JP2954376B2 - Rice peroxidase gene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a peroxidase gene which can be used as a marker gene in rice genome analysis, and more particularly to a rice green leaf peroxidase cDNA.
Regarding the IIB gene.

[0002]

2. Description of the Related Art Peroxidase [EC. 1.11.
1.7] is a redox enzyme having heme as a prosthetic group and transferring H of a substrate to H ₂ O ₂ of a receptor.
Peroxidase is particularly widely distributed in the plant kingdom, and its biochemical research has been conducted since the 1940s. Although the mechanism of the physiological role of peroxidase is not necessarily clear, generally, (1) secondary wall biosynthesis, (2) wound healing, (3) cell differentiation, (4) hormone metabolism, (5) ) It is said to be involved in disease resistance, resistance to drugs, and the like. So far, peroxidases derived from horseradish, tobacco, turnips, tomatoes, potatoes and the like have been studied. In addition, for peroxidases of these origins, approaches at the gene level have also been advanced [L. M. Lagrimini (LM La)
grimini) et al., The Proceedings of the National Academy of Sciences of the U
SA (Proc. Natl. Acad. Sci. USA), 84, 7542 (1987); Elizabeth (R. Eliz
abeth) et al., Molecular and General Genetics, Mol. Gen. Genet., 217, 233.
P. (1989), R.A. Elizabeth et al., Plant Molecular Mol. Biol.
1, page 15 (1988)], and the general structure of oxidase is also discussed [New Name, Cell Engineering, 7, 588 (1988)]. Generally, higher plants have multiple peroxidase isozymes or isoforms. Therefore, it is considered that there are a plurality of genes encoding peroxidase.

[0003]

There has been almost no report on peroxidase originating from rice, but Ida et al. Purified peroxidase from rice embryos in 1972 [Agriculture]. Cultural and biological chemistry (Agric. Biol. C
hem.), 36, 611 (1972)]. However, no progress has been reported at the genetic level.
There are a plurality of peroxidase genes in rice green leaves, all of which are considered to have similar structures. Therefore, if the structure of at least one of them is elucidated, it is possible to isolate the remaining gene using the probe as a probe. An object of the present invention is to isolate a peroxidase gene for isolating a plurality of peroxidase genes present in rice green leaves or to use as a marker gene for rice genome analysis and to analyze its structure.

[0004]

SUMMARY OF THE INVENTION In general, the present invention relates to a rice green leaf peroxidase cDNA IIB gene, which is represented by SEQ ID NO: 1 in the sequence listing,
The cDNAIIB gene is, for example, rice (Oryza sativa
L. cv. Nipponbare) It encodes a kind of peroxidase present in green leaves.

[0005] The cDNAIIB gene of the present invention can be prepared, for example, by preparing mRNA from rice green leaves, then synthesizing cDNA,
It can be obtained by screening a gene of interest from this cDNA library.

[0006] For screening of the peroxidase gene, for example, a probe may be prepared and plaque hybridization may be performed, for example. As a probe used for screening, there are a case where an oligo DNA synthesized based on a partial amino acid sequence in the target protein and a case where a DNA fragment of a heterologous organism encoding the target protein is used. In the case of peroxidase, the genes have already been isolated from several other sources and the characteristics of the sequence are clear, and at least two or more highly conserved sequences are present in the peroxidase protein. It is clear that One of them is considered to form the active center of the present enzyme, and the other is thought to be a ligand portion binding to heme.

FIG. 1 shows the general structure. That is, FIG. 1 shows a general structure of peroxidase, in which a represents an active center region, and b represents a heme binding site region. Examples of highly conserved sequences of the active center region include, for example, four amino acid sequences represented by SEQ ID NOs: 2 to 5 in the sequence listing, and these are the active center regions of peroxidase derived from tobacco, turnip, horseradish, and potato, respectively. The peroxidase derived from tomato also has the sequence of SEQ ID NO: 5 in the sequence listing. Examples of the highly conserved sequence of the heme binding site region include, for example, four types of amino acid sequences represented by SEQ ID NOs: 6 to 9 in the sequence listing, and these are peroxidases derived from tobacco, turnip, horseradish, and potato, respectively. Is the amino acid sequence of the heme binding site region, and there is also a sequence of SEQ ID NO: 9 in peroxidase derived from tomato.

[0008] Synthetic primers were prepared for two highly conserved regions in the peroxidase primary structure, and c
For example, by performing PCR using DNA as a material,
A part of the peroxidase gene present therein can be amplified and used as a probe. Examples of the primer include SEQ ID NOs: 10 and 11 in the sequence listing.
May be synthesized, purified and used.

A cDNA library prepared by introducing a cDNA synthesized from rice mRNA into an appropriate vector, for example, a plasmid or a phage vector, is used to obtain, for example, a DNA amplified by PCR using the above primer pair.
Is used as a probe for screening, a clone containing DNA having a sequence complementary to the probe is selected, DNA is purified from the clone, and this is analyzed in detail to obtain a gene encoding rice peroxidase, that is, rice green leaf The peroxidase cDNA IIB gene can be obtained. Using the whole region or a part of this gene as a probe to search for other peroxidase cDNA clones present in rice green leaves, or to search for chromosomal DNA encoding peroxidase, or to search for peroxidase in the rice genome Genes can be mapped, and the genes are useful in rice genetic engineering.

[0010]

The present invention will be described below in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 (a) Isolation of rice green leaf mRNA and synthesis of cDNA Rice (Oryza sativa L. cv. Nipponbare) seeds were allowed to absorb water overnight and germinated in a laboratory greenhouse at a constant temperature of 25 ° C. On the 25th day, green leaves were harvested. Liquid nitrogen was added to 30 g of the green leaf and crushed by a blender. To this
0 ml of guanidine thiocyanate solution was added, and the mixture was completely crushed with a homogenizer. This was filtered through a nylon membrane of 300 μm, and the filtrate was centrifuged at 15000 rpm for 10 minutes. The supernatant was layered on a tube containing the CsCl solution, and ultracentrifuged at 28,000 rpm for 24 hours. The obtained precipitate was washed with 70% ethanol, dried, and dissolved in 3 ml of Tris-HCl buffer (pH 8.0) to obtain total RNA. 50 μl of 5 M NaCl is added to 1 ml of the total RNA solution.
l, 2 ml of ethanol was added, and the mixture was allowed to stand at -80 ° C for 1 hour, and then the precipitate was collected by centrifugation. This precipitate was dissolved in an elution buffer [10 mM Tris-HCl (pH 7.5),
200 μl of 1 mM EDTA, 0.1% SDS] and 200 μl of Oligotex (Takara Shuzo Co., Ltd.),
After leaving it for 5 minutes, put it on ice, then add 40 μl of 5M NaCl, keep it at 37 ° C. for 15 minutes, dissolve the precipitate obtained by centrifugation further in 500 μl of elution buffer, and leave it at 65 ° C. for 5 minutes. Thereafter, a supernatant was obtained by centrifugation. 1 ml of ethanol was added to the supernatant, and the resultant was stored at −80 ° C. as an mRNA solution. Next, the precipitate obtained by centrifugation of the mRNA solution was dissolved in 15 µl of Tris-HCl buffer, and 2 µl of which was used to synthesize cDNA by the method exemplified below. 2 μl mRNA, 4 μl 5
× First strand buffer [250 mM Tris-H
Cl (pH 8.3), 250 mM KCl, 50 mM
MgCl ₂ , 5 mM DTT], 1 μl of sodium pyrophosphate, 1 μl of RNase inhibitor (20 U /
μl), 2 μl of each 10 mM dNTP mixture, 1 μl
1.5 μl of AMV reverse transcriptase (33 U / μl) was added to the reaction system containing the oligo (dT) _12-18 (1.6 mg / ml) primer, and reacted at 42 ° C. for 1 hour. 0.5 μl of second strand buffer [30 m
M Tris-HCl (pH 7.4), 75 mM MgC
l ₂ , 15 mM (NH ₄ ) ₂ SO ₄ , 15 mM KC
l, each 1 mM dNTP mixture], 1.5 μl RNa
seH (1 U / μl), 6 μl of DNA polymerase I
(4 U / μl), reacted at 12 ° C. for 1 hour, further reacted at 22 ° C. for 1 hour, then left at 70 ° C. for 10 minutes to inactivate the enzyme, and further added ₄ U of T ₄ polymerase. And reacted at 37 ° C. for 10 minutes.
Was synthesized. An equivalent amount of saturated phenol was added to 104 μl of the synthesized cDNA solution, mixed, and then centrifuged to obtain about 100 μl of an aqueous layer. Next, 100 μl of chloroform was added thereto, mixed, and centrifuged to obtain about 100 μl of an aqueous layer again. An equal volume of a 4 M ammonium acetate solution was added to the aqueous layer, and the mixture was precipitated with ethanol.
H8.0), 1 mM EDTA] to obtain 480 ng of a purified double-stranded cDNA.

(B) Preparation of probe for peroxidase gene screening A pair of synthesized primers (20 pmol each) represented by SEQ ID NOs: 10 and 11 in the sequence listing, and c synthesized with (a)
Tack buffer containing 1 μl of DNA and 16 μl of dNTP mixture [10 mM Tris-HCl (pH 8.3),
50 mM KCl, 1.5 mM MgCl ₂ , 0.00
1% gelatin] and Ampritac (Takara Shuzo) 2.5U
Was added and PCR was performed. The condition of PCR is DNA
Denaturation was performed at 94 ° C. for 1 minute, primer annealing was performed at 42 ° C. for 2 minutes, and primer extension reaction was performed at 72 ° C. for 2 minutes, and this was repeated 30 cycles. The DNA fragment thus obtained is about 400 bp long,
A was used as a probe.

(C) Preparation of cDNA library The cDNA synthesized in (a) was converted into a λ phage vector, λg
This was inserted into t11 to prepare a cDNA library. First, 4.5 μl of cDNA (200 ng) was added to EcoRI-
5 μl of Smal adapter, 76 μl of solution A and 9.5 μl of solution B in a DNA ligation kit (Takara Shuzo Co., Ltd.) were added, and the mixture was reacted at 16 ° C. for 1 hour.
Gel filtration was performed with the column No. 0 to remove unbound adapters. Next, 5 'of the cDNA with the adapter
The ends were 30 minutes phosphorylated at 37 ° C. with 100 mM ATP, of T ₄ polynucleotide kinase, followed by ethanol precipitation. The resulting precipitate was dissolved in TE buffer 5 [mu] l, and ligated using of T ₄ ligase in λgt11 of 1 [mu] g. This was introduced into phage particles (Stratagene) and infected with Escherichia coli Y1090 to prepare a cDNA library. The prepared library had a size of 5 × 10 ⁶ pfu / ml.

(D) Screening of cDNA and Selection of Clones Containing the Target Gene The phage cDNA library prepared in (c) was infected into Escherichia coli Y1090 to form plaques, which were plaqued with the probe prepared in (b). Hybridization was performed. About 10 ⁵ plaques were formed on the plate, and a nylon membrane (Hybond N ⁺ ) was placed on the plate and left for 1 minute. Next, the membrane was treated with 0.5M NaOH-
After denaturation in 1.5 M NaCl solution and neutralization in 0.5 M Tris-HCl (pH 7.5) -1.5 M NaCl solution, 2 × SSC solution (1 × SSC: 0.15
M NaCl-0.035M sodium citrate) and baked at 80 ° C for 30 minutes. This membrane was placed in a prehybridization solution and allowed to stand at 42 ° C. for 4 hours or more to block the unbound portion of the phage DNA on the membrane. Thereafter, a probe labeled with ³² P was added, and the membrane was incubated at 55 ° C. for 1 hour. After standing overnight, a recombinant phage having a nucleotide sequence homologous to the probe was detected. The labeling of the probe followed the random priming method. From the six candidate clones thus obtained, phage DNA
Is prepared and digested with EcoRI to obtain cDNA.
The resulting DNA fragment was obtained. This was further subcloned into the EcoRI site of the pUC18 vector, and the nucleotide sequence was determined. One of the six was named IIB clone and E. coli harboring it was named E. coli. coli HB101 / pUC. The nucleotide sequence of the IIB clone is shown in SEQ ID NO: 1 in the sequence listing. Thus, the gene cDNAIIB encoding peroxidase was isolated and its nucleotide sequence was determined. The above-mentioned probe was a probe that recognized base numbers 220 to 611 of SEQ ID NO: 1 in the sequence listing.

[0015]

According to the present invention, the gene structure of rice peroxidase has been elucidated. The gene is useful in the field of rice genetic engineering and the like.

[Sequence list]

SEQ ID NO: 1 Sequence length: 1348 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin: Oryza sativa Sequence characteristics: 4-1008 E CDS (rice green leaf) peroxidase) 220-243 E primer binding 592-611 E primer binding 1331-1348 E poly A site sequence: GGCATGGAGT ACGCTACTCG TGGAGATCGC ACGGCTAGCT GCCTGAGTTT CCTCTGCAAT 60 ATCGTCGTTC TGCTGGGCCT CGCCGCCGCG GCGGGCAGCG GGCAGCTGAC GGACGACTAC 120 TACGACTATT GCTGCCCCCA GGTTTACCGC ATCGTCCGGT CCCGCGTGGC CGCCGCGATG 180 AAGGCCGAGA TGCGCATGGG CGCCTCCCTG CTCAGGCTTC ACTTCCACGA CTGCTTCGTC 240 AATGGCTGTG ACGCGTCCAT CCTCCTTGAC GGCACAAACA GCGAGAAGTT CGCGGCGCCG 300 AACAACAACT CGGTGAGAGG GTACGAAGTC ATCGATGCGA TAAAGGCCGA CCTCGAGGGC 360 GCCTGCCCGG GAGTCGTCTC CTGCGCCGAC ATAGTAGCCC TTGCAGCTAA ATACGGAGTA 420 CTACTTAGTG GAGGACCTGA TTATGATGTC CTCCTGGGAA GAAGAGATGG TCTGGTGGCA 480 AATCAGACGG GGGCGAACAG TAACTTGCCT AGCCCTTTCG ATTCGATCAG CGTTATCACT 540 GCGAGGTTCA AGGATGTCGG TCTCAACGCA ACCGATGTTG TGGT CTTATC AGGGGCGCAC 600 ACGATCGGGC GATCTCGCTG CCTGCTGTTC AGCAACCGGC TGGCGAACTT CTCGGCGACC 660 AACTCCGTCG ACCCGACGCT GGACTCGTCG CTGGCGTCCA GCCTGCAGCA GGTGTGCCGC 720 GGCGGCGCTG ACCAGCTGGC GGCGCTGGAC GTCAACTCCG CCGACGCGTT CGACAACCAC 780 TACTACCAGA ACCTGCTGGC CAACAAGGGC CTCCTCGCCT CCGACCAGGG CCTCGTCTCC 840 AGCTCCGGCG ACCCCGCCGT CGCCGCCACC AAGGCGCTGG TGCAGGCCTA CAGCGCCAAC 900 GGCCAGCGCT TCTCCTGCGA CTTCGGCAAC TCCATGGTCA AGATGGGCAA CATCAGCCCT 960 CTCACCGGCT CTGCCGGCCA GATTCGCAAG AACTGCAGGG CCGTCAACTG ATGAGCAAAA 1020 AGGCAAAGAT TTTTTGCATC TGCCATGACC CCATCTTGGA TTTGCCAGAA GCTATATCGT 1080 CTTCTTGGAC TCAAGTGTGA ATCTGTCGTT TTTAATGTGT TGTGGAGCGC TACATGTTTC 1140 GTTTTGTTCA AGCTATCTAG GATTCTCTCT CCAATGCACG AGTAGAATAA GCAATTAGCA 1200 TGCAAAGTTG CTCGTCCTAC ACGAATCTGC AGCTGCATTT TCAGTGGGTG TATCACCAAT 1260 TATTAGAGCA CAGACAAACG CGCTGTGTCT ATCAGAGTAA AAGAAAGAAT ATGCAACTGC 1320 CATATGGTTT AAAAAAAAAA AAAAAAAA 1348 SEQ ID NO: 2 sequence length of: 20 Sequence type: number of amino acid chains: single-stranded topo Gee: linear Sequence type: peptide Fragment type: middle fragment Origin: Nicotiana tabacum Sequence: Ile Arg Leu His Phe His Asp Cys Phe Val Asn Gly Cys Asp Gly 1 5 10 15 Ser Ile Leu Leu Asp 20 SEQ ID NO: 3 Sequence length: 20 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide Fragment type: middle fragment Origin: Brassica napus Sequence: Ile Arg Leu His Phe His Asp Cys Phe Val Asn Gly Cys Asp Gly 1 5 10 15 Ser Leu Leu Leu Asp 20 SEQ ID NO: 4 Sequence length: 20 Sequence type: Amino acid Number of chains: 1 chain Topology: Linear Sequence type: Peptide Fragment type : Intermediate fragment Origin: Armoracia rusticana Sequence: Leu Arg Leu His Phe His Asp Cys Phe Val Asn Gly Cys Asp Ala 1 5 10 15 Ser Ile Leu Leu Asp 20 SEQ ID NO: 5 Sequence length: 20 Sequence type: amino acid Number of chains: 1 chain Topology: straight Type Sequence type: Peptide Fragment type: Middle fragment Origin: Solanum tuberosum Sequence: Ile Arg Leu His Phe His Asp Cys Phe Val Asp Gly Cys Asp Gly 1 5 10 15 Gly Ile Leu Leu Asp 20 SEQ ID NO: 6 Sequence length Length: 12 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide Fragment type: middle fragment Origin: Nicotiana tabacum SEQ ID NO: 7 Sequence length: 12 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide Fragment type: middle fragment Origin: Brassica napus SEQ ID NO: 8 Sequence length: 12 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide Fragment type: middle fragment Origin: Armoracia rusticana SEQ ID NO: 9 Sequence length: 12 Sequence type: amino acid Number of chains: 1-chain Topology: linear Sequence type: peptide Fragment type: middle fragment Origin: Solanum tuberosum SEQ ID NO: 10 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Hypothetical sequence: NO Antisense: NO Sequence: CAYTTCCAYG ACTGCTTYGT NAAT 24 SEQ ID NO: 11 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Hypothetical sequence: NO Antisense: YES Sequence characteristics: 1-20 E primer 15,16 E i sequence: CGTCCGATCG TGTGNNCTCC 20

[Brief description of the drawings]

FIG. 1 shows the general structure of peroxidase.

────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Akira Obayashi 3-4-1, Seta, Otsu City, Shiga Prefecture Inside Takara Shuzo Co., Ltd. Central Research Laboratory (58) Field surveyed (Int.Cl. ⁶ , DB name) C12N 15/09 C12N 9/08 CA (STN) GenBank / EMBL / DDBJ / GeneSeq REGISTERRY (STN)

Claims (1)

(57) [Claims] 1. A rice green leaf peroxidase cDNA IIB gene represented by SEQ ID NO: 1 in the sequence listing.