CN102994511A - Cloning and application for bovine slaughter trait related gene CMYA4 - Google Patents
Cloning and application for bovine slaughter trait related gene CMYA4 Download PDFInfo
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Abstract
The invention relates to cloning and an application for bovine slaughter trait related gene CMYA4. An SNP (single nucleotide polymorphism) marker site related to bovine slaughter trait is formed at the 8th exon 1092bp of the gene CMYA4, which is subjected to T/C mutation. The cloning preparation for the gene CMYA4 comprises the following steps of: obtaining the mRNA (messenger ribonucleic acid) sequence of the gene CMYA4; performing chromosomal localization, genetic structure analysis and protein characteristics analysis for the gene CMYA4; designing a primer to amplify a genomic DNA (deoxyribonucleic acid) containing the SNP site; and performing RFLP-PCR (restriction fragment length polymorphism-polymerase chain reaction) polymorphism detection, and taking the SNP marker related to bovine slaughter trait in the gene CMYA4 as a breeding and genetic marker for Simmental beef molecules. Via the cloning for the bovine slaughter trait related gene CMYA4 disclosed by the invention, new information is provided for a bovine genome database, and reference is provided for utilization for high-quality trait genes; and via the application for the SNP polymorphic site disclosed by the invention, a new technical index is provided for bovine slaughter trait detection, and a new marker is provided for bovine marker-assisted breeding.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to clone and the application of a kind of ox slaughter trait genes involved CMYA4.
Background technology
Muscle in the animal body can be divided into three major types: skeletal muscle, cardiac muscle and unstriated muscle.The 40-60% of skeletal muscle percentage of liveweight is determining the meat yield of domestic animal.Skeletal muscle is comprised of a large amount of myofiber (myofiber), and each myofiber is exactly a myocyte.Myofiber can be divided into according to metabolic characteristic: red muscle fiber (slowly aerobic shrinkage type) and white muscle fiber (fast glycolytic shrinkage type), also have in addition the contained enzyme activity of a kind of myofiber to occupy between this two classes myofiber, pinkiness is intermediate muscle fiber.The composition of the muscle tissue of CMYA family gene and animal has close relationship.The process of growth of skeletal muscle relates to the expression of large quantities of genes and transcribes regulation and control with translation skill, the meat-producing traits difference of Different Individual or different breeds of cattle and embryonic stage and to give birth to the expression regulation of late gene closely related.CMYA is primary cardiomyopathy associated protein (cardiomyopathy associated protein, CMYA), and present research about the CMYA family gene mainly concentrates on the interaction to this gene and myoprotein.
The CMYA4 gene has another name called UNC-45B(unc-45homolog B) or skeletal muscle UNC-45(Skeletalmuscle UNC-45).It is relevant with the assembling of crin to draw this gene by genetic experiment, finds that it has the effect of molecular chaperones.Nematode UNC-45 molecular size range is 107kDa, and aminoterminal contains 34 amino acid whose tumor-necrosis factor glycoproteinss, the central zone of about 400 amino-acid residues and UNC-45/Cro1/She4p (UCS) structural domain.UNC-45 expresses in muscle tissue, and immunofluorescence dyeing shows that it and heavy meromyosin are positioned muscle of trunk A-band crin altogether.Research finds to have 2 kinds of UNC-45 albumen in mammalian body, is respectively GC UNC-45 and SM UNC-45.The homology about 74% of these two kinds of gal4 amino acids generally, and mouse and people are the highest, reach respectively 95% and 98%.Detected respectively them organizes in the distribution situation demonstration GC of adult mice different tissues UNC-45 wide expression in each, and SM UNC-45 is only at skeletal muscle and Expression in Myocardium, and utilize hybridization in situ technique to detect them in the expression of embryo different development stage, the result showed that SM UNC-45 gene only expresses beginning in the 8.75th day of Mouse Embryo Development in heart, and GC UNC-45 gene just begins wide expression the 8th day of fetal development.In the C2C12 clone of cultivating, the GC-UNC45 gene is the highest at the proliferation period expression amount, and SM UNC-45 expressed in differential period, participates in myocyte's maturation and the formation of myotube.Drawing SM UNC-45 by above as a result author plays an important role in muscle development.Zebra fish knocked out occur the unusual of paralysis and heart function behind the SM UNC-45 gene, further research finds that paralysis is because the shortage of myosin filament in the muscle of trunk sarcomere.Above experimental result further illustrates the vital role of SM UNC-45 in muscle development.
In real work, people attempt to seek a kind of method of easy evaluation carcass quality, and to reduce financial loss, therefore large quantity research is devoted to seek the optimum prediction index that trunk forms.And at present development and the application of dna molecular marker technology, for the rapid evaluation of carcass quality has been opened up new way.Molecular genetic marker then may overcome this shortcoming and earlier kind of an ox be selected, and therefore suitable genetic marker is accelerated genetic progress, and realized that finally molecular breeding is extremely important for carrying out marker assisted selection.In addition, the polymorphism of research mutational site in colony, and carry out the strong means that the proterties association analysis is the research gene function.In colony, seek the gene relevant with the ox important economical trait by the proterties association analysis, carry out molecular breeding and be all the time the important and difficult task of of breeder.
The CMYA4 gene is an important muscle growth development related gene, be used in the selection of molecule aid mark this gene significant, separating clone ox slaughter trait genes involved CMYA4 also carries out the association analysis of its polymorphism and slaughter trait, can develop the new technology for detection of ox slaughter trait index, for the marker-assisted breeding of ox provides new mark.
By the retrieval to domestic publication document, do not retrieve the Patents document with ox slaughter trait genes involved CMYA4 technology.
Summary of the invention
The object of the invention is to Separation of Bovine slaughter trait genes involved CMYA4, obtain CMYA4 gene mRNA sequence, realization is to the clone of ox slaughter trait genes involved CMYA4, by the RFLP polymorphic detection to clone gene, the association analysis of RFLP polymorphism and carcass trait, set up the RFLP polymorphism for detection of the technology of ox slaughter trait index, and provide new mark for the marker-assisted breeding of ox.
The present invention solves its technical problem and takes following technical scheme to realize:
A kind of ox slaughter trait genes involved CMYA4 has a SNP marker site relevant with the ox slaughter trait at the 8th exons 1 092bp place of CMYA4 gene, is the T/C sudden change.
The clone of a kind of ox slaughter trait genes involved CMYA4, preparation process is as follows:
The first step, by the inquiry ncbi database (
Http:// www.ncbi.nlm.nih.gov/guide/) and the Ensembl database (
Http:// asia.ensembl.org/index.html) utilize the comparative genomics method to obtain CMYA4 gene mRNA sequence:
⑴ Query Database obtains Homo sapiens cardiomyopathy-associated protein 4 (CMYA4), mRNA sequence NM_173167.2 and Mus musculus cardiomyopathy-associatedprotein 4 (CMYA4), mRNA sequence NM_178680.4;
⑵ utilize NCBI BLAST instrument to utilize Highly similar sequences (megablast) comparison ox expressed sequence tag EST, choose homology greater than 80% expressed sequence tag sequence .EDY048664.1, EE373716.1, EE381791.1, EE376524.1, DV933195.1, DV930911.1, DV797178.1, CB438141.1, by DNAstar(Madison, WI, USA) program splicing acquisition CMYA4mRNA full length sequence 3430bp;
Second step, according to CMYA4 gene mRNA sequence, utilize the method for comparing to carry out chromosomal localization and gene structure analysis and the albumen characteristic analysis of CMYA4 gene:
The 3rd goes on foot, and finds out the SNP site of CMYA4 gene, and the design primer amplification contains the genomic dna in SNP site;
⑴ find out a SNP site of CMYA4 gene;
⑵ go out the nucleotide fragments at SNP to be measured place according to gene order design primer amplification;
The 4th step, the RFLP-PCR polymorphic detection.
And, the ⑵ in the 3rd step of described preparation process in the step design primer amplification go out the primer sequence of nucleotide fragments at SNP to be measured place shown in SEQ ID NO:3 ~ 4.
The application of a kind of ox slaughter trait genes involved CMYA4 is as beef molecular breeding genetic marker with SNP mark relevant with the ox slaughter trait among the gene C MYA4.
And, SNP mark relevant with the ox slaughter trait among the gene C MYA4 is as follows as the step of ox molecular breeding genetic marker:
The first step, the association analysis of ox CMYA4 gene HindIII-RFLP genotype and Part Traits;
Second step determines that gene C MYA4 and SNP site thereof are for detection of carcass traits such as individual gross weight, carcass weight, trunk is long, trunk is dark, tare weights.
Advantage of the present invention and effect are:
1, Separation of Bovine slaughter trait genes involved CMYA4 of the present invention; utilize the comparative genomics method to obtain CMYA4 gene mRNA sequence; and the structure of the chromosomal localization of definite CMYA4 gene and gene; can be for cow genome group database provides new information, for the utilization of high-quality character gene provides reference.
2, the CMYA4 gene is an important muscle growth development related gene, be used in the selection of molecule aid mark this gene significant, the present invention chooses a SNP site of CMYA4 gene, design the nucleotide fragments that primer amplification goes out SNP to be measured place according to gene order, carry out RFLP-PCR with HindIII.In the cows of analyzing, utilize pair of primers for the mutational site, detect genotype and the gene frequency in SNP site by PCR-RFLP.And carry out the proterties association analysis with 130 13 months large F3 for Simmental, and confirm available ox molecular breeding genetic marker, can develop the new technology for detection of ox slaughter trait index, for the marker-assisted breeding of ox provides new mark.
Description of drawings
Fig. 1 uses online software among the present invention
Http:// cn.expasy.org/tools/The prediction CMYA4 protein function territory figure that predicts the outcome;
Fig. 2 is T1092-C1092 loci gene type demonstration figure of the present invention.
Embodiment
The present invention is further described below in conjunction with specific embodiments, and its specific embodiments only is construed as illustrating, and is not determinate, can not illustrate to limit protection scope of the present invention with following.
Technological line of the present invention is: obtain CMYA4 gene mRNA sequence, utilize the method for comparing to carry out chromosomal localization and gene structure analysis and the albumen characteristic analysis of CMYA4 gene, design the association analysis that primer amplification contains genomic dna, RFLP polymorphic detection, RFLP polymorphism and the carcass trait in SNP site by inquiry ncbi database and Ensembl data base manipulation comparative genomics method, affirmation can be used as beef molecular breeding genetic marker SNP site.
Clone's step of a kind of ox slaughter trait genes involved CMYA4 is as follows:
The first step, by the inquiry ncbi database (
Http:// www.ncbi.nlm.nih.gov/guide/) and the Ensembl database (
Http:// asia.ensembl.org/index.html) utilize the comparative genomics method to obtain CMYA4 gene mRNA sequence:
⑴ Query Database obtains Homo sapiens cardiomyopathy-associated protein 4 (CMYA4), mRNA sequence NM_173167.2 and Mus musculus cardiomyopathy-associatedprotein 4 (CMYA4), mRNA sequence NM_178680.4;
⑵ utilize NCBI BLAST instrument to utilize Highly similar sequences (megablast) comparison ox expressed sequence tag EST, chooses homology greater than 80% expressed sequence tag sequence.EDY048664.1, EE373716.1, EE381791.1, EE376524.1, DV933195.1, DV930911.1, DV797178.1, CB438141.1 passes through DNAstar(Madison, WI, USA) program splicing acquisition CMYA4mRNA full length sequence 3430bp, sequence is shown in SEQ ID NO:1
The CMYA4mRNA sequence: SEQ ID NO:1 is as follows:
ctgggggttcacacacagaggagccgcaccccaggaggactgaccgagaacattgtccttgagagaaaatggccgaggcggaagcgatgcagctgaaggagga
ggggaatcagcatttccagctccaggactacaaggctgccaccaaaagctacagccaggccctgaagctgaccaaggataaggccctgctggccacactctatcg
gaaccgggcggcctgtggcctgaaaatggagagctatgttcaggcggcttctgatgcctcaagagccattgatatcaactcctcggacatcaaggctctgtaccggc
gatgccaggcactggagcacctgggcaagctggaccaggccttcaaggatgtgcagcgctgtgccactctggagccacagaaccagagcttccaggagacactg
aggaggctcaataccagcatccaggagaagctccgtgtgcagttctccactgactcgagggttcagacgatgtttgagattctcctggacagaaacagtgaagcggat
aaactagagaaggccgccaacaacctcattgtcctcagccgtgaggaagcgggggccgagaggatcttccagaacaacggcgtggccctgctgatgcagcttttg
gacacgaagaggcctgagctgatgctggctgccgtgcggaccttgtcaggcgtgtgcagtagccaccgagctagggccacagcgaccctccacgcagtccggat
agaccgaatctgcagcctcatggcggtggagagcgaggagatgtctctggccgtctgcaacctgctgcagggcatcatcgacgctctgtctggagaggacaaacag
gagcatcgggggaaggaagaggccctggttctggactccaagaaggacctgaagcagatcaccaaccacctgctcgacatgctggtcagtaagaaggtgtctggc
cagggtagggatcaggccctgaacctgctcaataagaacgtccccaggaaggacctcgccattcacgacaactcccgcaccatctacgtggttgataacggcctaa
ggaagatcctgaaggtggtgggacaggtcccagatctgccgtcctgcctgcccttgaccgacaacacccgcatgctggcctcaatcctcatcaacaagctttacgac
gacctgcgctgtgaccccgagcgcgaccacttccgcaagatctgcgaggagcacatcacgggcacgtttgacccccaggacatggacaagaatgtgattgccatcc
agacggtgtcggggatcctgcagggcccctttgacctgggcaatcagctgctgggactgaaaggtgtgatggagatgatggtggctctgtgcggctcggagcgtga
gacggaccagctggtggccgtggaggccctcatccacgcctccaccaagctcagccgtgccaccttcatcatcaccaacggtgtgtcactgctcaaagagatctaca
agaccaccaaaaacgagaagatcaagatccgcgcactggtgggactctgtaaacttggctcggcgggcggcacagattacgctctcaggcagtttgctgaaggctc
aacagaaaagctggccaaacagtgtcgcaagtggctgtgcaatgcatccatagacacccggacccggaaatgggcagtggagggcctggcctacctcacgctgg
acgccgacgtgaaggacgactttgttcaggacatccctgccctgcaggccatgttcgagctggccaagacccccgacaagaccatcctgtactcggtggccaccac
cctggtgaactgcaccaacagctacgatgtcaaggaggtcatccccgagctggtgcagctggccaagttctccaagcagcatgtgcctgaggagcaccccaagga
caagaaggactttgtggacatgcgagtgaagcggcttctgaaggccggcgtcacctcggcgctggtctgcatggtgaaagcagacaatgccatcctcactgaccag
accaaagagctgctggccagggtattcctggcactgtgtgacaacccaaaggaccgaggcaccattgtggctcaaggtggtggtaaggccctgattcccctggcttt
ggagggcacagacgtgggcaaggtgaaggcagcccacgccctagcgaagatcgccgcggtctccaacccagacatcgccttccccggggagcgggtgtacga
ggtggtgcggcccctcgtgagtctcctgaacacacagagggatggcctccagaactatgaggctctcctaggcctcaccaacctgtctgggcggagtgacaagctc
cgaaagaagatctttaaggagaaggccttgccggacattgagaactacatgtttgagaaccatgaccagctgcggcaggcggccactgagtgcatgtgcaacatggt
ggtgaacaaggaggtacaggagaggttcctggccgatggcaacgaccggctgaagctggtggtgctgctctgtggcgaggatgacgacaagctgcagaacgcgg
ctgcgggggccctggccatgctgacagcagcgcataagaagctgtgcttcaagatgactgaagtgacaactcagtggttggagattctacagcggctttgcttgcatg
accggctgtcagttcaacaccggggcctggtcattgcctacaacctgctggcggctgatgctgagttggctaagaagctggtggagagtgagctgctggagatcctg
acggtggtgggcaagcaagagccggacgagaagcgggcagcagtggttcagacagctcgggaatgtctcatcaaatgcatggattatggcttcattaaaccagtgg
cttagacaggggcccagagggagactggggctgctcctgtaactgtgcagagttccgaactgagaactcttggggtgtcagttccattagggatcattgcaaccacga
tgaggtgtccatgtaaaagaaggactgtggatagtcccctgagttgtacatcttgccttttgttctaggaaagtttgcatatttcattagctctcttgctccttaacatctgtcaa
gttgcagaaattcctagaataattttcatctgtgattaggaagacaggttggataattctttctgtacccattccaaaggccgggataatgggctggagttctttacattttgg
aaaatagattgtgtggtgaagtaggagctaacaggtcctactttaaatatgtaaagtagtgtgactataaagtttagtgctggtgtacctggatgctgatctgtctggttttta
tatcctgtttcccctgctgtttgactgcggctgcctggaaatgcatgtttcagcagcagtgcccgtcccagacgcgtgcgcattcctttcacttaactatgatggaaataca
gctgatgttcagt
Second step, according to CMYA4 gene mRNA sequence, utilize the method for comparing to carry out chromosomal localization and gene structure analysis and the albumen characteristic analysis of CMYA4 gene:
The genome sketch of ox is completed, and we have obtained CMYA4 gene mRNA sequence, so we have utilized the method for comparison to carry out the structural analysis of chromosomal localization and the gene of CMYA4 gene;
⑴ CMYA4 is positioned at karyomit(e) Chromosome 19:15233409-15265756bp, 18 introns of nineteen exon are arranged, the long 929residues of the long 3430bp aminoacid sequence of transcript, derivation isoelectric points of proteins (Isoelectric point) out: 7.7698, molecular weight (Molecular weight): 103,549.83g/mol.The CMYA4 protein sequence: sequence shown in SEQ ID NO:2,
The CMYA4 protein sequence: SEQ ID NO:2 is as follows:
MAEAEAMQLKEEGNQHFQLQDYKAATKSYSQALKLTKDKALLATLYRNRAACGLKMESYVQAASDAS
RAIDINSSDIKALYRRCQALEHLGKLDQAFKDVQRCATLEPQNQSFQETLRRLNTSIQEKLRVQFSTDSRV
QTMFEILLDRNSEADKLEKAANNLIVLSREEAGAERIFQNNGVALLMQLLDTKRPELMLAAVRTLSGVCS
SHRARATATLHAVRIDRICSLMAVESEEMSLAVCNLLQGIIDALSGEDKQEHRGKEEALVLDSKKDLKQIT
NHLLDMLVSKKVSGQGRDQALNLLNKNVPRKDLAIHDNSRTIYVVDNGLRKILKVVGQVPDLPSCLPLT
DNTRMLASILINKLYDDLRCDPERDHFRKICEEHITGTFDPQDMDKNVIAIQTVSGILQGPFDLGNQLLGL
KGVMEMMVALCGSERETDQLVAVEALIHASTKLSRATFIITNGVSLLKEIYKTTKNEKIKIRALVGLCKLGS
AGGTDYALRQFAEGSTEKLAKQCRKWLCNASIDTRTRKWAVEGLAYLTLDADVKDDFVQDIPALQAMFE
LAKTPDKTILYSVATTLVNCTNSYDVKEVIPELVQLAKFSKQHVPEEHPKDKKDFVDMRVKRLLKAGVTS
AVCMVKADNAILTDQTKELLARVFLALCDNPKDRGTIVAQGGGKALIPLALEGTDVGKVKAAHALAKI
AAVSNPDIAFPGERVYEVVRPLVSLLNTQRDGLQNYEALLGLTNLSGRSDKLRKKIFKEKALPDIENYMFE
NHDQLRQAATECMCNMVVNKEVQERFLADGNDRLKLVVLLCGEDDDKLQNAAAGALAMLTAHKKL
CFKMTEVTTQWLEILQRLCLHDRLSVQHRGLVIAYNLLAADAELAKKLVESELLEILTVVGKQEPDEKRA
AVVQTARECLIKCMDYGFIKPVA
⑵ use online software
Http:// cn.expasy.org/tools/Prediction CMYA4 protein function territory:
As shown in Figure 1, gene structure territory predictive display CMYA 4 albumen contain a TPR-like superfamily structural domain, and an ARM repeats the superfamily structural domain, and a myosin is in conjunction with the division center territory.
The 3rd goes on foot, and finds out the SNP site of CMYA4 gene, and the design primer amplification contains the genomic dna in SNP site;
⑴ find out two SNP sites of CMYA4 gene:
Choose a SNP site SNP:rs109057450 of CMYA4 gene by inquiring about ncbi database, be positioned at Chromosome 19:15250730bp, CMYA4 gene the 8th exons 1 092bp is T/C same sense mutation site (T1092-C1092);
⑵ go out the nucleotide fragments at SNP to be measured place according to gene order design primer amplification:
1. primer sequence is as follows:
SEQ?ID?NO:37450-F?GCTGACGGTGCCGTTTCTGTG
SEQ?ID?NO:47450-R?GGCCGGCATCTTACGCTTGCT
2. pcr amplification condition:
PCR reaction cumulative volume 20 μ L, wherein the about 100ng of cow genome group DNA contains 1 * buffer (Promega); 1:5mmol/L MgCl2; the dNTP final concentration is 150 μ mol/L, and the primer final concentration is 0:2 μ mol/L, 2U Taq archaeal dna polymerase (Promega).The pcr amplification program is: 94 ℃ of 4min, and the 94 ℃ of 45s that circulate 5 times, 62 ℃ of 45s, 72 ℃ of 1min, and then the 94 ℃ of 45s that circulate 30 times, 57 ℃ of 45s, 72 ℃ of 1min, last 72 ℃ are extended 5min.The PCR reaction product detects with 2% agarose gel electrophoresis, obtains the long purpose fragment of 467bp;
The 4th step, the RFLP-PCR polymorphic detection:
⑴ select suitable restriction endonuclease to carry out RFLP-PCR for the SNP site:
RFLP-PCR is carried out with HindIII in the T1092-C1092 site, and PCR product endonuclease reaction volume is 15 μ L, 1 * buffer, 1.5 μ L wherein, and PCR product 3 ~ 5 μ L, restriction enzyme are 0.5 μ L (5U), use H
2O supplies 15 μ L, and with centrifugal behind the sample blending, 37 ℃ of water-bath 4h detect enzyme with 2% agarose gel electrophoresis and cut the result, and the record genotype is taken pictures under ultraviolet lamp, and the T1092-C1092 loci gene type as shown in Figure 2;
To single nucleotide polymorphism the distribution in cows add up:
In the cows of analyzing, utilize pair of primers for the mutational site, by genotype and the gene frequency in PCR-RFLP detection SNP site, statistics is as shown in table 1;
Table 1.SNP rs109057450 (T1092-C1092) allelotrope gene frequency
Can find out the T1092-C1092 site by table, in the cows body, the CC type is higher than CT type and TT type, and C allelotrope is protogene, and its frequency is greater than T allelotrope.
The application of a kind of ox slaughter trait genes involved CMYA4, step is as follows:
At first, we determine that the proterties association analysis carries out for Simmental with 130 13 months large F3, cows are according to the farm of national feeding standard NY/T 815-2004 raising in Horqin, the Inner Mongol, the raising term harmonization of all oxen, trunk and Meat Quality are surveyed and are carried out according to national beef segmentation standard GB/T 17238-2008
The association analysis of ox CMYA4 gene HindIII-RFLP genotype and Part Traits:
Utilize the clone of ox slaughter trait genes involved CMYA4, the HindIII-RFLP genotype detection result who obtains, the target cows are carried out association analysis between genotype and carcass trait and Meat Quality, eliminating kind, butcher batch and sex between difference after, simple mean and the standard error analysis of proterties the results are summarized in table 2 between genotype, found that, SNP rs109057450 has proterties related with ox part slaughter trait: SNP rs109057450 and gross weight (kg) utmost point significant correlation (p=0.008＜0.01), the CT type gross weight utmost point is significantly higher than the TT type, exceeds 36.8Kg; With carcass weight (kg) significant correlation (P=0.033＜0.05), CT type carcass weight is significantly higher than the TT type, exceeds 17.3Kg; With long (cm) significant correlation (P=0.023＜0.05) of trunk, CT type trunk length is significantly higher than the TT type, exceeds 3.3cm; With dark (cm) significant correlation (P=0.016＜0.05) of trunk, CT type trunk is significantly higher than the TT type deeply, exceeds 2.02cm; With tare weight (kg) significant correlation (P=0.023＜0.05), CT type tare weight is significantly higher than the TT type, exceeds 3.01Kg; SNP rs109057450 can be used as the genetic marker of ox molecular breeding, be C or T according to the 1092nd Nucleotide of CMYA4 gene the 8th exon, thereby determine that this individuality is in the allelotype in this mutational site, and assess the difference of the proterties such as this individuality gross weight, carcass weight, trunk are long, trunk is dark, tare weight with this, can be applied in the middle of the molecular mark of ox;
Table 2.SNP rs 109057450 proterties association analysiss
Annotate: lowercase mark expression significant difference between different genotype in the table, P＜0.05; Capitalization mark expression difference is extremely remarkable, P＜0.01.
In sum, the carcass traits such as ox slaughter trait genes involved CMYA4 of the present invention and SNP site thereof can be used for detecting individual gross weight, carcass weight, trunk is long, trunk is dark, tare weight, thereby for the molecular breeding of ox provides a new genetic marker, and will in the breeding of ox, play a significant role.
Claims (5)
1. an ox slaughter trait genes involved CMYA4 is characterized in that: at the 8th exons 1 092bp place of CMYA4 gene a SNP marker site relevant with the ox slaughter trait is arranged, be the T/C sudden change.
2. the clone of an ox slaughter trait genes involved CMYA4, it is characterized in that: preparation process is as follows:
The first step, by the inquiry ncbi database (
Http:// www.ncbi.nlm.nih.gov/guide/) and the Ensembl database (
Http:// asia.ensembl.org/index.html)Utilize the comparative genomics method to obtain CMYA4 gene mRNA sequence:
⑴ Query Database obtains Homo sapiens cardiomyopathy-associated protein 4 (CMYA4), mRNA sequence NM_173167.2 and Mus musculus cardiomyopathy-associatedprotein 4 (CMYA4), mRNA sequence NM_178680.4;
⑵ utilize NCBI BLAST instrument to utilize Highly similar sequences (megablast) comparison ox expressed sequence tag EST, choose homology greater than 80% expressed sequence tag sequence .EDY048664.1, EE373716.1, EE381791.1, EE376524.1, DV933195.1, DV930911.1, DV797178.1, CB438141.1, by DNAstar(Madison, WI, USA) program splicing acquisition CMYA4mRNA full length sequence 3430bp.
Second step, according to CMYA4 gene mRNA sequence, utilize the method for comparing to carry out chromosomal localization and gene structure analysis and the albumen characteristic analysis of CMYA4 gene:
The 3rd goes on foot, and finds out the SNP site of CMYA4 gene, and the design primer amplification contains the genomic dna in SNP site:
⑴ find out a SNP site of CMYA4 gene;
⑵ go out the nucleotide fragments at SNP to be measured place according to gene order design primer amplification;
The 4th step, the RFLP-PCR polymorphic detection.
3. the clone of ox slaughter trait genes involved CMYA4 according to claim 2 is characterized in that: the ⑵ in described the 3rd step of preparation process in the step design primer amplification go out the primer sequence of nucleotide fragments at SNP to be measured place shown in SEQ ID NO:3 ~ 4.
4. the application of an ox slaughter trait genes involved CMYA4 is characterized in that: with SNP mark relevant with the ox slaughter trait among the gene C MYA4 as Simmental beef molecular breeding genetic marker.
5. the application of ox slaughter trait genes involved CMYA4 according to claim 4 is characterized in that: SNP mark relevant with the ox slaughter trait among the gene C MYA4 is as follows as the step of beef molecular breeding genetic marker:
The first step, the association analysis of ox CMYA4 gene HindIII-RFLP genotype and Part Traits;
Second step determines that gene C MYA4 and SNP site thereof are for detection of carcass traits such as individual gross weight, carcass weight, trunk is long, trunk is dark, tare weights.
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| CN113862380A (en) * | 2021-11-22 | 2021-12-31 | 广东海洋大学 | Molecular marker related to pH of slaughtered meat of yak Wnt3a gene and application |
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| CN104059963A (en) * | 2013-11-29 | 2014-09-24 | 吉林大学 | Detection method of Chinese simmental cattle carcass and meat quality trait genetic markers |
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| CN114058711B (en) * | 2021-09-06 | 2024-09-17 | 广东海洋大学 | Method for evaluating pH and cooking loss rate of beef quality traits of Sichuan yak beef 45min after slaughter |
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| CN115044680A (en) * | 2022-04-22 | 2022-09-13 | 四川省畜牧科学研究院 | SNP molecular marker related to slaughter traits of meat rabbits, detection primer set, detection kit and application thereof |
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