JP2955060B2 - Hybrid plasmid - Google Patents

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 that confers crown gall disease resistance to plants.

[0002]

BACKGROUND OF THE INVENTION The phytopathogenic fungus Agrobacterium tumefaciens is known to cause crown gall disease, an intractable disease, in many plants. Crown gall disease is a disease that forms a hump-shaped carcinoma on the roots and stems of plants, and is particularly damaging to permanent crops such as trees and fruit trees, inhibiting growth and reducing yields. Is causing. In the United States, Europe, Australia, etc.,
Diseases during seed production are a problem.

[0003] The carcinoma of the plant is caused by the fact that an oncogene present in the T region of the Ti plasmid of Agrobacterium tumefaciens is introduced into plant cells and expressed, resulting in abnormal cell proliferation. The oncogene consists of the genes iaaM and iaaH, which encode the enzyme that synthesizes the plant hormone auxin, and the gene ipt, which encodes the enzyme that synthesizes cytokinin, and causes overproduction of plant hormones in carcinoma cells. ing.

Agrobacterium radiobacterium K84, a strain of non-pathogenic Agrobacterium, is an agrosine 8 antibiotic that is lethal to pathogenic strains.
Secretes 4. Therefore, biological control of pathogenic Agrobacterium tumefaciens has been attempted using this method, but this method has a limited scope because not all pathogenic strains are necessarily sensitive to agrosine 84. It has not yet been fully commercialized. Therefore, development of a more effective and universal method 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 genetic recombination technique and introduced into dicotyledonous plants .

That is, the present invention relates to Agrobacterium
Two Hinds of the gene aaaM encoding auxin synthase from the Ti plasmid of Tumefaciens
A hybrid plasmid obtained by incorporating a DNA fragment containing a portion flanked by III cleavage sites into a binary vector plasmid of a plant, and a dicotyledon transformed with the hybrid plasmid and having crown gall disease resistance
Related to plants .

[0007] The disease resistance gene imparts disease resistance to plants by suppressing the expression of oncogenes introduced into plant cells by Agrobacterium tumefaciens, a pathogen of crown gall disease. Suppression of oncogene expression is controlled by a disease resistance gene having a DNA fragment of the auxin synthase gene iaaM, which is one of the oncogenes involved in the synthesis of the 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.

[0008] The disease resistance gene is introduced into dicotyledonous plants by the hybrid plasmid of the present invention prepared as follows.

[0009] From the PO22 strain of Agrobacterium tumefaciens isolated from a carcinoma of P.
i plasmid DNA was extracted, and this was
The DNA fragment containing the auxin synthase gene aaaM is cloned into the pUC13 plasmid. The gene aaaM has a HindIII site 143 bp and 840 bp downstream from the start codon (J. Gielen et al, The EMBO
Journal, vol. 3,835-846,198
4). The fragment sandwiched between these two Hind III cleavage sites corresponds to the 41st DNA fragment obtained by digesting T37T-DNA with Hind III (MW Beva).
n et al, Ann. Rev .. Genet. , Vo
l. 16, 357-384, 1982). The Hind
The DNA fragment sandwiched between the III cleavage sites is taken out and Kl
After blunting the ends using the enow fragment, p
Re-cloning into the SmaI site of the UC19 plasmid.

[0010] The base sequence of the DNA fragment inserted into this plasmid was determined by the dideoxy method, and the gene aaaM
To obtain two clones whose DNA fragments have different orientations with respect to the pUC19 plasmid.

On the other hand, the plant binary vector plasmid pBI121 has a kanamycin resistance gene (NPT II) that can be expressed in plant cells. The BamHI-SacI fragment containing the HindIII fragment was removed from the pUC19 plasmid,
A hybrid plasmid is obtained by inserting the plasmid pBI121 into the BamHI / SacI site.

[0012] This hybrid plasmid is prepared by inserting a DNA fragment of the gene iaaM between the 35S promoter of the cauliflower mosaic virus and the polyA recognition signal of the novalin synthase gene of the Ti plasmid in the same direction (sense) or reverse direction as the original gene iaaM. (Antisense).

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

[0014]

Next, the present invention will be described in detail with reference to examples. Example 1 Agrobacterium isolated from a carcinoma of a Japanese ash
Ti plasmid D from the PO22 strain of Tumefaciens
NA was extracted. Next, this was cleaved with the restriction enzyme PstI, and the D in which the auxin synthase gene
The NA fragment was cloned into the pUC13 plasmid.
Sandwiched Hind III cleavage sites of the gene iaaM
The obtained DNA fragment was taken out, the end was blunt-ended using a Klenow fragment, and then cloned again into the SmaI site of the pUC19 plasmid.

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

Next, from the pUC19 plasmid, the Hi
The BamH I-Sac I fragment containing the nd III fragment was extracted, and this was expressed in a kanamycin resistance gene (NPT
II) B of the binary vector plasmid pBI121 with
A hybrid plasmid was obtained by insertion into the amH I / Sac I site.

Example 2 The hybrid plasmid obtained in Example 1 was subjected to freeze-thawing to obtain LBA of Agrobacterium tumefaciens.
The strain was introduced into strain 4404, and leaf disks of tobacco were infected with Agrobacterium tumefaciens.
Next, this is Murashig containing kanamycin and hormones.
The cells were cultured in an e-Skoog medium, and the recombinant individuals having the kanamycin resistance gene that had differentiated into buds were selected.

A recombinant tobacco plant having the DNA fragment of the gene iaaM in antisense and sense directions was conditioned and cultivated in an artificial weather machine. The Agrobacterium tumefaciens P22R1 strain was infected to each leaf disk of a normal individual and a recombinant individual, and was placed on the Murashige-Skoog medium. As a result, formation of carcinoma was observed on leaf disks of normal individuals 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 remarkably prevented carcinoma formation and had crown gall disease resistance. In addition, in the tobacco recombinant individual (ACR) having the DNA fragment of the gene iaaM in the antisense direction, abnormal carcinomas that form multi-blasts as well as carcinomas were observed.

According to Southern analysis of the transgenic tobacco individuals, the above SCR individual had a DN of 4 copies of the gene iaaM.
The A fragment was integrated in the sense direction, and it was confirmed that one copy of the gene iaaM DNA fragment was integrated in the antisense direction in the ACR individual. In addition, No
rthern analysis revealed that the DNA in both directions was both transcribed.

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

[0022]

Industrial Applicability The hybrid plasmid obtained according to the present invention can confer crown gall disease resistance to plants. Therefore, crown gall disease resistant varieties can be easily prepared by introducing the hybrid plasmid into plants.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Kihachiro Oshima 5-21-1, Oji, Kita-ku, Tokyo Jujo Paper Co., Ltd. Central Research Laboratory (72) Inventor Kunio Hata 5-2-1-1, Oji, Kita-ku, Tokyo No. Jujo made of paper, Ltd. center within the Institute (72) inventor Hiroshi Sano Akita Prefecture Minamiakita District Ogata south 2-2 Akita Prefectural agricultural College of biological Engineering, the laboratory (58) investigated the field (Int.Cl. ^6, DB Name) C12N 15/09 A01H 1/00 A01H 5/00 C12N 5/10 BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A hybrid plasmid in which a DNA fragment containing a portion between two HindIII cleavage sites of a gene iaM encoding an auxin synthase derived from Agrobacterium tumefaciens Ti plasmid is incorporated into a binary vector plasmid of a plant. . 2. A DNA fragment containing a portion held two Hind III cleavage sites of the gene iaaM encoding auxin synthesis enzyme, are those derived from a Ti plasmid of Agrobacterium Tsumefashensu PO22 strains, binary plant Vector plasmid is binary
The hybrid plasmid according to claim 1, which is a vector plasmid pBI121. 3. It is transformed with the hybrid plasmid according to claim 1 and has crown gall disease resistance.
Dicot plants . 4. The hybrid plasmid according to claim 1,
Plant tissue transformed with E. coli .