JP2006067928A - Method for genetically diagnosing mastitis resistance of cow - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for surely and rapidly detecting and diagnosing mastitis resistance of a cow. <P>SOLUTION: A DNA containing a mutated site of a mastitis resistance gene of the cow and an oligonucleotide primer consisting of ≥10 nucleotides for amplifying a domain containing the mutated site capable of existing in the mastitis resistance gene of the cow by a gene amplification reaction are provided. The method for genetically diagnosing the mastitis resistance of the cow comprises a process (a): obtaining a nucleic acid specimen which is an object of the diagnosis, process, (b): obtaining nucleic acid fragments in which the domain containing the mutated site capable of existing in the mastitis resistance gene of the cow is amplified by subjecting the nucleic acid specimen obtained in the process (a) under a gene amplification reaction, and process (c): examining the presence of the mutation in the nucleic acid fragments obtained by the process (b). A kit used in the diagnostic method, a genetic diagnostic reagent for diagnosing the mastitis resistance, and a probe for examining the presence of the mutation capable of existing in the mastitis resistance gene are also provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a genetic diagnosis method for bovine mastitis resistance, and more particularly to a method for diagnosing a bovine mastitis resistance gene.

  Mastitis is an infectious disease found in milking cows such as Holstein breeds, and indigenous bacteria such as Streptococcus aureus invade from bovine papilla and cause inflammation in the mammary gland tissue. In general, susceptibility and resistance to infectious diseases have many genetic aspects, but since DNA information regarding mastitis has not been clarified, measures are taken exclusively from the aspect of feeding management.

Milk from cattle affected by mastitis is discarded as a commodity because it contains many bacteria. In addition, milking cows once suffering from mastitis have significant economic losses because they repeatedly develop mastitis. In Japan, the annual damage caused by mastitis is estimated at 60 billion yen.
Diagnosis of mastitis is performed by the diagnostic method described in Non-Patent Document 1, but means for diagnosing resistance is not known.
Therefore, in order to reduce the occurrence of mastitis in the future, it has been desired to develop a means for detecting (screening) and diagnosing mastitis-resistant cows simply, reliably and quickly.

“Bovine mastitis: clinical and effective treatment” by Tadayoshi Arita (Chikusan Publishing Co., Ltd.)

  Therefore, an object of the present invention is to provide a means for detecting and diagnosing bovine mastitis resistance simply, reliably and rapidly.

In order to achieve the above object, the present inventors considered that it is necessary to establish a method (detection method) for diagnosing bovine mastitis by genetic engineering.
Therefore, as a result of repeated research, the inventors successfully identified a mastitis resistance gene (FEZL gene), which is a gene closely related to bovine mastitis. Mastitis resistance is a quantitative trait involving multiple genes, one of which is the FEZL gene.
Analysis of the FEZL gene revealed that it encodes a transcription factor containing a zinc finger domain. In addition, the mutation of 3 bp insertion into the translation region of the FEZL gene resulted in the insertion of the glycine at the 107th amino acid residue at the 107th protein of the protein FEZL purified by transcribing and translating the gene. As a result, the transcriptional activity of FEZL From the decrease, it was found that this amino acid insertion mutation causes a decrease in mastitis resistance.
From these findings, if the presence or absence of a 3 bp insertion mutation in the FEZL gene is detected by PCR (polymerase chain reaction) method using an oligonucleotide primer containing a specific oligonucleotide primer set on the FEZL gene, mastitis resistance As a result, the present inventors have found that it is possible to easily, reliably and quickly diagnose sex.

That is, the present invention and its embodiments are shown below.
(1) DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing.
(2) The DNA according to claim 1, wherein the DNA is any one of a genomic DNA sequence and a cDNA sequence.
(3) The DNA according to claim 1 or 2, wherein the DNA is either a synthetic sequence or a semi-synthetic sequence.

(4) 10 or more nucleotides for amplifying a region containing a mutation site that may be present in a bovine mastitis resistance gene represented by the following (a), (b) or (c) by a gene amplification reaction An oligonucleotide primer comprising:
(A) An oligonucleotide primer comprising the whole or a part of the base sequence shown in SEQ ID NO: 1 in the sequence listing or its complementary strand.
(B) A region containing a mutation site that may be present in a bovine mastitis resistance gene is amplified by a gene amplification reaction by hybridizing at 50 to 60 ° C. with the base sequence shown in SEQ ID NO: 1 in the sequence listing or its complementary strand. Oligonucleotide primer consisting of any base sequence that can be used for
(C) An oligonucleotide primer comprising a base sequence derived from the sequences (a) and (b) for degeneracy of the gene code.
(5) (a) an oligonucleotide having a base sequence corresponding to an arbitrary region of the base sequence shown in SEQ ID NO: 1 in the sequence listing; and (b) a base sequence shown in SEQ ID NO: 1 in the sequence listing. The oligonucleotide primer according to claim 4, wherein the oligonucleotide primer is selected from the group consisting of oligonucleotides having a complementary base sequence for an arbitrary region.
(6) (a) an oligonucleotide having a base sequence corresponding to the region on the 5 ′ end side of the base sequence shown in SEQ ID NO: 1 in the sequence listing; and (b) shown in SEQ ID NO: 1 in the sequence listing. 6. The oligonucleotide primer according to claim 4, wherein the oligonucleotide primer is selected from the group consisting of oligonucleotides having a complementary base sequence to the 3′-end region of the base sequence.
(7) The oligonucleotide primer according to any one of claims 4 to 6, wherein the oligonucleotide primer comprises an oligonucleotide consisting of 10 to 50 nucleotides.
(8) The oligonucleotide primer according to any one of claims 4 to 7, wherein the oligonucleotide primer comprises an oligonucleotide consisting of 15 to 35 nucleotides.
(9) It is selected from the group consisting of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 of the sequence listing and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 of the sequence listing. The oligonucleotide primer in any one of.

(10) Step (a): A step of obtaining a bovine nucleic acid sample to be diagnosed,
Step (b): A step of subjecting the nucleic acid sample obtained in step (a) to a gene amplification reaction to obtain a nucleic acid fragment in which a region containing a mutation site that may be present in a bovine mastitis resistance gene is amplified. And (c): a method for genetic diagnosis of bovine mastitis resistance, comprising the step of examining the nucleic acid fragment obtained in step (b) for the presence of mutation.
(11) The genetic diagnosis method according to claim 10, wherein the region containing a mutation site that may be present in a bovine mastitis resistance gene is a region containing the base sequence represented by SEQ ID NO: 1 in the sequence listing.
(12) The gene diagnosis method according to claim 10 or 11, wherein the gene amplification reaction is performed by a polymerase chain reaction method.
(13) The genetic diagnosis method according to any one of (10) to (12), wherein the presence of the mutation is examined by detecting a nucleic acid fragment length type amplified by a gene chain reaction.
(14) The genetic diagnosis method according to any one of claims 10 to 13, wherein the nucleic acid sample is a sample containing genomic DNA, cDNA, or mRNA.
(15) The gene diagnosis method according to any one of claims 10 to 14, wherein a gene amplification reaction is performed using the oligonucleotide primer according to any one of claims 4 to 9.

(16) A kit for diagnosing bovine mastitis resistance, comprising the oligonucleotide primer according to any one of claims 4 to 9.
(17) A genetic diagnostic reagent for diagnosis of bovine mastitis resistance, comprising the oligonucleotide primer according to any one of claims 4 to 9.

(18) a mutation that may be present in a bovine mastitis resistance gene comprising a region containing a mutation site that may be present in a bovine mastitis resistance gene, comprising 56 to 79 nucleotides and labeled at the end; Probe to check for existence.
(19) A method for elucidating the base sequence of a region containing a mutation site that may be present in a bovine mastitis resistance gene and / or isolating the region using the probe according to claim 18.

  According to the present invention, a mastitis resistance gene having a mutation site related to mastitis resistance reduction is provided, and mastitis resistance is genetically simply, reliably and rapidly using the gene. Oligonucleotides, genetic diagnostic methods, and probes for diagnosis and detection are provided.

  Hereinafter, the present invention will be described in detail.

[1] Bovine mastitis resistance gene (FEZL gene)
In order to investigate genes related to bovine mastitis resistance, the present inventors conducted a genome linkage analysis of families including mastitis resistant cattle and susceptible cattle, and used a method called positional cloning method. A bovine mastitis resistance gene (FEZL gene) was isolated. Here, the positional cloning method is a method of mapping a causal locus on a linkage map and then isolating the causative gene from its chromosomal region. Details of this method are described in the Animal Genetics Breeding Dictionary (Japan Society for Animal Husbandry Technology). Issuance).

The entire base sequence of cDNA encoding the bovine FEZL gene is as shown in SEQ ID NO: 1 in the sequence listing. This determination of the base sequence (sequencing) can be performed by a chemical decomposition method (Maxam & Gilbert method), a chain terminator method (Sanger dideoxy method), or the like.
The DNA consisting of the base sequence shown in SEQ ID NO: 1 in this sequence listing is a translation region of the bovine FEZL gene and a region encoding the bovine protein FEZL. In the case of mastitis-sensitive cattle, a mutation was found in which a 3 bp portion was inserted at position 319 in the base sequence shown in SEQ ID NO: 1 in the sequence listing (FIG. 1).

Since the DNA consisting of the base sequence shown in SEQ ID NO: 1 in the Sequence Listing is a part of the genomic DNA on the gene that causes a decrease in resistance to mastitis, using the mutation on the DNA, By comparing the base sequence of the FEZL gene from the tissue, it can be determined whether it is mastitis resistant or sensitive.
Further, as described in [2] below, the DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing amplifies a region containing a mutation site that may be present in a bovine mastitis resistance gene by a gene amplification reaction. It can be used as an oligonucleotide primer.
Further, as described in [3] below, it can also be used in a genetic diagnosis method (detection method) for mastitis resistance based on a mutation on DNA consisting of the base sequence shown in SEQ ID NO: 1 in the Sequence Listing.
In this case, the DNA may be any of a genomic DNA sequence and a cDNA sequence, or any of a synthetic sequence and a semi-synthetic sequence.

[2] Oligonucleotide primer In the present invention, the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the above sequence listing amplifies a region containing a mutation site that may be present in a bovine mastitis resistance gene by a gene amplification reaction. This can be used to design oligonucleotide primers for the purpose.
As such an oligonucleotide primer, it is represented by the following (a), (b) or (c), for amplifying a region containing a mutation site which may be present in a bovine mastitis resistance gene by a gene amplification reaction An oligonucleotide primer characterized by comprising 10 or more nucleotides can be mentioned.
(A) An oligonucleotide primer comprising the whole or a part of the base sequence shown in SEQ ID NO: 1 in the sequence listing or its complementary strand.
(B) A region containing a mutation site that may be present in a bovine mastitis resistance gene is amplified by a gene amplification reaction by hybridizing at 50 to 60 ° C. with the base sequence shown in SEQ ID NO: 1 in the sequence listing or its complementary strand. Oligonucleotide primer consisting of any base sequence that can be used for
(C) An oligonucleotide primer comprising a base sequence derived from the sequences (a) and (b) for degeneracy of the gene code.

  As such an oligonucleotide primer, (a) an oligonucleotide having a base sequence corresponding to an arbitrary region of the base sequence represented by SEQ ID NO: 1 in the sequence listing, desirably a region on the 5 ′ end side And (b) one selected from the group consisting of oligonucleotides having a complementary base sequence to any region of the base sequence shown in SEQ ID NO: 1 in the sequence listing, preferably the region on the 3 ′ end side, particularly At least one of each of the above (a) and (b) can be used.

  Here, in the present specification, “oligonucleotide” means a relatively short single-stranded or double-stranded polynucleotide, preferably polydeoxynucleotide, and known methods such as triester method, phosphite, etc. It can be chemically synthesized by a method such as a method, a phosphoamidite method, a phosphonate method. It is known that synthesis can usually be performed conveniently on a modified solid support, for example, using commercially available automated synthesizers. In addition, the synthesis of the oligonucleotide can be performed by outsourcing to others. The oligonucleotide may contain one or more modified bases, for example, a non-naturally occurring base such as inosine, or a tritylated base.

In the present invention, the number of nucleotides in the oligonucleotide primer can be 10 or more, preferably 15 or more. The upper limit of the number of nucleotides is desirable because the longer the specificity, the higher the specificity. However, in view of the cost of synthesis, it can be 50 or less, more preferably 35 or less.
As such an oligonucleotide primer, specifically, for example, a group consisting of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 of the sequence listing and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 of the sequence listing More specifically, an oligonucleotide (FEZL-F) consisting of the base sequence shown in SEQ ID NO: 2 in the sequence listing and an oligonucleotide (FEZL-F) consisting of the base sequence shown in SEQ ID NO: 3 in the sequence listing ( The combination of both of FEZL-R) can be mentioned. FEZL-F is an oligonucleotide corresponding to a base sequence consisting of a region on the 5 ′ end side of the base sequence shown in SEQ ID NO: 1 in the Sequence Listing (the portion surrounded by the upper square in FIG. 1). An oligonucleotide corresponding to a base sequence composed of a complementary base sequence for the region on the 3 ′ end side of the base sequence shown in SEQ ID NO: 1 in the Sequence Listing (the part enclosed by the lower square in FIG. 1).

[3] Genetic diagnosis method for bovine mastitis resistance (detection method)
Bovine mastitis resistance can be genetically used for diagnosis and detection by utilizing a mutation on DNA consisting of the base sequence shown in SEQ ID NO: 1 in the Sequence Listing.
As a genetic diagnosis method (detection method) of such bovine mastitis resistance,
Step (a): obtaining a bovine nucleic acid sample to be diagnosed,
Step (b): A step of subjecting the nucleic acid sample obtained in step (a) to a gene amplification reaction to obtain a nucleic acid fragment in which a region containing a mutation site that may be present in a bovine mastitis resistance gene is amplified. And step (c): an embodiment comprising a step of examining the presence of mutation in the nucleic acid fragment obtained in step (b) is exemplified.

First, step (a) will be described. In step (a), a bovine nucleic acid sample to be diagnosed is obtained.
The bovine nucleic acid sample used in the present invention is particularly limited as long as it has a base sequence encoding the protein FEZL purified by transcribing and translating the bovine mastitis resistance gene (FEZL gene). Rather, nucleic acids derived from appropriate cells or tissues (including total genomic DNA and cDNA transcribed from total cellular RNA), such as genomic DNA, cDNA (DNA transcribed from total cellular RNA), Examples include mRNA. The bovine nucleic acid sample is prepared by a known method such as Molecular cloning, a laboratory manual (3rd edition), J. Org. Sambrook & D. W. Russell (Ed.), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001).

Second, step (b) will be described. In step (b), a nucleic acid fragment obtained by subjecting the nucleic acid sample obtained in step (a) to a gene amplification reaction to amplify a region containing a mutation site that may be present in a bovine mastitis resistance gene is obtained. obtain.
The gene amplification reaction used in this step (b) is not particularly limited as long as it is a method capable of amplifying a region containing a mutation site that may be present in a bovine mastitis resistance gene, but the polymerase chain reaction method ( PCR method), nucleic acid amplification methods such as nucleic acid amplification methods using RNA polymerase and strand displacement amplification methods can be used, and among these, the PCR method is preferably used.
As a region containing a mutation site that may be present in the bovine mastitis resistance gene to be amplified, a region containing a mutation that causes a decrease in bovine mastitis resistance in the base sequence of the bovine FEZL gene, specifically Specifically, if it is a region containing DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing, the exon part of the translation region encoding the protein FEZL, the exon part of the adjacent non-translation region, those An intron portion and a region involved in the control of the expression of the gene may be included.

  In the present specification, the “polymerase chain reaction” or “PCR method” generally refers to a method as described in US Pat. No. 4,683,195, for example, a desired base sequence is enzymatically converted in vitro. Refers to a method for amplification. In general, the PCR method includes repeating the cycle of performing primer extension synthesis using two primers that can preferentially hybridize with a template nucleic acid. Typically, in the PCR method, a primer complementary to the base sequence of the region to be propagated inside the template can be used, for example, the nucleotide sequence to be amplified is complementary at both ends. Some or those adjacent to the nucleotide sequence to be amplified can be preferably used.

  The PCR method is described in R.A. K. Saiki et al. , Science, 239, 487-491 (1988); A. Frohman et al. , Proc. Natl. Acad. Sci. USA, 85, 8998-9002 (1988), etc., or a modification or modification thereof. In addition, the PCR method can be carried out using a commercially available kit suitable for the PCR method, and in that case, it can also be carried out according to a protocol clarified by the kit manufacturer or the kit distributor.

In a gene amplification reaction such as PCR, a primer used for amplifying a region containing a mutation site that may be present in a bovine mastitis resistance gene by a gene amplification reaction is the oligonucleotide primer described in [2] above. It is preferable to use it.
The conditions for gene amplification reaction such as PCR are not particularly limited as long as they can amplify a region containing a mutation site that may be present in a bovine mastitis resistance gene, and may be known conditions that are usually performed. For example, it can be selected with reference to the description in the literature. For example, in the PCR method, one cycle consisting of heat denaturation of a DNA strand, annealing of a primer, and synthesis of a complementary strand by a polymerase is repeated a plurality of times, usually 10 to 80 times, preferably 20 to 35 times.

Third, step (c) will be described. In step (c), the nucleic acid fragment obtained in step (b) is examined for the presence of mutation.
The method for detecting the presence of the mutation is not particularly limited, but a method for determining the nucleotide sequence using the nucleic acid fragment itself amplified by the gene amplification reaction in the above step (b) as a template, or an amplified nucleic acid is suitable. A method for determining a nucleotide sequence by cloning into a simple vector and a detection method for examining a nucleic acid fragment length type can be used, and a detection method for detecting and examining a nucleic acid fragment length type is particularly preferred.
As a method for examining the length of a nucleic acid fragment, a nucleic acid fragment is separated by electrophoresis on a polyacrylamide gel or an agarose gel, for example, based on the mobility of a marker DNA fragment of a known molecular weight based on its mobility. The method of identifying the DNA fragment of is preferred. Furthermore, an appropriate DNA fragment that contains a region containing a mutation site that may be present in a bovine mastitis resistance gene or that corresponds to the region, preferably hybridizable under conditions of 50 to 60 ° C., is used as a probe. A known mutation detection method such as a hybridization method can also be used.

Oligonucleotide primers, probes, etc. can be labeled with a label component to facilitate detection. The label component can be detected by spectroscopic means, optical means, biochemical means, immunological means, enzymatic chemistry means, radiochemical means and the like. Examples of the label component include enzymes such as peroxidase and alkaline phosphatase, isotopes such as ³² P, biotin, fluorescent dyes, luminescent substances, and coloring substances.
Such a probe includes a region containing a mutation site that may be present in a bovine mastitis resistance gene, is composed of 56 to 79 nucleotides, and ends (both ends or one end) are labeled with the label component described above. What is made can be used.
In addition to the genetic diagnosis method of the present invention, such a probe can be used for clarifying a sequence containing a mutation that may be present in the FEZL gene, or for isolating a region containing the mutation. Is possible.

According to the bovine mastitis resistance genetic diagnosis method (detection method) of the present invention, mastitis resistance and the degree of sensitivity can be diagnosed quickly and accurately.
The oligonucleotide primer described in the above [2] includes a kit for diagnosing bovine mastitis resistance in combination with the probe exemplified in the above step (c), and a bovine mastitis resistance diagnosing gene. It can be put to practical use as a diagnostic reagent.
The bovine mastitis resistance referred to in the present invention means that it is genetically resistant to mastitis, and it should be interpreted in a broad sense to include carriers regardless of the presence or absence of symptoms.

EXAMPLES Hereinafter, although this invention is demonstrated further in detail with an Example and an experiment example, this invention is not limited by these.
In the examples, J.I. Sambrook & D. W. The method described in Russell (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001), or a modified and / or modified method thereof Shall. In addition, when using commercially available reagent kits or measuring devices, unless otherwise explained, it is assumed that the test was performed according to the instructions of the attached protocol.

Example 1
In Example 1, the base sequence of the bovine FEZL gene was determined, and the relationship with disease was examined by expressing the gene.
(1) Determination of base sequence of FEZL gene of mastitis resistant cow and susceptible cow First, mastitis resistant cattle (297 cattle) and susceptible cattle (181 cattle) from a group of milking cows who are granddaughters of a specific Holstein bull. The region of bovine chromosome linked to mastitis resistance (mastitis resistance region) was examined using a polymorphic DNA marker based on the positional cloning method.
That is, first, among the DNA markers on the linkage map, the marker that was most strongly linked to mastitis resistance was isolated, and as a result, the mastitis resistance region was determined to be chromosome 22.
From a separately prepared bovine artificial bacterial chromosome (BAC) library, 35 BAC clones existing in the mastitis resistant region were isolated. Genome Analysis: Genetic and Physical Mapping "Kay E. Davis & Shirley M. Tilghman editors (Cold Spring Harbor Laboratory Press) developed many DNA markers, Terwilliger, JD (1995) A powerful likelihood method for the analysis The DNA marker most strongly linked to mastitis resistance was searched for by the method of linkage disequilibrium between trait loci and one or more polymorphic marker loci.Am.J.Hum.Genet.56, 777-787. The flame resistant region could be narrowed and up to 5 candidate genes could be narrowed down.

  In order to decipher the base sequence of the region encoding the protein of these candidate genes, the entire base sequence was determined by reading the sequence of the BAC clone containing the region. When this was compared between mastitis resistant cattle and susceptible cattle, among the candidate genes, a part of the cDNA of the FEZL gene (position 319 in the base sequence described in SEQ ID NO: 1 in the sequence listing) Mutations were found in which 3 bp encoding specific amino acids were inserted. Hereinafter, a gene having an amino acid insertion mutation is sometimes referred to as a sensitive genotype, and a gene having no mutation is sometimes referred to as a resistance genotype.

(2) Expression of FEZL gene In which tissue the FEZL gene is expressed, homozygous mastitis-resistant cattle (susceptibility genotype homozygous individuals) and mastitis-susceptible / resistant cattle heterozygous individuals (sensitivity / resistance genes) Whether or not there is a difference in the expression level of the gene with the heterozygous type).
Using fresh brain, kidney, lung, and mammary gland tissues collected from cows (one homozygous homozygous individual) as a material, total RNA was prepared using Trizol (manufactured by Invitrogen), a reagent for RNA preparation. . Using this total RNA fraction as a template, quantitative PCR for FEZL gene expression was performed using TaqMan reagent (Applied Biosystems) and Genetic Analyzer HT-7900 (Applied Biosystems).
As a result, it was found that the FEZL gene was expressed in the brain, kidney, and mammary gland. In particular, expression in the mammary gland was observed, suggesting a relationship with mastitis resistance.
Further, when the expression level of the FEZL gene in the mammary gland tissues of the susceptibility genotype homozygous and the susceptibility / resistance genotype heterozygous individual was examined, no difference was found between the two.

(3) Effect of FEZL gene function and amino acid insertion mutation It was investigated whether there was a difference in the expression of the FEZL gene due to the function of the FEZL gene and the amino acid insertion mutation.
First, a construct in which a HIS tag is linked to cDNA of FEZL gene (susceptible genotype and resistant genotype) is put into a vector (manufactured by Invitrogen), cell line OCUB-M derived from human breast cancer and green monkey kidney origin It was introduced into the cell line COS-7 and expressed. Each cell into which the FEZL gene was introduced was fixed in formalin according to a conventional method, and a chromatin immunoprecipitation assay was performed with a CHIP assay kit (manufactured by Upstate) to determine the FEZL consensus element, which is the base sequence on the genome recognized by FEZL. . The luciferase gene construct connected with the genomic fragment containing this element is introduced into the above cell line together with the FEZL gene resistance genotype and sensitive genotype cDNA construct, and the expression of the luciferase gene is measured for the activity of the luciferase enzyme Quantified with.
As a result, it was found that the transcriptional activity of the susceptibility genotype of FEZL was significantly lower than that of the resistance genotype. From this, it was considered that an amino acid insertion mutation in the FEZL gene resulted in a decrease in transcriptional activity, resulting in a decrease in mastitis resistance.

Example 2
Whether there was an insertion mutation on the FEZL gene on bovine genomic DNA was confirmed.
First, oligonucleotide primers FEZL-F and FEZL-R were synthesized to amplify a DNA fragment containing a 3 bp insertion site found in mastitis-sensitive cattle. The base sequences of the primers FEZL-F and FEZL-R are as shown in SEQ ID NO: 2 and SEQ ID NO: 3 respectively in the sequence listing, and both are designed from a part of the FEZL gene (see FIG. 1). .

  On the other hand, as a nucleic acid sample of cattle (220 susceptibility genotype homozygous individuals and 92 susceptibility genotype homozygous individuals and susceptibility / resistance genotype heterozygous individuals), which are diagnostic controls, (Including EDTA and heparin), each genomic DNA was prepared using an automatic nucleic acid separator Na-1000 (manufactured by Kurabo Industries). DNA preparation is described in Molecular cloning, a laboratory manual (3rd edition), J. MoI. Sambrook & D. W. Russell (Ed.), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001).

Using these genomic DNAs as templates, PCR was performed using primers FEZL-F and FEZL-R. That is, using La Taq (manufactured by Takara Bio Inc.), a reaction of 60 cycles was performed with a process consisting of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 minute as one cycle.
The nucleotide sequences of the nucleic acid fragments (PCR products) obtained by PCR were compared using a 3700 fluorescent DNA sequencer (Applied Biosystems) to examine the presence of mutations. The result of the mutated portion of each PCR product is shown in FIG.

As is clear from FIG. 2, the 69th base of the DNA fragment amplified by PCR using the resistance genotype of the FEZL gene derived from a resistant bovine (resistant genotype homozygous individual) is adenine (A, When the susceptibility genotype derived from susceptible cattle (susceptible genotype homozygous individuals) was used as a template, the 69th base was cytosine because 3 bp was inserted at the 67th position. (C, susceptibility genotype).
From this, it was clarified that the resistance genotype and the sensitive genotype of the FEZL gene can be distinguished by comparing the mutation site (the 69th base) that may exist in the FEZL gene.
In addition, as described above: Step (a) for obtaining a bovine nucleic acid sample to be diagnosed, the nucleic acid sample obtained in step (a) is subjected to a gene amplification reaction and is present in the bovine mastitis resistance gene. Genetic diagnosis can be performed accurately by the step (b) of obtaining a nucleic acid fragment in which a region containing a possible mutation site is amplified, and the step (c) of examining the presence of mutation in the nucleic acid fragment obtained in the step (b) Became clear.

  According to the present invention, a mastitis resistance gene having a mutation site related to mastitis resistance reduction is provided, and mastitis resistance is genetically simply, reliably and rapidly using the gene. Oligonucleotides, genetic diagnostic methods, and probes for diagnosis and detection are provided.

It is a figure which shows the example of a design of the variation part and primer of a mastitis resistance gene (FEZL gene). It is a figure which shows the presence of the insertion mutation in a FEZL gene.

Explanation of symbols

In FIG. 1, the part enclosed by a square shows the part used as the basis of primer design (the upper stage is FEZL-F, the lower stage is FEZL-R). The arrow indicates the direction of the primer from 5 'to 3'.
In FIG. 2, the upper (a) shows a resistance genotype PCR product, and the lower (b) shows a sensitive genotype PCR product.

Claims (19)

  DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing.   The DNA according to claim 1, wherein the DNA is any one of a genomic DNA sequence and a cDNA sequence.   The DNA according to claim 1 or 2, wherein the DNA is either a synthetic sequence or a semi-synthetic sequence. It is represented by the following (a), (b) or (c), and consists of 10 or more nucleotides for amplifying a region containing a mutation site that may be present in a bovine mastitis resistance gene by a gene amplification reaction. An oligonucleotide primer characterized by
(A) An oligonucleotide primer comprising the whole or a part of the base sequence shown in SEQ ID NO: 1 in the sequence listing or its complementary strand.
(B) A region containing a mutation site that may be present in a bovine mastitis resistance gene is amplified by a gene amplification reaction by hybridizing at 50 to 60 ° C. with the base sequence shown in SEQ ID NO: 1 in the sequence listing or its complementary strand. Oligonucleotide primer consisting of any base sequence that can be used for
(C) An oligonucleotide primer comprising a base sequence derived from the sequences (a) and (b) for degeneracy of the gene code. (A) an oligonucleotide having a base sequence corresponding to any region of the base sequence shown in SEQ ID NO: 1 in the sequence listing; and (b) any of the base sequences shown in SEQ ID NO: 1 in the sequence listing. The oligonucleotide primer according to claim 4, wherein the oligonucleotide primer is selected from the group consisting of oligonucleotides having a complementary base sequence to the region. (A) an oligonucleotide having a base sequence corresponding to the 5 ′ end region of the base sequence shown in SEQ ID NO: 1 in the sequence listing, and (b) a base sequence shown in SEQ ID NO: 1 in the sequence listing 6. The oligonucleotide primer according to claim 4, wherein the oligonucleotide primer is selected from the group consisting of oligonucleotides having a complementary base sequence for the region on the 3 ′ end side.   The oligonucleotide primer according to any one of claims 4 to 6, wherein the oligonucleotide primer is composed of an oligonucleotide consisting of 10 to 50 nucleotides.   The oligonucleotide primer according to any one of claims 4 to 7, which comprises an oligonucleotide consisting of 15 to 35 nucleotides.   9. The method according to any one of claims 4 to 8, which is selected from the group consisting of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 of the sequence listing and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 of the sequence listing An oligonucleotide primer according to 1. Step (a): obtaining a bovine nucleic acid sample to be diagnosed,
Step (b): A step of subjecting the nucleic acid sample obtained in step (a) to a gene amplification reaction to obtain a nucleic acid fragment in which a region containing a mutation site that may be present in a bovine mastitis resistance gene is amplified. And (c): a method for genetic diagnosis of bovine mastitis resistance, comprising the step of examining the nucleic acid fragment obtained in step (b) for the presence of mutation.   The genetic diagnosis method according to claim 10, wherein the region containing a mutation site that may be present in a bovine mastitis resistance gene is a region containing the base sequence represented by SEQ ID NO: 1 in the Sequence Listing.   The gene diagnosis method according to claim 10 or 11, wherein the gene amplification reaction is performed by a polymerase chain reaction method.   The genetic diagnosis method according to any one of claims 10 to 12, wherein the presence of the mutation is examined by detecting a nucleic acid fragment length type amplified by a gene chain reaction.   The genetic diagnosis method according to any one of claims 10 to 13, wherein the nucleic acid sample is a sample containing genomic DNA, cDNA, or mRNA.   The gene diagnostic method according to any one of claims 10 to 14, wherein a gene amplification reaction is performed using the oligonucleotide primer according to any one of claims 4 to 9.   A kit for diagnosing bovine mastitis resistance, comprising the oligonucleotide primer according to any one of claims 4 to 9.   A genetic diagnostic reagent for diagnosing bovine mastitis resistance, comprising the oligonucleotide primer according to any one of claims 4 to 9.   A region containing a mutation site that may be present in a bovine mastitis resistance gene, comprising 56 to 79 nucleotides and end-labeled for the presence of a mutation that may be present in a bovine mastitis resistance gene Probe for. A method for elucidating the base sequence of a region containing a mutation site that may be present in a bovine mastitis resistance gene and / or isolating the region using the probe according to claim 18.

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