Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1997-02-26
2004-06-15
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S279000
Reexamination Certificate
active
06750381
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for efficiently producing a useful foreign protein in a plant. The present invention also relates to a method for expressing a foreign protein in a plant so as to provide the plant with a new property, and to the breeding of a plant. More specifically, the present invention relates to a plant containing a gene encoding an anti-bacterial peptide derived from a Diptera insect and which confers resistance to bacterial and fungal pathogens.
2. Description of the Related art
In the past, it has been reported that when a foreign gene is introduced into the nucleus of a plant cell, the productivity of a peptide, which is a foreign gene product, is at most 0.1% to 0.3% of the total proteins. Particularly, in the case where the product of the foreign gene is a small peptide (hereinafter, referred to as “a short peptide”) with a molecular weight of 7,000 or less, the product is likely to be degraded in a plant and is difficult to be accumulated in a great amount. Thus, there has been a demand for a method for efficiently expressing a short peptide.
When a foreign gene is introduced and expressed in a plant, a constitutive promoter, which is constantly expressed, such as the CaMV 35S promoter is mostly used. However, some possibilities are considered; for example, this expression method using the constitutive promoter may impose a burden at least on the plant's ability to produce a protein and the expressed protein may adversely affect the plant. In fact, attempts to produce a protein encoded by a gene that affects the growth of a plant, such as a gene involved in the synthesis of a plant hormone, often result in failures. This adverse tendency is especially outstanding when one attempts to increase the productivity of the protein encoded by the gene introduced into the plant.
On the other hand, a study has been made on a technique for introducing a gene encoding an anti-bacterial peptide into a plant and expressing the anti-bacterial peptide so as to provide the plant with resistance to bacteria. In particular, it has been disclosed in Japanese Laid-Open Patent Publication No. 7-250685 that Sarcotoxin
1
a
, which is an anti-bacterial peptide derived from a Diptera insect, was expressed in a plant in such a manner that the plant obtained anti-bacterial properties. However, since a short peptide such as Sarcotoxin 1a is expected to be unstable in plants, it is necessary to stabilize the peptide by producing it as a fusion protein with PR-1a which is an pathogenesis related protein of tobacco.
Furthermore, no plants have been discovered that produce an anti-bacterial peptide derived from a Diptera insect having anti-fungal activity, and confer resistance to pathogenic fungi.
SUMMARY OF THE INVENTION
According to the present invention, a plant which confers resistance to pathogenic fungi, includes a gene encoding an anti-bacterial peptide.
In one embodiment of the present invention, the pathogenic fungi are
Rhizoctonia solani, Pythium aphanidermatum
, and
Phytophthora infestans.
In another embodiment of the present invention, the anti-bacterial peptide is derived from the Diptera insect.
In another embodiment of the present invention, the anti-bacterial peptide derived from the Diptera insect is Sarcotoxin 1a.
In another embodiment of the present invention, a gene encoding the anti-bacterial peptide derived from the Diptera insect is introduced into a plant in a form selected from the group consisting of: a recombinant gene containing the gene encoding the anti-bacterial peptide derived from the Diptera insect; an expression cassette in which the recombinant gene is bound to a plant promoter; and an expression vector composed of the expression cassette and a drug resistant gene linked to a plant promoter which is constitutively expressed.
In another embodiment of the present invention, the recombinant gene which encodes the anti-bacterial peptide derived from the Diptera insect is bound to a plant gene via a hinge region of tobacco chitinase.
In another embodiment of the present invention, the Sarcotoxin 1a is bound to a signal sequence of a plant protein.
In another embodiment of the present invention, the plant promoter is the inducible promoter of the tobacco PR-1a gene.
In another embodiment of the present invention, the expression cassette has a terminator derived from the tobacco PR-1a gene.
In another embodiment of the present invention, the expression vector further has a T-DNA region and a drug resistant gene.
In another embodiment of the present invention, the drug resistant gene is expressed by the Cauliflower mosaic virus 35S promoter.
According to the present invention, a plant with resistance to pathogenic bacteria, includes a gene selected from the group consisting of: a recombinant gene in which a gene encoding an anti-bacterial peptide is bound to a plant gene via a hinge region of tobacco chitinase, an expression cassette in which the recombinant gene is bound to a plant promoter, and a gene having the expression cassette and a drug resistance gene linked to a plant promoter which is constitutively expressed.
In one embodiment of the present invention, the pathogenic bacteria is
Pseudomonas syringae
pv.
tabaci
or
Erwinia carotovora
subsp.
carotovora.
In another embodiment of the present invention, the anti-bacterial peptide is Sarcotoxin 1a derived from a Diptera insect.
According to the present invention, a recombinant gene, in which a gene encoding an anti-bacterial peptide is bound to a plant gene via a hinge region of tobacco chitinase gene, is provided.
In one embodiment of the present invention, the gene encoding an anti-bacterial peptide is a gene encoding an anti-bacterial peptide derived from the Diptera insect.
In another embodiment of the present invention, the anti-bacterial peptide derived from the Diptera insect is Sarcotoxin 1a.
In another embodiment of the present invention, an expression cassette, in which the above-mentioned recombinant gene is bound to a plant promoter, is provided.
In another embodiment of the present invention, an expression vector for introducing the above-mentioned expression cassette into a plant is provided.
According to the present invention, a plant which confers resistance to pathogenic fungi and bacteria, includes a gene encoding a peptide which has anti-fungal and anti-bacterial activity.
The gene encoding the fusion protein of the present invention was introduced into a plant with the expression vector and expressed. As a result, the fusion protein was constitutively produced, and the productivity thereof increased by the induction of salicylic acid or the like. In addition, the fusion protein was cleaved at its linked site by the proteolytic activity of an unidentified protein which was induced by salicylic acid and TMV infection. This led to the induction of producing the respective free proteins from the fusion protein.
By using the recombinant gene, the expression cassette, or the expression vector of the present invention, a foreign protein can be constitutively produced as a fusion protein and the respective free proteins can be produced. Furthermore, the anti-bacterial peptide is expressed as a fusion protein with a plant gene by the method of the present invention, and thus, it is possible to obtain a plant which confers resistance to pathogenic fungi as well as pathogenic bacteria.
Thus, the invention described herein makes possible the advantages of (1) increasing the stability of a target foreign protein (peptide) in a plant by expressing the foreign protein as a fusion protein with other proteins; (2) providing an expression cassette or an expression vector containing the expression cassette, wherein the expression cassette expresses a recombinant gene encoding the fusion protein at a constitutively constant level, and the expression level inductively increases, thereby increasing the productivity of a foreign protein in a plant; (3) cleaving proteins once expressed as a fusion protein at its fusion si
Mitsuhara Ichiro
Ohashi Yuko
Ohshima Masahiro
Kubelik Anne
National Institute of Agrobiological Sciences
Nelson Amy J.
Townsend and Townsend / and Crew LLP
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