JPH11313680A - Cecropin gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain cecropin gene which can obtain plants having potentiated resistance to damage due to disease caused by plant bacteriogenous diseases, e.g. lettuce scab and the like, by coding a specific polypeptide. SOLUTION: This cecropin gene contains the base sequence that encodes (A) a polypeptide having the amino acid sequence of formula I or encodes (B) the amino acid sequence in which one or several amino acids are deleted, substituted or added to the amino acid sequence of formula I, and has antimicrobial activity and is represented by formula II. Further, it is favorable to produce transgenic plants having resistance to damage due to disease by cultivating or growing the plant host obtained by means of introducing the recombinant vector containing this cecropin gene into the host of plants, e.g. lettuce (botanical name: Lactuca scariola L. var. sativa), tabacco (botanical name: Nicotiana tabacum L.), pimento (botanical name: Capsicum annuum L. var. angulosum) or rice plant (botanical name: Oryza saliva L).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a gene encoding cecropin, which is an antibacterial polypeptide, a recombinant vector containing the gene, a transformed plant containing the vector, and a method for producing the transformed plant.

[0002]

2. Description of the Related Art Lettuce rot is a disease caused by Pseudomonas cichorii and the like. Since lettuce rot develops symptoms inside the head, detection of the onset of the disease is delayed, and it is difficult to obtain the effect of a drug sprayed from outside. In addition, lettuce rot is a bacterial disease, and thus has a problem that there is no drug that exhibits an excellent effect.
Furthermore, at present, there is no lettuce variety that shows absolute resistance to rot caused by P. cichorii. In addition, since the use of chemicals is restricted from the viewpoint of environmental considerations, rot easily occurs, and it is difficult to suppress the spread of damage once it occurs. Therefore, lettuce rot is an intractable disease among various plant diseases.

[0003] In order to control the above-mentioned rot, cultivation of varieties having relatively high resistance, thorough hygiene management in fields, and removal of weeds are performed. For example, since P. cichorii is polygenic, removal of weeds, which are intermediate hosts, and the like have been performed as cultivated pest control methods. However, the effect is not enough.

[0004] By the time the lettuce rot develops, the bacterial density of P. cichorii in the lettuce leaves is 10 ⁵ colony forming units.
/ g Fresh weight (cfu / g) or higher. At densities of 10 ⁵ cfu / g or less (10 ³ to 10 ⁴ cfu / g), P. cichorii does not form lesions because it lives inhabited on lettuce leaves (Ann. Phytopath . Soc. Jap. 62: 141-14
6). In addition, it has been shown that P. cichorii invading lettuce leaves proliferates in the intercellular space of the epidermis and mesophyll (An
n. Phytopath. Soc. Jap. 62: 125-129).

[0005] Accordingly, it is considered that the onset of disease can be suppressed by suppressing the growth of bacteria in the lettuce cell space. So far, attempts have been made to confer bacterial disease resistance by introducing the cecropin gene into tobacco plants. However, it was difficult to obtain stable results (Plant
Cell Report 13: 295-299).

[0006]

DISCLOSURE OF THE INVENTION The present invention relates to a gene encoding cecropin, which is an antibacterial polypeptide, a recombinant vector containing the gene, a transformed plant containing the vector, and having enhanced disease resistance. An object of the present invention is to provide a method for producing the transformed plant.

[0007]

Means for Solving the Problems The present inventors have made intensive studies to solve the above problems, and as a result, designed and synthesized a gene encoding a derivative of cecropin B, and introduced the gene into a plant. As a result, the present inventors succeeded in producing a plant imparted with resistance to plant diseases such as bacterial diseases, and completed the present invention.

[0008] That is, the present invention is a cecropin gene encoding the following polypeptide (a) or (b). (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (b) an amino acid sequence comprising one or more amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and an antibacterial activity The above-mentioned gene includes a gene containing the base sequence represented by SEQ ID NO: 1.

Further, the present invention is a recombinant vector containing the gene. Furthermore, the present invention is a transformed plant comprising the recombinant vector and imparted with disease resistance. As the plant, a plant, plant organ, plant tissue or plant culture cell of a plant (for example, lettuce, tobacco, pepper, rice, etc.) belonging to any family selected from the group consisting of Asteraceae, Solanaceae, and Gramineae Is mentioned. Furthermore, the present invention is a method for producing a transformed plant imparted with disease resistance, which comprises introducing the recombinant vector into a plant host and culturing or cultivating the obtained plant host. Hereinafter, the present invention will be described in detail.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a gene synthesized based on a known amino acid sequence of cecropin using codons frequently used in plants. Also,
The present invention relates to a transgenic plant to which disease resistance (eg, bacterial disease resistance) has been imparted by introducing the gene linked to a vector into Agrobacterium and infecting the resulting Agrobacterium into a plant host. It is characterized by producing.

1. Synthesis of cecropin gene of the present invention Cecropin gene of the present invention (referred to as SHIVA-1 gene)
Is the known amino acid sequence of cecropin (Plant Science
89: 43-53), and can be obtained by synthesis using codons frequently used in plants. The codon usage frequency uses that of Codon Usage Tabulated from GenBank. The synthesis of the gene is performed using a usual chemical synthesizer.

SEQ ID NO: 1 shows the nucleotide sequence of the gene of the present invention, and SEQ ID NO: 2 shows the amino acid sequence of the polypeptide encoded by the gene, provided that the polypeptide comprising this amino acid sequence has antibacterial activity. Mutations such as deletion, substitution, and addition may occur in one or several amino acids in the amino acid sequence.

The mutation was introduced by adding Kunkel and Gapped dup to the gene having the nucleotide sequence represented by SEQ ID NO: 1.
A known method such as the lex method or a method similar thereto can be used. For example, a mutation introduction kit (Mutant-K (T
AKARA), Mutant-G (TAKARA), TAKARA LA P
Mutation is introduced using a CR in vitro Mutagenesis series kit).

2. Preparation of Recombinant Vector and Transformant (1) Preparation of Recombinant Vector It is possible to construct the recombinant vector of the present invention by introducing the SHIVA-1 gene prepared as described above together with a promoter into an appropriate vector. it can. The vector for inserting the SHIVA-1 gene is not particularly limited as long as it can be replicated in a host, for example, plasmid DNA,
Phage DNA and the like.

[0015] Plasmid DNA (binary vector)
Is the alkaline extraction method from E. coli and Agrobacterium (B
irnboim, HC & Doly, J. (1979) Nucleic acid Res 7: 1
513) and the like. Examples of the binary vector include pBI121, pBI101 and the like. As commercially available plasmids, for example, pUC118 (manufactured by Takara Shuzo), pUC119 (manufactured by Takara Shuzo), pBluescript SK + (Strat
agene), pGEM-T (Promega), pCR2.1 (Invitr
ogen).

As phage DNA, for example, M13mp18,
M13mp19, M13tv18 and the like. To insert a promoter into a vector, first, the purified SHIVA-1 gene is cut with an appropriate restriction enzyme, inserted into an appropriate vector DNA restriction enzyme site or a multiple cloning site, and ligated to a vector. Adopted. Examples of the promoter include a cauliflower mosaic virus 35S promoter, an actin promoter, a promoter of a nopaline synthase gene, and the like.

Here, the SHIVA-1 gene to be expressed needs to be incorporated into a vector so that the function of the gene is exhibited. Therefore, in addition to the above promoter and the above genes, a signal peptide gene, a terminator, a drug resistance gene and the like can be incorporated into the recombinant vector of the present invention. In this case, as the signal peptide gene, tomato (Lycopersicon escu
lentum) -derived β-1,3 glucanase signal peptide gene, etc., as the terminator, a nopaline synthase gene terminator, etc., as an enhancer, tobacco mosaic virus Ω sequence, etc., as a drug resistance gene. Include kanamycin resistance gene and hygromycin resistance gene.

More specifically, as shown in FIG.
A tobacco mosaic virus Ω sequence (Ω) and a signal peptide gene (Signal) are ligated upstream of the gene (Cecropin SHIVA-1) (referred to as SHIVA-1 gene cassette). Next, SHI
VA-1 gene cassette in cauliflower mosaic virus 35
Connect downstream of S promoter (CaMV 35S Pro). The nopaline synthase gene terminator (NOS-te
r). In addition, a cassette in which a kanamycin resistance gene (NPT II) is connected between the promoter and the terminator of the nopaline synthase gene is produced (referred to as NPT II cassette). Then, the NPT II cassette is ligated upstream of CaMV 35S Pro to obtain the recombinant vector of the present invention (FIG. 1).

(2) Preparation of Transformed Plant The transformed plant of the present invention comprises the recombinant vector of the present invention,
It can be obtained by introducing it into a plant host so that the target gene can be expressed. For example, Agrobacterium tumefaciens (Agrobacterium
Tumefaciens) T-DNA in Ti plasmid during infection
A method utilizing the transfer of chromosomes into plant cells and chromosomal integration is used.

Here, the plant host means the whole plant, plant organs (eg, leaves, petals, stems, roots, seeds, etc.), plant tissues (eg, epidermis, phloem, parenchyma, xylem, vascular bundle, etc.) Or any of cultured plant cells. Plants used for transformation include, for example, plants belonging to the Asteraceae, Solanaceae or Gramineae. Plants belonging to these families include, for example, those described below, but are not limited to such plants, as long as they can impart disease resistance by introducing the recombinant vector of the present invention. It can be arbitrarily selected. Asteraceae: Lettuce (Lactuca sativa L) Solanaceae: Tobacco (Nicotiana tabacum), Bell Pepper (Capsi
cum annuum) Poaceae: Rice (Oryza sativa)

When a plant, a plant organ or a plant tissue is used as a host, transformation can be carried out by introducing the recombinant vector of the present invention into a collected plant section by the Agrobacterium method. It can also be introduced into protoplasts by an electroporation method, or can be introduced into a plant organ or plant tissue by a particle bombardment method.

The tumor tissue, shoot or hairy root obtained as a result of the transformation can be used as it is for cell culture, tissue culture or organ culture, or by using a conventionally known plant tissue culture method. Plants can be regenerated and cultivated by administration of plant hormones at an appropriate concentration.

When a plant culture cell is used as a host,
Transformation is carried out by introducing the recombinant vector for expression of the present invention into cultured cells by electroporation or Agrobacterium binary vector method, etc., and thereafter, cell culture, tissue culture, organ culture and cultivation in the same manner as described above. To obtain a transformed plant to which disease resistance has been imparted.

3. Confirmation of Disease Resistance Acquisition Whether or not disease resistance has been imparted to the transformed plant obtained as described above can be confirmed by conducting an inoculation test of various pathogenic bacteria on the grown transformed plant.
In the present invention, the disease resistance is imparted, as a result of observing the enlargement of the lesion 4-7 days after inoculating the plant with the causative bacteria of the disease, the enlargement is the lesion of a non-transformed plant (mm) Means less than 50%, preferably less than 10%. The diseases that can be controlled in the present invention include, for example, lettuce rot, lettuce spot bacterial disease, and the like, but are not limited to these diseases.

[0025]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. [Example 1] Synthesis of cecropin SHIVA-1 gene The nucleotide sequence of cecropin SHIVA-1 was the previously reported amino acid sequence of cecropin SHIVA-1 (Plant Science 89:
43-53), using a translation codon frequently used in plants (SEQ ID NO: 1). The amino acid sequence of the polypeptide encoded by the nucleotide sequence represented by SEQ ID NO: 1 is shown in SEQ ID NO: 2.

Based on the nucleotide sequence represented by SEQ ID NO: 1, the following primers A and B were synthesized. Primer A: CCGGA TCCAT ATGCC AAGAT GGAGA CTTTT
CAGAA GAATC GATAG AGTTG GAAAG CAAAT CAAGC AAGGA AT
CCT TAGAGC (SEQ ID NO: 3) Primer B: TCGAG CTCTT ATCCA ACAGC TCTAG CATCT C
CAAC AAGAG CGATA GCTGG TCCAG CTCTA AGGAT TCCTT GCT
TG ATTTGC (SEQ ID NO: 4)

Taq DNA polymerase (Takara Shuzo) 0.5 μl, ×
10 buffer 10 μl, 10 mM dNTP 2 μl, primers A and B
Was mixed with 85.5 μl of sterilized water and reacted at 94 ° C. for 1 minute. Next, 30 cycles of this reaction were performed at 98 ° C. for 20 seconds and 68 ° C. for 5 minutes as one cycle.
The reaction was performed for 10 minutes. The obtained reaction product is phenol /
After the chloroform treatment, ethanol precipitation was performed. The precipitate fraction is dissolved in sterile water, cut with restriction enzymes Bam HI and Sac I (Takara Shuzo), and then subjected to agarose electrophoresis.
Using 1 (Takara Shuzo), an approximately 130 bp fragment was gel-recovered.

Example 2 Synthesis of Signal Peptide Based on the amino acid sequence of a signal peptide (Plant Mol. Biol. 20: 513-527) of tomato (L. esculentum) which has already been reported, the following primers C and Using D, PCR was performed in the same manner as in the synthesis of the cecropin gene to synthesize a signal peptide gene. Primer C: CCGGA TCCAT ATGGC TTTTC TAAGT TCTCT C
TTAG CTTCC CTTTT ACTTG TTGGG CTTCT (SEQ ID NO: 5) Primer D: CTTAG CTTCC CTTTT ACTTT TTTGG GCTTC T
AATC CAAAT AACAG GAGCG CAGAG ATCT (SEQ ID NO: 6) The obtained reaction product was treated with phenol / chloroform and then precipitated with ethanol. The precipitate fraction was dissolved in sterile water, cut with restriction enzymes Bam HI and Bgl II (Takara Shuzo), and subjected to agarose gel electrophoresis to recover a fragment of about 90 bp by gel.

Example 3 Construction of Binary Vector pBI121 (Toyobo) was cut with Bam HI and Sac I, followed by phenol / chloroform treatment and ethanol precipitation. The precipitate fraction was dissolved in sterile water, treated with alkaline phosphatase (CIP, BOEHRINGER MANNHEIM), and then subjected to agarose electrophoresis to recover a gel fragment of about 11 kbp, followed by ethanol precipitation. After the cecropin SHIVA-1 gene collected from the gel was ligated to the precipitate fraction (DNA ligation kit, Takara Shuzo), Escherichia coli
DH5α). Plasmids were recovered from the transformants selected in the presence of kanamycin 25 mg / l to obtain a binary vector pIBRC SH in which the cecropin SHIVA-1 gene was inserted into Bam HI / Sac I.

After pIBRC SH was digested with Bam HI, CIP treatment was carried out, and a fragment of about 11 kbp was recovered by agarose gel electrophoresis, ligated with a signal peptide gene, and ligated with E. coli DH.
5α was transformed. Plasmid pIBRC SigSH was obtained by alkaline miniprep of the transformant. The obtained plasmid was treated with Hind III and Bam HI.

On the other hand, the plasmid E12ΩGUSN was replaced with the restriction enzyme Hin.
The fragment was cleaved with dIII (Takara Shuzo) and Bam HI, and a fragment of about 900 bp was gel-recovered by agarose gel electrophoresis. This fragment was combined with the pIBRC SigSH cut with Hind III and Bam HI.
Were linked to obtain pIBRCΩSigSH (FIG. 1).

Example 4 Binary vector Agrobacterium tumefaciens
faciens) The constructed binary vector is transferred to A. tumefaciens LBA44
04 was introduced by electroporation. A. tumefaciens LBA4404 culture solution cultured with shaking in LB liquid medium was collected by centrifugation, and suspended in 10% glycerol. 40
Add 40 ng of the binary vector to the sample cuvette (0.2 c between electrodes) in μl of A. tumefaciens LBA4404 suspension.
m), and after cooling, 12.5 kv / cm, 25 μFμ, 600
Electroplating with Ω (Gene Pulser, Bio Ra
d) was performed. Transfer the culture to 0.8 ml of SOC broth and add
Cultivated with shaking at 1 ° C for 1 hour, and kanamycin 25 mg / l was added.
The cells were spread on LB agar medium and cultured at 30 ° C. for 36 hours to obtain transformed colonies.

Example 5 Production of Lettuce Transformant The seeds of lettuce (Lactuca sativa L) variety "Success" were used.
Immerse in 70% ethanol for 5 seconds, 1% antiformin for 15 minutes, wash several times with sterile water, and grow in MS-1 medium (2% sucrose and 0.2% gellan gum in MS basic medium, pH 5.8) did. Growth temperature is 20 ℃, light condition is 4000 lux
(16 hr / day).

After growing, the tip and base of the cotyledon were cut off, immersed in an A. tumefaciens culture solution shake-cultured at 30 ° C. for 18 hours in an LB liquid medium for 5 minutes, drained with a sterile filter paper, and dried in an MS-2 medium. (0.1 mg / l benzylaminopurine (BA) in MS basic medium,
0.1 mg / l naphthaleneacetic acid (NAA) was added) for 2 days. The subsequent culture was performed at 25 ° C. and 4000 lux (16 hr / day).

A lettuce section inoculated with A. tumefaciens was placed in an MS-3 liquid medium (0.1 mg / l BA, 0.1 mg / l in MS basic medium).
After washing three times for 2 hours each in NAA, 300 mg / l cefataxime, and 100 mg / l kanamycin, transfer to MS-3 medium (1% INAAGAR added to MS-3 liquid medium) until callus formation Cultured. During the culture, lettuce sections were transferred to MS-3 medium every 7 days. The formed calli were transferred to MS-4 medium (MS basic medium at 0.
1 mg / l BA, 0.1 mg / l NAA, 300 mg / l cefataxime, 25
0 mg / l kanamycin was added) and cultured until adventitious bud formation. During the culture, lettuce sections were transferred to MS-4 medium every 7 days.

The adventitious shoots thus formed are transferred to an MS-5 medium (MS basic medium supplemented with 0.01 mg / l BA, 0.05 mg / l NAA, and 100 mg / l kanamycin), and the regenerated individuals become about 5 cm. Cultured until During cultivation, lettuce sections were transferred to MS-5 medium every 7 days. Transfer the regenerated individuals to MS-6 medium (MS basic medium), transplant the seeds to sterilized vermiculite after the roots elongate,
Raised at 4000 lux (16hr / day).

Example 6 Confirmation of the Introduced Cecropin Gene ISO PLANT (Nippon Gene) from transformed lettuce leaves
Was used to prepare genomic DNA. Using this genomic DNA as a template, the following primers E and F
R was performed to confirm that the cecropin gene had been introduced. Primer E: GAT GAC GCA CAA TCC CAC TAT CC (SEQ ID NO: 7) Primer F: GCA TCT CCA ACA AGA GCG ATA GC (SEQ ID NO: 8)

That is, 1 μm of genomic DNA from a regenerated individual
l, Amp Taq Gold (Takara Shuzo) 0.2μl, × 10 buffer 2.5μ
1, 2.5 mM MgCl ₂ 2.5 μl, 10 mM dNTP 2.5 μl, 20 μM each of primers E and F 2.0 μl, sterile water 14.8 μl, 9
After a reaction at 4 ° C. for 10 minutes, a reaction was performed at 94 ° C. for 45 seconds, 65 ° C. for 2 minutes and 72 ° C. for 3 minutes as one cycle, followed by a reaction at 72 ° C. for 10 minutes. This PCR
The product was subjected to agarose gel electrophoresis, and a 363 bp band was obtained.
It was confirmed that the signal peptide sequence had been introduced (FIG. 2). In FIG. 2, lanes 1-10 show the results of the test on the transformed lettuce, lane 11 shows the results of the test on the non-transformed lettuce, and lane M is a marker (100 bp ladder).

[Example 7] Lettuce rot resistance of the transformed lettuce The 10th developed leaf of the transformed lettuce in which introduction of the cecropin gene was confirmed was cut, and Pseudomonas chicory (P. c.
ichorii) (10 ⁶ cfu / ml) was inoculated injured, and the control effect was examined. Injured inoculations were performed at five locations on the left side of each leaf. The cut leaf of lettuce was allowed to stand still at 28 ° C. and in a humid condition, and the symptom was examined on the fourth day after inoculation.

As a result, lettuce showing almost no symptom and lettuce with suppressed symptom (enlargement of lesion) were obtained (FIG. 3). In FIG. 3, No. 1 was similar to the non-transformed lettuce, and the lesion was enlarged to about 50 mm. Did not show any symptoms. As in No.2 and No.3, the lesion diameter at the inoculation site is less than 5mm.
In some cases, symptoms were suppressed.

In the present invention, an individual whose symptom was suppressed such as No. 2 and No. 3 and an individual whose symptom hardly appeared such as No. 4 were determined to have been given resistance. As a result of performing such an experiment on 187 individuals, 47 individuals to which resistance was imparted could be obtained from 187 regenerated individuals.

[0042]

Industrial Applicability The present invention provides a gene encoding an antibacterial cecropin, a recombinant vector containing the gene, a transformed plant containing the vector, and a method for producing the transformed plant. According to the present invention, a plant individual having enhanced disease resistance to plant bacterial diseases such as lettuce rot can be obtained by introducing a gene SHIVA-1 encoding cecropin, which is an antibacterial polypeptide, into plants. .

[0043]

[Sequence list] SEQ ID NO: 1 Sequence length: 114 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Sequence ATG CCA AGA TGG AGA CTT TTC AGA AGA ATC GAT AGA GTT GGA AAG CAA 48 Met Pro Arg Trp Arg Leu Phe Arg Arg Ile Asp Arg Val Gly Lys Gln 1 5 10 15 ATC AAG CAA GGA ATC CTT AGA GCT GGA CCA GCT ATC GCT CTT GTT GGA 96 Ile Lys Gln Gly Ile Leu Arg Ala Gly Pro Ala Ile Ala Leu Val Gly 20 25 30 GAT GCT AGA GCT GTT GGA 114 Asp Ala Arg Ala Val Gly 35

SEQ ID NO: 2 Sequence length: 38 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Met Pro Arg Trp Arg Leu Phe Arg Arg
Ile Asp Arg Val Gly Lys Gln 15
10 15 Ile Lys Gln Gly Ile Leu Arg Ala Gly
Pro Ala Ile Ala Leu Val Gly 20 25
30 Asp Ala Arg Ala Val Gly 35

SEQ ID NO: 3 Sequence length: 81 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CCGGATCCAT ATGCCAAGAT GGAGACTTTT CAGAAGAATC GATAGAGTTG GAAAGCAAAT 60 CAAGCAAGGA ATCCTTAGAG C 81

SEQ ID NO: 4 Sequence length: 81 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence TCGAGCTCTT ATCCAACAGC TCTAGCATCT CCAACAAGAG CGATAGCTGG TCCAGCTCTA 60 AGGATTCCTT GCTTGATTTG C 81

SEQ ID NO: 5 Sequence length: 60 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence CCGGATCCAT ATGGCTTTTC TAAGTTTCCTCT CTT
AGCTTCC CTTTTACTTG TTGGGCTTCT 60

SEQ ID NO: 6 Sequence length: 59 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CTTAGCTTCC CTTTTACTTT TTTGGGCTTC TAATCCAAAT AACAGGAGCG CAGAGATCT 59

SEQ ID NO: 7 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence GATGACGCAC AATCCCACTA TCC 23

SEQ ID NO: 8 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence GCATCTCCAA CAAGAGCGAT AGC
23

[Brief description of the drawings]

FIG. 1 shows the binary vector pIBRCΩSigS of the present invention.
It is a construction diagram of H.

FIG. 2 is an electrophoretic photograph showing the results of detecting the cecropin SHIVA-1 gene.

FIG. 3 is a photograph showing the symptoms of transformed lettuce on day 4 after inoculation with lettuce rot (organism form).

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yasushi Hikiji 200 Monotsube, Nankoku City, Kochi Prefecture 29-1 Kochi University Dormitory (72) Inventor Yoshiko Nasu 1-2-8 Shimogawara Utsunomiya City, Tochigi 202

Claims (10)

[Claims] 1. A cecropin gene encoding the following polypeptide (a) or (b): (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (b) an amino acid sequence comprising one or more amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and an antibacterial activity A polypeptide having 2. A cecropin gene comprising the nucleotide sequence represented by SEQ ID NO: 1. 3. A recombinant vector containing the gene according to claim 1 or 2. 4. It comprises the recombinant vector according to claim 3,
A transformed plant to which disease resistance has been imparted. 5. The transformed plant according to claim 4, wherein the plant is a plant, a plant organ, a plant tissue or a plant cultured cell. 6. The transformed plant according to claim 4, wherein the plant is a plant belonging to any family selected from the group consisting of Asteraceae, Solanaceae, and Gramineae. 7. The transformed plant according to claim 6, wherein the plant belonging to the family Asteraceae is lettuce. 8. The transformed plant according to claim 6, wherein the plant belonging to the Solanaceae family is tobacco or pepper. 9. The transformed plant according to claim 6, wherein the plant belonging to Poaceae is rice. 10. A method for producing a transformed plant imparted with disease resistance, which comprises introducing the recombinant vector according to claim 3 into a plant host, and culturing or cultivating the obtained plant host.