CN102943076A - Cloning and application of beef quality-related gene CMYA1 - Google Patents

Abstract

The invention belongs to the technical field of animal genetic engineering, and particularly relates to cloning and application of a beef quality-related gene CMYA1. The mRNA (micro Ribonucleic Acid) sequence of the cloned CMYA1 gene is shown as SEQ ID NO: 1, the protein sequence of the cloned CMYA1 gene is shown as SEQ ID NO: 2, an SNP (single nucleotide polymorphism) marker locus is formed at the second exon 1058bp of the CMYA1 gene due to C/T mutation, an SNP marker locus is formed at the second exon 2168bp of the CMYA1 gene due to C/T mutation, and primer sequences for detection of SNP markers are shown as SEQ ID NO: 3 to SEQ ID NO: 6. The beef quality-related SNP marker in the gene CMYA1 is used as a genetic marker for molecular breeding of Simmental beef. New information is provided for a cattle genome database, and references are provided for application of genes with excellent traits. A new technical indictor is provided for detection of the beef quality due to the application of polymorphic sites of the SNP, and a new marker is provided for marker-assisted breeding of cattle.

Description

A kind of clone and application of beef qualitative correlation gene CMYA 1

Technical field

The invention belongs to gene engineering technology field, be specifically related to a kind of clone and application of beef qualitative correlation gene CMYA 1.

Background technology

Along with improving constantly of standard of living, people are also more and more higher to the requirement of meat quality.The index of tolerance meat quality mainly comprises the pH value, is waterpower, intramuscular fat content, tender degree, yellowish pink, diameter of muscle fiber, cold cuts rate, storage loss etc.Intramuscular fat content (Intramuscular fat, IMF) is an important indicator of tolerance meat quality.Research finds that suitably improving intramuscular fat content will make Meat Tenderness and succulence improve, thereby improves meat quality.Beef has the characteristics such as high protein, lower fat, low-cholesterol, and is nutritious, is the first-selection of world meat industry developed country consumption of meat always, and the beef consumption of China is also rising year by year.

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.In order to explore the molecular genetic mechanism of China and foreign countries' ox kind phenotypic difference on muscle growth and meat quality, our previous work adopts the mRNA differential display technique that two Representative Cultivars are grown up, and the summer Lip river is come and adult Mongolia Cattle longissimus dorsi muscle histological difference is expressed the EST(expressed sequence tag) analyze, wherein the expression amount of a new EST in Xia Luo comes is apparently higher than Mongolia Cattle.Further find that by upper people's nonredundancy (non redundancy, the nr) sequence library of comparison GenBank this EST is corresponding to people's primary cardiomyopathy associated protein 1 (cardiomyopathy associated 1, CMYA1) gene.Utilize the expression rule of in situ hybridization and Northern hybridization research CMYA1 gene, find that the mRNA level of CMYA1 gene is up-regulated expression in the dirty tissue of chicken blastophore of growing, subsequently the identified called after of this gene " cXin " or " heart ".Someone utilizes the cXin sequence to compare in the EST public database and has found the homology EST of this gene on the mouse, and this EST screened, identifies in the mice skeletal cDNA library as probe, after this be mXin α with upper this unnamed gene of mouse, also utilize simultaneously cXin to process the chicken embryo of hatching, found that the dirty aberrant morphogenesis of chicken blastophore, infer that thus cXin plays an important role in heart development.Have research to find that the homologous gene of mXin α on the people is CMYA1, by radiation hybrid with this assignment of genes gene mapping at 3p21.2-p21.3, the genomic dna sequencing result shows that this gene is positioned at 3p22.2.As probe, hang down rigorous Southern hybridization with the genomic dna of mouse with cXin cDNA sequence, found the existence of another one mXin β, the people upper mXin β be again the CMYA3 gene, is positioned at people's 2q24.3.CMYA1 is the new Actin muscle binding motif of a class, specifically expressing in voluntary muscle, Actin muscle (actin), TnC (troponinC), TnT (troponin T) and Actin muscle gelsolin (gelsolin) are its interact proteins, the product of its coding is positioned at the junction of adult animals heart intercalated disc, may participate in the formation of heart intercalated disc and the integrity of keeping sarcostyle.

In real work, people attempt to seek the method that a kind of easy live body is evaluated carcass quality, butcher the financial loss of bringing with minimizing, and 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 CMYA1 gene is an important muscle growth development related gene, be used in the selection of molecule aid mark this gene significant, separating clone beef qualitative correlation gene CMYA 1 also carries out the association analysis of its polymorphism and carcass trait, can develop the new technology for detection of beef matter index, for the marker-assisted breeding of ox provides new mark.

Summary of the invention

The object of the invention is to Separation of Bovine meat genes involved CMYA1, obtain CMYA1 gene mRNA sequence, realization is to the clone of beef qualitative correlation gene CMYA 1, 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 beef matter 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 beef qualitative correlation gene CMYA 1, this CMYA1 gene mRNA sequence be as described in the SEQ ID NO:1, and this CMYA1 gene protein sequence is as described in the SEQ ID NO:2.

And there is a SNP marker site at the exon 2 1058bp place of CMYA1 gene, for the C/T sudden change, at exon 2 2168bp place a SNP marker site is arranged, and is the C/T sudden change.

A kind of clone of beef qualitative correlation gene CMYA 1, 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 CMYA1 gene mRNA sequence in conjunction with the RT-PCR technology:

⑴ obtain Homo sapiens cardiomyopathy-associated protein 1 (CMYA1) by Query Database, mRNA sequence NM_194293.2 and Mus musculus cardiomyopathy-associatedprotein 1 (CMYA1), mRNA sequence NM_011724.3;

⑵ utilize NCBI BLAST instrument to compare ox expressed sequence tag EST with Highly similar sequences (megablast), choose homology greater than 80% expressed sequence tag sequences Design RT-PCR primer, fill up the vacancy between the expressed sequence tag;

⑶ utilize

Reagent (Invitrogen, USA) extract cor bovinum and the total RNA of muscle, RevertAidTM Premium Reverse Transcriptase (Fermentas) reverse transcription becomes the cDNA corresponding vacancy that increases, the RT-PCR product is reclaimed test kit (Solarbio, China) recovery connection with glue enter the order-checking of pUCM-T carrier.

Second step, according to CMYA1 gene mRNA sequence, utilize the method for comparing to carry out chromosomal localization and gene structure analysis and the albumen characteristic analysis of CMYA1 gene:

The 3rd goes on foot, and finds out two SNP sites of CMYA1 gene, and the design primer amplification contains the genomic dna in SNP site:

⑴ find out two SNP sites of CMYA1 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 ~ 6.

A kind of application of beef qualitative correlation gene CMYA 1, with in the gene CMYA 1 with the SNP mark of beef qualitative correlation as Simmental beef molecular breeding genetic marker.

And, with as follows as the step of beef molecular breeding genetic marker with the SNP mark of beef qualitative correlation in the gene CMYA 1:

The first step, the association analysis of ox CMYA1 gene Hin6I-RFLP genotype and Part Traits;

Second step, the association analysis of ox CMYA1 Gene A paI-RFLP genotype and Part Traits;

In the 3rd step, determine that gene CMYA 1 and SNP site thereof are for detection of the carcass traits such as lipid content and tare weight between individual flesh.

Advantage of the present invention and effect are:

1, Separation of Bovine meat genes involved CMYA1 of the present invention; utilize the comparative genomics method to obtain CMYA1 gene mRNA sequence in conjunction with the RT-PCR technology; and the structure of the chromosomal localization of definite CMYA1 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 CMYA1 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 two SNP sites of CMYA1 gene, design the nucleotide fragments that primer amplification goes out SNP to be measured place according to gene order, carry out RFLP-PCR with Hin6I and ApaI respectively.In the cows of analyzing, utilize two pairs of primers for different mutational sites, detect genotype and the gene frequency in each SNP site by PCR-RFLP.And carry out the proterties association analysis with 130 13 months large F3 for Simmental, and confirm available beef molecular breeding genetic marker, can develop the new technology for detection of beef matter 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 CMYA1 protein function territory figure that predicts the outcome;

Fig. 2 is C1058-T1058 of the present invention site and C2186-T2186 site pcr amplification and genotype demonstration figure, wherein Fig. 2 (a) is that C1058-T1058 site PCR and Hin6I-RFLP genotype show figure, and Fig. 2 (b) is that C2186-T2186 site PCR and ApaI-RFLP genotype show figure.

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: the total RNA extraction of ox muscular tissue, RT-PCR obtain full length cDNA sequence, obtain the mRNA sequence of CMYA1 gene, utilize the method for comparing to carry out chromosomal localization and gene structure analysis and the albumen characteristic analysis of CMYA1 gene, design the association analysis that primer amplification contains genomic dna, RFLP polymorphic detection, RFLP polymorphism and the carcass trait in SNP site, and affirmation can be used as beef molecular breeding genetic marker SNP site.

A kind of clone's step of beef qualitative correlation gene CMYA 1 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 CMYA1 gene mRNA sequence in conjunction with the RT-PCR technology:

⑴ obtain Homo sapiens cardiomyopathy-associated protein 1 (CMYA1) by Query Database, mRNA sequence NM_194293.2 and Mus musculus cardiomyopathy-associatedprotein 1 (CMYA1), mRNA sequence NM_011724.3;

⑵ utilize NCBI BLAST instrument to compare ox expressed sequence tag EST with Highly similar sequences (megablast), choose homology greater than 80% expressed sequence tag sequences Design RT-PCR primer, fill up the vacancy between the expressed sequence tag;

⑶ utilize Reagent (Invitrogen, USA) extract cor bovinum and the total RNA of muscle, RevertAidTM Premium Reverse Transcriptase (Fermentas) reverse transcription becomes the CDNA corresponding vacancy that increases, the RT-PCR product is reclaimed test kit (Solarbio, China) recovery connection with glue enter the order-checking of PUCM-T carrier; Its concrete steps are:

1. RNA extracting: Simmental cor bovinum and the muscle tissue of utilizing the laboratory to preserve are extracted total RNA, post transcription cloning CMYA1 gene;

Getting 50-100mg, to organize liquid nitrogen to be ground to Powdered, adds 1ml Trizol, and it is liquid to treat that Trizol reagent and tissue are melted into, and mixture, is added in the 1.5ml centrifuge tube after 10 minutes as for homogenate in the glass homogenizer, and room temperature leaves standstill 5min; 4 ℃ of 12000rpm * 5min are centrifugal, get supernatant and put into a new centrifuge tube; Add the 0.2ml chloroform, thermal agitation 15s leaves standstill 3min; 4 ℃ centrifugal, and 12000rpm * 10min gets supernatant; Add the 0.5ml Virahol, mixing leaves standstill 20-30min on ice.4 ℃ centrifugal, and 12000rpm * 10min abandons supernatant; Add 1ml 75% ethanol, washing precipitation 2 times.4 ℃, 7500g * 5min abandons supernatant; Room temperature place dry or super clean bench in dry up about 5min, add the Rnase-free H2O dissolving of 20 microlitres; Electrophoresis detection-RNA electrophoresis result;

2. reverse transcription: cDNA the first chain is synthetic:

In 0.2-ml PCR pipe, add following reagent: 5 μ l total RNA, 50pmol of random primer, 50pmol of oligo (dT) primer, 1 μ L dNTPs (10mM each), 70 ° of C temperature are bathed 5min.

Ice bath 10sec, the following reagent of centrifugal adding: 4.0 μ l 5*Reaction Buffer, 1.0 μ l dNTP Mix(10mmol/L), 1.0 μ l RNA enzyme inhibitors (20U/ μ l), 2.0 μ l RevertAidTM PremiumReverse Transcriptase, 20.0 μ l Total volume, 37 ° of C temperature are bathed 5min;

42 ° of C temperature are bathed 60min;

70 ° of C temperature are bathed 10min.Termination reaction;

Mentioned solution-20 ° C is preserved;

3. RT-PCR reaction system and step:

Reaction system: 1.25 μ L (50ng) of cDNA, 1 μ L of each primer (10 μ M) (such as table 1), 0.5 μ L dNTPs (10mM), 2.5 μ L of 10 * PCR buffer with 1.5mM MgCl2,0.625unitsof Taq DNA polymerase (CW, BioTech Co.Ltd, China), and 18.625 μ L nuclease-freewater, 25 μ L reaction mixture containing Total volume;

The PCR reactions steps: 94 degree denaturation 5min, 94 degree sex change 30s, (TmF+TmR/2) degree 30s, 72 degree extend (the extension time is according to PCR product size 1Kb/min) 35 circulations, and 72 degree extended 15 minutes;

Table 1.CMYA1 RT-PCR primer

4. product reclaims to connect and enters the order-checking of pUCM-T carrier:

Product reclaims:

Downcut the sepharose that contains target DNA under ultraviolet lamp, weighing gel weight is with 1mg=1 μ l conversion gel volume.The sol solution that adds 3 times of gel volumes; After suspending evenly in 55 ℃-65 ℃ heating 10min, during every 2-3min rock once, until gel piece melts fully; The DNA-agarose solution is added to one reclaims on the purification column, and pillar is contained in a clean 2ml collects in vitro, the centrifugal 1ml of 13000rpm discards effluent liquid.For the sample of volume greater than 700 μ l, then be added to respectively on the different pillars each 700 μ l; With the lavation buffer solution washing pillar of 700 μ l dehydrated alcohols dilutions, leave standstill 2-3min after adding in the pillar.The centrifugal 1min of 13000rpm under the room temperature; Abandon filtrate, wash again once with same method;

Pillar is contained on the clean 1.5ml centrifuge tube of sterilizing, and on base for post matter, the centrifugal 1min of 13000rpm is to elute DNA for the DNA elution buffer of adding 30-50 μ l or aseptic deionized water (65 degree preheating).Get 3 μ l samples, 1% agarose gel electrophoresis detects and reclaims the result.Reclaiming product-20 degree preserves.

The PCR product directly is connected with the T carrier:

In advance with PCR instrument (or the water in the ice chest) Temperature Setting at 16 ° of C, get one by one the 200ul Eppendorf tube of sterilization, add: 4 μ L goal gene, 1 μ L T carrier, 1 μ L ligase enzyme damping fluid, 10 x buffer, 1 μ L PGE, 1 μ L T4 dna ligase (5U/ul), 2 μ L aqua sterilisas.Of short duration centrifugal again after the above-mentioned mixed solution light shaking, then place 16 ° of C PCR Wen Yizhong incubated overnight (16h).Product after the connection is used for the transformed competence colibacillus cell immediately, and the picking mono-clonal utilizes universal primer M13R and M13F order-checking;

⑷ obtain CMYA1 mRNA total length;

Splicing NCBI expressed sequence tag EST:DV802652.1, DY086104.1, DY191701.1, DY051477.1, DY043391.1, BM287640.1, EE371471.1, DV791878.1 and RT-PCR product sequencing result, obtain Bos taurus cardiomyopathy-associated protein 1(CMYA1) mRNA, CMYA1 mRNA sequence 6272bp, sequence is shown in SEQ ID NO:1

CMYA1 mRNA sequence: SEQ ID NO:1 is as follows:

acagaccccgagctagggcagcgagaaggcgtccgacggagccagcagagtgagagcagcccaaacaagaggaaccgacagagccgcaggcctcacctgccgaagaccccaaaccccaacagccaggcagaacccgcagaaggatggccaatgcccagacgcagatggcccccaccccaaccatcccgatggcagctacagaggacctgcccctccctccaccacctgccctggaggatctaccaccgccgccacccaaggagtccttctccaagttccaccagcagcggcaagccagcgagctccgccgcctctacaagcacatccaccctgagctccgcaagaatctggctgaggccgtggccgaagacttggctgaggtcctgggttccgaggagcccaccgagggtgatgtccaatgcatgcgctggatctttgagaactggcggctcgatgccattggggaccatgagaagccacctgccaaggagtccgtgcctggtggcaacgtccaggccacctcccgcaagtttgaggaaggctcctttgctaatagtataaaccaggagccggctggacctcggccatctggaggggacgtgcgtgcagcccgccagctgtttgagacaaagccgctggatgcgctgacagttcgtgctgaagcatcagaggctacagtgagggagcctgcagccagtggagacgtccagggtaccaggatgctctttgagacacggccactggaccgccttggctcccgcccctccacccaggagcagagccccttggagctgcgctcagagatccaggagctgaaaggcgatgtgaagaagacagtgaagctattccaaacagagccgctgtgtgccatccaggactcagagggcgccatccacgaggtcaaggccgcctaccgggaggagatccaaagcaacgcagtgaggtctggccgttggctcttcgagaccaagcctctggatgccatcaaccgggaccccagccaggtgcgggtgatccgggggatctccctggaggaggcagcccggcctgatgtcagcgcgactcgctggatctttgagacacagcccctggatgcgatccgggagatcttagtggatgagcaggacttccagccatccccggaccttatccctcctggtccggatgttcagcagcagcggcgtctgtttgagacccgagcattagacactctcaagggggaagaggaggccggagcagaggccccacccaaagaggcagtggtccccggtgacgtccgctccaccctgtggctgtttgagacgaagcccctggacaccctcagagacaatgtccaagtgggtcatctccagcgggtgggtccccaggagggcgagaggttcacaaacgagcatctatccaatgctgacccctcagcaccaaccctctctcagggtgccccccagagggatggggtgaagggagatgtgaagaccttcaagaacctttttgagacccttcccctggacagtatcgggcagggggaagctttggcccctgggagcatatgcagagcagaaggaactgattctgccgggcagtcccaggacacagggtccccagtgtacgccatgcaggatggcaaaggtcacctccatgccctgacctccgtcagcagagaacaggtagtcggaggtgacgtgcagggttacaagtggatgtttgagacacagcccctagaccgactaggccgaagccccagtaccgtcgatgtggtgcggggtatcacccagcaggaagtggtggctggagatgtgggcactacccggtggctctttgagacacagcccctggaggtaatccaccaacgggagcagcaggaacgagaggaagaagaaggaaagcctcagggaggccctcagcccgaaataccccacaagggtgacgtgcagaccatccgctggttgttcgagacgtgcccgatgagtgagctggccgagaggcaggggtcagaggtcacagacctcacaagcaaggcacggtcgtgcacctggatgttcgcgccccaatccccggactggcctgaaggctccaaggagcagcaccttgaggtgagccaggtccaggctggggaaagacagacggagagacacgtctttgaaactgagcctctgcaggccgcgggccacccctgcggaagggggcccgtgcggtactgcagcagagtggacatcccctcggggcaggtgtctcgtcagaaggaggttttccaggctctggaggcaggcaagagagaagaccagggacccagggaaatccctgagcccatatcagcgggctcagtgcacaagttcacctggctctttgagaactgccccatgggctccctggcagctgagagcatccgagggggcaacctccaggaagagcagcccgtgggtccctcgggcaagggggtgccggagaggcaagagactgcggccgaggggaccctgcggacgctgcatgccacgcctggcgtcctgcaccatggaggcatcctcatggaggcccgagggccaggggagctctgcctcgccaggtacgtgctcccatgcccagggcaggacagcccccatgttcggaaggaggagctggtgtctggggaactccccaggattgtccgccaagtgctgcgccgggcagacgtggaccagcaggggctgctggtgcaggaggaccccgcgggccagcttcacctcaagccgctgaagctgccagcgccaggcagcagcgggagcatcgaagacatggaccctgagttccagcagttgctggcttgtggcctcgggacctcggcggggaggacggggctggtgatgcaggagacagagcggggcctggtggcgctgaccgcctactccctgcaaccctggctagccagcagggcccccgaaaggagcagtgtgcagctgctggccggctgcataaacaaaggagacctgagtggcctgcacaatctgcggtgggagccgccggctgactcaagtccagtgccggccagcaagggggcccagaagctgcccccagctgaaagcatcatccacgttcccccactggaccctggcaaggggatggggcatctgagagggccgggggccaccccctgcgccccacaggccgctggaaaggcagtctctctggctggggaagaaaagcaggaaagcaggtgcactgggcagaaagggacggcagctttaggaaagtcagagggagccatgactatgcccccaggacctaggtttccagccctccaggtcactatgcagagtcgaaggacgccaacagcccaagcccaaagcctgcagcagcaagctcggagcaagcacaagccgggccccacccctggggccgcctctctgcccacccaggatgggcttccgcaagcaccggccgcagggactgcccagagcaacagcaagcctctggcgggaggcaaccccaggatcccagcagcccccagcaaggtcagtggggaacagaaagcactgcctggagggctatctggggggtgggtgactattcaggatggcatctacactgctcatcccgccaggacctttgacctacccggggctgtctggccatctgggcaaggaccctttccactcctggaaggcctgggtcagagtctcgggcccgggcaagaggagctggggggccacacacagaaggcctgggagcctccagagaaggtgatggcaggactcggcccagggggcctccaagctgcagagaccaccctgaaggctgccttagcccaccacactcgggcctctgagcctcgggcggcaggtgccagtctgcactctcgtaacgcctctgttcctcctcctcctcctctcccagcttctgtgacgggaccggacttcccagcccaacccaggcatgatgagaattccatccggcagacttccaagcccacgcaagacccccttctccactcccacaacagccctgctggccagaaaagccctgggcagccacagacaaagaccctgaaaccggagcctcccacacacctgagaaaaaagccccagctgccccccaaacctgcacacttaagccagctcccccttcctcgctggctgtccaagcccgcagccctggctcacagcgccgctgaggagggggggcaagggaaacacaaacaaggtgagactgctacagccaaccacgaccctcggccacacagggtctccatcgctgcagaccagggccgagtatctctaccccagggccctgctggacagagccagcccagcccccagcatggccccagcaccgtggcccccagccccaccaagagccaggcgataggcagcaacaaccacagccctgaccccctcaggctctcagctctcagcagccaccccatctcactgcagcagggccctagcctcacaggagagaagtgctcggacagttcccagcaaggggcccccaagagccctgagattctgcagggaggccagcaagagctccagggcctcctgagccaggtgcaagctctggagaaggaggccgaaagcactgtggacgtgcaggcgctgcagaggctctttgaggctgtgccccagctaccaggagcccctccggctcccgctaccccccacaagcctgaggcctcggtggagcaggcctttggggagctgacgagggttagcacagaagtggcccggctgaaggaacagaccctggccaggctgctggacatcgaggaggctgtgcacaaggcactcagctccatgtctagcctccagactgggactaacaccaggggccattcccagggacccccaaaggaacacagtgctcaagagatcagtgtcacagacagcgggagagtcaggcccaactgctcaggccaggaggtcaaaagtcaaactctagtcaagagccaaactgaggttacaagccatactgaggtccagagtcaagccaaggtcagaaatcactcagaggccggaagccaagcagccccggccaccccttccaccaggaagctggaggccttgagagaagactcgcaccttccccaagcgttacctctcagcagggagtcaccctcctccccaacctttatctccatggagtcggccacaaggaagcttccggaggaggccagccccaggggcaaccctgagatctctgtgaagagggcacatttcacgcaggatgaatgccagactcagccccaccagaaagatatctggcacaaggctggagagaaagaggccccccagctctctggaccgccaccacctggccctgctgcagccagcgccctgcccaccaggcagaagagtgctctggagccgcagacagcgccgggtggctcccggcacgatggagccacaggagcagggactgagagggtgggccagtgcaggaccacagcgctcgtgtcccccaccacggtcactgagcccgccgagccacccaggggcccaggcccccacctcgggcaccacacttcccccttgatgaggcagtttctgcacagccagactgggctcagcaccggcctggcagaagctgagatgctgcgtgtgccctgtggccaccccacaccaactgcgcagtgagcccctccatctgccaccacagtcttccacccacctccagggacccgggacggaggtgggtgctcacctcctgcacgcacggagaaagaaccaaaaagaaagggcatgttctgagatccaggggcagtttccctttcatcagtagcatccatgggccaggttcttgcaaaaggcagggggggatggtagggaagaagccctccggagtagcccaagcccagcaacagaatgggggcttgtggctgcttgtggagcatataatgtgtacagaaacacacgcttctcacacaggtctggcccgcacgccaacacaagtgagtctgcatgccactgcctgcgattgagccccctgagcgaccttccactttttcaatctttatttctttaatacgagtcaaatgttctcagccttctgctctttaattcctagagaagacagggccagccggattggtctctgttacatgttgatgggccccagaagggccccgtcatcagcacctccctcattcagcgggtcatcctggcagcacaaatttcacacagacaaagtctacgatcaccaatgtccctagaagccaaagattctctccacccccacagcctctcactttgtaccccaaagaagaaggatacagtctaataaaccaaagttttattcccttc

Second step, according to CMYA1 gene mRNA sequence, utilize the method for comparing to carry out chromosomal localization and gene structure analysis and the albumen characteristic analysis of CMYA1 gene:

The genome sketch of ox is completed, and we have obtained CMYA1 gene mRNA sequence, so we have utilized the method for comparison to carry out the structural analysis of chromosomal localization and the gene of CMYA1 gene;

⑴ CMYA1 is positioned at karyomit(e) Chromosome 22:12549676-12558895bp, intron of two exons is arranged, the long 6272bp of transcript, the long 1820residues of aminoacid sequence, derivation isoelectric points of proteins (Isoelectric point) out: 6.4162, molecular weight (Molecular weight): 194,814.91g/mol the CMYA1 protein sequence: sequence is shown in SEQ ID NO:2

The CMYA1 protein sequence: SEQ ID NO:2 is as follows:

MANAQTQMAPTPTIPMAATEDLPLPPPPALEDLPPPPPKESFSKFHQQRQASELRRLYKHIHPELRKNLAEAVAEDLAEVLGSEEPTEGDVQCMRWIFEN?WRLDAIGDHEKPPAKESVPGGNVQATSRKFEEGSFANSINQEPAGPRPSGGDVRAARQLFETKPLDALTVRAEASEATVREPAASGDVQGTRMLFETRPLDRLGSRPSTQEQSPLELRSEIQELKGDVKKTVKLFQTEPLCAIQDSEGAIHEVKAAYREEIQSNAVRSGRWLFETKPLDAINRDPSQVRVIRGISLEEAARPDVSATRWIFETQPLDAIREILVDEQDFQPSPDLIPPGPDVQQQRRLFETRALDTLKGEEEAGAEAPPKEAVVPGDVRSTLWLFETKPLDTLRDNVQVGHLQRVGPQEGERFTNEHLSNADPSAPTLSQGAPQRDGVKGDVKTFKNLFETLPLDSIGQGEALAPGSICRAEGTDSAGQSQDTGSPVYAMQDGKGHLHALTSVSREQVVGGDVQGYKWMFETQPLDRLGRSPSTVDVVRGITQQEVVAGDVGTTRWLFETQPLEVIHQREQQEREEEEGKPQGGPQPEIPHKGDVQTIRWLFETCPMSELAERQGSEVTDLTSKARSCTWMFAPQSPDWPEGSKEQHLEVSQVQAGERQTERHVFETEPLQAAGHPCGRGPVRYCSRVDIPSGQVSRQKEVFQALEAGKREDQGPREIPEPISAGSVHKFTWLFENCPMGSLAAESIRGGNLQEEQPVGPSGKGVPERQETAAEGTLRTLHATPGVLHHGGILMEARGPGELCLARYVLPCPGQDSPHVRKEELVSGELPRIVRQVLRRADVDQQGLLVQEDPAGQLHLKPLKLPAPGSSGSIEDMDPEFQQLLACGLGTSAGRTGLVMQETERGLVALTAYSLQPWLASRAPERSSVQLLAGCINKGDLSGLHNLRWEPPADSSPVPASKGAQKLPPAESIIHVPPLDPGKGMGHLRGPGATPCAPQAAGKAVSLAGEEKQESRCTGQKGTAALGKSEGAMTMPPGPRFPALQVTMQSRRTPTAQAQSLQQQARSKHKPGPTPGAASLPTQDGLPQAPAAGTAQSNSKPLAGGNPRIPAAPSKVSGEQKALPGGLSGGWVTIQDGIYTAHPARTFDLPGAVWPSGQGPFPLLEGLGQSLGPGQEELGGHTQKAWEPPEKVMAGLGPGGLQAAETTLKAALAHHTRASEPRAAGASLHSRNASVPPPPPLPASVTGPDFPAQPRHDENSIRQTSKPTQDPLLHSHNSPAGQKSPGQPQTKTLKPEPPTHLRKKPQLPPKPAHLSQLPLPRWLSKPAALAHSAAEEGGQGKHKQGETATANHDPRPHRVSIAADQGRVSLPQGPAGQSQPSPQHGPSTVAPSPTKSQAIGSNNHSPDPLRLSALSSHPISLQQGPSLTGEKCSDSSQQGAPKSPEILQGGQQELQGLLSQVQALEKEAESTVDVQALQRLFEAVPQLPGAPPAPATPHKPEASVEQAFGELTRVSTEVARLKEQTLARLLDIEEAVHKALSSMSSLQTGTNTRGHSQGPPKEHSAQEISVTDSGRVRPNCSGQEVKSQTLVKSQTEVTSHTEVQSQAKVRNHSEAGSQAAPATPSTRKLEALREDSHLPQALPLSRESPSSPTFISMESATRKLPEEASPRGNPEISVKRAHFTQDECQTQPHQKDIWHKAGEKEAPQLSGPPPPGPAAASALPTRQKSALEPQTAPGGSRHDGATGAGTERVGQCRTTALVSPTTVTEPAEPPRGPGPHLGHHTSPLMRQFLHSQTGLSTGLAEAEMLRVPCGHPTPTAQ

⑵ use online software Http:// cn.expasy.org/tools/Prediction CMYA1 protein function territory:

As shown in Figure 1, gene structure territory predictive display CMYA1 albumen comprises the abundant district of a proline(Pro), a spiral corner spiral, 16 amino acid of eight Actin muscle bonding units repeat, and (T/S/G) X (R/K/T) WLFETXPLD of conserved sequence GDV (K/Q/R) is contained in these 16 amino acid repeating units.

The 3rd goes on foot, and finds out two SNP sites of CMYA1 gene, and the design primer amplification contains the genomic dna in SNP site;

⑴ find out two SNP sites of CMYA1 gene:

Choose two SNP site SNP rs110346467 and the SNP rs110575295 of CMYA1 gene by the inquiry ncbi database, wherein SNP rs110346467 is positioned at Chromosome 22:12554890bp, CMYA1 gene Second Exon 1058bp is C/T same sense mutation (C1058-T1058); SNPrs110575295 is positioned at Chromosome 22:12553762bp, and CMYA1 gene Second Exon 2186bp is C/T same sense mutation site (C2186-T2186);

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:3?6467-F?CCCGCAAGTTTGAGGAAG

SEQ?ID?NO:4?6467-R?CATTGGATAGATGCTCGTTTGT

SEQ?ID?NO:5?5295-F?CATCCGCTGGTTGTTCG

SEQ?ID?NO:6?5295-R?ACGGGCTGCTCTTCCTG

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 and takes pictures, such as Fig. 2 (a), 2(b) shown in;

The 4th step, the RFLP-PCR polymorphic detection:

⑴ select suitable restriction endonuclease to carry out RFLP-PCR for two different SNP sites:

RFLP-PCR is carried out with Hin6I and ApaI respectively in C1058-T1058 site and C2186-T2186 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, with centrifugal behind the sample blending, and 37 ℃ of water-bath 4h, detect enzyme with 2% agarose gel electrophoresis and cut the result, the record genotype is taken pictures under ultraviolet lamp, C1058-T1058 site and C2186-T2186 loci gene type, such as Fig. 2 (a), 2(b) shown in;

To single nucleotide polymorphism the distribution in cows add up:

In the cows of analyzing, utilize two pairs of primers for different mutational sites, detect genotype and the gene frequency in each SNP site by PCR-RFLP, statistics is shown in table 2 and table 3;

Table 2.SNP rs110346467 (C1058-T1058) allelotrope gene frequency

Table 3.SNP rs110575295 (C2186-T2186) allelotrope gene frequency

Can find out the C1058-T1058 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 C2186-T2186 site, in the cows body, the TT type is higher than the CT type, and without the CC type, T allelotrope is protogene, and its frequency is far longer than C allelotrope.

A kind of application of beef qualitative correlation gene CMYA 1, 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 first step, the association analysis of ox CMYA1 gene Hin6I-RFLP genotype and Part Traits:

Utilize the clone of beef qualitative correlation gene CMYA 1, the Hin6I-RFLP genotype detection result who obtains, the target cows are carried out association analysis between genotype and growth traits, carcass trait and Meat Quality, eliminated kind, butcher batch and sex between difference after, simple mean and the standard error analysis of proterties the results are summarized in table 4 between genotype, found that SNP rs110346467 does not have proterties related with ox part Meat Quality;

The association analysis of table 4.SNP rs110346467 proterties

Annotate: not remarkable without letter mark expression difference between each index genotype in the table, P〉0.05.

Second step, the association analysis of ox CMYA1 Gene A paI-RFLP genotype and Part Traits:

Utilize the clone of beef qualitative correlation gene CMYA 1, the ApaI-RFLP genotype detection result who obtains, the target cows genotype and growth traits have been carried out, association analysis between 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 5 between genotype, found that, (Intermuscular fatty acid) the QIB% significant correlation of lipid content between SNP rs110575295 and flesh (p=0.049＜0.05), lipid content is significantly higher than the CT type between TT type flesh, exceeds 0.303%; With tare weight utmost point significant correlation (p=0.009＜0.01), the TT type tare weight utmost point is significantly higher than the CT type, exceeds 4.00kg; SNP rs110575295 can be used as the genetic marker of beef molecular breeding, be C or T according to the 2186th Nucleotide of CMYA1 gene Second Exon, thereby determine that this individuality is in the allelotype in this mutational site, and with this difference of assessing lipid content and tare weight between this individuality flesh, can be applied in the middle of the molecular mark of ox;

The association analysis of table 5.SNP rs110575295 proterties

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, beef qualitative correlation gene CMYA 1 of the present invention and SNP site thereof can be used for detecting the carcass trait such as lipid content and tare weight between individual flesh, thereby for the molecular breeding of ox provides a new genetic marker, and will in the breeding of ox, play a significant role.

Claims (6)

1. beef qualitative correlation gene CMYA 1 is characterized in that: this CMYA1 gene mRNA sequence is as described in the SEQ ID NO:1, and this CMYA1 gene protein sequence is as described in the SEQ ID NO:2.

2. beef qualitative correlation gene CMYA 1 according to claim 1, it is characterized in that: there is a SNP marker site at the exon 2 1058bp place at the CMYA1 gene, for the C/T sudden change, at exon 2 2168bp place a SNP marker site is arranged, and is that C/T suddenlys change.

3. the clone of a beef qualitative correlation gene CMYA 1, 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 CMYA1 gene mRNA sequence in conjunction with the RT-PCR technology:

⑴ obtain Homo sapiens cardiomyopathy-associated protein 1 (CMYA1) by Query Database, mRNA sequence NM_194293.2 and Mus musculus cardiomyopathy-associatedprotein 1 (CMYA1), mRNA sequence NM_011724.3;

⑵ utilize NCBI BLAST instrument to compare ox expressed sequence tag EST with Highly similar sequences (megablast), choose homology greater than 80% expressed sequence tag sequences Design RT-PCR primer, fill up the vacancy between the expressed sequence tag;

⑶ utilize

Reagent (Invitrogen, USA) extract cor bovinum and the total RNA of muscle, RevertAidTM Premium Reverse Transcriptase (Fermentas) reverse transcription becomes the cDNA corresponding vacancy that increases, the RT-PCR product is reclaimed test kit (Solarbio, China) recovery connection with glue enter the order-checking of pUCM-T carrier.

Second step, according to CMYA1 gene mRNA sequence, utilize the method for comparing to carry out chromosomal localization and gene structure analysis and the albumen characteristic analysis of CMYA1 gene:

The 3rd goes on foot, and finds out two SNP sites of CMYA1 gene, and the design primer amplification contains the genomic dna in SNP site:

⑴ find out two SNP sites of CMYA1 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.

4. the clone of beef qualitative correlation gene CMYA 1 according to claim 3 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 ~ 6.

5. the application of a beef qualitative correlation gene CMYA 1 is characterized in that: with in the gene CMYA 1 with the SNP mark of beef qualitative correlation as Simmental beef molecular breeding genetic marker.

6. the application of beef qualitative correlation gene CMYA 1 according to claim 5 is characterized in that: with as follows as the step of beef molecular breeding genetic marker with the SNP mark of beef qualitative correlation in the gene CMYA 1:

The first step, the association analysis of ox CMYA1 gene Hin6I-RFLP genotype and Part Traits;

Second step, the association analysis of ox CMYA1 Gene A paI-RFLP genotype and Part Traits;

In the 3rd step, determine that gene CMYA 1 and SNP site thereof are for detection of the carcass traits such as lipid content and tare weight between individual flesh.

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