JPH0568574A - Hybrid plasmid - Google Patents

Abstract

PURPOSE:To obtain the subject plasmid capable of readily producing a variety resistant to crown gall diseases by integrating a DNA fragment containing a part sandwiched between HindIII cleavage sites of an auxin synthase gene derived from a Ti plasmid of a specific microorganism. CONSTITUTION:The objective hybrid plasmid is obtained by extracting a Ti plasmid DNA from a P022 line of Agrobacterium tumefaciens separated from a canker of a Populus sieboldi Miq., then cleaving the resultant extract with a restriction enzyme PstI, cloning a DNA fragment containing a gene iaaM capable of coding an auxin synthase gene into a pUC13 plasmid, taking out a DNA fragment containing a part sandwiched between two cleavage sites with a restriction enzyme HindIII of the gene iaaM, recloning the DNA fragment into an SmaI site of a pUC19 plasmid, taking out a BamHI-SacI fragment containing the HindIII fragment therefrom and integrating the fragment into the BamHI/SacI sites of a binary vector plasmid pBI 121 of a plant.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hybrid plasmid, and more particularly to a hybrid plasmid which imparts crown gall disease resistance to plants.

[0002]

2. Description of the Related Art The plant pathogenic fungus Agrobacterium tumefaciens is known to cause crown gall disease, which is a difficult control disease, in many plants. This crown gall disease is a disease that forms a lumpy carcinoma on the roots and trunks of plants, and it is particularly damaging to perennial crops such as trees and fruit trees, which hinders growth and reduces yields. Is causing. Also, in America, Europe, Australia, etc.
Disease at the time of seed production is a problem.

This plant carcinoma is caused by the expression of an oncogene present in the T region of the Ti plasmid of Agrobacterium tumefaciens when introduced into plant cells, resulting in abnormal cell growth. The oncogene consists of genes iaaM and iaaH, which encode the enzyme that synthesizes the plant hormone auxin, and ipt, which encodes the enzyme that synthesizes cytokinin, and causes overproduction of the plant hormone in carcinoma cells. ing.

Agrobacterium radiobacter K84, a strain of non-pathogenic Agrobacterium, is a virulent antibiotic agrosin 8 which is lethal to pathogenic strains.
Secrete 4. Therefore, biocontrol of pathogenic Agrobacterium tumefaciens has been attempted by utilizing this, but this method has a limited application range because all pathogenic strains are not necessarily sensitive to agrosin 84 It has not been fully commercialized. Therefore, the development of more effective and universal methods is desired.

[0005]

[Means for Solving the Problems] In order to impart crown gall disease resistance to plants, a disease resistance gene was prepared by a gene recombination method and introduced into plants.

That is, the present invention relates to Agrobacterium
A hybrid plasmid in which a DNA fragment containing a portion flanked by two Hind III cleavage sites of a gene iaaM encoding a auxin synthase derived from Ti plasmid of Tumefascens is incorporated into a binary vector plasmid of a plant, and transformed by the hybrid plasmid, The present invention relates to a plant having crown gall disease resistance.

[0007] The disease resistance gene imparts disease resistance to plants by suppressing the expression of oncogenes introduced into plant cells by Agrobacterium tumefaciens, which is a pathogen of crown gall disease. Suppressing the expression of oncogenes is a disease-resistant gene having a DNA fragment of auxin synthase gene iaaM, which is one of the oncogenes involved in the synthesis of plant hormone auxin in the antisense or sense direction, and Agrobacterium.・
This is due to the effect of interference with oncogenes that are introduced into plants by tumefaciens.

The disease resistance gene is introduced into a plant by the hybrid plasmid of the present invention prepared as follows.

[0009] From the PO22 strain of Agrobacterium tumefaciens isolated from the porcupine carcinoma, T
DN in which auxin synthase gene iaaM exists by extracting i plasmid DNA and cleaving it with restriction enzyme Pst I
The A fragment is cloned into the pUC13 plasmid. gene
iaaM is 143bp and 840bp downstream from the start codon
It has a Hind III site (J. Gielen et al, The EMBO
Journal, vol.3,835-846,1984). These two Hind II
The fragment sandwiched between the I cleavage sites was T37 T-DNA
It corresponds to the 41st DNA fragment cleaved with dIII (M.
W. Bevan et al, Ann. Rev. Genat., Vol.16,357-384,198
2). DNA containing a portion sandwiched between the Hind III cleavage sites
The fragment is removed, the ends are made blunt with the Klenow fragment and then recloned into the Sma I site of the pUC19 plasmid.

The nucleotide sequence of the DNA fragment inserted into this plasmid was determined by the dideoxy method, and the
Two clones were obtained in which the NA fragment was oriented differently to the pUC19 plasmid.

On the other hand, the plant binary vector plasmid pBI 121 has a kanamycin resistance gene (NPT II) which can be expressed in plant cells. The BamH I-Sac I fragment containing the above Hind III fragment was removed from the pUC19 plasmid,
A hybrid plasmid is obtained by inserting into the BamHI / SacI site of the plasmid pBI121.

In this hybrid plasmid, the DNA fragment of the gene iaaM is inserted between the 35S promoter of cauliflower mosaic virus and the poly A recognition signal of the novalin synthase gene of the Ti plasmid, in the same direction (sense) or in the opposite direction as the original gene iaaM. (Antisense) has.

A plant body having crown gall disease resistance can be obtained by transforming the hybrid plasmid containing the above-mentioned disease resistance gene by a conventional method.

[0014]

EXAMPLES Next, the present invention will be described in more detail by way of examples. Example 1 Agrobacterium isolated from a porcupine carcinoma
Ti plasmid D from PO22 strain of Tume fascens
NA was extracted. Then, this was cleaved with the restriction enzyme PstI, and the DNA fragment containing the auxin synthase gene iaaM was cloned into the pUC13 plasmid. This gene
A DNA fragment containing a portion flanked by the Hind III cleavage sites of iaaM was taken out, the ends were made blunt with Klenow fragment, and then recloned into the Sma I site of pUC19 plasmid.

The nucleotide sequence of the DNA fragment inserted into the pUC19 plasmid was determined by the dideoxy method, and two clones in which the DNA fragment of the gene iaaM had different orientations to the pUC19 plasmid were obtained.

Next, from the above pUC19 plasmid, the above Hi
The BamH I-Sac I fragment containing the nd III fragment was extracted, and this was expressed in a plant cell using a kanamycin resistance gene (NPT
B) of the binary vector plasmid pBI 121 with II)
A hybrid plasmid was obtained by inserting into the amHI / SacI site.

Example 2 The hybrid plasmid obtained in Example 1 was subjected to a freeze-thaw method to obtain LBA of Agrobacterium tumefaciens.
It was introduced into strain 4404, and leaf discs of tobacco were infected with Agrobacterium tumefaciens.
Then add this to Murashig containing kanamycin and hormones
Recombinant individuals having a kanamycin resistance gene that had differentiated into buds were selected by culturing in e-Skoog medium.

Recombinant tobacco individuals having a DNA fragment of the gene iaaM in the antisense and sense directions were acclimated and cultivated in an artificial weather device. The Agrobacterium tumefaciens P22R1 strain was infected to each leaf disk of a normal individual and a recombinant individual and placed on the Murashige-Skoog medium. As a result, formation of carcinoma was observed in the leaf discs of normal individuals that were infectious for root carcinoma.

On the other hand, it was confirmed that the recombinant tobacco (SCR) having the DNA fragment of the gene iaaM in the sense direction markedly inhibited the formation of carcinoma and was resistant to crown gall disease. Further, in the recombinant tobacco (ACR) having the DNA fragment of the gene iaaM in the antisense direction, abnormal carcinoma in which not only the formation of carcinoma but also the multiblast was observed.

According to Southern analysis of tobacco recombinant individuals, 4 copies of DNa of gene iaaM was found in the above SCR individuals.
It was confirmed that the A fragment was integrated in the sense direction, and that one copy of the DNA fragment of the gene iaaM was integrated in the antisense direction in the ACR individual. Furthermore, No
Rthern analysis revealed that DNA in both directions was transcribed together.

Next, in order to test the crown gall disease resistance in the plant, a stem of a tobacco recombinant individual having a height of 15 cm was perforated with an injection needle and inoculated with Agrobacterium tumefaciens P22R1. As a result, in the case of normal individuals, the formation of carcinoma, which is a characteristic of root carcinoma, was observed at almost all inoculation sites. On the other hand, SCR
In the case of individuals, almost no formation of carcinoma was observed, and buds were formed at a part of the inoculation site. In addition, in the case of ACR individuals, the formation of carcinoma was observed in about half, but the number was small and variation in size was also observed. In addition, SCR
The individuals and ACR individuals also showed almost complete resistance to crown cancer disease to other Japanese Agrobacterium tumefaciens strains.

[0022]

INDUSTRIAL APPLICABILITY The hybrid plasmid obtained by the present invention can impart crown gall disease resistance to plants. Therefore, by introducing this hybrid plasmid into a plant, a crown gall disease resistant variety can be easily prepared.

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[Procedure amendment]

[Submission date] September 21, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0009

[Correction method] Change

[Correction content]

[0009] From the PO22 strain of Agrobacterium tumefaciens isolated from the porcupine carcinoma, T
DN in which the auxin synthase gene iaaM is present by extracting i plasmid DNA and cleaving it with the restriction enzyme Pst I
The A fragment is cloned into the pUC13 plasmid. gene
iaaM is 143bp and 840bp downstream from the start codon
It has a Hind III site (J. Gielen et al, The EMBO
Journal, vol.3,835-846,1984). These two Hind II
The fragment sandwiched between the I cleavage sites was T37 T-DNA
It corresponds to the 41st DNA fragment cleaved with dIII (M.
W. Bevan et al, Ann. Rev. Genet. , Vol.16,357-384,198
2). DNA containing a portion sandwiched between the Hind III cleavage sites
The fragment is removed, the ends are made blunt with the Klenow fragment and then recloned into the Sma I site of the pUC19 plasmid.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location // C12N 5/10 15/52 (C12N 15/52 C12R 1:01) (72) Inventor Oshima Kihachiro, 5-21-1, Oji, Kita-ku, Tokyo Inside the Central Research Institute, Jujo Paper Co., Ltd. (72) Inventor, Kunio Hata, 5-21-1-1, Prince, Kita-ku, Tokyo Inside the Central Research Laboratory, Jujo Paper Co., Ltd. (72) Inventor Sano Hiroshi Akita Prefecture 2-2 Minami Ogata-mura, Minami-Akita District Akita Prefectural College of Agriculture Biotechnology Research Institute

Claims (3)

[Claims] 1. A hybrid plasmid in which a DNA fragment containing a portion flanked by two Hind III cleavage sites of a gene iaaM encoding an auxin synthase derived from Ti plasmid of Agrobacterium tumefaciens is incorporated into a plant binary vector plasmid. .. 2. A gene encoding an auxin synthase
Includes a part between two Hind III cleavage sites of iaaM D
NA fragment is Agrobacterium tumefaciens P
It is derived from the Ti plasmid of the O22 strain and is a plant binary vector plasmid pBI 121.
The hybrid plasmid described. 3. A plant having crown gall disease resistance, which is transformed with the hybrid plasmid according to claim 1.