CN104561279A - Method of improving quality of chicken semen, primer used for method, kit and using method of kit - Google Patents
Method of improving quality of chicken semen, primer used for method, kit and using method of kit Download PDFInfo
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Abstract
The invention discloses a primer for a gene closely related to the quality of chicken semen. The primer is prepared from the following components including a mixture of upstream and downstream primers PCR amplified by a gene of a vasoactive intestinal peptide receptor 1 (VIPR1), a mixture of upstream and downstream primers PCR amplified by a gene of a vasoactive intestinal peptide receptor 2 (VIPR2) and a mixture of upstream and downstream primers PCR amplified by a gene of a dopamine receptor 2 (DAR2). A method of improving the quality of the chicken semen comprises the following steps: during breeding screening, extracting DNA of a chicken genome of a sample to be tested, carrying out specific primer amplification on a gene sequence to obtain target fragments of 192, 250 and 270 bp, after SSCP analysis and gel electrophoresis on the target fragments, coloring the target fragments by virtue of silver staining to obtain DNA and SSCP maps, and selecting a genotype individual of three genes of TT/GG/OQ according to banding pattern selection in three maps. A kit comprises mixtures of upstream and downstream primers PCR amplified by genes of the VIPR1, the VIPR2 and the DAR2 and a reagent needed by PCR. A using method of the kit comprises the following steps: carrying out PCR amplification on a fragment located by an SNP site; carrying out PCR-SSCP detection; and carrying out SSCP analysis. The method is remarkable in effect and simple and rapid in detection method and is not influenced by external environment.
Description
Technical field
The present invention relates to the closely related gene diagnosis kit of high-quality chicken semen quality and using method thereof, belong to technical field of molecular biology, be applied to the early screening of cock.
Background technology
The mankind are to poultry production proterties from the unconscious history to selecting consciously to experience several thousand, and research from Phenotypic Selection to hereditary basis and genotype select this process to be only the thing of nearly decades.Outstanding stock cock is one of key of increasing economic efficiency, and can reduce production cost on the one hand, particularly plant the feeding cost of chicken.Take artificial insemination that the breeding ratio of chicken can be made to improve about 3 times, can reduce by the stock cock of 3/4, what this can be larger reduces costs, and greatly can improve the utilization ratio of outstanding stock cock on the other hand, enhance productivity and product quality.The current selection to cock still adopts conventional selection, although conventional breeding methods obtains certain progress in poultry genetic improvement, it will grow to the certain period at individual growth to the selection of quantitative character, and breeding cycle is long, costly.High-quality chicken semen quality proterties is quantitative character, only carries out individual selection result very reliably with individual or relatives' phenotypic number, therefore, needs genetic marker to carry out assisted Selection.Along with completing of human genome examining order, the screening of SNP and detection formally become the focus of investigator's extensive concern.This technology have specificity high, detect fast, highly versatile, the feature such as applied widely, with low cost, in the research of the assignment of genes gene mapping of complex disease, association analysis, individual disease susceptibility analysis and pharmacogenomics etc., play more and more important effect.
Through the retrieval to existing domestic and foreign literature and patent, so far there are no the report of VIPR1, VIPR2 and DAR2 gene polymorphism sites and Chicken Semen quality trait dependency.
Summary of the invention
The object of this invention is to provide a kind of raising Chicken Semen quality method and primer, test kit and using method thereof, the inventive method Be very effective, detection method simple and fast, and not by the impact of external environment.
The invention provides the closely related gene primer of a kind of Chicken Semen quality, comprise the upstream and downstream primer mixture of vip receptor 1 (VIPR1) gene PCR amplification, the upstream and downstream primer mixture of vip receptor 2 (VIPR2) gene PCR amplification, the upstream and downstream primer mixture of Dopamine Receptors 2 (DAR2) gene PCR amplification;
The upstream and downstream primer mixture middle and upper reaches primer sequence of described vip receptor 1 (VIPR1) gene PCR amplification: 5 '-gctcccgcagatatatggaa-3 '; Downstream primer sequence: 5 '-tgggaggagacaaaacaaca-3 ';
The upstream primer sequence of the upstream and downstream primer mixture of described vip receptor 2 (VIPR2) gene PCR amplification: 5 '-gtgttcacttttgggcacct-3 '; Downstream primer sequence: R:5 '-cagccaaaaacattggtgtg-3 ';
The upstream and downstream primer mixture of described Dopamine Receptors 2 (DAR2) gene PCR amplification, upstream primer sequence: 5 '-gttggggagtggaggttcag-3 '; Downstream primer sequence: 5 '-attgggctggagatggcaaa-3 '.
Present invention also offers a kind of method utilizing complex gene type to improve Chicken Semen quality, extract the chicken genomic dna of sample to be tested, the chicken genomic dna gene order of sample to be tested carries out pcr amplification SNP site place fragment through the closely related gene primer of Chicken Semen quality according to claim 1, amplification obtains 192, the object fragment of 250 and 270 bp, after the advanced performing PCR-SSCP of object fragment detects, with the colour developing of silver dye after gel electrophoresis, obtain the SSCP collection of illustrative plates of DNA respectively, the individuality of TT/GG/OQ tri-gene complex gene type is selected according to the banding pattern in 3 collection of illustrative plates, namely 73352, 36127, 201 and 208 places are respectively T, G, the allelic individuality of T and A/G.
Preferably, the reaction system 20 μ L of described pcr amplification SNP site place fragment: add upstream and downstream primer mixture 2.0 μ L, PCR reaction reagent 3 μ L, 50ng/ μ L DNA profiling 1.0 μ L in PCR reaction tubes, add ultrapure water to 20 μ L, fully of short duration centrifugal after mixing.
Preferably, the reaction conditions of described pcr amplification is first 94 DEG C of sex change 5min; Again through 35 circulations, each circulation comprises 94 DEG C of 30s, 60 DEG C of 15s, 72 DEG C of 30s; Then 72 DEG C extend 10min, last 4 DEG C of preservations.
Preferably, described PCR-SSCP detects and comprises: 2 μ L pcr amplification products add 7 μ L sample-loading buffers, 98 DEG C of sex change 10min, then ice bath 5min; The polyacrylamide concentration of 3 pairs of primers is 10%, and gel cross-linkage degree is 29:1, and voltage is all 150V, electrophoresed overnight.
In addition, invention further provides a kind of Chicken Semen quality three gene association effect diagnostic kit, comprise the liner in box body and box body, in the vestibule of liner, be provided with the upstream and downstream primer mixture P of described vip receptor 1 VIPR1 gene PCR amplification, the upstream and downstream primer mixture P of vip receptor 2 VIPR2 gene PCR amplification, the upstream and downstream primer mixture P of described Dopamine Receptors 2 DAR2 gene PCR amplification and PCR reaction reagent, ultrapure water, sample-loading buffer; Described PCR reaction reagent is the mixture of 10 × buffer, dNTP and Taq enzyme.
Invention further provides the using method of Chicken Semen quality three gene association effect diagnostic kit, comprise the following steps:
(1) pcr amplification SNP site place fragment: adopt 20 μ L reaction systems: add upstream and downstream primer mixture 2.0 μ L, PCR reaction reagent 3 μ L, 50ng/ μ L DNA profiling 1.0 μ L in PCR reaction tubes, add ultrapure water to 20 μ L, fully of short duration centrifugal after mixing;
(2) PCR reaction tubes is put into PCR instrument and carry out reaction amplification: first 94 DEG C of sex change 5min; Again through 35 circulations, each circulation comprises 94 DEG C of 30s, 60 DEG C of 15s, 72 DEG C of 30s; Then 72 DEG C extend 10min, last 4 DEG C of preservations.
(3) PCR-SSCP detects: 2 μ L pcr amplification products add 7 μ L sample-loading buffers, 98 DEG C of sex change 10min, then ice bath 5min.The polyacrylamide concentration of 3 pairs of primers is 10%, and gel cross-linkage degree is 29:1, and voltage is all 150V, electrophoresed overnight;
(4) object fragment is through sscp analysis, with the colour developing of silver dye after gel electrophoresis, obtain the SSCP collection of illustrative plates of DNA, select the genotype of VIPR1, VIPR2 and DAR2 gene to be respectively the individuality of TT/GG/OQ tri-gene complex gene type according to the banding pattern in 3 collection of illustrative plates, be namely respectively the allelic individuality of T, G, T and A/G at 73352,36127,201 and 208 places.
Compared with prior art, the present invention has following beneficial effect:
First, 1 pair of primer is designed respectively for VIPR1, VIPR2 and DAR2 gene, the amplified production of VIPR1 primer produces 3 kinds of genotype (CC, CT and TT) through sscp analysis, and direct Sequencing shows that TT type there occurs C → T at 73352 bp places and suddenlys change compared with CC type; The amplified production of VIPR2 primer produces 3 kinds of genotype (AA, AG and GG) through sscp analysis, and GG type and AA type there occurs A → G at 36127 bp places and suddenly change; The amplified production of DAR2 primer produces 5 kinds of genotype (OO, OP, OQ, PP and PQ) through sscp analysis, and QQ type detects that compared with OO type at 201 places, C → T occurring suddenlys change, and PP type is compared discovery 208 place A → G and suddenlyd change with OO type.The correlation analysis of 3 gene different composite genotype and high quality meat chicken cockscomb and semen quality shows, the semen quality of three gene complex gene type TT/GG/OQ genotype combination is best, and semen volume is significantly higher than TT/AA/PP, CC/AA/OO, CC/AA/OP and CT/AA/OO type individuality (P<0.05); Sperm concentration is significantly higher than CT/AA/PP, CC/GG/OP, CC/AA/OO, CC/GG/OO and CT/AA/OO type individuality (P<0.05); Motility of sperm is significantly better than CT/AA/OO and CC/AA/OP type individuality (P<0.05);
The second, the genotyping kit that the present invention utilizes the detection of the complex gene type of vip receptor 1 (VIPR1), vip receptor 2 (VIPR2) and Dopamine Receptors 2 (DAR2) gene to provide a kind of accuracy high, quick, cheap.Test kit of the present invention comprises the upstream and downstream primer mixture of VIPR1, VIP2 and DAR2 gene PCR amplification, reagent needed for PCR.When breeding is screened, extract the chicken genomic dna of sample to be tested, gene order obtains the object fragment of 192,250 and 270 bp through primer amplified, object fragment is through sscp analysis, with the colour developing of silver dye after gel electrophoresis, obtain the SSCP collection of illustrative plates of DNA, select the individuality of TT/GG/OQ tri-gene complex gene type according to the banding pattern in 3 collection of illustrative plates.The inventive method Be very effective, detection method simple and fast, and not by the impact of external environment.
Accompanying drawing explanation
Fig. 1 is the SSCP collection of illustrative plates of VIPR1 gene; 1,2:CC type in figure; 3,4:CT type; 5,6:TT type.
Fig. 2 is the sequencer map of VIPR1 gene.
Fig. 3 is the SSCP collection of illustrative plates of VIPR2 gene; 2,3,5:AA types in figure; 4,6:GG type; 1:AG type.
Fig. 4 is the sequencer map of VIPR-2 gene;
Fig. 5 is the SSCP collection of illustrative plates of DAR2 gene; 1:QQ type in figure; 2:PP type; 3:PQ type; 4,5:OQ type; 6,8:OP type; 7:OO type.
Fig. 6 is the sequencer map of DAR2 gene.
Embodiment
The closely related gene primer of a kind of Chicken Semen quality, comprises the upstream and downstream primer mixture of vip receptor 1VIPR1 gene PCR amplification, the upstream and downstream primer mixture of vip receptor 2VIPR2 gene PCR amplification, the upstream and downstream primer mixture of Dopamine Receptors 2 DAR2 gene PCR amplification.
The upstream and downstream primer mixture middle and upper reaches primer sequence of vip receptor 1VIPR1 gene PCR amplification: 5 '-gctcccgcagatatatggaa-3 '; Downstream primer sequence: 5 '-tgggaggagacaaaacaaca-3 '.
The upstream primer sequence of the upstream and downstream primer mixture of vip receptor 2VIPR2 gene PCR amplification: 5 '-gtgttcacttttgggcacct-3 '; Downstream primer sequence: R:5 '-cagccaaaaacattggtgtg-3 '.
The upstream and downstream primer mixture of Dopamine Receptors 2 DAR2 gene PCR amplification, upstream primer sequence: 5 '-gttggggagtggaggttcag-3 '; Downstream primer sequence: 5 '-attgggctggagatggcaaa-3 '.
Complex gene type is utilized to improve the method for Chicken Semen quality: the chicken genomic dna extracting sample to be tested, the chicken genomic dna gene order of sample to be tested carries out pcr amplification SNP site place fragment through the closely related gene primer of Chicken Semen quality according to claim 1, amplification obtains 192, the object fragment of 250 and 270 bp, after object fragment carries out PCR-SSCP detection, with the colour developing of silver dye after gel electrophoresis, obtain the SSCP collection of illustrative plates of DNA respectively, the individuality of TT/GG/OQ tri-gene complex gene type is selected according to the banding pattern in 3 collection of illustrative plates, namely 73352, 36127, 201 and 208 places are respectively T, G, the allelic individuality of T and A/G.
The reaction system 20 μ L of pcr amplification SNP site place fragment: add upstream and downstream primer mixture 2.0 μ L (the corresponding PCR reaction tubes of each upstream and downstream primer mixture), PCR reaction reagent 3 μ L, 50ng/ μ L DNA profiling 1.0 μ L in PCR reaction tubes, add ultrapure water to 20 μ L, fully of short duration centrifugal after mixing.
The reaction conditions of pcr amplification is first 94 DEG C of sex change 5min; Again through 35 circulations, each circulation comprises 94 DEG C of 30s, 60 DEG C of 15s, 72 DEG C of 30s; Then 72 DEG C extend 10min, last 4 DEG C of preservations.
PCR-SSCP detects and comprises: 2 μ L pcr amplification products add 7 μ L sample-loading buffers, 98 DEG C of sex change 10min, then ice bath 5min; The polyacrylamide concentration of 3 pairs of primers is 10%, and gel cross-linkage degree is 29:1, and voltage is all 150V, electrophoresed overnight.
A kind of Chicken Semen quality three gene association effect diagnostic kit, comprises box body 1, liner 2, VIPR1 primer mixture P3, VIPR2 primer mixture P 4, VAR2 primer mixture P 5, the mixture of PCR reaction reagent 6:10 × buffer, dNTP and Taq enzyme, ultrapure water 7, sample-loading buffer 8.
The upstream and downstream primer mixture of VIPR1 gene PCR amplification, upstream primer sequence 5 '-gctcccgcagatatatggaa-3 '; Downstream primer sequence: 5 '-tgggaggagacaaaacaaca-3 '.
The upstream and downstream primer mixture of VIPR2 gene PCR amplification, upstream primer sequence: 5 '-gtgttcacttttgggcacct-3 '; Downstream primer sequence: R:5 '-cagccaaaaacattggtgtg-3 '.
The upstream and downstream primer mixture of DAR2 gene PCR amplification, upstream primer sequence: 5 '-gttggggagtggaggttcag-3 '; Downstream primer sequence: 5 '-attgggctggagatggcaaa-3 '.
The closely related gene diagnosis kit of Chicken Semen quality, its working method is as follows:
1, pcr amplification SNP site place fragment: adopt 20 μ L reaction systems: add upstream and downstream primer mixture 2.0 μ L, PCR reaction reagent 3 μ L, 50ng/ μ L DNA profiling 1.0 μ L in PCR reaction tubes, add ultrapure water to 20 μ L, fully of short duration centrifugal after mixing.The corresponding PCR reaction tubes of each upstream and downstream primer mixture.
PCR reaction tubes is put into PCR instrument and carry out reaction amplification: first 94 DEG C of sex change 5min; Again through 35 circulations, each circulation comprises 94 DEG C of 30s, 60 DEG C of 15s, 72 DEG C of 30s; Then 72 DEG C extend 10min, last 4 DEG C of preservations.
PCR-SSCP detects: 2 μ L pcr amplification products add 7 μ L sample-loading buffers, 98 DEG C of sex change 10min, then ice bath 5min.The polyacrylamide concentration of 3 pairs of primers is 10%, and gel cross-linkage degree is 29:1, and voltage is all 150V, electrophoresed overnight.
Object fragment is through sscp analysis, with the colour developing of silver dye after gel electrophoresis, obtain the SSCP collection of illustrative plates of DNA, select the genotype of VIPR1, VIPR2 and DAR2 gene to be respectively the individuality of TT/GG/OQ tri-gene complex gene type according to the banding pattern in 3 collection of illustrative plates, be namely respectively the allelic individuality of T, G, T and A/G at 73352,36127,201 and 208 places.
Embodiment 1
One, VIPR-1, VIPR-2 and DAR2 tri-gene SNP detect
Detect 218 high quality meat chickens---the genotype of Shao uncle chicken three genes.Blood sample picks up from Jiangsu Inst. of Fowls Science, wing venous collection blood sample 1.5mL, heparin sodium anti-freezing ,-20 DEG C of preservations.Extract genomic dna by the method for phenol chloroform, be dissolved in TE, for subsequent use after concentration determination is carried out to genomic dna.
According to the operation steps of this test kit, as shown in Figure 1, (banding pattern shows to define 3 kinds of genotype the SSCP collection of illustrative plates of VIPR1 primer, and two kinds homozygous represents with CC and TT respectively, and heterozygous CT represents to show 3 kinds of genotype.Order-checking shows that TT type there occurs C → T at 73352 bp places and suddenlys change (Fig. 2) compared with CC type.
The SSCP collection of illustrative plates of VIPR2 primer as shown in Figure 3, (banding pattern shows to define 3 kinds of genotype to show 3 kinds of genotype, two kinds homozygous represents with AA and GG respectively, and heterozygous AG represents, order-checking shows that GG type there occurs at 36127bp place G → A prominent (Fig. 4).
DAR2 primer SSCP collection of illustrative plates as shown in Figure 5, (banding pattern shows to define 6 kinds of genotype to show 6 kinds of genotype, three kinds homozygous represents with OO, PP and QQ respectively, corresponding heterozygous OP, OQ and PQ represent, OO type is respectively C and A at the allelotrope at 201 and 208bp place, PP type is respectively C and A in corresponding site, and QQ type is respectively T and G in corresponding site, is defined as OO, OP, OQ, PP, PQ and QQ genotype respectively.Get the genotypic PCR primer fragment of OO, PP and QQ3 kind to check order.Found that: QQ and OO compares, at 201 places, C → T sudden change occurs, and A → G sudden change (Fig. 6) occurs at 208 places.
Two, VIPR1, VIPR2 and DAR2 gene complex gene type is to the effect analysis of high quality meat chicken cockscomb and semen quality
Coordinate following model analysis VIPR-1, VIPR-2 and DAR2 complex gene type on the impact of high quality meat chicken cockscomb and semen quality: the genotype effects+residual error effect of the genotype effects+DAR2 gene of the genotype effects+VIPR2 gene of Y=μ+VIPR1 gene.Comparing in table 1 of high quality meat chicken complex gene type TT/GG/OQ and different composite genotype cockscomb and semen quality.
Table 1 high quality meat chicken complex gene type TT/GG/OQ compares with different composite genotype semen quality
| Complex gene type | Density (hundred million/ml) | Semen volume (μ l) | Vigor | Abnormal rate % |
| TT/GG/OP | 35.98±1.02 a | 621±22 a | 8.33±0.13 a | 4.01±0.33 |
| CC/GG/OQ | 35.44±1.31 a | 582±25 ab | 8.31±0.18 a | 4.21±0.35 |
| TT/AA/PP | 35.12±1.23 a | 551±21 b | 8.12±0.15 ab | 4.12±0.28 |
| CT/AA/PP | 34.62±1.52 b | 579±24 ab | 8.30±0.16 a | 4.20±0.31 |
| CT/GG/OP | 35.02±1.21 ab | 584±23 ab | 8.24±0.23 a | 4.13±0.36 |
| CC/GG/OP | 34.48±1.22 b | 587±25 ab | 8.16±0.12 ab | 4.10±0.18 |
| CC/AA/OO | 34.07±1.23 b | 536±42 b | 7.50±0.15 b | 4.20±0.36 |
| CT/AA/OO | 34.54±1.28 b | 554±25 b | 7.86±0.19 b | 4.44±0.68 |
| CT/AA/OP | 35.04±1.36 ab | 583±34 ab | 8.11±0.18 ab | 4.18±0.47 |
| CC/AG/OO | 35.54±1.29 a | 587±28 ab | 8.11±0.31 ab | 4.18±0.39 |
| TT/GG/OO | 35.68±1.47 ab | 608±16 ab | 8.11±0.07 ab | 4.02±0.62 |
| CC/GG/OO | 34.59±1.39 b | 575±31 ab | 8.63±0.11 a | 4.20±0.58 |
| TT/AA/OO | 34.81±1.42 ab | 581±28 ab | 8.37±0.23 a | 4.21±0.39 |
| TT/AG/OP | 35.59±1.18 a | 606±37 a | 8.11±0.26 ab | 4.18±0.47 |
| CC/AA/OP | 34.24±1.37 b | 568±36 b | 7.99±0.29 b | 4.24±0.52 |
| TT/AA/OP | 35.68±1.31 a | 603±25 a | 8.31±0.18 a | 4.22±0.35 |
| CT/GG/OO | 35.12±1.23 ab | 572±21 ab | 8.12±0.15 ab | 4.02±0.28 |
Note: the different lowercase alphabet of colleague's data shoulder mark shows significant difference (P<0.05).
From table, the individual semen quality of three gene complex gene type TT/GG/OQ is best, semen volume 15.9% (P<0.05) more than CC/AA/OO genotype individuals, 12.1% (P<0.05) more than CT/AA/OO genotype individuals; The individuality of sperm concentration TT/GG/OQ than CC/AA/OO individuality the more 5.6% (P<0.05), many 5.1% (P<0.05) more individual than CC/AA/OP; The significant difference (P<0.05) of motility of sperm TT/GG/OP type individuality and CC/AA/OO, CT/AA/OO, CC/AA/OP individuality; Although difference is not remarkable between rate of teratosperm each genotype individuals, the abnormal rate of TT/GG/OQ type individuality is less than other genotype individuals.In colony, screen complex gene type TT/GG/OQ can improve high-quality chicken semen quality, so can be used as the method for high quality meat chicken cockscomb and semen quality ahead of time according to the SSCP collection of illustrative plates screening TT/GG/OQ tri-gene complex gene type of VIPR1, VIPR2 and DAR2 gene.
<110> Jiangsu Inst. of Fowls Science
<120> improves Chicken Semen quality method and primer, test kit and using method thereof
<160> 6
<210> 1
<211> 20
<212> DNA
<213> artificial sequence
<220>
The upstream primer of <221>VIPR1 gene PCR amplification
<223>
<400> 1
gctcccgcag atatatggaa 20
<210>2
<211> 20
<212> DNA
<213> artificial sequence
<220>
The downstream primer of <221> VIPR1 gene PCR amplification
<223>
<400> 2
tgggaggaga caaaacaaca 20
<210>3
<211> 20
<212> DNA
<213> artificial sequence
<220>
The upstream primer of <221>VIPR2 gene PCR amplification
<223>
<400> 3
gtgttcactt ttgggcacct 20
<210>4
<211> 20
<212> DNA
<213> artificial sequence
<220>
The downstream primer of <221>VIPR2 gene PCR amplification
<223>
<400> 4
cagccaaaaa cattggtgtg 20
<210>5
<211> 20
<212> DNA
<213> artificial sequence
<220>
The upstream primer of <221>DAR2 gene PCR amplification
<223>
<400> 5
gttggggagt ggaggttcag 20
<210>6
<211> 20
<212> DNA
<213> artificial sequence
<220>
The downstream primer of <221> DAR2 gene PCR amplification
<223>
<400> 6
attgggctgg agatggcaaa 20
Claims (7)
1. the closely related gene primer of Chicken Semen quality, comprises the upstream and downstream primer mixture of vip receptor 1VIPR1 gene PCR amplification, the upstream and downstream primer mixture of vip receptor 2VIPR2 gene PCR amplification, the upstream and downstream primer mixture of Dopamine Receptors 2 DAR2 gene PCR amplification;
The upstream and downstream primer mixture middle and upper reaches primer sequence of described vip receptor 1VIPR1 gene PCR amplification: 5 '-gctcccgcagatatatggaa-3 '; Downstream primer sequence: 5 '-tgggaggagacaaaacaaca-3 ';
The upstream primer sequence of the upstream and downstream primer mixture of described vip receptor 2VIPR2 gene PCR amplification: 5 '-gtgttcacttttgggcacct-3 '; Downstream primer sequence: R:5 '-cagccaaaaacattggtgtg-3 ';
The upstream and downstream primer mixture of described Dopamine Receptors 2DAR2 gene PCR amplification, upstream primer sequence: 5 '-gttggggagtggaggttcag-3 '; Downstream primer sequence: 5 '-attgggctggagatggcaaa-3 '.
2. utilize complex gene type to improve the method for Chicken Semen quality, it is characterized in that, extract the chicken genomic dna of sample to be tested, the chicken genomic dna gene order of sample to be tested carries out pcr amplification SNP site place fragment through the closely related gene primer of Chicken Semen quality according to claim 1, amplification obtains 192, the object fragment of 250 and 270 bp, after object fragment carries out PCR-SSCP detection, with the colour developing of silver dye after gel electrophoresis, obtain the SSCP collection of illustrative plates of DNA respectively, the individuality of TT/GG/OQ tri-gene complex gene type is selected according to the banding pattern in 3 collection of illustrative plates, namely 73352, 36127, 201 and 208 places are respectively T, G, the allelic individuality of T and A/G.
3. the method utilizing complex gene type to improve Chicken Semen quality according to claim 2, it is characterized in that, the reaction system 20 μ L of described pcr amplification SNP site place fragment: the upstream and downstream primer mixture 2.0 μ L adding the upstream and downstream primer mixture of vip receptor 1VIPR1 gene PCR amplification or the upstream and downstream primer mixture of vip receptor 2VIPR2 gene PCR amplification or the amplification of Dopamine Receptors 2 DAR2 gene PCR in PCR reaction tubes, PCR reaction reagent 3 μ L, 50 ng/ μ L DNA profiling 1.0 μ L, add ultrapure water to 20 μ L, of short duration centrifugal after abundant mixing.
4. the method utilizing complex gene type to improve Chicken Semen quality according to claim 2, is characterized in that, the reaction conditions of described pcr amplification is first 94 DEG C of sex change 5min; Again through 35 circulations, each circulation comprises 94 DEG C of 30s, 60 DEG C of 15s, 72 DEG C of 30s; Then 72 DEG C extend 10 min, last 4 DEG C of preservations.
5. the method utilizing complex gene type to improve Chicken Semen quality according to claim 2, it is characterized in that, described PCR-SSCP detects and comprises: 2 μ L PCR amplified productions add 7 μ L sample-loading buffers, 98 DEG C of sex change 10 min, then ice bath 5 min; The polyacrylamide concentration of 3 pairs of primers is 10 %, and gel cross-linkage degree is 29:1, and voltage is all 150V, electrophoresed overnight.
6. a Chicken Semen quality three gene association effect diagnostic kit, comprise the liner (2) in box body (1) and box body, it is characterized in that, the upstream and downstream primer mixture P(3 of vip receptor 1 VIPR1 gene PCR according to claim 1 amplification is provided with) in the vestibule of described liner (2), the upstream and downstream primer mixture P(4 of described vip receptor 2VIPR2 gene PCR amplification), the upstream and downstream primer mixture P(5 of described Dopamine Receptors 2 DAR2 gene PCR amplification) and PCR reaction reagent (6), ultrapure water (7), sample-loading buffer (8), described PCR reaction reagent (6) is the mixture of 10 × buffer, dNTP and Taq enzyme.
7. the using method of Chicken Semen quality three gene association effect diagnostic kit, is characterized in that, comprise the following steps:
(1) pcr amplification SNP site place fragment: adopt 20 μ L reaction systems: upstream and downstream primer mixture 2.0 μ L, PCR reaction reagent 3 μ L, the 50 ng/ μ L DNA profiling 1.0 μ L that add the upstream and downstream primer mixture of vip receptor 1VIPR1 gene PCR amplification or the upstream and downstream primer mixture of vip receptor 2VIPR2 gene PCR amplification or the amplification of Dopamine Receptors 2 DAR2 gene PCR in PCR reaction tubes, add ultrapure water to 20 μ L, fully of short duration centrifugal after mixing; Described PCR reaction reagent (6) is the mixture of 10 × buffer, dNTP and Taq enzyme;
(2) PCR reaction tubes is put into PCR instrument and carry out reaction amplification: first 94 DEG C of sex change 5min; Again through 35 circulations, each circulation comprises 94 DEG C of 30s, 60 DEG C of 15s, 72 DEG C of 30s; Then 72 DEG C extend 10 min, last 4 DEG C of preservations;
(3) PCR-SSCP detects: 2 μ L PCR amplified productions add 7 μ L sample-loading buffers, 98 DEG C of sex change 10 min, then ice bath 5 min; The polyacrylamide concentration of 3 pairs of primers is 10 %, and gel cross-linkage degree is 29:1, and voltage is all 150V, electrophoresed overnight;
(4) object fragment is through sscp analysis, with the colour developing of silver dye after gel electrophoresis, obtain the SSCP collection of illustrative plates of DNA, select the genotype of VIPR1, VIPR2 and DAR2 gene to be respectively the individuality of TT/GG/OQ tri-gene complex gene type according to the banding pattern in 3 collection of illustrative plates, be namely respectively the allelic individuality of T, G, T and A/G at 73352,36127,201 and 208 places.
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| CN111004851A (en) * | 2019-12-19 | 2020-04-14 | 南昌师范学院 | Detection method and application of correlation between VIPR1 gene and cock body quality and slaughter trait |
| CN111004851B (en) * | 2019-12-19 | 2021-05-11 | 南昌师范学院 | Detection method and application of correlation between VIPR1 gene and cock body quality and slaughter trait |
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