CN104830838B - The preparation method and application of the genetic marker of high intramuscular fat content in a kind of Baicheng fine breed of chicken with thick brownish feathers leg flesh - Google Patents

Abstract

The invention discloses a kind of preparation method and application of the genetic marker of high intramuscular fat content in Baicheng fine breed of chicken with thick brownish feathers leg flesh, the present invention chooses white 60, the plumage of a 120 age in days Baicheng fine breed of chicken with thick brownish feathers, male and female half and half, the content of intramuscular fat IMF in H FABP Second Exons polymorphisms and leg flesh is detected, and analyzes relation between the two.The present invention passes through experiment and understood, the high Baicheng fine breed of chicken with thick brownish feathers of IMF contents in genetic marker selection leg flesh prepared by the method for the invention.

Description

A kind of preparation method of genetic marker of high intramuscular fat content in Baicheng oil drumstick muscle and applications

technical field

The invention belongs to the technical field of poultry genetic markers, and relates to a preparation method and application of a genetic marker with high intramuscular fat content in the leg muscle of Baicheng oil chicken, in particular to a kind of high intramuscular fat content in the leg muscle of Baicheng oil chicken (white feather). Preparation and application of genetic markers for fat content.

Background technique

With the improvement of people's living standards, there are stricter requirements on the quality and taste of meat. Chicken is the main meat on the table, and the quality of chicken has increasingly become the focus of attention. Most of the standards for measuring the quality of chicken meat come from people's taste, and there is no unified standard at present. After analysis, the high intramuscular fat IMF in chicken is considered to be a main indicator affecting the meat quality traits of chicken and improving the tenderness of meat. When the high intramuscular fat content reaches more than 3.5%, good meat flavor can be obtained.

With the development of molecular biology, people try to use genetic markers in the selection of high intramuscular fat content. Genetic Marker refers to any gene that can trace chromosomes, a certain segment of chromosomes, and a certain locus in a family. a genetic trait. It has two basic characteristics, that is, heritability and identifiability, so any genetic mutants with different phenotypes can be used as genetic markers.

At present, an accurate and reliable genetic marker is needed for the selection of high intramuscular fat content in Baicheng oil drumstick muscle.

Contents of the invention

The purpose of the present invention is to overcome the defects in the prior art, and provide a preparation method and application of a genetic marker of high intramuscular fat content in Baicheng oil drumstick muscle. Its specific technical plan is:

A kind of genetic marker preparation method of high intramuscular fat content in Baicheng oil drumstick muscle, comprises the following steps:

I. Preparation of Polyacrylamide Gels

Use 12% polyacrylamide gel to separate the fragments to be tested. According to the different specifications of the glass plate, prepare 7ml and 10ml of glue respectively, the formula is:

II. Preparation of samples

1. Design primers, the sample fragment length should generally be between 150-300bp;

2. Amplify the target fragment by PCR and detect it on agarose gel;

3. Sample dilution: According to the test result of agarose gel, if the PCR product is very concentrated, dilute the PCR product 3 times; if the PCR product concentration is weak, do not dilute;

4. Take 1 μl of the diluted PCR product, add it to 9ul denaturation buffer, and mix well;

5. Denaturation buffer: bromophenol blue 5mg, 0.5M EDTA.Na2pH8.0400μl, formamide 9.6ml, mix well;

III. Transgender

1. Turn on the PCR instrument and set the program to 98°C for 11 minutes;

2. Put the sample mixed with formamide into the PCR instrument and start denaturation;

3. Prepare the ice box;

4. When denaturing for 10 minutes, turn on the PCR instrument, quickly insert the sample into ice, and keep it for 10 minutes;

IV. Loading electrophoresis

1. Before electrophoresis, the gel and electrophoresis buffer used must be pre-cooled;

2. Take 3 μl of samples, and place them in turn on the pre-prepared and refrigerated polyacrylamide gel; when applying the samples, be careful not to place the standard on the two holes on the far side of the gel, so as not to cause uneven gel production or other reasons. The type of the standard product is not good, and it cannot play an indicative role;

3. Put the electrophoresis tank in a 4°C refrigerator, connect it to the electrophoresis instrument, run at 250V for 10 minutes, and run at 50V for 16 hours;

4. Electrophoresis conditions are different for different fragments to be separated, so if the above conditions cannot separate different genotypes, try other conditions;

V. Staining

1) Fixation: Remove the glue, make a starting mark, put it into 80ml fixative solution and fix it on the shaker for 10min;

2) Staining: 60ml staining solution + 60μl formaldehyde, stain for 6min, wash once with distilled water;

3) Color development: 60ml color development solution + 120 μl formaldehyde, put on a shaker to develop color until the bands are clear. Pour off the color developing solution, add distillation to stop the color development.

The application of the genetic marker of the invention in the selection of high intramuscular fat content in Baicheng oil drumstick muscle.

Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention can be known through experiments that the genetic marker prepared by the method of the present invention selects Baicheng Youji (White Feather) with high IMF content in the leg muscles.

Detailed ways

The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments.

The present invention selects 60 120-day-old Baicheng oil chickens (white feathers), half male and half female, detects the second exon polymorphism of H-FABP and the content of intramuscular fat (IMF) in leg muscles, and analyzes both The relationship between.

PCR primers

5′-CGACAAGGCGACGGTGAA-3′(forward)

5′-TGGGGCAGGAAGGAGTTT-3′(reverse)

1) PCR reaction system:

2) PCR reaction program:

SSCP experimental steps

I. Preparation of Polyacrylamide Gels

In general, use 12% polyacrylamide gel to separate the fragments to be tested. According to the different specifications of the glass plates (1.0 and 1.5), prepare 7ml and 10ml of glue respectively. The formula is shown in Table 1:

Table 1

II. Preparation of samples

1. Design primers, the sample fragment length should generally be between 150-300bp.

2. Amplify the target fragment by PCR and detect it on agarose gel.

3. Sample dilution. According to the results of agarose gel detection, if the PCR product is very concentrated (as bright as the marker), then dilute the PCR product 3 times; if the PCR product concentration is weak, you can not dilute.

4. Take 1 μl of the diluted PCR product, add it to 9ul denaturation buffer, and mix well.

5. Denaturation buffer: bromophenol blue 5mg, 0.5M EDTA.Na2 (pH8.0) 400μl, formamide 9.6ml, mix well.

III. Transgender

1. Turn on the PCR instrument and set the program to 98°C for 11min

2. Put the sample mixed with formamide into the PCR instrument and start denaturation

3. Prepare ice box

4. When denaturing for 10 minutes, turn on the PCR instrument, quickly insert the sample into ice, and keep it for 10 minutes

IV. Loading electrophoresis

1. Before electrophoresis, the gel and electrophoresis buffer (1×TBE) used must be pre-cooled.

2. Take 3 μl of samples, and spot them in turn on the polyacrylamide gel prepared in advance and refrigerated. When spotting the sample, be careful not to point the standard product to the two holes on the far side of the glue, so as to avoid the poor classification of the standard product due to uneven glue production or other reasons, and it will not be effective as an indicator.

3. Put the electrophoresis tank in a refrigerator at 4°C, connect it to the electrophoresis instrument, run at 250V for 10min, and run at 50V for 16h

4. Note: Electrophoresis conditions are different for different fragments to be separated, so if the above conditions cannot separate different genotypes, you can try other conditions.

V. Staining

1) fixed. Remove the glue, make the initial mark, and put it into about 80ml of fixative solution (50ml of absolute ethanol, 2.5ml of glacial acetic acid, dilute to 500ml, refrigerated) and fix on a shaker for 10min.

2) Dyeing. 60ml of staining solution (0.75gAgNO3, dissolved in 500ml of water, protected from light) + 60μl of formaldehyde, stained for 6min. Wash once with distilled water.

3) Color development. 60ml chromogenic solution (7.5g NaOH, dissolved in 500ml water) + 120μl formaldehyde, placed on a shaker to develop color until the bands are clear. Pour off the color developing solution, add distillation to stop the color development.

1. T vector cloning and sequencing

1. PCR amplification

1) PCR reaction system:

3) 1% agrose gel electrophoresis, after confirming that there is specific amplification, then amplify two PCRs of 50 μl system, and recover the PCR products.

2. PCR product recovery

1) Add 4 times the volume of Buffer CP to a 1.5ml centrifuge tube;

2) Shake violently and centrifuge briefly;

3) Put the adsorption column in the collection tube;

4) Transfer the mixture in step 3 to the adsorption column (750 μl each time, one transfer cannot be completed, after centrifugation, pour off the waste liquid, and then transfer the remaining mixture to the adsorption column for centrifugation);

5) Centrifuge at 13000g for 1min, discard the filtrate;

6) Add 700 μl of eluent, centrifuge at 13000 g for 1 min, and discard the filtrate;

7) Add 500 μl of eluent, centrifuge at 13,000 g for 1 min, and discard the filtrate;

8) Centrifuge at 13000g for 2min, and shake off the ethanol on the adsorption column;

9) Transfer the adsorption column to a new 1.5ml centrifuge tube, add 30μl ddH ₂ O to the center of the adsorption column, and place it at room temperature for 1min; centrifuge at 13000g for 2min, and the filtrate is the recovered DNA;

10) The PCR product is connected to the T vector.

3. PCR product ligation and transformation

1) Prepare the connection system as follows:

2) Shake gently and centrifuge briefly.

3) Stand at room temperature for 5 minutes.

4) The ligation product is directly transformed.

5) The chemically competent DH5α cells were taken out and thawed in ice-water mixture.

6) Add the ligation product and place on ice for 30 minutes.

7) Put the tube in a water bath at 42°C for exactly 90 seconds without shaking.

8) Quickly move the tube to an ice bath for 2 minutes, and place it at room temperature for 5 minutes.

9) Add 800 μl of LB liquid medium without antibiotics to each tube, shake at 37° C. for 45 minutes to recover.

10) Centrifuge at 8000 rpm for 1 min, remove 800 μl of the supernatant, resuspend and spread evenly on the Amp resistance plate.

11) Place the plate at room temperature to dry it, place it upside down in a 37°C incubator, and culture for 12-16 hours to grow colonies.

12) Perform PCR colony identification and sequencing.

Three IMF content determination

1. Sample freeze-drying: Take an appropriate amount of sample, cut off the fat tissue, put it in a small aluminum box in a freeze dryer, and freeze it for about 3 days.

2. Operation steps: Weigh about 0.2-0.3g of freeze-dried sample, accurate to 0.0002g. Wrap it with filter paper, mark the number with a pencil, and weigh it. Put the filter paper bag into the extraction tube, add 60-100mL of anhydrous diethyl ether into the extraction tube, heat in a water bath at 60-75°C to reflux the ether, and control the reflux rate to about 10 times per hour until the extraction tube flows out After ether volatilizes and leaves no oil traces, it is the end point of the extraction, which takes about 8 hours. After the extraction is completed, take out the filter paper bag, dry in an oven at 105°C for 1 hour, cool in a desiccator for 30 minutes, weigh, dry for another 30 minutes, and weigh after cooling again.

3. Calculation of results: crude fat (%) = (W1-W2) ÷ W0 × 100

4. In the formula: W1-105℃ drying and pumping advance fat bag weight, g;

5. W2-105℃ drying and extraction of fat package weight, g;

6.W0-sample weight, g;

The results are shown in Table 2:

Table 2 Genotypes and genotype frequencies

Correlation between IMF and polymorphisms in breast muscle of Baicheng chicken

G939A mutation site produces AA type, G956A mutation site produces BB type

The IMF content of AA type was higher than that of AB type, significantly higher than that of BB type, so G939A can be used as a genetic marker to select Baicheng Youji (White Feather) with high IMF content in leg muscles.

The above is only a preferred specific embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any person familiar with the technical field within the technical scope disclosed in the present invention can obviously obtain the simplicity of the technical solution. Changes or equivalent replacements all fall within the protection scope of the present invention.

Claims (2)

1. the genetic marker preparation method of high intramuscular fat content in a kind of Baicheng fine breed of chicken with thick brownish feathers leg flesh, it is characterised in that including following Step：

I. the preparation of polyacrylamide gel

Fragment to be measured is separated with 12% polyacrylamide gel；According to the difference of glass plate specification, 7ml, 10ml are prepared respectively Glue, be formulated and be：

II. the preparation of sample

1st, primer is designed, sample fragment length typically should be between 150-300bp；

The primer is：

forward：5′-CGACAAGGCGACGGTGAA-3′

reverse：5′-TGGGGCAGGAAGGAGTTT-3′；

2nd, PCR expands purpose fragment, Ago-Gel detection；

3rd, sample dilutes：According to Ago-Gel testing result, if PCR primer is dense, PCR primer is diluted 3 times；If PCR primer concentration is weaker, then does not dilute；

4th, the PCR primer for taking 1 μ l to dilute, add in 9ul denaturation buffers, mix；

5th, denaturation buffer：Bromophenol blue 5mg, 0.5M EDTA.Na2 pH8.0 400 μ l, formamide 9.6ml, mix；

III. it is denatured

1st, PCR instrument is opened, program is arranged to 98 DEG C of 11min；

2nd, the sample for being mixed with formamide is put into PCR instrument, starts to be denatured；

3rd, ice chest is prepared；

4th, when being denatured 10min, PCR instrument is opened, rapidly inserts sample in ice, keeps 10min；

IV. loading electrophoresis

1st, before electrophoresis, glue used, electrophoretic buffer will first precooling；

2nd, the μ l of sample 3 are taken, are put successively in the polyacrylamide gel for making and freezing in advance；Standard items are paid attention to during point sample not Main points to the most side Shang Liangge holes of glue, in order to avoid due to glue is uneven or other reasonses caused by standard items parting it is bad, rise not To indicative function；

3rd, electrophoresis tank is put into 4 DEG C of refrigerators, connects electrophoresis apparatus, 250V runs 10min, 50V 16h；

4th, deposition condition is different and different because of fragment to be separated, if therefore conditions above can not be by different genotype point Open, it tries other conditions；

V. dye

1) it is fixed：Glue is unloaded, carries out start mark, is put into 80ml fixers and 10min is fixed on shaking table；

2) dye：The μ l formaldehyde of 60ml dyeing liquors+60, dyes 6min, and distilled water washed once；

3) develop the color：The μ l formaldehyde of 60ml nitrite ions+120, it is clear to band to be put into colour developing on shaking table；Nitrite ion is outwelled, adds distillation to stop Only develop the color.

2. application of the genetic marker described in claim 1 in Baicheng fine breed of chicken with thick brownish feathers leg flesh in high intramuscular fat content selection.