JPH07274966A - Ornithine carbamoyl transferase gene - Google Patents

Abstract

PURPOSE:To provide an ornithine carbamoyl transferase gene useful for the construction of a host-vector system by an arginine-requiring variant of Pleurotus ostreatus to facilitate the breeding of Pleurotus ostreatus. CONSTITUTION:This ornithine carbamoyl transferase gene is constructed of a DNA coding for the amino acid sequence of formula. The gene can clone a DNA fragment coding for OCT from a restriction enzyme-treated fragment of the chromosome DNA of Pleurotus ostreatus.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is directed to the basidiomycete oyster mushroom ( Pl.
a novel ornithine carbamoyl transferase (hereinafter referred to as "O") obtained from Eurotus ostreatus.
(Abbreviated as CT) gene and recombinant DNA containing the same. OCT is an enzyme that converts ornithine to citrulline in the arginine biosynthetic pathway in living organisms.

Pleurotus ostreatus is an edible mushroom that is in high demand,
Since it grows quickly and is advantageous for cultivation, it has been attempted to add a new function by improving the variety. Therefore, in oyster mushrooms that have not had an effective selection marker by the enzyme gene, a novel host-vector system was constructed using an arginine-requiring oyster mushroom mutant strain in which the gene was disrupted by UV irradiation or NTG treatment. It can be expected that breed improvement will be easy.

[0003]

2. Description of the Related Art So far, no host-vector system has been developed which uses edible mushrooms such as oyster mushrooms which grow rapidly. Furthermore, little genetic analysis of oyster mushrooms has been done. Therefore, the present inventors focused their attention on the OCT gene in constructing a novel oyster mushroom host-vector system. Several cases have been reported in which the base sequence has been determined by OCT other than basidiomycetes.

For example, regarding the mammalian OCT nucleotide sequence, the human OCT gene [Horwick (Horwick
k) et al .: Science, 224, 10
68], mouse OCT gene [Veres]
Et al .: Science, 237, 41
5], rat OCT gene [Takigut
i) et al .: Proceeding Natural Academic Society of USA (Proc.Na)
tl. Acad. Sci. USA) 81,7412],
Regarding the eukaryotic OCT gene, the koji mold (Uphall et al .: Molecular General, Genetics (Mol. Gen. Genet.) 20)
4,349], black koji mold [Buckston (Buxto
n) et al .: Gene 60, 255] 5-carbon sugar-assimilating yeast [Skrzypek et al .: Yeast (Yeas)
t) 6,141], baker's yeast [Vanhufer (Va
n Huffel) et al .: European Journal of Biochemistry (Eur. J. Bioche)
m. 205, 33], and Escherichia coli [Bencini et al .: Nucleic Acid Research (Nucleic. Aci) for the prokaryotic OCT gene.
ds. Res. ) 11,8509] and the like are already known.

However, no basidiomycete has been reported. Moreover, some host-vector systems using basidiomycetes have been reported [A. Munoz-Rivas, e
tal. , Mol. Gen. Genet. , 205, 1
03, (1986)], it has not yet been put to practical use.

[0006]

Accordingly, the present invention is intended to provide a novel gene having an effective control region necessary for the development of a host-vector system using the basidiomycete oyster mushroom.

[0007]

Means for Solving the Problems The present inventors have focused on the OCT gene of Pleurotus ostreatus and cloned a DNA fragment encoding OCT from a chromosomal DNA restriction enzyme-treated fragment of Pleurotus ostreatus to complete the present invention. Accordingly, the present invention provides an ornithine carbamoyl transferase gene containing a promoter region encoding the amino acid sequence of SEQ ID NO: 1.

The invention further provides a hybrid DNA, such as a plasmid, having the above-described DNA fragment, an origin of replication, preferably a bacterial origin of replication, and at least one selectable marker that is functional in the host, preferably in bacteria. To do. The invention further provides a host transformed with the above hybrid DNA, for example a bacterial host, in particular E. coli.

[0009]

[Detailed Description] The chromosomal DNA donor used in the present invention is oyster mushroom, for example, IFO6515 strain. To prepare chromosomal DNA from the strain, the method of Yelton et al. [Proc. Natl. Acad. Sci. US
A, 81, 1470 (1984)], etc.
The NA extraction method can be used.

Next, the obtained chromosomal DNA is treated with an appropriate restriction enzyme and partially digested, and then a fragment fraction of 12 kbp to 20 kbp is obtained by sucrose density gradient ultracentrifugation. The DNA fragment obtained above is inserted into a phage DNA treated with a restriction enzyme that produces the same sticky ends.
Examples of the phage DNA include EMBL3 [AM,
Frishauf, et al. J. Mol. Bio
l. 170,827 (1983)] is used. After insertion, packaging in vitro, chromosome D
Create an NA library. For subcloning, a commonly used cloning vector, preferably an Escherichia coli cloning vector, such as pBluescript cloning vector is used.

In the isolation of the gene from the chromosomal DNA library obtained as described above, there is no example of purifying OCT from basidiomycete and no example of determining the nucleotide sequence of the enzyme gene. A highly conserved region was determined from the amino acid sequences of OCT derived from other organisms that have been reported so far, and synthetic primers and Aspergillus nidu were prepared from them.
The present invention has been completed by performing plaque hybridization using the OCT gene pSa143 of L. ) as a probe.

The isolated fragment containing the oyster mushroom OCT chromosomal gene is represented by the restriction map shown in FIG. The above fragment is inserted into a suitable cloning vector, preferably a bacterial cloning vector, especially an E. coli cloning vector, such as the pBluescript cloning vector. The amino acid sequence deduced from the obtained DNA sequence was highly homologous to the amino acid sequences of ARGB of Aspergillus oryzae and OCT derived from other species, and a carbamoyl group-binding region specific to OCT was also recognized. Therefore, the chromosomal DNA fragment isolated this time is Pleurotus ostreatus IFO6.
515) OCT gene.

Further, the chromosomal OCT gene is divided by three introns as compared with OCT derived from other species, and two Ts are located upstream of the translation initiation point (188 bp).
There were ATA-box-like sequences and three CAAT-box-like sequences.

[0014]

EXAMPLES The present invention will be described in detail below with reference to examples. Example 1 . Preparation of Chromosomal Gene Library Agar pieces (diameter 5 mm) were punched out from plate agar culture of oyster mushroom (IFO6515 strain) with a cork borer, and GMY medium (Malt Extract 1%, glucose 0.
4%, yeast extract 0.4%, adjusted to pH 5.6 with phosphoric acid) to 200 ml, and cultivated at 28 ° C. for 7 days with rotary shaking. After collecting the cells, the cells were washed with 1 L of sterilized water and frozen with liquid nitrogen.

5 g of the frozen cells were crushed using a mortar.
The crushed cells were transferred to a centrifuge tube, and Lysis buffer solution (10
0 mM Tris (pH8), 100 mM EDTA, 100 mM
10 ml of NaCl and proteinase K were added so that the concentration would be 100 μg / ml), and the mixture was kept at 55 ° C. for 3 hours. After that, phenol extraction and chloroform extraction were performed, and ethanol was gradually added to the extracted aqueous layer portion to obtain DN.
When A was deposited, the chromosomal DNA was wound with a glass rod and suspended in a TE solution.

100 μg of the obtained chromosomal DNA was partially digested with the restriction enzyme Sau3AI and fractionated by 5-25% sucrose density gradient ultracentrifugation (22,500 rpm, 16 hours), and the 12-20 kbp fragment fraction was ethanol. It was recovered by precipitation. Toyobo Co., Ltd. Phage λ EMBL3-Bam
1 μg of HI arm and DNA50 after ethanol precipitation
0 ng was ligated by reacting at 16 ° C. for 16 hours in a ligation buffer containing 2.5 units of T4 DNA ligase. The obtained phage DNA was treated with STRATAGENE.
Packaged using Gigapack Gold II,
A chromosomal DNA library was created.

The DNA library was transformed into E. coli KW251.
The strain was infected and allowed to form plaques on LB agar plates. As a result, 5 × 10 ⁷ plaques / μg insert DNA could be obtained. Escherichia coli KW251 strain was infected with a phage so that 1,000 to 2,000 plaques would appear on a Petri dish with a diameter of 90 mm, and the gene was cloned.

Example 2 From chromosomal DNA library
Isolation of the OCT gene of Escherichia coli The selection of clones containing the ornithine carbamoyl transferase gene from the chromosomal DNA library was carried out by conventional plaque hybridization [Sambr
Look et al., "Molecular Cloning"
A Laboratory Manual / 2nd E
section (1989)]. That is, when the plaque was about 0.5 to 1 mm in size, a nylon membrane manufactured by Amersham was placed on the plate for 2 minutes to transfer the phage to the membrane. Repeat the same operation,
Two membranes were made from one plate.

The membrane was treated with a denaturing solution (0.5N NaO).
H, 1.5M NaCl) after alkaline denaturation, neutralization solution (0.5M Tris (pH 7.5), 1.5M NaCl)
Was twice neutralized, washed with a 2 × SSC solution, air-dried, and then UV-irradiated to fix the phage DNA to the nylon membrane. As probes for plaque hybridization, the following two synthetic oligomers and pSa143 radiolabeled with ³² P
One was used as a probe.

[0020]

[Chemical 1]

Subsequently, the two membranes were separately separated from each other by pre-hybridization solution (5 × SSC, 1 × Denhardt's solution, 1% SDS, denatured 100 μg / ml calf thymus DN.
The mixture was shaken in A) at 68 ° C. overnight, and one of the three probes was added to each membrane at 1 × 10 ⁶ cpm and hybridized at 40 ° C. for 24 hours. The membrane after hybridization was washed twice with 2 × SSC, air-dried, and then autoradiographed with an intensifying screen according to a conventional method. For one probe, those hybridizing at the same position on the two membranes prepared from the same plate were selected as quasi-positive clones.

This operation was carried out for each of the above three probes, and those showing quasi-positive in two or more were selected as positive clones. As a result, 2 positive clones could be selected from about 30,000 plaques. Phage DNA prepared by the plate lysate method from the positive clones was digested with various restriction enzymes according to a conventional method, and Southern hybridization was performed using the above-mentioned synthetic oligomer as a probe. As a result, a single DN was included in the fragment obtained by digestion with the restriction enzyme EcoRI.
As an A band, a clone hybridizing to 10 kbp was recognized.

The above 10 kbp EcoRI fragment was cut out by agarose gel electrophoresis and the E. coli vector pBl was cut out.
It is subcloned into the EcoRI site of uescriptIIKS ⁺ and transformed into Escherichia coli JM109 strain. Subcloned DNA was prepared in large quantities and purified by ultracentrifugation (50,000 rpm, 16 hr, 20 ° C),
The base sequence was determined. Determination of nucleotide sequence is United
This was performed using a Sequenase kit manufactured by States Biochemical.

The results are shown in SEQ ID NO: 2. Obtained D
The amino acid sequence deduced from the NA sequence is AR of koji mold.
High homology was observed with the amino acid sequences of OCT derived from GB and other species, and a carbamoyl group-binding region peculiar to OCT was also observed. Therefore, the chromosome D isolated this time
NA fragment is oyster mushroom (Pleurotus ostre)
atus IFO6515).

Further, the chromosomal OCT gene is divided by three introns by comparison with OCT derived from other species, and two TATA-box-like sequences and three CAATs are located upstream of the translation initiation point (188 bp). -Box
There was such a sequence. The obtained chromosome OCT
E. coli transformants containing the gene coli JM10
9 / pBSHR1 has been deposited as FERM P-14264 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

[0026]

[Sequence list]

SEQ ID NO: 1 Sequence length: 359 Sequence type: Amino acid Origin Biological name: Pleurotus ostrea
tus) Co., Ltd. Name: IFO6515 feature 1-359 P mat peptide sequences of SEQ: Met Ala Ala Ala Val Pro His Leu Met Thr Leu Ala Asp Leu Ser 5 10 15 Val Pro Gln Ile His Arg Ile Leu Val His Ser His Tyr Leu Lys 20 25 30 Gln Ile Ser Leu Pro Trp Leu Gln Pro Gly Lys Leu Trp Lys Gln 35 40 45 Lys Pro Ser Gln Ser Leu Asp Lys Lys Thr Val Ala Leu Leu Phe 50 55 60 Ser Lys Arg Ser Thr Arg Thr Arg Val Ser Ala Glu Thr Ala Ala 65 70 75 Thr Leu Leu Gly Gly Gln Ala Leu Phe Leu Gly Lys Glu Asp Ile 80 85 90 Gln Leu Gly Val Asn Glu Ser Pro Arg Asp Thr Ala Arg Val Ile 95 100 105 Gly Gly Met Cys Gln Gly Ile Phe Ala Arg Val Gly Asp His Ser 110 115 120 Glu Ile Glu Glu Leu Ala Lys Tyr Ser Pro Val Pro Val Leu Asn 125 130 135 Ala Leu Ser Ser Leu Trp His Pro Thr Gln Ile Leu Ala Asp Leu 140 145 150 Leu Thr Leu His Glu His Ala His Leu Phe Ser Pro Asn Asn Val 155 160 165 Thr Glu Tyr Lys Ser Ile Asp Asp Lys Arg Trp Thr Glu Leu Pro 170 175 180 Asp Val Arg Pro Leu Thr Val Ala T yr Val Gly Asp Ser Ala Asn 185 190 195 Val Leu His Asp Met Leu Val Thr Tyr Pro Arg Phe Gly His Ser 200 205 210 Val Ala Ile Ala Ser Pro Glu Asn Glu Arg Tyr Arg Ala Pro Asn 215 220 225 Gly Val Trp Asp Arg Val Val Glu Leu Glu Cys Asp Lys Lys Ile 230 235 240 Trp Trp Gly Ala Asp Pro Lys Glu Ala Val His Gly Ala Asp Val 245 250 255 Val Val Thr Asp Thr Phe Ile Ser Met Gly Gln Glu Ala Glu Lys 260 265 270 Glu Glu Arg Leu Arg Asp Phe Gln Gly Tyr Gln Val Thr Glu Gln 275 280 285 Leu Trp Lys Asp Gly Gly Ala Asn Pro Asp Trp Lys Phe Leu His 290 295 300 Cys Leu Pro Arg Lys Pro His Glu Val Asp Asp Glu Val Phe Tyr 305 310 315 Gly Pro Arg Ser Leu Val Phe Pro Glu Ala Asp Asn Arg Lys Trp 320 325 330 Thr Ile Met Ser Leu Phe Asp Leu Leu Phe Gly Lys Trp Asn Leu 335 340 345 His Ser Asp Lys Ile Thr Ala Ala Tyr Ala Leu Asp Arg Pro 350 355

SEQ ID NO: 2 Sequence length: 1452 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: genomic DNA Origin organism name: Pleurotus ostrea
tus) strain name: features of IFO6515 sequence 188-1429 P CDS 557-611 intron 1179-1232 intron 1307-1362 intron sequence: AAACTGTCGA AGCAGCAATG AAAAGTACGC ACGGTGCAGG ACTCGGTTAT 50 GTGCGAATGG GATATTCAAT GAACTGGAGG CAATTCAGTG GACCGGCGCT 100 AATTTGACCA CTTGTTGCTT TTCTCAGTTC TGTTCCATTC GACAGTAACT 150 TCGACGTTCC GGCAACTCAA ATATCCAGGA CTCGACCATG GCAGCCGCAG 200 TTCCCCACTT GATGACTCTT GCGGACCTTT CCGTTCCTCA AATTCATCGT 250 ATTCTTGTTC ATTCGCATTA CCTGAAGCAA ATCTCACTTC CGTGGCTGCA 300 GCCTGGCAAA CTGTGGAAAC AAAAGCCCTC GCAGTCACTG GACAAAAAGA 350 CTGTCGCTCT GCTGTTCTCA AAGCGAAGCA CTCGGACACG CGTCAGTGCC 400 GAAACAGCCG CGACACTCCT CGGGGGACAA GCTCTGTTTT TAGGCAAGGA 450 AGACATCCAG CTGGGCGTCA ACGAGAGCCC AAGGGATACT GCAAGGGTTA 500 TTGGGGGGAT GTGCCAGGGT ATCTTCGCGA GGGTCGGTGA TCACAGCGAG 550 ATTGAGGTCG TACCTTAGCC CGTTTTCCCT CGTTTCTGGA ACTGATACCG 600 TCTCTGAGTA GGAACTAGCC AAGTACTCGC CTGTGCCCGT GTTG AACGCG 650 CTGTCCTCCT TGTGGCACCC AACCCAGATA TTGGCAGACC TACTGACACT 700 GCACGAGCAC GCGCATCTTT TTAGCCCAAA CAATGTCACG GAATACAAGT 750 CCATCGACGA CAAGCGGTGG ACCGAGCTCC CAGACGTCCG CCCTCTCACA 800 GTCGCGTACG TAGGCGACTC GGCAAATGTG TTGCACGACA TGCTAGTGAC 850 CTATCCGCGA TTCGGGCATT CCGTAGCCAT CGCGAGCCCT GAGAATGAAC 900 GGTACCGTGC GCCGAACGGT GTTTGGGACC GTGTAGTGGA GCTGGAGTGT 950 GACAAGAAGA TTTGGTGGGG GGCTGACCCC AAGGAAGCGG TTCATGGCGC 1000 TGATGTCGTT GTCACGGATA CGTTTATATC AATGGGCCAG GAGGCGGAGA 1050 AGGAGGAAAG GCTGAGGGAT TTCCAAGGAT ATCAAGTCAC GGAACAACTT 1100 TGGAAGGACG GCGGTGCTAA CCCCGACTGG AAGTTCCTGC ACTGCCTGCC 1150 GCGGAAGCCT CACGAGGTTG ACGACGAGGT TCGGTTTCAC TGCACATTCA 1200 GTCAGGGATG CACTAATTTC ATCGACGCAT AGGTCTTCTA TGGCCCGAGG 1250 TCGCTGGTAT TCCCTGAAGC TGACAACCGA AAATGGACCA TCATGTCCCT 1300 CTTTGAGTGA GTAGAACAGT TGCTCCTGCA CTCTTCGACG AAGACTTGCT 1350 GACCTGCTTT AGTCTCCTGT TCGGAAAGTG GAATCTTCAC TCAGACAAAA 1400 TAACTGCTGC ATACGCTCTT GATCGTCCTT GATGATCAGC GCACACCAAA 1450 GT 1452

[Brief description of drawings]

FIG. 1 is a restriction map of a DNA fragment containing the ornithine carbamoyltransferase gene of the present invention.

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[Procedure amendment]

[Submission date] July 4, 1994

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 1

[Correction method] Change

[Correction content]

[Figure 1]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1: 645)

Claims (2)

[Claims] 1. An ornithine carbamoyl transferase gene consisting of DNA encoding the amino acid sequence of SEQ ID NO: 1. 2. A vector comprising the gene according to claim 1.