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
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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.
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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 |
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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 |
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| Title |
|---|
| 《GenBank》 20100330 Jiang,X.和Xu,N. GenBank登录号：FJ859911.1 , 2 * |
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Open date: 20101215 |