CN116287298A - Molecular marker related to Hu sheep immune traits, detection method and application thereof - Google Patents
Molecular marker related to Hu sheep immune traits, detection method and application thereof Download PDFInfo
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Abstract
The invention provides a molecular marker related to immune traits of Hu sheep, a detection method and application thereof. Through PCR amplification and sequence analysis of the Hu sheep PD-L1 gene, a G/A polymorphic site exists at the 135 th position of the amplified fragment, further KASPar primer is used for detecting the polymorphic site of 898 Hu sheep, a least squares model is built, the correlation analysis of genotype and growth traits is carried out, and finally the amplified PD-L1 gene fragment can be used as a molecular marker of immune traits related to the total number of red blood cells and the average concentration of red blood cell hemoglobin of the Hu sheep. The molecular marker can be used for disease-resistant breeding of the Hu sheep to ensure the health of the Hu sheep body and is beneficial to increasing economic benefit.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to a molecular marker related to immune traits of Hu sheep, a detection method and application thereof.
Background
The programmed death ligand 1 gene (PD-L1) is an important immune co-signaling molecule, belongs to type I transmembrane protein, and is known to assist tumor cells in "immune escape" (Xue et al 2018; zhang et al 2019). PD-L1 transmits an inhibition signal by binding to programmed death receptor 1 (PD-1) on immune cells such as T cells, thereby inhibiting proliferation and activation of T cells (Vargas et al 2020; wang et al 2020). Furthermore, the expression of the PD-L1 gene greatly enhances the migration and invasion of fibroblasts, leading to fibrosis of lung tissue (Geng et al 2019; xue et al 2018). Studies in cattle have demonstrated that PD-L1 is associated with immune dysfunction during Mycobacterium bovis infection, and that PD-L1 expression in diseased animals inhibits the production of interferon-gamma (IFN-gamma) by anti-mycoplasma specific cells in Peripheral Blood Mononuclear Cells (PBMC) (Goto et al 2017). However, the role of PD-L1 in the immune profile of Hu sheep is not yet clear, especially in terms of hematological indices. The correlation between PD-L1 genes and immunophenotype is not reported, so the invention takes the PD-L1 genes as candidate genes, and discusses the correlation between different genotypes and the immune-related characters of Hu sheep through PCR amplification, DNA sequencing and sequence analysis, thereby providing a novel molecular marker for improving the disease resistance breeding of Hu sheep.
Disclosure of Invention
The invention aims to provide a molecular marker related to immune traits of Hu sheep, a detection method and application thereof. The molecular marker is amplified from a Hu sheep PD-L1 gene, and the specific nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1. Through amplifying DNA sequence of the Hu sheep PD-L1 gene and sequencing, polymorphic sites of the PD-L1 gene are searched, correlation between different genotypes and immune traits of the Hu sheep is analyzed, a detection method of a molecular marker containing the polymorphic sites is established, and the molecular marker can be applied to cultivation of novel disease-resistant mutton sheep new varieties.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a molecular marker related to the immune trait of Hu sheep, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, the 135bp position R of the sequence shows G or A, and the G/A mutation of the 135 th base of the sequence leads to the G/A polymorphism of the Hu sheep PD-L1 gene at the site.
The invention provides a PCR primer pair for detecting the molecular marker, preferably comprising a primer F and a primer R, wherein the nucleotide sequence of the primer F is shown as SEQ ID NO.2, and the nucleotide sequence of the primer R is shown as RSEQ ID NO. 3;
F(SEQ ID NO.2)：5＇-GGCAAAACCAGCATCACCC-3＇，
R(SEQ ID NO.3)：5＇-CATCCTCTGTCATCCCCTT-3＇。
the invention provides a KASPar primer pair for detecting the molecular marker, which comprises a forward primer A1, a forward primer A2 and a reverse primer C, wherein the nucleotide sequence of the forward primer A1 is shown as SE Q ID NO.4, the forward primer 2 is shown as SEQ ID NO.5, and the reverse primer C is shown as SEQ ID NO. 6;
SEQ ID NO.4：5＇-GAAGGTGACCAAGTTCATGCTATTAGGTCATG AGGAAAACAACAC-3＇；
SEQ ID NO.5：5＇-GAAGGTCGGAGTCAACGGATTGATTAGGTCAT GAGGAAAACAACACA-3＇；
SEQ ID NO.6：5＇-GATGTTCAAAATATTACCTGGGATGACC-3＇。
a kit for detecting the molecular marker, wherein the kit comprises a PCR primer pair or a KASPar primer pair for detecting the molecular marker.
A method for detecting molecular markers related to the growth traits of Hu sheep, wherein the nucleotide sequence of the molecular markers is shown as SEQ ID NO.1, R at 135bp position of the sequence represents T or C, the method comprises the steps of detecting the genomic DNA of the Hu sheep by using the primer pair or the kit, and the specific detection method comprises the following steps:
a) Amplifying the Hu sheep genomic DNA by using the PCR primer pair, the KASPar primer pair or a kit comprising the primer pair;
b) Identifying the polymorphic site of the amplification product obtained in step a).
Wherein, in step b), the method of typing identification includes, but is not limited to, direct sequencing method, probe method, gene chip method, high resolution dissolution profile method.
The method for detecting the molecular marker related to the growth traits of the Hu sheep by using the primer pair comprises the following steps:
a) Extracting genome DNA from Hu sheep blood as a sample, and performing high-throughput water bath PCR amplification by using primer pairs with nucleotide sequences shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6;
b) After amplification, fluorescence signals are detected by using a BMG PHERAstar instrument and the typing result is checked.
The molecular marker is detected in the genome DNA of the Hu sheep to be detected, and the type of the polymorphic site is analyzed, so that the immune trait of the Hu sheep can be determined, and the Hu sheep with disease resistance can be screened out.
The molecular marker and the polymorphism site thereof, the PCR primer pair, the KASPar primer pair or the kit are applied to auxiliary breeding of Hu sheep, and the primer pair or the kit is utilized to amplify and detect genomic DNA of the Hu sheep to determine the genotype of the PD-L1 gene of a sample to be detected, so that a disease-resistant Hu sheep variety can be bred from the sample.
Finding mutation sites of genes, finding the relationship between genes and traits through association analysis between the genes and the traits is an important means for researching gene functions and is also a basis for marker-assisted selection. The invention discovers that a G/A polymorphism site exists at the 135 th position of an amplified fragment by carrying out PCR amplification and sequencing on PD-L1 genes of Hu sheep representing a hu sheep variety, and determines a molecular marker related to the immune traits of the Hu sheep by detecting 898 hu sheep polymorphisms and establishing a least squares model.
According to the invention, the detection is carried out on the molecular marker by designing the KASPar primer required by competitive allele specific PCR (KASP), the detection method does not need to synthesize a specific fluorescent probe aiming at each SNP locus, and all locus detection is finally carried out by using a universal fluorescent primer based on the unique ARM PCR principle, so that the cost of reagents is greatly reduced, the accuracy is higher, and a simple, convenient, accurate and low-cost operation method is provided for the detection of the molecular marker.
The invention has the beneficial effects that:
the invention provides a molecular marker related to the immune traits of Hu sheep, in particular to a G/A polymorphic site at position 135 of a SEQ ID NO.1 fragment, and also provides an application of a primer pair or a detection kit or a detection method for detecting the molecular marker in the immune traits related detection of Hu sheep, and an effective detection means is provided for breeding of the disease-resistant Hu sheep by determining the polymorphism genotype of the molecular marker to effectively identify whether the Hu sheep is disease-resistant. The molecular marker and the detection of polymorphic loci can be used for selecting the GG homozygous Hu sheep as breeding sheep to improve the immunity of the Hu sheep and contribute to the improvement of the economic benefit of the Hu sheep breeding industry.
Drawings
FIG. 1 is a gel electrophoresis diagram of a Hu sheep PD-L1 gene fragment as a molecular marker.
FIG. 2 shows the sequencing result of the PD-L1 gene mutation site of Hu sheep in the invention.
FIG. 3 shows the result of KASPar SNP typing of a mutation site of a lake sheep PD-L1 gene g.11858G > A in the invention.
Detailed Description
The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions made to the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention.
Unless otherwise indicated, all technical means used in the examples are conventional means well known to those skilled in the art, and unless otherwise specified, all reagents used in the examples below are of analytical grade or above.
Example 1 amplification of PD-L1 Gene
A pair of primers were designed using Hu sheep PD-L1 gene DNA (GenBank accession number: NC_ 040253) as a template and Oligo7.0 software: comprising a forward primer F (SEQ ID NO. 2) and a reverse primer R (SEQ ID NO. 3), the sequences of which are as follows
F(SEQ ID NO.2)：5＇-GGCAAAACCAGCATCACCC-3＇，
R(SEQ ID NO.3)：5＇-CATCCTCTGTCATCCCCTT-3＇
(2) Amplification and sequencing of PD-L1 Gene
PCR amplification is carried out on genome DNA extracted from Hu sheep whole blood cells as a DNA template, and the amplification reaction is carried outThe total volume was 25. Mu.L, including 1.5. Mu.L of DNA template, 12.4. Mu.L of 2 XPCR Master Mix, 0.8. Mu.L of forward primer F (concentration 10. Mu. Mol/L), 0.8. Mu.L of reverse primer R (concentration 10. Mu. Mol/L) and ddH 2 O9.5. Mu.L. The conditions for the PCR amplification reaction were:
pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 54.5℃for 30s, extension at 72℃for 30s, and cycling for 35 times; finally, the extension is carried out for 10min at 72 ℃.
For the PCR amplification product obtained above, 5. Mu.L was subjected to electrophoresis using 1.5% agarose gel, and the results are shown in FIG. 1. Wherein, lane M: DL 2000marker, lanes 1-10: result of PD-L1 Gene amplification. The result shows that 484bp specific amplified fragment is obtained.
And sequencing the amplified PCR fragment, wherein the sequencing result is 484bp, and analyzing the sequencing result by means of chord (v2.3.0.0) and DNAMAN (6.0.3.99) software. As shown in SEQ ID NO.1, wherein a polymorphic site exists in the fragment, specifically R at the 335bp site is G or A, i.e., the amplified PD-L1 gene fragment has a G/A polymorphism at the 135bp site, which is at position 11858 in the NC_040253 sequence, i.e., g.11858G > A (see FIG. 2). Wherein, SEQ ID NO.1:
GGCAAAACCAGCATCACCCATTCTAAGAGGGAGGAAAAGCTTTTCAATGTGACCAGCACACTGAGAATCAACACAACAGCTGACAAAATTTTCTACTGCACTTTTCGGAGATTAGGTCATGAGGAAAACAACACRGCTGAGTTGGTCATCCCAGGTAATATTTTGAACATCTCAATTAAAATGTGCCTAGTATTGTCTCTTGGTATGTAGCCTGATGCCTGCTCATCATAAACTTTCATTGTTTGTTGAATGAATGAATGAATGAATAAACTATATTTATATGAATGAACTACATTTACAAAATGCATTCTTCTTCCTCACCTCCATTCATCTGAGTCATATTTCTACTTAGTAAACATAAGGACTGATGCTAAAGCTGAAACTCCAGTACTTTGGCCACCTCATGCAAAGAGTTGACTCATTGGAGAAGACTCTGATGCTGGGAGGGATTGGGGGCAGGAGGAGAAGGGGATGACAGAGGATG。
DNA sequence homology search identification:
the DNA sequences obtained after sequencing were compared for sequence homology with known physiological functional genes published in the GenBank database by BLAST software of the national center for Biotechnology information (NCBI, http:// www.ncbi.nlm.nih.gov) website to identify and obtain functional information for the DNA sequences. The search result shows that the homology of the sequenced sequence and the partial sequence of the Hu sheep PD-L1 gene DNA (GenBank accession number: NC-040253) reaches 99 percent.
Example 2 establishment of genotyping assay
1. Primer sequence design
A KASPar primer pair was designed for the G/a polymorphic site of the amplified fragment SEQ ID No.1 of example 1, the nucleotide sequence of which is:
the forward primer A1 for detecting AlleX has a nucleotide sequence shown as SEQ ID NO.4,
SEQ ID NO.4：5＇-GAAGGTGACCAAGTTCATGCTATTAGGTCATG AGGAAAACAACAC-3＇；
the forward primer A2 for detecting Alley is shown as SEQ ID NO.5,
SEQ ID NO.5：5＇-GAAGGTCGGAGTCAACGGATTGATTAGGTCAT GAGGAAAACAACACA-3＇
the general reverse primer C is shown as SEQ ID NO.6,
SEQ ID NO.6：5＇-GATGTTCAAAATATTACCTGGGATGACC-3＇。
the above primers were commissioned for synthesis by Beijing Biotechnology Co., ltd., each set of primers in the KASPar primer pair was diluted to 10. Mu. Mol/L, and the primers were prepared as described in primer A1: primer A2: the volume ratio of the universal reverse primer C is 12:12:30, and the universal reverse primer C is uniformly mixed for standby.
2. DNA quality control
The whole blood of Hu sheep is extracted with genome DNA and may be extracted with DNA extracting kit. The quality detection of genome DNA obtained by whole blood extraction of Hu sheep is carried out by respectively adopting 1% agarose electrophoresis and Nanodrop2100, and the qualified DNA is required to be reached: (1) Agarose electrophoresis showed a single DNA band without significant diffusion. (2) Nanodrop2100 detection a260/280 is between 1.8-2.0; a260/230 is between 1.8 and 2.0; there is no significant light absorption at 270 nm. And according to KASPar detection technology and genome size of LGC company in UK, the dosage of DNA is 10-20 ng/sample, and the diluted concentration of extracted genome DNA is 10-20 ng/mu L as DNA template for standby.
3. Genotyping assays
Firstly, a K-pette liquid separation workstation is utilized to take 1.5 mu L of diluted DNA template (10-20 ng/. Mu.L) of the whole blood of the Hu sheep to be detected and blank control (No template control, NTC, sterilized water is adopted) to be respectively added into 384-hole reaction plates, and the mixture is dried at 60 ℃ for 30min (a drying box, LGC company) to obtain DNA powder for standby.
Each of the above KASPar primer pairs was diluted to 10. Mu. Mol/L and was used as forward primer A1: forward primer A2: the universal reverse primer C is uniformly mixed according to the volume ratio of 12:12:30 to be used as a primer mixed solution for standby.
And then adding a mixed solution of 1 XMaster Mix (1536 microwell plates, product number: part No. KBS-1016-011) and a primer into each reaction well by using a Meridian sample adding workstation under a Krake operating system, putting the microwell plates on a Kube heat sealing instrument and a Fusion laser membrane sealing instrument in sequence for membrane sealing after Mix packaging is finished, and carrying out high-throughput water bath PCR amplification by using a hydroxyler. The PCR reaction is carried out in a high-flux water bath system, and the specific procedures are as follows:
pre-denaturation at 94 ℃ for 15 min;
94 ℃,20 seconds (denaturation) -61 ℃ -55 ℃,1 minute (renaturation & extension), 10 cycles of amplification in the touchdown order, 0.6 ℃ decrease per cycle;
amplification was continued for 26 cycles at 94℃for 20 seconds (denaturation) -55℃for 60 seconds.
After amplification, detecting fluorescent signals by using a BMG PHARMAStar instrument and checking parting conditions, wherein the specific result is shown in a figure 3, each dot in the figure represents a piece of material to be detected, and a red (dark gray) dot near the upper left side represents that the locus is homozygous genotype 'AA'; the green (light) dots near the middle indicate that the locus is heterozygous genotype "AG" or "GA"; the blue (dark grey) dot near the right indicates that the locus is homozygous genotype "GG".
4. Application of molecular marker in Hu sheep growth trait correlation analysis
The polymorphism of 898 Hu sheep is detected by the method, the genotype of the Hu sheep is determined, and the correlation analysis of the genotype and the immune trait is carried out. And establishing a least squares model as described below, and carrying out correlation analysis on genotype and immune traits.
Y ijkl ＝μ+Genotype i +P j +F k +M l +ε ijkl
Wherein Y is ijkl Is the observed value of growth characteristics, mu is the overall average, genotype i For genotypic effect, P j For batch effect, F k For the father effect, M l Epsilon as the maternal effect ijkl For random error, assume ε ijkl Independent of each other, obeys the N (0, σ2) distribution.
The immune index in the blood of six month old male hu sheep was detected by a blood routine detector (ProCyte Dx, IDEXX, westbrook, ME, USA). Detailed hematology parameter characteristics of 898 animals were obtained by testing, and the results showed that hematology parameters of all sheep remained within normal ranges. Genotype test results showed 505 AA genotypes, 303 AG genotypes, and 90 GG genotypes in 898 individuals. The results of genotype and trait association analysis are shown in Table 1, wherein RBC in the table represents the number of red blood cells in M/. Mu.L; HGB represents hemoglobin concentration in g/dL; HCT represents hematocrit in units; MPV represents the average platelet volume in fL; PLT represents the platelet count in K/. Mu.L; WBC represents the number of white blood cells in K/μl; LYMPH represents lymphocyte count in K/. Mu.L; mono represents the number of monocytes in K/. Mu.L; EO represents eosinophil count in K/. Mu.L; NEUT represents neutrophil count in K/. Mu.L; BASO represents the number of basophils in K/. Mu.L; MCHC represents the mean erythrocyte hemoglobin concentration in g/dL; MCV represents the average red blood cell volume in fL; MCH represents mean coronary hemoglobin in pg; RDW_SD represents the standard deviation of the distribution width of the volume of the flat red blood cells, and the unit is fL; RDW_CV represents the coefficient of variation of the width of the distribution of the volume of the flat red blood cells, expressed in%.
TABLE 1
Note that: the subscript different lower case letters between the same row of data indicate significant difference (P < 0.05), and the same letter indicates insignificant difference (P > 0.05).
Many new red blood cells replace aged red blood cells, and maintain the dynamic balance of the number of red blood cells to maintain the normal needs of body metabolism. There are a number of causes that can result in imbalance in erythropoiesis and destruction, with the result that on the one hand the number of erythrocytes is reduced or increased, thus causing anemia or polycythemia; on the other hand, the erythrocytes are changed in quality. Hemoglobin levels are normal or high, indicating that iron is abundant in the body. The low content of hemoglobin indicates iron deficiency in vivo, thereby affecting the synthesis of hemoglobin and the bearing capacity of oxygen, resulting in anemia and preventing normal physiological activities of animals. As shown by the results of the above table, the G/A polymorphic site shown at position 135 in the amplified fragment SEQ ID NO.1 is significantly correlated with Hu sheep red blood cell number (RBC) and average red blood cell hemoglobin concentration (MCHC) (P < 0.05), wherein RBC and MCHC of Hu sheep carrying GG genotype are significantly higher than those of Hu sheep carrying AA and AG genes (P < 0.05). The normal range of RBCs is: the normal range of MCHC is: 310-370 g/L, selecting GG genotype according to the requirement for breeding, and hybridizing with other sheep by using GG genotype as breeding sheep. In particular, the GG genotype ram semen is adopted for artificial insemination, so that the GG genotype ram semen can be used for sheep disease-resistant breeding, the sheep organism health is ensured, and the economic benefit is increased.
Claims (10)
1. A molecular marker related to the immune trait of Hu sheep is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, the 135bp position R of the sequence represents G or A, and the mutation leads to the G/A polymorphism of the Hu sheep PD-L1 gene at the position.
2. The PCR primer pair for detecting the molecular marker of claim 1, which is characterized by comprising a primer F and a primer R, wherein the nucleotide sequence of the primer F is shown as SEQ ID NO.2, and the nucleotide sequence of the primer R is shown as SEQ ID NO. 3.
3. A KASPar primer pair for detecting the molecular marker according to claim 1, which comprises a forward primer A1, a forward primer A2 and a reverse primer C, wherein the nucleotide sequence of the forward primer A1 is shown as SEQ ID NO.4, the nucleotide sequence of the forward primer A2 is shown as SEQ ID NO.5, and the nucleotide sequence of the reverse primer C is shown as SEQ ID NO. 6.
4. A detection kit comprising a PCR primer pair according to claim 2 or a KASPar primer pair according to claim 3.
5. A method for detecting molecular markers related to immune traits of Hu sheep, wherein the nucleotide sequence of the molecular markers is shown as SEQ ID NO.1, and the 135bp position base R of the molecular markers represents G or A, and the method comprises the steps of amplifying and detecting sheep whole blood genome DNA by using the PCR primer pair according to claim 2 or the KASPar primer pair according to claim 3; and carrying out typing identification on polymorphic sites of the obtained amplification product.
6. The method of claim 5, wherein the genotyping method is direct sequencing, probe, gene chip, or KASP genotyping.
7. The method according to claim 5, characterized in that the method comprises the steps of:
s1, extracting genome DNA by taking Hu sheep whole blood as a sample, carrying out PCR amplification on the extracted genome DNA by utilizing a PCR primer pair shown as SEQ ID NO.2 and SEQ ID NO.3,
s2, sequencing and sequence analysis are carried out on the PCR amplification product, so that the genotype of the polymorphic site is determined.
8. The method of claim 5, wherein the steps of:
a) Extracting genome DNA from Hu sheep whole blood as a sample, and performing high-throughput water bath PCR amplification by using primer groups shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6;
b) After amplification, a KASP genotyping technology is adopted, and a BMG PHARMAStar instrument is used for detecting fluorescent signals and checking the genotyping result.
9. Use of the molecular marker related to the immune trait of hu sheep according to claim 1, the PCR primer pair according to claim 2, the KASPar primer pair according to claim 3 or the detection kit according to claim 4 or the method according to any one of claims 5-8 in hu sheep assisted breeding.
10. The use according to claim 9, wherein the use is for selection of high immunity Hu sheep breeds.
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| CN117721221A (en) * | 2024-01-25 | 2024-03-19 | 中国农业科学院兰州畜牧与兽药研究所 | Cloning and application of SNP molecular marker related to Tibetan sheep immune traits |
| CN117904318A (en) * | 2024-01-25 | 2024-04-19 | 中国农业科学院兰州畜牧与兽药研究所 | SNP molecular marker related to Tibetan sheep immune traits and application thereof |
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|---|---|---|---|---|
| CN117721221A (en) * | 2024-01-25 | 2024-03-19 | 中国农业科学院兰州畜牧与兽药研究所 | Cloning and application of SNP molecular marker related to Tibetan sheep immune traits |
| CN117904318A (en) * | 2024-01-25 | 2024-04-19 | 中国农业科学院兰州畜牧与兽药研究所 | SNP molecular marker related to Tibetan sheep immune traits and application thereof |
| CN117721221B (en) * | 2024-01-25 | 2024-06-11 | 中国农业科学院兰州畜牧与兽药研究所 | Cloning and application of SNP molecular marker related to Tibetan sheep immune traits |
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