CN107385061A - One gene mutation site associated with sheep reproductive trait and its application - Google Patents
One gene mutation site associated with sheep reproductive trait and its application Download PDFInfo
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Abstract
The invention discloses a gene mutation site associated with sheep reproductive trait and its application, the mutational site is located at 414, area of sheep FSHR gene core promoters nucleotides, and the site is A>G is mutated, and is significantly associated with sheep litter size, wherein AA types and AG type individual litter sizes are significantly higher than GG types, and AA type promoter region Gene Transcription in vitro is significantly higher than GG types.Livestock reproduction character is a low-heritability traits, it is slower using traditional breeding method genetic progress, because the mutational site significantly associates with sheep reproductive performance, the site is detected using the genotyping technique of simplicity, prolificacy genotype individuals can be selected and remain, low reproductive capacity genotype individuals are eliminated, there is important economic value to accelerating prolificacy sheep colony or strain breeding progress.
Description
Technical field
The invention belongs to biology field, be related to a gene mutation site associated with sheep reproductive trait and its
Using.
Background technology
Follicle-stimulating hormone acceptor (Follicle stimulating hormone receptor, FSHR) is that influence domestic animal is more
The major gene resistance of tire character, regulation and control gonad granulocyte state (propagation and apoptosis), follicular development and ovulation.FSHR is the female food in one's mouth
A kind of important receptor protein in newborn animal ovary Granulosa Cell Membrane, mediation follicle-stimulating hormone (Follicle stimulating
Hormone, FSH) function.FSH signal transductions must be by being combined and could be made with the acceptor FSHR on gonad granulocyte film
For ovary, so as to promote follicular development, regulation and control gamete to be formed, therefore necessary to FSHR is dam reproduction and reproductive performance.
Research find FSHR transcriptional levels and sheep reproductive capacity (such as litter size, number of eggs ovulated), mutational site polymorphism with
It is in close relations between sheep Fecundity traits.FSHR gene transcription levels are significantly higher than in prolificacy sheep individual folliculus ovarii
Low reproductive capacity individual.FSHR genes are in Sheep Ovary tissue different size (4-7mm) and different conditions (dominant follicle, inferior position ovum
Bubble and leuteinization ovarian follicle) the different expression of ovarian follicle mean deviation, illustrate FSHR gene expression doses and sheep follicular development, advantage and
Ovulate relevant.Meanwhile the high expression of FSHR is considered as the main machine that FecB mutation influence sheep prolificacy in ovary or ovarian follicle
System.FSHR gene 5'- control regions (such as g.-200G>A、g.-197G>A、g.-98T>C and g.-47C>) and code area SNP site T
(as c.1235T>C polymorphism) significantly associates with sheep Fecundity traits (such as litter size).In addition, pig follicle is ripe with ovulating
The expression of FSHR genes is relied on, 3 base mutations are (c.74C on its gene coding region>G、c.532G>A and c.1166T>C) significantly
Pig number of eggs ovulated is influenceed, FSHR is considered as the major gene resistance for influenceing pig reproductive trait.Ox FSHR genes base mutation is (as c.337C
>G、c.871A>G、c.1973C>G and G-278A) significantly associated with superfecundation character.These results absolutely prove FSHR genes
On mutational site and livestock reproduction character it is closely related, but at present not yet obtain One function and Sheep Reproductive Characters
The mutational site significantly associated.
The content of the invention
Significantly associated with sheep litter size the invention provides one and significantly affect the mutation position of Gene Transcription in vitro
Point, mutational site detection technique, and the method for screening prolificacy sheep individual.
Another object of the present invention is to provide above-mentioned mutational site in assist-breeding prolificacy sheep new lines
Using.
The purpose of the present invention can be achieved through the following technical solutions：
One mutational site for significantly being associated with sheep litter size and significantly affecting Gene Transcription in vitro, the mutational site
At -414, area of sheep FSHR gene core promoters nucleotides, the site is A>G is mutated, and is significantly closed with sheep litter size
Connection, wherein AA types and AG type individual litter sizes are significantly higher than GG types, and AA type promoter region Gene Transcription in vitro is significantly higher than GG
Type.The nucleotide sequence in described sheep FSHR gene core promoters area is as shown in SEQ ID NO.4.
ctcttaaaagtgccccctaccatctgtccaggggctcactaacccactgcctgtcttctgagctgcmccatgtttgg ttgttaattccagcaagaaagagaatcagtgactttgacccagaagttctggttttgtatcaagcagcctggaggaa gacattgacaccaagactggaaacaggtccctgaccttcactgaggaactctcttaatgatgtttcacactgcagat tgcatctgttttggagaaagtcaagcgtgtcactctgttttgagaaaaaaaaaatagtgacccacagggacagtctt acagcgaatttaatataagctattctagacatgcatcaagtttcaatttgcaaacccaaccaaaaaaggtaaaggga cagcgtatcttgccacgccctctacctctcccaccccacccccaccaaagtcactgctgtcactcagaaattctgct atttgtctggaagtgaccgataaaaaagaaaaaaaggaaagcggccctgggcgggtcacgtgaccctaccagctccc
aatgcagacctcttctcaaaagggctcagtgtggagcctctgaaatctgggcaggattgtgtctgcagaagcagaag
caagcaggtggatggataagtaaacatg(SEQ ID NO.4).Wherein, m is mutational site, and the mutational site is located at lower stroke
LineaccRise at -414 nucleotides,atgFor initiation codon.
A kind of to be used to detect the specific primer pair that whether there is said gene mutational site in sheep genome, its is positive and negative
To primer as shown in SEQ ID NO.1 and SEQ ID NO.2.
It is a kind of to be used to detect the method that whether there is said gene mutational site in sheep genome, utilize the special of design
Property primer pair SEQ ID NO.1 and SEQ ID NO.2 enter performing PCR amplification, direct Sequencing is carried out to amplified production, identifies different bases
Because of type.
A kind of method for screening prolificacy sheep individual, using specific primer to SEQ ID NO.1 and SEQ ID
NO.2 enters performing PCR amplification to sheep genomic DNA, to pcr amplification product sequencing and typing, selects and remain AA types and AG types individual as high
Reproductive capacity sheep individual.
Application of the above-mentioned gene mutation site in the assisted selection of prolificacy sheep new lines.
Above-mentioned specific primer is to the application in the mutational site described in test right requirement 1.
Above-mentioned specific primer is to the application in the assisted selection of prolificacy sheep new lines.
Beneficial effects of the present invention：
The invention provides upper one of sheep FSHR gene core promoters area work(that is new, significantly being associated with reproductive trait
Energy property mutational site and its detection technique, assisted Selection and prolificacy sheep new lines available for prolificacy sheep individual
Assistant breeding.
Brief description of the drawings
Fig. 1 sheep FSHR genes -414A>G sites sequencing and typing peak figure.
Fig. 2 sheep FSHR genes -414A>G sites different genotype litter size.
Fig. 3 sheep FSHR genes -414A>G sites different genotype promoter region activity.
Embodiment
With reference to example, the present invention is further illustrated.
Embodiment one
1st, experiment material
The sheep ewe that selection health is disease-free, body condition is good, records its litter size.Collection ear tissue sample is clamped with overbit, is put
In -20 DEG C of preservations, the extraction for genomic DNA.
2nd, extracting genome DNA
Using phenol/chloroform extraction method extraction sheep ear tissue genomic DNA of routine, -20 DEG C of preservations.
3rd, design of primers
According to ovine genome sequence (GenBank ID：NC_019460.1), using Primer5.0 Software for Design 1 to drawing
Thing, forward primer sequence are：5'-CGG GGT ACC CCA AAA GCA AGG GCA ATC-3'(SEQ ID NO.1), reversely
Primer sequence is：5'-CCC AAG CTT CCT GTT TCC AGT CTT GGT GTC-3'(SEQ ID NO.2).Primer by
Shanghai Ying Jun Bioisystech Co., Ltd synthesizes.
4th, PCR is expanded
PCR amplification system is 10 μ L, is mainly included：0.5 μ L sheep genomic DNAs, 0.5 μM of positive trip primer, 0.5 μM anti-
To primer, 5 μ L 2 × Premix rTaq archaeal dna polymerases (TaKaRa companies), 10 μ L are settled to distilled water.PCR expands journey
Sequence is：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 32 circulations, last 72 DEG C extend
5min.The PCR instrument that the present invention uses is TaKaRa grads PCR instrument.
5th, sequencing and typing
Pcr amplification product after purification, directly delivers Shanghai English using purification kit (Beijing Tiangeng biology Co., Ltd)
Fine horse Bioisystech Co., Ltd is sequenced, and the series of pcr amplification product is as shown in SEQ ID NO.3.Peak figure is sequenced to use
Chromas v2.3 softwares are analyzed, and judge genotype according to peak figure, wherein only unimodal A for AA types, only unimodal G's
For GG types, and bimodal is then AG types (accompanying drawing 1).
6th, association analysis
It is as follows using two Factor Models, structure Least square analysis model according to genotype effects and parity effect：
Y=μ+Gi+Pj+eij
Wherein：Y is character observation value；μ is colony's average；Gi is genotype effects；Pj is parity effect；Eij is random
Residual error effect.
It whether there is significant difference using litter size between SPSS v18.0 software analysis different genotype sheep individuals, tie
Fruit is represented using average ± standard error.As a result find AA types individual between AG type individuals litter size (be respectively 2.05 ±
0.10 and 2.03 ± 0.19) without significant difference, but GG types individual (1.54 ± 0.21) is all remarkably higher than, illustrate FSHR bases
Because of -414A>Significantly associated between G site mutations and sheep litter size, AA type promoter region Gene Transcription in vitro is significantly higher than GG
Type (such as Fig. 2 and 3).
Livestock reproduction character is a low-heritability traits, slower using traditional breeding method genetic progress, due to this hair
The bright mutational site is significantly associated with sheep reproductive performance, and the site, Bian Kexuan are detected using the genotyping technique of simplicity
Prolificacy genotype individuals are stayed, eliminate low reproductive capacity genotype individuals, to accelerating prolificacy sheep colony or strain breeding
Progress has important economic value.
<110>Agricultural University Of Nanjing
<120>One gene mutation site associated with sheep reproductive trait and its application
<160> 4
<210> 1
<211> 27
<212> DNA
<213>Artificial sequence
<220>
<223>Forward primer sequence
<400> 1
cggggtaccc caaaagcaag ggcaatc 27
<210> 2
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<223>Forward primer sequence
<400> 2
cccaagcttc ctgtttccag tcttggtgtc 30
<210> 3
<211> 353
<212> DNA
<213>Artificial sequence
<220>
<223>Pcr amplification product sequence
<400> 3
cggggtaccc caaaaggcaa gggcaatctt cagagactgg aaatgagaga agccagaaag 60
gacatgtgac agagggtggg atagttggtg tctactaagc ccagtgaaaa cccagcccga 120
cctgttggtc actcagctga gtcagttatc tctgtagact actctcttaa aagtgccccc 180
taccatctgt ccaggggctc actaacccac tgcctgtctt ctgagctgca ccatgtttgg 240
ttgttaattc cagcaagaaa gagaatcagt gactttgacc cagaagttct ggttttgtat 300
caagcagcct ggaggaagac attgacacca agactggaaa caggaagctt ggg 353
<210> 4
<211> 644
<212> DNA
<213>Sheep
<220>
<223>The nucleotide sequence in sheep FSHR gene core promoters area
<400> 4
ctcttaaaag tgccccctac catctgtcca ggggctcact aacccactgc ctgtcttctg 60
agctgcmcca tgtttggttg ttaattccag caagaaagag aatcagtgac tttgacccag 120
aagttctggt tttgtatcaa gcagcctgga ggaagacatt gacaccaaga ctggaaacag 180
gtccctgacc ttcactgagg aactctctta atgatgtttc acactgcaga ttgcatctgt 240
tttggagaaa gtcaagcgtg tcactctgtt ttgagaaaaa aaaaatagtg acccacaggg 300
acagtcttac agcgaattta atataagcta ttctagacat gcatcaagtt tcaatttgca 360
aacccaacca aaaaaggtaa agggacagcg tatcttgcca cgccctctac ctctcccacc 420
ccacccccac caaagtcact gctgtcactc agaaattctg ctatttgtct ggaagtgacc 480
gataaaaaag aaaaaaagga aagcggccct gggcgggtca cgtgacccta ccagctccca 540
atgcagacct cttctcaaaa gggctcagtg tggagcctct gaaatctggg caggattgtg 600
tctgcagaag cagaagcaag caggtggatg gataagtaaa catg 644
Claims (7)
- A 1. mutational site for significantly being associated with sheep litter size and significantly affecting Gene Transcription in vitro, it is characterised in that：Should Mutational site is located at -414, area of sheep FSHR gene core promoters nucleotides, and the site is A>G is mutated, with sheep lambing Digital display writes association, and wherein AA types and AG type individual litter sizes are significantly higher than GG types, and AA type promoter region Gene Transcription in vitro shows Work is higher than GG types.
- 2. a kind of be used to detect the specific primer pair that whether there is mutational site described in claim 1 in sheep genome, its It is characterised by：Its forward and reverse primer is as shown in SEQ ID NO.1 and SEQ ID NO.2.
- 3. a kind of be used to detect the method that whether there is mutational site described in claim 1 in sheep genome, its feature exists In：Enter performing PCR amplification to SEQ ID NO.1 and SEQ ID NO.2 using the specific primer of design, amplified production is carried out straight Sequencing is connect, identifies different genotype.
- A kind of 4. method for screening prolificacy sheep individual, it is characterised in that：Using specific primer to SEQ ID NO.1 and SEQ ID NO.2 enter performing PCR amplification to sheep genomic DNA, to pcr amplification product sequencing and typing, select and remain AA types and AG types Body is as prolificacy sheep individual.
- 5. application of the gene mutation site in the assisted selection of prolificacy sheep new lines described in claim 1.
- 6. the specific primer described in claim 2 is to the application in the mutational site described in test right requirement 1.
- 7. the specific primer described in claim 2 is to the application in the assisted selection of prolificacy sheep new lines.
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Cited By (4)
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| CN108753985A (en) * | 2018-04-27 | 2018-11-06 | 湖北多羔生物育种科技有限公司 | It is a kind of improve sheep the first-born litter size genetic marker and its application |
| CN109207602A (en) * | 2018-08-14 | 2019-01-15 | 南京农业大学 | One for eliminating gene mutation site and its application of low reproductive capacity sheep |
| CN109337987A (en) * | 2018-10-18 | 2019-02-15 | 广西大学 | Molecular labeling relevant to Nubia goat yeaning traits and combinations thereof application |
| CN109735633A (en) * | 2019-02-20 | 2019-05-10 | 新疆农业大学 | The detection method and its application of FSHR gene specific SNP marker, the black sheep litter size character in Turfan |
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| CN108753985A (en) * | 2018-04-27 | 2018-11-06 | 湖北多羔生物育种科技有限公司 | It is a kind of improve sheep the first-born litter size genetic marker and its application |
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| CN109337987A (en) * | 2018-10-18 | 2019-02-15 | 广西大学 | Molecular labeling relevant to Nubia goat yeaning traits and combinations thereof application |
| CN109735633A (en) * | 2019-02-20 | 2019-05-10 | 新疆农业大学 | The detection method and its application of FSHR gene specific SNP marker, the black sheep litter size character in Turfan |
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Application publication date: 20171124 |