WO2000037647A1

WO2000037647A1 - Abc transporter and genes encoding the same

Info

Publication number: WO2000037647A1
Application number: PCT/JP1999/007079
Authority: WO
Inventors: Sohei Kanno; Eiichiro Kimura; Kazuhiko Matsui; Tsuyoshi Nakamatsu
Original assignee: Ajinomoto Co., Inc.
Priority date: 1998-12-18
Filing date: 1999-12-16
Publication date: 2000-06-29
Also published as: AU1687800A; JP2002293797A

Abstract

ABC transporter constituents of Brevibacterium lactofermentum having the amino acid sequences represented by SEQ ID NOS: 8, 9 and 10; and DNAs encoding the same. These DNAs are usable in breeding corynebacteria.

Description

Specification

TECHNICAL FIELD The present invention relates to a novel ABC transporter and a gene encoding a protein which is a component thereof. The gene can be used for breeding microorganisms having altered cell membrane transport of amino acids. There are several known mechanisms by which substances such as amino acids and ions can penetrate cell membranes. One of them is the ATP binding cassette, a superfamily one (ABC transporter). It is known (CF Higgins, Ann. Rev. Cell Biol., 8, 67 (1992)).

The ATP-binding cassette is a group of proteins having an ATP-binding domain including a transmembrane domain, and its physiological function is mainly the uptake of substances into cells, but it is also involved to some extent in the excretion of substances. Is believed to be Bacteria often contain membrane proteins (membrane components), proteins that are inside the membrane and have ATPase activity, and binding proteins that are outside the membrane and bind to transported substances. The membrane protein and the protein having ATase activity form a multimeric complex. It is said that the substance emission system lacks a binding protein that binds to the substance to be transported (Reizer, J. et al., Prot. Sci. 1, 1326 (1992)).

Since the ABC transporter or its components are involved in the transport of substances, it is thought that by modifying the expression of these genes, it is possible to modify the properties of the cells relating to the substance transport.

In bacteria such as Escherichia coli, the structure of various ABC transport genes has been analyzed, and it is known that each gene encoding a component of the ABC transporter forms an operon. However, in coryneform bacteria, amino acids Many of the genes encoding the ABC transporter and its components related to membrane transport are unknown. DISCLOSURE OF THE INVENTION The present inventors aimed to breed coryneform L-glutamic acid-producing bacteria by using an enzyme involved in one of the L-glucaminic acid biosynthesis pathways, such as glumine-mino-oxoglurate-aminotransferase (glutamate The gene coding for “GO GAT” was cloned. In the process, they accidentally found that the DNA fragment containing the gene encoding the GOGAT (gltBD) contained the gene encoding the ABC transporter, which is thought to be involved in amino acid transport. Reached.

That is, the present invention relates to proteins that are components of the ABC transporter, and DNAs that encode them.

The first component of the ABC transporter of the present invention is a protein represented by the following (A) or (B).

(A) A protein having the amino acid sequence described in SEQ ID NO: 8 in the sequence listing.

(B) In the amino acid sequence of SEQ ID NO: 8 in the sequence listing, the amino acid sequence comprises one or several amino acid substitutions, deletions, insertions, additions, or inversions, and comprises an ABC transporter. The proteins that make up one.

The second component of the ABC transporter of the present invention is a protein represented by the following (C) or (D).

(C) A protein having the amino acid sequence described in SEQ ID NO: 9 in the sequence listing.

(D) an amino acid sequence represented by SEQ ID NO: 9 in the sequence listing, which comprises an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids; A protein having an ATPase activity.

The third component of the ABC transporter of the present invention is a protein represented by the following (E) or (F).

(E) a protein having the amino acid sequence of SEQ ID NO: 10 in the sequence listing,

(F) In the amino acid sequence of SEQ ID NO: 10 in the sequence listing, one or several A protein comprising an amino acid sequence containing amino acid substitutions, deletions, insertions, additions, or inversions, and constituting an ABC transporter.

The present invention also provides a DNA encoding a protein that is a component of each of the above ABC transporters.

The invention further provides an operon encoding the ABC transporter. Hereinafter, the present invention will be described in detail.

The DNA of the present invention has been found as an ORF existing in the vicinity of the g1tBD gene from Brevibacterium and Lactophamentum, and can be obtained as follows.

Brevibacterium lactofermentum, such as Brevibacterium lactobacillus ATCC13869 chromosomal DNA, is Escherichia coli K — 12 (Gene, Vol. 60, 1: L: 1 1987) and yeast (Saccharomyces' Celepiche, GenBank accession No. X89221). A DNA fragment of about 1.4 kb is obtained by PCR (polymerase chain reaction) using a primer having the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing. Pre-Bacterium 'Lacto Amentum ATCC13869 is available from the ATCC (American Type Culture Collection: 20852, United States, Maryland 20852, Rockville, Park Live 10301) be able to.

Next, the PCR-amplified fragment obtained as described above was used as a probe, and a colony hybridization of the chromosomal DNA library of Brevibacterium 'lactofermentum ATCC13869 was performed.The DNA fragment to be hybridized to the probe was subjected to colony hybridization. By obtaining, the DNA of the present invention can be obtained together with the gitBD gene. When chromosomal DNA cut with Hindi II is used for preparing a chromosomal DNA library, the above-mentioned DNA fragment can be obtained as a fragment having a size of about 14 kb.

The above DNA fragment contains the g1tBD gene, downstream of which g There are two open reading frames (ORFs) opposite to the 1 t BD gene. These ORFs correspond to the second and third ORFs in the base sequence shown in SEQ ID NO: 7.

As shown in Examples described later, there is a possibility that the two ORFs form an operon together with another ORF existing upstream of these two ORFs. This ORF corresponds to the first ORF in ΔRF contained in the nucleotide sequence shown in SEQ ID NO: 7. The first ORF has a chromosomal DNA of Brevibacterium lactofamentum, for example, Brevibacterium lactofamentum ATCC13869, and has a base sequence having the nucleotide sequence shown in SEQ ID NO: 5 in the sequence listing and the sequence in the sequence listing. It can be obtained as a DNA fragment of about 1.8 kb by PCR using a primer having the nucleotide sequence shown in No. 6. This DNA fragment has a region presumed to be a promoter region upstream of the target ORF.

The nucleotide sequence shown in SEQ ID NO: 7 links the above-described nucleotide sequence (1.3 kb) in the approximately 14 kb DNA fragment with the nucleotide sequence (1.1 kb) in the approximately 1.8 kb DNA fragment. It was done.

Since the nucleotide sequence and the nucleotide sequence of the adjacent region of each ORF have been clarified, it can also be obtained by PCR using oligonucleotides prepared based on those nucleotide sequences as primers. .

Methods for preparing chromosomal DNA, preparing chromosomal DNA libraries, hybridization, PCR, preparing plasmid DNA, cutting and ligating DNA, transforming, and setting oligonucleotides used as primers are known to those skilled in the art. The usual methods well known in the art can be employed. These methods are described in Sambrook, J., Frisch, E.F., Maniatis, T., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1.21 (1989) and the like.

The second ORF and the amino acid sequence encoded thereby were compared with a known sequence for homology. The databases used were EMBL and SWISS-PR0T. As a result, these sequences were found to have homology to the previously reported ATPase protein constituting the ABC transporter responsible for amino acid transport and the gene encoding the same, as shown in Table 1. The three ORFs including this are operons May form, Table 1

The genes encoding the components of the ABC transporter of the present invention may be substituted or deleted by one or several amino acids at one or more positions, as long as the properties of each encoded protein are not impaired. ATP binding tan with insertion, addition or inversion It may be one that encodes protein. Here, "several" differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein. It is derived from the fact that some amino acids, such as isocyanate isine and palin, have highly related amino acids, and such amino acid differences do not significantly affect the three-dimensional structure of the protein.

DNA encoding a protein substantially the same as the components of the ABC transporter as described above can be obtained by substituting, deleting, inserting, adding, or substituting an amino acid residue at a specific site by, for example, site-directed mutagenesis. Alternatively, it can be obtained by modifying the nucleotide sequence to include an inversion. The modified DNA as described above can also be obtained by conventionally known mutation treatment. Examples of the mutation treatment include a method in which DNA encoding each protein is treated in vitro with hydroxylamine, etc., and a method in which a microorganism having a DNA encoding each protein, such as a bacterium belonging to the genus Escherichia, is irradiated with ultraviolet light or Examples of the method include treatment with a mutagen that is commonly used for mutagenesis, such as N, -nitro-N-nitrosoguanidine (NTG) or nitrous acid.

In addition, such substitutions, deletions, insertions, additions, or inversions of bases as described above include mutations that occur naturally, such as those based on individual differences of microorganisms carrying each component and differences in species and genera. mutant or variant) is also included.

By expressing the DNA having the above mutation in an appropriate cell and examining the properties of the expression product, a DNA encoding a protein substantially identical to the components of the ABC transporter can be obtained. In addition, a DNA encoding each protein having a mutation or a cell carrying the same, or a base sequence encoding each component or a probe prepared from the same base sequence. A nucleotide sequence consisting of nucleotide numbers 1117 to 1725 in SEQ ID NO: 7 or a probe prepared from the nucleotide sequence, which encodes a protein that hybridizes under stringent conditions and has properties of each component. Isolation of DNA also provides DNA that encodes a protein substantially identical to the components of the ABC transporter. The term “stringent conditions” used herein refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Say. Although it is difficult to quantify these conditions clearly, one example is that DNAs with high homology, for example, DNAs with homology of 40% or more, hybridize and have lower homology 60 ° C, 1 XSSC, 0.1% SDS, preferably 0.1 lxSSC, 0.1% SDS, which is the condition under which DNA does not hybridize with each other, or the conditions for washing ordinary Southern hybridizations. Conditions for hybridization at the corresponding salt concentration are mentioned.

Some of the genes that hybridize under these conditions include those with a stop codon in the middle and those that have lost their activity due to mutations in the active center, but these are expressed using a commercially available activity expression vector. It can be easily removed by characterizing the product.

The DNA encoding the components of the ABC transporter of the present invention and the operon of the ABC transpouse (hereinafter, these may be simply referred to as “genes of the present invention”) are used for breeding coryneform bacteria. Can be used for That is, since the ABC transport protein of the present invention or its component is considered to be involved in amino acid transport, the expression of these genes is modified to modify the properties of cells for amino acid transport. It is thought that it can be done.

Coryneform bacteria to which the present invention can be applied include bacteria that were previously classified into the genus Brevipacterium, but are now integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 1, 255 ( 1981)), and also includes bacteria of the genus Brevibacterium closely related to the genus Corynebacterium. Examples of such coryneform bacteria include the following.

Corynebacterium acetoacidophilum

Corynebacterium

Corynebacterium alkanolicam

Corynebacterium carnaye

Corynebacterium guru mimic

Corynebacterium lilium (Corynebacterium .gulpus micam) Corynebacterium melasecola

Corynebacterium ■ Thermoaminogenes Escherichia coli Herculis

Brevibacte) um Divaricatam (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium -Guruyumicam) Brevibacte) ゥ mimmariofilam

Brevibacte ') lactofermentum (corynebacterium gulumicum)

Brevi Bacte J. Rosem

Brevibacterium Saccharolyticum

Brevibacterium choge squirrel

Brevi Bacte 'J ゥ Album

Brevibacterium Serinum

Microbacterium Ammonia Filam

Specifically, the following strains can be exemplified.

Corynebacte 'J-Amcetacetofilam ATCC 13870

Corynebacterium acetoglutamicum ATCC 15806

アルアルアルアル力力 CC ATCC2 15 1 1

Dream Carnaye AT C C 1599 1

Corynebacterium glutamicum ATCC 13020, 13032, 3

060

Corynebacterium Jumiumium (Corynebacterium glutamicum) AT C C 15990

Corynebacte J ゥ me Merasecora ATCC 17965

π 'J ゥ m Thermoaminogenes AJ 1 2340 (FERM BP

-1539)

Corynebacterium, Hercullis ATCC 13868

Previbacterium divaricatum (Corynebacterium glutamicum) AT CC 14020

Brevipacterium 'Flavum (Corynebacterium' glutamicum) AT CC 13826, ATCC 14067 Brevipacterium immariophyllum ATCC 14068 Brevipacterium lactofermentum (corynebacterium glutamicum): AT CC 13665, AT CC 13869,

Brevi pacificum / mouth theme ATCC 13825

Brevi Pacterium Saccharolity Cam A T C C 14066

Brevi Pacterium Choge Niyu Squirrel ATCC 19240

Brevibacterium · Album AT C C 15 1 1 1

Brevi Pacterium Serinum ATCC 15 1 12

Micropacterium, ammonia phyllum ATCC 15354

Methods for modifying the genes encoding the ABC transporter or its components include amplification or disruption of these genes. Amplification of a gene or the like can be performed by transforming a coryneform bacterium with a recombinant vector obtained by ligating a gene to a vector such as a plasmid. At that time, the efficiency of amplification can be increased by using a multi-copy type vector. Examples of such vectors include plasmids that can autonomously replicate in coryneform bacteria, such as the following.

AM 330 See JP-A-58-67699.

p HM 15 19 See JP-A-58-77895

p A J 655 Special Edition 58—See 192900

p A J 6 1 1 Same as above

p A J 1844 Same as above

pCG 1 See JP-A-57-134500

See pCG 2 Special Publication 58-35197

See pCG 4 Special Publication 57-183799

pCG 1 1 Same as above

Transformation of a coryneform bacterium can be performed by an electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791).

Gene amplification can also be achieved by allowing the gene of the present invention to exist in multiple copies on the chromosomal DNA of the host. Target on chromosome DNA of coryneform bacteria In order to introduce a gene in multiple copies, homologous recombination is performed by utilizing a sequence present in multiple copies on chromosomal DNA (Experiments in Molecular Genetics, Cold Spring Harbor Laboratory press (1972); Matsuyama, S and Mizushima, S., J. BacterioL, 162, 1196 (1985)). In addition, as a sequence present in multiple copies on chromosomal DNA, it is possible to use reactive DNA and inverted repeats present at the end of a transposable element. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, a gene of interest can be mounted on a transposon, transferred and transfected into chromosomal DNA in multiple copies.

In addition, the expression of a gene that is originally present on a chromosome can also be modified by replacing an expression control sequence such as a promoter with a strong or weak function.

On the other hand, gene disruption by homologous recombination has already been established, and can be performed by a method using linear DNA or a method using temperature-sensitive plasmid. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically with reference to examples.

(1) Cloning of gltBD gene of Brevibacterium lactofermentum ATCC 13869

A region with high amino acid sequence homology was selected between the g1tB gene products of Escherichia coli and yeast, the nucleotide sequence was deduced from the sequence, and the oligonucleotides shown in SEQ ID NO: 1 and SEQ ID NO: 2 were synthesized. Separately, chromosomal DNA of Brevipacterium 'lactofermentum AT CC 13869 was prepared using the Bacterial Genomic DNA Purification Kit (manufactured by Advanced Genetic Technologies Corp.). Using this chromosomal DNA as type I, PCR was performed under standard reaction conditions described on page 8 of PCR Technology (Henry and Erich, Stockton Press, 1989) using the oligonucleotide as a primer. Was done. Agarose gel electrophoresis of the PCR product revealed that a DNA fragment of approximately 1.4 kb had been amplified. Was.

The nucleotide sequences at both ends of the obtained DNA were determined using the oligonucleotides shown in SEQ ID NO: 1 and SEQ ID NO: 2. For DNA sequencing, use the DNA Sequencing Kit

(Applied Biosystems) using the method of Sanger (J. Mol. Biol., 143, 161).

(1980)). When the determined nucleotide sequence was translated into an amino acid sequence, and compared with the amino acid sequence expected from the g1tB gene of E. coli and yeast, the homology was high. It was determined to be part of the gltB gene of Brevibacterium lactofermentum ATCC 13869. Using this PCR-amplified DNA fragment as a probe, a fragment obtained by cutting the chromosome DNA of Brevibacterium 'lactofarmentum ATCC 13869 prepared by the above method with EcoRI, BamHK Hindll Pstl, Sail (Takara Shuzo) by a standard method, Southern hybridization was performed using DIG DNA Labeling and Detection Kit (manufactured by Behringer Mannheim). As a result, it was found that a fragment of about 14 kb cut with Hindlll hybridized with probe DNA. Therefore, the agarose gel electrophoresis of the Hindi II fragment of brevipacterium 'lactofermentum ATCC 13869 chromosomal DNA prepared by a standard method was performed, and a DNA fragment of about 10 kilobases or more was recovered using glass powder, and the recovered DNA fragment was recovered. PMW219 (manufactured by Takara Shuzo) digested with restriction enzyme Hindlll (manufactured by Takara Shuzo) was ligated using a ligation kit (manufactured by Takara Shuzo) to obtain Escherichia coli KM.

Transformation was performed using 109 combi-tent cells (Takara Shuzo). Transformants were treated with I PTG (isopropyl-I /?-D-thiogalactopyranoside) 10〃g / m

1, X-Gal (5-bromo-14-cloth-3-indolyl /?-D-galactoside) L medium containing 40 zg / ml and kanamycin 25 / g / ml (pact tryptone 10 g / l, Pactoist extract 5 g / l, NaC 15 g / l, agar agar 15 g / l, pH 7.2) After culturing overnight, the white colonies that appear are picked up, separated into single colonies, and approximately 1,000 Was obtained.

The obtained transformant was prepared by the alkaline method (Biotechnology Experiment Book, edited by the Society of Biotechnology, Japan, 1

(P. 05, Baifukan, 1992). SEQ ID No. 3 and SEQ ID No. 3 were prepared based on the portion of the DNA sequence used as the probe whose base sequence was determined. PCR was performed under the above conditions using the above-mentioned plasmid as a primer and a synthetic oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 4 as a primer. Using this primer, Brevipacterium, Lactofamentum ATCC 13869 A transformant having a plasmid capable of obtaining an amplified fragment having a size of about 1.3 kilobases, which is the same as the DNA fragment amplified when PCR was performed using the chromosome as type I, was selected. (2) Determination of the entire nucleotide sequence of the DNA fragment containing the Brevibacterium lactofermentum ATCC 13869 1 tBD gene and isolation of the ABC transporter overnight gene

Plasmid DNA prepared from the transformant obtained in the above (1) by an alkaline method contained a DNA fragment of about 14 kilobases derived from the chromosome of Brevibacterium lactofermentum ATCC 13869. In the same manner as described above, the entire nucleotide sequence of a DNA fragment of about 14 kilobases derived from the chromosome of Brevipacterium lactofermentum ATCC 13869 of the obtained plasmid was determined. As a result, the obtained DNA fragment contained the entire length of the g1 tBD gene, but an open reading frame of 500 bp or more was directed downstream from the end of the g1 tBD gene. It was found that there was a sequence that was presumed to be one minute and one minute downstream of these open reading frames. However, since the open reading frame lacked the promoter region, the upstream portion was cloned as follows.

Brevipacterium lactofermentum ATCC 13869 LA PCR in vitro cloning Kit (Takara Shuzo) from a DNA fragment digested with the restriction enzyme BamHI using the primers shown in SEQ ID NO: 5 and SEQ ID NO: 6. Cloning was performed using). As a result of performing PCR using the above primer, a MA fragment of about 1.8 kilobases was amplified. The nucleotide sequence of this DNA fragment was determined in the same manner as described above. As a result, the amplified DNA fragment contains an open reading frame of about 350 amino acids located upstream of the above two open reading frames, and a promoter region further upstream. It was also found that there was an area estimated to be. Thus, these three open reading 'frames could be operons. The nucleotide sequence of these open reading frames is as shown in SEQ ID NO: 7 in the sequence listing. The sequence of SEQ ID NO: 7 also shows the amino acid sequence of the product deduced from the nucleotide sequence. Of these, base numbers 1 to 1101 are the first open reading frame, 1117 to 1725 are the second open reading frame, and 1759 to 2367 are the third open reading frame. The methionine residue at the N-terminus of the protein encoded by each open 'reading' frame is derived from the initiation codon ATG, and is often unrelated to the original function of the protein. It is well known that it is removed by the action of a peptidase, and it is possible that methionine residues have been removed also in the case of each of the above proteins. In addition, since the promoted evening area and the evening / mine / one-night sequence estimated here are the result of analysis using a computer, an open 'reading' frame is actually upstream or downstream. Exists and may be expressed together.

The homology was compared with the known sequence for each of the nucleotide sequence and the amino acid sequence. The data bases used were EMBL and SWISS-PR0T. As a result, it was revealed that the DNA shown in SEQ ID No. in the sequence listing and each protein encoded therewith were novel genes and proteins in the genus Corynebacterium. Of these, the second open reading frame and the protein encoded by it have high homology to the previously reported ATP-binding protein of ABC transporter and the gene encoding it, and coryneforms. In Pacterium bacteria, it was found to be a gene encoding a novel ATP binding protein. INDUSTRIAL APPLICABILITY The present invention provides the components of the ABC transporter of Brevipacterium lactofarmentum and the DNA encoding them. The gene of the present invention can be used for breeding coryneform bacteria.

Claims

The scope of the claims

1. A protein shown in (A) or (B) below.

(A) a protein having the amino acid sequence of SEQ ID NO: 8 in the sequence listing,

(B) the amino acid sequence described in SEQ ID NO: 8 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids; and The proteins that make up one.

2. DNA encoding the protein shown in (A) or (B) below.

(A) a protein having the amino acid sequence of SEQ ID NO: 8 in the sequence listing;

(B) the amino acid sequence described in SEQ ID NO: 8 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids; and Proteins that make up.

3. The DNA according to claim 2, which is a DNA shown in (a) or (b) below.

(a) DNA containing a base sequence consisting of base numbers 1 to 1101 of SEQ ID NO: 7 in the sequence listing.

(b) hybridizes under stringent conditions to a probe prepared from the base sequence consisting of base numbers 1 to 1101 or the base sequence of SEQ ID NO: 7 in the sequence listing, and encodes a protein constituting the ABC transporter DNA.

4. The DNA according to claim 3, wherein the stringent condition is a condition in which washing is performed at 60 ° C. at a salt concentration corresponding to 1 XSSC and 0.1% SDS.

5. Protein shown in (C) or (D) below.

(C) a protein having the amino acid sequence of SEQ ID NO: 9 in the sequence listing;

(D) In the amino acid sequence of SEQ ID NO: 9 in the sequence listing, the amino acid sequence comprises substitution, deletion, insertion, addition, or inversion of one or several amino acids, and comprises an ATPase of ABC transport A protein having activity.

6. DNA encoding the protein shown in (C) or (D) below. (C) a protein having the amino acid sequence of SEQ ID NO: 9 in the sequence listing;

(D) In the amino acid sequence of SEQ ID NO: 9 in the sequence listing, the amino acid sequence comprises one or several amino acid substitutions, deletions, insertions, additions, or inversions. A protein having one ATPase activity.

7. The DNA according to claim 6, which is a DNA shown in (c) or (d) below. (c) DNA containing a base sequence consisting of base numbers 1117 to 1725 of SEQ ID NO: in the sequence listing.

(d) hybridizing under stringent conditions with a nucleotide sequence consisting of nucleotide numbers 11 17 to 1725 of SEQ ID NO: 7 in the sequence listing or a probe prepared from the nucleotide sequence, and DNA encoding a protein with ATPase activity of the transporter.

8. The DNA according to claim 7, wherein the stringent condition is a condition in which washing is performed at 60 ° C. at a salt concentration corresponding to l × SSC and 0.1% SDS.

9. A protein shown in (E) or (F) below.

(E) a protein having the amino acid sequence of SEQ ID NO: 10 in the sequence listing;

(F) In the amino acid sequence of SEQ ID NO: 10 in the sequence listing, the amino acid sequence comprises one or more amino acid substitutions, deletions, insertions, additions, or inversions, and the ABCC transport sequence is included. The proteins that make up one.

10. DNA encoding the protein shown in (E) or (F) below.

(F) In the amino acid sequence of SEQ ID NO: 10 in the sequence listing, the amino acid sequence comprises one or several amino acid substitutions, deletions, insertions, additions, or inversions, and is an ABCC transport sequence. The proteins that make up one.

1 1. The DNA according to claim 10, which is a DNA represented by the following (e) or (f).

(e) DNA containing a base sequence consisting of base numbers 175 to 2367 of SEQ ID NO: 7 in the sequence listing. (f) a nucleotide sequence consisting of nucleotide numbers 1759 to 2367 of SEQ ID NO: 7 in the sequence listing or a probe prepared from the nucleotide sequence and hybridizing under stringent conditions, and constituting an ABC transporter DN A to code.

12. The DNA according to claim 11, wherein the stringent condition is a condition in which washing is performed at 60 ° C. at a salt concentration corresponding to 133 ° and 0.1% SDS.

13. A base sequence encoding a protein having the amino acid sequence of SEQ ID NO: 8, a base sequence encoding a protein having the amino acid sequence of SEQ ID NO: 9, and a protein having the amino acid sequence of SEQ ID NO: 10. DNA containing a coding base sequence.

14. The DNA according to claim 13, which has the nucleotide sequence of SEQ ID NO: 7.