KR101683086B1 - Prediction method for swine fecundity using gene expression level and methylation profile - Google Patents

Abstract

The present invention relates to a method for predicting the number of pigs, and more particularly, to a method for predicting the number of pigs by using a gene expression profile and methylation. Industrial Applicability According to the present invention, it is possible to effectively predict the number of black pigs and to establish a system of black pigs having a large number of pigs.

Description

Description: TECHNICAL FIELD The present invention relates to a method for predicting the number of pigs by using a methylation profile,

TECHNICAL FIELD The present invention relates to a method for predicting the number of pigs, and more particularly, to a method for predicting the number of pigs using a gene expression amount and a methylated profile.

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 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.

Numerous cited documents and patent documents are referred to and cited throughout this specification. The disclosures of the cited documents and patents are incorporated herein by reference in their entirety to more clearly describe the state of the art to which the present invention pertains and the content of the present invention.

Korean Registered Patent No. 0444160 (2004.08.02)

Under these technical backgrounds, the present inventors have made intensive efforts to accomplish the present invention. DISCLOSURE OF THE INVENTION The object of the present invention is to analyze the methylation of placenta from sows having an inferior number of sows and an infertile sow to secure a DMR in order to improve a black pork variety having a high number of pigs, By establishing the linkage with the number of living organisms using the relevant genes, it is possible to provide a diagnosis technology for the composition of the black pork system having a high number of living organisms.

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

According to one aspect of the present invention there is provided a method for the treatment and / or prophylactic treatment of pigs such as PTGS1 (Prostaglandin-endoperoxide synthase 1), PCDH1 (protocadherin 1), SPP1 (secreted phosphoprotein 1), TRIM3 (tripartite motif containing 3), F10 a carrier family 38 member 10), and an agent for measuring the methylation level of at least one gene selected from the group consisting of:

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

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

According to another aspect of the present invention, there is provided a method for quantifying the expression level of each gene by extracting mRNA from two or more pigs, And at least one gene of PTGS1 (Prostaglandin-endoperoxide synthase 1) and SPP1 (secreted phosphoprotein 1) is expressed in an amount higher than the average expression level of the gene, or PCDH1 (protocadherin 1), SPP1 the expression level of at least one of phosphoprotein 1, TRIM3 (tripartite motif containing 3), F10 (coagulation factor X) and SLC38A10 (solute carrier family 38 member 10) is lower than the average expression level of the gene, A method of predicting the number of pigs to be predicted can be provided.

According to the present invention, it is possible to effectively predict the number of hatchlings of pigs, and to form a pig system having an excellent number of hatchlings.

Figure 1 shows the correlation of methylation levels according to the number of hatchings.
Figure 2 shows the relative distribution of the hypomethylated region and the hypomethylated region according to the Circos plotting of the predicted DMRs and the region of the gene.
FIG. 3 shows the relationship between fold change of DEG, the number of DEGs ensured, and the number of litters according to the number of the living quarters.
Figure 4 shows the profiles of genes that correlate with CG-DMR and DEG by analysis according to the number of hatchlings.

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

According to one aspect of the present invention there is provided a method for predicting the number of pigs in a pig, comprising the step of determining the level of expression and methylation of one or more genes selected from the group consisting of PTGS1, PCDH1, SPP1, TRIM3, F10 and SLC38A10 of pigs May be provided.

Here, the 'measurement of the expression level' may be to measure the level of mRNA or protein.

Measuring the level of mRNA hereinabove can be analyzed by any of the known methods including RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, Northern blotting, DNA microarray and the like. 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.

In the present invention, the above-mentioned "measurement of the level of the protein" may be performed using any of known protein measurement or detection methods. 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 length of the probe is capable of specifically binding to the target nucleic acid sequence, and includes polynucleotides of any length. For example, the length of the probe may be a length of 7 to 200 nucleotides, 7 to 150 nucleotides, 7 to 100 nucleotides, 7 to 50 nucleotides, or a single strand of a full-length gene, but is not limited thereto.

The probe may be labeled with a detectable label. The detectable label includes a fluorescent label such as Cy3 or Cy5, a radioactive label, an enzyme for converting the substrate into a coloring material, and the like, but the present invention is not limited to these examples.

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

1. methylation specific PCR

When the genomic DNA is treated with bisulfite, 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 And then,

When genomic DNA is converted into bisulfite and PCR is performed using the above two types of primers, PCR products are produced by using primers corresponding to the methylated nucleotide sequence when methylated, and in the case of unmethylated PCR products are produced from primers corresponding to unmethylated primers and 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 bisulfate sequencing for the detection of methylated nucleic acids.

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

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 another aspect of the present invention, there is provided a method for quantifying the expression level of a gene from two or more pigs and obtaining an average expression amount thereof; Checking whether the pig is methylated; And a method for predicting the number of pigs in a pig, comprising determining a pig belonging to the case where at least one gene of the following Table 1 is 1 to 6 as a pig having a higher number of pigs than a non-pig.

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.

As used herein, the term " hypermethylation "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 an increased methylation state compared to the amount of 5-methylcytosine.

As used herein, the term " hypomethylation "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 analyzes the information on the amount of expression of the gene obtained from a specimen and the information on methylation to predict the number of the living organisms.

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. Construction of DEG Information

1. Placental sampling and mRNA sequencing

The placenta was collected immediately after delivery for 12 higher litter size and 7 lower litter size and the sequencing was carried out with 2x100 bp lead length for RNA with reference to the lambs association chart from black pig sows.

2. Analysis of RNA sequencing results

RNA was isolated from each of the three strains of low abundance (TN1307R3720) and high abundance (TN1308R4083) and subjected to RNA-seq after pooling. As a result, the total number of leads was 47,641,439 (low abundance) and 45,940,962 (61.17%) and 28,078,596 (61.12%) of proper paired read, 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, 278 genes were found to be significantly involved in DEG. Compared to the low - abundance group of high - abundance genes, there were 37 high - expression genes and 241 low - expression genes. As a result of the analysis of the read gene, the total genes detected in both groups were 19,545, among which 14,824 and 14,511 genes were mapped in the low and high expression groups, respectively . No new gene was found.

4. Number of Sanjas  Analysis of expression changes of related genes

Analysis of 278 DEG data was performed based on the results of NCBI data analysis. As a result, up-regulated genes were associated with 13 genes and down-regulated genes were associated with 108 genes.

II . DMR  Building information

1. Placental Sampling and Genome Bisulfite ( 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) Immediately after delivery, the placenta was collected, 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).

The total number of raw reads obtained by sequencing the bisulfite-treated genomic DNA was 396,699,088 and 422,836,798, respectively. The mapped reads were 72.26% and 70.55%, respectively, and the number of uniquely mapped reads was 260,135,422 (65.56%) and 269,776,674 (63.80%). Both samples showed similar data yields and mapping ratios.

DNA methylation occurs in the cytosine. CG, CHG, and CHH, and the average sequence depth was 5.63 ~ 6.64. CG, CHG, and CHH were 81.16 ~ 84.07% of the reference sequence.

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

As a result of counting the sites where methylation occurred, methylation occurred predominantly by 18,955,910 to 19,270,603 (81.60 to 82.96%) of CG. Relatively, CHG or CHH showed a very low methylation tendency, ranging from 2.28 to 2.76%. The group with low number of embryos was higher than that with high CG methylation by 314,693. However, CHG and CHH showed high levels of 238,512 and 605,263, respectively, as a result of contradictory results.

The average methylation level of CG was 53.64 and 50.27%, respectively, and CHG and CHH were about 0.50 ~ 0.56%, which is very low average level. CG methylation was found to be 3.57% higher than that of the low -

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

The similarity between the methyl cytosines corresponding to the depth &lt; RTI ID = 0.0 &gt; 5 &lt; / RTI &gt; In this hierarchical clustering, yellow has a low degree of methylation and blue has a high degree of methylation. As shown in FIG. 1, pearson correlation values between two samples for methylated sites were highly correlated to 0.946 and 0.913 for the entire genome CG and gene sites, respectively. In particular, it can be seen that the DMR due to methylation of the gene region is more apparent than the entire CG methylation (Fig. 1). These results suggest that there is DMR in the genes between the two groups, which can be predicted to have a direct correlation with gene expression.

4. Mapped  Methylation CG  ( ^m CG ) Comprehensive analysis of results

Analysis of the ^m CG mapped results showed that methylation was slightly higher in the high population group in the region associated with the coding of the gene. In particular, the highest number of leads was 10,690, 7,088, and 20,435, respectively, in the upstream 1 kb from TSS, 5 UTR, and coding exon groups. However, methylation analysis of the non-genic region revealed that it was as high as 78,893 in the low population group.

The average CG methylation level for the gene region was found to be lowered around the TSS (transcription start site) and 50% in the gene body region. 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.

5. Number of Sanjas  Depending on the group DMR Analysis of

When DMR was analyzed on the basis of a group of high abundance, hypomethylated DMR was analyzed in a uniform manner throughout the genome (FIG. 2). Hypermethylated regions showed higher numbers of high abundance, whereas low methylated regions were higher in the low abundance group. There were 5001 low-methylated DMRs and 850 hypermethylated DMRs (Fig. 2). The DMR density of genes was lower than that of hypermethylated DMRs. The density of DMR was mainly low in the 5 'and 3' side of the gene and relatively high in the gene body.

III . To the number of Sanjas  Following CG - DMR and DEG Correlation analysis result

DMR and DEG information were compared and analyzed to analyze the expression patterns of genes following DNA methylation.

As a result of analyzing the correlation of the number of living quarters with respect to DEG information, FIG. 3 shows the result. Of the total 278 DEGs, 122 genes were associated with the number of living organisms. As a result of comparing and analyzing CG-DMR and DEG information, 15 genes having correlation with each other were detected as shown in FIG. Among these genes, six genes that are highly related to the number of living organisms were analyzed as shown in Table 2.

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 (5)

(PTGS1), PCDH1 (protocadherin 1), SPP1 (secreted phosphoprotein 1), TRIM3 (tripartite motif containing 3), F10 (coagulation factor X) and SLC38A10 (solute carrier family 38 member 10) A composition for predicting swine populations, comprising an agent for measuring an expression level. 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. Extracting mRNA from two or more pigs, quantifying the expression level of each gene, and obtaining an average expression level; And
In the quantified genes, the genes of PTGS1 (Prostaglandin-endoperoxide synthase 1) and SPP1 (secreted phosphoprotein 1) were expressed higher than the average expression levels of the respective genes, and PCDH1 (protocadherin 1), SPP1 (secreted phosphoprotein 1) (TRIM3), F10 (coagulation factor X), and SLC38A10 (solute carrier family 38 member 10) are expressed in an amount less than the average expression level of each gene, Wherein the method comprises the steps of:

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