JP5995232B2 - Efficient method for fusion of Neisseria gonorrhoeae cell lines - Google Patents

Description

  The present invention relates to a method for increasing the fusion efficiency of an Aspergillus cell line such as Aspergillus oryzae, a method for simply analyzing the fusion process of an Aspergillus cell line, and the like.

Aspergillus oryzae (A. oryzae), a type of Aspergillus spp., Is a filamentous fungus that has been used for more than 1000 years in the production of Japanese traditional sake, miso, and soy sauce. It is a microorganism that is said to be a “national fungus” in Japan (Kitamoto, 2002). In addition, A. oryzae is recognized as a highly safe bacterium by the World Health Organization (WHO) and the International Food and Agriculture Organization (FAO) (Barbesgaard et al., 1992), and is an enzyme for food, chemicals and medical products. Drugs and metabolic compounds are widely distributed internationally. In addition, A. oryzae, which has excellent protein secretion ability and safety, and a post-translational modification mechanism common to eukaryotes, is used as a host for mass production of useful proteins (Ito et al. , 2007; Nakajima et al., 2006; Tsuchiya et al., 1992, 1994).

As a genetic engineering technique for A. oryzae, a transformation system was developed in 1987 (Gomi et al., 1987), and various selection markers using auxotrophy and drug resistance have been developed to date. (Gomi et al., 1987; Kubodera et al., 2000; Mattern et al., 1987; Yamada et al., 1997). A four-auxotrophic host / vector system for A. oryzae has also been constructed using gold, and up to five genes can be introduced or destroyed simultaneously (Jin et al., 2004b). In recent years, disruption of the ligD gene and ku70 gene involved in non-homologous recombination repair has created host strains capable of high-frequency homologous recombination, making it easy to create gene-disrupted strains (Takahashi et al ., 2006; Escano et al., 2009; Mizutani et al., 2008; Maruyama and Kitamoto, 2008). In addition, multiple gene disruption using the pyrG marker recycling technology has become possible (Maruyama and Kitamoto, 2008). By using marker recycling technology, a protease gene multiple disruption strain has been created and the production of heterologous proteins has been successfully improved (Yoon et al., 2009, 2011).

A. oryzae has no sexual generation and is classified as a Deuteromycete. And mating is not possible, and it is difficult to efficiently breed strains with excellent phenotypes of multiple strains. Moreover, it was difficult to apply classical genetics using mating, and genetic research was delayed.

In recent years, however, A. oryzae has been shown to be capable of sexual reproduction with the completion of genome decoding (Machida et al., 2005; Galagan et al., 2005). On the other hand, RIP (Repeat-induced point mutation) has been reported in A. oryzae (Montiel et al., 2006). RIP is a phenomenon in which base substitution from C: G to T: A occurs in homologous sequences at different sites on the genome during sexual reproduction, and the same applies to strains in which sexual generation is found in other filamentous fungi Reported RIP (Braumann et al., 2008). This suggests the possibility that A. oryzae had performed sexual reproduction in the past.

On the other hand, sexual generations have been found in other Aspergillus species. For example, in A. fumigatus and A. flavus, which is considered to be very closely related to A. oryzae, inside a structure called mature cleistothecium or sclerotium, respectively. Confirms the existence of ascospores. Here, the sclerotium is a thick-filmed small particle mass in which mycelium is repeatedly fused and closely resembles the ascending shell which is a sexual generation structure (Geiser, 2009; Dyer, 2007).

A. flavus, an ancestor of A. oryzae in the genus Aspergillus that has been found to have a sexual generation, is known to have a higher ability to form sclerotia than A. oryzae (Murakami et al. ., 1971). In recent years, it has been thought that A. flavus lost its pathogenicity in the process of domestication and differentiated into A. oryzae (Chang et al., 2010; Ichishima, 2003) ) Probably, the ability to form nuclei was also lost in the course of such evolution. On the other hand, the discovery of the sexual generation of A. flavus was revealed by the confirmation of ascospores in the mature sclerotia (Chang et al., 2010; Horn et al., 2009a). The discovery of the sexual generation in oryzae is also thought to be due to the confirmation of ascospores formed in the sclerotia. A. oryzae is considered to be capable of heterothalic sexual reproduction (Wada et al., 2012) .After nucleation between mycelia of different zygotic strains, sexual reproduction occurs within the nuclei. It is expected to complete and eventually form ascospores.

  Therefore, the search for culture conditions and factors for promoting nucleation is considered to be an important issue, and conditions such as the carbon / nitrogen ratio and amino acid composition of medium components have a significant effect on nucleation. Have been studied so far (McAlpin and Wicklow, 2005; Agnihotri, 1968). It has been advanced. In addition, Ssp1, which is specifically expressed in the mycorrhizal tissue, is shared by these plant pathogens and A. oryzae and A. flavus that form mycorrhiza in the genus Aspergillus that is systematically distant from Sclerotinia sclerotiorum Has also been clarified in the latest research (Amselem et al., 2011).

In addition, Jin et al. First identified a novel transcription factor SclR ( Scl erotium r egulator) of the basic-helix-loop-helix type highly conserved in Aspergillus in A. oryzae (Jin et al., 2009 ), It was suggested that this transcription factor SclR may be involved in the control of mycelial morphology in A. oryzae (Jin et al., 2011). Furthermore, overexpression of SclR was also reported to promote the formation of mycorrhiza by promoting the formation of entangled hyphae compared to the wild type strain (Jin et al., 2011).

As described above, even in A. oryzae, mycelial fusion is repeated between different zygotic strains in the sclerotia, and if nuclei from each zygote are present in the same cell, Ascospores are expected to form through meiosis, but knowledge about mycorrhizal formation and mycelial fusion in relation to sexual reproduction in A. oryzae, and the relationship between mycorrhizal formation and mycelial fusion The knowledge about is very scarce.

Jin FJ, Takahashi T, Matsushima K, Hara S, Shinohara Y, Maruyama J, Kitamoto K, & Koyama Y (2011) SclR, a basic helix-loop-helix transcription factor, regulates hyphal morphology and promotes sclerotial formation in Aspergillus oryzae. Eukaryot Cell 10: 945-955 Wada R, Maruyama J, Yamaguchi H, Yamamoto N, Wagu Y, Paoletti M, Archer DB, Dyer PS, & Kitamoto K (2012) Presence and Functionality of Mating-Type Genes in the Supposedly Asexual Filamentous Fungus Aspergillus oryzae.Appl Environ Microbiol in press

In order to initiate sexual reproduction in this heterothallic species, A. oryzae, after cells of different mating type strains are fused, nuclei from different strains exist within the same cell. It is considered essential that a heterozygous heterozygote (heterokaryon) is formed, and then nuclei derived from different mating strains are fused (Dyer, 2007). It is necessary to have a method for discriminating completion. Furthermore, if the fusion efficiency of such cell lines can be increased, a great contribution can be expected in basic research and practical use.

  As a result of diligent research to solve the above problems, the present inventor conducts mycelial fusion experiments and sexual reproduction experiments for cell fusion and heterokaryon formation, which has hardly been observed or studied so far in A. oryzae. One of the two strains is given uridine / uracil requirement and the nucleus is visualized with red fluorescence (H2B-mDsRed, ΔpyrG), the other strain is given adenine requirement and the nucleus is By visualizing with green fluorescence (H2B-EGFP, ΔadeB), we found that cell fusion and heterokaryon formation can be easily observed and identified visually based on phenotype.

Furthermore, in order to attempt to induce sexual reproduction in A. oryzae, overexpression of the nucleation promoting factor SclR, which is considered to be an essential process when A. flavus and A. oryzae perform sexual reproduction As a result, it was confirmed for the first time that the mycelium (cell) fusion ability was increased in the process of producing the strain.

The present invention has been made based on the above new findings.
That is, the present invention includes the following aspects.
[Aspect 1] In the fusion of two Aspergillus cell lines (mycelia), the fusion efficiency of the Aspergillus cell line is increased by increasing the expression of the nucleation promoting factor SclR in at least one of the fused cell lines. How to make.
[Aspect 2] The method according to Aspect 1, wherein the Aspergillus cell line is Aspergillus oryzae.
[Aspect 3] The expression of the nucleation-promoting factor SclR in the cell line is increased by transforming an Aspergillus cell line with a plasmid vector containing a sclR gene linked under the control of a promoter. Method.
[Aspect 4] The method according to any one of Aspects 1 to 3, wherein the Aspergillus genus cell line (mycelium) is fused in the sclerotia or in the process of sclerotia formation.
[Aspect 5] The aspect according to any one of Aspects 1 to 4, wherein the Aspergillus cell line (mycelium) is fused in an opposing culture of the Aspergillus cell line at 25 ° C. in the dark and under aeration conditions using MEA medium. Method.
[Aspect 6] Further, two Aspergillus cell lines (mycelia) to be fused are transformants that can be distinguished from each other by different phenotypes, and the phenotypes are complemented in the fused cells, and / or Alternatively, the method according to any one of aspects 1 to 5, wherein the fused cell is identified based on the fact that the fused cell has two phenotypes.
[Aspect 7] The method according to Aspect 6, wherein the phenotype is based on the expression of a transformation marker and / or a labeling substance.
[Aspect 8] The method according to Aspect 7, wherein the phenotype based on the transformation marker is auxotrophy.
[Aspect 9] The method according to Aspect 8, wherein the phenotype is complemented in fused cells by culturing in an agar CD minimal medium.
[Aspect 10] The method according to Aspect 7, wherein the labeling substance is a fluorescent protein.
[Aspect 11] The method according to Aspect 10, wherein it is confirmed by observing a nucleus with a fluorescence microscope that a fused cell has two labeling substances.
[Aspect 12] In the fusion of two types of Aspergillus cell lines (mycelia), the two Aspergillus cell lines to be fused are transformants that can be distinguished from each other by different phenotypes. A method of identifying a fused cell based on the type being complementary and / or that the fused cell has two phenotypes.
[Aspect 13] The DNA of the following (a), (b), (c), (d) or (e) is contained, and the cell line is transformed by transduction or transformation into the host Aspergillus genus cell line. A recombinant vector having a function of increasing the fusion efficiency:
(A) DNA comprising the base sequence represented by SEQ ID NO: 1,
(B) a protein that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) and having a function of increasing the fusion efficiency of an Aspergillus cell line DNA encoding,
(C) a DNA comprising a nucleotide sequence having a sequence homology of 90% or more with the DNA comprising the nucleotide sequence of (a), which encodes a protein having a function of increasing the fusion efficiency of an Aspergillus cell line,
(D) DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, or (e) an amino acid in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence of (d) A DNA encoding a protein comprising a sequence and having a function of increasing the fusion efficiency of an Aspergillus cell line.
[Aspect 14] An Aspergillus cell line, which is transduced or transformed with the recombinant vector according to Aspect 13 and has an increased fusion efficiency compared to the parent strain.

Boundary line-specific formation occurs in two types of gonococcal cell lines with increased expression of SclR or in combination with two types of gonococcal cell lines, including strains with increased expression of SclR. It was confirmed that the number of fungal nuclei was significantly increased. Furthermore, as a result of complementation of the auxotrophy of gonococcal cell lines in the sclerotia and observation of the nuclei by fluorescence microscopy, phenotypes of different strains appeared in the same strain. Alternatively, it was shown that the mycelium was fused during the formation of mycorrhiza.

An outline of the construction of NPlD-HR1 (H2B-mDsRed) is shown. An outline of the construction of NPlD-HG1 (H2B-EGFP) is shown. An outline of the production of a nucleation promoting factor SclR overexpression strain is shown. An outline of production of a control strain for a strain overexpressing the nucleation promoting factor SclR is shown. The process of conversion from uridine / uracil requirement to adenine requirement in transformants is shown. The result of the confirmation of the conversion of auxotrophy using CD minimum medium is shown. The chromosome structure of the strain | stump | stock in which auxotrophy was converted is shown. The systematic diagram of the strain | stump | stock in which auxotrophy was converted is shown. Fig. 2 shows the formation of sclerotia in a strain that overexpresses the sclerotium promotion factor SclR and a control strain. A comparison of growth between the nucleation-promoting factor SclR overexpression strain and the control strain is shown. The sclerotial formation on the boundary line of strains with different auxotrophy is shown. The number of microbial nuclei in borderline specific nucleation is shown. The complementation of auxotrophy in the sclerotia formed specifically on the border line in CD minimal medium is shown. The number of myconuclei considered to have undergone boundary-specific hyphal fusion. The result of observing with a fluorescence microscope the mycelium which grew in the agar CD minimum culture medium which does not contain uridine / uracil and adenine among the mycelia specifically formed in the boundary line of the strain | stump | stock from which an auxotrophy differs is shown.

In the fusion of two types of Aspergillus cell lines (mycelium), the present invention increases the fusion efficiency of the Aspergillus cell line by increasing the expression of the nucleation promoting factor SclR in at least one of the fused cell lines. It is about the method to make it.

  The nucleation-promoting factor SclR is already known, and the base sequence (SEQ ID NO: 1) of the sclR gene is also disclosed in the Aspergillus oryzae genome database, etc., so that those skilled in the art can easily obtain information on SclR from them. I can do it.

The genus Aspergillus consists of approximately 250 species of bacteria (Samson and Varga, 2010), but many of them are industrially useful, and conversely, many are pathogenic to humans and plants. In the life cycle of fungi, there are generally a life cycle that forms sexual spores and a life cycle that forms asexual spores, but in Aspergillus, 64% do not have a sexual life cycle and asexual life. It grows on rings only (Dyer et al., 2012). The sexual life cycle refers to a process in which an individual who has spent a single-phase nucleus generates a genetically new individual through fusion and meiosis. In contrast, asexual reproduction refers to the process of producing genetically identical individuals only through somatic division.

Fungi are classified based on the form of sexual reproduction structure (fruiting body), so that only the asexual life cycle is known and the sexual life cycle is not found Classified as imperfect bacteria. In the genus Aspergillus, sexual life cycles have been found in A. nidulans, A. fumigatus, A. flavus, A. parasiticus, A. nomius, etc., but most other Aspergillus species including A. oryzae Only the asexual life cycle that repeats the offspring → mycelium → conidia is known and classified as an incomplete fungus.

  Therefore, as the Aspergillus genus cell line targeted for the method of the present invention, a strain classified as an incomplete bacterium such as Aspergillus oryzae is preferable.

In the method of the present invention, the means for increasing the expression of the nucleation promoting factor SclR in the cell line is not particularly limited and can be carried out by any method known to those skilled in the art. For example, by transforming an Aspergillus cell line in advance with a plasmid vector containing a sclR gene linked to the promoter to be expressed under the control of a suitable constitutive or inducible promoter, the SclR in the transformant It is possible to increase the expression of

The mycorrhiza is a durable dormant structure that is asexually formed and is observed in some filamentous fungi belonging to Ascomycota and Basidiomycota (Willets and Bullock, 1992; Erentala et al., 2008). The hyphae are formed by repeatedly branching, fusing and adhering to form a hard wall with melanin deposition. The diameter is on the order of 1 to 3 mm, but some exceed 3.5 cm in diameter (Bolton et al., 2006). From the outside, it consists of cortical rind consisting of thick colored cells rich in melanin and medulla medulla consisting of woven hyphae, and germinates under good environmental and nutritional conditions.

In the method of the present invention, it is preferable that the Aspergillus genus cell line (mycelium) is fused in such a sclerotia or in the process of sclerotia formation.

In the method of the present invention, these cell lines can be fused by culturing two types of Aspergillus cell lines (mycelia) under appropriate conditions. Such culture conditions can be appropriately selected by those skilled in the art according to the type of cell line used. For example, as described in the Examples of the present specification, it is preferable to use a medium known to those skilled in the art such as MEA medium at a temperature of 25 ° C. to 30 ° C. in a dark place and under aeration conditions. In addition, as a culture method, an appropriate method known to those skilled in the art, such as counter culture using a solid medium and liquid culture, can be used.

In the method of the present invention, the two Aspergillus cell lines (mycelium) to be fused are preferably transformants that can be distinguished from each other by different phenotypes. In this way, two fused Aspergillus cell lines (mycelia), i.e., based on the fact that the phenotype is complemented in fused cells and / or has two phenotypes. Formation of heteronuclear coexistence (heterokaryon) by a cell line and / or subsequent fusion of nuclei can be easily and easily identified. Therefore, the present invention is a transformant in which two Aspergillus cell lines can be distinguished from each other by different phenotypes in the fusion of two types of Aspergillus cell lines (mycelia). It also relates to a method for identifying fused cells on the basis that the phenotypes are complemented and / or that the fused cells have two phenotypes.

Such phenotypes are, for example, drug resistance and auxotrophy based on a number of transformation markers, including any drug resistance marker and auxotrophic marker known to those skilled in the art, and any known to those skilled in the art. This is based on the expression of a labeling substance such as a fluorescent protein. Examples of auxotrophic markers include the argB gene related to the arginine biosynthesis pathway, the pyrG gene related to the pyrimidine biosynthesis pathway, and the adeA gene and adeB gene related to the adenine biosynthesis pathway.

The transformant used in the method of the present invention preferably has two different types of such phenotypes as described in the examples of the present specification.

More specifically, for example, in two fused Aspergillus cell lines, complementation of the phenotype (auxotrophy) is confirmed using the fact that only fused cells can grow in culture on agar CD minimal medium. I can do it.

If the phenotype is a fluorescent protein, the two Aspergillus cell lines (mycelia) are fused by observing the nucleus with a fluorescence microscope according to standard methods known to those skilled in the art. It can be easily confirmed.

Furthermore, the present invention includes the following DNA (a), (b), (c), (d) or (e), and the cells can be obtained by transduction or transformation into a host cell line of Aspergillus. Recombinant vectors having the function of increasing the fusion efficiency of a strain:
(A) DNA comprising the base sequence represented by SEQ ID NO: 1,
(B) a protein that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a), and having a function of increasing the fusion efficiency of an Aspergillus cell line DNA encoding,
(C) a DNA consisting of a base sequence showing 90% or more sequence homology with DNA consisting of the base sequence of (a), which encodes a protein having a function of increasing the fusion efficiency of an Aspergillus cell line,
(D) DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, or (e) an amino acid in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence of (d) A DNA comprising a sequence and encoding a protein having an activity having a function of increasing the fusion efficiency of an Aspergillus cell line.

The present invention also relates to an Aspergillus cell line which is transduced or transformed with the above-described recombinant vector and has an increased fusion efficiency compared to the parent strain.

  The “DNA comprising the nucleotide sequence represented by SEQ ID NO: 1” in the present invention encodes the nucleation promoting factor SclR, and comprises a nucleotide sequence consisting only of the amino acid-encoding region (ie, only the exon part is bound). CDNA comprising a base sequence) is also included. Such cDNA can be obtained by PCR or the like using gonococcal mRNA as a template using an appropriate primer prepared based on the nucleotide sequence information disclosed in this specification by any method known to those skilled in the art. Is possible. The DNA of the present invention can also be chemically synthesized by any method known to those skilled in the art.

  Furthermore, the DNA of the present invention includes DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA, and a sequence that is about 90% or more, preferably about 95% or more with the DNA. DNA comprising a base sequence exhibiting homology, which encodes a protein having a function of increasing the fusion efficiency of an Aspergillus cell line.

  Here, hybridization is performed by, for example, Molecular cloning third.ed. (Cold Spring Harbor Lab.Press, 2001), or Current protocols in molecular biology (edited by Frederick M Ausubel et al., 1987), etc., etc., which are known in the art, or a method analogous thereto, and when using a commercially available library, the method described in the attached instruction manual Can be done according to.

  In this specification, “stringent conditions” means, for example, at a temperature of 60 ° C. to 68 ° C., a sodium concentration of 15-900 mM, preferably 15-600 mM, more preferably 15-150 mM, pH 6-8. Can be mentioned.

  Therefore, as a DNA that can hybridize under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 1, for example, the DNA having homology with the entire base sequence of the DNA Examples thereof include DNA containing a base sequence having a degree of about 90% or more, preferably about 95% or more in average on the whole.

  In addition to the protein consisting of the amino acid sequence encoded by the DNA consisting of the base sequence represented by SEQ ID NO: 1, the protein of the present invention has one or several amino acids deleted or substituted in such an amino acid sequence. Or a protein comprising an added amino acid sequence (amino acid sequence having sequence homology with the above amino acid sequence) and having a function of increasing the fusion efficiency of an Aspergillus cell line.

  In order to determine sequence homology between two base sequences or amino acid sequences, the sequences are pre-processed to the optimal state for comparison. For example, by making a gap in one sequence, the alignment with the other sequence is optimized. Thereafter, the amino acid residues or bases at each site are compared. When the same amino acid residue or base as the corresponding site in the second sequence is present at a site in the first sequence, the sequences are identical at that site. Sequence homology between the two sequences is expressed as a percentage of the total number of sites (all amino acids or all bases) of the number of sites that are identical between the sequences.

  In accordance with the above principles, sequence homology between two base or amino acid sequences can be determined, for example, by the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990 and Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993). A BLAST program or FASTA program using such an algorithm is mainly used to search a database for a sequence showing high sequence homology with a given sequence. These are available, for example, on the Internet website of the National Center for Biotechnology Information.

  DNA that exhibits the sequence homology of the base sequence as described above or the sequence homology of the encoded amino acid sequence can also be obtained using hybridization as an index as described above, and functions obtained by genome base sequence analysis etc. It is also easy to find out from unknown DNA groups or public databases by a method commonly used by those skilled in the art, for example, by searching using the aforementioned BLAST software. Furthermore, the gene of the present invention can also be obtained by various known mutagenesis methods.

  The recombinant vector of the present invention can be prepared by ligating the above DNA into an appropriate vector using an appropriate genetic engineering means known to those skilled in the art. As a vector, the above DNA is inserted at an appropriate position on the genome of the host microorganism to be transduced or transformed, and as a result, the fusion efficiency of the Aspergillus cell line can be increased compared to the parent strain. If it is a thing, there will be no restriction | limiting in particular in the structure, a kind, etc. For example, vectors such as plasmids, cosmids, phages, viruses, chromosomal integration types, or artificial chromosomes can be used.

  The vector may contain any marker gene known to those skilled in the art, such as AosC, to allow selection of transformed cells. In addition, the recombinant vector is inserted with a promoter capable of expressing the gene of the present invention in a host cell and other control sequences (for example, an enhancer sequence, terminator sequence, polyadenylation sequence, etc.) and further the desired DNA. It is desirable to include multiple cloning to do this. As each element contained in these recombinant vectors, those known to those skilled in the art can be used as appropriate.

  Transduction or transformation can be performed by a known appropriate method. For example, a method using polyethylene glycol and calcium chloride after protoplastization (Mol. Gen. Genet., 218, 99-104 (1989)) can be used. In particular, it is preferred to use Gateway technology as described in the examples.

  The Aspergillus cell line transduced or transformed with the recombinant vector of the present invention has an increased expression level of the nucleation promoting factor SclR. As a result, the Aspergillus cell line has a fusion efficiency of 20 compared to the parent strain. % Or more, preferably 2 to 3 times the function. The cell line fusion efficiency can be measured and evaluated by the method as shown in the examples of the present specification.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example. Unless otherwise specified in the present specification, various conditions, means, methods, and the like can be appropriately set and implemented by those skilled in the art according to technical common sense in the technical field.

Nuclear visualization with nuclear histone H2B-mDsRed and H2B-EGFP fusion protein expression strains <br/> For nuclear visualization, a fusion protein consisting of nuclear histone H2B fused with red fluorescent protein mDsRed and green fluorescent protein EGFP, respectively. Two plasmids were designed to be expressed in (pgHHDRS, pgHHGS; FIG. 1A, FIG. 2A). As a selection marker for transformation, an sC gene derived from A. oryzae (hereinafter referred to as AosC) was used.

In A. oryzae NSPlD1 strain (Maruyama and Kitamoto, 2008), both pgHHDRS and pgHHGS plasmids were inserted into the AosC locus in a single copy by transformation (FIGS. 1B and 2B). Southern analysis confirmed the single copy insertion of these plasmids into the AosC locus, and confirmed the visualization of nuclei by the respective fluorescent proteins with a fluorescence microscope (FIG. 1C, FIG. 2C). Strains whose nuclei were visualized with red fluorescent protein and green fluorescent protein were named NPlD-HR1 (H2B-mDsRed) and NPlD-HG1 (H2B-EGFP), respectively.

Production of nucleation- promoting factor SclR overexpression strain and control strain <br/> niaD genes of two strains, NPlD-HR1 (H2B-mDsRed) and NPlD-HG1 (H2B-EGFP), obtained by visualization of the nucleus A plasmid (pamy215; FIG. 3A) for overexpressing SclR under the amyB promoter was inserted into the locus, and overexpression of SclR was attempted for each strain. After transformation, it was confirmed by Southern analysis using the construct shown in FIG. 3B that a strain in which the SclR overexpression plasmid was inserted at a single copy at the niaD locus was confirmed. The two SclR-overexpressing strains obtained from the parent strains NPlD-HR1 and NPlD-HG1 are PlD-HR1-sR (H2B-mDsRed, SclR OE), PlD-HG1-sR (H2B-EGFP, SclR OE) Named.

Similarly, a plasmid (pUNA; FIG. 4A; Yamada et al., 1999) having only the amyB promoter was inserted into the niaD locus of two strains of NPlD-HR1 and NPlD-HG1 obtained by nuclear visualization. An attempt was made to create a control strain for overexpression of SclR. After the transformation, it was confirmed by Southern analysis using the construct shown in FIG. 4B that a strain in which the pUNA plasmid was inserted in a single copy at the niaD locus was confirmed. The obtained control strains were named PlD-HR1-c (H2B-mDsRed, Ctrl) and PlD-HG1-c (H2B-EGFP, Ctrl) with respect to the two strains of parent strains NPlD-HR1 and NPlD-HG1, respectively.

Conversion of auxotrophy
Conversion from uridine / uracil requirement to adenine requirement
In A. oryzae, many transformation markers including drug resistance markers and auxotrophic markers and vector systems having them have been developed so far. As for auxotrophy, the argB gene related to the arginine biosynthesis pathway, the pyrG gene related to the pyrimidine biosynthesis pathway, and the adeA gene and adeB gene related to the adenine biosynthesis pathway have been developed as auxotrophic markers (Jin et al., 2004a, 2004b; Kubodera et al., 2000). Of these auxotrophic marker genes, ADEA gene in NSPlD1 strain mutated by UV irradiation was introduced into ADEA locus argB locus on chromosome 3 of the original chromosome 6 ADEA ^- Gene And the normally functioning adeA gene is finally present at the pyrG locus on chromosome 7. In order to prevent complications, the adeB gene is used as a gene that can confer auxotrophy in addition to the above argB gene, and the adeB gene present at the first chromosome adeB locus of uridine / uracil auxotrophic strain with ΔpyrG is expressed by the pyrG gene. By substituting, it was decided to convert to adenine requirement with ΔadeB.

The adeB gene on chromosome 1 of the PlD-HG1-sR strain (H2B-EGFP, SclR OE, ΔpyrG) and the control strain PlD-HG1-c strain (H2B-EGFP, Ctrl, ΔpyrG) A strain in which uridine / uracil requirement was converted to adenine requirement was prepared by replacing the DNA fragment containing 1.0 kb upstream and 1.0 kb downstream of the adeB gene and the pyrG gene shown in FIG. 5A with the pyrG gene. The target gene replacement was confirmed by Southern analysis (FIG. 5B), and the conversion of auxotrophy was confirmed using a CD minimal medium (FIG. 6). The obtained adenine-requiring strains were named AblD-HG1-sR (H2B-EGFP, SclR OE, ΔadeB) and AblD-HG1-c (H2B-EGFP, Ctrl, ΔadeB), respectively.

The upstream 1.0 kb and downstream 1.0 kb of the adeB gene each contain a part of the ORF of the preceding and succeeding genes (AO090009000332 and AO090009000334). Therefore, there is a possibility that an unintended mutation may be introduced into the strain obtained by replacing the adeB gene with the pyrG gene. Therefore, the presence or absence of mutations in the 1.0 kb region upstream and downstream of the adeB gene on the chromosomal DNA of AblD-HG1-sR and AblD-HG1-c was confirmed by sequencing. As a result, no mutation was confirmed in the region due to transformation. 7 and 8 show the chromosomal structure and systematic diagram of the strain prepared above.

Optimization of medium conditions for nucleation of SclR overexpressing strains
The culture medium conditions suitable for sclerotia formation of the strain which overexpressed SclR under the amyB promoter were examined. In other Aspergillus species, A. parasiticus and A. flavus, which is closely related to A. oryzae, MCA (Mixed Cereal Agar; baby cereal for infants dissolved in water and agar added) The sexual generation has been discovered by culturing different mating types using culture media (O'Gorman et al., 2009; Horn et al., 2009a, 2009b; experimental materials and methods). On the other hand, Jin et al. Used MEA (Malt Extract Agar) medium containing malt extract for the identification and functional analysis of the nucleation promoting factor SclR. Therefore, the conidial suspensions of the SclR overexpressing strain and the control strain were inoculated into three media, MCA medium, MEA medium and PD medium with PD medium added to these media, and cultured in the dark and aerated conditions. . At this time, although the number of conidia which inoculate each agar medium with 10 ^3/5 [mu] l, A. flavus and that this is the number of conidia inoculation amount is too large sclerotium formation speed is reduced A. Based on experimental results reported in parasiticus (Brown et al., 2008, 2009). When cultured for about 2 weeks, the number of mature black sclerotia formed was highest in the MEA medium, and some nuclei tended to be formed in the PD medium (FIG. 9). This is thought to be because the expression of SclR under the amyB promoter was strongly induced by maltose contained in malt extract. In addition, when comparing the nucleation in the dark and aerated conditions at 30 ° C and 25 ° C, there was little change in PD and MCA media. Then, conidia formation was suppressed and an increase in the number of mycorrhizal formation was observed. Under the same conditions, sclerotia was hardly formed in any medium at 20 ° C or lower.
Based on the above results, in order to promote sclerotia formation and induce sexual reproduction, and to evaluate the effect of SclR on the mycelial fusion ability of A. oryzae, culture conditions at 25 ° C, dark place, and aeration using MEA medium It was decided to use.

Phenotypic analysis using SclR overexpressing strains In Aspergillus spp., There is a negative correlation between conidia formation and fungal nucleation in A. flavus etc. (Brown et al., 2008, 2009) . In the ΔpyrG (uridine / uracil auxotrophic) strain, when the conidia formation amount was compared between the SclR overexpressing strain and the control strain, the control strain showed about 7 times more conidia in the PD medium than the SclR overexpressing strain. Formed. In addition, the SclR overexpression strain had a lower colony formation rate. The decrease in colony formation rate was more pronounced with CD medium. In the CD minimal medium using glucose as the carbon source, the growth of the SclR-overexpressing strain was slightly reduced compared to the control strain at the early stage of growth (30 ° C, 3 days culture), and in the CD minimal medium using maltose as the carbon source. It was observed that the growth of the SclR-overexpressing strain was remarkably reduced compared to the control strain at the same early stage of growth (FIG. 10). Similar results were also obtained in the ΔadeB (adenine-requiring) strain, but the number of nuclei formed by the SclR overexpressing strain of ΔadeB was slightly decreased compared to the ΔclrG SclR overexpressing strain. There were many things that did not mature from sclerotia to black sclerotia. The adenine biosynthetic pathway is inhibited in the middle of the process, and adenine auxotrophy is conferred. It is considered that this adenine biosynthetic pathway may affect the change in the color of the sclerotia.

Effect of SclR overexpression on nucleation on the boundary of strains with different auxotrophy
Boundary-specific nuclei formation Among the SclR overexpressing strains and control strains prepared above, ΔpyrG has uridine / uracil requirement and nuclei are visualized with red fluorescence, ΔadeB has adenine requirement A cell line whose nuclei were visualized with green fluorescence was used. The strains having different auxotrophy in the MEA medium, and the SclR overexpressing strains and the control strains were face-to-face cultured at 25 ° C. in a dark place at aeration (FIG. 11A). After about 7 days of culture, each strain begins to form black or white sclerotia in the colony. Then, the formation of microbial nuclei specific to the boundary line of strains having different auxotrophy was observed after about 14 days of culture (FIG. 11B, red line). The microbial nuclei formed on the boundary line were observed to form later than the microbial nuclei formed inside the colony, and many of them were slightly larger in size. In addition, the sclerotia formed inside the colony matured black in about 7 days in the initial culture, whereas the nuclei specifically formed on the boundary remained white even after long-term culture for more than 1 month. Met.

Effect of SclR overexpression on borderline-specific nucleation In FIG. 11B, a difference was observed in the number of nuclei formed between the borderline between SclR overexpressing strains and the borderline between control strains (FIG. 11B, (In red line). Therefore, the number of fungal nuclei specifically formed at the boundary line in the combination of strains shown in FIG. 12 was compared. As a result, compared to the combination of the control strains, the number of bacterial nuclei formed specifically in the boundary line was significantly increased in the case of the overexpression strain or the combination containing either of the overexpression strains (FIG. 12).

Complementation of auxotrophy within the sclerotia formed specifically at the borderline
Confirmation of complementation of auxotrophy in CD minimal medium <br/> Unlike the sclerotia formed in the colonies of each strain, the sclerotia formed specifically on the borderline of strains with different auxotrophy Are closely intertwined with each other's hyphae. It is also expected that some of the mycelia of strains with different auxotrophy are fused inside the mycorrhiza. If the mycelium and cells in these mycelium are fused to form heterokaryons between mycelia of strains having different auxotrophy, the possibility that the auxotrophy is complemented is predicted. Therefore, when the sclerotia specifically formed between the overexpressing strains and between the control strains was shifted to an agar CD minimal medium not containing uridine / uracil and adenine, colonies and hyphae centered on the shifted sclerotia. Growth was observed (Figure 13). In addition, the mycorrhiza shifted from the inside of the colonies of each strain did not show hyphal growth (FIG. 13). From this, it was suggested that the mycelium was fused in the sclerotia formed on the boundary line or in the process of nucleation. However, a difference was observed in the number of fungal nuclei that showed hyphal growth after the medium shift among the overlying strains and the control strains among the bacterial nuclei formed at each boundary line (FIG. 13).

Therefore, 8 bacterial nuclei formed specifically on the borderline were randomly selected per plate and shifted to agar CD minimal medium without uridine / uracil and adenine. Then, the mycelial fusion efficiency was evaluated by using as an index how many of the eight fungal nuclei that were replaced became capable of hyphal growth by complementation of auxotrophy. As a result, the number of fungal nuclei considered to have undergone mycelial fusion within the fungal nucleus or in the process of forming the fungal nucleus may be significantly increased by about 20% in other combinations compared to the control strain x control strain combination. Suggested (Figure 14).

Observation of nuclei with fluorescence microscope Among SclR overexpressing strains and control strains prepared, strains that have uridine / uracil requirement with ΔpyrG and nuclei are visualized with red fluorescence, those with adenine requirement with ΔadeB and nuclei with green fluorescence The strain that is visualized is used. In the previous section, the complementation of auxotrophy was confirmed, suggesting that mycelial fusion was carried out inside the sclerotia formed on the boundary line of strains with different auxotrophy or in the process of formation of sclerotia. . Of the sclerotia formed specifically on the borderline of the auxotrophic strains by the combination of the mating type and the combination of the SclR overexpressing strain and the control strain, uridine / uracil and adenine are not included in FIG. The mycelium that grew on the agar CD minimal medium was observed with a fluorescence microscope. As a result, in most mycelia, the color of the nucleus in one mycelium was observed with both red and green fluorescence (FIG. 15).

Strains used, medium and transformation

[Used strain]
Escherichia coli
E. coli DH5α (supE44 ΔlacU169 (Ф80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1) was used to obtain the E. coli recombinant plasmid.

Aspergillus oryzae
A list of A. oryzae strains used in this study is shown in Table 1. NSPlD1 strain (niaD ^- sC ^- adeA ^- ΔargB :: adeA ^- ΔligD :: argB ΔpyrG :: adeA) (Maruyama and Kitamoto, 2008) was used as a parent strain for transformation.

[Used medium]
[For E. coli DH5α]
LB medium : 1% Bacto tryptone, 0.5% Yeast Extract, 0.5% NaCl (The plate agar medium contains 2.0% Agar. If necessary, kanamycin or ampicillin was added to a final concentration of 50 mg / ml. .)
SOC medium : 2% Tryptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ , 20 mM Glucose (mixed and then filter sterilized)

[For A. oryzae]
The following were added to each medium according to the auxotrophy of each strain.
Uridine / uracil requirement; 0.5% uridine + 0.2% uracil
Adenine requirement; 0.01% adenyl sulfate
DPY medium : 2% Dextrin, 1% Polypeptone, 0.5% Yeast Extract, 0.5% KH ₂ PO ₄ , 0.05% MgSO ₄ .7H ₂ O (pH 5.5)
CD medium : 0.3% NaNO ₃ , 0.2% KCl, 0.1% KH ₂ PO ₄ , 0.05% MgSO4 · 7H2O, 0.002% FeSO4 · 7H ₂ O, 2% Glucose or Maltose (pH 5.5)
M medium : 0.2% NH ₄ Cl, 0.1% (NH ₄ ) ₂ SO ₄ , 0.05% KCl, 0.05% NaCl, 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ .7H ₂ O, 0.002% FeSO ₄ .7H ₂ O, 2% Glucose (pH 5.5)
M + Met medium : 0.2% NH ₄ Cl, 0.1% (NH ₄ ) ₂ SO ₄ , 0.05% KCl, 0.05% NaCl, 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ .7H ₂ O, 0.002% FeSO _4. 7H ₂ O, 2% Glucose, 0.15% methionie (pH 5.5)
PD medium : “Nissui”, a potato dextrose agar medium for fungi, manufactured by Nissui Pharmaceutical Co., Ltd. was prepared according to the usage method (composition: 39.0 g, potato leachate powder 4.0 g · Glucose 20.0 g · Agar 15.0 g).
MCA medium
5% Gerber mixed grain cereal (Gerber Products Co., Freemont, Michigan), 2.0% Agar
MEA medium
8.0% Malt Extract (Oriental Yeast Industry Co., Ltd. Bio Division Microorganism Medium, Medium Malt Extract), 2.0% Agar, 0.002% CuSO ₄ , 0.00004% Na ₂ B ₄ O ₇ / 10H ₂ O, 0.00087% FePO ₄ , 0.00095% MnSO ₄ , 0.0008% Na ₂ MoO ₄ , 0.008% ZnSO ₄ (pH 6.0)

[Gene manipulation]
[Plasmid preparation]
The primers used in this study are shown in Table 2, and the plasmids are shown in Table 3. Of these plasmids, pgHHDRS, pgHHGS, and pgABpG were prepared using the MultiSite Gateway ™ cloning system.

pgHHDRS
Plasmid pgHHDRS for labeling nuclei with red fluorescence is 5 'entry clone (pg5'PH), center entry clone (pgH2B), 3' entry clone (pg3'DRM-CF) and AosC marker as a selection marker for transformation Destination vector (pgDSO) containing LR was mixed and prepared.
pgHHGS
Plasmids for labeling nuclei with green fluorescence are pgHHGS, 5 'entry clone (pg5'PH), center entry clone (pgH2B), 3' entry clone (pg3'E) and AosC marker as a selection marker for transformation. A destination vector (pgDSO) containing the mixture was mixed to prepare an LR reaction.

pgABpG
AdeB gene upstream 1.0 kb is amplified with primers attB4-5aB-F and attB1-5aB-R, and adeB gene downstream 1.0 kb is amplified with primers attB2-3aB-F and attB3-3aB-R. By inserting the amplified product into the 5 'entry vector (pDONR P4-P1R) by BP reaction, the 5' entry clone (pg5'AB4) and the amplified product of the DNA fragment downstream of the adeB gene are converted into the 3 'entry vector (pDONR P2R-P3 3 'entry clone (pg3'AB4) was prepared by inserting into) by BP reaction. Furthermore, 5 'entry clone (pg5'AB4), 3' entry clone (pg3'AB4), center entry clone (pgEpG) and destination vector (pDEST R4-R3) were mixed to perform LR reaction. Thus, the plasmid pgABpG for adeB gene disruption was prepared. A gene fragment for adeB gene disruption was amplified by PCR using pgABpG as a template and primers attB4-5aB-F and attB3-3aB-R.

[PCR conditions]
For cloning of each gene, PrimeSTAR (TaKaRa) was used as a polymerase. Chromosomal DNA of A. oryzae RIB40 strain was used as a template. In addition, KOD FX Neo (TOYOBO) was used for colony PCR of A. oryzae. The composition of the reaction solution was in accordance with the attached instructions.

Prime STAR temperature conditions
98 ℃ 2 min, {98 ℃ 10 sec, (annealing temperature) 15 sec, 72 ℃ 1 kb / min; 30 cycle}, 4 ℃
KOD FX Neo temperature conditions
94 ℃ 5 min, {98 ℃ 10 sec, (annealing temperature) 30 sec, 68 ℃ 2 kb / min; 45 cycle}, 4 ℃
As a template, 2 μl of a platinum loop suspension in 50 μl TE per 20 μl scale was used.

[Preparation of plasmid DNA]
E. coli plasmid DNA was prepared using an alkaline SDS method or a kit from Promega (Cat. No. A1222).

[DNA sequence]
An order sequence was made by Fasmac.

[Transformation of E. coli]
The following method was used for transformation using the MultiSite Gateway (trademark) system. For other transformations, the method of Hanahan (Hanahan, 1983) was followed.

Transformation for obtaining plasmids produced with the MultiSite Gateway system
Add 5 μl of enzyme reaction solution (BP reaction and LR reaction) to a 100 μl competent cell, mix, leave on ice for 30 minutes, incubate at 42 ° C for 1 minute, and quickly transfer to ice.
Add 250 μl of SOC medium and incubate at 37 ° C for 1 hour with shaking, then apply to LB selective medium (LB + kanamycin or ampicillin). Colonies are obtained by overnight culture at 37 ° C.

[A. oryzae transformation method]
Protoplast PEG method The parent strain is cultured with shaking in 100 ml of DPY liquid medium at 30C for 18 to 20 hours, and the cells are collected using Miracloth (CALBIOCHEM).
The collected cells are washed with sterilized water and treated with 10 ml TF Solution 1 (1% Yatalase (TaKaRa), 0.6 M (NH ₄ ) ₂ SO ₄ , 50 mM Maleate buffer (pH 5.5)) at 30 ° C for 3 hours. To protoplast.
Protoplasts are collected using Miracloth, 10 ml TF Solution 2 (1.2 M Sorbitol, 50 mM CaCl ₂ , 35 mM NaCl, 10 mM Tris-HCl (pH 7.5)) is added, and the precipitate is collected by centrifugation. In the same way, wash the protoplasts twice more and suspend in TF Solution 2 to 1.0-5.0 × 10 ⁷ cells / ml.
Add 10 μl of 1-5 μg / 10 μl of DNA for transformation to 200 μl of protoplast suspension, and leave on ice for 30 minutes.
Add TF Solution 3 (60% PEG4000, 50 mM CaCl ₂ , 10 mM Tris-HCl (pH 7.5)) in three portions of 250, 250, and 850 μl, and let stand at room temperature for 20 minutes.
After adding 5 ml of TF Solution 2 and centrifuging, the pellet was resuspended in 500 μl of TF Solution 2, and the suspension was pre-warmed at 45 ° C. Top agar (1.2 M Sorbitol, 0.8% Agar-containing selective medium) ) And layer on the lower layer medium (selective medium containing 1.2 M Sorbitol, 1.5% Agar). The transformant is obtained by culturing at 30 ° C. for 3-7 days, and the trait is stabilized by inoculating it about 1 to 3 times with a selective medium.

[Southern blotting analysis]
25 μg of the chromosomal DNA of the collected transformant was treated with a restriction enzyme overnight, and then electrophoresed on a 0.8% agarose gel. Subsequent operations were performed according to the instructions using ECL direct nucleic acid labeling and detection systems (Healthcare Bioscience) and Positively charged Nylon transfer membrane Hybond-N + (Healthcare Bioscience). For detection of the band, a lumino image analyzer LAS-1000plus (Fujiflim) was used. Transfer was carried out overnight, and dry heat fixation of the membrane was carried out at 100 ° C. for 2 hours, prehybridization 1 hour and hybridization 8 hours or overnight.

[Mycelium fusion experiment]
Confrontation culture on agar medium
10 ³ pieces / 5 μl of conidia of different mating type strains were inoculated on the MEA medium at intervals of 4 cm. After culturing at 25 ° C, dark place, and aeration conditions for 10 to 14 days, sclerotia formed on the boundary line of strains with different auxotrophy is sterilized in CD minimal medium without uridine / uracil adenine The mycorrhiza was shifted with tweezers and the subsequent growth was observed. After the shift, the mycelium of the strain that had been cultured at 30 ° C. for about 3-5 days and showed mycelia growth was observed for the color of the nucleus at red and green wavelengths using a fluorescence microscope.

[Microscopic observation method]
[Used equipment]
In the observation of EGFP fluorescence and mDsRed fluorescence with a confocal laser microscope, an IX71N inverted microscope (Olympus) equipped with an objective lens Neofluor 40x, 100x (Olympus) and a confocal scanner system CSU22 (Yokogawa) was used. The analysis software was Andor iQ (Andor Technology PLC). In addition, an Andor iXon cooled digital charge-coupled-device camera (Andor Technology PLC) was used to acquire a fluorescent image.
[Culture method]
A glass base dish (IWAKI Glass-base dish) was sterilized by UV irradiation for 15 minutes or more, and then 100 μl of the medium was dropped to inoculate conidia or mycelium. Thereafter, the glass base dish was sealed with parafilm, and stationary culture was performed.

When sexual reproduction is confirmed for a strain classified as an incomplete bacterium, such as Aspergillus oryzae, among the Aspergillus genus cell lines, the fusion efficiency of the cell line can be increased by using the method of the present invention. It becomes possible to increase or facilitate confirmation of fusion. Therefore, the present invention greatly contributes to the development of a technique for efficiently breeding a strain having excellent phenotypes of a plurality of strains in Aspergillus oryzae cells such as Aspergillus oryzae.

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Claims (9)

Two types of Aspergillus oryzae cell lines (mycelia) are cultured oppositely, and are the same by fusion of the two types of Aspergillus oryzae cell lines in the nuclei specifically formed on the boundary line or in the formation process of the sclerotia A method for producing a fusion mycelium that is a heteronuclear coexistence body (heterokaryon) in which nuclei from different cell lines are present in cells,
The two types of Aspergillus oryzae cell lines are transformants that can be distinguished from each other by different phenotypes, and at least one of the two types of Aspergillus oryzae cell lines expresses the nucleation promoting factor SclR. Said method, characterized by increasing the fusion efficiency of said two types of Aspergillus oryzae cell lines by increasing. The following DNA (a), (b), (c), (d) or (e) linked under the control of a promoter:
(A) DNA comprising the base sequence represented by SEQ ID NO: 1,
(B) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence of (a), and has the function of increasing the fusion efficiency of the Aspergillus oryzae cell line DNA encoding a protein,
(C) a DNA comprising a nucleotide sequence having a sequence homology of 90% or more with the DNA comprising the nucleotide sequence of (a), which encodes a protein having a function of increasing the fusion efficiency of an Aspergillus oryzae cell line ,
(D) DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, or
(E) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (d), and encodes a protein having a function of increasing the fusion efficiency of an Aspergillus oryzae cell line DNA to
The method according to claim 1, wherein the expression of the nucleation promoting factor SclR in the cell line is increased by transforming an Aspergillus oryzae cell line with a plasmid vector comprising The method according to claim 1 or 2, wherein the Aspergillus oryzae cell line (mycelium) is fused in an opposing culture under a condition of 25 ° C, dark place and aeration using MEA medium. And further comprising identifying heterokaryon formation in the fused cell based on the phenotype being complemented in the fused cell and / or the fused cell having two phenotypes. Item 4. The method according to any one of Items 1 to 3. The method according to any one of claims 1 to 4, wherein the phenotype is based on expression of a transformation marker and / or a labeling substance. 6. The method of claim 5, wherein the phenotype based on the transformation marker is auxotrophic. The method according to claim 6, wherein the phenotype is complemented in the fused cells by culturing in an agar CD minimal medium. The method according to claim 5, wherein the labeling substance is a fluorescent protein. The method according to claim 8, wherein the fused cells are confirmed to have two labeling substances by observing nuclei with a fluorescence microscope.