotropicpolypeptide, GIP) GCG).GLP2R is as the specific receptors of GLP2, and GLP2 and its combination regulate intestinal mucosa cells to breed, break up and reparation etc.In addition, Luo etc. (2012) build F with Large White and Min pig
2for Resource family, F
2, there are 455 pigs in generation, gene chip used is PorcineSNP60K, determine the intramuscular fat content of longissimus dorsi muscle, marble grain, yellowish pink etc., whole-genome association (Genome-WideofAnalysisofStudy, GWAS) research shows, the GLP2R gene of pig and intramuscular fat content significant correlation.GLP2R is positioned at pig No. 12 karyomit(e)s, mouse No. 11 karyomit(e)s, people's No. 17 karyomit(e)s.At present, GLP2R in rat, people and the mouse etc. clonal expression out, but, both at home and abroad to the rarely seen report of research of pig GLP2R gene.
Summary of the invention
Technical problem to be solved by this invention is: for above-mentioned the deficiencies in the prior art, the molecular cloning of the GLP2R gene fragment providing a kind of pig flesh characters to be correlated with and application, by clone pig GLP2R gene cDNA molecule, find the mutational site of GLP2R gene, and detect GLP2R gene polynorphisms, be the molecule marker that pig flesh characters marker assisted selection provides.
In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of GLP2R gene fragment relevant to pig flesh characters, its nucleotide sequence is as described in SEQIDNO:1, there is the base mutation of 1 G/A at the 244bp place of this SEQIDNO:1 sequence, causes PCR-RFLP-BstE II polymorphism.
The present invention with the GLP2R gene order of people (the GenBank number of including is for NM_004246.1) for Seed Sequences, BLASTN (BasicLocalAlignmentSearchToolNucleotide) is utilized in GenBank, special primer is designed with reference to the pig EST (ExpressionSequenceTags) of its homology more than 80%, utilize RACE (RapidAmplificationofcDNAEnd), clone pig GLP2R gene cDNA total length, the DNA sequence dna of this pig GLP2R gene, as shown in SEQIDNO:1, is shown in Fig. 3.GLP2R gene polynorphisms utilizes comparative genomics method according to the GLP2R gene order design primer of people, and forward primer is 5 '-CGGGAACTCCTGTTGTGC-3 ', and reverse primer is 5 '-TGAAGCGGTGAGAATGGTA-3 '; Again with the genomic dna of pig for template amplification, amplified fragments utilizes order-checking screening SNP, and utilize PCR-RFLP to carry out genotyping technique, find that there is the base mutation of an A/G at 244bp place, cause PCR-RFLP-BstE II polymorphism, there are 3 kinds of genotype in A/G site by analysis, AA genotype (746bp), AG type (746bp, 506bp, 240bp) and GG type (506bp, 240bp).
The pig molecule mark of above-mentioned preparation can be utilized to detect external pig kind and place of china kind, for marker assisted selection (MAS) establishes solid basis, improve Swine Production economic benefit, providing theoretical foundation for instructing the breeding practice of pig.
Accompanying drawing explanation
Fig. 1 is the electrophoresis result figure of the pcr amplification product of GLP2R gene fragment of the present invention.
In figure: M:100bpDNALadderMarker, 1-6 represent the 1st to the 6th swimming lane, represent Large White the 1 to No. 6 sample respectively.
Fig. 2 is the BstE II restriction enzyme digestion and electrophoresis result in GLP2R of the present invention Gene A/G site.
In figure: swimming lane 1-3:GG genotype; Swimming lane 4,5,8,10:AA genotype; Swimming lane 6,7,9:AG genotype; M:100bpDNALadderMarker.
Fig. 3 is the DNA sequence dna of pig GLP2R gene of the present invention, which show mutated site.
Embodiment
Below in conjunction with specific embodiment, the present invention is explained further, but concrete enforcement does not do any restriction to the present invention.
The clone of GLP2R gene:
With the GLP2R gene order of people (the GenBank number of including is for NM_004246.1) for Seed Sequences, BLASTN is utilized in GenBank, design special primer with reference to the pig EST (ExpressedSequenceTags) of its homology more than 80%, utilize RACE technology to clone pig GLP2R gene cDNA total length.
Test materials: 25 age in days Shaziling pig are provided by Shaziling pig resource field, xiangtan, hunan province city, get longissimus dorsi muscle-80 DEG C preservation, 3 '-RACESMARTer
tMrACEcDNAAmplificationKit test kit is purchased from Clontech company.
The synthesis of Total RNAs extraction and cDNA the 1st chain: carry out the synthesis of gene first chain cDNA to total serum IgE with SUPERSCRIPTIIRT enzyme and Auele Specific Primer 3'445-1, uses the cDNA of RNaseMix to synthesis to go RNA process, finally carries out purifying to cDNA.
Design of primers: according to GLP2R gene (accession number: NM_004246.1) the sequence conservation design primer of GenBank people, the software of design of primers is PrimerPremier5.0, the principle of design of primers is that Auele Specific Primer length is at 23-28 Nucleotide, GC content is at 50%-70%, annealing temperature, at 65-70 DEG C, uses nest-type PRC to increase.
Encoding sequence pcr amplification: with the pig cDNA synthesized for template, at conserved regions design primer through cloning and sequencing, obtains this Gene Partial CDS (codingdomainsequence) sequence.
3 '-RACE amplification: with 3 ' 445-1 primer and UPM (UniversalPrimerAMix) (see table 1), and the cDNA of above-mentioned synthesis is that template carries out PCR first round amplification; After PCR first round amplified production is diluted 50 times, carry out nest-type PRC second and take turns amplification, amplified production is carried out electrophoresis and cuts glue to object band reclaiming purifying; Finally the PCR primer after purifying is connected with pMD18-T carrier, picking positive colony order-checking after transforming.
Table 1 primer sequence
After intermediate segment and 3 '-RACE sequence assembly, obtain the long cDNA sequence for 1868bp, thus complete the clone of GLP2R gene of the present invention.
The screening of GLP2R gene molecule marker:
GLP2R gene polynorphisms utilizes comparative genomics method according to the GLP2R gene order design primer of people, with the genomic dna of pig for template amplification, amplified fragments utilizes order-checking screening SNP, finds that there is the base mutation of an A/G at 244bp place, causes PCR-RFLP-BstE II polymorphism.
Test materials: carrying out pig kind Large White is in addition subjects, gets fritter pig ear tissue and extracts DNA.
Utilize comparative genomics method according to the intron between the 5th exon of the GLP2R gene of people and the 6th exon, BLAST is made at GenBank with this gene, GLP2R gene candidate SNPs site is filtered out after sequence alignment, choose GLP2R gene candidate SNPs site, with reference to GenBank sequence NC_010454.3 primers, use this site of PCR-RFLP technical identification.
The primer of the detection above-mentioned SEQIDNO:1 sequence gene fragment sudden change of design is: forward primer is 5 '-CGGGAACTCCTGTTGTGC-3 ' (see SEQIDNO:6), and reverse primer is 5 '-TGAAGCGGTGAGAATGGTA-3 ' (see SEQIDNO:7).
PCR reaction conditions: PCR reaction system (cumulative volume 20 μ L), comprises 10 × Buffer2 μ L, 2mmol/LdNTPs1.6 μ L, 20mmol/LMgCl
21.6 μ L, Taq DNA polymerase (5U/ μ L) 0.4 μ L, DNA profiling (100ng/ μ L) 1 μ L, upstream and downstream primer (10pmol/ μ L) each 0.4 μ L and ddH
2o12.6 μ L.
PCR response procedures: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 61 DEG C of annealing 30s, 72 DEG C extend 40s, totally 35 circulations; 8min is extended, last 4 DEG C of preservations after 72 DEG C.
Get 10 μ LPCR amplified productions, after adding 2 μ L tetrabromophenol sulfonphthalein sample-loading buffer mixings, point sample, on the sepharose (containing 0.05% ethidium bromide) of 1%, then puts 6 μ L100bpDNAMarkers as reference, 5V/cm electrophoresis 0.5-1.0h.In gel imaging system, observe amplification after electrophoresis terminates and take pictures, result as shown in Figure 1.By the PCR primer Song Boshang biotech company order-checking after purifying.
BstE II enzyme of PCR primer is cut: in 10 μ LPCR products, add 0.8 μ L10 × restriction enzyme damping fluid, 0.2 μ L restriction enzyme and 1 μ L distilled water, cumulative volume is 12L, 37 DEG C of digestion 4-10h.With 2% agarose gel electrophoresis analysis, 5V/cm electrophoresis 0.5h, observations under ultraviolet lamp is also taken pictures, enzyme cuts result as Fig. 2, amplified production checks order, and sequence is as shown in SEQIDNO:1, and am-plified fragments is the segment between the 5th exon and the 6th exon, 746bp, is PCR-RFLP-BstE II molecule marker altogether.
Comprise this SNP primer by design amplification, the sudden change in A/G site adopts BstE II to carry out enzyme and cuts detection polymorphism by analysis.In the fragment of the 746bp of amplification, there is the restriction enzyme site of 1 BstE II, there are 3 kinds of genotype in the A/G site that electrophoresis detection result is presented at GLP2R gene, GG type (506bp, 240bp), AG type (746bp, 506bp, 240bp) and AA genotype (746bp).
BstE II polymorphism of PCR-RFLP technology for detection GLP2R gene:
With pig GLP2R gene for research object, using 4 local pig breeds (Daweizi pig, Shaziling pig, Ning Xiang Swine, the black pig in the Land of Peach Blossoms) and external pig kind (Large White) as experimental subjects, get fritter pig ear tissue and extract DNA, wherein, Large White 293, the black pig in the Land of Peach Blossoms 74, Shaziling pig 25, Ning Xiang Swine 62 and Daweizi pig 45.GLP2R Gene A/the gene frequency in G site, genotype frequency are detected, and analyzes its genetic construction.
The distribution situation of PCR-RFLP-BstE II polymorphism in each kind is as shown in table 2, as shown in Table 2, in these 3 local variety of the black pig in Ning Xiang Swine, Daweizi pig and the Land of Peach Blossoms, G gene is advantage allelotrope, this gene frequency of the black pig in the Land of Peach Blossoms is up to 0.987, and the frequency of Ning Xiang Swine and Daweizi pig reaches 1; And in adventive (Large White), A gene frequency is advantage allelotrope, this gene frequency is 0.6433.From genotype distribution frequency, GG type is preponderated in distribution, and AA type individuality is less, only in Shaziling pig, finds 1, in the black pig in the Land of Peach Blossoms, detects 15, but GG type less (41) and AA type and AG type have 125 and 127 respectively in Large White.
The gene frequency in table 2 different varieties GLP2R Gene A/G site and genotype frequency distribution
Claims (2)
1. detect the primer pair of the base mutation of the GLP2R gene fragment relevant to pig flesh characters, the nucleotide sequence of this GLP2R gene fragment is as described in SEQIDNO:1, there is the base mutation of 1 G/A at the 244bp place of this SEQIDNO:1 sequence, cause PCR-RFLP-BstE II polymorphism, it is characterized in that, the sequence of this primer pair is as follows:
Forward primer: 5 '-CGGGAACTCCTGTTGTGC-3 ',
Reverse primer: 5 '-TGAAGCGGTGAGAATGGTA-3 '.
2. the application in the primer pair according to claim 1 molecular marker assisted selection of being correlated with at pig flesh characters.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410237979.4A CN103966231B (en) | 2014-05-30 | 2014-05-30 | Pig flesh characters is correlated with the molecular cloning of GLP2R gene fragment and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410237979.4A CN103966231B (en) | 2014-05-30 | 2014-05-30 | Pig flesh characters is correlated with the molecular cloning of GLP2R gene fragment and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103966231A CN103966231A (en) | 2014-08-06 |
| CN103966231B true CN103966231B (en) | 2016-02-24 |
Family
ID=51236237
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410237979.4A Expired - Fee Related CN103966231B (en) | 2014-05-30 | 2014-05-30 | Pig flesh characters is correlated with the molecular cloning of GLP2R gene fragment and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103966231B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450729B (en) * | 2014-12-31 | 2015-10-28 | 湖南农业大学 | Pig flesh characters is correlated with the clone of WNT10B gene molecule marker and application |
| CN105861738A (en) * | 2016-06-23 | 2016-08-17 | 湖南农业大学 | Ningxiang pork detection kit |
| CN110484540B (en) * | 2019-09-12 | 2022-11-29 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Duck uaa gene and application thereof in duck MHC haplotype breeding |
-
2014
- 2014-05-30 CN CN201410237979.4A patent/CN103966231B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| Sus scrofa breed mixed chromosome 12， Sscrofal0.2;bio;《Genbank登录号：NC_010454.3（region：57455384..57456129）》;20130929;全文 * |
| 胰高血糖素样肽－2受体在新生仔猪部分内脏器官中的分布;齐珂珂，等;《营养饲料》;20130415;第49卷(第7期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103966231A (en) | 2014-08-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105755131B (en) | Genetic marker associated with pork quality traits and carcass traits | |
| CN106544412B (en) | Molecular marker related to boar sperm motility character and application thereof | |
| CN101824416B (en) | Pork quality trait related gene DGAT1 and application thereof in porcine marker-assisted selection | |
| CN104450729B (en) | Pig flesh characters is correlated with the clone of WNT10B gene molecule marker and application | |
| CN103966231B (en) | Pig flesh characters is correlated with the molecular cloning of GLP2R gene fragment and application | |
| CN104774836A (en) | Polygene pyramiding early-breeding method for raising litter size of sheep | |
| CN101440399A (en) | Molecular marking method for indicating and identifying litter size in pigs by MMP23 gene | |
| CN101633943A (en) | Identification method taking growth hormone receptor gene as Chinese grassland red bull excellent slaughter trait molecular marker and application thereof | |
| Riaz et al. | Molecular marker assisted study of kappa-casein gene in Nili-Ravi (buffalo) breed of Pakistan | |
| CN103725790A (en) | Molecular marker relevant to growth of fenneropenaeus chinensis and application of molecular marker | |
| CN104004750B (en) | A kind of SNP marker relevant to mitten crab sexual prematurity proterties and application thereof | |
| CN106191253B (en) | Beijing duck based on GBS technology simplifies gene order surveying method | |
| CN109439763B (en) | Molecular marker for identifying 817 broiler chickens and yellow-leg free-range chickens and application | |
| CN103739696B (en) | Cynoglossus semilaevis female specificity CSW3 protein as well as gene and application thereof | |
| Latifah et al. | Genetic analysis using partial sequencing of melanocortin 4 receptor (MC4R) gene in Bligon goat | |
| CN105112425A (en) | Molecular cloning and application of pork quality character related gene Myo6 (Myosin 6) | |
| Mahmoud et al. | Nucleotide sequence polymorphism within exon 3 region of leptin and prolactin genes in Herri sheep | |
| CN103224941B (en) | A kind of detect the relevant molecular marker of pig flesh characters and application | |
| Yuan et al. | Novel SNPs polymorphism of bovine CACNA2D1 gene and their association with somatic cell score | |
| CN102649958B (en) | Genetic marker related to growth rate of pig and application of genetic marker | |
| CN112094917B (en) | Western pig blood marginal infiltration site SNP marker related to body length in local pig in China and application | |
| CN114921568A (en) | SNP molecular marker related to Qinchuan cattle body ruler and meat quality traits and application thereof | |
| CN103333897A (en) | Clone and application of pork quality character correlation POSTN gene molecular marker | |
| CN103509099B (en) | Female cynoglossus semilaevis specific expression gene CSW2 and applications thereof | |
| CN103333899B (en) | Cloning and application of CDC16 gene molecular marker related to pork quality character |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160224 Termination date: 20170530 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |