JP2002125670A - New pathogenic gene (abva gene) of agrobacterium tumefaciens - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for improving an Agrobacterium tumefaciens vector system used for transducing an exogenote into a plant. SOLUTION: The pathogenic gene of the Agrobacterium tumefaciens, characterized by containing the new pathogenic gene (abvA gene) of the Agrobacterium tumefaciens or a gene homologous to the gene. Namely, the pathogenic gene encoding the next peptide: MMLRLLIAAD WPTWRSTRKA ARCAAPWPPE QVSRSRSMTK IWSETICTVS KRSRNPGGETSSRSSDSHRR RPASCRMKTP VQSPTIGMPA AARSFRKRSA SGGRALWSLI SPPTTTI or homologous to the peptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to Agrobacterium.
Tumefaciens is a vector system that has been used to introduce foreign genes into plants. It relates to the improvement of this vector system.

[0002]

BACKGROUND ART Agrobacterium tume
Faciens is widely used to introduce foreign genes into plants, but has problems such as problem A: limited host range and problem B: low transfection efficiency.

[0003]

The novel pathogenic gene discovered this time relates to the introduction of a foreign gene into a plant chromosome, and aims at improving problems A and B of the prior art.

[0004]

When this gene is inactivated, Agrobacterium tumefacie is inactivated.
ns discovered that no foreign gene could be introduced into the plant at all. Therefore, if the expression of this gene is promoted, it is expected that the host range of transformation will be expanded and the efficiency will be increased.

[0005]

[Function] This gene introduces a foreign gene into the chromosome of a plant, and causes the plant to express a new trait.

[0006]

EXAMPLES The present gene ( abvA gene) was replaced with Agroba.
Example which isolate | separated from Cterium tumefaciens .

[0007] Example 1 (1) transposon (Tn5) insertion mutagenesis by the separation of the mutant strain (Figure 1): A. tumefaciens (A2
08 strain, parent strain, pathogenic) to transposon (Tn
5) was inserted to isolate 5000 mutant strains. This mutant strain is called Kalanchoe p.
inna ) leaves, Kalanchoe pinnata ( Kala)
Nchoe pinnata ) and carrot ( Daucus carota ) roots were inoculated and examined for virulence. One of the mutants (provisionally called M-1 mutant)
Did not show any pathogenicity and did not induce a goal at all against Komochibenshisui and Kyouronkeisou,
It showed weak pathogenicity to carrots.

(2) Cloning of a gene fragment into which Tn5 has been inserted from M-1mutant (FIG. 2): M-1mu
During Tant, only one Tn5, not chromosome but Ti-
It was found that it was inserted into the plasmid. This T
The DNA fragment inserted with n5 was cloned, and the nucleotide sequence of the DNA portion linked to Tn5 was determined. When the homology of this sequence was searched using a computer,
No homology was found with the sequence of a known homologous gene.

(3) The parent strain (A20) of the gene before the insertion of the gene in which Tn5 has been inserted in M1mutant
Cloning (Fig. 3a): Ti-plasmid was isolated from A208 strain, and a library was constructed from this. This library was selected using a probe prepared from the above DNA site adjacent to Tn5.
A clone having 3 kb of DNA was obtained. This 2.3 kb
DNA was introduced into M-1 mutant.
The pathogenicity of 1 mutant was completely restored.

(4) Determination of nucleotide sequence of 2.3 kb DNA (FIG. 3b, FIG. 4): When the nucleotide sequence of 2.3 kb DNA was determined, three open reading frames were detected. M-1mutant
In this case, Tn5 was inserted into the first open reading frame ( designated as abvA gene). This a
When only the bvA gene was inserted into M-1 mutant, the pathogenicity of M-1 mutant was restored. However, the recovery was not complete and a slightly smaller goal was induced compared to the parent stock (A208).

(5) Distribution of abvA gene (FIG. 5): a
the bvA gene in the probe, was examined by Southern hybridization analysis, this gene is nopaline type Ti- plasmid, octopine type Ti- plasmid, A. Tumefaciens was found to be present in the chromosome.

The function of the abvA gene (FIG. 6): M-1m
A binary vector having an intron in the reporter gene (GUS gene) was introduced into utant. This m
utant is inoculated into the leaves of Koimochibenchisou, and GUS
When the activity was measured, a high GUS enzyme activity was detected. From these results, it was found that although M-1 mutant was non-pathogenic, it could transfer T-DNA to the nucleus of host plant cells. But,
The T-DNA is not integrated into the chromosome of the host plant cell because no goal can be achieved. Therefore, abv
The A gene converts the foreign gene in the T-DNA into the chromosome D of the plant.
It is concluded that it has a function to incorporate into NA.

As described above, when the present gene is highly expressed, it is expected that the host range for plant transformation is expanded and the efficiency is increased.

[0013]

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1: Assay for pathogenicity of M-1 mutant on KODAKARAKENISOU leaves, KAIRON KONIDOO stems and carrot roots.

FIG. 2. Determination of the site of Tn5 insertion in M-1 mutant

FIG. 3 DNA cloned into M-1 mutant
Recovery of virulence when fragments are introduced

FIG. 4. Agrobacterium tumefac
nucleotide and amino acid sequence of the new virulence gene of iens (abvA gene)

FIG. 5: Distribution of abvA gene

FIG. 6. TD in which M-1 mutant contains a foreign gene
Results showing that NA can be transferred into the nucleus of the cells of Kodakarabensiso

 ──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Majumdar Parimoru 1-4-1 Jyoiri, Ueda-shi, Nagano Prefecture, No.7 Higashimachiso F-term (reference) 4B024 AA08 CA03 DA05 EA04 FA20 GA11 GA19 HA12 4H045 AA10 BA21 CA11 EA05 FA71

Claims (2)

[Claims] 1. Agrobacterium tume
Agrobacterium Tumefacie characterized in that it contains a new virulence gene (AbvA gene) or a gene homologous genes faciens
ns virulence gene. 2. The following peptide: MMLRLLIAD WPWRSTRKA ARC
AAPWPPEQVSRSRSMTK IWSETIC
TVS KRSRNPGGETSSRSSSDHRR
RPASCRMKTP VQSPTIGMPAARS
FRKRSA SGGRALWSLI SPPTTI
2. The virulence gene according to claim 1, which encodes a peptide having homology thereto.