CN107354216A - A kind of molecule labelling method of beef cattle fat color trait - Google Patents
A kind of molecule labelling method of beef cattle fat color trait Download PDFInfo
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Abstract
The invention discloses a kind of molecule labelling method of beef cattle fat color trait.This method includes 4 cow genome group DNA extraction, the extron design of primers of the dioxygenase of bata-carotene 9 ', 10 ' (BCO2) gene the 3rd, amplification in vitro and genotype detection steps.The present invention can be used for the assisted Selection of fatty color trait in the breeding of ox, the early stage seed selection of kind of ox can be achieved, the superior genotypes of BCO2 genes can be settled out by a generation can with the gene pyramiding method, the excellent genes of the fatty color trait of one beef cattle strain are reached homozygous by the seed selection i.e. through a generation, substantially reduce generation inteval, accelerating selection process.The present invention is easy to operate, and PCR process conditional is less demanding, and expanding fragment length is shorter (525bp), and amplification is easier to, and improves amplification efficiency and judges the accuracy of genotype.
Description
Technical field
The present invention relates to a kind of molecule labelling method of beef cattle fat color trait.
Background technology
Fatty color is top grade beef most intuitively one of index, and the most important components of various countries' beef classification.
Nineteen ninety-five Hayes yellow fats in the trunk that Australia once reported in outlet can reduce by 10% grade, if
50% yellow fat, the income of Australian 9,200,000 dollars of beef raising is reduced per annual meeting；China Liu Xiao is herded etc. in Shandong Province part
The investigation of meat producing plant, in per kilogram top grade beef, 40 yuan lower than white adipose or so of the meat price of yellow fat.According to phase
It is mainly caused by β-carotenoid in adipose tissue to close report Yellow Fat of Beef, and carotenoid is found in plant
One group of chemical complex, predominance is accounted in fat is beta carotene and lutein, and carrot is known as a variety of differences
Isomr, content full cis-beta-carotene is main in most common isomers, and ox body-internal-circulation in forage crop
The carotenoid wanted.In recent years in, the detection of the QTL progress to candidate gene that may be associated with beef fat color, and
Effective QTL is not found.Kruk research thinks have during the deposition of Jersey ox beta carotenes causes yellow fat
One major gene resistance in action, but does not find out this major gene resistance.Because beta carotene is the main of Yellow Fat of Beef
Contributor, the enzyme in being metabolized to beta carotene are studied to explore the genetic mechanism of Yellow Fat of Beef.Inventor is right
Found in beta carotene metabolic process in the research of the main dioxygenase of digestive enzyme bata-carotene 9 ', 10 ' (BCO2), should
One of enzyme is mutated the expression that can significantly affect the gene, and then influences the fatty color of beef.Thus by using molecule
Mark is detected to the mutation, and the fatty color perfecting of beef cattle can be predicted.
The existing detection that carried out to beef cattle fat color has no special method, mainly to the beef carcass after butchering
Body surface fat is directly detected with color difference meter, and then evaluates its fat grade.
Conventional beef cattle fat color detection needs to be butchered, so being difficult to adopt in the breeding production of beef cattle, institute
The cost needed is too high, and fatty color trait can not be selected in Genetic Improvement of Beef Cattle.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of molecule labelling method of beef cattle fat color trait.The party
Method is realized to the dioxygenase of beef cattle bata-carotene 9 ', 10 ' ((beta-carotene oxygenase 2, be abbreviated as BCO2) base
Because of the detection of the 3rd exons mutation body.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is a kind of point of beef cattle fat color trait
Sub- labeling method, comprises the following steps：
(1) cow genome group DNA extraction；
(2) the extron design of primers of the dioxygenase of bata-carotene 9 ', 10 ' (BCO2) gene the 3rd, the primer are SEQ
The anti-sense primer shown in sense primer and SEQ ID No.2 shown in ID No.1；
(3) amplification in vitro
Using PCR, the extron of the dioxygenase of ox bata-carotene 9 ', 10 ' (BCO2) gene the 3rd is obtained
Amplification in vitro product；
(4) genotype detection
Reacted using digestion with restriction enzyme, obtain digestion products；Restricted of polymorphic site is carried out to digestion products
Section digestion polymorphism (RLFP) detection, selects wherein band to be reserved seed for planting for GG type individuals；
The extron Bsr1 digestion with restriction enzyme of ox bata-carotene 9', 10' dioxygenase (BCO2) gene the 3rd can be with
Detect to co-exist in 3 kinds of genotype that (GG genotype is 391 and 134bp, AA genotype are 391,107 and 27bp, AG genotype
For 391,134,107 and 27bp), M swimming lanes are 50bp loader Marker.
Preferably, amplification in vitro is PCR, concrete operation step is as follows：
(5) PCR system is prepared
By μ l of 4 × dNTP2.5mmol/l deoxynucleoside triphosphates 2,10mmol/ml sense primers and 10mmol/ml downstreams
Each 0.5 μ l of primer, μ l of 10U/ μ l Taq polymerases 0.4, concentration 50ng/ μ l μ l of cow genome group DNA solution 2, containing 20mmol/l
The μ l of 10 × PCR (PCR) buffer solution 2.5 of magnesium chloride (MgCl2), add the μ l of water 17.1 to the μ l of final volume 25；And
It is well mixed, 25 μ l PCR systems are made；
(6) landing-type PCR condition
25 μ l PCRs (PCR) systems are put into Polymerized human serum albumin device, reaction condition is as follows：
1. 95 DEG C of pre-degenerations 4 minutes,
2. 95 DEG C are denatured 30 seconds,
3. annealing temperature 60 DEG C 30 seconds,
4. 72 DEG C extend 30 seconds,
5. repeating the 2.~4. step, 10 circulations are repeated, the annealing temperature often circulated reduces by 1 DEG C.
6. entering normal circulation, 95 DEG C are denatured 30 seconds,
7. annealing temperature 50 DEG C 30 seconds,
8. 72 DEG C extend 30 seconds,
9. repeating the 6.~8. step, 24 circulations are repeated,
10. 72 DEG C extend 5 minutes, 4 DEG C of preservations are finally cooled to, obtain the dioxygenase of ox bata-carotene 9 ', 10 ' (BCO2)
The amplification in vitro product of the extron of gene the 3rd；The dioxygenase of ox bata-carotene 9 ', 10 ' (BCO2) of the amplification in vitro product
The bit base of the 3rd extron of gene the 111st sports A by G.
Preferably, the concrete operations of genotype detection are as follows：
(7) digestion with restriction enzyme reacts, polymorphic site restriction fragment digestion polymorphism (RLFP) detection
Because the bit base of the 3rd extron of dioxygenase (BCO2) gene of ox bata-carotene 9 ', 10 ' the 111st is sported by G
A, result in the digestion polymorphism site of a Bsr1 restriction enzyme, and specific endonuclease reaction operating procedure is：Take ox β recklessly
The μ l of amplification in vitro product 10 of the extron of the dioxygenase of radish element 9 ', 10 ' (BCO2) gene the 3rd are in PCR
(PCR) in pipe, the 10U/ μ l μ l of 1 μ l, Bsr1 restriction enzymes enzyme buffer liquid of Bsr1 restriction enzymes 2 is separately added into, add water
7 μ l, mix, 37 DEG C of temperature is reacted 3 hours, obtains digestion products；
(8) polymorphic site restriction fragment digestion polymorphism (RLFP) detects
By digestion products 2.5% agarose gel electrophoresis 1 hour, deposition condition：Normal temperature, voltage 120V, obtain containing enzyme
Cut the gel of product；Gel containing digestion products is placed under gel imaging system and observes genotype, outside ox BCO2 genes the 3rd
Showing can detect to co-exist in 3 kinds of genotype that (GG genotype is 391 Hes in sub- Bsr1 digestion with restriction enzyme image
134bp, AA genotype are 391,107 and 27bp, AG genotype are 391,134,107 and 27bp, and in the general gel pictures of 27bp
Can not show), M swimming lanes are 50bp loader Marker；AA genotype individuals are fatty color trait superior genotypes, choosing
It is that AA type individuals are reserved seed for planting to select wherein band, can improve beef fat color trait score, improve the fatty whiteness for selecting and remain colony.
The beneficial effects of the invention are as follows：
Can be used for the assisted Selection of fatty color trait in the breeding of ox, the early stage that kind of ox can be achieved chooses seeds, or even
Ox can be selected and remain exactly when being just born, and can pass through a generation can by BCO2 with the gene pyramiding method
The superior genotypes of gene settle out, i.e., the seed selection through a generation is i.e. by the fatty color trait of a beef cattle strain
Excellent genes reach homozygous, and conventional breeding methods will pass through substantial amounts of performance test and progeny testing, and want subculture
Seed selection more than multiple generations can be only achieved the effect of needs, substantially reduce generation inteval, accelerating selection process.
This method is easy to operate, and PCR process conditional is less demanding, and expanding fragment length is shorter
(525bp), amplification is easier to, and is improved amplification efficiency and is judged the accuracy of genotype.
Embodiment
In order to realize the detection to the exons mutation body of the dioxygenase of beef cattle bata-carotene 9 ', 10 ' (BCO2) gene the 3rd,
Present embodiments provide a kind of beef cattle fat color trait molecule labelling method.
The present embodiment devises the extron primer of beef cattle BCO2 genes the 3rd, establishes the 3rd extron w80x site mutations
The detection method of body, and the correlation between its polymorphism and fatty color trait is analyzed, establishing influences fatty color
Superior genotypes, for the selection to beef cattle fat color trait, it is big to solve the blindness of traditional seed choosing method, seed selection
The problem of accuracy is low.
The concrete operation step of the molecule labelling method of the present embodiment beef cattle fat color trait is as follows：
1st, cow genome group DNA extraction；
2nd, the extron design of primers of the dioxygenase of bata-carotene 9 ', 10 ' (BCO2) gene the 3rd, the primer draw for upstream
Thing and anti-sense primer, its sequence are as follows：
Sense primer：5 '-AACCCATCCCACTTCCTTATCT-3 ',
Anti-sense primer：5’-GCTGAAATCAAACCCCAAAG-3’；
3rd, amplification in vitro
Using PCR, the extron of the dioxygenase of ox bata-carotene 9 ', 10 ' (BCO2) gene the 3rd is obtained
Amplification in vitro product, concrete operation step are as follows：
(1) PCR system is prepared
By μ l of 4 × dNTP2.5mmol/l deoxynucleoside triphosphates 2,10mmol/ml sense primers and 10mmol/ml downstreams
Each 0.5 μ l of primer, μ l of 10U/ μ l Taq polymerases 0.4, concentration 50ng/ μ l μ l of cow genome group DNA solution 2, containing 20mmol/l
The μ l of 10 × PCR (PCR) buffer solution 2.5 of magnesium chloride (MgCl2), add the μ l of water 17.1 to the μ l of final volume 25；And
It is well mixed, 25 μ l PCR systems are made；
(2) landing-type PCR condition
25 μ l PCRs (PCR) systems are put into Polymerized human serum albumin device, reaction condition is as follows：
1. 95 DEG C of pre-degenerations 4 minutes,
2. 95 DEG C are denatured 30 seconds,
3. annealing temperature 60 DEG C 30 seconds,
4. 72 DEG C extend 30 seconds,
5. repeating the 2.~4. step, 10 circulations are repeated, the annealing temperature often circulated reduces by 1 DEG C.
6. entering normal circulation, 95 DEG C are denatured 30 seconds,
7. annealing temperature 50 DEG C 30 seconds,
8. 72 DEG C extend 30 seconds,
9. repeating the 6.~8. step, 24 circulations are repeated,
10. 72 DEG C extend 5 minutes, 4 DEG C of preservations are finally cooled to, obtain the dioxygenase of ox bata-carotene 9 ', 10 ' (BCO2)
The amplification in vitro product of the extron of gene the 3rd；The dioxygenase of ox bata-carotene 9 ', 10 ' (BCO2) of the amplification in vitro product
The bit base of the 3rd extron of gene the 111st sports A by G.
4th, genotype detection
Reacted using digestion with restriction enzyme, obtain digestion products；Restricted of polymorphic site is carried out to digestion products
Section digestion polymorphism (RLFP) detection, selects wherein band to be reserved seed for planting for GG type individuals.
The concrete operations of genotype detection are as follows：
(1) digestion with restriction enzyme reacts, polymorphic site restriction fragment digestion polymorphism (RLFP) detection
Because the bit base of the 3rd extron of dioxygenase (BCO2) gene of ox bata-carotene 9 ', 10 ' the 111st is sported by G
A, result in the digestion polymorphism site of a Bsr1 restriction enzyme, and specific endonuclease reaction operating procedure is：Take ox β recklessly
The μ l of amplification in vitro product 10 of the extron of the dioxygenase of radish element 9 ', 10 ' (BCO2) gene the 3rd are in PCR
(PCR) in pipe, the 10U/ μ l μ l of 1 μ l, Bsr1 restriction enzymes enzyme buffer liquid of Bsr1 restriction enzymes 2 is separately added into, add water
7 μ l, mix, 37 DEG C of temperature is reacted 3 hours, obtains digestion products；
(2) polymorphic site restriction fragment digestion polymorphism (RLFP) detects
By digestion products 2.5% agarose gel electrophoresis 1 hour, deposition condition：Normal temperature, voltage 120V, obtain containing enzyme
Cut the gel of product；Gel containing digestion products is placed under gel imaging system and observes genotype.
It can detect to co-exist in 3 kinds of bases in the extron Bsr1 digestion with restriction enzyme images of ox BCO2 genes the 3rd
Because of type, (GG genotype is 391 and 134bp, AA genotype are 391,107 and 27bp, AG genotype are 391,134,107 and
27bp, and can not be shown in the general gel pictures of 27bp), M swimming lanes are 50bp loader Marker；AA genotype individuals are fat
Color trait superior genotypes, select wherein band to be reserved seed for planting for AA type individuals, beef fat color trait score can be improved, improve
Select and remain the fatty whiteness of colony.
The embodiments of the present invention described above are not intended to limit the scope of the present invention.It is any in the present invention
Spirit and principle within the modifications, equivalent substitutions and improvements made etc., should be included in the claim protection model of the present invention
Within enclosing.
SEQUENCE LISTING
<110>Husbandry ＆ Veternity Research Inst. of Anhui Prov. Agriculture Science Academy
<120>A kind of molecule labelling method of beef cattle fat color trait
<130> NO
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 12
<212> DNA
<213>Artificial sequence
<400> 1
AACCCATCCC ACTTCCTTAT CT 12
<210> 2
<211> 10
<212> DNA
<213>Artificial sequence
<400> 2
GCTGAAATCA AACCCCAAAG 10
Claims (3)
1. a kind of molecule labelling method of beef cattle fat color trait, comprises the following steps：
(1) cow genome group DNA extraction；
(2) the extron design of primers of 9 ', 10 ' dioxygenase gene of bata-carotene the 3rd, the primer are shown in SEQ ID No.1
Sense primer and SEQ ID No.2 shown in anti-sense primer；
(3) amplification in vitro
Using PCR, the amplification in vitro production of the extron of 9 ', 10 ' dioxygenase gene of ox bata-carotene the 3rd is obtained
Thing；
(4) genotype detection
Reacted using digestion with restriction enzyme, obtain digestion products；Polymorphic site restriction fragment enzyme is carried out to digestion products
Polymorphic detection is cut, selects wherein band to be reserved seed for planting for GG type individuals.
2. molecule labelling method according to claim 1, it is characterised in that：The amplification in vitro is polymerase chain reaction
Should, concrete operation step is as follows：
(5) PCR system is prepared
By μ l of 4 × dNTP2.5mmol/l deoxynucleoside triphosphates 2,10mmol/ml sense primers and 10mmol/ml anti-sense primers
Each 0.5 μ l, μ l of 10U/ μ lTaq polymerases 0.4, concentration 50ng/ μ l μ l of cow genome group DNA solution 2, magnesium chloride containing 20mmol/l
(MgCl2) the μ l of 10 × PCR (PCR) buffer solution 2.5, add the μ l of water 17.1 to the μ l of final volume 25；And mix equal
It is even, 25 μ l PCR systems are made；
(6) landing-type PCR condition
25 μ l PCRs (PCR) systems are put into Polymerized human serum albumin device, reaction condition is as follows：
1. 95 DEG C of pre-degenerations 4 minutes,
2. 95 DEG C are denatured 30 seconds,
3. annealing temperature 60 DEG C 30 seconds,
4. 72 DEG C extend 30 seconds,
5. repeating the 2.~4. step, 10 circulations are repeated, the annealing temperature often circulated reduces by 1 DEG C；
6. entering normal circulation, 95 DEG C are denatured 30 seconds,
7. annealing temperature 50 DEG C 30 seconds,
8. 72 DEG C extend 30 seconds,
9. repeating the 6.~8. step, 24 circulations are repeated,
10. 72 DEG C extend 5 minutes, 4 DEG C of preservations are finally cooled to, obtain the dioxygenase of ox bata-carotene 9 ', 10 ' (BCO2) gene
The amplification in vitro product of 3rd extron；The dioxygenase of ox bata-carotene 9 ', 10 ' (BCO2) gene of the amplification in vitro product
The bit base of 3rd extron the 111st sports A by G.
3. molecule labelling method according to claim 1, it is characterised in that：The concrete operations of the genotype detection are such as
Under：
(7) digestion with restriction enzyme reacts, the detection of polymorphic site restriction fragment digestion polymorphism
The endonuclease reaction operating procedure is：Take the amplification in vitro of the extron of 9 ', 10 ' dioxygenase gene of ox bata-carotene the 3rd
For the μ l of product 10 in PCR pipe, Bsr1 restriction enzymes 1 the μ l, Bsr1 for being separately added into 10U/ μ l are restricted interior
The μ l of enzyme cutting buffer solution 2, add the μ l of water 7, mix, 37 DEG C of temperature is reacted 3 hours, obtains digestion products；
(8) polymorphic site restriction fragment digestion polymorphism detects
By digestion products 2.5% agarose gel electrophoresis 1 hour, deposition condition：Normal temperature, voltage 120V, obtain producing containing digestion
The gel of thing；Gel containing digestion products is placed under gel imaging system and observes genotype.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881247A (en) * | 2017-12-12 | 2018-04-06 | 吉林省农业科学院 | The related molecular labeling of Red Steppe fat content and its acquisition methods and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106498083A (en) * | 2016-12-21 | 2017-03-15 | 西北农林科技大学 | A kind of RFLP methods of detection cattle PCAF gene mononucleotide polymorphisms and test kit |
-
2017
- 2017-08-09 CN CN201710678279.2A patent/CN107354216A/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106498083A (en) * | 2016-12-21 | 2017-03-15 | 西北农林科技大学 | A kind of RFLP methods of detection cattle PCAF gene mononucleotide polymorphisms and test kit |
Non-Patent Citations (3)
| Title |
|---|
| LIETZ, G 等: "Importance of beta,beta-carotene 15,15 "-monooxygenase 1 (BCMO1) and beta,beta-carotene 9 ",10 "-dioxygenase 2 (BCDO2) in nutrition and health", 《MOLECULAR NUTRITION & FOOD RESEARCH》 * |
| 刘晓牧等: "牛肉黄脂研究进展", 《中国牛业科学》 * |
| 徐磊等: "西门塔尔牛BCO2基因多态性及与脂肪颜色性状的相关性研究", 《中国牛业科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881247A (en) * | 2017-12-12 | 2018-04-06 | 吉林省农业科学院 | The related molecular labeling of Red Steppe fat content and its acquisition methods and application |
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