CN104878005B - Influence FAK gene molecule markers and its application of breeding oxen reproductive performance - Google Patents
Influence FAK gene molecule markers and its application of breeding oxen reproductive performance Download PDFInfo
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Abstract
The present invention discloses a kind of FAK gene molecule markers for influenceing breeding oxen reproductive performance and application, g.129462_129463insGAA molecular labeling is.By determining in breeding oxen genomic DNA FAK gene molecule markers g.129462_129463insGAA; according to the insertion mutation that tri- bases of GAA whether occur between the 129462nd and 129463 sites; predict the reproductive performance height of breeding oxen; when the insertion of the bases of GAA tri- does not occur between breeding oxen FAK genes the 129462nd and 129463 sites, breeding oxen reproductive performance is best.The present invention illustrates the FAK gene functions molecular labeling g.129462_129463insGAA correlation with breeding oxen reproductive performance first, there is provided the method for prediction breeding oxen reproductive performance, also provides relevant primer pair and the kit comprising the primer pair.
Description
Technical field
The invention belongs to domestic animal technical field of molecular biology, is related to the FAK that screening influences breeding oxen reproductive performance height
Gene function molecular labeling, authentication method and its kit.
Background technology
In modern milk cow population genetic improvement system, breeding oxen is the principal element for influenceing milk cows hereditary quality.Kind
The contribution rate that bull is in progress to milk cow population genetic is up to more than 70%, and the reproductive performance of breeding oxen is to influence it at utmost
An important factor for playing hereditary potential.Therefore, the breeding oxen with high reproductive performance how is screened, is the heat in cattle breeding field
Point and difficulties.The reproductive performance of breeding oxen is influenceed by many factors such as heredity, season, feeding management, temperature, at present milk
One outstanding breeding oxen of industry developed country can produce 5 ten thousand to 10 ten thousand doses of jelly essence every year, reach as high as 250,000 doses, and China only 20,000
Agent or so.Therefore, exploring influences the Genetic Mechanisms of breeding oxen reproductive performance, has to the high-quality breeding oxen of breeding high-yield extremely important
Meaning.The volume of semen of breeding oxen, fresh smart vigor, jelly essence solution Frozen semen activity, fresh precision and rate of teratosperm are to weigh kind of a public affairs
The important indicator of ox reproductive performance.Early stage improves cow reproduction performance generally use and introduces new breeds and improve the side such as raise competence
Method, and use the report of genetics research spermatogenesis-related gene progress assisted Selection seldom.With Protocols in Molecular Biology
Fast development, candidate gene approach (Candidate gene approach) plays an important role in Animal Genetics,
People can study the candidate gene for influenceing reproductive performance or the molecular labeling with its close linkage on DNA level, pass through mark
Remember assisted Selection (Marker assisted selected, MAS) breeding, reference is provided to cultivate high yield and high quality breeding oxen.
Focal adhesion kinase (Focal adhesion kinase, FAK) is a kind of cytosolic non-receptor protein tyrosine kinase
(PTKs) protein tyrosine kinase (Protein tyrosine kinase) superfamily, is belonged to, FAK is in cell signalling
Play a significant role, it is the maincenter that intracellular external signal comes in and goes out, and mediates more signal paths.FAK can receive from integrin,
The signal path such as the signal of growth factor and mechanical stimulus etc., activation intracellular PI3K/Akt, Ras/MAPK, regulation cell life
It is long.There are some researches show FAK also participates in the process such as the release of sperm and the capacitation of sperm.Smart ovum fertilization needs to complete a series of things
Part, be related to signal transduction pathway, participate in these approach a series of LCKs (PTKs) expression apparently higher than
Other cells.FAK is to be present in a kind of kinases in testis with the cell adhesion of beta 1 integrin mediation.SABC shows
Show, during essence is arranged, FAK is present in the last moment of sperm release, shows that FAK albumen plays important work in sperm deenergized period
With.FAK is almost expressed in all cells and organization type, but the high expression in testis and brain.Speculate that FAK genes may accordingly
It is a candidate gene for influenceing breeding oxen reproductive performance, its SNP (mutation) is to cause different breeding oxen individuals
Between reproductive performance produce the genetic base of difference, but it is numerous using its mutation with the relation of breeding oxen reproductive performance to detect breeding oxen
The kit for growing performance height has no report.
The content of the invention
For above-mentioned prior art, the purpose of the present invention is to propose to one kind to utilize FAK gene functions molecular labeling early stage
The method for screening the breeding oxen of high reproductive performance, and pass through the association analysis of this molecular labeling and breeding oxen reproductive performance, there is provided
A kind of kit for the breeding oxen that can screen high reproductive performance.The present invention provides new for the marker assisted selection of breeding oxen
Functional molecular marker, selection gist is provided for the early screening of breeding oxen.
To realize this purpose, one proposed by the present invention new breeding oxen FAK gene function molecular labeling
G.129462_129463insGAA, it is to detect milk cow from GenBank Accession No NC_007312.5
Whether sent out between the 129462nd and 129463 sites in (2124036..2316975, FAK gene start codon ATG conducts+1)
The insertion mutation of tri- bases of raw GAA, and gene of the breeding oxen in this site is differentiated using molecular biology correlation technique
Type.Show that the mutation is related with breeding oxen reproductive performance to the association analysis of breeding oxen reproductive performance using the mutation, selection has
The genotype individuals that the bases of GAA tri- are not inserted between 129462nd and 129463 sites of profit are reserved seed for planting.
To achieve the above object, the invention provides a kind of molecular labeling predicted or aid in prediction breeding oxen reproductive performance
G.129462_129463insGAA, the feature of the molecular labeling be breeding oxen FAK genes the 129462nd and 129463 sites it
Between whether there is occur tri- bases of GAA insertion mutation, wherein, nucleotide position numbering be based on GenBank Accession No
NC_007312.5 (2124036..2316975, FAK gene start codon ATG conducts+1).
To achieve the above object, present invention also offers it is a kind of predict or auxiliary prediction breeding oxen reproductive performance method,
Comprise the following steps：The blood or seminal fluid genomic DNA of breeding oxen are extracted, determines FAK genes the 129462nd and 129463 sites
Between the insertion mutations of tri- bases of GAA whether occurs, g.129462_129463insGAA kind of a public affairs are predicted according to its molecular labeling
The reproductive performance height of ox.
When the insertion of the bases of GAA tri- does not occur between breeding oxen FAK genes the 129462nd and 129463 sites, breeding oxen
Reproductive performance is best；When the insertion mutation that the bases of GAA tri- occur between breeding oxen FAK genes the 129462nd and 129463 sites
When, breeding oxen reproductive performance is worst.
G.129462_129463insGAA, present invention also offers a kind of FAK gene mutation sites to screen classifying method, bag
Include following steps：
(1) breeding oxen blood or seminal fluid are gathered, extracts blood or seminal fluid genomic DNA, genomic DNA dilution is standby；
(2) for being g.129462_129463insGAA mutated, breeding oxen FAK gene-specific primers is designed, enter performing PCR
Reaction, obtains the amplified production 1 of specific fragment, such as sequence table SEQ ID NO：Shown in 1；Described gene-specific primer has
Such as sequence table SEQ ID NO：2 and sequence table SEQ ID NO：Sequence shown in 3；
(3) detected using the method for pcr amplification product direct Sequencing in amplified production and whether there is following mutation：
g.129462_129463insGAA；
Wherein, nucleotide position numbering is based on GenBank call numbers：GenBank Accession No NC_007312.5
(2124036..2316975, FAK gene start codon ATG conducts+1).
In the step (1), the method for extracting genomic DNA is：Phenol/chloroform extraction process is respectively adopted and high salt method carries
Blood or seminal fluid genomic DNA are taken, it is standby that genomic DNA is diluted into 50ng/ μ L.
In the step (2), the PCR reaction conditions are：94 DEG C of denaturation 5min, 94 DEG C of denaturation 30s, 60 DEG C of annealing 30s,
72 DEG C of extension 30s, 35 circulations；72 DEG C of extension 10min, 4 DEG C of preservations；The described length of amplified production 1 is 631bp.
To achieve the above object, the present invention also provides detection and can predict or aid in predict the molecule of breeding oxen reproductive performance
The primer pair of mark, is made up of forward primer and reverse primer, and its nucleotide sequence is as follows：
Forward primer F：TTTGACTGACGGCTGCTTGG, as shown in sequence table SEQ ID NO.2；
Reverse primer R：AGTTTGGAGGTGCTCTGGTA, as shown in sequence table SEQ ID NO.3.
The primer pair specific amplification goes out the nucleotide sequence shown in the SEQ ID NO.1 of the gene containing FAK.
To achieve the above object, present invention also offers prediction or the kit of auxiliary prediction breeding oxen reproductive performance, bag
Include forward primer described in above-mentioned primer pair and direction primer.
The kit also includes Taq PCR MasterMix and ddH2O。
The kit is specially 100 ox detection dosage, and the composition of kit is as follows：100 μm of ol/L forward primer
50μL；
The 100 μm of ol/L μ L of reverse primer 50；
2×Taq PCR MasterMix 3mL；
ddH2O 3mL。
To achieve the above object, the invention provides following any applications：
(1) application of the above-mentioned molecular labeling in predicting or aiding in prediction breeding oxen reproductive performance, in actual applications,
Present invention determine that influence breeding oxen reproductive performance molecular labeling can be used as breeding oxen breeding assisted Selection mark.FAK bases
Because the reproductive performance of the breeding oxen individual for the genotype that the insertion of the bases of GAA tri- does not occur between the 129462nd and 129463 sites is high
In other genotype individuals.
(2) application of the above-mentioned molecular labeling in the breeding oxen of seed selection different reproductive performance；
(3) application of the above-mentioned primer pair in predicting or aiding in prediction breeding oxen reproductive performance；
(4) application of the above-mentioned primer pair in the kit for preparing prediction or auxiliary prediction breeding oxen reproductive performance；
(5) application of the above-mentioned primer pair in the breeding oxen of seed selection different reproductive performance；
(6) application of the above-mentioned kit in predicting or aiding in prediction breeding oxen reproductive performance；
(7) application of the above-mentioned kit in the breeding oxen of seed selection different reproductive performance；
(8) application of the above-mentioned method in predicting or aiding in prediction breeding oxen reproductive performance；
(9) application of the above-mentioned method in the breeding oxen of seed selection different reproductive performance.
The positive effect of the present invention is：
(1) insertion mutation of tri- bases of GAA is whether there is between breeding oxen FAK genes the 129462nd and 129463 sites, this
Mutation is through international genome database and document patent retrieval, it was demonstrated that is new mutational site.
(2) through above-mentioned molecular labeling is g.129462_129463insGAA individual with 218 Chinese Holstein breeding oxens
Reproductive performance carry out correlation analysis, do not occur between FAK genes the 129462nd and 129463 sites the bases of GAA tri- insertion
The reproductive performance of the breeding oxen individual of genotype is higher than other genotype individuals.It can be used for the early screening of breeding oxen, so as to
Lower feeding cost, increase value-added content of product.
(3) kit invented can be used for early detection to improve detection efficiency, reduce testing cost.
Brief description of the drawings
Fig. 1：FAK gene molecule markers g.129462_129463insGAA amplified production sequencing and typing result.
Fig. 2：Cell transient transfection experimental verification is g.129462_129463insGAA to bta-miR-21 and breeding oxen FAK
The influence that gene 3'UTR areas combine.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part or according to the condition proposed by manufacturer.
The present invention conducts in-depth research for breeding oxen FAK genes, finds 1 new molecular labeling g.129462_
129463insGAA, carry out Genotyping for this mutational site and carry out correlation point with the reproductive performance of breeding oxen
Analysis, wherein statistic analysis result is shown, the insertion of the bases of GAA tri- does not occur between FAK genes the 129462nd and 129463 sites
The reproductive performance of the breeding oxen individual of genotype is higher than other genotype individuals.Therefore this molecular labeling can be used as detection kind public
The specific molecular marker of ox reproductive performance height, the marker assisted selection for breeding oxen.This hair is completed on this basis
It is bright.
The detection of the breeding oxen FAK genes of embodiment 1 g.129462_129463insGAA molecular labeling
The present invention carries out examination using PCR amplifications and sequencing technologies to breeding oxen FAK gene mutations, and sequencing result is carried out
Compare analysis, it is determined that g.129462_129463insGAA mutational site.
(breeding oxen blood or seminal fluid used in the present invention are derived from the milk cow of Beijing for 1.1 collection breeding oxen blood or seminal fluid
The heart, Guangming Hesitan Livestocks Co., Ltd. Shanghai, Shandong AUX Breeding bull station etc.), respectively in accordance with phenol/chloroform extraction process and
High salt method blood or seminal fluid genomic DNA, it is standby that DNA sample is diluted into 50ng/ μ L.Test ox totally 218.
1.2 design of primers
According to the gene order for the FAK that Genbank accession number is NC_007312.5 (2124036..2316975), use
Primer Premier5.0 software Design primers, using breeding oxen blood or seminal fluid genomic DNA as template, expand FAK genes.
The sequence of designed primer is as follows
F:TTTGACTGACGGCTGCTTGG
R:AGTTTGGAGGTGCTCTGGTA
1.3PCR amplification
25 μ L reaction system：The μ L of template (50ng/ μ L) 1, each 1 μ L, 2 × Taq PCR of 10 μm of ol/L upstream and downstream primers
MasterMix 12.5 μ L, ddH2O 4.5μL。
PCR reaction conditions：94 DEG C of denaturation 5min, 94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, 35 are followed
Ring；72 DEG C of extension 10min, 4 DEG C of preservations.
The detection in 1.4 mutational sites and parting
PCR primer, it is sequenced with ABI 3730DNA sequenators, the FAK gene sequences that sequencing result provides with Genbank
Arrange (accession number：NC_007312.5) it is compared, finds there is a GAA between FAK genes the 129462nd and 129463 sites
The insertion mutation of three bases.As a result it is as shown in Figure 1.Different genotype is distinguished according to sequencing result, site of analysis it is polymorphic
Implementations.
G.129462_129463insGAA, embodiment 2FAK gene molecule markers divide with the correlation of breeding oxen reproductive performance
Analysis
Experiment bull is respectively provided with complete reproductive performance testing result, and Testing index includes：It is volume of semen, fresh smart vigor, fresh
Precision and Frozen semen activity etc..
Using the statistical softwares of SAS 9.0, GLM programs are called, with least square fitting linear model, compare breeding oxen
FAK genes different genotype and the correlation of reproductive performance (volume of semen, fresh smart vigor, fresh precision, Frozen semen activity).Its model
For：
Yijk=μ+Gi+Yj+Hk+eijk
YijkFor the observed value of breeding oxen reproductive trait；μ is community average；Gi:The fixed effect of genotype；Hk：Play
Fixed effect；Yj：The fixed effect in season；eijk：Random residual effect.
Molecular labeling g.129462_129463insGAA different genotype and the progress of bull reproductive trait to FAK genes
Association analysis, the results showed that：The breeding oxen of the bases of GAA tri- insertion does not occur between FAK genes the 129462nd and 129463 sites
The volume of semen and fresh precision of individual are significantly higher than genotype individuals (P ＜ 0.05) (table that tri- base insertion mutations of GAA occur
1)。
The association analysis with breeding oxen reproductive trait of the FAK gene molecule markers of table 1 g.129462_129463insGAA
Note：Significant difference (P ＜ 0.05) between the average value of different lowercases.
As shown in Table 1, there is the base that the insertion of the bases of GAA tri- does not occur between FAK genes the 129462nd and 129463 sites
Because the reproductive performance of the breeding oxen individual of type is higher than other genotype individuals.This has latent in breeding oxen seed selection and breed improvement
Application value.By the way that to breeding oxen FAK genes, g.129462_129463insGAA site carries out Genotyping, if finding
The insertion of the bases of GAA tri- does not occur between the 129462nd and 129463 sites for some individual, then the individual is with carrying other bases
Compared because of the individual of type and expect to have higher reproductive performance.Accordingly, breeder can instruct the early stage of breeding oxen to sieve accordingly
Choosing, so as to reduce feeding cost, increase value-added content of product.It can be seen that breeding oxen FAK gene molecule markers are g.129462_
129463insGAA has important application prospect in the marker assisted selection of breeding oxen.
The Function Identification of the breeding oxen FAK genes of embodiment 3 g.129462_129463insGAA molecular labeling
G.129462_129463insGAA, breeding oxen FAK gene mutation sites are located at the 3'UTR areas of gene.Utilize miRNA
On-line prediction software TargetScan (http://www.targetscan.org/index.htmL) predict and FAK genes 3'
The miRNAs that UTR region combines.As a result show that g.129462_129463insGAA the mutational site on FAK genes 3'UTR is located at
Bta-miR-21 seed zone, the bta- when the insertion mutation of tri- bases of GAA occurs between the 129462nd and 129463 sites
MiR-21 can be combined with FAK gene 3'UTR fragments, and the free energy ratio combined after insertion with bta-miR-21 inserts preceding and bta-
The free energy that miR-21 is combined diminishes.Free energy is smaller under normal circumstances, and binding ability is stronger, therefore, the 129462nd He
Breeding oxen FAK gene 3'UTR areas and bta-miR-21 knot after the insertion mutation of generation tri- bases of GAA between 129463 sites
Conjunction ability may be than strengthening before insertion.
In order to preferably verify this molecular labeling g.129462_129463insGAA to the shadow of bta-miR-21 binding abilities
Ring, above-mentioned prediction result is verified using cell transient transfection experiment.The insertion bases of GAA tri- will be contained respectively and be not inserted into
The FAK gene 3'UTR fragments of the bases of GAA tri- are connected with pMIR carriers, build 3'UTR luciferase expression plasmids, and corresponding life
Entitled pMIR-3'UTR-GAA and pMIR-3'UTR-0；Meanwhile build bta-miR-21 plasmids.Then by 3'UTR luciferases
Expression plasmid pMIR-3'UTR-GAA and pMIR-3'UTR-0, respectively with bta-miR-21 plasmids, internal reference β-gal plasmid co-transfections
Mouse Leydig Cells (MLTC-1), after cultivating 36 hours, analyzed by double luciferase reporter systems, as a result such as Fig. 2
It is shown.Plasmid pMIR-3'UTR-GAA activity is less than plasmid pMIR-3'UTR-0, and this is consistent with above-mentioned prediction result.Explanation
After insertion, bta-miR-21 is stronger than before insertion with breeding oxen FAK gene 3'UTR areas binding ability, so that FAK gene expression amounts
Reduce.The low expression of FAK genes is unfavorable for the raising of breeding oxen reproductive performance, therefore again it can be assumed that when the 129462nd He
The genotype that the insertion of the bases of GAA tri- does not occur between 129463 sites is favourable molecular labeling, contains its genotype individuals
Breeding oxen reproductive performance is higher than other genotype individuals.
The detection kit of embodiment 4
The kit of high breeding breeding oxen is g.129462_129463insGAA screened based on FAK gene mutation sites, strictly according to the facts
Apply described in example 1, being g.129462_129463insGAA mutated for FAK genes is closely related with the reproductive performance of breeding oxen.Therefore,
This mutational site design FAK gene-specific primers can be based on to be expanded and detected.
The kit (100 times) of detection breeding oxen reproductive performance height is prepared, composition is as shown in table 2：
Table 2
Breeding oxen blood or seminal fluid are gathered, extracts STb gene, enters performing PCR reaction by the methods described of embodiment 1.By kit
In PCR primer be diluted to 10 μm of ol/L, performing PCR is entered with above-mentioned kit as template using the DNA that is extracted and reacted.PCR reacts
Condition：94 DEG C of denaturation 5min, 94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, 35 circulate；72 DEG C of extension 10min,
4 DEG C of preservations.25 μ L reaction system：The μ L of template (50ng/ μ L) 1, each 1 μ L, 2 × Taq PCR of 10 μm of ol/L upstream and downstream primers
MasterMix 12.5 μ L, ddH2O 4.5μL。
PCR primer is detected using 1.0% agarose gel electrophoresis, is sequenced with ABI 3730DNA sequenators, according to
Sequencing result determines the insertion mutation of tri- bases of GAA is whether there is between the 129462nd and 129463 sites, distinguishes different genes
Type, the polymorphic implementations of site of analysis.
The present invention has the illustration of practicality：
1) g.129462_129463insGAA the present invention screens FAK gene mutation sites, and this molecular labeling can be used for early
Phase diagnoses the height of breeding oxen reproductive performance, in favor of cultivating the breeding oxen of prolificacy.
2) the nucleotide sequence primer for the detection FAK gene mutation sites that the present invention establishes and detection breeding oxen reproductive performance
The molecular labeling of height, can high sensitivity, the specifically kit applied to prediction breeding oxen reproductive performance height.
As described above, it was therefore concluded that, the polymorphism of FAK gene mutation sites g.129462_129463insGAA is public with kind
The reproductive performance tool significant correlation of ox.Therefore, according to the present invention, determine this polymorphism and can be used for carrying out gene diagnosis.
Invention describes the new mutation site for the FAK genes for influenceing breeding oxen reproductive performance height.According to the present invention, only
A small amount of DNA sample is needed just to be enough to determine FAK gene pleiomorphisms.As a result, the invention provides one kind to determine breeding oxen reproductive ability
The gene diagnosis method in the mutational site of energy height.
Although above-mentioned the embodiment of the present invention is described with reference to accompanying drawing, model not is protected to the present invention
The limitation enclosed, one of ordinary skill in the art should be understood that on the basis of technical scheme those skilled in the art are not
Need to pay various modifications or deformation that creative work can make still within protection scope of the present invention.
Claims (6)
1. a kind of method predicted or aid in prediction breeding oxen reproductive performance, methods described are not belonging to diagnosis and the treatment side of disease
Method, it is characterized in that：Comprise the following steps：Extract the blood or seminal fluid genomic DNA of breeding oxen, measure FAK genes the 129462nd
And 129463 insertion mutations for whether occurring tri- bases of GAA between site, according to its molecular labeling g. 129462_
The reproductive performance height of 129463insGAA prediction breeding oxens, when between breeding oxen FAK genes the 129462nd and 129463 sites
When the insertion of the bases of GAA tri- not occurring, breeding oxen individual reproduction performance highest.
2. the method as described in claim 1, it is characterised in that specifically comprise the following steps：
（1）Using the blood of breeding oxen to be measured or seminal fluid genomic DNA as template；
（2）It is mutated for g. 129462_129463insGAA, designs breeding oxen FAK gene-specific primers, it is anti-to enter performing PCR
Should, the amplified production 1 of specific fragment is obtained, such as sequence table SEQ ID NO：Shown in 1；Described gene-specific primer has such as
Sequence table SEQ ID NO：2 and sequence table SEQ ID NO：Sequence shown in 3；
（3）Detected using the method for pcr amplification product direct Sequencing in amplified production and whether there is following mutation： g.
129462_129463insGAA。
3. application of the molecular labeling of breeding oxen reproductive performance in predicting or aiding in prediction breeding oxen reproductive performance is influenceed, it is described
Molecular labeling inserts tri- bases of GAA between FAK genes the 129462nd and 129463 sites, when breeding oxen FAK genes
When the insertion of the bases of GAA tri- does not occur between 129462 and 129463 sites, breeding oxen individual reproduction performance highest.
4. influence application of the molecular labeling of breeding oxen reproductive performance in the breeding oxen of seed selection different reproductive performance, the molecule
Labeled as tri- bases of GAA are inserted between FAK genes the 129462nd and 129463 sites, when breeding oxen FAK genes the 129462nd
And 129463 when the insertion of the bases of GAA tri- not occurring between site, breeding oxen individual reproduction performance highest.
5. application of any described method in predicting or aiding in prediction breeding oxen reproductive performance in claim 1-2.
6. application of any described method in the breeding oxen of seed selection different reproductive performance in claim 1-2.
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Non-Patent Citations (4)
| Title |
|---|
| FAK的基因多态性与冠心病的关系;王丽;《中国医药科学》;20130331;第3卷(第5期);第22-23，85页 * |
| XM_010834804;Genbank;《Genbank》;20141231;序列部分 * |
| 粘着斑激酶FAK基因功能研究进展;马勇 等;《家畜生态学报》;20150228;第36卷(第2期);第2.2节 * |
| 荷斯坦牛精液品质相关基因分子标记研究;于俊娜;《中国优秀硕士学位论文全文数据库农业科技辑》;20100715(第07期);摘要 * |
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