CN103146697A - Single nucleotide polymorphism of Blackett black cow H-FABP gene and detecting method thereof - Google Patents
Single nucleotide polymorphism of Blackett black cow H-FABP gene and detecting method thereof Download PDFInfo
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Abstract
The invention discloses a polymorphic site of the second intron sequence of a Blackett black cow H-FABP gene and a detecting method thereof. The single nucleotide polymorphism sequence of the Blackett black cow H-FABP gene is obtained by the following steps of: based on a Blackett black cow whole genome DNA (deoxyribonucleic acid) sequence including an H-FABP gene to be detected as a template and a primer pair P as a primer, amplifying the second intron of the Blackett black cow H-FABP gene through polymerase chain reaction (PCR); implementing single stranded conformational polymorphism (SSCP) detection to the PCR product and typing the gene; and sequencing a homozygote of genetic typing. On the basis of PCR-SSCP and sequencing result analysis, the second intron of the H-FABP is either G or C on 872nd site. The H-FABP gene has a great significance on beef tenderness, and intramscular fat content and marbling enrichment degree of AA genotype are remarkably better than those of an AB genotype and a BB genotype, so that the AA genotype can be used as a molecular marker of high-quality meat cow variety breeding, thereby providing an important technical support for breeding of the Blackett black cow excellent variety.
Description
Technical field
The invention belongs to the molecular genetics field, relate to gene mononucleotide polymorphism and detection thereof, particularly the black ox H-FABP gene mononucleotide polymorphism of a kind of cloth Lay Kate and detection method thereof.
Background technology
Cloth Lay Kate is black, and ox is on the basis of modern biotechnology and conventional improved crossing method, by Introduced from Japan and ox, Luxi Yellow cattle and Bohai Black Cattle is carried out the good beef cattle germ plasm resource (Li Xingfang, 2010) that the genetic material improvement obtains.It is with fine and tender taste, and marbling is abundant and famous.Intramuscular fat (Intramuscular Fat, IMF) content is the important indicator (Sun Haoxue etc., 2006, Zhu Qi etc., 2005) that affects the ediblenesies such as tenderness of beef utilizing, local flavor, succulence and mouthfeel.The IMF content of 10% left and right can produce desirable marbling, forms top grade beef (ZHU GUO-li, et al, 2005).Abundant marbling not only can reduce the variation of beef tender degree in gastronomical process, and is the important references of estimating Quality Beef and grade.Fatty acid binding protein (Fatty acid-binding proteins, FABPs) participate in lipid acid transportation, adjusting fatty acid concentration in myocardial cell and adipocyte, promote the deposition of triglyceride level, the effect (Veerkamp JH, 1993 that increase IMF content are arranged; Veerkamp JH, et al, 1995; Cameron N D, et al, 1991).Gerbens etc. (1999) are to H-FABP (Heart fatty acid-binding protein, H-FABP) gene studies discovery, and the H-FABP gene can be used as the candidate gene that affects IMF content.Li Xingfang etc. (2010) measure the sequence of the black ox H-FABP gene of cloth Lay Kate.In view of above result of study, this experimental study is take the variant sites of H-FABP gene intron2 as research object, adopt the PCR-SSCP method, to intramuscular fat content between the different genotype effect value and marbling grade scoring significance test of difference, the cultivation of deceiving the ox new variety for cloth Lay Kate provides reference in conjunction with the least square linear model.
The variations such as single nucleotide polymorphism (SNP) refers to change on a certain specific nucleotide position in genomic dna, transversion, insertion or disappearance.Single nucleotide polymorphism has that quantity is many, the characteristics such as wide and genetic stability that distribute, and is a kind of good means of carrying out molecular genetic breeding.
Summary of the invention
Technical problem to be solved by this invention is to provide the detection method of the black ox H-FABP gene mononucleotide polymorphism of a kind of cloth Lay Kate, searches out the SNP relevant to the black beef matter proterties of cloth Lay Kate, for its molecular marker breeding lays the foundation.
The present invention is achieved by the following technical solutions:
A kind of cloth Lay Kate deceives ox H-FABP gene mononucleotide polymorphism sequence, is in the 872nd nucleotide polymorphisms sequence for C or G of the black ox H-FABP gene intron 2 of cloth Lay Kate.
The detection method of the black ox H-FABP gene mononucleotide polymorphism of cloth Lay Kate comprises the following steps:
Deceive ox complete genome DNA sequence as template take the cloth Lay Kate to be measured who comprises the H-FABP gene, take primer pair P as primer, pcr amplification cloth Lay Kate deceives ox H-FABP gene intron 2; The PCR product is carried out that SSCP detects and to gene type; Homozygote order-checking to gene type obtains the black ox H-FABP gene mononucleotide polymorphism sequence of cloth Lay Kate.
Described primer pair P is:
Upstream primer: 5 '-TTCCTCCAGCAGCACCTTTC-3 ';
Downstream primer: 5 '-CCTCCTCATTCCCATTCCTC-3 '.
Described PCR reaction system is: 10 * Buffer2.0 μ L, 20 μ mol/L upstream and downstream each 0.15 μ L of primer, 2mmol/LdNTPs2 μ L, 5U/ μ LTaq enzyme 0.2 μ L, the masterplate DNA1 μ L of 50ng/L, the Mg of 25mmol/L
2+2 μ L, ultrapure water 12.5 μ L amount to 20 μ L.
Described pcr amplification program is: 95 ℃ of denaturation 5min, and 95 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 30 circulations, 72 ℃ are extended 10min again, are down at last 4 ℃.
Described SSCP detects application 6% polyacrylamide gel electrophoresis and detects.
The black ox H-FABP gene mononucleotide polymorphism sequence of described cloth Lay Kate and detection method can also comprise the detection to the allelic gene frequency of pleomorphism site and genotype frequency.
The black ox H-FABP gene mononucleotide polymorphism sequence of described cloth Lay Kate and detection method can also comprise the correlation analysis of H-FABP gene genetic polymorphism and Meat Quality, preferably the SPSS17.0 statistical software.
The invention has the beneficial effects as follows:
Find first the base mutation of C → G at the black ox H-FABP gene intron 2 872bp of cloth Lay Kate, the AA(CC homozygote) genotype and AB(CG heterozygote), the BB(GG homozygote) genotype compares at intramuscular fat content and marbling and enriches significant difference on degree (P＜0.05), and AB, BB genotype difference not significantly (P〉0.05), namely the genotypic intramuscular fat content of AA and marbling enrich degree and significantly are better than AB genotype and BB genotype.Hence one can see that, the variation in H-FABP gene intron 2 site affects tenderness of beef utilizing, this variant sites has impact more significantly to meat, therefore in the black ox breeding of cloth Lay Kate, can pass through to select the AA genotype as the assisted Selection foundation that improves intramuscular fat content and marbling demeanour degree.
For the black ox H-FABP gene SNP polymorphism of above-mentioned cloth Lay Kate, detection method provided by the invention is simple, quick, with low cost, tolerance range is high.
Description of drawings
Fig. 1 is the black ox complete genome DNA sepharose detected result (the Far Left swimming lane is DL15000DNA Marker) of cloth Lay Kate;
Fig. 2 is H-FABP gene intron 2 Partial Fragment amplification (the Far Left swimming lane is DL100DNA Marker);
Fig. 3 is H-FABP gene intron 2 PCR-SSCP polymorphic detection result;
Fig. 4 a and Fig. 4 b are respectively the black ox H-FABP gene intron 2 amplified fragments AA of cloth Lay Kate and BB genotype order-checking peak figure.
Embodiment
The present invention will be further described below in conjunction with accompanying drawing.
1. extracting genome DNA
Take respectively 35 adult cloth Lay Kates to deceive the ears of an ox or cow sample, use the tissue gene group DNA extraction test kit of TIANGEN Biotech (Beijing) Co., Ltd. to extract genomic dna from the black the ears of an ox or cow tissue of cloth Lay Kate, concrete steps are as follows:
(1) take the black the ears of an ox or cow sample tissue of 10mg cloth Lay Kate and be put in the 1.5mL centrifuge tube, shred with the operating scissors after sterilization as far as possible, get 200 μ L damping fluid GA and add wherein, concussion makes its thorough suspension.
(2) add the Proteinase K solution of 20 μ L, mixing.Put into 56 ℃ of water-bath 1.5h of water-bath, during put upside down the mixing sample 2-3 time, to complete digestion, then brief centrifugal to remove the globule of cap wall.
(3) add 200 μ L damping fluid GB, put upside down mixing, place 10min in 70 ℃ of water-baths, it is limpid that solution becomes, and brief centrifugal removing managed the globule in lid.
(4) get 200 μ L dehydrated alcohols and add mentioned solution, vibration mixing 15sec on vibrator, the brief centrifugal interior globule of pipe lid of removing.
(5) previous step gained solution and floss are all joined in an adsorption column CB3, adsorption column is put into collection tube, in the centrifugal 30sec of whizzer 12000rpm, adsorption column is put back in collection tube again after outwelling waste liquid.
(6) add in the adsorption column 500 μ L to add the damping fluid GD of dehydrated alcohol, the centrifugal 30sec of 12000rpm, abandon waste liquid after adsorption column be put in collection tube.
(7) the rinsing liquid PW that adds 700 μ L to add dehydrated alcohol carries out rinsing, the centrifugal 30sec of 12000rpm, abandon waste liquid after adsorption column put into again collection tube.
(8) repetitive operation step 7, post rinse one time.
(9) after adsorption column was put in collection tube, the centrifugal 2min of 12000rpm after outwelling waste liquid, was put in room temperature 10 minutes with adsorption column, thoroughly dried rinsing liquid remaining in material, took a smell without the smell of ethanol.
(10) above-mentioned adsorption column is put into a clean centrifuge tube that cuts off upper cover, unsettled to adsorption column adsorption film middle part dropping 100 μ L elution buffer TE, room temperature is placed 2-5min, the centrifugal 2min of 12000rpm.
(11) for increasing the yield of genomic dna, with solution obtained above unsettled joining on the adsorption column adsorption film again, room temperature is placed 2min, the centrifugal 2min of 12000rpm.
(12) the genomic dna solution that obtains is transferred in a clean centrifuge tube, got 2 these solution of μ L and detect its purity and concentration with 0.8% agarose gel electrophoresis, preserve in ℃ refrigerator of remaining being put in-20.
Portion gene group DNA electrophoresis result is extracted the genomic dna band clear as shown in Figure 1 as can be known, without serious conditions of streaking, illustrates that the DNA integrity is better, and concentration is higher, and the masterplate that can be used as PCR uses, and meets the follow-up test requirement.
2. design of primers is with synthetic
Sequence (Acc.No.:NW_001494693.2) according to the common ox H-FABP gene of announcing on ncbi database, use Primer Premier5.0 to carry out design of primers to H-FABP gene intron 2 partial sequence, purpose fragment overall length is 333bp, and carries out the synthetic of primer by Shanghai living work biotechnology company limited.
Primer sequence is:
Upstream primer (SEQ ID No.1): 5 '-TTCCTCCAGCAGCACCTTTC-3 '
Downstream primer (SEQ ID No.2): 5 '-CCTCCTCATTCCCATTCCTC-3 '
3.PCR amplification
The PCR reaction system is: 10 * Buffer2.0 μ L, 20 μ mol/L upstream and downstream each 0.15 μ L of primer, 2mmol/L dNTPs2 μ L, 5U/ μ LTaq enzyme 0.2 μ L, the masterplate DNA1 μ L of 50ng/L, the Mg of 25mmol/L
2+2 μ L, ultrapure water 12.5 μ L amount to 20 μ L.
The pcr amplification program is: 95 ℃ of denaturation 5min, and 95 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 30 circulations, 72 ℃ are extended 10min again, are down at last 4 ℃.
The PCR product detects PCR product specificity with 1.5% agarose gel electrophoresis, the part amplification as shown in Figure 2, the product size is consistent with the expectation clip size, and specific band nothing but, can be used for follow-up test.
4.PCR-SSCP analyze
Above-mentioned pcr amplification product is added the sex change sample-loading buffer according to volume ratio 1:1 ratio, and the sex change after product carries out single-strand conformation polymorphism analysis (SSCP) in 6% polyacrylamide gel.And carry out the band statistics, and then to its gene type, selecting wherein, homozygote carries out cloning and sequencing.
4.1 polyacrylamide gel electrophoresis
(1) sheet glass is processed: supporting sheet glass, comb and edge strip are cleaned dried, wipe one time with cotton ball soaked in alcohol, after drying, draw a small amount of (approximately 1000 μ L) and peel off silane on thieving paper, rapidly comprehensive wiping notch board (upper glass plates), room temperature is placed 5-10min; Then draw the affine solution of silane of a small amount of dilution on thieving paper, rapidly comprehensive wiping dull and stereotyped (lower glass plate) a time is treated 2-3min, then draws a small amount of wiping one time that room temperature is placed 5-10min.
(2) assembling sheet glass: flat board is lain against on a horizontal medium higher than desktop, and edge strip is put on dull and stereotyped both sides well, covers notch board, and plate both sides fix with 12 clip symmetries.
(3) preparation of poly-propionic acid amide gel: take out 30% acrylamide, 10% ammonium persulphate and the TEMED that have prepared from 4 ℃ of refrigerators, pipette 10mL30% acrylamide, 10mL5 * TBE, 30mL distilled water with transfer pipet, pipetting 350 μ L10% ammonium persulphates, 20 μ L TEMED with pipettor, rapidly stirring and evenly mixing.Attention: acrylamide, ammonium persulphate and TEMED all have neurotoxicity, will wear gloves and mouth mask when taking.
(4) encapsulating: with pipette, extract above-mentioned coagulant liquid, slowly inject from sheet glass recess one end, simultaneously with rubber suction bulb tapping board upper surface gently, make glue rapidly mobile and evenly in plate, until be full of glue in plate.
(5) insert comb: slowly insert the comb butt end from notch end, to avoid Bubble formation as far as possible, if generation bubble, to take away bubble several clips on one side, slowly lever up sheet glass with little spades, until bubble collapse, then put down gently sheet glass, clip clip, add from the another recess the other end glue that lacks.In glue is full of plate and when there is no bubble, the room temperature water placing flat treats that gelling is solid.
(6) prerunning: after gelling is good, the comb butt end is slowly extracted, inserted rapidly nose of comb, broach inserts in glue approximately 1-2mm.Take off plate both sides clip, sheet glass is moved in Vertial electrophorestic tank, both sides fix, then pour appropriate 1 * tbe buffer liquid in the electrophoresis chamber of up and down, with syringe absorption electrophoresis liquid cleaning point sample hole, to remove bubble and broken glue wherein, be plugged at last electrode, 300V electrophoresis 30min.
(7) the PCR product is processed: add sex change sample-loading buffer (deionized formamide 98mL, EDTA(0.5M according to volume ratio 1:1 ratio in the PCR product, pH is 8.0) 2mL, tetrabromophenol sulfonphthalein 0.25g, dimethylbenzene cyanogen 0.25g, mixing), then 98 ℃ of sex change 10min in the PCR instrument insert rapidly ice bath 10min in ice.
(8) point sample: draw PCR product after 2.5 μ L sex change with pipettor, click and enter successively in the point sample hole.
(9) electrophoresis: 4 ℃, 300V electrophoresis 5min then changes 200V electrophoresis 10h into.
(10) finish electrophoresis: close electrophoresis apparatus, reclaim electrophoretic buffer, turn on the screw of electrophoresis chamber both sides fastening glass panels, taking off sheet glass is put on the horizontal medium of desktop, pull out the edge strip on comb and both sides, Yi Bian carefully lever up notch board with little spades from sheet glass flat mouth bottom, prepare silver and dye.
4.2 polyacrylamide gel silver dyes
(1) fixing: the sheet glass that will remove the top notch board is put in and fills 2L and fix/tank of stop buffer in, fixing 10min, during vibrate several times back and forth gently.Then take out sheet glass and be put in the tank that fills 2L distilled water, rinse gently sheet glass twice, rinse out unnecessary stationary liquid and not securing DNA.
(2) dyeing: sheet glass is put in the 20min that dyes in the tank that fills the 2L staining fluid, during gently back and forth the vibration several times.Then clean twice rapidly in distilled water, wash out the unnecessary staining fluid in glue surface.
(3) colour developing: sheet glass is put into rapidly the tank colour developing that fills 2L nitrite ion (now adding before use 3mL formaldehyde), during constantly vibrate gently, until clearly band occurs.General 5-10min, the time is unsuitable long, and the background that prevents from developing the color is excessively dark.
(4) stop: the sheet glass that will occur clear band is put into rapidly fixedly color development stopping in the fixing of use/stop buffer of the first step.
(5) reclaim each silver-colored transfection reagent, and sheet glass is pulled out from fixing/stop buffer, be put on horizontal plane and naturally dry.
(6) the good sample sequence number of mark, take pictures with gel imaging system.
The PCR product presents different bands after polyacrylamide gel electrophoresis silver dyes, result as shown in Figure 3.As shown in Figure 3, two allelotrope (A and B) detected in H-FABP gene First Exon, 3 kinds of genotype (AA, BB and AB).
4.3 band statistics
Dye band quantity and the position of each sample of interpretation of result according to silver, the genotype of establishing each sample, and each genotypic number of samples.
5. homozygote cloning and sequencing
5.1PCR amplification
Each primer amplification fragment is selected two homozygote genotype samples after sscp analysis, it is multiple that each genotype is selected two individual weights, according to above-mentioned pcr amplification reaction system and response procedures, carries out pcr amplification.Get 3 μ L amplified productions, add 1 μ L6 * Loading buffer mixing, point sample in 2% sepharose, 120V electrophoresis 25min.
5.2PCR product is cut glue and is reclaimed
Use the plain agar sugar gel DNA recovery test kit of TIANGEN Biotech (Beijing) Co., Ltd. to cut glue recovery purifying to pcr amplification product, step is as follows:
(1) each primer is got above-mentioned 4 2% agarose gel electrophoresis detected results pcr amplification product preferably, join in a new little centrifuge tube, add 15 μ L6 * Loading buffer, mixing, adding the point sample hole width is in 1.5% sepharose of 2cm, 120V electrophoresis 30min.Put into the EB solution 18min that dyes.
(2) after centrifuge tube is weighed, put on label, under ultraviolet lamp, cut the target DNA fragment band, cut away the gel that does not contain the DNA band as far as possible, put into clean centrifuge tube, weigh.
(3) add the sol solutions PN of 3 times of volumes in the centrifuge tube, 10min are placed in 50 ℃ of water-baths, during constantly the upset centrifuge tube several times, guarantee that gel all dissolves.
(4) balance of adsorption column: adsorption column is put into collection tube, add 500 μ L balance liquid BL, the centrifugal 1min of 12000rpm after outwelling waste liquid, then puts back to adsorption column in collection tube.
(5) step (2) gained solution is cooled to room temperature after, join in the good adsorption column of balance, room temperature is placed 2min, then the centrifugal 60sec of 12000rpm, outwell waste liquid, then adsorption column put back in collection tube.
(6) add 600 μ L to add the rinsing liquid PW of dehydrated alcohol in adsorption column, after standing 3min, the centrifugal 60sec of 12000rpm outwells waste liquid, then adsorption column is put back in collection tube.
(7) repetitive operation step 5.
(8) adsorption column is put back to collection tube after, the centrifugal 2min of 12000rpm.Adsorption column is positioned over room temperature 10min left and right, thoroughly dries, affect follow-up test to prevent residual rinse liquid.
The adsorption column that (9) will dry is put in a centrifuge tube of cutting upper cover, and to the unsettled dropping 40 μ L elution buffer EB of adsorption film middle position, room temperature is placed 2min, and the centrifugal 2min of 12000rpm obtains target DNA solution.
(10) the centrifugal target DNA solution that obtains of upper step is added drop-wise on adsorption film again, the standing 2min of room temperature, the centrifugal 2min of 12000rpm collects target DNA solution in a new centrifuge tube.
(11) get above-mentioned purpose DNA solution 5 μ L, add 1 μ L6 * Loading buffer, be splined on electrophoresis detection in 1.5% sepharose, remaining target DNA solution is put in-20 ℃ and saves backup.
5.3 being connected of target DNA and T-carrier, conversion
(1) Bechtop sterilization: first with 75% alcohol with table surface and the instrument wiping that will use a time, the article such as rifle head, centrifuge tube, centrifuge tube shelf of sterilization are put into Bechtop, then open ultra violet lamp 20min, then open blower fan and blow 5min and get final product.
(2) take out the T-carrier from-20 ℃ of refrigerators and connect test kit, be put in dissolving on ice.On the Bechtop after sterilization, add successively following linked system in the 200 μ L centrifuge tubes: Solution I 2.5 μ L, PCR purified product 2.0 μ L(50ng/ μ L), pMD19-T Vector(50ng/ μ L) 0.5 μ L, total amount is 5 μ L.Mixing, 16 ℃ of standing and reacting 30min.
(3) taking out the TOP10 competent cell from-80 ℃ of refrigerators is placed on ice, when it just thaws (approximately 5-10min), above-mentioned linked system full dose 5 μ L are joined in 50 μ L competent cells after mixing, sample is changed in the centrifuge tube of 1.5mL, then be put in rapidly ice bath 30min in ice.
(4) bath of fetching boiling water, temperature setting is set to 42 ℃, opens in advance simultaneously constant incubator and constant-temperature table, and Temperature Setting is 37 ℃.
(5) put into rapidly 42 ℃ of water-bath heat shock 45sec after ice bath finishes, then put into rapidly ice ice bath 2min, during do not rock sample, handle with care.
Take out in (6) 4 ℃ of refrigerators and do not add the LB liquid culture medium of penbritin (Amp), and add 450 μ L not contain the liquid LB substratum of Amp, 37 ℃ of 200rpm shaking culture 90min in the sample that takes out in Xiang Congbing.
(7) repetitive operation step 1.
(8) getting the 100 above-mentioned cultures of μ L evenly coats on good solid-state LB substratum with spreader and (is added with Amp
+, ITPG and X-gal), remaining culture can be kept in 4 ℃ of refrigerators, reuses when bad in order to the coated plate effect.
(9) plate that coats just is being put in 37 ℃ of constant incubators and is cultivating 30min, then is inverted overnight incubation (approximately 14-16h).
5.4 the evaluation of recombinant plasmid
(1) take out the culture dish of overnight incubation, observe the upgrowth situation of dull and stereotyped upper bacterium.As stand density, can adjust as required the bacterium liquid measure of next coated plate; The bacterium distribution situation, whether what be coated with in the time of can judging coated plate accordingly is even; Positive bacterium colony, positive bacterium colony is hickie, negative bacterium colony is locus coeruleus; The bacterium colony size can be adjusted dull and stereotyped incubation time accordingly.
(2) cultured flat board is sealed with sealed membrane, and be put in 4 ℃ of refrigerators and preserved 8 hours, choose bacterium when blue hickie is more obvious.
(3) in the Bechtop after sterilization, the single colony inoculation of picking white is in the liquid LB/Amp that fills 500 μ L37 ℃ preheatings from the culture dish of overnight incubation
+In the 1.5mL centrifuge tube of substratum, with being put in 200rpm/min overnight incubation in 37 ℃ of shaking tables after the sealed membrane sealing.
(4) preserve being put in 4 ℃ of refrigerators after the flat board sealing, unsuccessfully again choose bacterium in order to checking order.
(5) bacterium liquid PCR detects: operate in the Bechtop of sterilization, the PCR reaction system is: 10 * Buffer2.0 μ L, 20 μ mol/L upstream and downstream each 0.15 μ L of primer, 2mmol/L dNTPs2 μ L, 5U/ μ LTaq enzyme 0.2 μ L, bacterium liquid 1 μ L, the Mg of 25mmol/L
2+2 μ L, ultrapure water 12.5 μ L amount to 20 μ L.
The pcr amplification program is: 95 ℃ of denaturation 5min, and 95 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 30 circulations, 72 ℃ are extended 10min again, are down at last 4 ℃.
(3) result is identified: bacterium liquid PCR result detects through 1.5% agarose gel electrophoresis, has or not and size according to amplified fragments, identifies whether the external source target DNA inserts in plasmid.
5.5 cloned sequence sequencing
Above-mentioned positive colony bacterium liquid is served sea living work biotechnology company limited check order, each sample send surveys 3 repetitions, to guarantee the accuracy that checks order.
Two kinds of homozygous PCR product cloning sequencing results of AA and BB such as Fig. 4 a and 4b find B allelotrope in the sudden change of H-FABP gene intron 2 872bp place's generation C → G, and the AA genotype is the CC homozygote, and the BB genotype is the GG homozygote.The genotypic sequence of AA is as shown in SEQ ID No.3, and the genotypic sequence of BB is as shown in SEQ ID No.4.
6. Meat Quality is measured
Choose when butchering that between trunk the 12nd～13 rib, longissimus dorsi muscle is the measuring point, for mensuration and the marbling grade scoring of IMF.The mensuration of IMF content adopts the soxhlet extraction method, measures with reference to GB/T9695.7-2008.Marbling grade scoring is with reference to 7 grades of scorings of NY/T676-2003(People's Republic of China's agricultural industry evaluation criteria marbling) evaluation.
7. data statistic analysis
In conjunction with SSCP collection of illustrative plates and sequencing result, genotype frequency and gene frequency are carried out statistical computation.Because individual of sample is selected from same kind, same plant, same fatten batch, so press Y
i=μ+g
i+ e
iModel carries out the analysis of genotype effect value, Y
iBe the measured value of the individual Meat Quality of i kind genotype, μ is the least square average of Meat Quality, g
iBe that i kind genotype is to the effect value of Meat Quality, e
iRandom residual effect for measured value.Adopt the dependency of SPSS17.0 statistical software analysis H-FABP gene genetic polymorphism and Meat Quality.
The distribution of the black ox H-FABP genotype frequency of table 1 cloth Lay Kate and gene frequency
Least square average and the standard error of the black ox H-FABP different genotype of table 2 cloth Lay Kate and Meat Quality
Annotate: have significant difference (P＜0.05) between different letter shoulder target mean values
As can be seen from Table 2, the genotypic intramuscular fat content of AA will be significantly higher than AB, BB genotype (P＜0.05), and AB genotype and BB genotype difference not significantly (P〉0.05).In marbling grade evaluation NY/T676-2003 standard, number of degrees is higher, and the marbling richness is poorer, and in associative list 2, the marbling significance as can be known, AA genotype marbling is than AB, BB genotype significantly (P＜0.05), difference not significantly (P〉0.05) between AB and BB.
Claims (9)
1. the black ox H-FABP gene mononucleotide polymorphism sequence of cloth Lay Kate, is characterized in that, is the 872nd nucleotide polymorphisms sequence for C or G of the black ox H-FABP gene intron 2 of cloth Lay Kate.
2. the detection method of the black ox H-FABP gene mononucleotide polymorphism of cloth Lay Kate, is characterized in that, comprises the following steps:
Deceive ox complete genome DNA sequence as template take the cloth Lay Kate to be measured who comprises the H-FABP gene, take primer pair P as primer, pcr amplification cloth Lay Kate deceives ox H-FABP gene intron 2; The PCR product is carried out that SSCP detects and to gene type; Homozygote order-checking to gene type obtains the black ox H-FABP gene mononucleotide polymorphism sequence of cloth Lay Kate.
3. the detection method of the black ox H-FABP gene mononucleotide polymorphism of cloth Lay Kate according to claim 2, is characterized in that, described primer pair P is:
Upstream primer: 5 '-TTCCTCCAGCAGCACCTTTC-3 ';
Downstream primer: 5 '-CCTCCTCATTCCCATTCCTC-3 '.
4. cloth Lay Kate according to claim 2 deceives the detection method of ox H-FABP gene mononucleotide polymorphism, it is characterized in that, described PCR reaction system is: 10 * Buffer2.0 μ L, 20 μ mol/L upstream and downstream each 0.15 μ L of primer, 2mmol/LdNTPs2 μ L, 5U/ μ LTaq enzyme 0.2 μ L, the masterplate DNA1 μ L of 50ng/L, the Mg of 25mmol/L
2+2 μ L, ultrapure water 12.5 μ L amount to 20 μ L.
5. cloth Lay Kate according to claim 2 deceives the detection method of ox H-FABP gene mononucleotide polymorphism, it is characterized in that, described pcr amplification program is: 95 ℃ of denaturation 5min, 95 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 30 circulations, 72 ℃ are extended 10min again, are down at last 4 ℃.
6. the detection method of the black ox H-FABP gene mononucleotide polymorphism of cloth Lay Kate according to claim 2, is characterized in that, described SSCP detects application 6% polyacrylamide gel electrophoresis and detects.
7. the detection method of the black ox H-FABP gene mononucleotide polymorphism of cloth Lay Kate according to claim 2, is characterized in that, also comprises the detection to the allelic gene frequency of pleomorphism site and genotype frequency.
8. the detection method of the black ox H-FABP gene mononucleotide polymorphism of cloth Lay Kate according to claim 2, is characterized in that, also comprises the correlation analysis of H-FABP gene genetic polymorphism and Meat Quality.
9. the detection method of the black ox H-FABP gene mononucleotide polymorphism of cloth Lay Kate according to claim 8, is characterized in that, the correlation analysis of described H-FABP gene genetic polymorphism and Meat Quality adopts the SPSS17.0 statistical software.
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| CN103966209A (en) * | 2014-05-02 | 2014-08-06 | 华中农业大学 | SNP molecular marker related to intramuscular fat content characters of pigs and application of SNP molecular marker |
| CN104830838A (en) * | 2015-05-15 | 2015-08-12 | 塔里木大学 | Preparation method and application of genetic marker for high intramuscular fat (IMF) content in crureus of Baicheng fatty chicken |
| CN104830837A (en) * | 2015-05-15 | 2015-08-12 | 塔里木大学 | Preparation method of genetic marker for high intramuscular fat (IMF) content in breast muscle of Hetian black chicken and application of genetic marker |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103866002A (en) * | 2014-01-17 | 2014-06-18 | 甘肃农业大学 | Yak meat tenderness candidate gene detection kit and detection method thereof |
| CN103866002B (en) * | 2014-01-17 | 2015-08-26 | 甘肃农业大学 | Carnis Bovis grunniens tenderness candidate gene detection kit and detection method thereof |
| CN103966209A (en) * | 2014-05-02 | 2014-08-06 | 华中农业大学 | SNP molecular marker related to intramuscular fat content characters of pigs and application of SNP molecular marker |
| CN103966209B (en) * | 2014-05-02 | 2016-04-06 | 华中农业大学 | The SNP marker relevant to pig intramuscular fat content proterties and application |
| CN104830838A (en) * | 2015-05-15 | 2015-08-12 | 塔里木大学 | Preparation method and application of genetic marker for high intramuscular fat (IMF) content in crureus of Baicheng fatty chicken |
| CN104830837A (en) * | 2015-05-15 | 2015-08-12 | 塔里木大学 | Preparation method of genetic marker for high intramuscular fat (IMF) content in breast muscle of Hetian black chicken and application of genetic marker |
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| CN106577517A (en) * | 2016-12-14 | 2017-04-26 | 吉林省农业科学院 | Beef cattle fattening method adopting meat quality as goal orientation |
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