JP2775405B2 - New thionin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to thionin which has a pharmacological action such as an inhibitory activity on the growth of microorganisms, animal cells or cultured cells thereof (for example, cancer cells) or a blood pressure lowering action. Regarding thionine. The present invention also relates to the preparation of a plant having the antibacterial activity based on the thionin.

[0002]

BACKGROUND OF THE INVENTION Thionine was first found as a substance having the action of inhibiting the growth of yeast present in flour (PWAllen, ed., W. Jago et al., "Industrial Fermentatio
n ", p. 128, (1926)), and subsequently a basic protein known to act as a poison also on microorganisms other than yeast, animals or cultured cells thereof. Thionine or a protein having a primary structure similar thereto (For example, mistletoe
(Losothaceae) -derived biscotoxin) has antibacterial action,
It is thought to have various pharmacological actions such as hypotensive action (Wada and Ozaki, Plant Cell Engineering, 3 (3), 200-207
P. (1991)). In general, thionine has a strong basicity and is stable to acids and heat.

[0003] Purothionine is known as a typical thionin. Purothionine obtained from wheat endosperm consists of 45 amino acid residues and has four disulfide bonds. Of the 45 amino acid residues, the acidic amino acid is only one aspartic acid residue,
It has 10 residues of lysine and arginine, which are thought to be the cause of strong basicity (Hase, T, Matsubara, H
Et al., J. Biochem., 83, 1671-1678, (1978)).

[0004] Various types of thionin are known, and their amino acid sequences are also known. For example, the above-mentioned plant cell engineering, 3 (3), pp. 200-207, (1991), introduces eight types of thionins and their amino acid sequences shown in Table 1 below.

[0005]

[Table 1]

[0006] Thionine, derived from cereal leaves, is a cysteine-rich protein, generally consisting of 46 amino acid residues and having a molecular weight of about 5 kDa (Bohlmann, H. and Ape).
l, K., Mol. Gen. Genet., 207, pp. 446-454, (198
7)). Among them, those derived from barley leaves are known to have antibacterial activity against various phytopathogenic fungi in vitro (Bohlmann, H. et al., EMBO J., 7, 1).
559-1565, (1988)).

[0007]

As described above, thionin has a pharmacological effect on various organisms and cells, but a novel thionin having less toxicity and higher pharmacological action is required. .

[0008] The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a novel leaf-derived thionin having excellent antibacterial properties and its gene, and furthermore, to express these genes. An object of the present invention is to provide a method for producing a useful plant having high anti-phytopathogenic bacterial activity and anti-phytopathogenic fungal activity and a means related thereto.

[0009]

SUMMARY OF THE INVENTION The present invention provides a novel oat leaf-derived thionin. More specifically, the following amino acid sequence represented by SEQ ID NO: 1 Lys Ser Cys Cys Lys Asp Ile Met Ala Arg Asn Cys Tyr Asn Val Cys 5 10 15 Arg Ile Pro Gly Thr Pro Arg Pro Val Cys Ala Thr Thr Cys Arg Cys 20 25 30 Lys Ile Ile Ser Gly Asn Lys Cys Pro Lys Asp Tyr Pro relates to thionine having 35 40 45 (hereinafter referred to as thionin of the present invention).

The present invention also relates to a mature form of the thionine of the present invention, from the N-terminus, the 7th isoleucine, 8th methionine, 15th valine, 18th isoleucine, 19th
Thionine having a proline at position 21, a threonine at position 21, a proline at position 22, a threonine at position 29, an asparagine at position 38, and having one or more deletions and / or substitutions.

Further, the present invention relates to a DNA sequence encoding the above-mentioned thionin of the present invention. Preferably, the DNA sequence of the present invention has the following nucleotide sequence represented by SEQ ID NO: 2 AAG AGT TGC TGC AAG GAT ATC ATG GCA AGA AAC TGC TAC AAC GTG TGC 48 Lys Ser Cys Cys Lys Asp Ile Met Ala Arg Asn Cys Tyr Asn Val Cys 5 10 15 CGT ATT CCT GGT ACT CCT AGG CCT GTC TGT GCA ACT ACA TGT CGT TGC 96 Arg Ile Pro Gly Thr Pro Arg Pro Val Cys Ala Thr Thr Cys Arg Cys 20 25 30 AAA ATC ATT AGT GGA AAT AAG TGC CCA AAG GAT TAT CCT AAA 138 Lys Ile Ile Ser Gly Asn Lys Cys Pro Lys Asp Tyr Pro Lys 35 40 45

Furthermore, the present invention relates to a vector having a DNA sequence encoding the thionin of the present invention.

As used herein, the term "expression cassette" refers to a DNA sequence encoding the thionin and various regulatory elements such as a promoter and an enhancer for regulating the expression so that the thionin can be expressed. Refers to a DNA fragment that is operably linked in a host cell.

[0014] The term "vector" refers to a medium for transmitting the above expression cassette into a host cell.

It is well known in the art that the type of the above-described vector and the type of each regulatory element used can be changed depending on the host cell.

A preferred type of the vector of the present invention is a plasmid vector. Preferred vectors of the present invention are those capable of transmitting the above-described expression cassette to microorganisms, animal cells, or plant cells. Those capable of transmitting to plant cells are particularly preferred.

The present invention also relates to a cell transformed with the vector. Preferred host cells are plant cells. Particularly preferred are higher plants, for example, cells of tobacco, rice, wheat, potato, corn, etc., for which the tissue culture method and the technique of cell or callus regeneration have been established.

The present invention also relates to a method for producing thionin, comprising the steps of culturing transformed cells transformed with the vector and recovering thionin expressed in the cells.

In the above-mentioned transformed cells, the thionin of the present invention can be produced either by a mature thionin, a thionin precursor, or a fusion of the thionin with another protein. The thionin of the present invention can also be produced by a chemical synthesis method (for example, the melifilt method).

Hereinafter, the present invention will be described in detail.

The present inventors have found that, unlike commercial cereal plants such as barley, oats are plants to which few artificial manipulations (breeding) have been added, and that the leaf-derived thionin, which can be specifically present in oats, Pharmacological action (especially antibacterial activity)
Have conceived that they are superior to those conventionally known, and have completed the present invention by isolating cDNA encoding a novel thionin from oat (Avena sativa L).

The thionin of the present invention has the following amino acid sequence shown in SEQ ID NO: 1 Lys Ser Cys Cys Lys Asp Ile Met Ala Arg Asn Cys Tyr Asn Val Cys 5 10 15 Arg Ile Pro Gly Thr Pro Arg Pro Val Cys Ala Thr Thr Cys Arg Cys 20 25 30 Lys Ile Ile Ser Gly Asn Lys Cys Pro Lys Asp Tyr Pro Lys 35 40 45

The thionin of the present invention has the following amino acid sequence shown in SEQ ID NO: 3 in a natural plant: Met Gly Ser Ile Lys Gly Leu Lys Ser Val Val Ile Cys Val Leu Val 5 10 15 Leu Gly Ile Val Leu Glu Gln Val Gln Val Glu Gly Lys Ser Cys Cys 20 25 30 Lys Asp Ile Met Ala Arg Asn Cys Tyr Asn Val Cys Arg Ile Pro Gly 35 40 45 Thr Pro Arg Pro Val Cys Ala Thr Thr Cys Arg Cys Lys Ile Ile Ser 50 55 60 Gly Asn Lys Cys Pro Lys Asp Tyr Pro Lys Leu His Gly Asp Pro Asp 65 70 75 80 Ala Gly Thr Pro Asn Ala Ile Glu Phe Cys Asn Thr Gly Cys Met Ser 85 90 95 Ser Ile Cys Asp Asn Met Asn Asn Ala Tyr Asn Val Glu Asp Lys Glu 100 105 110 Ile Asp Val Glu Leu Cys Gly Asn Ala Cys Thr Ser Phe Cys Asn Gln 115 120 125 Ile Ile Val Arg Ala Ser Val Ala Ala 130 135 Expressed as a thionin precursor protein You.
Of the thionin precursor, the leader peptide region is from position 1 (methionine) to position 28 (glycine), counting from the N-terminus of the amino acid sequence, and the position is 74 from position 29 (lysine).
The region up to position (lysine) is the mature thionine region, and the region from position 75 (leucine) to position 137 (alanine) is the acidic protein region.

The thionin of the present invention can be prepared as follows.
Can be produced.

The practice of the present invention can be practiced, unless otherwise indicated, using conventional techniques of molecular biology, biochemistry, genetics, genetic engineering, and plant breeding.

(1) Preparation of cDNA Library mRNA is extracted and purified from leaves and roots of oat (Avena sativa L) by a conventional method. After synthesizing a cDNA using the obtained mRNA as a template, various DNA adapters (for example, EcoRI adapter) or linkers are ligated and cloned into a cloning vector such as lambda phage (for example, λgt10) to prepare a cDNA library.

(2) Isolation of DNA fragment encoding thionin and nucleotide sequence determination A set of primers was prepared based on the known sequence of thionin and disclosed in a conventional manner from oat (Avena sativa L) leaves. PCR using genomic DNA extracted by
(Polymerase chain reaction) (Saiki et al., Science, 230, 1
350-1354, (1988)) to obtain an amplified partial gene fragment of the thionin region (hereinafter, referred to as a PCR fragment).

A positive clone is isolated from the cDNA library obtained in the above (1) by a hybridization assay using the obtained PCR fragment as a probe. CDNA is obtained from the obtained positive clone, and a nucleotide sequence analysis method known in the art (for example, Sang
The base sequence of each cDNA can be determined by the dideoxy method of E. et al.) or a commercially available automatic sequencer (for example, ABI model 373A based on the Dye Terminater method).

(3) DNA sequence encoding the amino acid sequence of thionin The DNA encoding the amino acid sequence of thionin of the present invention is obtained by using the entire chain length of the above isolated cDNA fragment, that is, the precursor of thionin of the present invention. A DNA sequence containing the entire coding region, or a DNA sequence in which a part thereof has been deleted, can be used. Alternatively, only the sequence corresponding to the mature thionin shown in SEQ ID NO: 1 may be cut out and used. A DNA fragment chemically synthesized by a known DNA synthesis method (for example, a phosphoramidite method) so as to encode the amino acid sequence of thionin of the present invention can also be used. Genomic DNA sequences encoding the thionin of the present invention, obtained using these sequences as probes, can also be suitably used.

These are used as they are or by connecting an appropriate linker to construct an expression cassette shown below.

In the preparation of the DNA encoding the amino acid sequence of thionin of the present invention, there are provided
Deletion and / or substitution of one or more amino acid residues or addition of one or more amino acid residues within the above-obtained native amino acid sequence of thionin of the present invention may also be contemplated. These modifications can also be included in the technical scope of the present invention, as long as the antibacterial activity of the thionin of the present invention is maintained. Such changes may be made by techniques well known prior to the filing of the present application, such as PCR or site-directed mutagenesis using complementary primers containing nucleotide sequence mismatches that are limited to obtain the desired change. Can be achieved by

(4) Construction of thionin expression cassette and vector DNA encoding thionin of the present invention or its precursor
The construction of vectors containing sequences and various regulatory elements as described above (including promoters, ribosome binding sites, terminators, enhancers, various cis elements controlling expression levels) is well understood in the art. Techniques for restriction of polynucleotides by various restriction enzymes and ligation of polynucleotide fragments may be used.

The type of regulatory element used for the vector and its construction depends on the type of host cell.

An example of construction of a thionin expression cassette and a vector when a plant cell is used as a host is described below.

As a preferable high expression promoter, a cauliflower mosaic virus (CaMV) 35S promoter, a nopaline synthesis promoter and the like are generally used, but not limited thereto. A plant site-specific expression promoter, and a promoter whose expression is induced by a certain kind of stress on the plant or cell (such as physical injury or stimulation by a chemical substance) can also be suitably used. The latter preferred promoter is disclosed in
No. 109992, which are promoters of tobacco-derived infection-specific protein (PR) 1a and 1b genes (Ohshima et al., The Plant Cell, 2, 95-106,
(1990)).

[0036] The enhancer region is important in optimizing expression. A preferred enhancer region that can be used in the practice of the present invention is one in which one or more enhancer regions (En35S) containing a sequence upstream of the 35S promoter are tandemly linked (Mitsuhara et al., "CaMV35").
High expression promoter cassette based on S1 and tobacco PR1a gene promoters, '' Annual Meeting of the Japanese Society of Plant Physiology 1992 and 32nd Symposium, 92 pages, (199
2)).

The terminator that can be used in the practice of the present invention is not particularly limited. The terminator of the PR1a gene can be suitably used.

The DNA sequence encoding the thionin of the present invention prepared in the above (3) can be used alone or ligated to a DNA sequence encoding another protein suitable for preparing a fusion protein with thionin. Then, each of the above regulatory elements is operably linked in a host cell to prepare an expression cassette.

[0039] The expression cassette may contain a selectable marker gene in addition to the target gene. Selectable marker genes are used to select only those host cells that have the expression cassette. A commonly used selectable marker gene is neomycin phosphotransferase II (NTPII), which confers kanamycin resistance.
But is not limited to this.

The above-mentioned expression cassette is introduced into a plasmid capable of introducing the expression cassette into a plant via a bacterium belonging to the genus Agrobacterium such as a pBI system, or a pUC system plasmid to construct a vector.

(5) Transformation of host cell Depending on the host cell to be used, the host cell is transformed with the above vector using a standard technique suitable for the cell.

In the case of prokaryotic cells, Cohen, SN et al. (Pr
oc. Natl. Acad. Sci. USA, 69, 2110, (1972)) can be used. The electroporation method (electroporation) can be applied to both plant cells and microorganisms in addition to animal cells.

In certain higher plant cells, Agrobacterium tumefaciens (Agrobacterium tum
efaciens) (Shaw, CH et al., Gene, 23, 3)
15 (1983)) is widely used. This method, for example,
According to the method of Nagel et al., The constructed vector (pBI system or the like) is first introduced into Agrobacterium tumefaciens by the above-described electroporation, and then the transformed bacteria are subjected to the leaf disk method (Horsch, R. .
B. et al., Science, 227, pp. 1229-1231, (1985)) introduces the above expression cassette into host plant cells.

A plasmid vector particularly preferably used for constructing an expression cassette was constructed by the present inventors and reported at the 1993 Japan Breeding Congress.
It is a vector derived from pBI121 (Clontech) containing a high expression promoter. This plasmid map is shown in FIG. In the figure, P _NOS represents the promoter region of the nopaline synthetase gene, NPTII represents the neomycin phosphotransferase II coding region, and T _NOS represents the terminator region of the nopaline synthetase gene. En35S represents the enhancer region of the CaMV35S promoter (corresponding to positions -417 to -90), and in this vector, the region is tandemly linked (J. Biotechnology, 14, 333, (1990)). P35S
Is the 35S promoter region (corresponding to positions -90 to -1). Ω represents the omega sequence of the tobacco mosaic virus (Gene, 217, 217, (1987)). The HindIII / BamHI fragment containing P35S in the vector of FIG. 1 is a preferred promoter sequence of the expression cassette for practicing the present invention.

(6) Production of Thionine of the Present Invention by Transformed Cells By culturing the transformed cells obtained by each of the above steps, the thionine of the present invention can be produced in a large amount. A preferred culture form is the transformed plant cell line or a differentiated or undifferentiated plant tissue culture system derived from the transformed plant cell. These cultures vary depending on the type of plant cell used, and may be, for example, a general MS (Murashige & Sk) by a method well known in the art.
oog) It can be carried out by solid or liquid culture using a medium.

The thionin of the present invention can be obtained by, for example, suspending a culture in a buffer and physically disrupting the tissue by sonication or the like, or treating the cell wall with an enzyme to destroy the tissue. , And is then recovered by filtering or centrifuging the extract to remove culture residues. The recovered thionine can be further purified by performing a general purification step such as ammonium sulfate fractionation (salting out), dialysis, various types of chromatography, and gel filtration.

(7) Production of the Thionine of the Present Invention in the Regenerated Transformed Plant The thionin of the present invention can also be obtained by subjecting the transformed plant cell to a known regeneration method (for example, tobacco) corresponding to the plant species. Where Horsch, RB et al., Science, 227, 1229-12, supra.
31 (1985)) and can be produced in a transformed plant established by redifferentiation.

The transformed plant expressing the thionin of the present invention can have a wide range of anti-phytopathogenic bacterial activities and anti-phytopathogenic fungal activities due to the antibacterial properties of the expressed thionin.

The present invention will be described in more detail with reference to the following examples, which do not limit the present invention in any way.

[0050]

EXAMPLES Example 1 (Preparation of cDNA library from oat) Oat (Avena sa) cultivated under natural conditions until the second leaf development
tiva L) from the second leaves and roots of the variety "Advance" and the first leaves of the same variety germinated and grown under dark conditions
RNA was extracted by a conventional method. 5 μg of the obtained mRNA
Was used to synthesize cDNA using a cDNA synthesis kit (manufactured by Pharmacia) according to the attached instruction manual.
The cDNA fragment obtained by using this kit is ligated to both ends with EcoRI adapter. CDNA synthesized above
Using about one-fifth of the total amount, EcoRI of λgt10 phage
About 4 × 10 ⁸ pfu cDNA
A library was made.

Example 2 (Isolation of DNA fragment encoding novel thionin from oat genomic DNA) The above oat (forward) was grown under natural conditions using green leaves cultivated until the second leaf was developed, by a conventional method. Genomic DNA was isolated. The following sequence represented by SEQ ID NOs: 5 and 6 synthesized based on the conserved regions (5'-side region and acidic protein region) of the known thionin base sequence: (Forward): 5 'AATCAAGCTTCCAAGTAGAAGGCAAGAGTTGC 3' ( Reverse): 5 ′ AGTCTCTAGAGTCCATGTTGTCACAGACGG 3 ′, or the following sequence represented by SEQ ID NOs: 7 and 8 synthesized based on a conserved region of the amino acid sequence of known thionin (C-terminal side of leader region and thionin region) : (Forward): 5 'GTTCCTGCAGTCATACTGGGTTTAGTTCTGGAGCC
3 '(Reverse): 5' GTAGTCTAGAATTCAGTTTAGGATAGTCMCTAGRGC
Using two sets of primers having 3 ′ (M is A or C, R is G or A), PCR was performed using the isolated genomic DNA as a template.

The PCR at this time was performed in a 50 μl reaction system. That is, 0.5 μm of Taq DNA polymerase was used.
5 μl of 10 × buffer, 2.5 mM MgCl ₂
, 4 μl of dNTP (10 mM), and 1.25 μl of any one of the above primers (10 pmol)
Each and about 500 ng of genomic DNA were added, and the total volume of the reaction solution was adjusted to 50 μl with distilled water. Table 2 shows the reaction conditions and the number of times of the PCR.

[0053]

[Table 2]

As a result of this PCR, the expected target fragment was amplified from the primer prepared from the amino acid sequence. As a result of sequencing this PCR fragment, it was confirmed that the primer used had features on the thionin sequence, although a change was observed at the 3 ′ end of the primer. On the other hand, in the reaction system using the other set of primers represented by SEQ ID NOS: 5 and 6, amplification products were detected only when the annealing temperature shown in Table 2 was reduced to 40 ° C. and repeated.

Example 3 (Isolation of the thionine cDNA of the present invention) From the approximately 150,000 clones of the cDNA library obtained in Example 1, the PCR fragment obtained in Example 2 was used as a digoxigenin-labeled probe. To perform a hybridization assay. The hybridization reaction is 5 × S
SC, 5 × Denhardet, 0.5% SDS, 0.2 mg / m
This was performed at 60 ° C. for 20 hours under the conditions of 1 salmon sperm DNA. The washing conditions were 2 × 30 minutes at room temperature under 2 × SSC and 0.1% SDS, then 2 minutes at 60 ° C. for 30 minutes.
I went there. Thereafter, 17 independent positive clones were obtained in the primary and secondary screenings by the fluorochrome method.

Example 4 (Sequencing of Thionine cDNA of the Present Invention) Of the seventeen positive clones obtained above, three clones (LTH22) in which the inside of the cloned cDNA fragment was not digested even after treatment with the restriction enzyme EcoRI. , LTH31, LTH
92) was isolated. After subcloning these three clones into pUC18, sequencing was performed using ABI DNA sequencer model 373A according to the manufacturer's instructions.

As a result, each of the clones contained 1
ORFs spanning 411 bases and with a molecular weight of 15,000
It was found to encode 0 protein. Of the three clones obtained, the cDNA sequence of clone LTH92 having the longest chain length had the nucleotide sequence represented by SEQ ID NO: 4.

Based on the amino acid sequence determined from the ORFs of these clones, the three clones isolated encode the same thionin precursor consisting of three regions, leader, thionin, and acidic protein. (See SEQ ID NO: 3). From the N-terminus of the amino acid sequence, positions 1 (methionine) to position 28 (glycine) are the leader peptide region, positions 29 (lysine) to position 74 (lysine) are the mature thionine region, and position 75 (leucine). To 137 (alanine) is the acidic protein region.

Table 3 shows a comparison between the amino acid sequence of thionin of the present invention and the amino acid sequence of known thionin.

[0060]

[Table 3]

The upper row of Table 3 shows thionin derived from leaves, and the lower row shows thionin derived from seeds. LAVT in the table is a novel thionin of the present invention, DB4, DC4, DG3, HOTα and HOTβ are barley-derived, VSA2 is from mistletoe, and AVTα and AVTβ are known thionin from oats.

The homology between clone LTH92 having the longest chain length and barley leaf-specific thionin (clone DB4) was compared at the amino acid level.
%, About 75% in the leader region, and about 7 in the thionine region.
The homology was 1%, and about 41% in the acidic protein region, indicating that the thionin was completely different from the known leaf-specific thionin. The isoelectric point (pI) calculated based on the amino acid sequence of the thionin precursor of the present invention is approximately 9.0 in the thionin region and approximately 4.0 in the acidic protein region. This bipolarity with respect to pI is thought to contribute to the stability of the thionin moiety in the cell.

In particular, the thionine of the present invention is preferably composed of isoleucine at the seventh position from the N-terminal, methionine at the eighth position, valine at the 15th position, isoleucine at the 18th position, proline at the 19th position,
It is characteristic that thirteenth is threonine, 22nd is proline, 29th is threonine, and 38th is asparagine, and amino acid residues at these positions are DB4, DC4, DG3, HOTα and HOT derived from barley.
β, AVTα and AVTβ derived from oat seeds were completely different.

Example 5 (Northern Analysis for Thionine of the Present Invention) Poly (A) + RN from the second leaves, roots, and seedlings of the oat (advanced) plant cultivated in a greenhouse and germinated and grown under dark treatment conditions
A was extracted and 2 μg each was electrophoresed on a 1% denaturing agarose gel. After the electrophoresis, high bond N with 20 × SSC
(Amersham) The RN transferred to the membrane and transferred onto the membrane using a commercially available UV crosslinker
A was cross-linked and fixed. Prehybridization is 5x
SSC, 7% SDS, 2% blocking agent, 0.02%
Lauryl sarcosine, 50% formaldehyde conditions,
Performed at 50 ° C. overnight. Hybridization was performed by using, in the above reaction buffer, a fragment obtained by amplifying the thionin region of the cDNA clone LTH92 obtained in Example 3 by PCR and digoxigenin labeling as a probe.
Add to a final concentration of 100 μg / ml,
For 20 hours. Then 2 × S
Washed twice at room temperature under SC and 0.1% SDS twice for 15 minutes, and further washed twice at 68 ° C. under 0.1 × SSC and 0.1% SDS at 15 minutes each. did. The detection was based on the fluorescent coloring method.

When the expression of the native gene encoding thionin of the present invention was analyzed by Northern analysis,
In the leaves of the plant germinated and grown under dark conditions, mRNA having a size of about 750 bases, which is considered to correspond to the mRNA of the thionin gene, was observed. Such a signal is not observed in the lane extracted from the leaves and roots of the plant body under the normal circadian cultivation environment.

Next, leaf pieces cut out from the above-mentioned plants were
The cells were placed in a 0 μM jasmonic acid aqueous solution and incubated at room temperature. As a result, an increase in the mRNA was observed 6 hours after the start of the incubation, and the maximum was observed 24 hours later. On the other hand, such an increase in mRNA was not observed in incubation with water alone as a control. Jasmonic acid is a substance that is deeply involved in injury stress induction in plant gene expression. Therefore, the above results show that the expression of the native gene encoding thionin of the present invention is inherently wound stress-inducing.

Example 6 (Southern Analysis on Thionine of the Present Invention) Genomic Southern analysis was performed on the above-mentioned oat varieties and two near-green species. The hybridization assay conditions are the same as those described in Example 2 above, but the washing step is further performed twice under 30 minutes for 30 minutes under 1 × SSC at 0.5 ×.
Twenty 30 minutes were performed under SSC conditions.

As a result, a strong positive signal band was present in all species. In the above-mentioned Southern analysis in the case of digestion with the same restriction enzyme, all species showed similar band patterns. This indicates that the genomic gene encoding thionin of the present invention exists in one copy per genome and is well conserved in oats.

Example 7 (Construction of Vector Containing Thionine Expression Cassette) From the above isolated cDNA clone LTH92, a normal PC
A fragment having a BamHI cleavage site at the 5 'end and a SacI cleavage site at the 3' end of the region encoding the thionin precursor was prepared by the R method. The obtained PCR fragment was ligated to a vector obtained by treating the pBI-derived vector shown in FIG. 1 with BamHI and SacI enzymes.

Example 8 (Introduction of Expression Cassette into Tobacco) (1) Transformation of A. pulmodium tumefaciens A. pulmorum tumefaciens LBA4404 strain (purchased from Clontech) at 250 μg / ml Streptomycin and 50
The cells were cultured at 28 ° C. in an L medium containing μg / ml of rifampicin, and Nagel et al. (Microbiol. Lett., 67, 325, (199)
According to the method of 0)), a cell suspension was prepared, and the plasmid vector prepared in Example 7 was introduced into the above strain by electroporation. 28 ° C, 3
Culture was performed for one day to obtain a transformant.

(2) Transformation of tobacco The transformed A. pacterium tumefaciens L obtained in the above (1)
After shaking culture (28 ° C., 200 rpm) of the BA4404 strain in a YEB medium (DNA cloning volume 2, page 78), the strain was diluted 20-fold with sterile water, and tobacco (Nicotiana Tabacum Sammsun) was diluted.
NN) were co-cultured. Two to three days later, the bacteria are removed with a medium containing antibiotics, passaged with a selection medium every two weeks, and transformed with or without kanamycin resistance due to NPTII expression from the introduced expression cassette. Was selected, calli were induced by a conventional method, and redifferentiated into plants.

[0072]

According to the present invention, there is provided a novel thionin derived from oat leaves having an amino acid sequence different from that of known thionin, and a DNA encoding the same. Since the thionin of the present invention has excellent antibacterial properties, plant varieties that express the thionin of the present invention and have high antibacterial activity by introducing DNA encoding the thionin of the present invention into plants are introduced. Provided.

[0073]

[Sequence list]

[0074]

[SEQ ID NO: 1] Sequence length: 46 Sequence type: Amino acid Sequence type: Protein sequence Lys Ser Cys Cys Lys Asp Ile Met Ala Arg Asn Cys Tyr Asn Val Cys 5 10 15 Arg Ile Pro Gly Thr Pro Arg Pro Val Cys Ala Thr Thr Cys Arg Cys 20 25 30 Lys Ile Ile Ser Gly Asn Lys Cys Pro Lys Asp Tyr Pro Lys 35 40 45

[0075]

[SEQ ID NO: 2] Sequence length: 138 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA sequence AAG AGT TGC TGC AAG GAT ATC ATG GCA AGA AAC TGC TAC AAC GTG TGC 48 Lys Ser Cys Cys Lys Asp Ile Met Ala Arg Asn Cys Tyr Asn Val Cys 5 10 15 CGT ATT CCT GGT ACT CCT AGG CCT GTC TGT GCA ACT ACA TGT CGT TGC 96 Arg Ile Pro Gly Thr Pro Arg Pro Val Cys Ala Thr Thr Cys Arg Cys 20 25 30 AAA ATC ATT AGT GGA AAT AAG TGC CCA AAG GAT TAT CCT AAA 138 Lys Ile Ile Ser Gly Asn Lys Cys Pro Lys Asp Tyr Pro Lys 35 40 45

[0076]

[SEQ ID NO: 3] Sequence length: 137 Sequence type: amino acid Sequence type: protein sequence Met Gly Ser Ile Lys Gly Leu Lys Ser Val Val Ile Cys Val Leu Val 5 10 15 Leu Gly Ile Val Leu Glu Gln Val Gln Val Glu Gly Lys Ser Cys Cys 20 25 30 Lys Asp Ile Met Ala Arg Asn Cys Tyr Asn Val Cys Arg Ile Pro Gly 35 40 45 Thr Pro Arg Pro Val Cys Ala Thr Thr Cys Arg Cys Lys Ile Ile Ser 50 55 60 Gly Asn Lys Cys Pro Lys Asp Tyr Pro Lys Leu His Gly Asp Pro Asp 65 70 75 80 Ala Gly Thr Pro Asn Ala Ile Glu Phe Cys Asn Thr Gly Cys Met Ser 85 90 95 Ser Ile Cys Asp Asn Met Asn Asn Ala Tyr Asn Val Glu Asp Lys Glu 100 105 110 Ile Asp Val Glu Leu Cys Gly Asn Ala Cys Thr Ser Phe Cys Asn Gln 115 120 125 Ile Ile Val Arg Ala Ser Val Ala Ala 130 135

[0077]

[SEQ ID NO: 4] Sequence length: 745 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA sequence GCAGGGCTTG CATGCCTGCA GGTCGACTCT AGAGGATCCC CGGGTACCGA GCTCGATTCG 60 CGGNCGATCA AGAAACTAGC GCCAACCAAC CGATGGA AGT ATC AAA GGT CTT AAG 115 Met Gly Ser Ile Lys Gly Leu Lys 5 AGT GTA GTC ATT TGT GTC CTT GTA TTG GGG ATA GTT CTG GAG CAG GTC 163 Ser Val Val Ile Cys Val Leu Val Leu Gly Ile Val Leu Glu Gln Val 10 15 20 CAA GTA GAG GGA AAG AGT TGC TGC AAG GAT ATC ATG GCA AGA AAC TGC 211 Gln Val Glu Gly Lys Ser Cys Cys Lys Asp Ile Met Ala Arg Asn Cys 25 30 35 40 TAC AAC GTG TGC CGT ATT CCT GGT ACT CCT AGG CCT GTC TGT GCA ACT 259 Tyr Asn Val Cys Arg Ile Pro Gly Thr Pro Arg Pro Val Cys Ala Thr 45 50 55 ACA TGT CGT TGC AAA ATC ATT AGT GGA AAT AAG TGC CCA AAG GAT TAT 307 Thr Cys Arg Cys Lys Ile Ile Ser Gly Asn Lys Cys Pro Lys Asp Tyr 60 65 70 CCT AAA CTG CAT GGC GAC CCC GAT GCC GGT ACA CCA AAT GCC ATC GAG 355 Pro Lys Leu His Gly Asp Pro Asp Ala Gly Thr Pro Asn Ala Ile Glu 75 80 85 TTC TGC AAC ACG GGA TGT ATG TCC TCC ATC TGC GAC AAC ATG AAT AAC 403 Phe Cys Asn Thr Gly Cys Met Ser Ser Ile Cys Asp Asn Met Asn Asn 90 95 100 GCT TAT AAC GTT GAA GAT AAA GAA ATA GAT GTG GAA CTC TGC GGC AAT 451 Ala Tyr Asn Val Glu Asp Lys Glu Ile Asp Val Glu Leu Cys Gly Asn 105 110 115 120 GCA TGT ACC AGT TTC TGT AAC CAG ATC ATC GTC CGT GCA TCA GTT GCA 499 Ala Cys Thr Ser Phe Cys Asn Gln Ile Ile Val Arg Ala Ser Val Ala 125 130 135 GCC TAATCATGCA TGCATGTGTA TGATGTCGAA GGCCAAGGTC ATGTTGTGGT 552 Ala TACCAGCGAA TAAATTTGGA TCCCATCAGT CACAACCAAG CCCGTGTGTC GTGTTAATTA 612 TGTATGTGTC AATGTTTGTG TGTTTTCCTT CCATGTTGCA ATAAAGGTTA CCATGATAAG 672 GATGTACTTG TACCCCTGGC CAAATACGAA CATGGTGGGA TGTCCAAAAA AAAAAAAAAA 732 AAAAAAAAAA AAA 745

[0078]

[SEQ ID NO: 5] Sequence length: 32 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (primer) Sequence AATCAAGCTT CCAAGTAGAA GGCAAGAGTT GC 32

[0079]

[SEQ ID NO: 6] Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (primer) Sequence AGTCTCTAGA GTCCATGTTG TCACAGACGG 3
0

[0080]

[SEQ ID NO: 7] Sequence length: 35 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (primer) sequence GTTCCTGCAG TCATACTGGG TTTAGTTCTG GAGCC 35

[0081]

[SEQ ID NO: 8] Sequence length: 36 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (primer) sequence GTAGTCTAGA ATTCAGTTTA GGATAGTCMC TAGRGC 36

[Brief description of the drawings]

FIG. 1 shows a vector derived from pBI121.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 5/10 C12N 5/00 C C12P 21/02 A61K 37/02 ABU // A01H 5/00 ADU A01N 63/00 ADX (C12P 21/02 C12R 1:91) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 15/00-15/90 C07K 14/00-14/825 C12N 5/00-5/28 GenBank / EMBL / DDBJ (GENETYX) BIOSIS (DIALOG) WPI (DIALOG)

Claims (7)

(57) [Claims] 1. A thionin having an amino acid sequence of positions 1 to 46 of SEQ ID NO: 1. 2. An isoleucine at the seventh position, a methionine at the eighth position, a methine at the eighth position, a valine at the fifteenth position, an isoleucine at the eighteenth position in the amino acid sequence from the first to the 46th position of SEQ ID NO: 1.
Thionine having the proline at position 21, threonine at position 21, proline at position 22, threonine at position 29, asparagine at position 38, and having one or more deletions and / or substitutions. 3. A DN comprising a sequence encoding thionin having the amino acid sequence from position 1 to position 46 of SEQ ID NO: 1.
A. 4. The sequence according to claim 1, wherein the sequence is 1 to 13 of SEQ ID NO: 2.
The DNA according to claim 3, which is an eighth DNA sequence. A thionine expression cassette comprising the DNA according to claim 3. 6. A cell transformed with a vector containing the expression cassette according to claim 5. 7. A method for producing thionin, comprising a step of culturing the transformed cell according to claim 6 and a step of collecting thionin expressed in the transformed cell.