CN103966231A - Molecular cloning of GLP2R gene fragments associated with pork quality traits and application of GLP2R gene fragments - Google Patents
Molecular cloning of GLP2R gene fragments associated with pork quality traits and application of GLP2R gene fragments Download PDFInfo
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Abstract
The invention belongs to the technical field of livestock molecular biology and particularly relates to a molecular cloning of GLP2R gene fragments associated with pork quality traits and application of GLP2R gene fragments. The human GLP2R gene sequence is taken as a seed sequence, specific primers are designed by BLASTN in GenBank and referring to swine EST of which the homology is above 80% and swine GLP2R gene cDNA full-length is cloned by RACE. DNA sequence of the swine GLP2R gene fragments is shown in SEQ ID NO:1. GLP2R gene polymorphism is determined by designing a primer based on human GLP2R gene via comparative genomics method, amplifying by swine genomic DNA as template, screening SNP from amplification fragment via sequencing, and genotyping by PCR-RFLP. A/G base mutation resulting in PCR-RFLP-BstEII polymorphism is found at 244bp, a new molecular marker is provided for swine marker-assisted selection and the situation of pig breeds at home and abroad can be detected by the marker.
Description
Technical field
The invention belongs to domestic animal technical field of molecular biology, be specifically related to a kind of nucleic acid molecule that comprises pig gene nucleotide as shown in SEQ ID NO:1.The invention still further relates to the mononucleotide polymorphism site in molecular cloning method and the polynucleotide sequence of the GLP2R gene as shown in SEQ ID NO:1 and detect as described in the method for mononucleotide polymorphism site.
Background technology
Pork is the main source of animal protein, along with the increase of people to meat requirement, to the requirement of meat also corresponding raising.Affecting pork qualitative factor has heredity 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, thereby meat is better.Meat Quality belongs to quantitative character mostly, by minor-polygene control.Many Meat Qualities can only could be measured after animal slaughtering, so conventional herd breeding can only carry out compatriot or half-sibs test, must strengthen like this seed selection cost, and genetic progress are slow.The structure of pig genome sketch, making to find the molecule marker that affects the key-gene of Meat Quality or be 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 effectively approach.
Glucagon-like peptide 2 (Glucagon-like peptide-2, GLP2) be the special somatomedin of a kind of enteric epithelium of discovered in recent years, contribute to enteron aisle to grow, can promote to repair the pathology damage of intestinal mucosa, improve intestinal barrier function, suppress intestinal cells apoptosis, accelerate the transhipment of enteric cavity nutritive substance, strengthen the absorption of enteron aisle to 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-like peptide-2recepter, GLP2R) belong to g protein coupled receptor family, with glucagon-like-peptide-1 (Glucagon-like peptide-1, GLP1), hyperglycemic-glycogenolytic factor (Glucagon, GCG) and glucose-dependent-insulinotropic polypeptide (Glucose-dependent insulinotropic polypeptide, GIP) be subject to height homology.GLP2R is as the specific receptors of GLP2, GLP2 and it is in conjunction with regulating intestinal mucosa cells propagation, differentiation and reparation etc.In addition, Luo etc. (2012) builds F with Large White and Min pig
2for resource family, F
2, there are 455 pigs in generation, gene chip used is PorcineSNP60K, intramuscular fat content, marble grain, yellowish pink of longissimus dorsi muscle etc. are measured, whole-genome association (Genome-Wide of Analysis of Study, GWAS) research shows, the GLP2R gene of pig and intramuscular fat content significant correlation.GLP2R is positioned at No. 12 karyomit(e)s of pig, No. 11 karyomit(e)s of mouse, No. 17 karyomit(e)s of people.At present, GLP2R in rat, people and mouse etc. clonal expression out, still, the rarely seen report of research to pig GLP2R gene both at home and abroad.
Summary of the invention
Technical problem to be solved by this invention is: for above-mentioned the deficiencies in the prior art, molecular cloning and the application of the GLP2R gene fragment that a kind of pig flesh characters is relevant are provided, by clone pig GLP2R gene cDNA molecule, find the mutational site of GLP2R gene, and detect the polymorphism of GLP2R gene, for pig flesh characters marker assisted selection provides the molecule marker of use.
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 SEQ ID NO:1, there is the base mutation of 1 G/A at the 243bp place of this SEQ ID NO:1 sequence, causes PCR-RFLP-BstE II polymorphism.
The present invention is taking people's GLP2R gene order (the GenBank number of including is as NM_004246.1) as Seed Sequences, in GenBank, utilize BLASTN (Basic Local Alignment Search ToolNucleotide), pig EST (Expression Sequence Tags) design special primer with reference to its homology more than 80%, utilize RACE (Rapid Amplification of cDNA End), clone pig GLP2R gene cDNA total length, the DNA sequence dna of this pig GLP2R gene, as shown in SEQ ID NO:1, is shown in Fig. 3.The polymorphism of GLP2R gene is to utilize comparative genomics method according to people's GLP2R gene order design primer, and forward primer is 5 '-CGGGAACTCCTGTTGTGC-3 ', and reverse primer is 5 '-TGAAGCGGTGAGAATGGTA-3 '; Again taking the genomic dna of pig as template amplification, amplified fragments utilization 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 is 3 kinds of genotype, AA genotype (746bp), AG type (746bp, 506bp, 240bp) and GG type (506bp, 240bp) in A/G site by analysis.
Can utilize the pig molecule mark of above-mentioned preparation to detect external pig kind and place of china kind, establish solid basis for marker assisted selection (MAS), improve Swine Production economic benefit, for the breeding practice that instructs pig provides theoretical foundation.
Brief description of the drawings
Fig. 1 is the electrophoresis result figure of the pcr amplification product of GLP2R gene fragment of the present invention.
In figure: M:100bp DNA Ladder Marker, 1-6 represents the 1st to the 6th swimming lane, represents respectively the 1st to No. 6 samples of Large White.
Fig. 2 is the BstE II restriction enzyme digestion and electrophoresis result in GLP2R Gene A/G of the present invention 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:100bp DNA Ladder Marker.
Fig. 3 is the DNA sequence dna of pig GLP2R gene of the present invention, has wherein shown sudden change position.
Embodiment
Below in conjunction with specific embodiment, the present invention is done to explain further, but concrete enforcement do not done any restriction to the present invention.
The clone of GLP2R gene:
Taking people's GLP2R gene order (the GenBank number of including is as NM_004246.1) as Seed Sequences, in GenBank, utilize BLASTN, pig EST (Expressed SequenceTags) design special primer with reference to its homology more than 80%, utilizes RACE technology to clone pig GLP2R gene cDNA total length.
Test materials: 25 pig resource fields, sand ridge, Zhu You xiangtan, hunan province city, age in days sand ridge provide, and get longissimus dorsi muscle-80 DEG C preservation, 3 '-RACE SMARTer
tMrACE cDNA Amplification Kit test kit is purchased from Clontech company.
Total RNA extracts synthetic with cDNA the 1st chain: total RNA is carried out to the synthetic of gene the first chain cDNA with SUPERSCRIPT II RT enzyme and Auele Specific Primer 3'445-1, use RNase Mix to go RNA processing to synthetic cDNA, finally cDNA is carried out to purifying.
Design of primers: according to GenBank people's GLP2R gene (accession number: NM_004246.1) sequence conserved regions design primer, the software of design of primers is Primer Premier5.0, 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, is used nest-type PRC to increase.
Encoding sequence pcr amplification: taking the pig cDNA that synthesizes as template, through cloning and sequencing, obtain this Gene Partial CDS (coding domain sequence) sequence at conserved regions design primer.
3 '-RACE amplification: with 3 ' 445-1 primer and UPM (Universal Primer A Mix) (in table 1), and above-mentioned synthetic cDNA is that template is carried out PCR first round amplification; By after 50 times of PCR first round amplified production dilutions, carry out nest-type PRC second and take turns amplification, amplified production is carried out to electrophoresis and object band cut to glue and reclaim purifying; Finally the PCR product after purifying is connected to picking positive colony order-checking after transforming with pMD18-T carrier.
Table 1 primer sequence
By after intermediate segment and 3 '-RACE sequence assembly, obtain the long cDNA sequence for 1868bp, thus the clone who completes GLP2R gene of the present invention.
The screening of GLP2R gene molecule marker:
The polymorphism of GLP2R gene is to utilize comparative genomics method according to people's GLP2R gene order design primer, taking the genomic dna of pig as template amplification, amplified fragments utilization 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: taking external pig kind Large White as subjects, get fritter pig ear tissue and extract DNA.
Utilize comparative genomics method according to the intron between the 5th exon of people's GLP2R gene and the 6th exon, make BLAST with this gene at GenBank, after sequence alignment, filter out GLP2R gene candidate SNP s site, choose GLP2R gene candidate SNP s site, with reference to GenBank sequence NC_010454.3 primers, use this site of PCR-RFLP technical identification.
The primer of the above-mentioned SEQ ID of the detection NO:1 sequence gene fragment sudden change of design is: forward primer is 5 '-CGGGAACTCCTGTTGTGC-3 ' (seeing SEQ ID NO:6), and reverse primer is 5 '-TGAAGCGGTGAGAATGGTA-3 ' (seeing SEQ ID NO:7).
PCR reaction conditions: PCR reaction system (cumulative volume 20 μ L), comprises 10 × Buffer2 μ L, 2mmol/LdNTPs1.6 μ L, 20mmol/L MgCl21.6 μ L, Taq archaeal dna polymerase (5U/ μ L) 0.4 μ L, DNA profiling (100ng/ μ L) 1 μ L, the each 0.4 μ L of upstream and downstream primer (10pmol/ μ L) and dd H2O12.6 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 are extended 40s, totally 35 circulations; After 72 DEG C, extend 8min, last 4 DEG C of preservations.
Get 10 μ L pcr amplification products, after adding 2 μ L tetrabromophenol sulfonphthalein sample-loading buffers and mixing, point sample is upper in 1% sepharose (containing 0.05% ethidium bromide), then puts 6 μ L100bp DNA Markers as reference, 5V/cm electrophoresis 0.5-1.0h.After electrophoresis finishes, in gel imaging system, observe amplification and take pictures, result as shown in Figure 1.By the PCR product Song Boshang biotech company order-checking after purifying.
The BstE II enzyme of PCR product is cut: in 10 μ L PCR 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 voltage electrophoresis 0.5h, observations taking pictures under ultraviolet lamp, enzyme is cut result as Fig. 2, amplified production checks order, and sequence is as shown in SEQ ID NO:1, and amplification segment is the segment between the 5th exon and the 6th exon, 746bp, is PCR-RFLP-BstE II molecule marker altogether.
Increased and comprised this SNP primer by design, 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 increasing, there is the restriction enzyme site of 1 BstE II, there is 3 kinds of genotype, GG type (506bp, 240bp), AG type (746bp, 506bp, 240bp) and AA genotype (746bp) in the A/G site that electrophoresis detection result is presented at GLP2R gene.
The BstE II polymorphism of PCR-RFLP technology for detection GLP2R gene:
Taking pig GLP2R gene as research object, using 4 local pig breeds (Daweizi pig, sand ridge 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, 293 of Large Whites, 45 of 74 of the black pigs in the Land of Peach Blossoms, 25 of sand ridge pigs, 62 of Ning Xiang Swines and Daweizi pigs.Gene frequency, genotype frequency to GLP2R Gene A/G site detect, and analyze 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 the pig of sand ridge, 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 respectively 125 and 127 in Large White.
The gene frequency in table 2 different varieties GLP2R Gene A/G site and genotype frequency distribute
Claims (4)
1. a GLP2R gene fragment relevant to pig flesh characters, its nucleotide sequence is as described in SEQ ID NO:1, and there is the base mutation of 1 G/A at the 243bp place of this SEQ ID NO:1 sequence, causes PCR-RFLP-BstE II polymorphism.
2. test right requires the primer pair of the base mutation of the GLP2R gene fragment relevant to pig flesh characters described in 1, it is characterized in that, the sequence of this primer pair is as follows:
Forward primer: 5 '-CGGGAACTCCTGTTGTGC-3 ',
Reverse primer: 5 '-TGAAGCGGTGAGAATGGTA-3 '.
3. the application of GLP2R gene fragment claimed in claim 1 in the molecular marker assisted selection relevant to pig flesh characters.
4. the application of primer pair claimed in claim 1 in the relevant molecular marker assisted selection of pig flesh characters.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450729A (en) * | 2014-12-31 | 2015-03-25 | 湖南农业大学 | Clone and application of pig meat quality character relevance WNT10B gene molecular mark |
| CN105861738A (en) * | 2016-06-23 | 2016-08-17 | 湖南农业大学 | Ningxiang pork detection kit |
| CN110484540A (en) * | 2019-09-12 | 2019-11-22 | 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 its application in duck MHC haplotype breeding |
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| BIO: "Sus scrofa breed mixed chromosome 12， Sscrofal0.2", 《GENBANK登录号：NC_010454.3（REGION：57455384..57456129）》, 29 September 2013 (2013-09-29) * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450729A (en) * | 2014-12-31 | 2015-03-25 | 湖南农业大学 | Clone and application of pig meat quality character relevance WNT10B gene molecular mark |
| 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 |
| CN110484540A (en) * | 2019-09-12 | 2019-11-22 | 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 its application in duck MHC haplotype breeding |
| 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 |
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