CN101914544A - Clone of porcine thyrotropin-releasing hormone receptor (TRHR) gene and application thereof - Google Patents
Clone of porcine thyrotropin-releasing hormone receptor (TRHR) gene and application thereof Download PDFInfo
- Publication number
- CN101914544A CN101914544A CN 201010220407 CN201010220407A CN101914544A CN 101914544 A CN101914544 A CN 101914544A CN 201010220407 CN201010220407 CN 201010220407 CN 201010220407 A CN201010220407 A CN 201010220407A CN 101914544 A CN101914544 A CN 101914544A
- Authority
- CN
- China
- Prior art keywords
- gene
- trhr
- pig
- seq
- sequence
- 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.)
- Pending
Links
- 108090001094 Thyrotropin-releasing hormone receptors Proteins 0.000 title claims abstract description 32
- 102000004852 Thyrotropin-releasing hormone receptors Human genes 0.000 title claims abstract description 30
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 27
- 239000002299 complementary DNA Substances 0.000 claims abstract description 13
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 11
- 230000035772 mutation Effects 0.000 claims abstract description 11
- 241001465754 Metazoa Species 0.000 claims description 3
- 108700026244 Open Reading Frames Proteins 0.000 claims description 3
- 238000010353 genetic engineering Methods 0.000 claims description 3
- 101150054439 TRHR gene Proteins 0.000 abstract description 16
- 239000003550 marker Substances 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 238000012098 association analyses Methods 0.000 abstract description 6
- 238000009395 breeding Methods 0.000 abstract description 5
- 230000001488 breeding effect Effects 0.000 abstract description 4
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 abstract 2
- 238000010367 cloning Methods 0.000 abstract 1
- 239000003147 molecular marker Substances 0.000 abstract 1
- 230000008859 change Effects 0.000 description 15
- 238000011144 upstream manufacturing Methods 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 9
- 238000013461 design Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 241000282887 Suidae Species 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 210000003205 muscle Anatomy 0.000 description 7
- 238000011160 research Methods 0.000 description 6
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 239000005556 hormone Substances 0.000 description 4
- 238000007918 intramuscular administration Methods 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 3
- 102100032251 Pro-thyrotropin-releasing hormone Human genes 0.000 description 3
- 101800004623 Thyrotropin-releasing hormone Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 210000000003 hoof Anatomy 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 3
- 230000001646 thyrotropic effect Effects 0.000 description 3
- 229940034199 thyrotropin-releasing hormone Drugs 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 229940035722 triiodothyronine Drugs 0.000 description 3
- 230000004584 weight gain Effects 0.000 description 3
- 235000019786 weight gain Nutrition 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 2
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 2
- 206010062767 Hypophysitis Diseases 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- YDTFRJLNMPSCFM-YDALLXLXSA-M levothyroxine sodium anhydrous Chemical compound [Na+].IC1=CC(C[C@H](N)C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 YDTFRJLNMPSCFM-YDALLXLXSA-M 0.000 description 2
- 239000003471 mutagenic agent Substances 0.000 description 2
- 230000001817 pituitary effect Effects 0.000 description 2
- 239000003488 releasing hormone Substances 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical class O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 101150090724 3 gene Proteins 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 241000218645 Cedrus Species 0.000 description 1
- 241000252233 Cyprinus carpio Species 0.000 description 1
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- XNSAINXGIQZQOO-UHFFFAOYSA-N L-pyroglutamyl-L-histidyl-L-proline amide Natural products NC(=O)C1CCCN1C(=O)C(NC(=O)C1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-UHFFFAOYSA-N 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 208000007913 Pituitary Neoplasms Diseases 0.000 description 1
- 102000003946 Prolactin Human genes 0.000 description 1
- 108010057464 Prolactin Proteins 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 102100040756 Rhodopsin Human genes 0.000 description 1
- 108090000820 Rhodopsin Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 102100029337 Thyrotropin receptor Human genes 0.000 description 1
- 239000000627 Thyrotropin-Releasing Hormone Substances 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- 230000009102 absorption Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 210000004754 hybrid cell Anatomy 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003601 intercostal effect Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 208000010916 pituitary tumor Diseases 0.000 description 1
- 235000015277 pork Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 229940097325 prolactin Drugs 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 239000005495 thyroid hormone Substances 0.000 description 1
- 229940036555 thyroid hormone Drugs 0.000 description 1
- 108040006218 thyroid-stimulating hormone receptor activity proteins Proteins 0.000 description 1
- 229940034208 thyroxine Drugs 0.000 description 1
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses clone of a porcine thyrotropin-releasing hormone receptor (TRHR) gene and application thereof. Overall length of porcine TRHR gene cDNA expressed as a sequence table SEQ ID NO: 1 and DNA sequences expressed as sequence tables SEQ ID NO: 2 and SEQ ID NO: 3 are obtained by cloning. Association analysis shows that a T442-C442 base mutation and a G626-T626 base mutation in the sequence table SEQ ID NO: 2 are remarkably associated with multiple porcine production traits. The clone provides a new molecular marker for porcine marker-assisted breeding.
Description
Technical field
The invention belongs to tame pig molecule assist-breeding technical field, be specifically related to the detection method of a kind of clone of porcine thyrotropin-releasing hormone receptor (TRHR) gene and single nucleotide polymorphism thereof and use as the molecular genetic marker of pig production character.
Background technology
Along with the raising of living standards of the people, the raising of pork demand and meat quality has been caused producers and consumers's extensive concern.Pig is the important goods source of China people meat product consumption, and it also is the human heteroplastic important animal model of research and may becomes suitable transplant organ source of supply.Pig industry has critical role in China's livestock industry and national economy, the carcass quality evaluation has become the problem that heredity, breeding scholar, the producer and human consumer generally are concerned about.Be used for the pig marker assisted selection so separating clone influences the new gene of pig growth and carcass quality proterties and seeks important molecule marker, to quickening the seed selection of pig new variety, the development of accelerating China's pig industry has extremely important value.
Synthetic and the excretory throtropin releasing hormone (Thyrotropin-releasinghormone of hypothalamus, TRH) act on hypophysis, trh receptor (Thyrotropin-releasing hormone receptor with the hypophysis surface, TRHR) in conjunction with activating courier's path in the born of the same parents, the secretion of promotion thyrotropic hormone and prolactin and synthetic.Wherein thyrotropic hormone is circulated to Tiroidina through blood and combines with being positioned at its surperficial thyrotropin receptor, starts courier's path in the born of the same parents, promotes Tiroidina to the running and the picked-up of iodine and Triiodothyronine is synthetic and secretion.And the Triiodothyronine effect spreads all over whole body, and major function promotes three major nutrient and energy metabolism for promoting tissue differentiation, growth and maturation, stress and keep individual normal growth growth etc.In addition, Triiodothyronine has calorigenic action in body, influences the metabolism of protein, fat, sugar, VITAMIN and water salt, and to digestion, absorption, cardiovascular, sexual gland, bone and neural system all play an important role (Chen Shouliang, 1996).Experiment shows, adds thyroxine and can improve individual food consumption and not influence meat ratio (feed institute of Chongqing City's feed company, 1996) in pig feed, effectively improves the speed of growth of pig.Therefore, the thyrotropic hormone of regulation and control thyroid hormone secretion level and throtropin releasing hormone and acceptor gene thereof are the critical function candidate genes that influences pig growth and carcass trait.Yet it is very few about the research of the relevant regulatory gene of pig thyroid gland hormone secretion both at home and abroad at present.Recently, the variation that exists in the human trh receptor TRHR gene that studies show that of Liu etc. is the major cause that causes human individual's lean mass difference.
TRHR belongs to g protein coupled receptor superfamily (G protein-coupled receptor superfamily, rhodopsin GPCR)/receptor, family (Family A).Nineteen ninety, obtain first TRH acceptor from mouse pituitary tumor cDNA library clone.Subsequently, its vertical homoreceptor also obtains the clone in other species, comprising: rat, ox, chicken, sheep, Africa xenopus, white carp and the mankind etc., but do not see the report of relevant pig TRHR gene.Applicant utilize technology such as radiation hybrid cell line location with the pig TRHR assignment of genes gene mapping to No. 4 karyomit(e)s of pig, find that there are a plurality of day weight gain, trunk is long, the thickness of backfat is relevant with the meat proterties QTL of influencing in respective regions, and shows that pig TRHR gene is an important growth and carcass quality genes involved.But it is up to the present, still blank to the research of pig TRHR gene both at home and abroad.The polymorphism of research mutational site in colony, and carry out the very favorable means that the proterties association analysis is the research gene function.So the applicant has cloned the cDNA sequence and the partial dna sequence of this gene and has carried out polymorphic research and association analysis, in selecting in the hope of the function that can disclose it and the molecule aid mark that applies to pig.
Summary of the invention
The objective of the invention is to overcome the prior art defective, a kind of clone and application thereof of porcine thyrotropin-releasing hormone receptor (TRHR) gene are provided.The cDNA encoding sequence total length and the dna sequence dna of the thyrotropin-releasing hormone receptor (TRHR) gene that clone of the present invention is relevant with pig production character, and utilize of the application of the pleomorphism site of this gene as the molecule marker of pig production character, for the breeding of pig provides a kind of new marker assisted selection.
Purpose of the present invention realizes by following technology: a kind of porcine thyrotropin-releasing hormone receptor (TRHR) gene, its cDNA sequence are the sequence shown in the SEQ ID NO:1, and it has the gene order shown in SEQ ID NO:2 and the SEQ ID NO:3.
This cDNA sequence length is 1469bp, comprises the open reading frame of 1198bp in the sequence, the 5 ' non-translational region of 233bp and the 3 ' non-translational region of 38bp.There is a T442-C442 base mutation at the 442bp place of sequence table SEQ ID NO:2, and there is a G626-T626 base mutation at the 626bp place.
The one above-mentioned application of gene TRHR in pig molecule mark assisted Selection and animal genetic engineering.
The invention has the beneficial effects as follows, the cDNA encoding sequence total length and the dna sequence dna of the thyrotropin-releasing hormone receptor (TRHR) gene that clone of the present invention is relevant with pig production character, and utilize of the application of the pleomorphism site of this gene as the molecule marker of pig production character, for the breeding of pig provides a kind of new marker assisted selection.
Description of drawings
Fig. 1 is that the present invention designs and the Tetra primersARMS-PCR method principle schematic of the quick sudden change detection technique institute foundation optimized; Among the figure, upstream and downstream outer primer (black arrow is represented) amplification comprises the big fragment of this polymorphic site, a kind of in upstream and downstream inner primer (red and blue arrow is represented) two allelotrope that increase respectively;
Fig. 2 is the somatotype electrophoretogram at pig TRHR gene T442-C442 polymorphism seat among the present invention; Among the figure, the M swimming lane is a dna molecular amount standard;
Fig. 3 is the somatotype electrophoretogram at pig TRHR gene G626-T626 polymorphism seat among the present invention; Among the figure, the M swimming lane is a dna molecular amount standard.
Embodiment
The present invention at first clones and obtains porcine thyrotropin-releasing hormone receptor (TRHR) gene, its cDNA sequence is as described in the sequence table SEQ ID NO:1, this sequence total length is 1469bp, comprises the open reading frame of 1198bp in the sequence, the 5 ' non-translational region of 233bp and the 3 ' non-translational region of 38bp.Promptly in this sequence, the 1-233bp place is 5 ' UTR, 234-1431 position CDS, and the 1432-1469 position is 3 ' UTR.
On this basis, the partial dna sequence that has obtained pig TRHR gene is as described in sequence table SEQ ID NO:2 and the SEQ ID NO:3.Wherein the dna fragmentation length of SEQ ID NO:2 is 2188bp, and the 1-159 position is the 1st exon sequence, and the 160-711 position is the 1st intron, and the 712-1582 position is the 2nd exon, and 1583-2188 is part the 2nd intron sequences; The dna fragmentation length of SEQ ID NO:3 is 947bp, and the 1-539 position is the 2nd intron partial sequence, and the 540-947 position is the 3rd exon.
Suddenly change just like the described T442-C442 base mutation of sequence table SEQ ID NO:2 and a G626-T626 in the pig TRHR Gene Partial dna sequence dna that is obtained.Based on tetra primers ARMS-PCR method principle design and optimize the fast typing technology be used for detecting two sudden changes of sequence table SEQ ID NO:2 and specifically see the embodiment part.
Clone of molecule marker and preparation method thereof carries out according to following steps among the present invention:
(1) utilizes that known pig TRHR partial dna sequence (959bp) and people TRHR gene DNA sequence and mRNA sequence compare in the GeneBank database, obtain pig TRHR gene the 2nd exon partial sequence; The design 3 ' RACE primer, wherein pig TRHR gene specific primer sequence shown in SEQ IDNO:4 and SEQ ID NO:5,
Upstream outer primer: GGTGTCTTTTATGTTGTGCCAAT
Upstream inner primer: TCAACAGCACAGTATCTTCAAGG
Extract the pig pituitary total tissue RNA; 3 ' RACE pcr amplification and purifying reclaim product, clone and order-checking; Obtain as the described sequence of SEQ ID NO:1 by sequential analysis.According to this sequences Design a pair of primer, can increase from the pig pituitary tissue cDNA one long is 1256bp, the PCR product of coding pig trh receptor albumen complete sequence, this product can be applicable to the animal genetic engineering field; The primer sequence is shown in SEQ ID NO:6 and SEQ ID NO:7:
Upstream primer: TTTCAGAGAAACCTCAAGCCACT
Downstream primer: TCTTTGTCATACATTTTCTTCTACTC
The pcr amplification condition is: 94 ℃ of pre-sex change 3min; 94 ℃ of 30s, 58 ℃ of 45s, 72 ℃ of 1min 30s, 40 circulations; 72 ℃ are extended 10min.
(2) according to the described cDNA sequence of sequence table SEQ ID NO:1, search for a plurality of pig genome databases, screening pig DNA sequence fragment.Design primer then and carry out pcr amplification and sequencing analysis, conclusive evidence obtains as sequence table SEQ ID NO:2 and the described pig TRHR of SEQ ID NO:3 gene DNA sequence at last.The relevant primer sequence is shown in SEQ ID NO:8~SEQ ID NO:17:
F0:TTGGAAAGGGCTGTGAGGGTTTAG,
R0:AGGCTGTGATTGAACAAGAGGAG;
F1:TTGAGAGGAAAGGAGGCAGA,
R1:AGGCTGAAGCTGTGTTTGGT;
F2:GGGAGAGAACCACTGCGATA,
R2:ATGGATGTGAAAGCCCAGAC;
F3:ATCTGTCACCCCATCAAAGC,
R3:ATTCTGGTTTTGCCATCAGC;
F4:CACTTTTGGAGCCGTGAGTAAAC,
R4:GGAATTTCTGGGACATGAGATTG。
Describe the present invention in detail with embodiment with reference to the accompanying drawings below, it is more obvious that purpose of the present invention and effect will become.
Embodiment 1:
(1) diagnostic method of the described T442-C442 base mutation of setting up based on Tetra primers ARMS-PCR principle of detection sequence table SEQ ID NO:2.
Gather generation and survey individual blood sample or all extraction of ear genome, genomic dna adopts traditional phenol/chloroform extraction method to extract, and after UV spectrophotometer measuring DNA purity and concentration, is diluted to 10ng/ μ l with sterile purified water.By the online design of primers program of tetra primers ARMS-PCR (
Http:// cedar.genetics.soton.ac.uk/public html/primer1.html) design sudden change serotype specific primer.The serotype specific primer of T442-C442 base mutation is shown in SEQ ID NO:18~SEQ ID NO:21:
Upstream inner primer: GATAGCAGATATTGTTTAGGTTTTTTCAAT
Downstream inner primer: AATGCTCCCTGTTTTGAGAGTGCTAAG
Upstream outer primer: GAAATTGTTCTCTGTTGGGTCTGTAAG
Downstream outer primer: AGTAATTGGGTTCATAGGTACTCCTGAA
The pcr amplification program is: 94 ℃ of pre-sex change 3min; 94 ℃ of 30s, 60 ℃ of 1min, 72 ℃ of 1min, 35 circulations; 72 ℃ are extended 10min.Amplification system is 10 μ l, template DNA 20ng wherein, rTaq 0.4U (Takara company), 10mM dNTPs 0.2 μ l, 10 * Buffer, 1.0 μ l, the upstream outer primer 0.08 μ l of 10 μ M, downstream outer primer 0.12 μ l, upstream inner primer 0.4 μ l, downstream inner primer 0.4 μ l.The PCR product detects with 2% sepharose.
The result is as shown in Figure 2: not mutated specific band is 443bp, promptly no matter which kind of mutator gene type individuality is, all can obtain the band of 443bp; The special product of allelotrope T is 200bp, if promptly there is the T442 sudden change, the band of 200bp can occur; The special product of allele C is 300bp, if promptly there is the C442 sudden change, the band of 300bp can occur.If individuality is T442 and C442 heterozygote, then can obtain 200bp and 300bp band simultaneously; If its corresponding sudden change band then only appears in homozygote.
(2) diagnostic method of the described G626-T626 base mutation of setting up based on Tetra primers ARMS-PCR principle of detection sequence table SEQ ID NO:2.
Extracting genome DNA and primer design method are the same.Shown in the serotype specific primer SEQ ID NO:22~SEQ ID NO:25 of G626-T626 base mutation:
Upstream inner primer: ACCCAATTACTGCAGATAAATGGAAT
Downstream inner primer: GTAACTCTCACATCCTCTCTTTTCGTC
Upstream outer primer: CTTCTTTATTGTACTTTTCCCAAGCATC
Downstream outer primer: TCACTTACTGTCTCGTTTTCCATCTTTA
The pcr amplification program is: 94 ℃ of pre-sex change 3min; 94 ℃ of 30s, 60 ℃ of 1min, 72 ℃ of 1min, 35 circulations; 72 ℃ are extended 10min.Amplification system is 10 μ l, template DNA 20ng wherein, rTaq 0.4U (Takara company), 10mM dNTPs 0.2 μ l, 10 * Buffer, 1.0 μ l, the upstream outer primer 0.08 μ l of 10 μ M, downstream outer primer 0.08 μ l, upstream inner primer 0.8 μ l, downstream inner primer 0.4 μ l.The PCR product detects with 2% sepharose.
The result is as shown in Figure 3: not mutated specific band is 460bp, promptly no matter which kind of mutator gene type individuality is, all can obtain the band of 460bp; The special product of allelotrope G is 296bp, if promptly there is the G626 sudden change, the band of 296bp can occur.The special product of allelotrope T is 217bp, if promptly there is the T626 sudden change, the band of 217bp can occur.If individuality is G626 and T626 heterozygote, then can obtain 296bp and 217bp band simultaneously; If its corresponding sudden change band then only appears in homozygote.
Embodiment 2:
The distribution of pig TRHR gene pleiomorphism in different swinerys: 4 external pig kinds (Pietrain, long white, Du Luoke, Yorkshire) and 2 Chinese Pigs kinds (pig is deceived in Jinhua pig and Jiaxing) in totally 228 pigs employing embodiment 1 described sudden change detection technique carry out the SNP somatotype.Detected result is as shown in table 1.T442-C442 is in the several pig kinds that detected, and the black pig in external swinery and Jiaxing is T allelotrope and preponderates, and preponderates with C allelotrope in the pig of Jinhua.G626-T626 is in the several pig kinds that detected, and the Chinese Pigs kind is bigger with external pig kind gene frequency difference, and external pig kind is preponderated for T allelotrope, and black pig in Chinese Pigs kind Jiaxing and Jinhua pig all are fixed as allelotrope G.
The genotype and the gene frequency of TRHR gene pleiomorphism in six pig kinds of table 1.
Embodiment 3:
The association analysis of the pig TRHR gene pleiomorphism mark and the production traits: the applicant has selected 354 Jinhua pigs * Pietrain pigs F2 generations as the association analysis test materials, the proterties of analysis comprises that day weight gain, trunk are long, nose heave, carcass weight, back leg is heavy, the back leg muscle is heavy, back leg fatty heavy, hoof is heavy, leaf fat is heavy, 4 thickness of backfats (shoulder thickness, 6-7 intercostal, aft rib and waist are recommended the junction), the average thickness of backfat, eye muscle area, be waterpower, intramuscular moisture content, intramuscular albumen, intramuscular fat and after pH value, electric conductivity and the temperature of leg muscle and eye muscle.The SNP somatotype adopts embodiment 1 described sudden change detection technique to carry out.Detected result adopts SAS statistical software (SAS Institute Inc, Version 9.0) GLM program to carry out single mark variance analysis, and the model that is adopted is:
Y
ijklmn=μ+F
i+D
j+S
k+M
1l+M
2m+M
1l*M
2m+β*X
ijklmn+e
ijklmn
Wherein, Y
IjklmnBe the proterties observed value, μ is a mean value, F
iThe male parent effect, D
jBe maternal effect, S
kBe sex effect, M
1lBe polymorphic seat T442-C442 genotype effect, M
2mBe polymorphic seat G626-T626 genotype effect, M
1l* M
2mBe two seat interactions, β * X
IjklmnExpression is a concomitant variable with live-weight before killing, e
IjklmnBe the residual error effect.
Genotype detection result shows: 354 Jinhua pig * Pietrain pigs F2 that detected for individuality in, the TT genotype of polymorphic seat T442-C442 accounts for 53.14%, the CC genotype accounts for 8%, the TC genotype accounts for 38.86%; The GG genotype of polymorphic seat G626-T626 accounts for 51.76%, and the TT genotype accounts for 27.06%, and the TG genotype accounts for 21.18%.The association analysis result shows that polymorphic seat T442-C442 is with back leg fat weight, leaf fat weight, the shoulder thickness of backfat, hoof is heavy and intramuscular moisture content remarkable related (P<0.05); And polymorphic seat G626-T626 and day weight gain, back leg muscle are heavy, fatty heavy, the back leg muscular temperature of back leg, hoof are heavy, all 4 thickness of backfats and the average thickness of backfat all have remarkable related (P<0.05).The effect of doing mutually at two seats has remarkable related (P<0.05) with fatty heavily, the back leg muscle pH value of back leg, eye muscle pH value and carcass weight.Pig TRHR gene polymorphic sudden change among presentation of results the present invention can be used as the molecule assisted Selection mark of the above production traits.
Claims (5)
1. a porcine thyrotropin-releasing hormone receptor (TRHR) gene is characterized in that, its cDNA sequence is the sequence shown in the SEQ ID NO:1.
2. cDNA sequence according to claim 1 is characterized in that, this cDNA sequence length is 1469bp, comprises the open reading frame of 1198bp in the sequence, the 5 ' non-translational region of 233bp and the 3 ' non-translational region of 38bp.
3. a porcine thyrotropin-releasing hormone receptor (TRHR) gene is characterized in that, it has the gene order shown in SEQ IDNO:2 and the SEQ ID NO:3.
4. dna sequence dna according to claim 3 is characterized in that: there is a T442-C442 base mutation at the 442bp place of sequence table SEQ ID NO:2, and there is a G626-T626 base mutation at the 626bp place.
5. the application of the described gene TRHR of claim 1-6 in pig molecule mark assisted Selection and animal genetic engineering.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 201010220407 CN101914544A (en) | 2010-07-06 | 2010-07-06 | Clone of porcine thyrotropin-releasing hormone receptor (TRHR) gene and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 201010220407 CN101914544A (en) | 2010-07-06 | 2010-07-06 | Clone of porcine thyrotropin-releasing hormone receptor (TRHR) gene and application thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CN101914544A true CN101914544A (en) | 2010-12-15 |
Family
ID=43322185
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN 201010220407 Pending CN101914544A (en) | 2010-07-06 | 2010-07-06 | Clone of porcine thyrotropin-releasing hormone receptor (TRHR) gene and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN101914544A (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5288621A (en) * | 1990-12-14 | 1994-02-22 | Cornell Research Foundation, Inc. | Pituitary TRH receptor |
-
2010
- 2010-07-06 CN CN 201010220407 patent/CN101914544A/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5288621A (en) * | 1990-12-14 | 1994-02-22 | Cornell Research Foundation, Inc. | Pituitary TRH receptor |
Non-Patent Citations (1)
Title |
---|
《GenBank》 20100330 Jiang,X.和Xu,N. GenBank登录号:FJ859911.1 , 2 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Amills et al. | Identification of three single nucleotide polymorphisms in the chicken insulin-like growth factor 1 and 2 genes and their associations with growth and feeding traits | |
Sacchi et al. | Casein haplotype structure in five Italian goat breeds | |
CN106498052B (en) | Molecular labeling, detection method and its application of one influence goat early growth | |
CN109371143B (en) | SNP molecular marker associated with pig growth traits | |
Kai et al. | Effect of genetic variations of the POU1F1 gene on growth traits of Nanyang cattle | |
CN109371144B (en) | SNP molecular marker associated with pig growth traits | |
US20030219819A1 (en) | Method for improving efficiencies in livestock production | |
Dvořáková et al. | Effect of the missense mutation Asp298Asn in MC4R on growth and fatness traits in commercial pig crosses in the Czech Republic. | |
Lee et al. | Novel single nucleotide polymorphisms of bovine SREBP1 gene is association with fatty acid composition and marbling score in commercial Korean cattle (Hanwoo) | |
CN111926086B (en) | Molecular marker influencing oblique growth of chicken body and application thereof | |
Li et al. | A further survey of the quantitative trait loci affecting swine body size and carcass traits in five related pig populations | |
CN112195253A (en) | SNP (Single nucleotide polymorphism) locus for increasing content of fatty acid C14:0 in chicken and method for breeding high-quality chicken strain by using SNP locus | |
CN101113470B (en) | SLC39A7 gene as genetic label of pig fat deposition description and application thereof | |
Purwantini et al. | Prolactin gene polymorphisms and associations with reproductive traits in Indonesian local ducks | |
Yuan et al. | Genetic diversity and population structure of Tongcheng pigs in China using whole-genome SNP chip | |
Wang et al. | Molecular cloning, tissue distribution and ontogenetic expression of the amino acid transporter b0,+ cDNA in the small intestine of Tibetan suckling piglets | |
Kurdistani et al. | Evaluation of insulin-like growth factor-I gene polymorphism on growth traits and yearling fleece weight in goats | |
CN104087582A (en) | Pig fat deposition character SNP genetic marker and application thereof | |
CN105200146A (en) | SNP genetic marker correlated to pig fat deposition property and application | |
Li et al. | Evaluation of coat color inheritance and production performance for crossbreed from Chinese indigenous Chenghua pig crossbred with Berkshire | |
CN111477272A (en) | Method for assisting in selecting high-yield-litter-size rex rabbits by using SNPs (single nucleotide polymorphisms) | |
CN110438243A (en) | The relevant FABP4 gene SNP molecular labeling of Yan-Bian yellow cattle meat, primer pair, kit and its application | |
Li et al. | Deletion of TTTTA in 5′ UTR of goat MSTN gene and its distribution in different population groups and genetic effect on bodyweight at different ages | |
CN103320429A (en) | Method for detecting Qinchuan cattle Wnt7a gene single nucleotide polymorphism, and application thereof | |
CN103849618B (en) | The SNP marker relevant to hog on hook and Meat Quality and application |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20101215 |