CN109897904A - Molecular labeling and application based on PRLR identified for genes Large White reproductive trait - Google Patents
Molecular labeling and application based on PRLR identified for genes Large White reproductive trait Download PDFInfo
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Abstract
The invention discloses a kind of molecular labeling based on PRLR identified for genes Large White reproductive trait and applications.The molecular labeling is located on the 10th exon of pig PRLR gene, i.e., at 260,362,527,540,584,673,745,765,934bp, it is denoted as g.260C > G, g.362C > T, g.527C > G, g.540A > G, g.584A > G, g.673A > T, g.745A > G, g.765C > T and g.934A > G.The invention also discloses application of the above-mentioned molecular labeling in pig kind breeding.The present invention provides a kind of new labeling method for the molecular marker assisted selection of pig, provides reference for the molecular breeding of pig.
Description
Technical field
The invention belongs to animal molecular marker breeding technical fields, specifically, being related to a kind of based on PRLR identified for genes
The molecular labeling of Large White reproductive trait and application.
Background technique
Pork is one of most important meat economic animal in the world today, and China consumes big country as pork, and pig is deposited
Column amount ranks first in the world.To improve productivity effect, the reproductive performance of pig be always the concern of major breeding enterprise important indicator it
One, wherein the total yield coefficient of pig and litter size living are particularly critical.
Molecular marker assisted selection is effectively prevented from tradition as one of modern domestic animal breeding programs important means
The drawbacks of breeding technique based on phenotype, shorten genetic interval phase, the selection accuracy of raising.Molecular labeling auxiliary choosing
The premise selected is to determine relevant to required phenotypic character candidate gene, purposefully these genes are detected so that it is determined that
Breeding direction.
Prolactin (Prolactin, PRL) is a kind of proteohormone secreted by anterior pituitary gland acidophil, with tire
Disk hormone, growth hormone belong to a family.PRL has extensive physiologic sexual function for animal body, is mainly manifested in: maintaining
The development of breast duct and acinar cells, influences mating behavior, provides good microenvironment for gestation, influence gestation maintenance with
The development of fetus promotes parent to generate maternal behavior, follows further for intake amino acid, synthesis milk and immune system, blood
Loop system etc. all has important influence.
Prolactin receptor gene (Prolactin receptor gene, PRLR) is a kind of albumen secreted by adenohypophysis
Matter parahormone belongs to cell factor/Growth Hormone/Prolactin receptor superfamily, by intracellular region, extracellular regions and trans-membrane region
It constitutes, wherein the variation of Intracellular domain results in the diversity of receptor structure.There are two kinds of receptors in mammal: elongated
And short.Studies have shown that the PRLR assignment of genes gene mapping of pig on No. 16 chromosomes, includes 10 exons and 9 intrones, wherein
Contain compared with multimutation in 10 exon regions.
Prolactin will exercise its function, it is necessary to combine with its receptor, act on target cell by receptor.Prolactin
(PLR) it is combined with hprl receptor (PRLR), is divided into extracellular regions activation and intracellular region activates two processes.It is extracellular to activate
Journey is exactly the process of dimerization, and two PRLR are integrated to the two basic change site of a PRL up, and it is compound to form a trimerization
Body；Activation process intracellular is exactly activation and the Phosphorylation events of PRLR of tyrosine kinase, final that PRLR is activated to enter transduction way
Diameter.The region SH2 of the tyrosine residue of phosphorylation and signal transduction and activating transcription factor forms new polymer, polymer into
Enter nucleus, to activate promoter, regulate and control the expression of target gene, PRL is promoted to play biological function.Such as stimulation mammary gland is sent out
It educates, promotes the generation and secretion of milk；Prolactin also can be done directly on the granular cell of ovary, inhibit the fragrance of granular cell
Enzymatic activity reduces synthesis estradiol, inhibits ovulation.Meanwhile prolactin also has the function of molten corpus luteum.Many data are shown, are urged
Newborn element can also influence animal reproduction, adjust immune, maintenance water salt and the balance of osmotic pressure etc..
Summary of the invention
In view of this, the present invention provides a kind of molecular labeling based on PRLR identified for genes Large White reproductive trait and answering
With.
In order to solve the above-mentioned technical problem, the invention discloses a kind of based on PRLR identified for genes Large White reproductive trait
Molecular labeling, the molecular labeling are located on the 10th exon of pig PRLR gene, i.e., 260,362,527,540,584,
673,745,765, at 934bp, be denoted as g.260C > G, g.362C > T, g.527C > G, g.540A > G, g.584A > G, g.673A >
T, g.745A > G, g.765C > T and g.934A > G.
The invention also discloses a kind of for detecting the primer pair of above-mentioned molecular labeling, including PRLR-E10-F and
PRLR-E10-R, nucleotide sequence is respectively as shown in SEQ ID NO.2 and SEQ ID NO.3.
The invention also discloses a kind of reagent or kit containing above-mentioned primer pair.
The invention also discloses a kind of methods for detecting above-mentioned molecular labeling, which comprises with Large White gene
Group DNA is that template utilizes above-mentioned primer pair progress PCR amplification acquisition PCR product；Ago-Gel is carried out to the PCR product
Electrophoresis.
Optionally, pcr amplification reaction system are as follows: 2 × Taq Master Mix, 15 1.5 μ L of μ L, DNA, 10 μM of upstreams are drawn
Object 1.5 μ L, 10 μM of downstream primers 1.5 μ L, ddH2O 10.5μL。
Optionally, pcr amplification reaction program: 94 DEG C of initial denaturation 1.5min；94 DEG C of denaturation 20s, 50-60 DEG C of annealing 20s, 72
DEG C extend 1Kb/60s, recycle 30 times；Last 72 DEG C re-extend 5min, 4 DEG C of preservations.
The invention also discloses a kind of application of above-mentioned molecular labeling in pig kind breeding.
Compared with prior art, the present invention can be obtained including following technical effect:
It is a discovery of the invention that co-exist in 9 mutational sites on the 10th exon of PRLR gene, this 9 mutational sites with
There are correlations for the reproductive trait of pig especially total yield coefficient and litter size living, and are easy detection.Therefore the present invention provides one
A molecular labeling relevant to the reproductive trait of pig based on PRLR gene, and provide the detection method of this molecular labeling
And its application in reproductive trait selection, a kind of new labeling method is provided for the molecular marker assisted selection of pig, is
The molecular breeding of pig provides reference.
Certainly, it implements any of the products of the present invention it is not absolutely required to while reaching all the above technical effect.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes a part of the invention, this hair
Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is the 10 exon region mutational site C260G sequencer map of pig PRLR gene of the present invention；
Fig. 2 is the 10 exon region mutational site C362T sequencer map of pig PRLR gene of the present invention；
Fig. 3 is the 10 exon region mutational site C527G sequencer map of pig PRLR gene of the present invention；
Fig. 4 is the 10 exon region mutational site A540G sequencer map of pig PRLR gene of the present invention；
Fig. 5 is the 10 exon region mutational site A584G sequencer map of pig PRLR gene of the present invention；
Fig. 6 is the 10 exon region mutational site A673T sequencer map of pig PRLR gene of the present invention；
Fig. 7 is the 10 exon region mutational site A745G sequencer map of pig PRLR gene of the present invention；
Fig. 8 is the 10 exon region mutational site C765T sequencer map of pig PRLR gene of the present invention；
Fig. 9 is the 10 exon region mutational site A934G sequencer map of pig PRLR gene of the present invention；
Figure 10 is 10 exon pcr amplification product gel electrophoresis figure of pig PRLR gene of the present invention；Wherein, the hole 1-8 with
And the hole 10-17 is the 10 exon region amplified production of PRLR gene of Large White, clip size 1251bp, the 9th hole
For 2000DNA marker；
Figure 11 is the pig PRLR gene exon10 nucleotide sequence that invention expands, clip size
For 1251bp, dash area is intron sequences in figure.Shown by box is base mutation, and underscore part is primer sequence
Column.
Specific embodiment
Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, whereby to the present invention how application technology hand
Section solves technical problem and reaches the realization process of technical effect to fully understand and implement.
Embodiment 1: the acquisition of pig PRLR genetic fragment and SNPs detection
PRLR gene exon10 core relevant to pig reproductive trait of the present invention as molecular labeling application
Nucleotide sequence, nucleotide sequence as shown in SEQ ID NO.1, wherein 260bp, 362bp of sequence, 527bp, 540bp,
There are gene mutations at 584bp, 673bp, 745bp, 765bp, 934bp.The above SNPs and pig reproductive trait (total yield coefficient,
Litter size living) there is certain correlation, and be easy to detect, it is assisted using the above site as molecule relevant to pig reproductive trait
Label selection.
201 different lines (U.S. system, pellet are plus are) Large White is collected from Sichuan Province Leshan Tian Ren agriculture and animal husbandry farm
Coat sample and the breeding record of production (primiparity and total yield coefficient and litter size living through producing), and 201 are extracted with kit
The complete genome DNA of Large White.
According to the PRLR gene nucleotide series (HM209402.1) announced in GenBank database, PRLR is compared online
10 exon region in gene carries out specific primer design to three above region using Primer5.0 software and (is detailed in table
1), and by template of genomic DNA pcr amplification reaction is carried out.Reaction system are as follows: 2 × Taq Master Mix 15 μ L, DNA
1.5 μ L, 1.5 μ L of upstream primer (10 μM), downstream primer (10 μM) 1.5 μ L, ddH2O 10.5μL.Response procedures: 94 DEG C of pre- changes
Property 1.5min；94 DEG C of denaturation 20s, 50-60 DEG C of annealing 20s, 72 DEG C of extension 1Kb/60s are recycled 30 times；Last 72 DEG C re-extend
5min.4 DEG C of preservation amplified productions.
1 PRLR gene primer information table of table
It takes 4 μ L to carry out agarose gel electrophoresis in every pipe PCR product, send the PCR product containing single band according to after glue
To Chengdu, Qing Ke biotech firm is sequenced.Sequencing result is compared respectively using BioEdit software, finds purpose piece
Section present in mutational site, and record it is each individual present in corresponding site (C260G, C362T, C527G, A540G,
A584G, A673T, A745G, C765T, A934G) mutation type (mutational site corresponds to sequencer map as shown in figs 1-9).PRLR
The information such as 10 exon nucleotide sequence of gene, corresponding primer and mutational site difference is as shown in figure 11.
In 201 detected Large Whites, 9 mutational site genes of PRLR gene and genotype frequency are shown in Table 2.
The genotype frequency and gene frequency in 9 sites SNPs in 2 PRLR gene of table
As shown in Table 2,9 mutational sites of PRLR gene show as three kinds of genotype respectively in 201 Large Whites.Its
The genotype frequency of the middle site C260G and the site C527G in each strain Large White is all the same, and the two sites are big in pellet system
CC genotype is all only shown as in white pig, in U.S. system and plus is that CC > CG > GG trend is then shown as in great Bai；The site C362T
Genotype frequency shows as CT > TT > CC trend in three kinds of strain Large Whites；The site A540G in pellet system Large White only
A kind of genotype i.e. AA type is shown as, in U.S. system and to add be that two kinds of genotype and AA > AG are shown as in great Bai；The site A584G exists
It is that AG > AA > GG trend is shown as in great Bai that pellet, which is and adds, is that AA > AG > GG is shown as in great Bai in U.S.A；The site A673T is in beauty
AA > AT > TT is shown as in system and pellet system great Bai, is to show as AT > AA > TT in great Bai adding；The site A745G is in pellet system and plus is
Two kinds of genotype and GG > AG are shown as in great Bai, are that GG > AG > AA is shown as in great Bai in U.S.A；CC, CT, the TT in the site C765T
Genotype frequency AA, AG, GG genotype frequency with the site A934G respectively, and in U.S. system and to add be in great Bai with CT and AG base
Because of type frequency highest, two sites in the big Bai Zhongjun of pellet system only there are two types of genotype, and CC > CT/AA > AG.
Embodiment 2: application of the molecular labeling in Large White reproductive trait
For the relationship established between ESR gene and Large White reproductive trait, chooses 201 and producing Large White (Leshan sichuan day
People's animal husbandry) it is used as experimental material, 201 Large White Reproduction records are acquired, and using the SNPs detection mentioned in embodiment 1
Mode detects experimental population.Using the GLM model in SAS8.0 software to existing in the PRLR gene of every Large White
9 sites SNPs and its being associated property of reproductive trait analysis, as a result indicated with LSM ± standard error.
To in PRLR gene there are the site SNPs and reproductive trait (primiparity and total yield coefficient and litter size living) through producing into
Row association analysis, using model are as follows: Yij=μ+Gi+Pj+eij, wherein YijFor reproductive trait record value, μ is group's mean value, GiFor
Genotype effects, PjFor parity effect, eijFor random residual effect.
Influence of the 3 PRLR gene different loci genotype of table to reproductive trait
As shown in Table 3, in 201 Large White groups, at the site C362T in 10 exon of PRLR gene, CC base
Because primiparity life birth son number of the type in U.S. system individual is significantly higher than TT genotype (p < 0.05), CT genotype is in pellet system individual
Primiparity life birth son's number is significantly higher than TT genotype (p < 0.05)；At the site A584G, primiparity of the AG genotype in red system's individual
Life birth son's number is significantly higher than AA genotype (p < 0.05)；At the site C765T, CC genotype is primiparity total yield in individual adding
Young number is significantly higher than CT genotype and TT genotype (p < 0.05)；At the site A934G, AA genotype is first in individual adding
It produces total yield coefficient and is significantly higher than AG genotype and GG genotype (p < 0.05)；Between other characters otherness it is not significant (P >
0.05)。
Embodiment 3: haplotype analysis
Haplotype is carried out to from 9 SNP in 10 exon of PRLR gene that detected in 201 Large White individuals
Analysis, obtains following result:
4 PRLR gene of table, 10 exon haplotype type and frequency
As shown in Table 4,9 mutational sites detected constitute 7 kinds of haplotypes, wherein highest three haplotypes of frequency
For H1, H2 and H3, respectively 0.417,0.228 and 0.195.
Above description has shown and described several preferred embodiments of invention, but as previously described, it should be understood that invention is not
It is confined to form disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations, modification
And environment, and can be carried out within that scope of the inventive concept describe herein by the above teachings or related fields of technology or knowledge
Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then it all should be in the appended power of invention
In the protection scope that benefit requires.
Sequence table
<110>Sichuan Agricultural University
<120>molecular labeling relevant to the reproductive trait of Large White based on PRLR gene and application
<130> 2019
<141> 2019-04-26
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1251
<212> DNA
<213>prolactin receptor gene (Prolactin receptor gene, PRLR)
<400> 1
taagtagtgg gatgttagga agccgacagc tgtcttgcct tgtgtcttta acagaaaggc 60
aagtccgaag agcttctgag cgccctggga tgccaagact tcccccccac ttccgactgc 120
gaggacctgc tggtggagtt cctggaggtg gatgacagcg aggaccagca gctgatgcca 180
gcccactcca aggaacaccc gagccaaggc cggaagccca cacacctgga tcccgacagc 240
gattccggcc ggggcagctg tgacagccct tcccttttgt ccgaaaagtg cgatgaaccg 300
cgggccaatc cccccaagtt tcacactccc gagggcatcg agaagccagg ggatcctgaa 360
acaaaccttc cccgtcccca ggaccctcag agcacaagcg tggaaagcaa gctgccctat 420
tttcacgcgg atggatccaa gtcttcgact tggcctttgc cgcagccccc gatcctgcac 480
gatccgggat cttcttacca caaccttgcc gacgtgggtc agctggtcct gggcaccgca 540
ggggccaccg ccgctttgct ggaaaaaacg gaccgacatg ctttcaaccc tccgaaaacc 600
accgagactg gcggggaagg aagggcaacc gagcagaagg agtcggagag cttccactcc 660
aagactgacc aaggcacagt gtggctgctg ccccaggaca aggggccctt tgtctcccct 720
aagcccatgg agtacgtgga gatacacaag gtcagcaaag atggagcact ggcgttgctc 780
ccaaaacagc aggagaacgg cgaccggccg gagaaggctg gcgcccctga aaccagcaag 840
gaatacgccc aggtgtcccg ggtgatggat aaccacatcc tggtgttagt gcaggatccg 900
cgagctcgaa acgtggctcc gtttgaagaa ccaaccaagg agaccccgcc atcccggccg 960
cagaatccag ctgcgaaaga cctggccggc ttcaccacgg ccccgggcca ctgcagacac 1020
ccgctgggtg ggctggatta cctcgatccc gcaggcttta tgcactcctt tcagtgagag 1080
cttggttcat gggatgatgg gttacaaggt ggggtttttt tcaggtcgca ctacgtgaaa 1140
tgcactctac cagagaaagc tcgaaaatgg ggttagaatg acactaccca aactcacagt 1200
tcactcctct tcatgctcca ttttcaacca gttgcctctt tctccacaac a 1251
<210> 2
<211> 21
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 2
taagtagtgg gatgttagga a 21
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
tgttgtggag aaagaggc 18
Claims (7)
1. a kind of molecular labeling based on PRLR identified for genes Large White reproductive trait, which is characterized in that the molecular labeling
On the 10th exon of pig PRLR gene, i.e., at 260,362,527,540,584,673,745,765,934bp, it is denoted as
G.260C > G, g.362C > T, g.527C > G, g.540A > G, g.584A > G, g.673A > T, g.745A > G, g.765C > T and
g.934A>G。
2. for detecting the primer pair of molecular labeling described in claim 1, which is characterized in that including PRLR-E10-F and
PRLR-E10-R, nucleotide sequence is respectively as shown in SEQ ID NO.2 and SEQ ID NO.3.
3. reagent or kit containing primer pair described in claim 2.
4. a kind of method for detecting molecular labeling described in claim 1, which is characterized in that the described method includes: with Large White
Genomic DNA is that template utilizes primer pair progress PCR amplification acquisition PCR product as claimed in claim 2；To the PCR product
Carry out agarose gel electrophoresis.
5. according to the method described in claim 4, it is characterized in that, pcr amplification reaction system are as follows: 2 × Taq Master Mix
15 μ L, DNA 1.5 μ L, 10 μM of upstream primers 1.5 μ L, 10 μM of downstream primers 1.5 μ L, ddH2O 10.5μL。
6. according to the method described in claim 4, it is characterized in that, pcr amplification reaction program: 94 DEG C of initial denaturation 1.5min；94
DEG C denaturation 20s, 50-60 DEG C of annealing 20s, 72 DEG C of extension 1Kb/60s, circulation 30 times；Last 72 DEG C re-extend 5min, 4 DEG C of preservations.
7. application of the molecular labeling described in claim 1 in pig kind breeding.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5935784A (en) * | 1996-07-19 | 1999-08-10 | Iowa State University Research Foundation, Inc. | Prolactin receptor gene as a genetic marker for increased litter size in pigs |
| TW201100551A (en) * | 2009-06-19 | 2011-01-01 | Univ Nat Taiwan | Methods for identifying reproduction performance of sows based on prolactin gene and applications thereof |
| CN105087820A (en) * | 2015-09-25 | 2015-11-25 | 江苏农林职业技术学院 | FSHR (follicle stimulating hormone receptor) gene based molecular marker related to porcine reproduction traits as well as detection method and application of molecular marker |
-
2019
- 2019-04-26 CN CN201910342803.8A patent/CN109897904B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5935784A (en) * | 1996-07-19 | 1999-08-10 | Iowa State University Research Foundation, Inc. | Prolactin receptor gene as a genetic marker for increased litter size in pigs |
| TW201100551A (en) * | 2009-06-19 | 2011-01-01 | Univ Nat Taiwan | Methods for identifying reproduction performance of sows based on prolactin gene and applications thereof |
| CN105087820A (en) * | 2015-09-25 | 2015-11-25 | 江苏农林职业技术学院 | FSHR (follicle stimulating hormone receptor) gene based molecular marker related to porcine reproduction traits as well as detection method and application of molecular marker |
Non-Patent Citations (3)
| Title |
|---|
| C DROGEMULLER等: "Candidate gene markers for litter size in different German pig lines", 《J ANIM SCI》 * |
| 曹果清等: "猪PRLR基因多态性及其与产仔性能的关联分析", 《畜牧兽医学报》 * |
| 陈耀闽: "3个猪种的PRLR和ESR基因多态性及其与繁殖性能的关联分析", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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