JP2004329086A - Method for discriminating yeast for brewing using yil169c gene, yol155c gene or muc1 gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for distinguishing yeast, in detail, a method for discriminating yeast for brewing by using YIL169C gene, YOL155C gene or MUC1 gene. <P>SOLUTION: The genome DNA of yeast for brewing is amplified by using a primer for amplifying a part or the whole of YIL169C gene, YOL155C gene or MUC1 gene by a PCR method. The amplified gene fragment is treated by direct agarose gel electrophoresis or with a restriction enzyme (e.g. Rsal), then subjected to agarose gel electrophoresis and the yeast is discriminated by difference of electrophoretic patters. The accurate classification and discrimination of yeast for brewing are rapidly and readily carried out. The discrimination method has high reproducibility, provides a result in an extremely short period, not only discriminates different uses of yeast such as wine yeast from yeast of refined rice wine but also discriminates yeast of the same use of wine such as wine No.1 yeast from wine No.3 yeast of the association. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００３０】
なお、表中、各記号はそれぞれ次のことを表わす。
Ｋ２： 鹿児島酵母
ＳＨ４： 協会焼酎酵母
Ｍｋ： 宮崎酵母
Ａｗ： 泡盛酵母
ブ１： ブドウ酒用協会１号酵母
ブ３： ブドウ酒用協会３号酵母
ブ４： ブドウ酒用協会４号酵母
【００３１】
上記から明らかなように、プライマーの組み合わせを各種選択し、増幅されるＤＮＡ断片の有無、及び／又は増幅されるＤＮＡ断片の長さを各種検討することによって、各酵母を判別することができる。
【００３２】
【発明の効果】
本発明によれば、小仕込試験、薬剤や培養条件を変えて判別する等従来の方法に比して、短時間に判別できるという著効が奏され、しかも明確に判別することができ、安定的な結果が得られ、再現性を有するものであり、操作も簡単という著効が奏される。
【００３３】
更に本発明によれば、異なった醸造用酵母間の判別はもとより、非常に困難でデリケートな同一の醸造用酵母間の判別、例えばブドウ酒酵母間の判別も可能であって、本発明は、酒類製造業、試験研究機関等において、短期間に簡易にして正確な酵母の分類・同定・判別を可能とするものであり、野生酵母ともろみ中の酵母の明確な判別も可能である。
【００３４】
【配列表】

【図面の簡単な説明】
【図１】プライマーＡ〜Ｏの塩基配列を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to discrimination of yeast, and more particularly, to discrimination, classification, and identification of yeast for brewing.
[0002]
[Prior art]
As brewer's yeast, several types of yeast such as wine yeast, sake yeast, and shochu yeast are used. Further, among these yeasts, for example, as a wine yeast, for example, Association Wine Yeast No. 1, No. 3, No. 4, etc. are used, and sake yeast is also used by Association No. 7, No. 9, AW No. 10, and shochu yeast is also used by Shochu Yeast SH4, Kagoshima Yeast K2, Miyazaki Yeast MK, Awamori Yeast, etc. Have been.
[0003]
However, all of these yeasts for brewing are classified into Saccharomyces cerevisiae, belong to the same species, and are used for brewing. It is extremely difficult to accurately and accurately distinguish between the yeasts in a short time. Therefore, at this time, the yeast must be distinguished by a method such as performing a small preparation test on the yeast and discriminating it from its brewing characteristics, or by discriminating the yeast by changing the drug and culture conditions. Is the fact. However, these discrimination methods require actually culturing the yeast or actually preparing the yeast and confirming its brewing characteristics, and it is inevitable that a considerable time is required for the discrimination. Are required to be improved.
[0004]
On the other hand, as genes derived from Saccharomyces cerevisiae S288C strain, the MUC1 gene, YIL169C gene, and YOL155C gene are known per se, and although the MUC1 gene is involved in the cohesiveness of yeast, the other two genes are either However, its function is not clear (for example, see Non-Patent Document 1).
[0005]
As for the primer, the LA-1 primer is known, but the determination of yeast using this is not performed (for example, see Non-Patent Document 2). No successful example has been reported other than Japanese Patent Application No. 2002-45773 (filed on Feb. 22, 2002) previously filed by the present inventors.
[0006]
[Non-patent document 1]
Lye, G .; , Bowan, S.A. Churcher, C .; , Internet <URL: http: // genome-www. Stanford. edu / Saccharomyces / >>
[0007]
[Non-patent document 2]
Miguel B. L. Alison, S.M. Paul, A. H, and Peter L. Applied and Environmental Microbiology, 1996, p. 4514-4520
[0008]
[Problems to be solved by the invention]
Thus, establishment of an accurate and simple yeast discrimination system in a short period of time has been an important issue in actual liquor production sites and also in research laboratories. In view of the current state of the art, the present invention is not only between yeasts of different uses such as wine yeast and shochu yeast, wine yeast and sake yeast, but also very delicate and difficult to distinguish, for example, Association Grape Even among yeasts of the same use, such as sake yeast No. 1, No. 3, and No. 4, a new technical task, which is extremely difficult to solve, is to be developed, which is a system that can accurately and easily determine them in a short period of time. The present inventors have succeeded in solving this technical problem, and have just filed a patent application for the result (Japanese Patent Application No. 2002-45773). It has been made for the purpose of further improvement.
[0009]
[Means for Solving the Problems]
The present invention has been made in order to solve the above-mentioned problems. First, the outline of the above-mentioned prior invention relating to the development of the present inventors is as follows.
[0010]
That is, the prior application invention was made by paying attention to a method using a gene, and the present inventors have already reported Non-Patent Document 2 (Miguel. BL AIson, S. Paul). , AH, nd Peter L. Applied and Environmental Microbiology (1996) pp. 4514-4520) and amplified by PCR method using genomic DNA of Shochu yeast using LA-1 primer (gcgacgggtgtactaac). Characteristic was observed in the band of the amplified DNA in the vicinity. When this DNA fragment was further cloned and its nucleotide sequence information was determined, it was found for the first time that it was a part of the YIL169C gene. Focusing on this new finding, the present inventors have further studied and found that the YIL169C gene is different in brewery yeast.
[0011]
As described above, the invention of the prior application has discovered that the YIL169C gene is different in brewing yeast, and has been completed as a result of further investigation based on these useful new findings, and has been completed based on the YIL169C gene. It is also involved in mother identification, confirmation and detection systems. Although the YIL169C gene itself is known (Non-Patent Document 1: Literature titles Lye, G. Bowman, S. and Churcher, C. unpublished, but published on the Internet), yeasts that focus on the YIL169C gene are identified. Furthermore, it has not been reported that the YIL169C gene differs between brewer's yeasts. Furthermore, there has been no report at all that it is possible to discriminate finely even yeasts of the same type as well as yeasts of different types for brewing, and the invention of the earlier application is the earliest.
[0012]
That is, the prior application invention uses the genomic DNA of a brewing yeast belonging to the genus Saccharomyces cerevisiae (cerevisiae) to amplify a part or all of the YIL169C gene by PCR, and to determine the length of the amplified fragment, The basic technical idea is to determine the brewer's yeast based on the difference in the number.
[0013]
In the invention of the prior application, the genomic DNA of the yeast for brewing is amplified by a PCR method using primers for amplifying a part or all of the YIL169C gene, and the amplified gene fragments are directly subjected to agarose gel electrophoresis or a restriction enzyme. Since the electrophoresis pattern may be observed by agarose gel electrophoresis after the treatment, the operation is easy and simple. In addition, the use of primers for amplifying a specific gene (in this case, a part or all of the YIL169C gene) has many advantages such as a stable type of DNA fragment obtained by amplification by PCR. .
[0014]
This time, the present inventors have made improvements to the above-mentioned prior application invention and conducted further research. As a result, in addition to the YIL169C gene, the YOL155C gene and the MUC1 A new gene was found. All of these genes are derived from Saccharomyces cerevisiae S288 strain (sold by Invitrogen Co.), but it is known that the MUC1 gene is involved in yeast aggregation, but the other genes are not. The function of is unknown. Further, as a result of further research on the YIL169C gene, it was newly found that primers other than the primers according to the prior application can also be used for discriminating yeast by the PCR method.
[0015]
The present inventors have studied from various aspects, and as a result, have noticed that the YOL155C gene has high homology with the YIL169C gene, and for the first time, from the viewpoint of whether or not the YIL169C gene has the diversity property of the YIL169C gene, When an experiment was performed from this viewpoint, it was first confirmed that the YOL155C gene can be distinguished from yeast by the PCR method.
[0016]
In addition, from a different point of view, the YOL155C gene and the YIL169C gene were examined in detail from other viewpoints, not homology. As a result, it was found that both genes are not essential genes for yeast growth, and that the YOL155C and YIL169C proteins are repeated in the molecule. For the first time, they found that they had common characteristics such as having a short amino acid sequence, and searched for genes having these characteristics from genes having no homology with the YOL155C and YIL169C genes. As a result of heading and experiments, it was also confirmed that the method can be used for discriminating yeast for brewing.
[0017]
From the above results, the present inventors have common features such as that the gene is not an essential gene for the growth of yeast among the yeast gene sequences, and that the protein has a repetitive sequence in the protein molecule encoded by the gene. For the first time, it was confirmed that it could be used for the method of discriminating yeast for brewing.
[0018]
The present invention has been made on the basis of these useful new findings, and uses the genomic DNA of a brewing yeast belonging to the genus Saccharomyces cerevisiae by PCR to obtain one of the YIL169C, YOL155C, and MUC1 genes. The basic technical idea is to amplify a part or the whole, and to discriminate the yeast for acid production based on a difference in length and / or number of fragments to be amplified.
[0019]
That is, the present invention has discovered that the YIL169C, YOL155C, and MUC1 genes are different in yeast for brewing, and based on these useful new findings, have been completed as a result of further studies. The YIL169C, YOL155C, and MUC1 genes have been completed. It is also involved in a brewer's yeast discrimination, confirmation, and detection system based on this.
[0020]
In the present invention, the genomic DNA of the yeast for acid production is used with primers for amplifying a part or all of the YIL169C, YOL155C, and MUC1 genes. The procedure is easy and simple, since the amplification pattern can be observed by directly agarose gel electrophoresis of the amplified gene fragments by the PCR method. In addition, the use of primers for amplifying specific genes (parts or all of the YIL169C, YOL155C, and MUC1 genes) stabilizes the types of DNA fragments obtained by amplification by the PCR method. There are many excellent points.
[0021]
In practicing the present invention, primers A to O (the base sequences of which are shown in SEQ ID NOs: 1 to 15 (FIG. 1) in the sequence listing), which were successfully developed for the first time by the present inventors, are used as primers. Selection and use are performed, and PCR is carried out using yeast genomic DNA as a template. As the primer, the above-mentioned primer may be synthesized and used. The YIL169C, YOL155C, and MUC1 genes can be obtained from the laboratory yeast S288C strain (purchased from Invitrogen Co., Ltd.), and may be cut out therefrom. The primer may be appropriately selected and used in accordance with the yeast to be discriminated. Depending on the yeast, a primer other than A to O may be designed from the YIL169C, YOL155C, and MUC1 genes, synthesized, and used. . PCR may be performed according to a conventional method, and as a result, a target gene fragment can be obtained.
[0022]
The gene fragment obtained by amplification in this way can be discriminated from yeast by directly agarose gel electrophoresis and observing the electrophoresis pattern. By selecting the type and combination of primers, a clear migration pattern unique to yeast can be obtained. If desired, the gene fragment grown by PCR is digested with a restriction enzyme (for example, RsaI), electrophoresed, and its migration pattern is observed. Can be done clearly.
[0023]
Therefore, according to the present invention, it is possible to discriminate and identify various yeasts for brewing by changing the type of at least one of a primer, a genomic DNA used as a template, an amplified DNA fragment, and a restriction enzyme digest thereof. Or, conversely, in order to identify and identify a specific yeast for brewing, it is only necessary to select primers and the like, and if a variation design of the primers is made so that the yeast variation appears or corresponds to it. Good.
[0024]
In this way, according to the present invention, at least one of the identification, identification, and classification of yeast for brewing can be performed.Therefore, a kit for identification, identification, and classification of yeast using at least one of the above-described primers and the like can be assembled. Can be. Therefore, for example, using the primers A and B or the PCR products of these primers, a kit for discriminating, identifying, and classifying the Association Wine Yeast No. 3 can be assembled.
[0025]
In the present invention, any yeast can be used as the yeast as long as it belongs to the genus Saccharomyces (Saccharomyces) cerevisiae. For example, sake yeast (Kyoto No. 7 yeast, Kyokai No. 9 yeast, Kyokai No. 10) Yeast, etc.), wine yeast (Kyoto Wine No. 1 yeast, Kyokai Wine No. 3 yeast, Kyokai Wine No. 4 yeast, etc.), shochu yeast (Kagoshima yeast K2, Miyazaki yeast MK, Kyosho shochu yeast SH-4, Awamori yeast) No. 1 etc.). The present invention is not only capable of discriminating between yeasts for different uses such as discriminating sake yeast and wine yeast, discriminating wine yeast and shochu yeast, and the same wine yeast, and the same wine yeast No. 1 yeast. The present invention also has a remarkable effect that it is possible to discriminate between yeasts of the same use, such as the same yeast, the third yeast and the fourth yeast. In particular, regarding the latter, discrimination itself is difficult and very delicate requirements are required, and a method that can be satisfied with a simple and accurate method has not been reported conventionally.
[0026]
Hereinafter, embodiments of the present invention will be described.
Embodiment 1
(Method of discriminating yeast for brewing)
Takara Shuzo from Association Saccharomyces cerevisiae (Saccharomyces cerevisiae), Association Wine No. 1 yeast, Association Wine No. 3, Yeast Wine No. 4, Kagoshima Yeast K2, Miyazaki Yeast MK, Association Shochu Yeast SH-4, Awamori Yeast No. 1 Genomic DNA purified using "Juntoru Kimi" of Co., Ltd. was amplified by PCR with each combination of primers A to O (SEQ ID NOS: 1 to 15, FIG. 1), and then agarose gel electrophoresis. Electrophoresis was performed. (Electrophoresis results are shown in Table 1).
[0028]
PCR was performed on the genomic DNA using the primers. The reaction conditions are as follows.
(PCR conditions)
1 cycle at 94 ° C for 3 minutes or less 25 cycles at 94 ° C for 1 minute 59 ° C for 1 minute 72 ° C for 2 minutes or less 1 cycle at 72 ° C for 5 minutes
[Table 1]

[0030]
In the table, each symbol represents the following.
K2: Kagoshima yeast SH4: Association shochu yeast Mk: Miyazaki yeast Aw: Awamori yeast brew 1: Wine brewing association No. 1 yeast brewing 3: Wine brewing association No. 3 yeast brewing 4: Wine brewing association No. 4 yeast
As is clear from the above, each yeast can be distinguished by variously selecting a combination of primers and variously examining the presence or absence of the DNA fragment to be amplified and / or the length of the DNA fragment to be amplified.
[0032]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, compared with the conventional method, such as a small preparation test, a determination by changing a drug or culture conditions, a remarkable effect that determination can be performed in a short time is achieved, and furthermore, a clear determination can be made, The results are reproducible, and the operation is simple.
[0033]
Furthermore, according to the present invention, it is possible to distinguish between different brewing yeasts, as well as extremely difficult and delicate distinguishing between the same brewing yeasts, for example, between wine yeasts. In the liquor manufacturing industry, testing and research institutes, etc., it is possible to easily and accurately classify, identify and discriminate yeast in a short period of time, and it is also possible to clearly discriminate yeast in mash from wild yeast.
[0034]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows the nucleotide sequences of primers A to O.

Claims (5)

The genomic DNA of the brewing yeast is amplified by a PCR method using primers for amplifying a part or all of at least one of YIL169C, YOL155C and MUC1 genes, and the amplified gene fragments are directly subjected to agarose gel electrophoresis. Or a restriction enzyme treatment, followed by agarose gel electrophoresis, and discriminating the yeast based on a difference in the electrophoresis pattern. The method according to claim 1, wherein the gene is not essential for the growth of yeast and / or has a repeated sequence in the encoded protein molecule. The yeast for brewing is at least one of wine yeast, shochu yeast, awamori yeast and sake yeast, and the discrimination is discrimination between the same use and / or between different uses. 3. The method according to 1 or 2. At least one of the DNAs of primers A to O for brewing yeast discrimination represented by the nucleotide sequences of SEQ ID NOS: 1 to 15, respectively. A yeast discriminating kit for brewing, comprising the primer DNA according to claim 4 or a PCR product using the primer.

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