A kind of genetic marker relevant to goat growth trait and applicationTechnical field
The invention belongs to animal molecular marker technical field, be specifically related to a kind of genetic marker relevant to goat growth trait and application.This genetic marker clone is from the gene fragment of a SKIP.
Background technology
Along with the raising of China's expanding economy and living standards of the people, the demand of mutton increases year by year.Produce compared to market pig, the shortcomings such as Native Breed of Goats ubiquity individuality is little, the speed of growth is slow, dressing percentage is low.Research is cultivated the fast growth that meets needed for modern market and is had the green goat new variety of lacol flavor, and sets up efficient breeding technique system, becomes the new trend of Mutton Sheep Industry.Along with the development of Protocols in Molecular Biology, molecular marker assisted selection being combined with traditional breeding way is that current Application of Animal Genetic improves one of main method that engineering takes.Therefore find the molecular genetic marker mutually chain with goat growth trait locus, being applied to molecular marker assisted selection and cultivating mutton sheep new variety, is emphasis and the urgent problem of for some time goat molecule field of biology research at present and in the future.
First SKIP (5 ' inositol monophosphate enzyme that skeletal muscle and kidney are rich in) gene is found by (2000) such as Ijuin and is separated, it is wide expression in each tissue, especially at heart, high expression level (Ijuin in skeletal muscle and kidney, T., etal., Identificationandcharacterizationofanovelinositolpolypho sphate5-phosphatase.JournalofBiologicalChemistry, 2000.275 (15): 10870-10875.).SKIP is accredited as by hydrolysis PI (3,4,5) P3 is PI (3,4) P2 and regulate and control the 5'-lipid phosphatase (Ijuin of insulin signaling, T.andT.Takenawa, SKIPnegativelyregulatesinsulin-inducedGLUT4translocation andmembraneruffleformation.MolCellBiol, 2003.23 (4): 1209-1220.).PI (3, 4, 5) the downstream Akt signal of P3 plays an important role in myogenic differentiation process: the progress (Boonstra of G1 → S phase in the adjustable cell cycle, J., IdentificationofarestrictionpointattheM/G1transitionduri ngtheongoingcellcycle.AdvEnzymeRegul, 2007.47:208-221.), control the expression of the early stage myogenin of muscle cells differentiate, maturation (the Rotwein of myotube, P.andE.M.Wilson, DistinctactionsofAkt1andAkt2inskeletalmuscledifferentiat ion.JCellPhysiol, 2009.219 (2): 503-511.) etc.In addition, the downstream targets mTOR of Akt is to the initial also very important (Hribal of myogenic differentiation, M.L., etal., Regulationofinsulin-likegrowthfactor – dependentmyoblastdifferentiationbyFoxoforkheadtranscript ionfactors.jCellbiol, 2003.162 (4): 535-541.).In overexpression SKIP gene inhibition sarcoplast, short differentiation factor IGF2 expresses and the report of PI3K-AKT signal further illustrates the myogenic differentiation (Ijuin that SKIP take part in Skeletal Muscle Cell, T.andT.Takenawa, Roleofphosphatidylinositol3,4,5-trisphosphate (PIP3) 5-phosphataseskeletalmuscle-andkidney-enrichedinositolpo lyphosphatephosphatase (SKIP) inmyoblastdifferentiation.JBiolChem, 2012.287 (37): 31330-31341.).The report of several species all shows muscle specific gene SKIP and myofiber is grown closely related.The weight of SKIP genetic heterozygosis mutant mice soleus muscle and quadriceps muscle of thigh significantly improves (Ijuin, T., etal., IncreasedinsulinactioninSKIPheterozygousknockoutmice.Mol ecularandCellularBiology, 2008.28 (17): 5184-5195.).The QTL of the location of this gene also many distribution influence correlated character in the economic animal such as pig, ox genome, as pig myofiber number with thigh is heavy and calf Birth weight.And this intragenic polymorphic can remarkably influenced Large white×Meishan pig F2 for the multinomial carcass trait (Xiong such as longissimus dorsi muscle height of colony, Q., etal., CharacterizationofporcineSKIPgeneinskeletalmuscledevelop ment:polymorphisms, associationanalysis, expressionandregulationofcellgrowthinC2C12cells.MeatSci, 2012.92 (4): 490-497.).But the research of SKIP gene in goat still belongs to blank, the polymorphism information of gene has not yet to see report.
Consider the Main Function that SKIP grows myofiber, the research carrying out SKIP gene in goat is necessary.The research polymorphism of gene mutation site in colony, and carry out trait associations analysis and be the relation between molecule marker and proterties of excavating, study very important means of gene function.So applicant has carried out polymorphism research and association analysis to this gene in goat, for applicant better uses this gene to provide important theories integration in naked eyed test improvement.
Summary of the invention
The object of the invention is to the genetic marker that acquisition one is relevant to goat growth trait, by the specific DNA fragment of cloned goat SKIP gene, and through association analysis, obtain a genetic marker detected for goat growth trait, this mark belongs to a kind of mark newly, can be applied in the association analysis of the marker assisted selection of goat.
The present invention is realized by following technical proposal:
Applicant is by screening, and obtain one for detecting the genetic marker of goat growth trait, the nucleotide sequence of this mark is as follows:
GGCTTCCCTTCTCAGCATACATGTCGTGACGTGGAATGTGGCCTCTGCCGCACCCCCTCCAGACCTCAGTGATCTGCTTCAGCTGAACAACCTGAACCTGAACCTGGACATATATGTCATTGGGTAGGTGTCCACAGGACCCGTCACGCATCCCTGTGTCTGAAATCAGGGTCAGGTAAGCCTGGCCAGTCCCAAATTGTCCCCACGGGGAACCACTATAACCTGTTCGCCCTTGCCCTGGAGTGTGAAGGCCAAGAGCTTTCAGCCTGCATCAGAGCCAGGCCACATCTGTTGTCCTGGGTCAGCAGTGGCAAGGGGCTGGCGAGCGACAGCCACAGGGGAGGGAGAACCCAGGGCTAGACAGTCGGCTGAGGGCAGTGCTGGATGTCGGAGCAGAGGCAGGGCCCTCCAGRCCAGGGGGCAGACCTCTGATCTGGGACGGGCAGTTTGCAGGAACTGAACTGTGGGATCATGAGCCTCCTTTCCGACACTGCCTTTGAAGACCCATGGAGCAGTTACTTCATGGACGTGCTTTCCCCTCTGAGCTTCGTCAAGGTAACTTGCAAGTTCATGGGCAATTGGGAAATGTGTATCACCTCTTATTACTAATCCCTAGTCCTTTGTCTCACAGTCCAGCTTTCATGCTATTACCCTTTTATTTAAAGACCCAAACCCCAGTT
" R " (i.e. allelic sudden change at 413bp place) shown in said gene fragment (sequence) is G or A, and this sudden change causes Bg1I-RFLP polymorphism.
Applicant devises a kind of goat SKIP gene and detect the primer pair primer pair of genetic marker of the present invention (be also increase) of this gene fragment sudden change of increasing, and the DNA sequence dna of this primer pair is as follows:
Forward primer: GGCTTCCCTTCTCAGCATA,
Reverse primer: AACTGGGGTTTGGGTCTTT.
We establish a kind of preparation method of the genetic marker relevant to goat growth trait, and the method is to comprise the following steps:
Be information probes with sheep SKIP genomic dna and cDNA, do homologous sequence screening, obtain the genome sequence of SKIP on goat relative chromosome.Genomic dna is extracted from Goat Blood, according to SKIP gene order design primer, the DNA sequence dna of this primer pair is as shown in sequence table SEQ IDNO:2 and SEQIDNO:3, in goat genomic dna, pcr amplification is carried out with the primer pair shown in sequence table SEQ IDNO:2 and SEQIDNO:3, PCR primer purifying, cloning and sequencing, obtain as sequence table SEQ IDNO:1.
Genetic marker of the present invention can be applied in goat growth trait detection.Wherein designed primer pair also can be applicable in the detection of goat growth trait.
More detailed technical scheme is as described in " embodiment ".
Accompanying drawing explanation
Sequence table SEQ IDNO:1 be clone with the gene order fragment of goat growth trait genes involved SKIP.Sequence length is 680bp, wherein replaces (allelic mutation, we are by this mutational site writing " R ") at 413bp place existence G/A of this sequence.
Sequence table SEQ IDNO:2 and 3 is the nucleotide sequences of the primer pair that the present invention designs, and it is positioned at 1-19 position and the 662-680 position of SEQIDNO:1 sequence.
Fig. 1: be the present invention clone with the partial nucleotide sequence of goat growth trait genes involved SKIP, show 1 pleomorphism site in figure.In figure: underscore part is primer sequence, the base in bracket is base mutation, and this sudden change is with R representative in claims of the present invention, and the two is same implication.
Fig. 2: the gene mutation site order-checking color atlas that in the present invention, the SKIP partial genome sequence direct Sequencing of pcr amplification detects.
Fig. 3: the agarose gel electrophoresis figure being the SKIP partial genome sequence that pcr amplification obtains, description of symbols in figure: swimming lane: M is DL2000Marker.
Fig. 4: goat SKIP gene PCR-Bg1I-RFLP detected result.Description of symbols in figure: swimming lane M is DL2000Marker; AA genotype, 680bp; AG genotype, 680bp, 410bp, 270bp; GG genotype, 410bp, 270bp.
Embodiment
Embodiment 1:
(1) clone of .SKIP Gene Partial genome sequence and the foundation of pleiomorphism detecting method
(1) design of primers:
Be information probes with sheep SKIP genomic dna (the GenBank number of including: NC_019468.1), utilize the BLAST instrument in NCBI in GenBank goat Genomes database, do homologous sequence screening, it is the homology SKIP genome sequence of 97% that acquisition is positioned at similarity on goat No. 19 karyomit(e)s.According to goat SKIP genome sequence design of amplification primers (forward primer 5'GGCTTCCCTTCTCAGCATA3'; Reverse primer 5'AACTGGGGTTTGGGTCTTT3').With Macheng black goat and boer goat for test colony, extract the poba gene group DNA of goat, using the DNA mixing constructed dna pond of gained two Goats Breeds as template amplification goat SKIP portion gene segment.
Through PCR primer purifying and order-checking, and obtain the nucleotide sequence (see Fig. 1) as shown in sequence table SEQ IDNO:1 by sequential analysis; By the color atlas obtained as shown in Figure 2 that checks order, find existence 1 place single nucleotide polymorphism (SNP).That is: the base mutation of a G/A is had at the 413bp place of SEQIDN0:1.
(2) purifying, the Cloning and sequencing of PCR primer
Pcr amplification: the sequence shown in SEQIDNO:1.Reaction cumulative volume 25 μ L:10 × Tapbuffer2.5 μ L, dNTP final concentration is 200 μMs, and forward and reverse primer is respectively 0.2 μM, 1UTaqDNA polysaccharase (purchased from precious biotechnology Dalian company limited).Pcr amplification program is: 94 DEG C of 4min, and then circulate 35 94 DEG C of 40s, 59 DEG C of 45s, 72 DEG C of 30s, and last 72 DEG C extend 10min.PCR reaction product 1.5% agarose gel electrophoresis detects.
The sequence of above-mentioned forward and reverse primer is as follows:
Forward primer: GGCTTCCCTTCTCAGCATA,
Reverse primer: AACTGGGGTTTGGGTCTTT.
The purifying of PCR primer: cut the gel containing object fragment from low melting-point agarose gel under ultraviolet lamp, put into 1.5mL centrifuge tube, reclaim test kit (purchased from Shanghai Sheng Gong biotechnology company limited) purifying DNA fragment with sanprep pillar DNA glue.Concrete steps are as follows: add 700 μ L sol solutionses, 50-60 DEG C of water-bath is thoroughly melted to glue, and when adding hot melt adhesive, every 2min mixing once, is cooled to room temperature; Centrifugal column is put into collection tube, mixed solution is moved to centrifugal column, room temperature places 2min; The centrifugal 1min of 9000r/min, now DNA is adsorbed on post; Outwell waste liquid in collection tube, centrifugal column is put into same collection tube, add 700 μ L elutriants, the centrifugal 1min of 12000r/min; Outwell the waste liquid in collection tube, the centrifugal 1min of 12000r/min; Centrifugal column is put into a preprepared sterilizing 1.5mL centrifuge tube, add 40 μ L elutriants or distilled water (pH>7.0), room temperature or 37 DEG C place 2-3min (improve eluting temperature to the 55-80 DEG C of elution efficiency being conducive to improving DNA, can wash-out twice.); The centrifugal 1min of 12000r/min, the liquid in centrifuge tube is the DNA fragmentation of recovery, can use immediately or be stored in-20 DEG C for subsequent use.
(3) foundation of PCR-Bg1I-RFLP pleiomorphism detecting method
Above-mentioned PCR primer is through order-checking (order-checking entrusts Shanghai Sheng Gong biotechnology company limited to carry out), and the size obtaining fragment is 680bp, and its nucleotide sequence is as shown in sequence table SEQ IDNO:1.Carry out sequence alignment through ClusterW software, find that being wherein arranged in this fragment 413bp place (intron 2) G/A variation causes the change of Bg1I restriction enzyme site (GCCNNNN ↓ NGGC) polymorphism.
Endonuclease reaction system is 10 μ L, wherein comprises: 10 × buffer1 μ L, restriction enzyme (10U/ μ L) 0.1 μ L, PCR primer 3.9 μ L, ddH2o5 μ L, puts 37 DEG C of isothermal reactions and spends the night, and by the sepharose gel electrophoresis analysis of 1.8%, observes and records enzyme cut genotyping result at gel imaging system.This site of Bg1I nonrecognition when the base at 452bp place is all A, be designated as AA type (680bp), when mutational site base is all G, this site of Bg1I enzyme identification, is designated as GG type (410bp+270bp), is designated as AG type 680bp+410bp+270bp when A and G exists).The enzyme of the PCR-Bg1I-RFLP of goat SKIP gene cuts genotyping result as Fig. 4.
Embodiment 2:
For association analysis test materials with 318 boer goats of Animal Husbandry and Veterinary Inst., Hubei Academy of Agricultural Sciences's sheep stud and Macheng black goat filial generation individual.The proterties analyzed is mainly growth traits.Growth traits comprises and records the body weight, height, Body steep length, chest measurement, circumference of cannon bone etc. of goat individuality at 3,6,12,18,24 monthly ages respectively.First the PCR-Bg1I-RFLP method adopting embodiment 1 to set up carries out individual gene type, adopt SAS8.0 statistical software (SASInstituteInc, Version8Edition) to data analysis, GLM program carries out marking and association analysis between proterties, the additive effect of REG program computation gene and dominant effect, and carry out test of significance, analyze the correlationship of goat SKIP gene Bg1I-RFLP different genotype and goat growth trait.Set up statistical model, except stochastic effect, other factors are fixed effect, adopt model as follows:
Yijklm=μ+Si+Yj+Gk+Fl+Mm+eijklm, wherein Yijklm is proterties observed value, and μ is population average, and Si is seasonal effect, and Yj is time effect, and Gk is genetic effect, and Fl is paternal effect, and Mm is maternal effect, and eijklm is random residual.Additive effect represents AA, AG, GG respectively with-1,0,1; Dominant effect is with 0, and 1,0 represents AA, AG, GG respectively.
Increase day by day SKIP gene polymorphic and the goat 3-6 month chest measurement as can be seen from Table 1, March chest measurement, December circumference of cannon bone, December body weight, the tenth August height in significantly or pole significant correlation, wherein GG genotype is preponderant genotype, relative to AA genotype, GG genotype individuals has higher chest measurement in March, December circumference of cannon bone, December body weight and the tenth height in August (see table 1).
The relation of the G/A sudden change of table 1SKIP gene and goat growth trait
Note: increase day by day the DCHC1:3-6 month chest measurement; CHC1: the March chest measurement; CC3: the December circumference of cannon bone; BW3: the December body weight; BH4: the ten August height.Significant difference (* P ﹤ 0.05) between the least square average (± standard error) containing different letter.