KR20160054245A - Novel SNP marker for discriminating level of amylase within porcine blood and use thereof - Google Patents

Abstract

The present invention relates to an SNP marker for determining amylase levels in porcine blood, a composition comprising the SNP marker for determining amylase levels in porcine blood, a kit comprising the composition for determining amylase levels in porcine blood, a method for determining amylase levels in porcine blood by using a microarray, the SNP marker, and the composition, a method for producing pigs whose blood amylase level is maintained within the normal range, and a method for maintaining the blood amylase level in pigs within the normal range. Also, the present invention relates to a composition comprising the SNP marker for detecting pancreatitis, pancreatic pseudocyst, pancreatic cancer, cholecystitis, renal failure, sialadenitis, obesity or diabetes in pigs, and a method using the same for diagnosing hyperamylasemia, macroamylasemia, pancreatitis, pancreatic pseudocyst, pancreatic cancer, cholecystitis, sialadenitis, renal failure, hepatitis, cirrhosis, obesity or diabetes in pigs.

Description

SNP markers for determining the level of amylase in the blood of pigs and their use [

The present invention relates to a composition for judging the level of amylase in the blood of a pig, a composition for determining the level of amylase in the blood of a pig containing the SNP marker, a kit for determining the level of amylase in the blood of the pig, A method for determining the level of amylase in the blood of a pig using the microarray and the SNP marker or composition thereof, a method for producing a pig in which the level of amylase in the blood of the pig is maintained at an amylase level in the blood of a normal pig, To the level of amylase in the blood.

The present invention also relates to a composition for diagnosing a porcine pancreatitis, a pancreatic pseudocyst, a pancreatic cancer, a cholecystitis, a renal failure, a salivary gland, an obesity or a diabetes mellitus comprising the SNP marker and a hyperamylasemia of the pig, macro amylase, pancreatitis, Pancreatic cancer, cholecystitis, salivary gland disease, renal failure, hepatitis, cirrhosis, obesity or diabetes mellitus.

Since breeding in the eastern part of India about 9,000 years ago, pigs have been raised as the most basic animal for the worldwide consumption of protein that people need, according to their age, situation and people's preferences.

In addition, pigs have been raised for a long period of time to increase feed efficiency and growth rate, resulting in stress-sensitive pigs and deterioration of meat quality. As a result, efforts are being made to improve the health status of pigs and to select good individuals without metabolic disturbances to establish a good group.

Amylase is one of the digestive enzymes that break down starch into maltose and dextrin and is known to be present in pancreatic juice, saliva, blood, urine, and the like. In particular, amylase in the blood is elevated mainly in pancreatitis, pseudocyst of pancreas, pancreatic cancer, etc., and when salivary glands are inflamed, elevation also appears as a marker for the disease. In addition, it has been reported that high levels of amylase in the blood can lead to pancreatitis and low levels of the metabolic syndrome ( JOP, J. Pancreas 2005; 6 (6): 536-551 Pancreatic Hyperenzymia: Clinical Significance and Diagnostic Approach ", Cardiovasc Diabetol . 2011; 10: 34. " Low serum amylase in association with metabolic syndrome and diabetes: A community-based study ").

However, no gene or SNP involved in the level of amylase in the blood of pigs has been reported, and the level of amylase in the blood is determined using the SNP, and the level of amylase in the blood is determined by fixing the allelotype at the SNP It has not been reported to keep it at normal level.

In recent years, efforts have been made to select healthy individuals that do not have metabolic disorders in the pig breeding business and to establish a good quality breeder group. Therefore, it is necessary to develop technology for selecting individuals by early judgment of the health condition of the individual. to be.

Under these circumstances, the inventors of the present invention have conducted intensive researches to determine the level of amylase in the blood of pigs and to maintain a normal level of amylase in blood. As a result, it has been found that the SNP of the gene involved in the amylase level in the blood of pigs , The level of amylase in the blood of genetically determined pigs can be determined and maintained at a normal level. Thus, the present invention has been completed.

It is an object of the present invention to provide a composition for judging the level of amylase in the blood of a pig.

Another object of the present invention is to provide a kit for judging the level of amylase in the blood of pigs, comprising a composition for judging the level of amylase in the blood of the pig.

It is another object of the present invention to provide a method for determining the level of amylase in the blood of a pig.

Another object of the present invention is to provide a method for producing pigs in which the level of amylase in the blood of pigs is maintained at an amylase level in the blood of normal pigs.

It is a further object of the present invention to provide a method of maintaining the level of amylase in the blood of a pig at the level of amylase in the blood of a normal pig.

Another object of the present invention is to provide a composition for the diagnosis of hyperamylasemia of a pig, macro amylase, pancreatitis, pancreatic pseudocyst, pancreatic cancer, cholecystitis, renal failure, salivary gland, renal failure, hepatitis, liver cirrhosis, obesity or diabetes mellitus.

Another object of the present invention is to provide a method for diagnosing hyperamylasemia of a pig, macro amylase, pancreatitis, pancreatic pseudocyst, pancreatic cancer, cholecystitis, salivary gland, renal failure, hepatitis, cirrhosis, obesity or diabetic disease.

It is another object of the present invention to provide a SNP marker for determining the level of amylase in the blood of a pig.

It is still another object of the present invention to provide a microarray for determining the level of amylase in the blood of a pig, which comprises the SNP marker.

In one embodiment of the present invention, a polynucleotide derived from an AMY2 (amylase alpha 2B) gene comprising the nucleotide sequence of SEQ ID NO: 1 is C or T at position 177 or AMY2 The polynucleotide derived from the AMY2 gene having the nucleotide sequence of SEQ ID NO: 3 is A or G in the polynucleotide derived from the AMY2 gene, and the nucleotide sequence of SEQ ID NO: 3 is the polynucleotide derived from the AMY2 gene wherein the 99th base is deleted or the 98th base is deleted and the 99th base is C, 2 of the AMY2 gene-derived polynucleotide consisting of the 177th base of the AMY2 gene-derived polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, the 99th base of the AMY2 gene-derived polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: Consisting of 5 to 1000 contiguous bases comprising a base Provides a level determined composition of the polynucleotide or comprising a formulation which is capable of detecting or amplifying a complementary polynucleotide thereof, porcine blood amylase (amylase).

In the present invention, the polynucleotide derived from the AMY2 (amylase alpha 2B) gene comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 is a polymorphic sequence comprising the polymorphic site of the gene involved in the amylase level of the porcine ), Wherein the polymorphic sequence means a sequence comprising a polymorphic site comprising a SNP in the polynucleotide sequence. The polynucleotide sequence may be DNA or RNA.

The term "polymorphism " of the present invention means a case where two or more alleles exist in one locus, and among polymorphic sites, only one single base is different from the single It is called single nucleotide polymorphism (SNP). Polymorphic markers may have two or more alleles with a frequency of occurrence of 1% or more, specifically 5% or 10% or more in the selected population.

The term "allele " of the present invention refers to various types of a gene existing on the same locus of a homologous chromosome. Alleles are also used to represent polymorphisms, for example, SNPs have two kinds of bialles.

Specifically, the pig to be the subject of the amylase level in the blood of the pig may be a hybrid of Jeju native pig, land lace or Jeju native pig and land lace.

In one embodiment of the present invention, genes and SNP markers related to amylase were analyzed for the cross reference group of Korean traditional pig and land race, and the SNP having the highest significance with respect to amylase was identified as ASGA0022387, Was further extracted and the sequence was analyzed to identify base mutations affecting the level of amylase. Specifically, in the polynucleotide derived from the AMY2 gene in which the 177th base of the nucleotide sequence of SEQ ID NO: 1 is C or T, or the polynucleotide derived from the AMY2 gene consisting of the nucleotide sequence of SEQ ID NO: 2, the 99th base is deleted from the T or the 98th base, Or a polynucleotide derived from the AMY2 gene consisting of the nucleotide sequence of SEQ ID NO: 3 showed that the content of amylase differs depending on whether the 482nd nucleotide is A or G (Tables 3 to 5).

The term "agents capable of detecting or amplifying SNP markers" in the present invention refers to agents capable of specifically recognizing binding to a polymorphic site containing a SNP in a gene and capable of amplifying the polymorphic site Specifically, a probe capable of specifically binding to a polymorphic site containing the SNP, a polynucleotide comprising the SNP marker, or a primer capable of specifically amplifying a complementary polynucleotide thereof.

More specifically, it is possible to amplify a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 4 and SEQ ID NO: 5 and the nucleotide sequence of SEQ ID NO: 2 capable of amplifying the polynucleotide comprising the nucleotide sequence of SEQ ID NO: A primer pair consisting of the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8, and a pair of primers consisting of the nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 10 capable of amplifying the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: But are not limited to, one or more primer pairs.

In the present invention, a probe used to recognize and bind to a SNP marker includes a sequence complementary to a polynucleotide sequence including a SNP, and may be a DNA, RNA, or DNA-RNA hybrid form . Further, fluorescent markers, radiation markers, and the like can be additionally attached to the 5 'or 3' ends of the probe so as to be visually recognizable.

The term " primer " of the present invention means a base sequence having a short free 3 'hydroxyl group and can form base pairs with a complementary template, It means a short sequence functioning as a point. The primers used in the present invention for the amplification of SNP markers can be amplified by PCR using appropriate conditions in suitable buffers (for example, 4 different nucleoside triphosphates and polymerase such as DNA, RNA polymerase or reverse transcriptase) Stranded oligonucleotide that can serve as a starting point for the directed DNA synthesis. The appropriate length of the primer may vary depending on the purpose of use, but it may be generally used in a size of 15 to 30 nucleotides. The primer sequence is not necessarily completely complementary to the polynucleotide comprising the SNP marker or its complementary polynucleotide, and can be used if it is sufficiently complementary to hybridize.

The primers can also be modified, for example, by methylation, capping, substitution of nucleotides or modifications between nucleotides, for example, uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate, Carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

The term " amylase " in the present invention refers to hydrolysis of starch as one of digestive enzymes and is present in pancreatic juice, saliva, blood, urine and the like. In addition, high levels of amylase in the blood lead to pancreatic diseases such as pancreatitis and salivary gland disease, and when it is too low, it causes obesity and diabetes due to metabolic disorders. Amylase in the blood, also called serum amylase, has been reported to cause pancreatitis when it is higher than the normal level, and cause metabolic syndrome when it shows low level. In the present invention, genes and SNPs involved in the content of amylase in the blood are identified, and the amylase level in the blood is determined using the same, and a good pork having a good health condition can be selected and manufactured using the same.

In one embodiment of the present invention, AMY2 and SNP ASGA0022387, which are genes involved in the amylase level in the blood of pigs, were identified (Fig. 1 and Table 1). In addition, when the allele genotype is C / C type at the 177th position of the base sequence of the porcine AMY2 gene represented by SEQ ID NO: 1 including the intron 1 region, the content of amylase in the blood is high and it is confirmed that it is low at the T / T type (Table 3 and FIG. 6). When the allele genotype at the 99th position in the exon 3 region of the AMY2 gene shown in SEQ ID NO: 2 was C / C type, the content of amylase in blood was high and it was low at T / T type (Table 4). Amylase content in the blood was high when the allele was at the 482nd position of the base sequence of the porcine AMY2 gene represented by SEQ ID NO: 3 including the 3'-UTR region, and the G / G-type (Table 5).

Since there are many reports that the content of amylase in blood is related to pancreatitis, salivary gland disease, metabolic disorder, etc., it is possible to maintain the normal level of amylase by fixing the allelotype to the desired trait, and furthermore, It will be apparent to those skilled in the art that the present invention can be applied to compositions and methods capable of diagnosing the diseases from the genotypes.

Another embodiment of the present invention provides a kit for determining the level of amylase in the blood of pigs comprising the composition. Specifically, the kit may be, but not limited to, a PCR (Polymerase Chain Reaction) kit or a DNA analysis kit (for example, a DNA chip).

The kit of the present invention can determine the level of amylase in the blood of a pig by amplifying a gene involved in the level of amylase in the blood of the pig and then confirming the band appearance by cutting with a restriction enzyme. The PCR kit may comprise a test tube or other appropriate container, a reaction buffer (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), and the like, in addition to the respective primer pairs specific for polynucleotides comprising SNP markers or their complementary polynucleotides. ), Enzyme such as Taq polymerase, DNase, RNAse inhibitor, DEPC-water, sterilized water and the like. Specifically, the PCR kit may be a kit for performing PCR-restriction fragment length polymorphism (PCR-RFLP).

In the present invention, the "PCR-RFLP" method is a technique for analyzing the restriction fragment length polymorphism (PCR) of gene fragments among various DNA analysis techniques for improving the genetic characteristics and capability of livestock. the presence of restriction enzyme recognition sites in the point mutation region allows the use of the fact that the lengths of the fragments generated by restriction enzymes vary according to the genotype difference of each individual, . In addition, the amylase level can be determined by detecting a frame shift mutation due to deletion and mutation of bases in the exon 3 region in the AMY2 gene using a PCR kit.

In particular, the kit of the present invention may be a kit for determining the level of amylase in the blood of pigs, which includes essential elements necessary for carrying out a DNA chip. DNA chip kits are those in which nucleic acid species are attached in a gridded array on a generally flat solid support plate, typically a glass surface not larger than a slide for a microscope, and nucleic acids are uniformly arranged on the chip surface, Hybridization reaction occurs between the nucleic acid on the surface of the chip and the complementary nucleic acid contained in the treated solution on the surface of the chip to enable the mass parallel analysis, which can be used to determine the level of amylase in the blood of pigs.

(A) a polynucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3 or a complementary polynucleotide thereof (polymorphic site ); And (b) determining the base of each amplified polymorphic site of step (a). ≪ IMAGE > At this time, DNA of the separated sample can be obtained from a sample isolated from an individual.

The term " individual " of the present invention means a pig to which the level of amylase in the blood is to be determined, and the level of amylase in the blood of the pig can be determined by analyzing the SNP using the sample obtained from the pig. As the specimen, DNA can be obtained from hair, urine, blood, various body fluids, separated tissues, isolated cells, or saliva, but is not limited thereto. In particular, the present invention can be used to early determine the level of amylase in the blood of pigs in a non-invasive manner, and to select pigs that have a particularly high or low amylase level and maintain a normal level of amylase content.

The step of amplifying the polymorphic site of the SNP marker from the DNA of step (a) or hybridizing with the probe may be performed by any method known to those skilled in the art. For example, the target nucleic acid can be obtained by PCR amplification and purification thereof. Other ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sequence amplification based on nucleic acids (NASBA) can be used as well as self-sustaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874 (1990)).

Determination of bases in the polymorphic site of step (b) of the above method can be carried out by sequencing, hybridization by microarray, allele specific PCR, dynamic allele-specifichybridization, DASH), PCR extension analysis, PCR-SSCP, PCR-RFLP analysis or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (e.g. MassenRAY system of Sequenom), mini- But are not limited to, the BioRad system, the CEQ and SNPstream system (Beckman), the Molecular Inversion Probe array technology (e.g. Affymetrix GeneChip), and BeadArray Technologies (e.g. Illumina GoldenGate and Infinium assay) . One or more alleles in a polymorphic marker, including microsatellite, SNP or other types of polymorphic markers, can be identified by such methods or by other methods available to those skilled in the art to which this invention pertains. The determination of the base of such a polymorphic site can be carried out specifically through the SNP chip.

The term " SNP chip " means one of DNA microarrays capable of confirming each base of several hundred thousand SNPs at a time.

The TaqMan method comprises the steps of: (1) designing and constructing a primer and a TaqMan probe to amplify a desired DNA fragment; (2) labeling probes of different alleles with FAM dyes and VIC dyes (Applied Biosystems); (3) performing PCR using the DNA as a template and using the primer and the probe; (4) after completion of the PCR reaction, analyzing and confirming the TaqMan assay plate with a nucleic acid analyzer; And (5) determining the genotype of the polynucleotide of step (1) from the analysis result.

Sequencing analysis can be performed using conventional methods for sequencing and can be performed using an automated gene analyzer. The allele-specific PCR means a PCR method in which a DNA fragment in which the SNP is located is amplified with a primer set including a primer designed with the base at the 3 'end at which the SNP is located. The principle of the above method is that, for example, when a specific base is substituted by A to G, an opposite primer capable of amplifying a primer containing the A as a 3 'terminal base and a DNA fragment of an appropriate size is designed, In the case of performing the reaction, when the base at the SNP position is A, the amplification reaction is normally performed so that a band at a desired position is observed. When the base is substituted with G, the primer can complementarily bind to the template DNA , And 3 'ends do not perform complementary binding, so amplification reaction is not performed properly. DASH can be carried out by a conventional method, specifically, by the method by Prince et al.

On the other hand, in the PCR extension analysis, first, a DNA fragment containing a base in which a SNP is located is amplified as a primer pair, and then all nucleotides added to the reaction are deactivated by dephosphorylation, and SNP specific extension primer, dNTP mixture , Digoxinucleotide, reaction buffer, and DNA polymerase to perform primer extension reaction. At this time, the extension primer has a base immediately adjacent to the 5 'direction of the base in which the SNP is located at the 3' terminus, the nucleic acid having the same base as the didyxin nucleotide is excluded in the dNTP mixture, and the didyoxynucleotide indicates the SNP Base type. ≪ / RTI > For example, when dGTP, dCTP and TTP mixture and ddATP are added to the reaction in the presence of substitution from A to G, the primer is extended by the DNA polymerase in the base in which the substitution has occurred, The primer extension reaction is terminated by ddATP at the position where the base first appears. If the substitution has not occurred, the extension reaction is terminated at the position, so that it is possible to discriminate the type of the base representing the SNP by comparing the lengths of the extended primers.

As the detection method, when the extension primer or the dioxynucleotide is fluorescently labeled, the SNP is detected by detecting fluorescence using a gene analyzer (for example, Model 3700 of ABI Co., Ltd.) used for general nucleotide sequence determination And when the unlabeled extension primer and the didyxin nucleotide are used, the SNP can be detected by measuring the molecular weight using MALDI-TOF (matrix assisted laser desorption ionization-time of flight) technique.

Specifically, in the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, the allele of the 177th nucleotide, which is the polymorphic site, is all C, or the polynucleotide consisting of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 is deleted, The allele of the 99th base is all C or the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 is all the allele of the 482nd nucleotide, which is the polymorphism site, in the case of A, it is judged that the level of amylase in the blood of the pig is increased .

More specifically, in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, all the alleles of the 177th base, which is a polymorphic site, are all T or a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 It can be judged that the level of amylase in the blood of the pig is decreased when the allele is all T, or when the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 is all G allele of the polymorphism site 482 base. The pig may be a hybrid of Jeju native pig, land lace or Jeju native pig and land lace.

In another embodiment of the present invention, there is provided a method for fixing an SNP trait of a gene involved in the level of amylase in a blood of a pig, comprising the steps of: (a) isolating an allele of the 177th base of a polynucleotide comprising the nucleotide sequence of SEQ ID NO: Or the allele of the 99th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 is fixed to the T / C type, or the allele of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: Wherein the amylase level in the blood of the pig is maintained at an amylase level in the blood of a normal pig, including the step of fixing the gene to the A / G type.

In another embodiment of the present invention, there is provided a method for fixing an SNP trait of a gene involved in the level of amylase in a blood of a pig, comprising the steps of: (a) isolating the allele of the 177th base of the polynucleotide comprising the nucleotide sequence of SEQ ID NO: Or the allele of the 99th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 is fixed to the T / C form, or the polynucleotide comprising the nucleotide sequence of the 482nd nucleotide of the nucleotide sequence of SEQ ID NO: 3 A method of maintaining amylase levels in the blood of a pig at an amylase level in the blood of a normal pig, comprising the step of fixing the allele in an A / G form.

In particular, the step of immobilizing the SNP trait may be carried out by crossing an individual having all of the SNP traits, and selecting an individual having the desired trait. More specifically, the pig may be a hybrid of Jeju native pig, land lace or Jeju native pig and land lace.

In the present invention, the 98th base in the exon 3 region of the 177th position and the exon 3 region in the base sequence of the porcine AMY2 gene represented by SEQ ID NO: 1, which contains the intron 1 region, Amylase content was different according to the allele genotype at position 482 in the base sequence of the porcine AMY2 gene shown in SEQ ID NO: 3 including '-UTR. Specifically, when the allele genotype at position 177 in the base sequence of the porcine AMY2 gene represented by SEQ ID NO: 1 including the intron 1 region is C / C type, amylase content is high, and when T / T type is amylase content is low (Table 3). When the 98th base of the exon 3 region represented by SEQ ID NO: 2 was deleted and the allele genotype at the 99th position was C / C type, the amylase content was high. When the T / T type was amylase (Table 4). When the allele genotype at the 482nd position in the base sequence of the porcine AMY2 gene represented by SEQ ID NO: 3 including the 3'-UTR was A / A type, the average amylase content And the average amylase content of G / G type was low (Table 5). In addition, it was found that the alleles of the C / T type, the T / C type or the A / G type showed the average level of amylase in each of the above positions.

As a result, the amylase level in the blood can be maintained at an average level by fixing the base at the SNP position involved in the amylase level in the blood in the AMY2 gene, so that the amylase level in the blood of the pig is maintained at the amylase level in the blood of the normal pig And the level of amylase in the blood of the pig can be maintained at the amylase level in the blood of the normal pig.

In another embodiment of the present invention, the polynucleotide derived from the AMY2 gene comprising the nucleotide sequence of SEQ ID NO: 1 is C or T at position 177, or the polynucleotide derived from the AMY2 gene consisting of the nucleotide sequence of SEQ ID NO: Is T or the 98th base is deleted and the 99th base is C or the polynucleotide derived from the AMY2 gene consisting of the nucleotide sequence of SEQ ID NO: 3 is A or G, and the AMY2 gene consisting of the nucleotide sequence of SEQ ID NO: 1 A polynucleotide derived from AMY2 gene consisting of the nucleotide sequence at position 177 of the polynucleotide derived from the AMY2 gene, the 99th base of the AMY2 gene-derived polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: A polynucleotide consisting of a continuous base or a polynucleotide consisting of Amylase, pancreatitis, pancreatic pseudocyst, pancreatic cancer, cholecystitis, salivary ganglion, renal failure, hepatitis, cirrhosis, obesity or diabetes mellitus, which comprises an agent capable of detecting or amplifying a complementary polynucleotide To a diagnostic composition.

Specifically, the preparation can amplify a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 4 and SEQ ID NO: 5 capable of amplifying a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, and a polynucleotide comprising the nucleotide sequence of SEQ ID NO: A primer pair consisting of the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8, and a pair of primers consisting of the nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 10 capable of amplifying the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: Lt; RTI ID = 0.0 > 1, < / RTI >

Formulations, primers and the like that can be detected or amplified are as described above.

Another specific embodiment of the present invention is a composition for the diagnosis of hyperamylasemia, maco amylase, pancreatitis, pancreatic pseudocyst, pancreatic cancer, cholecystitis, renal failure, salivary gland, renal failure, hepatitis, liver cirrhosis, obesity or diabetes mellitus of said pig The present invention relates to a diagnostic kit.

The kit of the present invention can include not only agents capable of detecting SNPs at specific positions in the AMY2 gene in a sample of a sample but also one or more compositions, solutions or devices suitable for the above analysis. The kit may be a PCR kit or a kit for DNA analysis (e.g., a DNA chip).

(A) a polynucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3 or a complementary polynucleotide thereof (polymorphic site ); And (b) determining the base of the polymorphic site amplified in the step (a). The method of claim 1, wherein the polymorphic site is selected from the group consisting of a hyperamylasemia, hyperamylasemia, macro amylase, pancreatitis, pancreatic pseudocyst, pancreatic cancer, cholecystitis, , Hepatitis, cirrhosis, obesity or diabetes.

Specifically, in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, the allele of the 177th base, which is the polymorphic site, is all C, or the 98th base is deleted from the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, The allele of the second base is C or the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 is all the A allele of the 482nd base, which is a polymorphism site, is selected from the group consisting of hyperamylase, macro amylase, pancreatitis, pancreatic pseudocyst, , A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, all the alleles of the 177th nucleotide, which is a polymorphic site, are all T, or a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, The allele of the 99th base is all T, Or a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 can be diagnosed as renal failure, hepatitis, cirrhosis, obesity or diabetes mellitus when the allele of the 482nd nucleotide, which is a polymorphic site, is all G.

The individual, polymorphic site is as described above.

It has been reported that amylase in the blood, which is also called serum amylase, does not maintain normal level and shows a level higher than normal or low level, respectively. Specifically, when amylase in the blood exhibits a level higher than normal, it has been reported to induce hyperamylaseemia, macro amylaseemia, pancreatitis, pancreatic pseudocyst, pancreatic cancer, cholecystitis, and salivary glanditis ( JOP J. Pancreas 2005; 6 (6): 536-551 " Pancreatic Hyperenzymemia: Clinical Significance and Diagnostic Approach "). The pancreatitis includes chronic pancreatitis and acute pancreatitis, particularly acute pancreatitis.

In the present invention, the term " hyperamylaseemia "refers to an increase in amylase in the blood, and may be accompanied by pancreatic diseases such as acute or chronic pancreatitis, pancreatic pseudocyst, pancreatic damage and the like.

The term " macroamylasemia " in the present invention means a disease in which amylase in the blood binds to blood proteins, polysaccharides and immunoglobulins to form a complex to increase the size thereof, resulting in limitation of filtration in the kidney.

In addition, it has been reported that amylase in the blood exhibits lower levels than normal, leading to metabolic syndrome and diabetes (Cardiovasc Diabetol. 2011; 10: 34. "Low serum amylase in association with metabolic syndrome and diabetes: A community- based study "). Specifically, it can cause obesity, diabetes, kidney failure, and liver damage. More specifically, the liver damage may be hepatitis, liver cirrhosis.

In one embodiment of the present invention, the amylase content is high when the allele genotype at the 177th position in the base sequence of the porcine AMY2 gene represented by SEQ ID NO: 1 including the intron 1 region is C / C type, and when the amylase content (Table 3). When the 98th base of the exon 3 region shown in SEQ ID NO: 2 was deleted and the allele genotype at the 99th position was C / C type, the amylase content was high and the T / T (Table 4). When the allele genotype at the 482nd position in the nucleotide sequence of the porcine AMY2 gene represented by SEQ ID NO: 3 including the 3'-UTR was A / A type, it was confirmed that the amylase content was low Amylase content was high and the average amylase content was low when G / G type (Table 5).

From the above results, it can be determined that the level of amylase in the blood is high or low in the early stage by detecting the genotype at each mutation position in the gene that affects the content of amylase in the blood, and the amylase in the blood shows a high level It is possible to diagnose that amylase is elevated and amylase, macro amylase, pancreatitis, pancreatic pseudocyst, pancreatic cancer, cholecystitis or salivary gland can be induced. When amylase in blood shows low level, obesity, diabetes, It can be diagnosed that it can be induced.

Another embodiment of the present invention is a polynucleotide comprising the nucleotide sequence at position 177 of the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence of the 99th nucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or the polynucleotide comprising the nucleotide sequence of SEQ ID NO: Provides SNP markers for determining the level of amylase in the blood of pigs, located at base 482. Specifically, the pig may be a hybrid of Jeju native pig, land lace or Jeju native pig and land lace.

Regarding the SNP marker of the present invention, in the base sequence of the porcine AMY2 gene represented by SEQ ID NO: 1 including the intron 1 region, the 177th base is C or T, or the 98th base of the exon 3 region represented by SEQ ID NO: 2 In the base sequence of the porcine AMY2 gene shown in SEQ ID NO: 3, wherein the 99th base is T or C or 3'-UTR, the content of amylase differs depending on whether the 482nd base is A or G , And it is possible to determine the level of amylase according to the allelic genotype at the above position. Further, by fixing the specific base, the amylase can be maintained at a normal level, so that it can be used for producing and sorting pigs of good health. Also, as the number of applied SNP markers increases, the accuracy can be increased.

Another embodiment of the present invention provides a microarray for determining the level of amylase in blood of a pig, which comprises the SNP marker. The microarray may comprise DNA or RNA polynucleotides. The microarray comprises a conventional microarray except that the polynucleotide of the present invention is contained in the probe polynucleotide.

Methods for preparing microarrays by immobilizing probe polynucleotides on a substrate are well known in the art. The probe polynucleotide means a polynucleotide capable of hybridizing, and means an oligonucleotide capable of binding to the complementary strand of the nucleic acid in a sequence-specific manner. The probe of the present invention is an allele-specific probe, wherein a polymorphic site is present in a nucleic acid fragment derived from two members of the same species and hybridizes to a DNA fragment derived from one member but not to a fragment derived from another member Do not. In this case, the hybridization conditions show a significant difference in the hybridization intensity between the alleles, and it is preferable that the hybridization conditions are sufficiently strict so as to hybridize only to one of the alleles. This can lead to good hybridization differences between different allelic forms. The probe of the present invention can be used for a method of determining the level of amylase in the blood of a pig by detecting an allele. The determination method includes detection methods based on hybridization of nucleic acids such as Southern blots, and may be provided in a form pre-bonded to a substrate of a DNA chip in a method using a DNA chip.

The process of immobilizing the probe polynucleotide on the substrate associated with the determination of the level of amylase in the blood of the pig of the present invention can also be easily prepared using this conventional technique. In addition, hybridization of nucleic acids on a microarray and detection of hybridization results are well known in the art. The detection can be accomplished, for example, by labeling the nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent material, such as Cy3 and Cy5, and then hybridizing on the microarray and generating The hybridization result can be detected.

The present invention discloses SNP markers capable of judging the level of amylase in pig blood and can be used as a means of objectively evaluating amylase content affecting the health status of pigs, And thus can produce an excellent pig without metabolic disturbance, which is advantageous for effective breeding improvement.

FIG. 1 shows gene loci involved in amylase level in blood through GWAS (genome-wide association study) analysis in Jeju native pig and land race cross-reference group.
FIG. 2 shows the results of confirming AMY2 (amylase alpha 2B) gene located at 126.3 M in the QTL region.
FIG. 3 shows the structure of AMY2 (amylase alpha 2B) gene.
Fig. 4 shows the results of detecting the mutation in the intron 1 of the porcine AMY2 gene.
Figure 5 shows a sequence containing intron 1 of the porcine AMY2 gene associated with amylase content in blood.
6 shows the difference in amylase content in blood according to the difference in allele type due to base mutation of intron 1 of porcine AMY2 gene.
Fig. 7 shows the results of detecting the mutation in the exon 3 of the porcine AMY2 gene.
FIG. 8 shows the deletion of the 98th base in the exon 3 of the porcine AMY2 gene associated with the amylase content in blood and the sequence showing the mutable 99th base.
Figure 9 shows a sequence comprising the 3'-UTR of the porcine AMY2 gene associated with the amylase content in the blood.
Figure 10 shows that the alleles of A / A type, A / G type and G / G type are different according to the mutation of the 482nd base in the nucleotide sequence of SEQ ID NO: 3 including 3'-UTR of porcine AMY2 gene as A or G The results are shown in Fig.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  One. GWAS ( genome - wise association study ) In pig serum Amylase  Gene for content Seat  analysis

QTL analysis of the amylase content in the blood using the supersatellite marker and GWAS (genome-wide association study) analysis using the large-capacity SNP chip were performed in the reference group of Jeju native pig and land race.

Specifically, genotypes were read from 1100 pigs of 60k SNP Beadchip commercially available from illumina. Genotypes of all pig chromosomes were read, and this genotype was used for phenotype and association analysis. At this time, we used the plink program to derive the results by analyzing the association between SNPs and phenotypes assigned to each chromosome.

GWAS analysis showed that the amylase-related gene in the blood was present at the chromosome 4 end (Fig. 1).

In addition, when the list of the top 20 SNPs having high significance regarding the content of amylase in pig blood was extracted, the results are shown in Table 1 below.

The highest SNPs were observed at 126.7M of chromosome 4 on the chromosomal locus involved in amylase content in blood, and it was confirmed that the highest 20 SNPs appeared in the region of 120M to 127M when the SNPs were extracted. Amylase content in blood was the most significant in ASGA0022387 and it was found to be 126.7M.

In addition, AMY2 (amylase alpha 2B) gene was identified at 126.3M position in the QTL region and selected as a candidate gene. Through the complete mRNA sequencing of the gene, the porcine AMY2 gene was composed of 511 amino acids and exon exon) were all composed of 10 (Figs. 2 and 3). Furthermore, it was confirmed that nucleotide sequence variations appeared in the intron 1 region, the exon 3 region and the 3'-UTR region.

Example  2. Pig AMY2  Within the gene Intron  According to the variation in 1 Amylase  Confirmation of content change

The most significant SNP was ASGA0022387 in relation to the amylase content in blood in the reference group of Jeju native pig and land race, and was the highest at 126.7M position. AMY2 (amylase alpha 2B) was identified at 126.3M by screening the surrounding genes. As the amylase content and gene name coincided with the measured phenotype, the core gene was selected and the RNA sequence and DNA sequence were analyzed to decode the base sequence . The nucleotide sequence (SEQ ID NO: 1) is as shown in Fig.

Gene amplification primers were constructed to determine the relationship between the genotype and the amylase content in intron 1, using the base sequence of the AMY2 gene registered in GenBank as a template.

TG-ATG-TG-3 '(SEQ ID NO: 4): 5'-GGT-TGA-CAT-TGC-TCT-TGA-

5'-biotin-AGC-GGA-AAT-GTC-CTG-CTT-AAA-A- 3 '(SEQ ID NO: 5): reverse primer (AMY2_int1_R)

The sequence of intron 1 in AMY2 gene (AMY2_int1_Seq): 5'-TGC-CGT-GCT-TAG-AGT-AAA-AGG-3 '

In the sequence shown in FIG. 5, the portion indicated by red bold indicates the position of the primer, and the portion shown in red indicates the base mutation portion ([C / T]) for determining the type of muscle fiber. It is marked with a green underlined letter.

The content of amylase in the blood according to the genotype variation as described above was analyzed. As a result, the allele frequency was shown in Table 2 below.

In addition, when the average amylase content was determined according to the genotype in the base sequence of the porcine AMY2 gene shown in SEQ ID NO: 1 including the intron 1 region, the amylase content in the blood was 2806.19 ± 1033.07 U / L and 615.55 ± 501.07 U / L when the allele genotype was T / T (Table 3 and FIG. 6).

From the above results, it can be seen that the amylase content is high when the allele genotype is C / C type and the amylase content is low when it is T / T type according to whether the 177th base of the nucleotide sequence of SEQ ID NO: 1 shown in FIG. , And it was confirmed that the content of amylase in blood varies according to the base mutation at the above position.

Example  3. Pigs AMY2  Mutations in exon 3 in the gene Amylase  Confirmation of content change

The following primers were used to detect frame shift mutations in exon 3 through sequencing.

5'-biotin-TGA-AAT-GGA-ACC-TTT-GAC-TTT-CAG-3 '(SEQ ID NO: 7)

(SEQ ID NO: 8): 5'-TGC-TGA-GAA-GCA-CAG-ATA-GTT-CTT-G-

8, the reverse primer was prepared in the intron 3 region (SEQ ID NO: 2), the forward primer for analysis in the intron 2 region, and the reverse primer in the intron 3 region. Through analysis using the above primers, it was found that the 98th base in the exon 3 region was deleted and the 99th base was mutated to T or C.

As a result of the analysis of the average amylase content according to the genotype in the exon 3 region of the pig AMY2 gene as described above, when the allele genotype according to the 99th base mutation was G / G type, the average amylase content was 3022.03 ± 967.76 U / And the mean amylase content was low at 920.39 ± 920.58 U / L when the allele genotype was A / A type (Table 4).

From the above results, it was confirmed that the amylase content is high when the allele genotype is C / C type and the amylase content is low when T / T type, when the 98th base of exon 3 of SEQ ID NO: 2 is T or C, The content of amylase in the blood varies depending on the base mutation at the above position.

Example  4. Pig AMY2  The 3'- UTR  Depending on the variation in the region Amylase  Confirmation of content change

A primer capable of detecting base mutation in the 3'-UTR region in the porcine AMY2 gene was constructed as follows by carrying out a genotyping analysis using a Pyro-sequencer.

5'-TCG-CAA-ACA-ACA-GTT-TGT-CAG-3 '(SEQ ID NO: 9): forward primer (AMY2_3UF)

5'-Biotin-AAT-AAA-CCA-TGA-AAG-GGC-CTA-3 '(SEQ ID NO: 10)

The sequence of the 3'-UTR region in AMY2 gene (AMY2_3U_seq): 5'-GGC-CAT-GAA-CAC-AAT-CTG-CC-

In the nucleotide sequence of SEQ ID NO: 3 shown in FIG. 9, the region indicated by red letters is the region of exon 10, and the last TAA represents the stop codon. The primers for analysis in the 3'-UTR region after the termination codon were indicated by underlined blue lines in FIG.

As a result of the pyrosequencing analysis using the above primers, the mutation of the 482nd nucleotide in the nucleotide sequence of SEQ ID NO: 3 was A or G, A / G type and G / G type, respectively.

The present inventors also investigated the average amylase content of the porcine AMY2 gene according to the genotype in the 3'-UTR region. As a result, when the allele genotype according to the 482 base mutation was 2735.2 ± 1007.9 U / L , And the average amylase content was 98.6 ± 175.5 U / L when the allele was G / G type (Table 5).

Especially, the total average of 1080 offspring of second generation of reference group was 2538U, but the distribution according to genotype was 2735U for A / A genotype, 1861U for A / G genotype and 98.6U for G / G genotype. I could see a big thing.

From the above results, it can be seen that the 177th base is C or T in the base sequence of the porcine AMY2 gene represented by SEQ ID NO: 1 and the 98th base of the exon 3 region represented by SEQ ID NO: 2 is deleted The content of amylase differs depending on whether the 482nd nucleotide is A or G in the base sequence of the porcine AMY2 gene represented by SEQ ID NO: 3, wherein the 99th base is T or C or 3'-UTR.

In addition, it is possible to determine the level of amylase in the pig blood by analyzing the allelotype at the above position in the porcine AMY2 gene, and furthermore, by fixing the specific base, the amylase can be maintained at a normal level, It can be used for manufacturing and sorting.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Novel SNP marker for discriminating level of amylase within          porcine blood and use thereof <130> KPA140972-KR <160> 11 <170> Kopatentin 2.0 <210> 1 <211> 400 <212> DNA <213> porcine <220> <221> gene &Lt; 222 > (1) .. (400) <223> Y = C or T <400> 1 agtatgcccc acaaacccag tctggacgaa cgtctattgt ccatctgttt gaatggcgct 60 gggttgacat tgctcttgaa tgtgagcggt atttgggccc caaaggattt ggaggggtac 120 aggtgggtat ggttcgcagt ataaattgga gattttgccg tgcttagagt aaaaggyatt 180 gcgatctgtg tctgtgaagc tgggacgtta gttcacaggt taagtaggcg aagtaaaaca 240 gttttctgaa gaagaaacac acgtgaaatt gttggcaact cactgtttta tttctaatac 300 catatttttt aagcaggaca tttccgctgt gatattgata ttcttacctg gttgtaaaga 360 acggtggcag catatacaaa gattcagaaa agaaaaaaat 400 <210> 2 <211> 198 <212> DNA <213> porcine <220> <221> gene &Lt; 222 > (1) .. (198) <223> Y = C or T <400> 2 gtgcgtatat atgtggatgc tgtcattaat catatgtgtg gaagtggtgc agctgcagga 60 acgggcacca cttgtggaag ttattgcaac cctggaaaya gggagtttcc agcagtccca 120 tactctgctt gggattttaa cgatggtaaa tgtaaaactg caagtggagg aatcgagagc 180 tataatgatc cttatcaa 198 <210> 3 <211> 656 <212> DNA <213> porcine <220> <221> gene &Lt; 222 > (1) .. (656) <223> R = G or A <400> 3 gcaattatct tcaactttgc aaactggtct tcctggtggc acatactgtg atgttatctc 60 tggagataaa gttggtaaca gttgtacagg aattaaagtc tatgtttcca gtgatggcac 120 agctcagttt tctattagta actctgctga agatccattt attgcaattc atgctgaatc 180 caaattgtaa aatttgaatt aaatacatct tccaagagca ataagaagtg tttgtttcct 240 ttctttatgt atgcgacata taacttccta caattcatta gtttttggta aaagctatca 300 tcagcaataa acttggaaag ctcagaaaag catgaaaatg acatatatga ttataagatg 360 atcgcaaaca acagtttgtc aggtggcaga agactcacat tgacctcttt gtcaaaaaag 420 tttccacgaa ctcagagaag catgctgaca agtatttttt tggccatgaa cacaatctgc 480 cragctgtgt ctctctcatg tgtgcctgta tttcttccgt aaggtgtttc taacccttgg 540 gtaggggtag ctgcttttat gatgagtccc atacaaaggt ataaggtctg ggtgtttttc 600 acaatatata ggccctttca tggtttatta ggtaaaacaa taaaagacac taaaat 656 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> AMY2 intron1 forward primer <400> 4 ggttgacatt gctcttgaat gtg 23 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> AMY2 intron1 reverse primer <400> 5 agcggaaatg tcctgcttaa aa 22 <210> 6 <211> 21 <212> DNA <213> porcine <220> <221> gene <222> (1) <223> AMY2 Intron1 <400> 6 tgccgtgctt agagtaaaag g 21 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> AMY2 exon3 forward primer <400> 7 tgaaatggaa cctttgactt tcag 24 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AMY2 exon3 reverse primer <400> 8 tgctgagaag cacagatagt tcttg 25 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AMY2 3'UTR forward primer <400> 9 tcgcaaacaa cagtttgtca g 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AMY2 3 'UTR reverse primer <400> 10 aataaaccat gaaagggcct a 21 <210> 11 <211> 20 <212> DNA <213> porcine <220> <221> gene <222> (1) <223> AMY2 3'-UTR <400> 11 ggccatgaac acaatctgcc 20

Claims (20)

The polynucleotide derived from the AMY2 (amylase alpha 2B) gene consisting of the nucleotide sequence of SEQ ID NO: 1 is C or T at position 177, or the polynucleotide derived from the AMY2 gene consisting of the nucleotide sequence of SEQ ID NO: The base is deleted and the 99th base is C or the polynucleotide derived from the AMY2 gene consisting of the nucleotide sequence of SEQ ID NO: 3 is A or G at position 482,
An AMY2 gene-derived polynucleotide consisting of the 177th base of the AMY2 gene-derived polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, the 99th base of the AMY2 gene-derived polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: A composition for determining the level of amylase in the blood of pigs, comprising a preparation capable of detecting or amplifying a polynucleotide consisting of 5 to 1,000 consecutive bases comprising the 482 base of SEQ ID NO: 482 or a complementary polynucleotide thereof .
[Claim 4] The method according to claim 1, wherein the preparation comprises a primer pair consisting of a nucleotide sequence of SEQ ID NO: 4 and SEQ ID NO: 5 capable of amplifying a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 and a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: A primer pair consisting of the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8 and a pair of primers consisting of the nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 10 capable of amplifying the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;
The method according to claim 1,
Wherein the pig is a hybrid of Jeju native pig, land lace or Jeju native pig and land lace.
A kit for determining the level of amylase in a blood of a pig, comprising the composition of any one of claims 1 to 3.
(a) amplifying a polymorphic site of a polynucleotide consisting of the nucleotide sequence of any one of SEQ ID NOS: 1 to 3 or a complementary polynucleotide thereof from the DNA of the sample separated from the individual; And
(b) determining the base of the polymorphic site amplified in the step (a), wherein the level of amylase in the blood of the pig is determined.
6. The method of claim 5,
In the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, the allele of the 177th base, which is the polymorphic site, is all C, or the 98th base is deleted from the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, Wherein the allele of the base is all C or the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 is all the allele of the 482nd base, which is the polymorphic site, is judged to be an increase in the level of amylase in the blood of the pig. Way.
6. The method of claim 5,
In the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, the allele of the 177th nucleotide, which is the polymorphic site, is all T, or the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 has allele of the 99th base T or a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 is all G allele of the polymorphic site 482 base is judged to be decreased in the level of amylase in the blood of pigs.
6. The method of claim 5, wherein the pig is a hybrid of Jeju native pig, land lace or Jeju native pig and land lace.
The step of fixing the SNP trait of the gene involved in the level of amylase in the blood of pigs comprises fixing the allele of the 177th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 to C / T type, Of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: Or fixing the allele of the 482nd nucleotide of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 to the A / G form, the level of amylase in the blood of the pig is adjusted to the level of amylase in the blood of the normal pig &Lt; / RTI &gt;
10. The method of claim 9,
Wherein the step of immobilizing the SNP trait is carried out by crossing a subject having all of the SNP traits and selecting an individual having the trait of interest.
The step of fixing the SNP trait of the gene involved in the level of amylase in the blood of pigs comprises fixing the allele of the 177th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 to C / T type, Of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 is fixed to the T / C type, or alleles of the 482nd nucleotide of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 are fixed to the A / G type Wherein the level of amylase in the blood of the pig is maintained at an amylase level in the blood of the normal pig.
12. The method of claim 11,
Wherein the step of immobilizing the SNP trait is carried out by crossing a subject having all of the SNP traits and selecting an individual having the trait of interest.
In the polynucleotide derived from the AMY2 gene of SEQ ID NO: 1, the 177th base is C or T, or the 99th base is deleted from the AMY2 gene-derived polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, The base is C or the polynucleotide derived from AMY2 gene consisting of the nucleotide sequence of SEQ ID NO: 3 is A or G at position 482,
An AMY2 gene-derived polynucleotide consisting of the 177th base of the AMY2 gene-derived polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, the 99th base of the AMY2 gene-derived polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: Of a polynucleotide consisting of 5 to 1,000 contiguous bases comprising a 482 base of the 482 base or a preparation capable of detecting or amplifying a complementary polynucleotide of the polynucleotide or of a pancreatic islet, Pancreatic cancer, cholecystitis, salivary ganglion, renal failure, hepatitis, cirrhosis, obesity or diabetes.
14. The method according to claim 13, wherein said preparation comprises a primer pair consisting of a nucleotide sequence of SEQ ID NO: 4 and SEQ ID NO: 5 capable of amplifying a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 1, a polynucleotide comprising a nucleotide sequence of SEQ ID NO: A primer pair consisting of the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8 and a pair of primers consisting of the nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 10 capable of amplifying the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;
A kit for the diagnosis of hyperamylasemia of a pig, macro amylase, pancreatitis, pancreatic pseudocyst, pancreatic cancer, cholecystitis, salivary glanditis, renal failure, hepatitis, cirrhosis, obesity or diabetes mellitus comprising the composition of claim 13.
(a) amplifying a polymorphic site of a polynucleotide consisting of the nucleotide sequence of any one of SEQ ID NOS: 1 to 3 or a complementary polynucleotide thereof from the DNA of the sample separated from the individual; And
(b) determining the base of the polymorphic site amplified in the step (a). The method of claim 1, wherein the polymorphic site is selected from the group consisting of hyperamylasemia, macro amylase, pancreatitis, pancreatic pseudocyst, pancreatic cancer, cholecystitis, Methods for the diagnosis of cirrhosis, obesity or diabetes.
17. The method of claim 16,
In the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, the allele of the 177th nucleotide, which is the polymorphic site, is all C, or the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 is deleted from the polynucleotide, The polynucleotide comprising all the alleles of C or the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 is a polymorphic site in which the allele of the 482nd nucleotide is all A. In the case of alleles of Polynucleotide of Polynucleotide of Polynucleotide of Polynucleotide of SEQ ID NO: 3 or Polynucleotide of Polynucleotide of SEQ ID NO: &Lt; / RTI &gt;
17. The method of claim 16,
In the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, the allele of the 177th base, which is a polymorphic site, is all T, or the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 is all the allele of the 99th base , Or a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 is diagnosed as renal failure, hepatitis, liver cirrhosis, obesity or diabetes mellitus when the allele of the 482nd base, which is a polymorphic site, is all G.
A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, nucleotide 177 of SEQ ID NO: 2, nucleotide 99 of the polynucleotide of SEQ ID NO: 2, or nucleotide sequence of SEQ ID NO: 3, SNP markers for determining the level of my amylase.
19. A microarray for determining the level of amylase in the blood of a pig, comprising the SNP marker of claim 19.

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