CN117305432B - SNP markers associated with the activity of acetylcholinesterase in Chinese herpes simplex virus and their detection primers and applications - Google Patents

Abstract

The present invention relates to the field of molecular biology technology, and in particular to SNP markers related to the activity of Chinese domestic butyrylcholinesterase and detection primers and applications thereof. The SNP markers of the present invention have a significant correlation with the activity of butyrylcholinesterase in horse serum. At the same time, we found that the activity of butyrylcholinesterase in breeds with better production and athletic performance is lower, indicating that butyrylcholinesterase with low enzyme activity is significantly selected during the breeding process of modern horses. In the SNP markers, when the genotype of Chinese domestic horses is CC, it can be determined that the horses have higher serum butyrylcholinesterase activity, and when the genotype is CG or GG, it can be determined that the horses have lower butyrylcholinesterase activity. This method can improve the accuracy and efficiency of screening domestic horses based on serum butyrylcholinesterase activity.

Description

SNP markers associated with the activity of Chinese acetylcholinesterase and their detection primers and applications

Technical Field

The invention relates to the technical field of molecular biology, and in particular to a SNP marker related to the activity of Chinese martinylcholinesterase and a detection primer and application thereof.

Background Art

After long-term natural and artificial selection, domestic horses of different breeds around the world have formed stable phenotypic characteristics in production performance, body size, and athletic performance, which enables them to be better specialized. Whole genome sequencing and collection of related phenotypes of domestic horses of different breeds can provide a more comprehensive understanding of the molecular basis for the formation and maintenance of stable phenotypes.

The rapid development of high-throughput sequencing technology has enabled the completion of genome sequencing of most domestic animals and promoted the popularization of high-density SNP chips and whole-genome sequencing technology. In the context of the genome era, it has provided a foundation for using large-scale genomic data and various bioinformatics methods to study and analyze the phenotypic evolution of excellent animal characteristics.

Through the association analysis of phenotypic data and genomic data, the selected regions on the genome related to the phenotype, key genes and their causal mutation sites can be identified. This genomic level association analysis can more accurately discover the causal mutation SNP sites. SNP as a genetic marker has been widely used in research fields such as gene positioning, cloning, genetic breeding and genetic diversity.

Butyrylcholinesterase (BChE), also known as serum cholinesterase (ChE), plasma cholinesterase, and pseudocholinesterase, has been studied for decades. It is an alpha glycoprotein with a biological half-life of about 12 days in humans and can be found in many central and peripheral tissues of the human body, such as the brain, skin, plasma, and leg muscles. Since Broomfield et al. demonstrated in 1991 that BChE has a neuroprotective effect against neurotoxic agents, BChE has attracted widespread attention from researchers. Pretreatment with BChE has been shown to have a protective effect against the toxicity of nerve agents in a variety of animal experiments, making BChE resistant to chemical weapons and organophosphorus pesticides in military and agricultural applications. In addition, BChE has attracted much attention due to its versatility as a pharmacological target and a tool for studying the mechanisms of neurodegenerative diseases. In recent years, there has been an increasing number of studies on the role of BChE in regulating fat metabolism and affecting emotional behavior by hydrolyzing ghrelin, and its role in fat metabolism needs to be explored. At the same time, the high compatibility and multiple functions of BChE in the body indicate that whether cholinesterase activity has selective value in animal evolution and livestock breeding is a scientific issue worth exploring.

Summary of the invention

The invention provides a SNP marker related to the activity of Chinese martinylcholinesterase and a detection primer and application thereof.

The present invention provides a SNP marker associated with butyrylcholinesterase activity, wherein the SNP marker is located at position 9198989 of horse chromosome 19, and the base at the position is G or C. The SNP marker of the present invention has a significant correlation with the activity of butyrylcholinesterase in horse serum and the high altitude adaptability of horses, and at the same time, butyrylcholinesterase with low enzyme activity has a significant selection effect in breeding varieties with better production and movement traits.

According to the SNP marker, the nucleotide sequence of the SNP marker is shown as SEQ ID NO.1.

According to the SNP markers, horses corresponding to the CC genotype have higher serum butyrylcholinesterase activity, while horses corresponding to the CG or GG genotype have lower serum butyrylcholinesterase activity.

The present invention provides a primer for amplifying the SNP marker.

According to the primers, it includes: forward primer F: 5’-CTTGCGATTTCTTCTGCTC-3’ (SEQ ID NO.2) and reverse primer R: 5’-ATTACCACCAAAGGCTGC-3’ (SEQ ID NO.3).

The invention provides a detection product containing the primer.

The detection product is a detection reagent, a test kit or a gene chip.

The present invention provides the use of the SNP marker, the primer, or the detection product in identifying and/or screening the activity trait of martinylcholinesterase.

Preferably, horses having low butyrylcholinesterase activity are identified and/or screened.

Further preferably, the screening is early screening.

Preferably, the horse is a Chinese domestic horse breed.

According to the application, horses corresponding to the CC genotype of the SNP marker have higher serum butyrylcholinesterase activity, and horses corresponding to the CG or GG genotype of the SNP marker have lower serum butyrylcholinesterase activity.

According to the application, the following steps are included:

1) Extracting genomic DNA from the horses to be tested;

2) using the genomic DNA of the horse to be tested as a template and using the primers to perform a PCR amplification reaction;

3) Analyze the PCR amplification products.

Preferably, the reaction system used for PCR amplification in step 2) is:

The PCR reaction conditions were as follows: pre-denaturation at 94°C for 2 minutes; denaturation at 98°C for 10 seconds, annealing at 59°C for 30 seconds, extension at 68°C for 30 seconds, 40 cycles; and final extension at 68°C for 5 minutes.

Step 3) Analyze the PCR amplification products: horses corresponding to the CC genotype have higher serum butyrylcholinesterase activity, and horses corresponding to the CG or GG genotype have lower serum butyrylcholinesterase activity.

The present invention provides the use of the SNP marker, the primer, or the detection product in any of the following:

(1) Application in detecting or assisting in detecting horse serum butyrylcholinesterase activity;

(2) Application in early prediction of horse serum butyrylcholinesterase activity;

(3) Application of molecular marker-assisted breeding in Chinese domestic horses;

(4) Application in improving Chinese domestic horse germplasm resources;

(5) Application in BCHE genotyping of horses;

(6) Application in screening domestic horses that are adaptable to high altitudes.

The inventors have conducted a large number of studies on the correlation between genotype and butyrylcholinesterase activity of domestic horses of different breeds from China and the world and Przewalski's horses, and found that the SNP site provided by the present invention is significantly associated with butyrylcholinesterase activity in the serum of Chinese horses (P<0.003). Genotype CC is 100% homozygous in wild horses, and appears in large quantities in local breeds, with a frequency of up to 56%. The serum butyrylcholinesterase activity of horses with genotype CC is significantly higher than that of horses with genotypes CG and GG (P<1.55e-11), which is instructive for distinguishing and screening Chinese horses with low butyrylcholinesterase activity by genotype. When the genotype of Chinese horses is CC, it can be determined that the horses have higher butyrylcholinesterase activity, and the genotype is CG. When the genotype is GG, it can be determined that the horses have lower serum butyrylcholinesterase activity. This method can improve the accuracy and efficiency of high-altitude adaptability screening of domestic horses.

The nucleotide sequence (SNP marker) consisting of the SNP site and the flanking sequences at both ends thereof is shown in SEQ ID NO: 1, and there is a C/G mutation at the 75 bp position of the sequence.

By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

(i) The molecular markers provided by the present invention are not limited by the age, gender, etc. of the horse and can be used for the early breeding of Chinese horses with better production traits and athletic traits. They can even be accurately screened right after birth, which can greatly accelerate the breeding process of Chinese horses.

(II) The method for detecting single nucleotide polymorphisms of BChE gene in Chinese horses is accurate, reliable and easy to operate.

(III) The detection of SNP sites in the BChE gene of Chinese horses provides a scientific basis for marker-assisted selection of Chinese horses based on cholinesterase activity.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are some embodiments of the present invention.

FIG. 1 shows the typing results of the three genotypes of the SNP markers described in Example 1 of the present invention in different groups of 1405 domestic horses from China and the world.

FIG. 2 shows the results of butyrylcholinesterase activities corresponding to the three genotypes of the SNP markers described in Example 2 of the present invention in different populations.

FIG. 3 shows the results of butyrylcholinesterase activities corresponding to the three genotype individuals of the SNP markers described in Example 3 of the present invention.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

The present invention provides a SNP marker associated with serum butyrylcholinesterase activity in Chinese horses, wherein the SNP marker is located between 9,139,595 bp and 9,199,063 bp on chromosome 19 of Chinese horses (on the horse BChE gene), wherein the SNP site is a base at 9198989 bp, and the base is C or G. The sequence of the SNP marker is shown in SEQ ID NO: 1 (the SNP site is a base n at 75 bp, and n is c or g).

In the present invention, the nucleic acid sequence of the horse BChE gene can be found in GenBank, GeneID: 100033901: 9139595-9199063.

Example 1 Identification of polymorphic sites of BChE gene in Chinese horses

1. Extract genomic DNA from the blood of Chinese domestic horses to be tested

Blood samples were collected from more than 40 horse breeds in China and abroad, including 1405 domestic horses and 21 Przewalski's horses (all ages and genders were not limited, randomly selected, covering horses of different genders and ages) (Table 1), and genomic DNA was extracted from the blood using conventional methods.

Table 1 Sample collection table

2. Amplify the nucleotide fragment containing the SNP site

Primers were designed according to the sequence of the BChE gene locus (chr19:9139595-9199063) included in the NCBI database, including forward primer F: 5'-CTTGCGATTTCTTCTGCTC-3' and reverse primer R: 5'-ATTACCACCAAAGGCTGC-3', and the genomic DNA in step 1 was used as a template to amplify the nucleotide fragment where the SNP to be tested is located, as shown in SEQ ID NO: 1. The SNP site is located at 74 bp of the PCR amplified fragment, where the base is C or G.

The amplification system used in the PCR reaction is calculated as follows based on 25 μL:

The PCR reaction conditions were as follows: pre-denaturation at 94°C for 2 minutes; denaturation at 98°C for 10 seconds, annealing at 59°C for 30 seconds, extension at 68°C for 30 seconds, 40 cycles; and final extension at 68°C for 5 minutes.

3. Detect PCR amplified fragments, obtain SNP markers, and perform genotype determination

The PCR amplification products in step 2 were sequenced and tested. According to the signal distribution position of the sample, the genotype can be divided into CC type, CG type and GG type. If the genotype is CC, the horse to be tested belongs to a breed with higher serum cholinesterase activity, and if the genotype is GG, the horse to be tested belongs to a breed with lower serum butyrylcholinesterase activity. The typing results of the three genotypes are shown in Figure 1. The typing results show that all Przewalski's horses are CC type, corresponding to high-activity butyrylcholinesterase; the frequency of CC genotypes in the domestic horse population of local Chinese breeds varies greatly, ranging from 11.5% to 56.0%, while the frequency of CC genotypes in Chinese and foreign breeds is low, with an average of 23.07%, corresponding to low-activity butyrylcholinesterase. Therefore, it can be seen that the CC genotype is gradually selected in the process of breeding and breeding of domestic horses, and low-activity cholinesterase is more in line with the requirements of modern domestic horses for better production performance and sports performance breeding.

Example 2 Detection of serum butyrylcholinesterase activity in Chinese horses of different genotypes

Serum cholinesterase activity was detected in 173 Chinese horses (Table 2) of the five varieties in the typing results in Example 1, and the butyrylcholinesterase activity was measured using an automatic analyzer (Hitachi 7080, Japan) according to the manufacturer's detection protocol. Butyrylcholinesterase activity detection kits were all purchased from Mike Biotech Co., Ltd. It was found that although there were differences in the average levels of butyrylcholinesterase in the five Chinese horse populations, the butyrylcholinesterase activity of horses with a genotype of CC in the five populations was significantly higher than that of horses with a genotype of GG and CG (P<0.05) (Figure 2). This also fully illustrates that the mutation of this SNP marker has a consistent effect on the activity of butyrylcholinesterase in different populations, and the mutation of this SNP marker C to G significantly reduces the activity of butyrylcholinesterase.

Table 2 Sample table for butyrylcholinesterase activity determination

Example 3 Specific application of the method of the present invention

The SNP marker provided by the present invention was used to perform an expanded population analysis on the BChE gene locus of Chinese domestic horses. The chi-square test of R software was used for analysis. The SNP marker showed that in Chinese domestic horses, the butyrylcholinesterase activity of individuals with genotype CC was significantly higher than that of individuals with genotype GG (P<0.003). It was further verified that the genotype CC of the SNP locus was associated with the serum butyrylcholinesterase activity of Chinese domestic horses (Figure 3).

In the genome-wide association analysis of serum butyrylcholinesterase indicators in Chinese domestic horses, it was found that the polymorphic variation of the BChE gene was related to the serum butyrylcholinesterase activity of Chinese horses. The frequency distribution of the CC genotype in 1426 domestic and wild horses was significantly different in wild horses, local breeds and cultivated breeds, and the frequency of the genotype CC was significantly reduced in cultivated breeds. At the same time, it was verified in multiple Chinese horse breeds that Chinese horses with CC genotypes had higher serum cholinesterase activity, indicating that low butyrylcholinesterase activity was significantly selected during the breeding process of domestic horses.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (7)

1. Application of SNP markers, their amplification primers, or detection products containing their amplification primers in identifying and/or screening for the activity trait of martinylcholinesterase; The SNP marker is located at position 75 of SEQ ID NO.1, where the base is G or C; The horses are one, two, three or four of Guanzhong horses, Yanqi horses, Kazakh horses and Tanggula Mountain horses. 2. The use according to claim 1, characterized in that horses corresponding to the CC genotype of the SNP marker have higher serum butyrylcholinesterase activity, while horses corresponding to the CG or GG genotype have lower serum butyrylcholinesterase activity. 3. The use according to claim 1 or 2, characterized in that the amplification primers include: forward primer F: 5'-CTTGCGATTTCTTCTGCTC -3' and reverse primer R: 5'-ATTACCACCAAAGGCTGC -3'. 4. The use according to claim 1 or 2, characterized in that the detection product is a detection reagent, a kit or a gene chip. 5. The use according to claim 1 or 2, characterized in that the use is for identifying and/or screening horses with low butyrylcholinesterase activity. 6. The use according to claim 1 or 2, characterized in that it comprises the following steps: 1) extracting genomic DNA of the horse to be tested; 2) using the genomic DNA of the horse to be tested as a template and using the amplification primers of the SNP molecular marker to perform a PCR amplification reaction; 3) Analyze the PCR amplification products. 7. Use of SNP markers, their amplification primers, or detection products containing their amplification primers in any of the following: (1) Application in detecting or assisting in detecting horse serum butyrylcholinesterase activity; (2) Application in early prediction of horse serum butyrylcholinesterase activity; (3) Application in horse molecular marker-assisted breeding; (4) Application in horse germplasm improvement; (5) Application in horse BChE genotyping; The SNP marker is located at position 75 of SEQ ID NO.1, where the base is G or C; The horses are one, two, three or four of Guanzhong horses, Yanqi horses, Kazakh horses and Tanggula Mountain horses.