CN107475400B - Method for auxiliary detection of cattle growth traits through MYLK4 gene and special kit thereof - Google Patents
Method for auxiliary detection of cattle growth traits through MYLK4 gene and special kit thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting the growth traits of cattle with the assistance of MYLK4 gene and a special kit thereof. Taking cattle genome DNA as a template and a primer pair P as primers, and carrying out PCR amplification to obtain a cattle MYLK4 gene; digesting the PCR product by using a restriction enzyme HhaI, and performing agarose gel electrophoresis; and identifying the single nucleotide polymorphism at 61595 th site of the MYLK4 gene of the cattle according to the electrophoresis result. The method provided by the invention can detect the single nucleotide polymorphism of the MYLK4 gene of the cattle, can be used as a molecular genetic marker closely related to the growth traits of the cattle, is used for auxiliary selection and molecular breeding of the cattle, and accelerates the breeding speed of fine cattle.
Description
Technical Field
The invention belongs to the field of molecular genetics, relates to screening and detection of Single Nucleotide Polymorphism (SNP) of a cattle gene as a molecular genetic marker, and particularly relates to a method for detecting single nucleotide polymorphism of MYLK4 gene of cattle and application.
Background
A molecular genetic marker is a marker of genetic polymorphism at the DNA level, which can directly reflect genetic variation in a DNA sequence. Because the source of the DNA sequence is the difference of the DNA sequence, the DNA sequence is called as a molecular marker, and the DNA sequence has the characteristics of high genetic diversity, strong stability and small environmental influence in biological populations. The molecular marker assisted selective breeding is a main research content of modern animal molecular breeding, and directly selects the genotype of characters on the DNA level, so that the accuracy of seed selection is greatly improved, and the defects of the traditional animal breeding method are overcome.
Single Nucleotide Polymorphism (SNP) is an important molecular genetic marker, mainly refers to sequence polymorphism at the genome level caused by a change of a certain nucleotide in a DNA sequence, and mainly takes the form of single nucleotide transition and transversion. Compared with other molecular markers, SNP has the advantages of large genome coverage, high density, high resolution, genetic stability, easy realization of analysis automation and the like. Therefore, the method is widely applied to the research fields of biology, botany, medicine, animal breeding, biological evolution and the like.
Since the first proposal by Botestein et al of the concept of DNA restriction fragment length polymorphism, PCR-RFLP has been used in large quantities for SNP detection. The premise for this approach is that the site of the SNP must contain a site recognized by the appropriate restriction enzyme. The method is one of the most classical methods, in which the target fragment is cut by restriction endonuclease and then analyzed by gel electrophoresis to accurately identify the genotype of the SNP site.
Myosin (myosin) is an important protein constituting muscle, and is a hexamer composed of two heavy chains (heavy chain), two essential light chains (essential light chain) and two Regulatory Light Chains (RLC). Myosin has ATPase activity and, when bound to ATP, is capable of changing its conformation to drive the myofilament to slide, causing contraction of the muscle. Myosin light chain kinase (MYLK) is capable of phosphorylating the myosin Regulatory Light Chain (RLC) and thereby activating or modulating myosin ATPase activity. Studies have shown that MYLK is associated with cytoskeletal changes that in turn may regulate cellular motility, secretory activity, epithelial cell permeability changes, and changes in platelet morphology; it also promotes elongation of axonal growth cones and the development of cell tension fibers, which in turn affects cytokinesis and apoptosis and repair of cells. Plays an important role in the life activities and the growth and development process.
MYLK4 is one of the MYLK family members, and is a Ca2+/CaM independent constitutively active kinase. It was found that it may be a significant contributor to RLC phosphorylation in cardiomyocytes. At present, the study on the MYLK4 gene polymorphism at home and abroad mainly focuses on the correlation study on cardiovascular diseases, tumors and the like, and the study on the livestock MYLK4 gene single nucleotide polymorphism has not been reported yet.
Disclosure of Invention
The invention aims to provide a method for detecting the growth traits of cattle with the assistance of MYLK4 gene and a special kit thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting single nucleotide polymorphism of MYLK4 gene of cattle comprises the following steps:
taking cattle genome DNA as a template and a primer pair P as primers, carrying out PCR amplification on partial fragment of cattle MYLK4 gene, digesting the PCR amplification product by using restriction enzyme HhaI, carrying out agarose gel electrophoresis, and identifying the genotype of the 61595 th single nucleotide polymorphism site of the cattle MYLK4 gene according to the electrophoresis result;
the sequence of the primer pair P is as follows:
an upstream primer F: 5'-CGGACACTTGTTCTCAATCGGC-3'
A downstream primer R: 5'-GTGGTCTAGGAATCTGGGTG-3' are provided.
The reaction procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 25s for 36 cycles; extension at 72 ℃ for 5 min.
The agarose gel electrophoresis was performed using agarose gel with a mass concentration of 3%.
The electrophoresis result of the genotype of the 61595 th single nucleotide polymorphism site of the cattle MYLK4 gene is as follows: the AA genotype shows a strip of 208 bp; the AG genotype is represented by three bands of 208bp, 185bp and 23 bp; the GG genotype shows two bands of 185bp and 23 bp.
The detection method of single nucleotide polymorphism of the cattle MYLK4 gene is applied to cattle molecular marker-assisted selective breeding.
The detection method of single nucleotide polymorphism of the cattle MYLK4 gene is applied to auxiliary detection of cattle growth traits.
The growth trait is selected from body height or body slant length.
The cattle is selected from Qinchuan cattle, Pinus parviflora, Xianan cattle, Nanyang cattle or Chadamu cattle.
A special kit for auxiliary detection of cattle growth traits by MYLK4 gene comprises a primer pair P (sequence is described above) for typing of single nucleotide polymorphism sites 61595 of MYLK4 gene based on PCR-RFLP method.
The invention has the beneficial effects that:
aiming at the mutation at the 61595 th site, the invention discloses a detection method, which solves the problems of complexity and instability of the traditional PCR-SSCP method by designing a specific primer, amplifying a target fragment by PCR and then carrying out enzyme digestion identification by using a specific restriction endonuclease, and can simply, quickly, low-cost and high-precision identify the single nucleotide polymorphism of a gene.
The invention detects the genotype of the mutation site and analyzes the gene frequency, and the site is associated with the growth traits of five cattle breeds. The result shows that the single nucleotide polymorphism of the MYLK4 gene 61595 position of the cattle is G or A, which can be used as a marker for molecular genetic auxiliary breeding, thereby providing basic data for the molecular marker auxiliary selective breeding of the cattle and accelerating the germplasm resource improvement work of Chinese cattle.
Drawings
FIG. 1 is a sequence diagram of MYLK4 gene 61595 (AC _ 000180.1: G61595A) in cattle: boxed position indicates the site.
FIG. 2 is an electrophoretogram of a 208bp fragment PCR-amplified from the MYLK4 gene of cattle.
FIG. 3 shows the electrophoresis result of the HhaI enzyme digestion of the PCR products of different genotypes at 61595 position of MYLK4 gene of cattle: m is Marke I and is respectively 600bp, 500bp, 400bp, 300bp, 200bp and 100 bp.
Detailed Description
The invention is described in detail below with reference to the drawings and examples, which are illustrative and not restrictive of the invention.
The invention designs primers according to a cattle MYLK4 gene sequence, respectively takes genome DNA pools of 5 cattle varieties as templates, performs PCR amplification, and obtains a partial sequence of the cattle MYLK4 gene by product sequencing. Comparing with a reference sequence published on NCBI, finding that the mutation of A > G exists at 61595 position of MYLK4 gene of cattle, and detecting the mutation by using PCR-RFLP method. The single nucleotide polymorphism of the cattle MYLK4 gene is subjected to correlation analysis with the growth traits of cattle, so that the correlation analysis proves that the correlation analysis can provide a basis for cattle molecular breeding.
Cloning of partial sequence of cattle MYLK4 gene and polymorphism detection thereof
1. Sample collection and genomic DNA extraction
(1) Collection of blood samples
The invention adopts 5 cattle breeds, 535 cattle are taken as detection objects in total, and the collection method of blood is jugular vein blood collection. The area where the blood sample was collected and the data of each variety are shown in Table 1.
TABLE 1 Experimental animal Condition
(2) Extraction of genomic DNA from blood samples
① frozen blood sample (mainly blood cells) is thawed at room temperature, 500. mu.L of blood is sucked into a 1.5mL centrifuge tube, Phosphate Buffer Solution (PBS) with the same volume is added for even mixing, the mixture is gently shaken, centrifuged at 12000rpm for 5min at 4 ℃, the supernatant is discarded, and the steps are repeated until the supernatant is transparent.
② adding DNA extraction buffer solution 500 μ L into the centrifuge tube, gently blowing to make the blood cell precipitate to separate from the wall of the centrifuge tube, and bathing in water at 37 deg.C for 1 h.
③ adding proteinase K to 3 μ L (20mg/mL), mixing, digesting in 55 deg.C water bath overnight (about 16 h) until no flocculent precipitate is observed, clarifying the solution, and adding 1 μ L proteinase K, mixing, and digesting until it is clear.
④ the sample is taken out and added with 200 μ L of 6mol/L NaCl, the mouth bottom is shaken 15 times to mix the solution thoroughly, the solution is centrifuged at 12000rpm for 10min at 4 ℃, and the supernatant is taken out and put into a 2.0mL centrifuge tube.
⑤ adding 1mL Tris-saturated phenol, placing on ice, gently shaking for 20min, mixing well, centrifuging at 12000rpm for 10min at 4 deg.C, transferring the upper water phase into another sterilized 2.0mL centrifuge tube by using a pipette.
⑥ 0.5mL of Tris-saturated phenol and 0.5mL of chloroform were added, gently shaken on ice for 20min, centrifuged at 12000rpm for 10min at 4 ℃ and the upper aqueous phase was pipetted into another sterilized 2.0mL centrifuge tube.
⑦ adding 1mL of chloroform, placing on ice and gently shaking for 20min, centrifuging at 12000rpm for 10min at 4 ℃, and transferring the upper aqueous phase to another sterilized 1.5mL centrifuge tube by using a pipette.
⑧ adding 1mL of precooled absolute ethanol (-20 deg.C), shaking with gentle mouth bottom for several times until DNA is precipitated, standing at-20 deg.C for 30min, taking out, centrifuging at 4 deg.C and 12000rpm for 10min, and discarding ethanol.
⑨ adding 70% ethanol 1mL, gently shaking for 10min, centrifuging at 12000rpm for 10min at 4 deg.C, discarding ethanol, and repeatedly rinsing once (sucking out the residual ethanol from the bottom of the tube with a pipette).
⑩ standing at room temperature for 15min, drying in 60 deg.C oven for 30s to volatilize ethanol, adding ultrapure water 50 μ L, storing at 4 deg.C until DNA is completely dissolved, measuring concentration with spectrophotometer, and storing at-80 deg.C.
(3) Construction of DNA pools
And measuring the OD values of the DNA samples at 260nm and 280nm and the DNA content of the DNA samples by using an ultraviolet spectrophotometer. If OD is greater than the total260/OD280The ratio is less than 1.6, which indicates that the sample contains more protein or phenol, and purification is required; if the ratio is greater than 1.8, then RNA purification removal should be considered. After the DNA detection is finished, a certain amount of DNA is taken out and diluted to 10 ng/. mu.L, and then 5 DNA samples are taken outRandomly selecting 30 diluted samples from the cattle population, and uniformly mixing 2.5 mu L of each sample to construct a DNA pool.
2. Amplification primer design
By taking a bovine MYLK4 gene sequence (AC _000180.1) published by NCBI as a reference, the Oligo 7.0 software is used for designing a PCR primer capable of amplifying a 10 th intron region of a bovine MYLK4 gene, and the primer sequence is as follows:
upstream primer 10F: 5'-ATTCCGAGTGGCTTTTCTCA-3' (SEQ. ID. NO.1, 20nt)
Downstream primer 10R: 5'-CATAGGATGGTGGTCTAGGA-3' (SEQ. ID. NO.2, 20nt)
PCR amplification
(1) The PCR reaction system is shown in Table 2.
TABLE 2 PCR reaction System
(2) The PCR reaction procedure is shown in Table 3.
TABLE 3 PCR reaction procedure
Sequencing of PCR products
The PCR product amplified by using the mixed DNA pool as a template is sent to Beijing Okkomy Biotech limited for sequencing. The sequencing result of the target fragment of the cattle MYLK4 gene is compared with a reference sequence, and the 10 th intron region is found to have an A > G mutation (FIG. 1, AC _ 000180.1: G61595A), but cannot form any enzyme cutting site. Therefore, the primer mismatching method is adopted to introduce the mutant base so as to form the enzyme cutting site.
5. Design of primer P for introducing enzyme cutting site in PCR-RFLP analysis of MYLK4 gene of cattle
After DNA pool amplification sequencing, the mutation of the site (61595 th site of MYLK4 gene reference sequence) is found to have no proper enzyme cutting site, so that an HhaI enzyme cutting site is artificially constructed, the genotype judgment can be carried out by using a PCR-RFLP method, and the constructed primer pair P is as follows (the design completion time is 2016 (10 months)).
An upstream primer F: 5' -CGGACACTTGTTCTCAATCGGC-3’(SEQ.ID.NO.3,22nt)
A downstream primer R: 5'-GTGGTCTAGGAATCTGGGTG-3' (SEQ. ID. NO.4,20nt)
Wherein, mismatched bases, namely underlined bases, are introduced into the 2 nd position from the 3' end of the upstream primer.
6, PCR product restriction enzyme digestion and RFLP detection
And (3) PCR reaction system: referring to table 2, genomic DNA extracted from individual blood samples was used as a template.
PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 25s, for a total of 36 cycles; extension at 72 ℃ for 5 min.
As shown in FIG. 2, the PCR amplification result showed an amplified fragment size of about 208 bp.
And (3) carrying out restriction enzyme HhaI enzyme digestion on the PCR amplification products, and judging the SNP polymorphism according to the electrophoresis result. 12.5 μ L of HhaI enzyme cutting system is: 10 μ L of PCR product, 10 XBuffer Tango1.25 μ L, 0.3 μ L of HhaI (10U/. mu.L), 0.95 μ L of sterile double distilled water. Digesting the mixed sample in a constant temperature incubator at 37 ℃ for 12-16 h, carrying out electrophoresis on 3% agarose gel for 40min at the room temperature under the conditions of 110V and 55mA, and imaging by using a Bio-RAD gel imager. Genotype was judged from the image of the electrophoretic band (FIG. 3). Because the amplification product of the primer pair P does not contain other HhaI enzyme cutting sites, the PCR amplification product fragment containing the G is cut into two fragments of 185bp and 23 bp; the PCR product containing "A" was not recognized by HhaI and thus remained a 208bp fragment. Therefore, the AA genotype electrophoresis result shows that a strip of 208bp is formed; the AG genotype shows three bands of 208bp, 185bp and 23bp, and the GG genotype shows two bands of 185bp and 23 bp. However, since 23bp is too short to be seen in the electrophoretogram, at most two bands were observed in the actual electrophoretic analysis, but the typing was not affected.
Second, frequency statistics of SNP loci of MYLK4 gene of cattle and correlation analysis of SNP loci and growth traits
1. Gene and genotype frequency
Genotype frequency refers to the ratio between the various genotypes of a trait in a population. The calculation formula is as follows:
PBB＝NBB/N
wherein P isBBRepresents the BB genotype frequency of a certain site; n is a radical ofBBRepresenting the number of individuals in the population having a BB genotype; and N is the total number of detection groups.
Gene frequency refers to the relative ratio of a gene to its allele in a population. The calculation formula can be written as:
PB＝(2NBB+NBb1+NBb2+NBb3+NBb4+……+NBbn)/2N
in the formula, PBIndicates allele B frequency, NBBRepresenting the number of individuals in the population with the BB genotype, NBbiIndicates Bb in the populationiNumber of genotyped individuals, b1～bnIs n different multiple alleles of allele B.
The statistical results of the gene frequency and genotype frequency in each cattle variety are shown in table 4 below.
TABLE 4.5 genotypes and allele frequencies of MYLK4 gene in cattle breeds
2. Correlation analysis statistical model
The association of gene loci with growth traits was analyzed using SPSS (18.0) software. Firstly, performing descriptive statistical analysis on data to determine whether outliers exist, and analyzing the genotype effect by using t analysis, variance analysis or a multivariate linear model according to the data characteristics. In the data processing, according to different factors influencing growth and development indexes such as body size, weight and the like, considering environmental effects, age, genotype effects and related interaction effects, a fixed model is adopted for analysis, and meanwhile, selection is carried out according to actual conditions. The complete model is as follows:
Yijk＝μ+Gj+Eijk
wherein: y isijk(ii) recording the phenotype of the individual; μ is the population mean; gjThe genotype effect for each site; eijkIs a random error.
The method is used for counting the related data of each cattle variety group, and the statistical result of the related data of the Nanyang cattle is shown in a table 5.
TABLE 5 correlation analysis of different genotypes and growth traits of MYLK4 gene of Nanyang cattle
Note: letter differences indicate significant differences (P < 0.05).
The result shows that the correlation analysis of different genotypes on the 61595 th single nucleotide polymorphic site of the MYLK4 gene sequence of cattle and growth and development indexes such as the size, the weight and the like of the south Yang cattle shows that: at 6 months of age, the mutation site has obvious influence on the oblique length of the body, and the AG type is obviously higher than the AA type; at 12 months of age, the mutation site has extremely obvious influence on body height, the heights of GG type and AG type are obviously higher than those of AA type, and the body slant lengths of AG type and GG type are also obviously larger than those of AA type. This shows that the allele G is closely related to the growth traits of south-Yang cattle, such as body height and body slant length, and finally determines that the G allele can be used as a molecular breeding gene marker for early selection of the body height and body slant length of cattle.
Sequence listing
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Claims (6)
1. A method for detecting single nucleotide polymorphism of MYLK4 gene of cattle is characterized in that: the method comprises the following steps:
taking cattle genome DNA as a template and a primer pair P as primers, carrying out PCR amplification on partial fragment of cattle MYLK4 gene, digesting the PCR amplification product by using restriction enzyme HhaI, carrying out agarose gel electrophoresis, and identifying the genotype of the single nucleotide polymorphism site of the cattle MYLK4 gene according to the electrophoresis result;
the sequence of the primer pair P is as follows:
an upstream primer F: 5'-CGGACACTTGTTCTCAATCGGC-3'
A downstream primer R: 5'-GTGGTCTAGGAATCTGGGTG-3', respectively;
the electrophoresis result of the genotype of the single nucleotide polymorphism site of the cattle MYLK4 gene is as follows: the AA genotype shows a strip of 208 bp; the AG genotype is represented by three bands of 208bp, 185bp and 23 bp; the GG genotype shows two bands of 185bp and 23 bp.
2. The method for detecting single nucleotide polymorphism of MYLK4 gene of cattle according to claim 1, which is characterized in that: the reaction procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 25s for 36 cycles; extension at 72 ℃ for 5 min.
3. The method for detecting single nucleotide polymorphism of MYLK4 gene of cattle according to claim 1, which is characterized in that: the agarose gel electrophoresis was performed using agarose gel with a mass concentration of 3%.
4. The application of the detection method of single nucleotide polymorphism of MYLK4 gene of cattle in auxiliary detection of growth traits of cattle of claim 1, which is characterized in that: the growth trait is selected from body height or body slant length.
5. Use according to claim 4, characterized in that: the cattle is selected from Qinchuan cattle, Pinus parviflora, Xianan cattle, Nanyang cattle or Chadamu cattle.
6. A special kit for auxiliary detection of cattle growth traits by MYLK4 gene is characterized in that: the kit comprises a primer pair P for MYLK4 gene single nucleotide polymorphism locus typing based on a PCR-RFLP method, wherein the sequence of the primer pair P is as follows:
an upstream primer F: 5'-CGGACACTTGTTCTCAATCGGC-3'
A downstream primer R: 5'-GTGGTCTAGGAATCTGGGTG-3' are provided.
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