CN103497994A - Molecule marking method for pig backfat thickness property - Google Patents
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Abstract
The invention relates to a molecule marking method for pig backfat thickness property. The method comprises: extraction of pig genome DNA, design of a long-chain acyl-coenzyme Asynthetase 1 (ACSL1) gene seventh exon primer, amplification in vitro and genotype detection. For the first time, it is discovered that the polymorphism of the long-chain acyl-coenzyme Asynthetase 1 (ACSL1) gene seventeenth exon has significant correlation with the pig backfat thickness, and a method for detecting the restriction fragment polymorphism at mutation sites of the seventeenth exon is established. The molecule marking method is applicable to auxiliary selection on backfat thickness property during breeding of pigs, helps to realize early-stage breeding selection of boars, and even helps to accurately select breeding just when pigs are born, so that the generation interval is shortened, and the selection progress of the backfat thickness property is accelerated; and the method is simple in operation, condition requirements during a polymerase chain reaction are low, the length of amplified fragments is relatively short (292 bp), amplification is relatively easy, and the method helps to improve amplification efficiency and accuracy of genotype determining.
Description
Technical field
The invention belongs to the gene pleiomorphism technical field of animal, be specifically related to pig long-chain fatty acyl-CoA synthetase 1 (ACSL1) gene pleiomorphism.
Background technology
External source and endogenic lipid acid will enter its pathways metabolism and must be activated, catalyze and synthesize aliphatic alcohol (Acyl-CoA), this is the first step reaction that Mammals utilizes lipid acid, ACS (Acyl-CoA Synthetase) is in the situation that there are the synthetic Acyl-COA (Piccini etc. of catalysis free fatty acids in CoA and ATP, 1998), catalyzed reaction is irreversible, reaction is divided into two steps, the first step is that catalysis free fatty acids and Triphosaden (ATP) are combined into acyl adenylic acid (Acyl-AMP) and discharge a tetra-sodium (Fatty acid+ATP → acyl-AMP+PPi), second step is that catalysis Acyl-AMP and CoA are combined into Acyl-CoA, and discharges adenylic acid (AMP) (Acyl-AMP+CoA → acyl-CoA+AMP).Lipid acid by the known activation of Adipocyte Factor signal path (Pathway:ssc04920), be that aliphatic alcohol (Acyl-CoA) has three outlets: (1) generates triglyceride (DAG) through the phospho-glycerol approach, and then synthetic glycerine three esters (TAG); (2) synthetic ceramide; (3) be transported in plastosome and carry out β-oxidation, decompose energy supply.Therefore fatty acyl-CoA synthetase plays keying action in the metabolism of lipid acid composition and decomposition, the enzyme that belongs to the multigene family coding, have 25 member composition (Jia etc. at least, 2007), can be divided into overlength chain acyl CoA synthetic enzyme (VACSL according to the specificity of the carbon chain lengths of catalysis lipid acid, claim again FATP), long-chain fatty acyl-CoA synthetase (ACSL) and medium chain fatty acyl-CoA synthetase (MACSL) (Killenberg etc., 1971).
It is different that ACSL gene family member expresses in different tissues, has implied that every kind of ACSL member may bring into play different specific function (Li etc., 2010) in specific tissue.ACSL1 is at the tissue relevant with energy metabolism, as high expression level in liver, fatty tissue and muscle tissue, for synthetic glycerine three esters provide the most important long-chain acyl CoA synthetic enzyme (Muoio etc., 2000) of acyl CoA substrate.Hendrik etc. (2007) research rat heart muscle ACSL expresses and shows during enzymic activity, ACSL1 fetus period expression amount lower, after birth, expression amount increases gradually, reaches maximum when adult; ACSL3 mainly expresses at fetal period, and after birth, expression amount obviously reduces; ACSL4 is less in period from prenatal to postnatal difference, maintains higher level always; ACSL5 and ACSL6 period from prenatal to postnatal expression amount are all lower.The ACSL3 of rat and ACSL6 a large amount in nervous tissue is expressed (Oikawa etc., 1998; Basu-Modak etc., 1999).And that ACSL4 expresses in suprarenal gland is the highest, but also in liver and Steroidgenesis tissue, express (Kang etc., 1997).ACSL5 expression amount in the intestinal epithelial cell of Rat is the highest, and likely with synthetic triglyceride level, to form the chyle particle relevant for this, to the lipid acid in assimilating food property source play an important role (Oikawa etc., 1998).ACSL6 expression amount in brain the highest (Fujino etc., 1992), the ACSL6 albumen of coding is partial to the activation (Marszalek etc., 2005) of catalysis over-long chain fatty acid.Mercade etc. (2006) have reported that the ACSL4 gene pleiomorphism of pig and the speed of growth and oleic acid content exist dependency.
studied the dependency of DLY market pig ACSL4 gene (the mRNA Genbank number of logging in DQ144454) 1 SNP site (G2645A) polymorphism in 3 ' UTR district and 21 Meat Qualities Deng (2011), it is relevant that result shows to only have 6 proterties and this loci polymorphism to exist, and wherein the individual intramuscular fat content utmost point of GG type is significantly higher than AA type individual (P<0.01).
The ACSL1 expression amount of rat is subject to the impact of other physiologic factors, the food of Hi CHO and higher fatty acid daily ration will improve 7-8 times of (Suzuki etc. of expression amount in the ACSL1 liver, 1990), and lipopolysaccharides can reduce the expression amount (Memo etc., 1998) of ACSL1 in liver, muscle and fatty tissue.ACSL1 overexpression in liver can improve 3.7 times of ACSL1 enzymic activity, and improves the amount of oleic acid in triglyceride and phosphatide, but has reduced the resultant quantity (Li etc., 2006) of oleic acid in cholesterol.Adenovirus mediated ACSL1 is at liver cancer cell (HepG2) overexpression; cause the acyl-CoA synthetase vigor to improve 20 times; increased the ACSL1-CoA content in the cell; deposition (the Parkes etc. of oleic acid in triglyceride level have been improved; 2006); amount due to carnitine in this research does not have raising to cause the CPT-1 activity not increase; thereby more ACSL1-CoA can not be transported in plastosome and carry out β-oxidation; therefore the expression amount that improves ACSL1 can increase the deposition (Mashek etc., 2007) of fat in cell.
The research of at present relevant domestic animal ACSL mostly is the detection of gene pleiomorphism.Li etc. (2012) detect the ACSL1 gene pleiomorphism of the southern regions of the Yunnan Province microtia pig, Tibetan pig and Yorkshire, but do not do association analysis, show that the polymorphism between lard type and bacon hogs exists difference.Khandker (2009) has studied-757 polymorphic detections that sported T by C of ox ACSL3 gene, found that the gene frequency of saltant type T allelotrope in catalo (456), Angus (206) and summer Luo Lainiu (187) is respectively 0.216,0.296 and 0.056, find that in the hybridization cows TT genotype individuality exceeds 9.63% than the marble grain of CC type individuality, other proterties does not find that there is remarkable association.Shogo etc. (2008) select 6 genes (comprising ACSL1 and ACSL4) as the Candidate Gene Study of black japan ox intramuscular fat content the polymorphism of ACSL1 exon 2 and ACSL4 exon 2, only on the ACSL1 exon, find 3 SNP, allele distributions differs greatly, so do not carry out the correlation analysis of ACSL1 and phenotypic character.The people such as Bionaz study when variation is expressed with lactation period before Lactation of Dairy Cow by ACSL family and find, the ACSL1 gene peak of lactation (lactation 60 days) expression amount the highest, be significantly higher than other member's expression amount of family, and to 60 days stage A CSL1 expression amounts of lactation, continue to rise in 15 days from calving, lactation improved 5 times than antenatal 15 days expression amounts in the time of 60 days, but lactation after 60 days to 240 days slow decreasings; ACSL3, ACSL4, ACSL5 have downtrending from the first 15 days expression amounts to (60 days) stage peak of lactation of calving, but lactation is on the rise to 240 days after 60 days; ACSL6 expression amount before peak of lactation increases not obvious, within 60 days, raises rapidly afterwards, and 240 days expression amounts than antenatal 15 days of lactation have improved 11 times, and therefore in 5 kinds of fatty acyl-CoA synthetases (ACSLs), ACSL1 has material impact to milk fat content.
At present, the live body thickness of backfat when selection of fat thickness at back of pig (lean ratio) proterties mainly relies on ultrasonic instrument (A surpasses or B ultrasonic back fat instrument) to measure boar 85kg to 115kg body weight, select the thin individuality of back fat to be reserved seed for planting, this kind of system of selection, affected by extraneous factor more, as larger as the impact of feed and environment, cause the back-fat thickness of mensuration to differ and react truly surely the back fat of pig, cause the accuracy of seed selection to descend.Because the ACSL1 gene of pig is relevant with the product fat ability of pig, the inventor finds that the 46th bit base of the 17th exon of this gene sports C by T, cause this site to produce different effects to subcutaneous lipids (back fat), have not yet to see report about the association analysis between pig ACSL1 gene pleiomorphism and the thickness of backfat at present.
Summary of the invention
In order to realize the detection of pig long-chain fatty acyl-CoA synthetase 1 (ACSL1) gene the 17th exons mutation body, the invention provides a kind of molecule marking method of fat thickness at back of pig proterties.
The present invention has designed pig ACSL1 gene the 17th exon primer, set up the detection method of the 17th exon T46C site mutation body, and analyzed the dependency between its polymorphism and the thickness of backfat, established and affected the excellent genes of thickness of backfat type, for the selection to the fat thickness at back of pig proterties, the blindness that has solved traditional seed choosing method is large, the low problem of seed selection accuracy.
The concrete operation step of the molecule marking method of fat thickness at back of pig proterties of the present invention is as follows:
(1) extraction of pig genomic dna;
(2) long-chain fatty acyl-CoA synthetase 1 (ACSL1) gene the 17th exon design of primers, described primer is upstream primer and downstream primer, its sequence is as follows:
Upstream primer: 5 '-ACAGGCTCACTTCGCAGGTAGAT-3 ',
Downstream primer: 5 '-GTTGGCTTGGAACCACTATTTGC-3 ';
(3) amplification in vitro
Adopt polymerase chain reaction, obtain the amplification in vitro product of pig long-chain fatty acyl-CoA synthetase 1 (ACSL1) gene the 17th exon;
(4) genotype detection
The reaction of employing digestion with restriction enzyme, obtain enzyme and cut product; Enzyme is cut to product and carry out polymorphic site restriction fragment enzyme and cut polymorphism (RLFP) and detect, selecting wherein band is that TT type individuality is reserved seed for planting.
In ACSL1 gene the 17th exon Tas1 digestion with restriction enzyme Fig. 2 of pig, the 5th, 6,8 swimming lane bands are the TT type, and the 1st, 3,7 swimming lane bands are the CC type, and the 2nd, 4 swimming lane bands are the TC type, and the M swimming lane is DL2000Marker.
Described amplification in vitro is polymerase chain reaction, and concrete operation step is as follows:
A, preparation polymerase chain reaction system
By the pig DNA genome solution 2 μ l of each 0.5 μ l of 4 * dNTP2.5mmol/l deoxynucleoside triphosphate, 2 μ l, 10mmol/ml upstream primer and 10mmol/ml downstream primer, 10U/ μ l Taq polysaccharase 0.4 μ l, concentration 50ng/ μ l, containing 20mmol/l magnesium chloride (MgCl
2) 10 * polymerase chain reaction (PCR) damping fluid 2.5 μ l, add water 17.1 μ l to final volume 25 μ l; And mix, make 25 μ l polymerase chain reaction systems;
B, polymerase chain reaction condition
25 μ l polymerase chain reaction (PCR) systems are put into to the polymerase chain reaction instrument, and reaction conditions is as follows:
1. 95 ℃ of denaturations are 4 minutes,
2. 95 ℃ of sex change are 30 seconds,
3. 60 ℃ of annealing temperatures are 30 seconds,
4. 72 ℃ are extended 30 seconds,
5. repeat 2.～4. step 35 circulation,
6. 72 ℃ are extended 5 minutes, finally are cooled to 4 ℃ of preservations, obtain the amplification in vitro product of pig long-chain fatty acyl-CoA synthetase 1 (ACSL1) gene the 17th exon; Pig long-chain fatty acyl-CoA synthetase 1 (ACSL1) gene the 17th exon the 46th bit base of described amplification in vitro product sports C by T.
The concrete operations of described genotype detection are as follows:
(1) digestion with restriction enzyme reaction, polymorphic site restriction fragment enzyme is cut polymorphism (RLFP) and is detected
Because pig long-chain fatty acyl-CoA synthetase 1 (ACSL1) gene the 17th exon the 46th bit base sports C by T, caused the enzyme of a Tasl restriction enzyme to cut pleomorphism site, concrete endonuclease reaction operation steps is: get the amplification in vitro product 10 μ l of pig long-chain fatty acyl-CoA synthetase 1 (ACSL1) gene the 17th exon in the pipe of polymerase chain reaction (PCR), the Tasl restriction enzyme 1 μ l that adds respectively 10U/ μ l, Tasl restriction enzyme damping fluid 2 μ l, add water 7 μ l, mix, 37 ℃ of reactions of temperature 3 hours, obtain enzyme and cut product;
(2) polymorphic site restriction fragment enzyme is cut polymorphism (RLFP) detection
Enzyme is cut to 2.5% agarose gel electrophoresis 1 hour for product, deposition condition: normal temperature, voltage 120V, obtain cutting containing enzyme the gel of product; The gel that to cut product containing enzyme is placed under gel imaging system observes genotype, in ACSL1 gene the 17th exon Tasl digestion with restriction enzyme figure of pig, the 5th, 6,8 swimming lane bands are the TT type, 1st, 3,7 swimming lane bands are the CC type, 2nd, 4 swimming lane bands are the TC type, and the M swimming lane is DL2000Marker; TT type individuality is thickness of backfat proterties excellent genes type, and selecting wherein band is that TT type individuality is reserved seed for planting, and can reduce the thickness of backfat of pig, improves the lean ratio of the colony of selecting and remain.
Useful technique effect of the present invention embodies in the following areas:
1. the present invention, by the ACSL1 gene pleiomorphism of pig and the correlation analysis of fatty deposits, finds that ACSL1 gene the 17th exon the 46th loci polymorphism and fat thickness at back of pig exist significant correlation first.
2. can be for the assisted Selection of the breeding thickness of backfat proterties of pig, can realize the early stage seed selection of boar, even when just being born, pig just can be selected and remain exactly, use this gene pyramiding method just the excellent genes type of ACSL1 gene can be settled out through a generation, the excellent genes that is about to the thickness of backfat proterties of this strain through the seed selection of a generation reaches and isozygotys, and the conventional breeding method will be passed through a large amount of performance test and progeny testings, and want 5 of subculture seed selections just can reach the effect needed more than the generation, greatly shortened the generation interval, the accelerating selection process.
3. the method is easy and simple to handle, and polymerase chain reaction process conditional is less demanding, expanding fragment length shorter (292bp), and amplification is easier to, and has improved amplification efficiency and has judged genotypic accuracy.
The accompanying drawing explanation
Fig. 1 is long-chain fatty acyl-CoA synthetase 1 of the present invention (ACSL1) gene PCR amplification agarose gel electrophoresis figure.
Fig. 2 is long-chain fatty acyl-CoA synthetase 1 (ACSL1) gene the 17th exon restriction enzyme (Tasl) cleavage map.
Embodiment
Below by 2 embodiment in Huai Pig new line and Yorkshire swinery, and by reference to the accompanying drawings the present invention is further described.
Following examples material therefor source is described as follows:
In embodiment, material used is 287 of the Huai Pig new line pigs raised in Anhui Province Kexin Pig Farming and Breeding Co., Ltd., Huai Pig new line is the premium muscle type pig kind that academy of agricultural sciences, Anhui Province animal and veterinary institute cultivates, contain 25% Huaihe River pig blood lineage, the white blood lineage of 25% length and 50% Yorkshire blood lineage, relevant Huai Pig new line data is shown in: (1) " raising pigs " magazine the 4th phase in 2009, (2) " herding and forage science " magazine the 6th phase of 30 volumes in 2009, (3) " herding and forage science " magazine the 30th the 9th phase of volume in 2009, (4) " Agriculture of Anhui science " magazine the 25th phases of 35 volumes in 2007 etc.Other 1 material is the introduced variety Yorkshire Pigs of universally acknowledged lean meat species, also is collected in Anhui Province Kexin Pig Farming and Breeding Co., Ltd., and a number is 239.
A kind of concrete operation method of molecule marking method of fat thickness at back of pig proterties is as follows:
(1) gather the ear tissue sample
Be born the same day piglet, taked the ear-edge tissue 0.2 gram left and right of pig with pig overbit pincers, put into the centrifuge tube of 1.5ml, poured 75% alcohol 1ml left and right into, then put into-20 ℃ of Refrigerator stores stand-by;
(2) extracting genome DNA
Take out ear-edge tissue from refrigerator, with scissors, it is shredded, pour DNA tissue extract 600ul into, its DNA tissue extraction liquid formula is in Table 1, the extracting solution matching while using.Then put into 55 ℃ of water-bath temperature and bathe 12 hours, then add the saturated phenol 600 μ l of Tris, fluctuate and mix 15 minutes, use centrifugal 10 minutes of low-temperature and high-speed whizzer, centrifuge speed requires 12000 rev/mins; Take out supernatant liquor to sterilized 1.5ml centrifuge tube with the 1ml pipettor, then add the saturated phenol of isopyknic Tris, then fluctuate and mix 15 minutes, more centrifugal 10 minutes (12000 rev/mins); Get supernatant liquor, add the saturated phenol of isopyknic Tris: chloroform: primary isoamyl alcohol (25: 24: 1) mixes 10 minutes, centrifugal 10 minutes (12000 rev/mins).Get supernatant liquor and add isopyknic chloroform: primary isoamyl alcohol (24: 1) mixes 10 minutes, centrifugal 10 minutes (12000 rev/mins).Get the ice ethanol (about 1ml) that supernatant liquor adds 2 times of volumes, then NaAc (sodium-acetate) level that adds the approximately 60ml of 1/10 volume shakes, cotton-shaped, white DNA sample occurs as seen.Sample is placed in-20 ℃ freezing 30 minutes, take out or choose DNA or centrifugal (12000 rev/mins) 10 minutes, DNA is sunken to the pipe end.With 75% washing with alcohol DNA, after shaking washing, centrifugal 5-10 minute (12000 rev/mins).Discard 75% ethanol, after seasoning, add appropriate (about 50ml) DNA diluent (TE) to dissolve, put into-20 ℃ of refrigerators and preserve.
Table 1DNA tissue extraction liquid formula (cumulative volume 200ml)
(3) design of primers
1 pair of primer of ACSL1 gene the 17th exon the 46th two ends, site design with Primer5.0 software to pig, primer sequence is in Table 2.
Table 2ACSL1 gene the 17th exon primer sequence
(4) polymerase chain reaction (PCR)
(pig) DNA genomic templates solution 2ml that 25ml polymerase chain reaction system is 50ng/ml by 4 * dNTP2.5mmol/l deoxynucleoside triphosphate 2ml, 10mmol/ml upstream primer and each 0.5ml of 10mmol/ml downstream primer, 10U/ml Taq polysaccharase 0.4ml, concentration, containing 20mmol/l magnesium oxide (MgCl
2) 10 * polymerase chain reaction (PCR) damping fluid 2.5ml, add water 17.1ml to final volume 25ml; And mix.
The reaction conditions of polymerase chain reaction instrument
1. 95 ℃ of denaturations are 4 minutes;
2. 95 ℃ of sex change are 30 seconds;
3. 60 ℃ of annealing temperatures are 30 seconds;
4. 72 ℃ are extended 30 seconds;
5. repeat 2.～4. step 35 circulation;
6. 72 ℃ are extended 5 minutes, finally are cooled to 4 ℃ of preservations.
Obtain the amplification in vitro product of ACSL1 gene through above reaction, detect with 2% agarose gel electrophoresis.ACSL1 gene amplification fragment length is 292bp; See Fig. 1.Find out that by Fig. 1 band is single, specific amplification products, can carry out next step analysis nothing but;
(5) the restriction fragment enzyme of polymorphic site is cut the foundation of polymorphism (RLFP) detection method
Because the sudden change (T sports C) of the 17th exon the 46th bit base has caused the enzyme of a nucleic acid restriction endonuclease Tasl to cut pleomorphism site, it is to get the amplified production 10ml of this gene in the pipe of polymerase chain reaction (PCR) that concrete enzyme is cut step, add respectively the Tasl restriction enzyme 1ml that concentration is 10U/ml, Tasl restriction enzyme damping fluid 2ml, add water 7ml, enter after mixing under 37 ℃ of environment and react 3 hours, then use 2.5% sepharose under normal temperature environment, 120V voltage electrophoresis 1 hour, gel is placed under gel imaging system and observes, its Tasl digestion with restriction enzyme result, see Fig. 2, in ACSL1 gene the 17th exon Tasl digestion with restriction enzyme Fig. 2 of pig the 5th, 6, 8 swimming lane bands are the TT type, the 1st, 3, 7 swimming lane bands are the CC type, the 2nd, 4 swimming lane bands are the TC type, the M swimming lane is DL2000Marker.
By long-chain fatty acyl-CoA synthetase 1 (ACSL1) gene pleiomorphism to lean-type Chinese Huai pigs strain and Yorkshire swinery and the correlation analysis of thickness of backfat proterties, can obtain consistent result, be that the individual thickness of backfat of TT type is thinner, excellent genes type for Huai Pig new line and Yorkshire swinery, select TT type individuality to reserve seed for planting and can effectively reduce the thickness of backfat of pig, thereby improve the lean ratio of pig, the experiment of two kinds is described below:
Embodiment mono-:
287 Huai Pig new line II are four generation sow group's long-chain fatty acyl-CoA synthetase 1 (ACSL1) gene pleiomorphism detected results, in Table 3.The T gene frequency is that 0.472, C gene frequency is 0.528, and this swinery exists abundant polymorphism in this site.
Table 3 Huai Pig new line II is four generation swinery ACSL1 gene polymorphic site genetic parameters
The association analysis of Huai Pig new line long-chain fatty acyl-CoA synthetase 1 (ACSL1) gene pleiomorphism and the thickness of backfat
The live body thickness of backfat at the 1st rib place reciprocal while with the back fat determinator, measuring the 90kg body weight, utilize general linear model (GLM) program of SPSS13.0 software to carry out the least square analysis to the thickness of backfat of 287, and genetic effect is analyzed, the results are shown in Table 4.The individual average thickness of backfat of TT type is the thinnest is 11.28mm, significantly be thinner than TC type (P<0.05) and extremely significantly be thinner than CC type individual (P<0.01), so wild-type (TT type) individuality is the excellent genes type of this site thickness of backfat proterties.From the genetic effect analysis, dominant effect is Incomplete dominance, and the ACSL1 gene pairs thickness of backfat all shows negative additive effect; The allelic average effect value of T (thickness of backfat) is respectively-0.684, and the allelic average effect value of C (thickness of backfat) is respectively 0.612; The allelic average effect value of T allelic substitution C is-1.300.In the Huai Pig new line swinery, the TT genotype is the excellent genes type, and T allelotrope is good allelotrope, and the TT type of selecting and remain pig can effectively reduce the thickness of backfat of Huai Pig new line, thereby improves lean ratio.
Table 4ACSL1 gene pleiomorphism and thickness of backfat dependency and genetic effect analysis (mean ± standard error)
Annotate: with a line shoulder different letters of mark (a and b), mean significant difference (P<0.05); Indicate a and c and mean difference extremely significantly (P<0.01).
Embodiment bis-:
Yorkshire Pigs is world-renowned lean meat species kind, has the characteristics such as fast growth, back fat is thin, lean ratio is high, feedstuff-meat ratio is low, mainly for the production of the binary sow of hybridization system.
239 Yorkshire sow group long-chain fatty acyl-CoA synthetases 1 (ACSL1) gene pleiomorphism detected result, in Table 5.In the Yorkshire swinery, the T gene frequency is that 0.565, C gene frequency is 0.435, and this swinery exists abundant polymorphism in this site.
Table 5 Yorkshire swinery ACSL1 gene polymorphic site genetic parameter
The association analysis of Yorkshire Pigs long-chain fatty acyl-CoA synthetase 1 (ACSL1) gene pleiomorphism and the thickness of backfat
The live body thickness of backfat at the 1st rib place reciprocal while with the back fat determinator, measuring the 90kg body weight, utilize general linear model (GLM) program of SPSS13.0 software to carry out the least square analysis to the thickness of backfat of 239 Yorkshire Pigs, and genetic effect is analyzed, the results are shown in Table 6.The individual average thickness of backfat of TT type is the thinnest is 9.35mm, significantly be thinner than TC type (P<0.05) and extremely significantly be thinner than CC type individual (P<0.01), so wild-type (TT type) individuality is the excellent genes type of this site thickness of backfat proterties.From the genetic effect analysis, dominant effect is Incomplete dominance, and the ACSL1 gene pairs thickness of backfat all shows negative additive effect; The allelic average effect value of T (thickness of backfat) is respectively-0.551, and the allelic average effect value of C (thickness of backfat) is respectively 0.716; The allelic average effect value of T allelic substitution C is-1.267.Therefore, in the Yorkshire swinery, the excellent genes type is the TT type, and good allelotrope is T, and the TT type of selecting and remain individual pig can reduce the thickness of backfat of pig, reduces fatty deposition, thereby improves the lean ratio of Yorkshire Pigs.
Table 6ACSL1 gene pleiomorphism and thickness of backfat dependency and genetic effect analysis (mean ± standard error)
Annotate: with a line shoulder different letters of mark (a and b), mean significant difference (P<0.05); Indicate a and c and mean difference extremely significantly (P<0.01).
Conclusion
Correlation analysis by two embodiment is found, in Huai Pig new line and Yorkshire swinery, this site is significantly relevant to thickness of backfat proterties, in the Huai Pig new line swinery, the individual thickness of backfat of TT type is than the individual thin 2.63mm of CC type, and the individual thickness of backfat of TT type is than the individual thin 1.66mm of TC type; In the Yorkshire swinery, the individual thickness of backfat of proofreading and correct of TT type is than the individual thin 2.52mm of CC type, and the individual thickness of backfat of TT type is than the individual thin 1.20mm of TC type.In Huai Pig new line and Yorkshire Pigs colony, wild-type individual (TT type) shows stronger advantage on thickness of backfat proterties, and TT is the excellent genes type of thickness of backfat proterties; The allelic average effect value of T allelic substitution C is negative effect in Huai Pig new line and Yorkshire swinery, the good allelotrope that shows the thickness of backfat that T allelotrope is this colony, improve the allelic frequency of T and can reduce the thickness of backfat of this colony, can improve the lean ratio of two colony pigs simultaneously.
Claims (3)
1. the molecule marking method of a fat thickness at back of pig proterties is characterized in that comprising following operation steps:
(1) extraction of pig genomic dna;
(2) long-chain fatty acyl-CoA synthetase 1(ACSL1) gene the 17th exon design of primers, described primer is upstream primer and downstream primer, its sequence is as follows:
Upstream primer: 5 '-ACAGGCTCACTTCGCAGGTAGAT-3 ',
Downstream primer: 5 '-GTTGGCTTGGAACCACTATTTGC-3 ';
(3) amplification in vitro
Adopt polymerase chain reaction, obtain pig long-chain fatty acyl-CoA synthetase 1(ACSL1) the amplification in vitro product of gene the 17th exon;
(4) genotype detection
The reaction of employing digestion with restriction enzyme, obtain enzyme and cut product; Enzyme is cut to product and carry out polymorphic site restriction fragment enzyme and cut polymorphism (RLFP) and detect, selecting wherein band is that TT type individuality is reserved seed for planting;
In ACSL1 gene the 17th exon Tasl digestion with restriction enzyme Fig. 2 of pig, the 5th, 6,8 swimming lane bands are the TT type, and the 1st, 3,7 swimming lane bands are the CC type, and the 2nd, 4 swimming lane bands are the TC type, and the M swimming lane is DL 2000 Marker.
2. the molecule marking method of a kind of fat thickness at back of pig proterties according to claim 1, it is characterized in that: described amplification in vitro is polymerase chain reaction, and concrete operation step is as follows:
A, preparation polymerase chain reaction system
By the pig DNA genome solution 2 μ l of each 0.5 μ l of 4 * dNTP2.5 mmol/ l deoxynucleoside triphosphate, 2 μ l, 10 mmol/ml upstream primers and 10 mmol/ml downstream primers, 10U/ μ l Taq polysaccharase 0.4 μ l, concentration 50ng/ μ l, containing 20 mmol/l magnesium chloride (MgCl
2) 10 * polymerase chain reaction (PCR) damping fluid 2.5 μ l, add water 17.1 μ l to final volume 25 μ l; And mix, make 25 μ l polymerase chain reaction systems;
B, polymerase chain reaction condition
25 μ l polymerase chain reaction (PCR) systems are put into to the polymerase chain reaction instrument, and reaction conditions is as follows:
1. 95 ℃ of denaturations are 4 minutes,
2. 95 ℃ of sex change are 30 seconds,
3. 60 ℃ of annealing temperatures are 30 seconds,
4. 72 ℃ are extended 30 seconds,
5. repeat 2.～4. step 35 circulation,
6. 72 ℃ are extended 5 minutes, finally are cooled to 4 ℃ of preservations, obtain pig long-chain fatty acyl-CoA synthetase 1(ACSL1) the amplification in vitro product of gene the 17th exon; The pig long-chain fatty acyl-CoA synthetase 1(ACSL1 of described amplification in vitro product) gene the 17th exon the 46th bit base sports C by T.
3. the molecule marking method of a kind of fat thickness at back of pig proterties according to claim 1, it is characterized in that: the concrete operations of described genotype detection are as follows:
(1) digestion with restriction enzyme reaction, polymorphic site restriction fragment enzyme is cut polymorphism (RLFP) and is detected
Due to pig long-chain fatty acyl-CoA synthetase 1(ACSL1) gene the 17th exon the 46th bit base sports C by T, caused the enzyme of a Tasl restriction enzyme to cut pleomorphism site, concrete endonuclease reaction operation steps is: get pig long-chain fatty acyl-CoA synthetase 1(ACSL1) the amplification in vitro product 10 μ l of gene the 17th exon are in the pipe of polymerase chain reaction (PCR), the Tasl restriction enzyme 1 μ l that adds respectively 10U/ μ l, Tasl restriction enzyme damping fluid 2 μ l, add water 7 μ l, mix, 37 ℃ of reactions of temperature 3 hours, obtain enzyme and cut product,
(2) polymorphic site restriction fragment enzyme is cut polymorphism (RLFP) detection
Enzyme is cut to 2.5% agarose gel electrophoresis 1 hour for product, deposition condition: normal temperature, voltage 120V, obtain cutting containing enzyme the gel of product; The gel that to cut product containing enzyme is placed under gel imaging system observes genotype, in ACSL1 gene the 17th exon Tasl digestion with restriction enzyme figure of pig, the 5th, 6,8 swimming lane bands are the TT type, 1st, 3,7 swimming lane bands are the CC type, 2nd, 4 swimming lane bands are the TC type, and the M swimming lane is DL 2000 Marker; TT type individuality is thickness of backfat proterties excellent genes type, and selecting wherein band is that TT type individuality is reserved seed for planting, and can reduce the thickness of backfat of pig, improves the lean ratio of the colony of selecting and remain.
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