JPH06319547A - Dna and gene expression vector containing the same - Google Patents

Abstract

PURPOSE:To obtain a novel DNA useful for creating a vector adapted to the expression of a gene in a basidiomycete. CONSTITUTION:The DNA has a base sequence expressed by the formula. A fragment of 1.2kbp (Hindlll-XbaI), the remaining ras gene and a fragment of 4.0kbp, cleaved with the restriction enzyme XbaI and containing the upstream side region (about 2.6kbp) are separated from a product obtained by cleaving the whole DNA of Lentinus edodes Sing. MS-LE1610 (FERM P-12960) with the restriction enzyme XbaI to afford the DNA of 2573 bp, expressed by the formula and containing the first exon (corresponding to 4 amino acids) in the whole base sequence thereof.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a DNA and a gene expression vector containing the DNA, and more particularly to a control region DNA of a basidiomycete shiitake-derived gene and a vector containing the DNA and suitable for gene expression in basidiomycetes.

[0002]

2. Description of the Related Art Conventionally, only one gene expression vector capable of expressing an exogenous gene in basidiomycetes has been developed, and the control region DNA for gene expression has an Aspergillus nidulans (Aspergillus nidulans).
ans), and the expression of foreign genes is not so strong (Barrett et al., Appl. Microbio
l. Biotechnol., 33, 313-316 (1990); Marmeisse et a
L., Curr. Genet., 22, 41-45 (1992)).

[0003]

DISCLOSURE OF THE INVENTION An object of the present invention is to develop a gene transcription expression control region DNA derived from a gene of basidiomycete shiitake, and a vector containing the DNA and adapted for gene expression in basidiomycete. is there. The present inventors proceeded with gene cloning targeting basidiomycete shiitake,
DN which directs the initiation of gene transcription from the ras homologue gene isolated from basidiomycete and priA gene, which is highly expressed in primordium
From the latter, by isolating the terminator, which is the DNA region involved in the termination of transcription, from the promoter, which is the A region, and using them, we created a novel gene expression vector that enables the expression of foreign genes in basidiomycetes. The present invention has been reached after repeated studies on a method for doing so.

[0004]

The present invention relates to a DNA having a nucleotide sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing and a vector containing the DNA and suitable for gene expression in basidiomycetes. The present inventors
edodes) from the Harvey rat sarcoma virus ra
s gene (Barbacid, Annu. Rev. Biochem., 56, 779-827
(1987)) and hybridize with 5.6 kilobase pairs (hereinafter,
Abbreviated as "kbp". ) HindIII DNA fragment and 2.6
The Ecobp DNA fragment of kbp was isolated (Figs. 1, 2, 3).
reference). The former contains the ras gene itself (Hori et a
l., Gene, 105, 91-96 (1991)) (see FIG. 2), the latter is a novel gene (named priA) (Kajiwara et al., Gene, 11
4, 173-178 (1992)) (see FIG. 3).

The DNA of the present invention and the gene expression vector containing the DNA can be obtained by the method described in Examples below. For example, by separating the promoter of the ras gene (the DNA region that directs the initiation of transcription) and the terminator of the priA gene (the DNA region involved in the termination of transcription), and inserting the foreign gene DNA between both DNA regions. A gene expression vector for Basidiomycetes that enables the expression of foreign genes can be produced.

[0006]

EXAMPLES The present invention will now be described with reference to examples. Example 1 (1) First, the total DNA of shiitake necessary for obtaining the promoter of the ras gene and the terminator of the priA gene used in the present invention was prepared by the following method. L. edodes MS-LE1610 (deposited as FERM P-12960 at the Institute of Biotechnology, Institute of Biotechnology, AIST) with SMY liquid medium (1% sucrose, 1%).
Malt extract, 0.4% yeast extract, pH 5.6) 2
Shake culture for ~ 4 weeks.

The culture solution is collected by passing through four layers of gauze, and the bacterial cells (50 g in wet weight) are washed with deionized water. Next, liquefied nitrogen was put in a cup of a Waring blender (manufactured by Sakuma Seisakusho Co., Ltd.), and cells were put in the cup little by little and frozen, and the blender was crushed by rotating at high speed until it became a powder. This crushed product was placed in a 50 ml centrifuge tube up to the half position, and 20 ml of extraction buffer (0.1 M Tris-HCl buffer (pH 8.0), 0.1 M ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA). ), 0.25 M NaCl, 100 μg / ml proteinase K (manufactured by Sigma)) and rapidly suspended. Furthermore, sodium lauryl sulfate was added so that the final concentration was 1%, and the mixture was kept warm at 55 ° C for 2 hours, then 2 mg of proteinase K was added and kept warm at 37 ° C overnight, and then 10
After centrifugation at 00 rpm for 15 minutes, an equal amount of isopropanol was added to the resulting supernatant.

[0008] The resulting filamentous DNA was wrapped around a closed Pasteur pipette, and 99.5% ethanol and 70%
Sequentially washed with 10 ml of ethanol, and 10 ml of TE buffer (10 mM Tris-HCl buffer (pH 8.0), 1 mM)
(Containing EDTA). About crude DNA solution T
E-saturated phenol extraction, then TE-saturated phenol:
CIAA (chloroform: isoamyl alcohol = 2
4: 1) [= 1: 1] extraction was performed and the DNA was precipitated with ethanol. The DNA precipitate was dissolved in an appropriate amount of TE buffer, RNase A (Boehringer Mannheim) was added to 10 mg / ml, and the mixture was incubated for 30 minutes.
After extraction with TE-saturated phenol, the DNA was ethanol-precipitated and dissolved in an appropriate amount of TE buffer to obtain a total (chromosomal and mitochondrial) DNA sample.

(2) Isolation of a 5.6 kbp HindIII fragment containing most of the coding region of the ras gene is Hori
Et al. (Gene, 105, 91-96 (1991)). The outline of this method is shown below. Restriction enzyme HindI for all DNA
Degraded with II and subjected to agarose gel electrophoresis, 5-6kb
A DNA fraction with a length of p is obtained. This was transformed into E. coli plasmid vector pUC19 (Yanisch-Perron et al., Gen.
e, 33,103-119,1985), and E. coli JM109 (Yanis
ch-Perron et al., Gene, 33, 103-119, 1985).
About the obtained Escherichia coli transformant rat sarcoma virus
Colony hybridization using the ras gene as a probe (Grunstein and Hogness, Proc. Natl. Acad. Sc
i.USA, 72,3961-3965,1975), 5.6 kbp HindIII
E. coli carrying the pUC19 recombinant plasmid carrying the fragment were selected. Next, the selected Escherichia coli is cultured in an appropriate amount, the recombinant plasmid is extracted, and digested with HindIII,
5.6 kbp HindIII when subjected to agarose gel electrophoresis
A fragment (clone 1, see FIG. 2) can be obtained. The ras gene was 5.6 kbp EcoR as shown in FIG.
The I fragment can be obtained by the same method.

(3) 2.6 kbp EcoRI containing priA gene
Fragment isolation was performed by Kajiwara et al. (Gene, 114, 173-178 (19
92)). The outline of this method is shown below. Total DNA is digested with restriction enzyme EcoRI and subjected to agarose gel electrophoresis to obtain a DNA fraction having a length of 2-3 kbp. As in the case of the ras gene, this was ligated to pUC19 and introduced into E. coli. Escherichia coli harboring the pUC19 recombinant plasmid having the 2.6 kbp EcoRI fragment was selected by colony hybridization using the rat sarcoma virus ras gene as a probe, and plasmid DNA was selected.
To extract. It is digested with EcoRI and electrophoresed to give a 2.6 kbp EcoRI fragment. The priA gene can also be obtained as a 7.8 kbp HindIII fragment as shown in FIG.

Example 2 (1) Next, a method for separating the promoter region of the ras gene will be described. Most of the ras gene is 5.6 kbp as described above.
Since it is contained in the 1.2 kbp [HindIII-XbaI] fragment of the HindIII fragment (see FIG. 2), this was used as a probe to convert the restriction enzyme XbaI digestion product of total DNA from the digested product with the restriction enzyme XbaI. , 1.2 kbp [HindIII- Xba
I] fragment and a 4.0 kbp XbaI fragment (clone 2, see FIG. 2) containing the rest of the ras gene and the upstream region (about 2.6 kbp) are isolated. Of the entire base sequence, 2573 bp containing the first exon (corresponding to 4 amino acids) is shown in SEQ ID NO: 1 in the sequence listing. The transcription start point is nucleotide numbers 2359 and 23.
Since it is specified as 64th and 2406th, the promoter region has nucleotide numbers from 1 to 252.
Use the 8th. This region is separated by the polymerase chain reaction (PCR) method using the same sequence as the 1st to 29th sequence and a sequence complementary to the 2504th to 2527th sequences as primers. 4). There are multiple specific base sequences found in various promoters in this region.

(2) Next, isolation of the terminator region of the priA gene will be described. This region is prepared by cleaving the above 2.6 kbp EcoRI fragment containing the priA gene with XbaI,
1199 bp including the termination codon TAG of the priA gene [Xba
I-EcoRI] fragment (see FIG. 3). The entire base sequence is shown in SEQ ID NO: 2 in the sequence listing. In this region, the transcription termination and poly (A) signal consensus sequence TA of Saccharomyces cerevisiae
G ... TAGT (TATGT) ... TTT (Zaret and Sherm
an, Cell, 28, 563-573 (1982)), a sequence similar to that of nucleotides 389 to 417 was found, and a poly (A) addition site exists at the 422 (or 423) position.

Example 3 (1) Next, the structure of an expression vector for an exogenous gene using the promoter of the ras gene and the terminator of the priA gene and the expression of the gene in basidiomycetes are described here by the present inventors. A particularly detailed examination of the aminoglycoside-3'-phosphotransferase gene (APHI) from E. coli transposon Tn903 (Webster and
Dickson, Gene, 26, 243 (1983)) and oyster mushroom (Pleurotus o)
streatus) is used.

(2) As shown in FIG. 4, the method for constructing a gene expression vector is as follows. First, pUC containing a 1.2 kbp [XbaI-EcoRI] fragment as a terminator of the priA gene.
The 19 plasmid was linearized with XbaI, blunted at both ends, added with a BamHI linker, and then cut with BamHI. On the other hand, BamHI linkers were added to both ends of the promoter DNA fragment of the ras gene separated by the polymerase chain reaction method, and the fragment was cleaved with BamHI. Both of the DNAs were ligated and circularized, and from the obtained plasmids, those in which the promoter and the terminator were correctly connected in the transcription direction were selected. Of the two BamHI cleavage sites of the plasmid, the one upstream of the promoter was crushed, and the plasmid was named pLC1. Next, the pUC19 plasmid having a 1.7 kbp PvuII fragment containing the APHI gene was simultaneously cleaved with XhoI and PvuII, and both ends were blunted to Bam.
After adding the HI linker, it was cleaved with BamHI. This DNA
A gene expression vector was constructed by inserting the fragment into the BamHI site of pLC1 and named pLC1-gen.

(3) Next, a strain in which pLC1-gen was isolated from the wild: hiratake KS-200 strain (Shishido, the inventor)
It is stored by Kazuo and is freely available for sale upon request. ), A transformant resistant to G418 (Geneticin) (manufactured by Sigma), which is an aminoglycoside effective for basidiomycetes, was obtained. The transformed strains do not lose their G418 resistance even when subcultured in a natural medium,
It was stable. The above means that pLC1-gen is effective as a basidiomycete expression vector.

[0016]

INDUSTRIAL APPLICABILITY According to the present invention, the ras gene promoter of Shiitake mushroom and the terminator of the priA gene of Shiitake mushroom are used to decompose, for example, wood, synthetic fibers, plastics and the like, promote fruiting body (mushroom) formation and disease resistance in basidiomycetes. It is possible to create a vector that enables information expression of a foreign gene involved in enhancement or the like.

[0017]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 2573 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Origin Biological name: Lentinus edodes Strain name: MS-LE1610

Sequence 10 20 30 40 50 60 CTAGATAGGA AAAGTATGTT GTTTATGATG AAAATGGCTC AGAATACTGC ATAATAAATA 70 80 90 100 110 120 TGAAAGTATG GAGGCGCAGAC GCCCGCCACAAG 130 140 150 160 170 180 CTGTGAACCC TGCTGTAGCA CAGC 130GC 180GCGTGAACCCA TGCTGTAGCA TAAGGAAGTA CTGAGGAATC

[0019] 250 260 270 280 290 300 TGCATATAGT AAATGACTAC ATATGACTAC ATATGACATA TTATATCACA TGATCATATT 310 320 330 340 350 360 AGTTGGCATG TGAACAGCCG CATTCAACAG ACCAATCTTT TGATATTTTG GTGAAACTGA 370 380 390 400 410 420 CCTCAACAGG CTTGAGACAA TCTCGGAGAA GTGATACATA TTTATATTTT TGACCAGGTG 430 440 450 460 470 480 TTAAATAATA TGTTCGTTGA GGTCATCTTT TGTTGATTTC TGAATAGATT TGCTTACACT

[0020] 490 500 510 520 530 540 GTCAATCATA AGTTCTACAT ATATTGAAAA ACGCGCAGTC CAGTAATTGG TATGTACCAG 550 560 570 580 590 600 GTAATTGAGC AAATCCAAAG TCGGGTGGCG TGAGCTGCAG AAGATAATGT TCTCATGAAG 610 620 630 640 650 660 AGCATTCAAG GCATAGTGGG AGGAAGGGAG GGTGGCGAGG TTACGAGGCT GTAGTTTACG 670 680 690 700 710 720 GAAGTATTGT CCTTATGTAC GGTACTTCAT ACAAAAATAA TTTCCTTTTA TGGCAGTAGT

[0021] 730 740 750 760 770 780 TTCGCCTTAT TCGGCCAAAT GTAACACTGA GTTGAATTGT ATTGAATTCT TGTGACCGGC 790 800 810 820 830 840 ACGGATATCA CGGGACTGTG ACTCTGTCAA TTATTTTTTC TCGTCTCACA GCATCTCACT 850 860 870 880 890 900 CAGTTACCCC CTGTTTACTC TATAGATACT GTAATTCATG TATTTATTTA TTCTTAAAAA 910 920 930 940 950 960 ACATTCAATG TCGGCGCCTG TCGCCGTTTA TTTGATTCCA TAATATTTAA TAGCCCCCAA

[0022] 970 980 990 1000 1010 1020 AAAAGGAGCC GAGTTTTTGC CATGCACGGT GGTGCAGTGG CCTGCAATCG CATGTATTGG 1030 1040 1050 1060 1070 1080 AGATAACACA GGGCTAGCAC TATACTCTGC GGGTGCACTC AGACATGATA CCTGTTCCCG 1090 1100 1110 1120 1130 1140 TGTATACATA GAGGAGTGAT GACTGCGTGG CTGGACAGGC CTACCATTCG TGAAGAAAAC 1150 1160 1170 1180 1190 1200 AGATTTGGAC TTGTTCGCGG AGGGCGCATA AGGCAGGGAT CGATGTTGAG TTGTGTGTGA

[0023] 1210 1220 1230 1240 1250 1260 TTCAATTCAC ATCCATTGGT GAAGGGACAG TTAACTAGGG CAGTGTGGAG TTGTCTAGTA 1270 1280 1290 1300 1310 1320 GTAGCAGGGG GCATTCAATG CCCACCGACA AGAATTTCCC TCGCAGTGCC TACTGCCACA 1330 1340 1350 1360 1370 1380 TCACGATCCC CTGTGCAACG AGCTGACCGG AAGAGAATGC CAACGTAGCG AACAATGATA 1390 1400 1410 1420 1430 1440 TCGCGGTAGA TCAATATATT CCAGGATTTC GTTCATCATA TGATTATGAG TCCCCGTGGC

[0024] 1450 1460 1470 1480 1490 1500 GCCAGCATCA TGCTGAGTTA TGTCCTGCAT AAGCAGGGGT TAGAACTGTT CTGACCTATC 1510 1520 1530 1540 1550 1560 GGTACCGTGA AAGTCATCTC AAAAGAGTCT GAGATGTTCA TCTGTTACAT CAGCTAGCTT 1570 1580 1590 1600 1610 1620 CAAAGATGAA CGGACGTTGA TACGATGATC AAGCTTTTAA TCATTTCCTG ACATGAAGCA 1630 1640 1650 1660 1670 1680 GTTTGTTCAG AATGACAAGC AAGATGATTC TGGAGTCTCG ATTAAATGGT GAAATATTGA

[0025] 1690 1700 1710 1720 1730 1740 ATTCGACGTC TAGTGTGAAA AATTTCGTAG CTAGAAATTC GACGAACCAC AAAACGACTA 1750 1760 1770 1780 1790 1800 CCAAAGCTGT TGATTTTCCA CAGCCTCCCC CCTTCGCATA GCGGGATCAT ATGAATCATT 1810 1820 1830 1840 1850 1860 TCCAGATGGT TGACGTTCAT ATGTAAAGCG CTATTCAAAG GCACGCACAG ATACCGAACA 1870 1880 1890 1900 1910 1920 CGACTACGTA TGTATATAAC TGTCATTTAG TTTTCACTGA ACAAATTGAC AGCACTATCA

[0026] 1930 1940 1950 1960 1970 1980 CATACCTTAC ACCGACCAAG AAAGAAGATC TATTTTTCTC AAGTCCTTGA CTTCTCTACC 1990 2000 2010 2020 2030 2040 TAGCGATCCT CACACCTTTA ACTTCCCGTG TTCCTTGAGC ATGATTCAAC AAGTCGAAAC 2050 2060 2070 2080 2090 2100 CTTCTCGCGA ACGACGAGGA TGTGCAGCGT AAATCACCAA CCATCAAGAG GGGTTCAATT 2110 2120 2130 2140 2150 2160 TAGATGAGAG ATGCATCGAT GAGTGATATA ACATGTCGAG TTGGAAAAGG CGTGGCTTGA

[0027] 2170 2180 2190 2200 2210 2220 GACAACACGG ACACAGCTTC TTGAACCAGA ATAGGGAGCT TCGATGCAGC TGCGCACAGC 2230 2240 2250 2260 2270 2280 CCACACCCCC AGCCAGTGAT ACTATGTACT GTGATACTAT AAAACCTGTA TAAAACCATT ***** *** **** ** ***** 2290 2300 2310 2320 2330 2340 TTGCCGTGCT CGTCGTTGAT TAAGATCTTG GTCCTTGGCA AAATAAAATA AACCTCGTGC 2350 2360 2370 2380 2390 2400 CCGAACACGT GAATCCAACC CTCTTTTGGA TCTCGGAATT CAACGCCTTT TTAGGGTTCT ^{# #}

2410 2420 2430 2440 2450 2460 ACCCGCTTTT CCCATCTTCC CCTTTATTCT TTGGTTGTCC TTCTTCTTCT TCTTTTCCAA ^# 2470 2480 2490 2500 2510 2520 AAGACTTGAG GAGCCGACTG TTTCTGAATC CTCGAATCCCTCGAATCCCTCAT2560AATC2CAT2560ATCCCAT25ATCCCAT25ATCCCAT25ATCCCCT25ATCCC2525 2540 ---

^***** : promoter common sequence, ^# : transcription start point; ------: translation region

SEQ ID NO: 2 Sequence length: 1199 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Origin Biologic name: Lentinus edodes Strain name: MS-LE1610

Sequence 10 20 30 40 50 60 CTAGATTGTG GCTCTCCATC ATATCGACCC GGCAACGTTA TAGATTCTAC ATCTACTGAT --- 70 80 90 100 110 120 CGTACGGTCT TCAATCTTCC TATCATTTGC CCTACCTGTC TTTACTTCTC TTCTGATTCTCTCTCTCTCTCTCTCATTCTTTCATCTGTCTCATTCTCATCTCGACATGAC GATTTACCTT CTTGGACGCA GAGTACATAG GTCTTTATCG TTTTAGGCTT

250 260 270 280 290 300 TGATCTCGAT TGTTTTGCAT TATTACATCC GCGCGCTTCT ACACAATTTC TAGTGTCTGA 310 320 330 340 350 360 TAGTGTTCGC CAACTGTTCT TGTTCCTTTT GTTCCTCTCTACC = TCTGTGTCTC = TCTGTCTCTCTC = TCTGTCTCTTTC = TCTGTCTCGTTC = 410TG 450 460 470 480 TAAGCCTCGG CACAAATAAT CTGAGTAAAT AATTCTTAGT AGGCCAGTCC GATGCAATAA ⁺⁺

[0033] 490 500 510 520 530 540 TCCTATCCTT AGATCCGGAT ACACTGCGTC GCTCAAGCAA TACGCAAGTG CAAAAATACC 550 560 570 580 590 600 TCTGTCCGGA TGCCCTCCAT GGACGGTTAG TTTGCCCAGC ATTGATTGGT CGAGCTCGCT 610 620 630 640 650 660 CAGGATGTTA TTTAGAAAAA ATGTACTGTA CAGTTTTGAG TCCACTGGCC CCGGGACTTT 670 680 690 700 710 720 CATAATTTTC TGAGACGATT ATTGGATGTT ATACTTTAGA TATAGCCAAT ATATTATATT

[0034] 730 740 750 760 770 780 GCCCAATTGG AGAATAGATA TTGCATACCA GGAATGTTAC CTTCGAGGCT ACCTCTCTGG 790 800 810 820 830 840 CTTTCATCAA TCCTCCAGGA ACAATGAAGG AACAAAATTC AATGCTTGAA CAAGGTACGG 850 860 870 880 890 900 AGGTCCAAGA CAATATTCAT AAGATCCTAA ATATGTAGTT TTGAGACGTG ATGAGAGAGA 910 920 930 940 950 960 GTCGTATAAG TATATATTGA GTATGTATAT GTAAGTTACG CTTTGTTACG ATGTACATAC

[0035] 970 980 990 1000 1010 1020 ATCGTAACGT CTGTAGCAGA GTTGTGTGTG TAATTAGGTG AATGCGATGA TCAAAGCTGA 1030 1040 1050 1060 1070 1080 TCAAAGGTCC TAATAGATGG AGTACAACTA AGGAAGATTG GAAGAAAAAA GAGTGCAATT 1090 1100 1110 1120 1130 1140 ATAACCGGAC CACGGCAGAC CAGGTTGTAT ATCCTTGATT TTGCTTGGAT ATTGCCCGAC 1150 1160 1170 1180 1190 1200 ACGAGCAAAA AATTCACACT ATGTATGTGT ATCTATGTGT ATTTGATTGC AATTTGGGG

---: stop codon; ===: transcription termination / sequence similar to poly (A) signal common sequence; ⁺⁺ : poly (A) addition site

[Brief description of drawings]

FIG. 1 is a Southern blot hybridization pattern of a mushroom chromosomal DNA restriction enzyme digestion product using the Harvey rat sarcoma virus ras gene as a probe.
Lane 1 shows the EcoRI digest and lane 2 shows the HindIII digest.

FIG. 2 is a restriction map of restriction enzymes of 5.6 kbp HindIII fragment, 4.0 kbp XbaI fragment, and ras cDNA of the ras gene.

FIG. 3 is a restriction enzyme digestion map of the 2.6 kbp EcoRI fragment containing the priA gene and the priA cDNA.

FIG. 4 is a schematic diagram of a process for preparing a gene expression vector pLC1-gen.

[Explanation of symbols]

2 indicates an exon, and E, H, S, and X represent restriction enzyme EcoRI, HindIII, SalI, and XbaI cleavage sites, respectively. The black squares in FIG. 3 are exons, and E, P, Sa, Sm, and X are restriction enzymes EcoRI, PstI, SacI, and SmaI, respectively.
The XbaI cleavage site is shown. In FIG. 4, ➝ indicates gene orientation, and B, E, P, X, and Xh represent restriction enzymes BamH.
I, EcoRI, PvuII, XbaI, XhoI cleavage sites are shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12R 1: 645)

Claims (4)

[Claims] 1. A DNA having the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing. 2. A gene expression vector containing the DNA according to claim 1. 3. A DNA having the nucleotide sequence set forth in SEQ ID NO: 2 in the Sequence Listing. 4. A gene expression vector containing the DNA according to claim 3.