CN104830837A - Preparation method of genetic marker for high intramuscular fat (IMF) content in breast muscle of Hetian black chicken and application of genetic marker - Google Patents
Preparation method of genetic marker for high intramuscular fat (IMF) content in breast muscle of Hetian black chicken and application of genetic marker Download PDFInfo
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- CN104830837A CN104830837A CN201510257016.5A CN201510257016A CN104830837A CN 104830837 A CN104830837 A CN 104830837A CN 201510257016 A CN201510257016 A CN 201510257016A CN 104830837 A CN104830837 A CN 104830837A
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Abstract
The invention discloses a preparation method of a genetic marker for high intramuscular fat (IMF) content in breast muscle of a Hetian black chicken and an application of the genetic marker. The method comprises the following steps: selecting 60 120-day-old Hetian black chicken, which include 30 hens and 30 cocks; and detecting the second exon polymorphism of H-FABP (heart fatty acid-binding protein) and the IMF content in the breast muscle, and analyzing the relationship between the second exon polymorphism and the content. An experiment shows that the Hetian black chicken with high IMF content in breast muscle is selected by the genetic marker prepared by the method.
Description
Technical field
The invention belongs to bird Genetic Markers field, relate to preparation method and the application of the genetic marker of high intramuscular fat content in a kind of and the black thoracic muscle in field.
Background technology
Along with the raising of people's living standard, also have stricter requirement to meat quality and taste, chicken is as the main meat on dining table, and the quality of chicken more and more becomes the focus of people's concern.Criterion for chicken meat quality stems from the mouthfeel of people mostly, does not also have a kind of unified standard at present.By analysis, the high intramuscular fat IMF in chicken is considered to a kind of leading indicator of fresh and tender degree aspect affecting chicken Meat Quality and improve meat, when high intramuscular fat content reaches more than 3.5%, just can obtain good meat flavor.
Along with the development of molecular biosciences, people attempt to be applied in high intramuscular fat content is selected by genetic marker, any one hereditary property that genetic marker Genetic Marker refers to a certain sections of traceable karyomit(e), karyomit(e), certain locus transmits in family.It has two essential characteristics, i.e. inheritability and identifiability, and the genic mutation type of therefore biological any variant phenotype all can be used as genetic marker.
Current needs one accurately, reliably genetic marker to be used for and during in the black thoracic muscle in field, high intramuscular fat content is selected.
Summary of the invention
The object of the invention is to overcome the defect existed in prior art, preparation method and the application of the genetic marker of high intramuscular fat content in a kind of and black thoracic muscle in field is provided.Its concrete technical scheme is:
A genetic marker preparation method with high intramuscular fat content in the black thoracic muscle in field, comprises the following steps:
I. the preparation of polyacrylamide gel
Polyacrylamide gel with 12% is separated fragment to be measured.According to the difference of sheet glass specification, prepare the glue of 7ml, 10ml respectively, fill a prescription as follows:
II. the preparation of sample
1, design primer, sample fragment length is generally between 150-300bp;
2, pcr amplification object fragment, sepharose detects;
3, diluted sample: according to sepharose detected result, if PCR primer is dense, then dilutes 3 times by PCR primer; If PCR primer concentration is more weak, then do not dilute;
4, get the PCR primer that 1 μ l has diluted, add in 9ul denaturation buffer, mixing;
5, denaturation buffer: tetrabromophenol sulfonphthalein 5mg, 0.5M EDTA.Na2 pH8.0 400 μ l, methane amide 9.6ml, mixing;
III. sex change
1, opening PCR instrument, is 98 DEG C of 11min by programming;
2, the sample being mixed with methane amide is put into PCR instrument, start sex change;
3, ice chest is prepared;
4, as sex change 10min, open PCR instrument, rapidly sample is inserted in ice, keep 10min;
IV. loading electrophoresis
1, before electrophoresis, glue used, first precooling all wanted by electrophoretic buffer;
2, sample thief 3 μ l, puts successively and to make in advance and in the polyacrylamide gel freezed.Note during point sample standard substance not main points to the most Shang Liangge hole, limit of glue, in order to avoid due to the standard substance somatotype that glue is uneven or other reasons causes bad, do not have indicative function;
3, be put into by electrophoresis chamber in 4 DEG C of refrigerators, connect electrophoresis apparatus, 250V runs 10min, 50V 16h;
4, note: deposition condition is different because of the difference of fragment to be separated, if therefore above condition by different genotype separately, then can not can attempt other condition;
V. dye
1) fixing: glue to be unloaded, carries out start mark, put into about 80ml stationary liquid and fix 10min on shaking table;
2) dye: 60ml staining fluid+60 μ l formaldehyde, dyeing 6min.Distilled water wash once;
3) develop the color: 60ml nitrite ion+120 μ l formaldehyde, be put into shaking table develops the color clear to band.Outwell nitrite ion, add distillation and stop colour developing.
The application of genetic marker of the present invention in selecting with high intramuscular fat content in the black thoracic muscle in field.
Compared with prior art, beneficial effect of the present invention is: the present invention is known through experiment, and genetic marker prepared by the method for the invention selects the chicken black in field that in chest muscle, IMF content is high.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described in more detail.
The present invention chooses the black chicken of 120 ages in days and field 60, male and female half and half, detects the content of intramuscular fat (IMF) in H-FABP Second Exon polymorphism and chest muscle, and analyzes relation between the two.
Experimental procedure
PCR primer
5′-CGACAAGGCGACGGTGAA-3′(forward)
5′-TGGGGCAGGAAGGAGTTT-3′(reverse)
1) PCR reaction system:
2) PCR response procedures:
SSCP experimental procedure
I. the preparation of polyacrylamide gel
Generally, fragment to be measured is separated with the polyacrylamide gel of 12%.According to the difference of sheet glass specification (1.0,1.5 two kind), prepare the glue of 7ml, 10ml respectively, formula is as table 1:
Table 1
II. the preparation of sample
1, design primer, sample fragment length is general because of between 150-300bp.
2, pcr amplification object fragment, sepharose detects.
3, diluted sample.According to sepharose detected result, if PCR primer dense (equally bright with marker), then PCR primer is diluted 3 times; If PCR primer concentration is more weak, then can not dilute.
4, get the PCR primer that 1 μ l has diluted, add in 9ul denaturation buffer, mixing.
5, denaturation buffer: tetrabromophenol sulfonphthalein 5mg, 0.5M EDTA.Na2 (pH8.0) 400 μ l, methane amide 9.6ml, mixing.
III. sex change
1, opening PCR instrument, is 98 DEG C of 11min by programming
2, the sample being mixed with methane amide is put into PCR instrument, start sex change
3, ice chest is prepared
4, as sex change 10min, open PCR instrument, rapidly sample is inserted in ice, keep 10min
IV. loading electrophoresis
1, before electrophoresis, glue used, first precooling all wanted by electrophoretic buffer (1 × TBE).
2, sample thief 3 μ l, puts successively and to make in advance and in the polyacrylamide gel freezed.Note during point sample standard substance not main points to the most Shang Liangge hole, limit of glue, in order to avoid due to the standard substance somatotype that glue is uneven or other reasons causes bad, do not have indicative function.
3, be put into by electrophoresis chamber in 4 DEG C of refrigerators, connect electrophoresis apparatus, 250V runs 10min, 50V 16h
4, note: deposition condition is different because of the difference of fragment to be separated, if therefore above condition by different genotype separately, then can not can attempt other condition.
V. dye
1) fixing.Glue is unloaded, carries out start mark, put in about 80ml stationary liquid (constant volume, to 500ml, refrigerates for 50ml dehydrated alcohol, 2.5ml glacial acetic acid) and fix 10min on shaking table.
2) dye.60ml staining fluid (0.75gAgNO3 dissolves in 500ml water, keeps in Dark Place)+60 μ l formaldehyde, dyeing 6min.Distilled water wash once.
3) develop the color.60ml nitrite ion (7.5g NaOH is dissolved in 500ml water)+120 μ l formaldehyde, be put into shaking table develops the color clear to band.Outwell nitrite ion, add distillation and stop colour developing.
One .T carrier cloning order-checking
1.PCR increases
3) PCR reaction system:
4) PCR response procedures:
5) 1%agrose gel electrophoresis, after defining specific amplified, then the PCR of 2 the 50 μ l systems that increase, carry out PCR primer recovery.
2.PCR product reclaims
1) Buffer CP to the 1.5ml centrifuge tube of 4 times of volumes is added;
2) concuss, of short duration centrifugal;
3) adsorption column is placed in collection tube;
4) the mixture of step 3 proceed to adsorption column (each 750 μ l, once cannot not turn completely, wait centrifugal after, outwell waste liquid, then it is centrifugal that remaining mixture is proceeded to adsorption column);
5) the centrifugal 1min of 13000g, abandons filtrate;
6) add 700 μ l elutriants, the centrifugal 1min of 13000g, abandons filtrate;
7) add 500 μ l elutriants, the centrifugal 1min of 13000g, abandons filtrate;
8) the centrifugal 2min of 13000g, gets rid of the ethanol on adsorption column;
9) adsorption column is forwarded to a new 1.5ml centrifuge tube, add 30 μ l ddH at adsorption column center
2o, room temperature places 1min; The centrifugal 2min of 13000g, namely filtrate be the DNA reclaimed;
10) PCR primer connects carrier T.
3.PCR product connects and transforms
1) according to the form below preparation linked system:
2) shake up gently, of short duration centrifugal.
3) room temperature places 5min.
4) connect product directly to transform.
5) taking-up Competent cell DH5 α is put in mixture of ice and water and thaws.
6) add connection product, place 30min on ice.
7) pipe is put in 42 DEG C of lucky 90s of water-bath, can not shakes.
8) fast pipe is moved on to 2min on ice bath, room temperature places 5min.
9) often pipe adds 800 μ l and does not have antibiotic LB liquid nutrient medium, 37 DEG C of vibration 45min recovery.
10) the centrifugal 1min of 8000rpm, removes 800 μ l by supernatant liquor, resuspended rear even spread is in Amp resistant panel.
11) flat board is positioned over room temperature and dries up, and is inverted in 37 DEG C of incubators, cultivates 12-16h and grows bacterium colony.
12) PCR colony identification and order-checking is carried out.
Three IMF assays
1. sample freeze-drying: get appropriate amount of sample, cuts fatty tissue, to put in little aluminium box in Freeze Drying Equipment, freezes general 3 days.
2. operation steps: take freeze-drying sample about 0.2-0.3g, accurately to 0.0002g.Wrap with filter paper, and indicate numbering with pencil, weigh.Filter paper packet is put into extraction tube, in extraction tube, adds anhydrous diethyl ether 60-100mL, 60-75 DEG C of heating in water bath, make aether backflow, controlling return velocity is about 10 times per hour, until it is extracting terminal that the ether that extraction tube flows out does not leave oil stain after volatilizing, about needs about 8h.Take out filter paper packet after extracting, dry 1h, cool 30min, weigh, then dry 30min in moisture eliminator in 105 DEG C of baking ovens, same cooling is weighed, and it is constant weight that the difference of two inferior qualities is less than 0.001g.
3. result calculates: crude fat (%)=(W1-W2) ÷ W0 × 100
4. in formula: fatty Bao Chong, g before W1-105 DEG C of oven dry extracting;
Fatty Bao Chong, g after 5.W2-105 DEG C of oven dry extracting;
6.W0-sample weight, g;
Result is as shown in table 2:
Table 2
The dependency of IMF and polymorphism in Baicheng fine breed of chicken with thick brownish feathers chest muscle
G939A detected altogether, G982A and C1014T tri-mutational sites
G982A mutational site produces AA type, G939A and C1014T mutational site produces BB type
AA type IMF content, higher than AB type, is significantly higher than BB type, therefore the G982A chicken black in field that IMF content in chest muscle can be selected high as genetic marker.
The above; be only the present invention's preferably embodiment; protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the simple change of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.
Claims (2)
1. the genetic marker preparation method of high intramuscular fat content and in the black thoracic muscle in field, is characterized in that, comprise the following steps:
I. the preparation of polyacrylamide gel
Polyacrylamide gel with 12% is separated fragment to be measured; According to the difference of sheet glass specification, prepare the glue of 7ml, 10ml respectively, fill a prescription as follows:
II. the preparation of sample
1, design primer, sample fragment length is generally between 150-300bp;
2, pcr amplification object fragment, sepharose detects;
3, diluted sample: according to sepharose detected result, if PCR primer is dense, then dilutes 3 times by PCR primer; If PCR primer concentration is more weak, then do not dilute;
4, get the PCR primer that 1 μ l has diluted, add in 9ul denaturation buffer, mixing;
5, denaturation buffer: tetrabromophenol sulfonphthalein 5mg, 0.5M EDTA.Na2 pH8.0 400 μ l, methane amide 9.6ml, mixing;
III. sex change
1, opening PCR instrument, is 98 DEG C of 11min by programming;
2, the sample being mixed with methane amide is put into PCR instrument, start sex change;
3, ice chest is prepared;
4, as sex change 10min, open PCR instrument, rapidly sample is inserted in ice, keep 10min;
IV. loading electrophoresis
1, before electrophoresis, glue used, first precooling all wanted by electrophoretic buffer;
2, sample thief 3 μ l, puts successively and to make in advance and in the polyacrylamide gel freezed; Note during point sample standard substance not main points to the most Shang Liangge hole, limit of glue, in order to avoid due to the standard substance somatotype that glue is uneven or other reasons causes bad, do not have indicative function;
3, be put into by electrophoresis chamber in 4 DEG C of refrigerators, connect electrophoresis apparatus, 250V runs 10min, 50V 16h;
4, note: deposition condition is different because of the difference of fragment to be separated, if therefore above condition by different genotype separately, then can not can attempt other condition;
V. dye
1) fixing: glue to be unloaded, carries out start mark, put into about 80ml stationary liquid and fix 10min on shaking table;
2) dye: 60ml staining fluid+60 μ l formaldehyde, dyeing 6min; Distilled water wash once;
3) develop the color: 60ml nitrite ion+120 μ l formaldehyde, be put into shaking table develops the color clear to band; Outwell nitrite ion, add distillation and stop colour developing.
2. the application of genetic marker described in claim 1 in selecting with high intramuscular fat content in the black thoracic muscle in field.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226515A (en) * | 2020-07-15 | 2021-01-15 | 华南农业大学 | Application of chicken molecular marker combination as detection site for identifying chicken intramuscular fat width |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1408885A (en) * | 2001-09-25 | 2003-04-09 | 李宁 | Mononucleotide polymorphism analyzing method for detecting chicken ventral fat character |
| CN103146697A (en) * | 2013-03-27 | 2013-06-12 | 董雅娟 | Single nucleotide polymorphism of Blackett black cow H-FABP gene and detecting method thereof |
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2015
- 2015-05-15 CN CN201510257016.5A patent/CN104830837A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1408885A (en) * | 2001-09-25 | 2003-04-09 | 李宁 | Mononucleotide polymorphism analyzing method for detecting chicken ventral fat character |
| CN103146697A (en) * | 2013-03-27 | 2013-06-12 | 董雅娟 | Single nucleotide polymorphism of Blackett black cow H-FABP gene and detecting method thereof |
Non-Patent Citations (6)
| Title |
|---|
| "《广东黄鸡三十年》" * |
| YONG WANG ET AL.: "Correlation between Heart-type Fatty Acid-binding Protein Gene Polymorphism and mRNA Expression with Intramuscular Fat in Baicheng-oil Chicken", 《ASIAN AUSTRALAS.J.ANIM.SCI》 * |
| 李小兵等: "和田黑鸡遗传资源现状及保护对策", 《新疆畜牧业》 * |
| 杨霞等: "鸡H-FABP基因外显子2的PCR-SSCP分析", 《四川农业大学学报》 * |
| 王彦等: "鸡H-FABP基因多态性及其与肌内脂肪含量的相关研究", 《遗传育种》 * |
| 萨姆布鲁克等: "《分子克隆实验指南（第三版）》", 31 August 2002 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226515A (en) * | 2020-07-15 | 2021-01-15 | 华南农业大学 | Application of chicken molecular marker combination as detection site for identifying chicken intramuscular fat width |
| CN112226515B (en) * | 2020-07-15 | 2021-08-24 | 华南农业大学 | Application of chicken molecular marker combination as detection site for identifying chicken intramuscular fat width |
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Application publication date: 20150812 |