JP2000308491A - Pyrithiamin-resistant marker gene and transformant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the new subject marker gene comprising a pyrithiamin- resistant marker gene having a specific base sequence and expressing pyrithiamin resistance, and useful for creating a transformant using Aspergillus filamentous fungus and the like as a host cell. SOLUTION: This new pyrithiamin-resistant marker gene comprises a DNA comprising a base sequence shown by the formula or a DNA comprising a base sequence in which one or several bases are substituted, deleted or added in the base sequence shown by the formula and expressing a similar pyrithiamin resistance as that of the DNA shown by the formula. The marker gene is useful as a selection marker for effectively selecting a transformant in breeding Aspergillus oryzae and the like in which Aspergillus filamentous fungus is major and utilized for production of a fermented food or various kinds of useful substances. The marker gene is obtained by culturing Aspergillus oryzae in a thiamine-free medium, selecting a pyrithiamin-resistant strain after treating the generated conidium with a mutagenic agent and constructing and screening of its genomic library.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pyrithiamine resistance marker gene comprising a novel drug resistance gene, a transformant using this gene as a host of Aspergillus fungi and the like, and a method for producing the same.

[0002]

2. Description of the Related Art Aspergillus, mainly a filamentous fungus belonging to the genus Aspergillus, is widely used for producing fermented foods such as sake, shochu and soy sauce, or for producing various useful substances. In the breeding of filamentous fungi of the genus Aspergillus used for these industries,
Genetic engineering techniques are extremely effective in imparting desirable traits, and require selectable markers that can be used to efficiently select transformants.

[0003] Drug-resistant genes which can be used as dominant markers for transformants in filamentous fungi include phelom of Penicillium roqueforti.
ycin resistance gene (J. Biotccol 1996 0ct. 18; 51 (1):
97-105), Penicillium Icelandium (Penici)
llium islandicum) benomyl resistance gene (curr. Gene
t. 1995 Nov .; 28 (6) 580-4), Aspergillus niger (hereinafter abbreviated as A. niger).
Hygromycin B resistance gene (Gene. 1987; 56 (1): 11)
7-24) and oligomycin resistance gene (Curr. Genet. 1988 Ju
l; 14 (1): 37-42), Aspergillus nidulans (Asp
ergillus nidulans Hereafter abbreviated as A. nidulans. ) Aureobasidin A resistance gene (Japanese Unexamined Patent Publication No. 9-98
784) or Aspergillus flavus (Aspe
rgillus flavus) benomyl resistance gene (Appl. Enviro
n Microbiol 1990 Dec; 56 (12): 3686-92).

At present, Aspergillus oryzae A. Genes that complement auxotrophy are used as selectable markers in gene recombination in olise, such as argB (Agric. B
iol. Chem. 51 (9), 2549-2555, 1987), or niaD
(Gene 111 (2), 149-55, 1992 Feb 15).

[0005]

However, in order to use a selection marker that complements auxotrophy, ultraviolet irradiation,
It is necessary to produce an auxotrophic strain by mutagenesis with a mutagen or the like. And, according to this random mutagenesis, there is a high possibility that some influence will be exerted on the properties of the host strain.

[0006] Aspergillus, an important koji mold used in the brewing industry such as sake, soy sauce, miso or mirin.
Olease (Aspergillus oryzae, hereinafter abbreviated as A. oryzae) exhibits strong resistance to a wide range of drugs, and thus a drug resistance gene effective as a dominant marker has not yet been discovered.

[0007] For the above reasons, there has been a demand for A. orise for a drug resistance dominant selectable marker which can be genetically modified without affecting its properties.

Therefore, an object of the present invention is to provide a pyrithiamine resistance gene which can be used as a selection marker in genetic engineering use of a filamentous fungus of the genus Aspergillus. Another object of the present invention is to provide a transformant in which a vector containing such a drug resistance gene has been transformed into a phenotype useful for genetic analysis.

[0009]

Means for Solving the Problems The present inventors have found that a given filamentous fungus of the genus Aspergillus exhibits a sensitive sensitivity to pyrithiamine, a metabolic anti-analog of thiamine, and mutated the susceptible cells. A gene that gives pyrithiamine resistance to A. oryzae from the resistant cells by converting the cells into resistant cells (resistance gene)
And succeeded in isolating the present invention.

That is, in the first invention of the present application,
The above-mentioned problem has been solved by employing a means for setting a pyrithiamine resistance marker gene comprising the following DNA (a) or (b). (A) DNA comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing. (B) a base sequence in which one or several bases are substituted, deleted or added in the base sequence (a), and
A pyrithiamine resistance marker gene expressing the same pyrithiamine resistance as NA (a).

In the second invention of the present application, the first invention
The above-mentioned problem was solved by making a transformant which is transformed with the pyrithiamine resistance marker gene of the invention of the invention and expresses pyrithiamine resistance. In the above invention, the host transformed by introducing the pyrithiamine resistance marker gene is Aspergillus (Aspergillus).
lus) The above-mentioned problem can be solved also when the transformant is a filamentous fungus.

[0012] In a third aspect of the present invention, there is provided a method for creating a pyrithiamine-resistant transformant, comprising the following steps. (1) a step of replicating the vector containing the pyrithiamine resistance marker gene according to claim 1, (2) a step of integrating the replication vector obtained in the above step into a chromosome of a host,
(3) a step of selecting a host transformed to pyrithiamine resistance in the above step in the presence of pyrithiamine;

In the method for creating a pyrithiamine-resistant transformant, the replication vector may be a replication vector containing a heterologous gene other than the pyrithiamine-resistance marker gene shown in SEQ ID NO: 1.

The gene for imparting pyrithiamine resistance of the present invention, ie, the pyrithiamine resistance marker gene, expresses pyrithiamine resistance when integrated into the chromosome of a cell of a filamentous fungus of the genus Aspergillus. In addition, by placing a filamentous fungus of the genus Aspergillus into which a pyrithiamine resistance marker gene has been incorporated in a specific medium, a transformant having pyrithiamine resistance can be easily detected from these cells.

[0015] Such a pyrithiamine resistance marker gene is a phenotypic gene useful for genetic analysis, and is useful for genetic analysis even for a transformant having pyrithiamine resistance.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The pyrithiamine resistance marker gene of the present invention can be obtained by subjecting a pyrithiamine-sensitive cell, which is sensitive to pyrithiamine, which is a metabolite of thiamine, to mutation, as described above. This is converted into a resistant cell, and a gene (resistance gene) that confers pyrithiamine resistance to A. oryzae is isolated from the resistant cell.

Aspergillus fungi having a sensitive sensitivity to pyrithiamine include, as shown in Table 1,
A. olise, A. niger, Aspergillus kawachi (As
pergillus lutiensis mut.kawachi Below A. Abbreviated as Kawachi. ), Aspergillus aw
amori and below A. Abbreviated as Awamori. ), Aspergillus sojae (hereinafter abbreviated as A. soya), Aspergillus sojae (Aspergillus sojae)
usami mut. Shiro-usami Below A. Abbreviated as Shirosami. ) Or A. Nidoransu.

[0018]

[Table 1]

Specifically, it is sensitive to pyrithiamine
A. Olyse for Nitrosoguanidi
Ne and abbreviated as NTG. ), A genomic library is prepared for the obtained resistant strains, and a restriction enzyme map of FIG. 1 containing a pyrithiamine resistance gene (hereinafter abbreviated to ptrA) as a dominant mutation is prepared from the library. The DNA fragment to be isolated.

The pyrithiamine resistance gene (ptr
The DNA encoding A) includes SEQ ID NO: 1 in the sequence listing.
There is DNA represented by

The DN encoding ptrA of the present invention
A can be prepared by incorporating a suitable vector into a replication vector to form a replication vector, and transforming the host, whereby pyrithiamine resistance can be imparted to the host as a selection marker, and the transformant can be easily transformed by drug resistance using pyrithiamine. You can choose. As vectors for the filamentous fungi belonging to the genus Aspergillus of this replication vector, pDG3, pkBY2, pSa123, pTAex3
Can be used.

The replication vector into which the DNA encoding ptrA is incorporated can be stably retained in E. coli or the like. At this time, PUC-based vectors, Escherichia coli-Aspergillus genus fungi shuttle vectors and the like can be used as vectors.

The ptrA of the present invention is a marker gene capable of imparting pyrithiamine resistance also to mononuclear and polynuclear filamentous fungi of the genus Aspergillus, in particular, practical koji mold.
It is extremely useful for breeding A. oryzae, which is widely used for industrial production of shochu, soy sauce, and useful substances.

Furthermore, the ptrA of the present invention can be applied to koji molds other than A. oryzae, and is also useful for breeding other Aspergillus fungi and for genetic engineering applications. Further, the ptrA of the present invention can impart pyrithiamine resistance to A. olise, and the vector having DNA encoding ptrA is the first vector provided as a drug resistance imparting vector to A. olise.

[0025]

EXAMPLES [Example 1] A pyrithiamine-sensitive gene (ptrA) derived from A. oryzae was cloned in the following steps.

1-a) Pyriamine resistance of A. oryzes
A. olise H sensitive to mutant pyrithiamine at 0.1 ppm
The L-1034 strain was inoculated on a thiamine-free CD plate (Tuapec Docks medium, 1% glucose, 2% agar) and cultured at 30 ° C. for 5 days. 0.1% of conidia that have settled
After suspending in a Tween 80 solution, the suspension was filtered through a glass filter (3G3 type), and the filtrate was centrifuged at 8,000 rpm for 5 minutes to collect conidia.

The conidia were suspended in an NTG solution (4000 ppm NTG) at 2.4 × 10 ⁷ spores / ml, and the mutation was induced by reciprocal shaking at 50 rpm at 30 ° C. for 10 minutes. . The survival rate at this time was about 2.2%.

After the mutagenesis treatment, the washing solution (0.1% t
The conidia are washed three times with a ween 80 and 0.01 M phosphate buffer (pH 7.0), and a screening plate (Zapec Dox medium, 1% glucose, 1 ppm) is adjusted to 1.0 × 10 ⁷ spores / plate. (Pyrithiamine) and cultured at 30 ° C. for 4 days. Thus, six pyrithiamine-resistant strains were obtained.

Although this resistant strain exhibited resistance to 1000 ppm of pyrithiamine, it was sensitive to oligomycin, cycloheximide, amphotericin B, and clotrimazole in the same manner as the parent strain. No specific resistance to pyrithiamine.

1-b) Genomic DNA of a pyrithiamine-resistant strain
Preparation of Ibrary Genomic DNA was extracted and purified from the pyrithiamine-resistant strain PTR-26, which has particularly good growth, by the following method. That is, YPD liquid medium (0.5% yeast ext., 1.0%
Mycelia cultured overnight at 30 ° C. with bact pepton (2% dextrin) were collected with a glass filter (3G1 type) and washed with distilled water. The cells were dehydrated, frozen with liquid nitrogen, and ground using a mortar. After suspending the crushed cells in a TE solution (10 mM Tris-HCl (pH 8.0), 1 mM EDTA),
Equivalent lysis solution (2% SDS, 0.1 M NaCl, 10 mM ED
TA, 50 mM Tris-HCl (pH 7.5)). After standing at room temperature for 1 hour, the mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was recovered. An equal volume of phenol / chloroform / isoamyl alcohol (25/24/1) was added, the tube was slowly stirred up and down, and then centrifuged at 3000 rpm for 5 minutes to collect the upper layer. Add 2.5 volumes of -20 ° C ethanol,
After leaving at −80 ° C. for 10 minutes, the mixture was centrifuged at 3500 rpm for 15 minutes, and the precipitated DNA was dried. An RNase solution (1 μ / ml RNaseGS (Takara Shuzo), TE solution) was added, and the mixture was kept at 37 ° C. for 1 hour. An equal volume of phenol / chloroform / isoamyl alcohol was added, and the mixture was stirred slowly and centrifuged at 15000 rpm for 5 minutes to recover the upper layer.
1/10 amount of 3M sodium acetate (pH 5.2) and 2.5
A double volume of ethanol at -80 ° C was added, and the mixture was allowed to stand at -80 ° C for 10 minutes.
A was recovered.

10 μg of the purified genomic DNA was used as a restriction enzyme
After partial digestion treatment with 0.156 U of Sau3A I at 37 ° C. for 1 hour, the protein was removed with phenol / chloroform / isoamyl alcohol and precipitated with ethanol. Partially degraded DNA
Was subjected to 0.8% agarose electrophoresis to extract and purify DNA of a 4 to 10 kb region. PDHG25 vector completely digested with the obtained DNA and BamHI [gene, Vol. 98, No. 61]
Page-67 (1991)] using a DNA ligation kit (Takara Shuzo), and transformed Escherichia coli DH5 to prepare a genomic library of resistant strains.

Escherichia coli containing this genomic library was transformed into an LB medium containing 100 ppm ampicillin (1% bactotryptone, 0.5% bactoeast extract, 0.5%
After culturing overnight at 37 ° C. in 50 ml of sodium chloride (PH 7.2), the plasmid was recovered from Escherichia coli and purified.

1-c) Pyritiamine resistance gene (ptr
Expression and cloning of A) A plasmid derived from a genomic library of a pyrithiamine-resistant strain prepared as described above was prepared by the following method.
Transformation was performed into Nidorans FGSC A89 strain.

That is, A. After culturing Nidrance in a thiamine-free CD medium or the like with shaking at 30 ° C. for 2 days, hyphae were collected by filtration through a glass filter (3Gl type) and washed with sterile water. After sufficient dehydration, 10 cells
The suspension was then suspended in ml of a protoplast solution [20 mg / ml yatalase (Ozeki Shuzo), 0.8 M NaCl, 10 mM sodium phosphate buffer, pH 6.0]. The reaction was carried out at 30 ° C. for about 3 hours while shaking slowly. Glass filter 3G3
The protoplasts in the filtrate filtered in the above were collected by centrifugation at 2000 rpm for 5 minutes, and washed twice with 0.8 M NaCl. Protoplasts so that ^{2 × 10 8 / ml Sol 1} (0.
_{8M NaCl, 10mM CaCl 2, 10mM} Tris -HCl, pH
8.0) and then 0.2 volume of So12 (40%
(W / v) PEG4000, 50mM CaC1 2, 50mM Tris-HCl
, PH 8.0) and mixed well. 0.2 ml of the protoplast suspension was dispensed, 10 μg of the genomic library-derived plasmid was added, and mixed well. 30 at ice temperature
After standing for 1 minute, 1 ml of So12 was added and mixed well. 1 at room temperature
Let stand for 5 minutes, add 8.5 ml of Soll, mix well, then add 2 ml
Protoplasts were collected by centrifugation at 000 rpm for 5 minutes. Add 0.2 ml of Soll and add a minimum medium containing 2 ppm of pyrithiamine (Czapek Dox medium, 1% glucose, 0.8 M NaCl, 20 ppb biotin, 2% agar)
And placed on the center of a soft agar medium (Zapec Dox medium, 1% glucose, 0.8M NaCl,
5 ml of 20 ppb biotin, 2 ppm pyrithiamine, 0.5% agar) was overlaid. The cells were cultured at 30 ° C for 5 to 7 days.

The colonies grown on this plate were considered to have a plasmid containing the pyrithiamine resistance gene, and about 6 colonies formed on the pyrithiamine-containing medium. CD containing 2 ppm pyrithiamine
+ Bi medium (Czapek Dox medium, 1% glucose,
20ppb biotin), incubate at 30 ° C for 2 days,
Total DNA was recovered and purified from the grown cells according to the DNA extraction and purification method described in Example 1-b). This DNA
Was transformed into E. coli DH5 and applied to an LB medium containing 100 ppm ampicillin. Plasmid DNA was prepared from the resulting E. coli colonies.

This plasmid contained 2 kb of DNA and was named p142-13. A restriction map of the DNA fragment containing the pyrithiamine resistance gene ptrA is as shown in FIG. This fragment was subcloned into the pBIISK + vector and named pptrAEH. Its base sequence is as shown in SEQ ID NO: 1.

Here, as a result of comparing the ptrA and the base sequence of the wild-type gene of ptrA cloned from the pyrithiamine-sensitive strain HL-1034 which is the parent strain of PTR-26 based on the sequence, ptrA showed 670 in SEQ ID NO: 1. It was confirmed that the second adenine was substituted with guanine. From this, it was presumed that this single base substitution was the cause of resistance to pyrithiamine.

[Example 2] Transformation using Aspergillus filamentous fungus as host 2-a) Aspergillus using free plasmid
Transformation of filamentous fungi Plasmid p142-13 or pD obtained in Example 1-c)
The A. HG25 vector was transformed into A. coli by the transformation method described in Example 1-c). Olyse or A. Introduced to Nidolance. After placing on the center of a minimal medium (Zapec Dox medium, 1% glucose, 0.8M NaCl, 2% agar) containing each concentration of pyrithiamine, the soft agar medium containing each concentration of pyrithiamine (Zapec) was kept at 45 ° C. 5 ml of Dox medium, 1% glucose, 0.8 M NaCl, 0.5% agar) was overlaid. After culturing at 30 ° C. for 5 to 7 days, a pyrithiamine-resistant strain was obtained.

As shown in Table 2, A. Olise, A. Transformants with p142-13 in both Nidorans can grow at all concentrations of pyrithiamine. On the other hand, pDHG
Transformants with the 25 vector were also sensitive to 0.1 ppm of pyrithiamine. Therefore, it was confirmed that ptrA can be used as an effective selection marker for Aspergillus.

[0040]

[Table 2]

2-b) Using a chromosomal integration type plasmid
Transformation of Aspergillus filamentous fungi Plasmid pptrAEH or pB obtained in Example 1-c)
A. Using IISK + Olise, A. Nidrance or A. Niger was transformed by the method described in Example 1-c). Transformed protoplasts were prepared in Example 2.
-In the minimum medium supplemented with pyrithiamine at each concentration described in a),
After culturing at 30 ° C. for 5 to 7 days, a pyrithiamine-resistant strain was obtained.

[0042]

[Table 3]

As shown in Table 3, A.p. Olise, A. Nidrance or A. Niger transformants were able to grow at all pyrithiamine concentrations.
On the other hand, the transformant with pBIISK + was sensitive to 0.1 ppm of pyrithiamine. Therefore, ptrA
Has been confirmed to be usable as an effective selection marker for Aspergillus fungi.

[0044]

According to the invention of the present application, a pyrithiamine resistance marker gene consisting of the DNA having the nucleotide sequence represented by SEQ ID NO: 1 is provided. There is an advantage that a pyrithiamine resistance gene that can be used as a selection marker when used for genetic engineering such as genetic information analysis can be provided.

Further, the pyrithiamine resistance marker gene of the present invention can impart a drug resistance trait as a marker without affecting a host strain such as commercial Aspergillus or the like as in the case of mutation induction.

According to the invention of the present application, a transformant having introduced therein a chromosome recombination vector using pyrithiamine resistance as a selection marker, which is useful in the genetic recombination of Aspergillus spp. The resulting transformant is provided. These can be effectively used for creation, breeding, and the like of transformants for producing useful proteins.

In particular, A. Olyse is a koji mold that is not only widely used as industrial koji mold but also highly safe in genetic recombination. Therefore, A. The pyrithiamine resistance gene from Olyse is A. Breeding of olise,
It is very useful for preparing transformants for producing useful proteins.

[0048]

[Sequence list] SEQUENCE LISTING <110> Hakutsuru Shuzou Kabushiki kaisha <120> Pyrithiamine resistance marker gene and transformed cell <130> KPO5407-14 <160> 1 <210> 1 <211> 2028 <212> DNA <213> Aspergillus oryzae <400> 1 ggggatctga cagacgggca attgattacg ggatcccatt ggtaacgaaa tgtaaaagct 60 aggagatcgt ccgccgatgt caggatgatt tcacttgttt cttgtccggc tcaccggtca 120 aagctaaaga ggagcaaaag gaacggatag aatcgggtgc cgctgatcta tacggtatag 180 tgcccttatc acgttgactc aacccatgct atttaactca acccctcctt ctgaacccca 240 ccatcttctt ccttttcctc tcatcccaca caattctcta tctcagattt gaattccaaa 300 agtcctcgga cgaaactgaa caagtcttcc tcccttcgat aaacctttgg tgattggaat 360 aactgaccat cttctatagt tcccaaacca accgacaatg taaatacact cctcgattag 420 ccctctagag ggcatacgat ggaagtcatg gaatactttt ggctggactc tcacaatgat 480 caaggtatct taggtaacgt ctttggcgtg ggccggtgtt cgttcccagt catcgatgca 540 ttcacatgcc ctccctaagc tgggccctag actctaggat cctagtctag aaggacatgg 600 catcgatgga ctgggttcgt tctgagatta tacggctaaa actt gatctg gataatacca 660 gcgaaaaggg tcatgccttc tctcgttctt cctgttgatg gaatggctaa cagatgatag 720 tcattgcaac ttgaaacatg tctcctccag ctgccatcta cgaacccact gtggccgcta 780 ccggcctcaa gggtaaggtc gtggtttctg agaccgtccc cgttgaggga gcttctcaga 840 ccaagctgtt ggaccatttc ggtggcaagt gggacgagtt caagttcgcc cctatccgcg 900 aaagccaggt ctctcgtgcc atgaccagac gttactttga ggacctggac aagtacgctg 960 aaagtgacgt tgtcattgtt ggtgctggtt cctgcggtct gagcactgcg tacgtcttgg 1O20 ccaaggctcg tccggacctg aagattgcta tcgtcgaggc cagcgtctct cctggtcagt 1080 agtccatgat ggattgcctt gcactcagct ttccggaact aacgtgcaat aggtggcggt 1140 gcctggttgg gtggccaact cttttctgct atggtcatgc gccgtcccgc ggaagtcttc 1200 ctgaacgagc tgggtgttcc ttacgaagag gacgcaaacc ccaactacgt tgtcgtcaag 1260 cacgcctccc tgtttacctc gacactcatg tcgaaggttc tctccttccc caatgtcaag 1320 ctcttcaatg ctaccgctgt tgaggacttg atcacccgtc cgaccgagaa cggcaacccc 1380 cagattgctg gtgttgtcgt caactggacg ctggtcaccc ttcaccacga tgatcactcc 1440 tgcatggacc ccaacactat caacgctcct gtcatcatca gtaccactgg tcacg atggg I5OO ccattcggcg ccttctgtgc gaagcgcttg gtgtccatgg gcagcgtcga caagctaggt 1560 ggcatgcgtg gtctcgacat gaactcggcc gaggatgcca tcgtcaagaa cacccgcgag 1620 gttactaagg gcttgataat cggcggtatg gagctgtctg aaattgatgg ctttaaccgc 1680 atgggcccta ccttcggtgc catggttctc agtggtgtca aggctgccga ggaggcattg 1740 aaggtgttcg acgagcgtca gcgcgagtgt gctgagtaaa tgactcacta cccgaatggg 1800 ttcagtgcat gaaccggatt tgtcttacgg tctttgacga taggggaatg atgattatgt 1860 gatagttctg agatttgaat gaactcgtta gctcgtaatc cacatgcata tgtaaatggc 1920 tgtgtcccgt atgtaacggt ggggcattct agaataatta tgtgtaacaa gaaagacagt 1980 ataatacaaa caaagatgca agagcggctc atcgtcaccc catgatag 2028

[Brief description of the drawings]

FIG. 1. Restriction enzyme map of pyrithiamine resistance marker gene

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:66) (72) Inventor Toru Motoyoshi 4-5-5 Sumiyoshi Minamicho, Higashinada-ku, Kobe Shiraku Tsuru Shuzo Research and Development Room Co., Ltd. (72) Inventor Akira Nishimura 4-5-5 Sumiyoshi Minamicho, Higashinada-ku, Kobe Shiratsuru Sake Brewery Co., Ltd. Research and Development Room

Claims (5)

[Claims] 1. A pyrithiamine resistance marker gene comprising the following DNA (a) or (b): (A) DNA comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing. (B) a base sequence in which one or several bases are substituted, deleted or added in the base sequence (a), and
A pyrithiamine resistance marker gene expressing the same pyrithiamine resistance as NA (a). 2. A transformant which is transformed with the pyrithiamine resistance marker gene according to claim 1 and expresses pyrithiamine resistance. 3. The host transformed by introducing a pyrithiamine resistance marker gene is Aspergillus (Aspergillus).
lus) The transformant according to claim 2, which is a filamentous fungus of the genus. 4. A method for creating a pyrithiamine-resistant transformant, comprising the following steps: (1) a step of replicating the vector containing the pyrithiamine resistance marker gene according to claim 1, (2) a step of integrating the replication vector obtained in the above step into a chromosome of a host,
(3) a step of selecting a host transformed to pyrithiamine resistance in the above step in the presence of pyrithiamine; 5. The method for creating a transformant according to claim 4, wherein the replication vector is a replication vector containing a heterologous gene other than the pyrithiamine resistance marker gene according to claim 1.