CN103361440B - LCT gene polymorphism detection method for piglet lactase digestive symptoms and applications of the method - Google Patents
LCT gene polymorphism detection method for piglet lactase digestive symptoms and applications of the method Download PDFInfo
- Publication number
- CN103361440B CN103361440B CN201310339074.3A CN201310339074A CN103361440B CN 103361440 B CN103361440 B CN 103361440B CN 201310339074 A CN201310339074 A CN 201310339074A CN 103361440 B CN103361440 B CN 103361440B
- Authority
- CN
- China
- Prior art keywords
- piglet
- lct
- gene
- utr
- polymorphism
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a LCT gene polymorphism detection method for piglet lactase digestive symptoms. Main steps of the method comprise a step of extracting tissue DNA from pig ears, designing primers, obtaining a sequence of a LCT gene 5'UTR section, and performing a PCR product clone sequencing process; a step of directly sequencing PCR-SSCP and a PCR product to perform polymorphism detection; and a step of analyzing correlation between polymorphism and piglet lactase non-persistent diarrhea symptoms. Results show that the LCT gene 5'UTR polymorphism and the piglet lactase non-persistent diarrhea are significantly correlated. The invention also discloses two pairs of primer sequences used for amplifying the LCT gene 5'UTR polymorphism, three base mutation sites of the LCT gene 5'UTR gene segment, and detection, evaluation and applications of the polymorphism for the piglet lactase non-persistent diarrhea symptoms. The detection method provides a novel molecular mark for the pig marker-assisted selection and pig breeding.
Description
Technical field
The invention belongs to animal gene engineering technology field, specifically, the present invention relates to pig Sumylact L (
lCT) gene polymorphism sites and Diagnosis and Treat pig lactose intolerance diarrhoea in application.
Background technology
Sumylact L (lactase, LCT), also known as beta-galactosidase enzymes, extensively exists in animals and plants and microorganism, can be divided into extracellular enzyme and intracellular enzyme according to source difference.Lactose hydrolysis can be glucose sugar and semi-lactosi by Sumylact L under given conditions, and then by intestinal absorption.Mammals (particularly baby), Sumylact L is mainly present in mucous membrane of small intestine brush border, in stove block distribution, the highest with activity in jejunum.After most of Mammals wean, lactose is no longer the main source of food, and lactase activity reduces gradually, but still has part population lactase activity to continue into adult life.Under normal circumstances, lactose is hydrolyzed to semi-lactosi by Sumylact L and glucose just can be absorbed and used.When lactase deficiencies, unabsorbed lactose rests in enteron aisle, and osmotic pressure in enteric cavity is raised, and cause extracellular fluid moisture to flow into enteron aisle, in enteric cavity, amount of liquid increases, and promotes intestines peristalsis, causes the symptoms such as diarrhoea.Simultaneously, when indigested lactose arrives colon, a part is resolved into the gas such as the short chain fatty acids such as lactic acid, propionic acid, butyric acid and hydrogen, methane, carbonic acid gas by intestinal bacteria, thus cause the symptoms such as abdominal distension, borborygmus, stomachache, be called lactose intolerance (Lactase non-persistent, LNP).
Research shows, people's
lCTc/T polymorphism and LNP that gene is strengthening ﹣ 13910bp place, subarea have good dependency.In European colony, be positioned at
lCTin upstream region of gene enhanser region, the mononucleotide diversity of-13910*T can affect the activation of Sumylact L promotor, and genotype C/C-13910 and lactase activity are negative correlation, and genotype C/T-13910 and T/T-13910 and lactase activity are proportionate.And about pig
lCTthe research of gene in this is what for few.Find according to applicant's test in place, the grice diarrhoea of Native Pig Breeds and wild boar, after remedy measures such as antibacterium antiviral grade is invalid, by adding Sumylact L in food, its symptom can be eased, even completely dissolve.This result point out, also there is LNP in pig, its reason may be due to
lCTgene polynorphisms causes
lCTexpress too low and cause.Therefore, pig is inquired into
lCTthe relation that gene polynorphisms and lactose intolerance are suffered from diarrhoea is significant, and the breeding for disease resistance also carrying out piglet lactose intolerance for setting up marker assisted selection technology (MAS) lays the foundation.
Summary of the invention
The object of the present invention is to provide pig Sumylact L (
lCT) gene and polymorphism thereof and the piglet lactose intolerance dependency of suffering from diarrhoea, and detecting the purposes in piglet lactose intolerance diarrhoea.
The invention provides a kind of detect pig Sumylact L (
lCT) method of gene polynorphisms, comprise step:
A, determine the Nucleotide that LCT gene 5 ' UTR region-797bp ,-308bp and-301bp locate;
B, detect whether there is single nucleotide polymorphism in described position;
Described single nucleotide polymorphism is the polymorphism that there is G → A at 5 ' UTR region-797bp, namely in the polymorphism that there is G → A corresponding to sequence the 172nd bit base place shown in SEQ ID NO.1; The polymorphism of G → C is there is, namely in the polymorphism that there is G → C corresponding to sequence the 661st bit base place shown in SEQ ID NO.1 at 5 ' UTR region-308bp; The polymorphism of A → G is there is, namely in the polymorphism that there is A → G corresponding to sequence the 668th bit base place shown in SEQ ID NO.1 at 5 ' UTR region-301bp.
Present invention also offers a kind of isolating nucleic acid, it has the sequence shown in SEQ ID NO.1, and the 172nd of this sequence is A; 661st is C; 668th is G.
Present invention also offers one group of nucleic acid primer for PCR-SSCP, it is characterized in that, its amplimer sequence as shown in SEQ ID NO:4 and SEQ ID NO:5, and amplifies the amplified production of the 172nd single nucleotide polymorphism in sequence shown in the SEQ ID NO:1 containing LCT gene 5'UTR region specifically.
Present invention also offers one group of nucleic acid primer for PCR primer direct sequencing, it is characterized in that, its amplimer sequence as shown in SEQ ID NO:6 and SEQ ID NO:7, and amplifies the amplified production of the 661st and the 668th single nucleotide polymorphism in sequence shown in the SEQ ID NO:1 containing LCT gene 5'UTR region specifically.
Present invention also offers a kind of method for detecting lactose intolerance diarrhoea susceptibility, the method is the genomic dna of the tested pig of extracting, shown in the SEQ ID NO:1 detecting the TCL gene 5'UTR region of testee, the genotype of the mononucleotide polymorphism site of the 172nd, the 661st and the 668th in sequence, detects the susceptibility that testee suffers from diarrhoea to lactose intolerance.If testee is Native Pig or wild boar, the 172nd is GG genotype or the 661st and the 668th is GAGA genotype, and the lactose intolerance diarrhoea susceptibility of testee is high.
Present invention also offers a kind of method for detecting lactose intolerance diarrhoea susceptibility, the method is the genomic dna of the tested pig of extracting, 172nd, the 661st and the 668th haplotype formed in sequence shown in the SEQ ID NO:1 in the TCL gene 5'UTR region of detection testee, detect the susceptibility that testee suffers from diarrhoea to lactose intolerance.If testee is Native Pig or wild boar, haplotype is GGA, and the lactose intolerance diarrhoea susceptibility of testee is high; If other haplotypes, the lactose intolerance diarrhoea susceptibility of testee is lower.
The result of study of contriver all shows that the pleomorphism site of LCT gene 5'UTR is suffered from diarrhoea relevant to piglet lactose intolerance, and therefore this point can by the detection as lactose intolerance diarrhoea susceptibility.Be conducive to the allelotype of lactose digestion proterties by marker assisted selection (MAS) choice of technology LCT or haplotype individuality is reserved seed for planting, eliminate cause lactose intolerance suffer from diarrhoea genotype or haplotype individuality, significantly can improve the digestion ability of population sucking piglets to lactose, reduce the diarrhoea caused by lactose intolerance, reduce piglet mortality ratio.
Accompanying drawing explanation
Fig. 1 is the pig that the present invention differentiates
lCTthe sequence alignment peak figure in SNP G-797A mutational site, gene 5'UTR region;
Fig. 2 is the pig that the present invention differentiates
lCTthe sequence alignment peak figure in gene 5'UTR region SNP G-308C and A-301G mutational site;
Fig. 3 is the genotype process decision chart of LCT gene 5'UTR region SNP G-797A, and wherein swimming lane 3,5,6 genotype is GG, and swimming lane 8,9,10,12 genotype is AA, and swimming lane 1,2,4,7,11,13 genotype is GA;
Fig. 4 is that the fluorescent signal value of LCT gene 5'UTR Zonal expression carrier in Caco-2 clone compares.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated.Should be understood that following examples only for illustration of the present invention but not for limiting scope of the present invention.
The collection of embodiment 1, sample and DNA extraction
Western pig breeds involved in the present invention (great Bai, long in vain with Duroc) piglet sample picks up from Shenyang and just becoming original seed pig farm, Native Pig and wild boar (Hebao pig, people pig, Changbai mountain wild boar) piglet sample pick up from the holy wild extraordinary animal husbandry company in Liaoning.Wherein Large White 76, landrace 73, Duroc 75, Hebao pig 87, people pig 73, Changbai mountain wild boar 83, totally 467 sucking piglets individualities are experimental animal.When feeding piglet is to 10-14 age in days, observes and whether suffer from diarrhoea, and gather ight soil.Get 2g fresh excreta to add 37 DEG C of 5ml distilled water and stir centrifugal; Get supernatant, add plumbic acetate 0.3g, 1min is boiled in water-bath; Add 2ml 7.14mol/L ammonium hydroxide again, solution becomes oyster white immediately; Continue heated and boiled 3min, leave standstill.How much lactose sxemiquantitative is carried out: sediment-free according to pink precipitate thing, lactose-content is-; Suspicious person is designated as ±; Trace pink precipitate, be designated as+; A small amount of pink precipitate is designated as ++; A large amount of pink precipitate yellowish precipitation is designated as +++.Ight soil pH is measured with accurate pH test paper.When ight soil lactose >=++, simultaneously during pH < 5.5, determine that this sample is lactose intolerance diarrhoea.Whether the piglet for diarrhoea is individual, add Sumylact L 0.3g/ head, once a day, observe afterwards for three days on end and suffer from diarrhoea and gather ight soil in feed, again carries out ight soil lactose-content and pH mensuration.If ight soil is normal, lactose intolerance diarrhoea can be confirmed as further.
Phenol chloroform isoamyl alcohol method routinely extracts genomic dna from piglet ear tissue, and concentration correction is to 100ng/ μ l.
Embodiment 2, pig
lCTthe acquisition of gene 5'UTR region nucleic acid fragment and the examination of these region SNP site
2.1, pig is inquired about from NCBI website (http://www.ncbi.nlm.nih.gov/)
lCTgene 5'UTR sequence (comprising enhanser and promotor) (GenBank:Y08677.1), applies Oligo6.71 software design pair of primers, primer pair 1:F:5'-TTC CTG AGT TCC AAA GAG TG-3'(SEQ ID NO:2); R:5'-TAG GAA CTG TTA GGA GGT ATG-3'(SEQ ID NO:3) (F represents forward primer, and R represents reverse primer), primer is synthesized by Shanghai Sheng Gong company.
2.2, the amplification of PCR
The genomic dna of order-checking examination 6 kinds of domestic and international pig kinds (Large White 16, landrace 14, Duroc 16, Hebao pig 18 and people pig 14, Changbai mountain wild boar 13).PCR reaction is totally 25 μ l, and PCR reactive component is as follows: 10 × LA PCR Buffer II ((Mg
2+plus) 2 μ L, TaKaRa LA Taq (5 U/ μ L) 0.25 μ L, dNTP Mixture (each 2.5 mM) 4 μ L, primer (20 uM) each 1 μ L, DNA profiling 50ng, add sterile purified water to 25 μ L.
The response procedures of pcr amplification is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s; 55 DEG C of annealing 30s, 72 DEG C extend 30s, totally 30 circulations, and last 72 DEG C extend 10min, 4 DEG C of preservations.The clip size of pcr amplification product is 976bp.
2.3, PCR primer reclaims purifying rear clone, conversion and DNA sequencing
Reclaim purification kit (AxyGen biotech firm) by PCR primer and PCR primer is cut glue recovery purifying.The object fragment reclaiming purifying is connected with pMD18-T carrier, the product that connection is spent the night is joined competent cell DH5a and transforms.Converted product is evenly applied to LB agar plate.Picking positive monoclonal bacterium colony enlarged culturing also extracts plasmid DNA qualification.Precious biotechnology (Dalian) company limited the plasmid DNA of qualification is sent to carry out two ends order-checking.
DNAMan software compare of analysis is adopted to sequencing result, finds 3 single nucleotide polymorphism sites, the results are shown in Table 1 and Fig. 1,2.3 single nucleotide polymorphism sites all have the sequence shown in SEQ ID NO:1.
Table 1: the distribution of the pig LCT gene 5'UTR three kinds of SNP in district and allelotrope
| SNP | Region respectively | Allelotrope |
| G-797A | Enhanser | G/A |
| G-308C | Promotor | G/C |
| A-301G | Promotor | A/G |
By analysis, SNP G-308C and A-301G is closely linked relation, and when namely SNP G-308C is G, SNP A-301G is A, and when SNP G-308C position is C, SNP A-301G position is G, therefore forms 2 kinds of promotor polymorphism GA and CG.
Embodiment 3, test swinery
lCTthe detection of gene 5'UTR district SNP G-797A
Adopt PCR-SSCP(single-strand conformation polymorphism, SSCP single strand conformation polymorphism) method.Design primer pair 2(F:5'-TAA ACA AAG CCA AGG ACA TT-3'(SEQ ID NO:4); R:5'-CTG GGG TGT ATG TGC TTG TGG-3'(SEQ ID NO:5)) amplification genes of individuals group DNA.Pcr amplification product size is 147bp.After PCR primer 95 DEG C of 3min sex change of 147bp, application of sample is in polyacrylamide gel (being formulated as of 30ml working fluid: 12ml 30% acrylamide of 12%, 3ml 50% glycerine, 3ml 10 × TBE, 210 μ l 10% over cure amino acids, 12 μ l TEMED, add 12ml pure water), carry out native polyacrylamide gel electrophoresis (130V, electrophoresis 11-14 hour), electrophoresis terminates rear employing cma staining, and takes pictures.Specifically sentence type method as shown in Figure 3.
Embodiment 4, test swinery
lCTthe detection of gene 5'UTR district SNP G-308C and A-301G
Adopt the method for PCR primer direct Sequencing.Design primer pair 3(F:5'-AAA AAG TTT GGT AAG GAC CT-3'(SEQ ID NO:6); R:5'-GGA ACT GTT AGG AGG TAT GTG-3'(SEQ ID NO:7)), carry out DNA cloning, amplified production length 316bp by warm start and grads PCR reaction.96 hole Millipore MultiScreen are adopted to carry out purifying to PCR primer.After purifying, utilize ABI PRISM Big-Dye sequencing kit, respectively forward and reverse sequencing reaction is carried out to PCR primer.Order-checking product carries out capillary electrophoresis and sequencing at ABI3730 automatic sequencer after ethanol purification, obtains sequence chromatographs ABI file.By Phred-Phrap-PolyPHRED-Consed software package carry out check order peak figure multiple ratio to the interpretation to pleomorphism site.
Embodiment 5, pig
lCTthe correlation analysis that gene 5'UTR district's polymorphism and lactose intolerance are suffered from diarrhoea
5.1, pig
lCTthe correlation analysis that gene 5'UTR district's genotype and lactose intolerance are suffered from diarrhoea
Adopt above-mentioned PCR-SSCP and PCR primer direct sequencing to judge the genotype that 467 piglet individualities carry out SNP G-797A, G-308C and A-301G, the results are shown in Table 2.
Table 2 LCT gene pleiomorphism is testing gene and the genotype frequency of swinery
The colony of western pig breeds, Native Pig and wild boar is carried out respectively to the Hardy – Weinberg equilibrium law inspection of gene and genotype frequency, except the enhanser site of western pig breeds, be all in equilibrium state.Adopt SPSS's software package (version17.0)
the correlationship that inspection statistics model analysis genotype and lactose intolerance are suffered from diarrhoea, the GG genotype of visible SNP G-797A, the GAGA genotype of SNP G-308C and SNP A-301G are suffered from diarrhoea with the lactose intolerance of Native Pig and wild boar significant correlation respectively, and other genotype and lactose intolerance in wild boar/Native Pig or western pig breeds proterties of suffering from diarrhoea is relevant not remarkable.Therefore, in kind of pig breeding, the GG genotype of SNP G-797A or the GAGA genotype individuals of SNP G-308C and SNP A-301G can be eliminated, to reduce the incidence of lactose intolerance diarrhoea.
5.2, pig
lCTthe correlation analysis that gene 5'UTR district's haplotype and lactose intolerance are suffered from diarrhoea
The individual haplotype of G-797A, G-308C and A-301G in test colony LCT gene 5'UTR district is calculated by manual type.Because promotor polymorphism site G-308C and A-301G presents complete close linkage, therefore pig LCT gene pleiomorphism only has 4 kinds of haplotypes; Haplotype is testing the distribution of colony in table 3; Adopt SPSS's software package (version17.0)
the correlationship that inspection statistics model analysis haplotype and lactose intolerance are suffered from diarrhoea.
The correlation analysis that table 3 LCT gene 5'UTR district's haplotype and lactose intolerance are suffered from diarrhoea
Can be seen by table 3, in wild boar and native pig population, haplotype GGA and lactose intolerance are suffered from diarrhoea proterties pole significant correlation, and other 3 kinds of haplotypes are relevant not remarkable.In western pig breeds, only there are 2 kinds of haplotypes, and uncorrelated with lactose intolerance proterties of suffering from diarrhoea.Therefore, plant in pig breeding, eliminate LCT gene haplotype GGA individual, retain other haplotypes individual, swinery healthy state can be improved, reduce lactose intolerance Incidence of Diarrhea.
According to table 2,3 test-results, in the cross-breeding process of the pure breeding and Native Pig or wild boar and western pig breeds that carry out Native Pig or wild boar, by marker assisted selection (MAS), eliminate the GG genotype of SNP G-797A or the GAGA genotype of SNP G-308C and SNP A-301G or haplotype GGA individual, offspring can be avoided to occur LNP, significantly improve swinery piglet digestion lactose ability, reduce because LCT lacks the grice diarrhoea caused, reduce piglet mortality ratio.
The two fluorescence report system experimentation of embodiment 6, pig LCT 5'UTR district haplotype
Choose the individual DNA sample of LCT gene 5'UTR district G-797A, G-308C and A-301G site homozygous genotype as template, adopt primer pair 4(F:5'-CTA GCT AGC AAA AAG TTT GGT AAG GAC CT-3'(SEQ ID NO:8); R:5'-CGG AAG CTT GGA ACT GTT AGG AGG TAT GTG-3'(SEQ ID NO:9)) amplification SNP G-308C and SNP A-301G fragment 334bp, make G-308C and A-301G site comprise wherein; Adopt primer pair 5(F:5'-TCG GGG TAC CGA TAT GCA GAA ATA AAG GTA G-3'(SEQ ID NO:10); R:5'-TCG CGA GCT CTT TCA GTA TCT GCA AAA CAG T-3'(SEQ ID NO:11)) amplification SNP G-797A fragment 165bp, G-797A site is comprised wherein.
PCR reaction is totally 25 μ l, and PCR reactive component is as follows: 10 × LA PCR Buffer II ((Mg
2+plus) 2 μ L, TaKaRa LA Taq (5 U/ μ L) 0.25 μ L, dNTP Mixture (each 2.5 mM) 4 μ L, primer (20 uM) each 1 μ L, DNA profiling 50ng, add sterile purified water to 25 μ L.
The pcr amplification reaction program of promoter region SNP G-308C and SNP A-301G is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s; 55 DEG C of annealing 30s, 72 DEG C extend 40s, totally 35 circulations, and last 72 DEG C extend 10min, and 4 DEG C of preservations, the clip size of pcr amplification product is 316bp.
The response procedures strengthening the pcr amplification of subarea SNP G-797A is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s; 50 DEG C of annealing 30s, 72 DEG C extend 30s, totally 30 circulations, and last 72 DEG C extend 10min, 4 DEG C of preservations.The clip size of pcr amplification product is 145bp.
Amplification obtains the cloned sequence of ProGA and ProCG of promotor SNP G-308C and SNP A-301G, EnhG and EnhA of enhanser SNP G-797A respectively, 2 kinds of different promoters and 4 kinds of enhansers and promotor haplotype are inserted pGL3.0 Basic luciferase reporter gene carrier respectively, after clone, gets rid of sudden change through order-checking.Except blank group, build 6 kinds of recombinant plasmid: ProGA-pGL3.0 Basic, ProCG-pGL3.0 Basic, EnhG-ProGA-pGL3.0 Basic, EnhG-ProCG-pGL3.0 Basic, EnhA-ProGA-pGL3.0 Basic and EnhA-ProCG-pGL3.0 Basic, respectively transfected with human Colon and rectum adenocarcinoma cell Caco-2 cell.Adopt Dual-Glo
?the multi-functional microplate reader of Luciferase Assay System measures Firefly(F value) and Renilla(R value) fluorescent signal value.Namely F/R ratio represent the activity of enhanser and promotor.Each experimental group gets the mean value in 4 holes, and experimental data adopts the LSD methods analyst of SPSS software package (version17.0), the results are shown in Table 4 and Fig. 4.
In table 4 Caco-2 clone, the fluorescent signal value of LCT genetic enhancer and promotor compares
| Group | N | F/R ratio (Mean ± SEM) |
| pGL3.0 Basic | 4 | 0.2278 ± 0.0417 Cc |
| ProGA-pGL3.0 Basic | 4 | 0.4787 ± 0.2137 Bc |
| ProCG-pGL3.0 Basic | 4 | 1.5971 ± 0.0648 Aa |
| EnhA-ProGA-pGL3.0 Basic | 4 | 1.096 ± 0.1701 ABb |
| EnhA-ProCG-pGL3.0 Basic | 4 | 0.6089 ± 0.1442 Bc |
| EnhG-ProGA-pGL3.0 Basic | 4 | 0.2987 ± 0.0816 Cc |
| EnhG-ProCG-pGL3.0 Basic | 4 | 0.6229 ± 0.1871 BCc |
Note: digital upper right corner capitalization is different, represent difference extremely significantly (
p< 0.01); Lowercase is different, expression significant difference (
p< 0.05)
From table 5: promotor SNP G-308C compare with GA with CG of SNP A-301G difference extremely significantly (
p=0.001), promotor CG promotes genetic expression strongly, and GA can not promote genetic expression.The A of enhanser SNP G-797A, for promotor GA, plays enhancement really, make promotor GA active significantly to strengthen (
p﹤ 0.05); For promotor CG, be but suppress son, make promotor CG activity extremely significantly reduce (
p﹤ 0.01).No matter the G of enhanser SNP G-797A is any promotor, all rise significant restraining effect (
p﹤ 0.01).The haplotype that pig LCT 5'UTR district's enhanser and promotor polymorphism Sites Combination are formed promotes that the effect of genetic expression is in descending order: enhanser A ﹢ promotor GA ﹥ enhanser A/G ﹢ promotor CG ﹥ enhanser G ﹢ promotor GA.This result is consistent with the result of study of embodiment 5, show that the haplotype that pig LCT gene 5'UTR district enhanser SNP G-797A and promotor SNP G-308C and SNP A-301G is combined to form and lactose intolerance are suffered from diarrhoea significant correlation, the detection of lactose intolerance in pig genetics and breeding can be used as.
The present invention utilizes reported first pig
lCTthe dependency that the pleomorphism site of gene 5'UTR and piglet lactose intolerance are suffered from diarrhoea.Contriver passes through pig
lCTthe order-checking of gene important area, has found 3 SNPs.By research that is individual to the lactose intolerance diarrhoea of 6 kind piglet colonies and normal swinery, the two fluorescence report system of application simultaneously confirms in vitro, in wild boar and Native Pig, the GG genotype of SNP G-797A or the GAGA genotype of SNP G-308C and SNP A-301G or haplotype GGA all suffer from diarrhoea with lactose intolerance significant correlation, and therefore this result can be used for molecular marker assisted selection (MAS) as a molecule marker in kind of pig genetics and breeding.
The above, be only preferred embodiment of the present invention, not any formal and substantial restriction is done to the present invention, all those skilled in the art, do not departing within the scope of technical solution of the present invention, when utilizing disclosed above technology contents, and a little change made, modify with differentiation equivalent variations, be Equivalent embodiments of the present invention; Meanwhile, all according to substantial technological of the present invention to the change of any equivalent variations that above embodiment is done, modify and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (1)
1. the method for the identification of piglet LCT gene 5'UTR polymorphism, it is characterized in that, in wild boar and Native Pig, primer SEQ ID NO:4 and SEQ ID NO:5 is utilized to carry out piglet LCT genes amplification subarea pcr amplification, obtain enhanser-797bp loci gene type, utilize primer SEQ ID NO:6 and SEQ ID NO:7 to carry out piglet LCT gene 5'UTR promoter region pcr amplification, obtain promotor-308bp and-301bp loci gene type; When piglet LCT gene 5'UTR enhanser-797bp site is GG genotype, or promotor-308bp is GAGA genotype with-301bp site, or enhanser-797bp site, promotor-308bp site and promotor-301bp site composition haplotype when being GGA, all suffering from diarrhoea with piglet lactose intolerance is significant correlation; When piglet LCT gene 5'UTR enhanser-797bp, promotor-308bp and-301bp be other genotype or haplotype time, not significant correlation of all suffering from diarrhoea with piglet lactose intolerance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310339074.3A CN103361440B (en) | 2013-08-06 | 2013-08-06 | LCT gene polymorphism detection method for piglet lactase digestive symptoms and applications of the method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310339074.3A CN103361440B (en) | 2013-08-06 | 2013-08-06 | LCT gene polymorphism detection method for piglet lactase digestive symptoms and applications of the method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103361440A CN103361440A (en) | 2013-10-23 |
| CN103361440B true CN103361440B (en) | 2015-03-11 |
Family
ID=49363695
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310339074.3A Expired - Fee Related CN103361440B (en) | 2013-08-06 | 2013-08-06 | LCT gene polymorphism detection method for piglet lactase digestive symptoms and applications of the method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103361440B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108165640B (en) * | 2018-03-20 | 2020-12-25 | 东北农业大学 | SNP locus related to resistance or susceptibility of diarrhea of suckling piglet and molecular marker detection and application |
| CN108624679A (en) * | 2018-06-28 | 2018-10-09 | 遵义市播州区大坝兴旺养殖有限公司 | The LCT gene pleiomorphism detecting methods of piglet lactose digestion character |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008057265A2 (en) * | 2006-10-27 | 2008-05-15 | University Of Maryland | Single nucleotide polymorphisms and the identification of lactose intolerance |
-
2013
- 2013-08-06 CN CN201310339074.3A patent/CN103361440B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008057265A2 (en) * | 2006-10-27 | 2008-05-15 | University Of Maryland | Single nucleotide polymorphisms and the identification of lactose intolerance |
Non-Patent Citations (4)
| Title |
|---|
| 1 kb of the lactasephlorizin hydrolase promoter directs post-weaning decline and small intestinal-specific expression in transgenic mice;Troelsen JT,et al;《FEBS Lett》;19941231;第342卷;291-296 * |
| Common polymorphism in a highly variable region upstream of the human lactase gene affects DNA-protein interactions;Edward J Hollox et al;《European Journal of Human Genetics》;19991231(第7期);791-800 * |
| GENETICS OF LACTASE PERSISTENCE AND LACTOSE INTOLERANCE;Dallas M. Swallow;《Annu. Rev. Genet.》;20031231;第37卷;197-219 * |
| 猪乳糖酶基因5"非编码区多态性分析;杜海廷等;《畜牧与兽医》;20130810;第45卷(第8期);45-49 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103361440A (en) | 2013-10-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107779516B (en) | It is a kind of influence pig birth weight character SNP marker and its application | |
| CN101440399B (en) | Molecular marking method for indicating and identifying litter size in pigs by MMP23 gene | |
| CN101693923B (en) | HSP70A1A gene SNP loci, application and kit for selecting heat-resistant cows | |
| CN108220408A (en) | A kind of selection of grain-saving type blueness shank and recessive white meat-type chickens new lines | |
| CN105567865A (en) | SNP (single-nucleotide polymorphism) marker related to Marsupenaeus japonicus heat resistance and detection method thereof | |
| CN107177681A (en) | Application of the pig SNP marker in daily gain in pigs behavior study and/or pig breeding | |
| CN103361440B (en) | LCT gene polymorphism detection method for piglet lactase digestive symptoms and applications of the method | |
| CN105087820A (en) | FSHR (follicle stimulating hormone receptor) gene based molecular marker related to porcine reproduction traits as well as detection method and application of molecular marker | |
| CN101906422B (en) | LAGLS 9 gene as molecular marker relevant to birthweight characteristics of pigs as well as preparation method and application | |
| CN113699246B (en) | SNP molecular marker affecting pig feed conversion efficiency character and application thereof | |
| CN107267631A (en) | A kind of SNP marker for influenceing daily gain in pigs character and its application | |
| CN1995395A (en) | Molecular diagnosis of sex-linked dwarf chicken and its uses in dwarf chicken breeding | |
| CN112921101A (en) | Molecular marker related to sheep remaining feed intake and application thereof | |
| Dubey et al. | Association analysis of polymorphism in thyroglobulin gene promoter with milk production traits in riverine buffalo (Bubalus bubalis) | |
| CN106480174A (en) | A kind of genetic molecule labeling method and application for pig disease resistant breeding | |
| CN106119407A (en) | LAP3 gene is as the molecular marker of ovine growth character and application thereof | |
| CN116004856A (en) | Haplotype markers associated with porcine fat deposition and uses thereof | |
| CN105063037A (en) | Hu sheep prolactin gene SNP locus and application thereof | |
| CN113584207B (en) | Sesame fertility molecular marker, primer, kit, application and Gao Mufen element new sesame variety breeding method | |
| CN113699247B (en) | SNP molecular marker related to pig residual feed intake on pig chromosome 1 and application thereof | |
| CN101906470B (en) | Method for detecting ox FTO (Fat Mass and Obesity-associated) gene single nucleotide polymorphism (SNP) | |
| CN112746114B (en) | Molecular marker for early selection of local chicken feed conversion rate, and identification method and application thereof | |
| CN108624679A (en) | The LCT gene pleiomorphism detecting methods of piglet lactose digestion character | |
| Dige et al. | Association of single nucleotide polymorphism in leptin gene with growth traits of Jamunapari goat | |
| CN104342492A (en) | MSTN gene molecular marker selection method for goat growth characters |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150311 Termination date: 20160806 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |