KR101735075B1

KR101735075B1 - Composition and method for prediction of swine fecundity using genomic differentially methylated region

Info

Publication number: KR101735075B1
Application number: KR1020150139091A
Authority: KR
Inventors: 김철욱; 하정임; 김태완; 박화춘; 김일석; 박다혜; 황정혜; 권슬기; 강덕경; 강경희
Original assignee: 경남과학기술대학교 산학협력단
Priority date: 2015-10-02
Filing date: 2015-10-02
Publication date: 2017-05-12
Also published as: KR20170039918A

Abstract

The present invention relates to a composition for predicting the production of pigs by differentially methylated regions (DMRs) and a method for predicting the number of pigs, and more particularly, to a method for predicting the number of pigs by using the profile of a specific gene and a difference in methylation of genomic DNA A composition for prediction and a prediction method. According to the present invention described above, it is possible to effectively predict the number of the pigs to live at a low cost in a short period of time, and to provide a system of pigs having a large number of pigs.

Description

[Technical Field] The present invention relates to a composition for predicting the number of pigs and a method for predicting the number of pigs using the DMR,

The present invention relates to a composition for predicting the production of pigs by differentially methylated regions (DMRs) and a method for predicting the number of pigs, and more particularly, to a method for predicting the number of pigs by using the profile of a specific gene and a difference in methylation of genomic DNA A composition for prediction and a prediction method.

The number of pigs is estimated to be very high compared to other traits, but relatively low heritability and technical limitations make it difficult to improve. The number of spermatozoa is an extremely complex trait which is determined by various traits such as ovulation rate, survival rate of early embryo, survival rate of the fetus, capacity and capacity of the uterus, and number of nipples.

In order to increase the number of living organisms, proper nutrition, management of sows, and genetic factors have been made. Genetic selection has contributed greatly to production of fertile sows. The improvement of sows is mainly focused on reproductive ability and sowing robustness. The main items are the number of litter size, the number of start of breastfeeding, birth weight, 21 day old body weight, 21st birthday number.

The existing breeding techniques related to productivity and quality improvement are mostly in the field of breeding technique, and since breeding technology has a disadvantage of long-term and high cost, there is no development of technology for increasing the number of pigs.

In recent years, the importance of pork production capacity testing project to improve the number of living quarters and the number of reasons has been recognized. This group of sows with a large number of siblings in Korea as well as in Europe has been continuously cultivating excellent systems in the group, which is called the hyper-prolific line. In the developed countries such as USA, UK and Japan, many studies have been actively carried out on the improvement of the number of pigs by using Meishan species, a Chinese native species well known to have a large number of aborigines, The results are not getting.

Korean Patent No. 0444160 (Aug. 2, 2004)

It is an object of the present invention to provide a composition for predicting the number of pigs in a pig and a method for predicting the number of pigs capable of effectively predicting the number of pigs in a short period of time at a low cost and capable of forming a pig system excellent in the number of pigs.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

Under these technical backgrounds, the present inventors have made intensive efforts to accomplish the present invention. The present inventors obtained DMR by analyzing the methylation from the uterus of sows having an inferior number of sows and sows with poor numbers of sows to improve the genetic resources of the black pork varieties having high numbers of spermatozoa, By establishing the linkage with the number of living organisms using the gene, it provides a diagnostic technology for the composition of the black pork system having a high number of living organisms.

Thus, according to one aspect of the present invention, the present invention provides a method of treating a subject suffering from a disease or condition selected from the group consisting of CPXM2 (NC_010456.4), VTCN1 (NC_010446.4), SYT13 (NC_010444.3), CREG1 (NC_010446.4), and TFF2 And an agent for measuring the expression level and the methylation level of the mRNA of at least one gene selected from the group consisting of the genes of the present invention.

According to another aspect of the present invention, there is provided a kit for predicting the number of pigs in a pig, comprising the composition for predicting the number of pigs.

In one embodiment, the kit may be an RT-PCR kit, an oligonucleotide array chip, a microarray chip kit, or a protein chip kit.

According to yet another aspect of the present invention, Quantifying the expression level of the gene and obtaining an average expression level; And determining the pigs belonging to the case of 1 to 5 of at least one of the genes in Table 1 as pigs having a higher number of pigs than the non-pigs.

According to one embodiment of the present invention, it is possible to predict the number of pigs in a short period of time efficiently at a low cost, and to provide a system of pigs having a large number of pigs.

FIG. 1 is a diagram showing the difference in DMR by a heat map. Hypo- and hyper-methylation regions are shown in green and red, respectively.
Fig. 2 is a diagram showing the methylation level according to a gene region. Fig.
FIG. 3 is a diagram showing the results of classifying the genes in the region showing the methylation difference according to function.
FIG. 4 is a graph showing the relationship between DMR and DEG in a point graph.
5 is a graph showing the relationship between methylation and the expression level of CPXM2 (NC_010456.4), VTCN1 (NC_010446.4), SYT13 (NC_010444.3), CREG1 (NC_010446.4), and TFF2 (NC_010455.4) Methylation and expression levels, respectively.

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, there is provided a method of selecting pigs from the group consisting of CPXM2 (NC_010456.4), VTCN1 (NC_010446.4), SYT13 (NC_010444.3), CREG1 (NC_010446.4), and TFF2 (NC_010455.4) A composition for predicting the number of swine populations can be provided, which comprises an agent for measuring the expression level and the methylation level of the mRNA of at least one gene to be tested.

In the present invention, the 'measurement of expression level' may be a measure of the level of mRNA or protein.

In the above, 'measuring the level of mRNA' can be analyzed by any of the methods known in the art including RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, Northern blotting, DNA microarray, have. Preferably, mRNA isolated from the biological sample or cDNA derived therefrom is hybridized on a microarray in which a probe specific to one or more marker genes selected from the group consisting of the genes is immobilized, and the resulting degree of hybridization is measured . The hybridization degree can be measured by any measurement method known in the art such as fluorescence measurement and electrical measurement. In this case, the probe or the target nucleic acid may be labeled with a detectable appropriate label. Here, the cDNA may be directly amplified by RT-PCR using a pair of sense and antisense primers targeting at least one marker gene selected from the group consisting of the genes as primers.

Any of known protein measurement or detection methods known in the art can be used to measure the level of a protein. For example, an assay method using an antibody that specifically binds to a protein expressed from one or more marker genes selected from the group consisting of the genes may be used. Methods for analyzing proteins using antibodies include Western blotting, ELISA, radioimmunoassay, radial immunodiffusion, Oucheronin immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assays, complement fixation assays, FACS But are not limited to these examples. Such ELISAs include direct ELISA, indirect ELISA, direct sandwich ELISA, indirect sandwich ELISA, and the like. Western blotting refers to separation of whole proteins, electrophoresis, separation of proteins according to their size, transfer to a nitrocellulose membrane, reaction with the antibody, and quantification of the amount of the produced antigen-antibody complex by using labeled antibodies It is a way to confirm. In addition, methods for measuring protein levels include methods using enzymes, substrates, coenzymes, and ligands that specifically bind to target proteins.

In the present invention, the expression level of the gene is determined by measuring the amount of the amplification product obtained by nucleic acid amplification performed by RT-PCR, using RNA isolated from the sample as a template Lt; / RTI >

The composition may further include a reagent necessary for hybridization with the marker gene in the sample or the nucleic acid expression product expressed therefrom. In addition, the composition may further comprise a buffer, a solvent, etc., which stabilizes the probe and becomes a reaction medium.

Throughout this specification, the term " probe " is an oligonucleotide that is capable of binding to a target nucleic acid in a base-specific manner as a nucleic acid strand partially or completely complementary to the target nucleic acid. Preferably, it is an oligonucleotide that is completely complementary to the target nucleic acid. The probe includes not only nucleic acid but also any nucleic acid derivative known in the art which is capable of complementary binding including a peptide nucleic acid.

The binding of the probe to the target nucleic acid (generally, also referred to as hybridization) occurs in a sequence-dependent manner and can be performed under various conditions. Generally, the hybridization reaction occurs at a temperature about 5 ° C below the Tm for a particular sequence at a specific ionic strength and pH. The Tm means that 50% of the probe complementary to the target sequence is bound to the target sequence. An example of the hybridization reaction conditions may be a pH 7.0 to 8.3, 0.01 to 1.0 M Na + ion concentration. In addition, in order to enhance the specificity of the target nucleic acid and the probe, it is necessary to carry out the hybridization under the condition that the binding of the probe nucleic acid and the target nucleic acid becomes unstable, for example, in the presence of a high temperature, high concentration of a destabilizer (for example, formamide) .

The methylation detection of the present invention can utilize the following method.

1. methylation specific PCR

When bisulfite is treated with genomic DNA, the cytosine in the 5'-CpG-3 'region is methylated and remains cytosine as it is, and when it is unmethylated, it changes into uracil. Therefore, PCR primers that were methylated at the sites where the 5'-CpG-3 'base sequences were present in the transformed base sequence after bisulfite treatment, and two types of primers corresponding to the unmethylated primers When genomic DNA is converted into bisulfite and then PCR is carried out using the above two types of primers, a PCR product is produced by using a primer corresponding to a methylated base sequence in the case of methylation. In contrast, a non-methylated , PCR products are produced from primers corresponding to unmethylated primers, and thus can be qualitatively confirmed by agarose gel electrophoresis.

2. Real time methylation specific PCR

Real-time methylation specific PCR is the conversion of methylation-specific PCR method to real-time measurement method. The PCR primer corresponding to methylation of bisulfite treated with genomic DNA is designed and real-time PCR is performed using these primers . In this case, there are two methods of detection using a TanMan probe complementary to the amplified nucleotide sequence and detection using LCgreen. Therefore, real-time methylation-specific PCR can quantitatively analyze only methylated DNA.

3. Pyrosequencing

The pyrosequencing method is a method of converting the bisulfite sequencing method into quantitative real-time sequencing. As in the case of bisulfite sequencing, genomic DNA was transformed by treatment with bisulfite, and PCR primers corresponding to the 5'-CpG-3 'base sequence were prepared. The genomic DNA was treated with bisulfite, After amplification with the PCR primer, real-time sequencing analysis is performed using a sequencing primer to quantitatively analyze the amount of cytosine and thymine at the 5'-CpG-3 'site, thereby indicating the degree of methylation.

4. PCR or quantitative PCR using methylated DNA-specific binding protein and DNA chip

When a protein specifically binding to methylated DNA is mixed with DNA, only the methylated DNA can be selectively isolated because the protein binds specifically to the methylated DNA. After the genomic DNA is mixed with the methylated DNA-specific binding protein, only the methylated DNA is selectively isolated, amplified using PCR primers, and then methylated by agarose electrophoresis.

In addition, methylation can be measured using a quantitative PCR method. The methylated DNA separated by a methylated DNA-specific binding protein is labeled with a fluorescent dye and hybridized to a complementary probe-integrated DNA chip to measure methylation .

5. Methylation-sensitive restriction endonuclease

Detection of differential methylation can be accomplished by contacting the nucleic acid sample with a methylation sensitive restriction endonuclease that cleaves only the unmethylated CpG site and cleaving the unmethylated nucleic acid. Here, a "methylation-sensitive restriction endonuclease" is a restriction enzyme that contains CG at the recognition site and is active when C is methylated as compared to when C is not methylated (for example, SmaI). Non-limiting examples of methylation sensitive restriction endonuclease include MspI, HpaII, BssHII, BstUI, and NotI. These enzymes can be used alone or in combination. Other methylation sensitive restriction endonucleotides include, but are not limited to SacII and EagI. Isokimers of methylation-sensitive restriction endonucleases are restricted endonucleases with the same recognition sites as methylation-sensitive restriction endonuclease, which cleave both methylated CGs and unmethylated CGs, for example, MspI have.

6. Bisulfite Sequencing

Another method for detecting a nucleic acid containing methylated CpG comprises contacting a sample containing nucleic acid with an agent for modifying unmethylated cytosine and amplifying the CpG-containing nucleic acid of the sample using a CpG-specific oligonucleotide primer . Here, the oligonucleotide primer may be characterized in that methylated nucleic acid is detected by distinguishing modified methylated and unmethylated nucleic acids. The amplification step is optional, but is not necessary. The method relies on PCR reactions to distinguish between modified (e. G., Chemically modified) methylated and unmethylated DNA. Such a method is disclosed in U.S. Patent 5,786,146, which is described in connection with bisulfite sequencing for the detection of methylated nucleic acids.

According to another aspect of the present invention, there is provided a kit for predicting swine production including the composition for predicting the amount of pigs.

In one embodiment, the kit may be an RT-PCR kit, a microarray chip kit, or a protein chip kit.

At this time, the kit may further include reagents necessary for detection of the methylation.

According to yet another aspect of the present invention, Quantifying the expression level of the gene and obtaining an average expression level; And a method for predicting the number of pigs in a pig, which comprises determining a pig belonging to the case of 1 to 5 of at least one gene of Table 1 below as a pig having a higher number of pigs than the other pigs.

[Table 1]

In the above, the term 'upward regulation' means that the expression is significantly increased in the pigs showing a high number of animals. In other words, it can be predicted that the number of the pigs will be high if it can be statistically confirmed that the expression level of these genes is significantly increased when the gene profile of pigs of unknown pigs is obtained. Likewise, the term 'downward regulation' means a significant decrease in expression in pigs with a high number of pigs, which can also be used to predict the number of pigs.

It is obvious that the accuracy of prediction increases with the number of specimens in predicting the number of pigs in accordance with Table 1 above.

In the present invention, there is no particular limitation on the kind of pig, but black pig is preferable.

The term " hypermethylation " as used herein means that the amount of 5-methylcytosine in one or more CpG dinucleotides in the DNA sequence of a DNA sample being tested is found in the corresponding CpG dinucleotide in a normal control DNA sample Means an increased methylation state compared to the amount of 5-methylcytosine.

The term " hypomethylation " as used herein means that the amount of 5-methyl cytosine in one or more CpG dinucleotides in the DNA sequence of a DNA sample being tested is found in the corresponding CpG dinucleotide in a normal control DNA sample Means a methylated state that is reduced compared to the amount of 5-methylcytosine.

The present invention predicts the number of pigs by analyzing information on the amount of expression of a specific gene obtained from a specimen and information on methylation.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

I. DMR Building information

1. Uterine sampling and genome Bisulfite Sequencing results

Samples were collected from black pig sows with reference to the number of litter sizes and the average number of litter size was 12 (higher litter size> 12 and average 12.1) and 7 (lower litter size, <7, average 7) The uterus was harvested, cut into the same area, and rapidly frozen with liquid nitrogen. Genomic DNA was isolated from the collected high and low samples of the Burkhira strain using Wizard genomic DNA purification kit (promega, USA).

2. Methyl cytosine ( Methylcytosine ) Mapping Distribution of synthesis and methylation

As a result of counting sites where methylation occurred, methylation occurred predominantly in CG. Relatively, CHG or CHH showed a very low methylation tendency. The total DMS (differentially methylated sites) was found to be 2,481,194 at depth 3 and difference 0.1 and was 12% of the total genome. As shown in Table 2, there were 4,399 differences in the methylation among these, with the low methylation position 2,169 and hypermethylation position 2,230.

3. Number of Sanjas Total methylation between groups 메틸레이션 ) Correlation

The DMR corresponding to depth = 3, which is common between two samples, was analyzed. Fig. 1 was prepared based on the information of the gene existing at the methylation position. At the total methylation position, 175 methylated regions and 148 methylated regions were identified in the gene body. Table 3 and Table 4 show information corresponding to the methylation region and the low methylation region, respectively. As shown in FIG. 1, the red region is the hypermethylation region and the green region is the low methylation region. When these genes are analyzed by function, first, the hypermethylation region is composed of genes corresponding to cytoskeleton organization, protein tyrosine kinase activity, and regulation of synaptic transmission, The transcriptional regulator activity was mainly composed of genes corresponding to transcription regulator activity.

4. Mapped DMR Of methylated CG ( ^m CG ) Comprehensive analysis of results

As a result of measuring the average CG methylation level for the gene region, it was observed that the methylation level was lowered around the TSS (transcription start site) as shown in FIG. In the TTS (transcription termination site) and downstream 1 kb, the methylation frequency was slightly lower than that of the gene body. The distribution pattern of methylation frequency in the gene body according to the group of the organisms was generally similar.

When the DMR according to the number of the organisms is classified according to the function of the gene, the group having a higher number of the organisms than the group having the lower number of the organisms has a large group of biological process and molecular function The biologic processes were cytoskeleton organization, cell morphogenesis involved in differentiation and phosphorylation, and the molecular function of the cells was molecular function ) Groups were found to function as protein homodimerization activity.

On the other hand, genes with low methylation could be divided into functions corresponding to biological processes and cellular components. Among them, genes corresponding to biological processes are positive regulation of transcription transcription, embryonic morphogenesis, and membrane organization. In the cellular component, the plasma membrane part, cell-cell junction, Of the genes that function.

II. DEG Building information

1. Uterine sampling and mRNA Sequencing

The uterus was harvested after slaughter for a mean litter size of 12 (higher litter size) and 7 (lower litter size), and sequencing was performed on the RNA with a 2 × 100 bp lead length.

2. Analysis of RNA sequencing results

RNA was isolated from each of the three strains of low abundance (TN1410R3382) and high abundance (TN1410R3379) and pooled and RNA-seq was performed. As a result, the total number of leads was 38,312,494 (low number of westerners) and 52,584,638 (high number of westerners), and the proper paired read was 22,649,360 (59.12%) and 31,915,162 (60.69% Respectively.

In order to remove the low quality sequence, the lead containing the nucleotide represented by N in the sequence information of 10% or more of the entire sequence or the base of less than 40% of the base of Q20 was removed, and the lead having the average quality of less than Q20 was also removed Respectively. The entire filtering process was performed by an internally generated program. The reference genome used for sequence alignment and analysis was the information provided in Ensembl (Flicek P. et al., 2013) and version 72 was used. The filtered sequence was aligned to the genomic sequence using STAR 2.3.0e (Dobin et al, 2013) and the gene information of ensembl version 72 was used in the sequence alignment. The number of Sus scrofa genes by the reference genome was predicted to be 25,323, and the number of transcripts was 30,587.

3. DEG Analysis

The expression level was measured using Cufflinks v2.1.1 (Trapnell C. et al, 2010). In order to measure the expression level, the gene information of the ensembl 72 version was used, and the non-coding gene region was excluded from the expression amount measurement using the -mask option. In order to increase the accuracy of the expression measurement, multi-read-correction and frag-bias-correct options were additionally used, and other options were used as default values.

For specific expression gene analysis, the lead number of each gene was calculated using HTSeq-count v0.5.4p3 (Anders S. et al, 20140), and the intersection-nonempty rule and the pair- End (Paired-end) sequence. Specific gene expression analysis using TCC (Sun J. et al, 2013) was performed using the calculated lead number of each gene. The TCC option used the iDEGES / edgeR method with consideration of repetition, and the specific expression gene selection was set at a reference value of less than 0.05 based on the Q-value corrected for errors caused by multiple-testing.

As a result of DEG analysis, there were a total of 789 DEG genes. Among these genes, DEG was selected to satisfy p <0.01 and q <0.05, and 82 genes with higher expression compared to the case of the group with lower abundance among the genes of higher abundance group were 82, Were 17.

III. To the number of Sanjas However, DMR and DEG Correlation analysis result

DMR and DEG information were compared and analyzed to analyze the expression patterns of genes following DNA methylation. Information on the positions at which methylation was measured in the gene body in the entire DMR was plotted on the abscissa and the expression level was expressed on the vertical axis by DEG analysis of each gene (Fig. 4). Generally, it is well known that when the methylation level is low, the expression level is increased and when the methylation level is high, the expression level is decreased. Such a pattern of methylation is more effectively known when it is located in the promoter region that regulates gene expression. Table 1 shows the genes whose expression level is regulated by methylation while methylation is changed in the promoter position in the point graph representing DMR / DEG. The degrees of methylation and expression levels of five genes of CPXM2 (NC_010456.4), VTCN1 (NC_010446.4), SYT13 (NC_010444.3), CREG1 (NC_010446.4), and TFF2 (NC_010455.4) have.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims

CPXM2 (Carboxypeptidase X, M14 Family Member 2), VTCN1 (V-Set Domain Containing T Cell Activation Inhibitor 1), SYT13 (Synaptotagmin-13), Cellular repressor of E1A-stimulated genes 1, and TFF2 factor 2) and the methylation level of the mRNA of the gene of the present invention.

The composition according to claim 1, wherein the composition predicts the number of pigs in pigs.

A kit for estimating the number of pigs of a pig comprising the composition according to claim 1 or 2.

4. The kit according to claim 3, wherein the kit is an RT-PCR kit, a microarray chip kit or a protein chip kit.

Quantifying the expression level of the gene from two or more pigs and obtaining an average expression level;
Obtaining an average methylation level for a gene from two or more pigs; And
(VTCN1), SYT13 (Synaptotagmin-13), CREG1 (VTCN1), and VEGF2 (VTCN1) were found to be higher than that of CPXM2 (Carboxypeptidase X and M14 Family Member 2) The pigs belonging to the case where the gene expression of the gene of the cellular repressor of E1A-stimulated genes 1) and TFF2 (Trefoil factor 2) is higher than the average expression level or the average methylation level is lower than that of the non- A method for predicting the number of pigs in a pig, including those identified as pigs.