CN117487935A - Method for detecting SNP (Single nucleotide polymorphism) typing of chicken NLRP3 gene exons - Google Patents
Method for detecting SNP (Single nucleotide polymorphism) typing of chicken NLRP3 gene exons Download PDFInfo
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses a method for detecting chicken NLRP3 gene exon SNP typing, belongs to the technical field of biotechnology genes, and solves the problem that related researches on NLRP3 gene SNP closely related to inflammatory factor regulation do not have related reports yet, and comprises the following steps: step one, collecting blood samples of 4 varieties of chickens for extracting DNA; step two, taking a DNA solution, and measuring the concentration and purity by using a differential spectrophotometer; thirdly, performing PCR amplification on the extracted DNA by adopting a specific primer group, and judging the genotype by electrophoresis detection; and step four, identifying the amplified product. The genotype possibly related to the salmonella enteritidis resisting bacteria obtained through NLRP3 in the chickens can be used as a molecular genetic marker to carry out marker-assisted selection research on the salmonella enteritidis resisting bacteria of the chickens, and has a certain practical significance for developing the salmonella enteritidis resisting breeding.
Description
Technical Field
The invention relates to the field of biotechnology genes, in particular to a method for detecting chicken NLRP3 gene exon SNP typing.
Background
In the 21 st century, the demand of poultry meat and egg products is in a rigid growth state in the total demand of meat products in China, and meanwhile, the pursuit awareness of people on the quality of the poultry products is increasingly enhanced. The harm of the salmonellosis of birds is accompanied with the whole period of chicken raising industry, and immeasurable economic loss is caused to the production of birds; in particular, salmonella-infected chickens are able to transmit pathogens to humans via the food chain, which severely threatens human food safety (Foley & Lynne,2008;Callaway et al,2008;Suzuki,1994;Gantois et al,2009), and have attracted great attention from the poultry community, the public health community and the poultry disease control community. In long-term development, a brand new prevention and control strategy is sought, a new disease-resistant variety (strain) is cultivated by adopting a genetics method, the resistance of chicken groups to salmonella enteritidis is permanently improved in genetic quality, and cultivation of high-efficiency, high-quality and resistant chicken species is an urgent need for realizing sustainable development of poultry industry.
Single nucleotide polymorphisms (single nucleotide polymorphism, SNPs) are polymorphisms in DNA sequences caused by variation of a single nucleotide, including transitions, transversions, insertions or deletions. SNPs are an important basis for differences in biological properties such as disease susceptibility, manifestation, resistance, and drug responsiveness, and many diseases are associated with genetic mutations or genetic polymorphisms. The mononucleotide genetic diversity is widely applied to molecular genetic marker research, and the mutation of specific sites can change the structure and the function of the protein expressed by the gene, and some of the single nucleotide genetic diversity can even directly change the translated amino acid.
Through the search of the existing domestic and foreign documents and patents, the related research of NLRP3 gene SNP closely related to the regulation of inflammatory factors has not been reported. The research is to conduct research and analysis on SNP polymorphism of NLRP3 gene, and conduct correlation analysis on different genes, genotype frequency and salmonella enteritidis resistance by adopting Case-Control analysis, so that possible genotypes are obtained for genetic marker assisted selection.
Disclosure of Invention
The invention provides a method for detecting chicken NLRP3 gene exon SNP typing, which aims to solve the technical problem that related researches on the traditional NLRP3 gene SNP closely related to inflammatory factor regulation do not have related reports yet.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the application provides a method for detecting SNP typing of chicken NLRP3 gene exons,
(1) Carrying out PCR amplification on the sequences of 3 rd and 4 th exons of the NLRP3 gene by a specific primer group;
(2) Determining the nucleotides at positions 12, 169 and 306 of the sequences shown in exons 3 and 4 of the chicken NLRP3 gene;
(3) The nucleotides at positions 12, 169 and 306 are tested for the presence of single nucleotide genetic diversity.
Further, the sequences of the specific primer group are shown as SEQ ID No. 1 and SEQ ID No. 2.
Further, the nucleotides at positions 12, 169 and 306 of the sequences shown in exons 3 and 4 of the NLRP3 gene are A, T, A respectively.
Further, the method specifically comprises the following steps:
step one, collecting blood samples of 4 varieties of chickens for extracting DNA;
step two, taking a DNA solution, and measuring the concentration and purity by using a differential spectrophotometer;
thirdly, performing PCR amplification on the extracted DNA by adopting a specific primer group, and judging the genotype by electrophoresis detection;
and step four, identifying the amplified product.
Further, in the first step, the 4 breeds of chickens are respectively Daheng S01, daheng S03, AA+ and Tibetan chickens.
Further, in the third step, when PCR amplification is performed:
PCR reaction system: 1 μl of DNA; 2 μl of the specific primer group, wherein the concentration of 2 primers in the specific primer group is equal, and the primers Fi are calculated according to the volume ratio: mixing the primers ri=1:1; dNTP 1. Mu.l; 5 μl of Taq buffer; mgCl 2 5 μl; 0.5 μl of Taq enzyme; 35.5 μl of water;
PCR reaction procedure: pre-denaturation at 95℃for 3.5min, denaturation at 95℃for 35S, annealing at 56℃for 40S, extension at 70℃for 40S, repair extension at 72℃for 10min, and cycle for 40 times.
In the third step, electrophoresis detection was performed by 1% agarose electrophoresis, and electrophoresis was performed at 150V for 100mA for 20 min.
Further, in the fourth step, the sequencing result of the amplified sequence shows that the NLRP3 exons 12, 169 and 306 have mutations of G.fwdarw. A, C.fwdarw.T and G.fwdarw.A respectively and genotypes GG, GA and AA, CC, CT and TT, GG, GA and AA respectively.
The beneficial effects realized by the invention are as follows:
according to the method for detecting the SNP typing of the chicken NLRP3 gene exon, provided by the invention, the specific locus is amplified by PCR, the nucleotide change of the specific locus can be detected by using a direct sequencing method, the SNPS locus genetic polymorphism can be obtained by statistically analyzing the gene and genotype frequency of the locus, and the associated effect of the character, the gene and the gene is obtained by combining with phenotypic character for analysis. The genotype related to the salmonella enteritidis resistance obtained through NLRP3 in chickens can be used as a molecular genetic marker to carry out marker-assisted selection research on the salmonella enteritidis resistance of chickens, and has a certain practical significance for developing the salmonella enteritidis resistance breeding.
Drawings
FIG. 1 is a diagram showing the sequencing peaks of the genotype of the No. 12 locus of the NLRP3 exons 3 and 4 according to the invention;
FIG. 2 is a diagram showing the sequencing peaks of the NLRP3 exon 3 and exon 4 No. 169 genotypes of the present invention;
FIG. 3 is a diagram showing the sequencing peaks of the genotype at position 306 of the NLRP3 exons 3 and 4 according to the invention;
the drawings are for illustrative purposes only and are not to be construed as limiting the present patent; for the purpose of better illustrating the embodiments, certain elements of the drawings may be omitted, enlarged or reduced and do not represent the actual product dimensions; it will be appreciated by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted; the same or similar reference numerals correspond to the same or similar components; the terms describing the positional relationship in the drawings are merely illustrative and should not be construed as limiting the present patent.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be considered to be absent and not within the scope of protection claimed in the present invention.
The technical scheme of the invention is described in detail below with reference to the specific drawings.
1. Method for detecting SNP (Single nucleotide polymorphism) typing of chicken NLRP3 gene exons
Example 1 Tibet and Daheng broiler test groups
And 2 varieties (lines) of Daheng S01 (medium breeding) and AA+ chicken (high breeding) are selected for DNA extraction, and Tibetan chicken (non-breeding) and Daheng S03 (medium breeding) are selected for DNA extraction. 67 samples of 4 variety (line) dead individuals and 67 samples of the equivalent healthy Control group were randomly selected for Case-Control analysis.
Example 2 sample collection and DNA extraction
The method for extracting DNA from 4 varieties of blood samples collected in the early stage comprises the following steps:
(1) Dissolving frozen blood at room temperature;
(2) Transferring 1ml of blood into a sterile centrifuge tube, adding an equal volume of PBS phosphate buffer solution, fully mixing, centrifuging at 12000rpm for 5min, and discarding the supernatant;
(3) 667 μl of STE buffer [10mM Tris-HCl (pH=8.0), naCl, EDTA (pH=8.0) ],24 μl of 20% SDS (sodium dodecyl sulfate), and water bath at 37deg.C for 1 hr;
(4) Adding 10 mu l of proteinase K of 20mg/ml, uniformly mixing, and carrying out water bath digestion at 55 ℃ overnight (10-14 h);
(5) Adding the digested sample into an equal volume of Tris saturated phenol, shaking up fully, and centrifuging at 12000rpm for 10min;
(6) Transferring the upper aqueous phase into another sterile centrifuge tube; adding equal volume of Tris saturated phenol, shaking thoroughly, centrifuging at 12000rpm for 10min;
(7) Transferring the upper aqueous phase into another sterile centrifuge tube; adding equal volume of phenol, chloroform and isoamyl alcohol (25:24:1), and shaking by vortex until mixing completely, centrifuging at 12000rpm for 10min;
(8) Transferring the upper aqueous phase into another sterile centrifuge tube; adding equal volume of chloroform and isoamyl alcohol (24:1), shaking thoroughly, mixing well, centrifuging at 12000rpm for 10min;
(9) Transferring the supernatant to another sterile centrifuge tube; adding 2 times of glacial ethanol and 1/10 volume of sodium acetate, horizontally shaking until flocculent DNA is separated out;
(10) Freezing the sample in a refrigerator at-20 ℃ for 30min or ice bath for 15-20 min, taking out, and centrifuging at 12000rpm for 10min again to precipitate DNA;
(11) Picking the DNA into another sterile centrifuge tube by using a gun head, washing with a proper amount of 70% ethanol, and shaking to wash out impurities of the DNA;
(12) Pouring out ethanol, vacuum drying or naturally airing DNA, adding appropriate amount of TE buffer solution for dissolution back, and preserving at-20 ℃ for standby.
Example 3 detection of genomic DNA
The genomic DNA solution was taken and its concentration and purity were measured by using a differential spectrophotometer. mu.L of genomic DNA solution was taken, labeled with 1 kb NAMark in agarose gel (0.05% EB), electrophoretically separated (120V 30 min), detected, and photographed by a gel imaging system.
Example 4 PCR reaction and sequencing
Preparation of 50. Mu.l of PCR reaction System the following Table 1 shows the system:
TABLE 1 PCR reaction System 50. Mu.l System
Primer design:
for the 3 rd and 4 th exons of NLRP3, primer sets were designed, the primer set sequences are shown in Table 2 below:
TABLE 2 primer set
PCR reaction conditions:
pre-denaturation at 95℃for 3.5min, denaturation at 95℃for 35S, annealing at 56℃for 40S, extension at 70℃for 40S, repair extension at 72℃for 10min, and cycle for 40 times. 1% agarose electrophoresis, 150V, 100mA 20min electrophoresis observation. The genotype was determined by electrophoretic detection.
The PCR purified product was recovered using the kit and operated according to Shanghai Biotechnology SK 1141. And according to the PCR-SSCP result, selecting PCR products with different SSCP bands, and sending the PCR products to Shanghai workers to finish PCR sequencing.
Example 5 data analysis
According to the sequencing on the machine, the gene frequency and the genotype frequency in 4 varieties (lines) are counted, hardy-Weinberg equilibrium detection (P >0.01 shows that the equilibrium is reached) in each variety is carried out, the conditions between the genes and the different varieties with genotypes are calculated by using SPSS 19.0 software, and the genetic polymorphism condition is calculated.
SAS 9.1.3 statistical software performs Case-Control analysis on 67 non-diseased groups and 67 diseased groups, and performs logistic regression analysis to calculate ratio (OR), 95% confidence interval and P value of OR value, so as to judge susceptibility of salmonella enteritidis.
2. Results and analysis
(1) Sequencing results of amplified sequences
The samples were sequenced by Shanghai, and the sequencing peaks of the genotypes at positions 12, 169 and 306 of exons 3, 4 of NLRP3 are shown in FIGS. 1-3. NLRP3 exons 12, 169 and 306 have mutations G.fwdarw. A, C.fwdarw.T and G.fwdarw.A, respectively, and GG, GA and AA, CC, CT and TT, GG, GA and AA genotypes, respectively.
(2) SNP locus Gene frequency and genotype frequency of NLRP3 exons 12, 169 and 306
Statistical results for NLRP3 exons 3 and 4, exon 12 are shown in Table 3.1. P test of the 4 varieties (lines) shows that the great constant S03 and AA+ deviate from Hardy-Weinberg equilibrium extremely significantly (P < 0.01), and the great constant S01 and Tibetan chickens deviate from Hardy-Weinberg equilibrium significantly (P < 0.05).
TABLE 3.1 NLRP3 exon 3, 4 locus 12 Gene frequency and genotype frequency distribution
The NLRP3 exons No. 169 gene frequencies and genotype frequency statistics are shown in Table 3.2. All 4 varieties (lines) were in Hardy-Weinberg equilibrium (P > 0.05).
TABLE 3.2 NLRP3 exon No. 3, no. 4 No. 169 locus Gene frequencies and genotype frequency distribution
The statistics of this site at exon 306 of NLRP3 are shown in Table 3.3. Table 3.3 shows that the Dataheng S01 significantly deviates from Hardy-Weinberg equilibrium (P < 0.05), and that Tibetan chicken, dataheng S03 and AA+ are all at Hardy-Weinberg equilibrium (P > 0.05).
TABLE 3.3 NLRP3 exon 3, 4, no. 306 locus Gene frequency and genotype frequency distribution
(3) Genetic diversity analysis
Genetic diversity of the 4 variety NLRP3 gene at position 3 and exon 12 is shown in Table 4.1. Table 4.1 shows that AA+ is at a low polymorphism and that large constant S01 (0.450) is at a medium polymorphism. Others are highly polymorphic.
TABLE 4.1 analysis of genetic diversity of NLRP3 Gene No. 3, no. 4 exon 12
Genetic diversity of the No. 169 loci of the No. 3 and No. 4 exons of the NLRP3 genes of the 4 varieties (lines) is shown in a table 4.2,4.2, and the highest Tibetan chicken heterozygosity (0.664) is in a high polymorphism, and the lowest Daheng S03 (0.340) is in a medium polymorphism. Large constants S01 and aa+ are highly polymorphic.
TABLE 4.2 analysis of genetic diversity of exon No. 169 of NLRP3 Gene
The positions of the No. 306 exons 3 and 4 of the 4 variety (line) NLRP3 gene are shown in Table 4.3. From Table 4.3, daheng S01 was moderately polymorphic, while Daheng S03, AA+ and Tibetan chickens were highly polymorphic.
TABLE 4.3 analysis of genetic diversity of exon 3, 4, no. 306 of NLRP3 Gene
In tables 4.1-4.3, ho is the average observed heterozygosity, he is the average expected heterozygosity, and PIC is the average polymorphic information content.
(4) Analysis of Salmonella enteritidis susceptibility relationship
Case-Control design salmonella enteritidis susceptibility was analyzed at the 3 rd and 4 th exons 3 SNP (12, 169 and 306) sites of NLRP3 and the results are shown in table 5 below. The test samples are 67 enteritis dead individuals (Case group), 67 non-diseased individuals are randomly and equivalently selected as a Control group (Control group), and as a result, the genotype difference of the sites 12 and 168 is not obvious, the genotype difference of the site 306 is obvious, and the GG genotype is possibly related to salmonella resistance.
TABLE 5 SNP locus genotype OR values for 3 rd and 4 th exons of NLRP3
In the table, cases represents experimental groups, control represents Control groups, and OR value represents odds ratio.
By screening NLRP3 SNP sites at positions 3, 169 and 306 of exons 3, 4 with NCBI NLRP3 (XM_ 001233261.2), it was found that the deviation of the Hardy-Weinberg equilibrium was significant for both variety 12 and 4 of exons 3, 4, site 306S 01 was deviated from the Hardy-Weinberg equilibrium (P < 0.05), S03, AA+ and TBC were all at the Hardy-Weinberg equilibrium (P > 0.05), and both gene frequency and genotype frequency were at the Hardy-Weinberg equilibrium (P > 0.05). Indicating that each of the 3 sites is not identical, whether selected manually or naturally, it is possible that an insufficient number of samples are measured resulting in deviations from this balance. In particular, position No. 12 aa+ P value= 0.00102, indicating that manual selection resulted in a severe deviation from Hardy-Weinberg equilibrium, which was entirely likely to occur when other traits were selected. The NLRP3 gene is in low polymorphism, the No. 12 AA+ chicken is in medium polymorphism or high polymorphism, the selection pressure of the site is larger, the homozygosity of the site is increased and the heterozygosity is reduced due to certain characters during selection, and the site is possibly closely related to the characters such as production performance and the like, so that the special phenomenon needs to be further studied. Case-Control designed 3 SNP (12, 169 and 306) loci to analyze salmonella enteritidis susceptibility, found that the genotype differences of the 12 and 168 loci are not significant, and the 306 locus difference is significant, and combined with Tibetan chicken and S03 genotype analysis, GG genotype is considered to be related to salmonella resistance.
The foregoing embodiment numbers of the present application are merely for describing, and do not represent advantages or disadvantages of the embodiments. The foregoing description is only of the preferred embodiments of the present application, and is not intended to limit the scope of the claims, and all equivalent structures or equivalent processes using the descriptions and drawings of the present application, or direct or indirect application in other related technical fields are included in the scope of the claims of the present application.
Claims (8)
1. A method for detecting SNP (Single nucleotide polymorphism) typing of an exon of a chicken NLRP3 gene is characterized in that,
(1) Carrying out PCR amplification on the sequences of 3 rd and 4 th exons of the NLRP3 gene by a specific primer group;
(2) Determining the nucleotides at positions 12, 169 and 306 of the sequences shown in exons 3 and 4 of the chicken NLRP3 gene;
(3) The nucleotides at positions 12, 169 and 306 are tested for the presence of single nucleotide genetic diversity.
2. The method for detecting chicken NLRP3 gene exon SNP typing according to claim 1, wherein the sequences of the specific primer set are shown as SEQ ID No. 1 and SEQ ID No. 2.
3. The method for detecting chicken NLRP3 gene exon SNP typing of claim 1 wherein the nucleotides at positions 12, 169 and 306 of the sequences shown in exons 3 and 4 of the NLRP3 gene are A, T, A, respectively.
4. The method for detecting chicken NLRP3 gene exon SNP typing according to claim 1, comprising the following steps:
step one, collecting blood samples of 4 varieties of chickens for extracting DNA;
step two, taking a DNA solution, and measuring the concentration and purity by using a differential spectrophotometer;
thirdly, performing PCR amplification on the extracted DNA by adopting a specific primer group, and judging the genotype by electrophoresis detection;
and step four, identifying the amplified product.
5. The method for detecting SNP typing of exons of chicken NLRP3 gene according to claim 4, wherein in the first step, the 4 breeds of chicken are Hengsu S01, hengheng S03, AA+ and Tibetan chicken, respectively.
6. The method for detecting chicken NLRP3 gene exon SNP typing according to claim 4, wherein in the third step, PCR amplification is performed:
PCR reaction system: 1 μl of DNA; 2 μl of the specific primer group, wherein the concentration of 2 primers in the specific primer group is equal, and the primers Fi are calculated according to the volume ratio: mixing the primers ri=1:1; dNTP 1. Mu.l; taq buffer 5 μl; mgCl 2 5 μl; 0.5 μl of Taq enzyme; 35.5 μl of water;
PCR reaction procedure: pre-denaturation at 95℃for 3.5min, denaturation at 95℃for 35S, annealing at 56℃for 40S, extension at 70℃for 40S, repair extension at 72℃for 10min, and cycle for 40 times.
7. The method for detecting chicken NLRP3 gene exon SNP typing according to claim 4, wherein in the third step, electrophoresis detection is performed by 1% agarose electrophoresis, and electrophoresis observation is performed at 150V and 100mA for 20 min.
8. The method according to claim 4, wherein in the fourth step, the amplified sequence sequencing result shows that the NLRP3 exons 12, 169 and 306 have mutations of G.fwdarw. A, C.T and G.fwdarw.A, respectively, and GG, GA and AA, CC, CT and TT, GG, GA and AA genotypes, respectively.
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