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
1999-03-31
2001-10-16
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
C435S320100, C435S419000, C536S023600
Reexamination Certificate
active
06303847
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to techniques for controlling the expression of genes relating to biosynthesis of phenylpropanoid.
2. Description of the Background
With the advance of plant molecular biology in recent years, it has become possible to breed plants having useful characteristics, such as resistance to disease and insect damage or resistance to a herbicide, by using a sense gene or anti-sense gene. That is, the expression of a desired characteristic can be promoted or suppressed by linking, in a sense direction or anti-sense direction, a gene relating to the expression of the desired characteristic with a promoter permitting the expression in a plant to form a chimeric gene, and introducing the resulting chimeric gene to a plant as a vector. Based on such a technique, for example, a plant resistant to disease and insect damage to which an insecticidal BT toxin gene derived from
Bacillus thuringensis
has been introduced in a sense direction (D. A. Fischhoff et al.,
Bio/Technology
, 232: 738-743 (1987)) and an excellent storable tomato to which a polygalacturonase gene relating to over-ripening of tomato fruit has been introduced in an anti-sense direction (C. J. Smith et al., Nature, 334: 724-727 (1988)) have been produced.
When such a technique is used, the expression of the desired characteristic is promoted in a plant to which a sense gene (a gene which expresses a desired characteristic and is fused to a promoter in a sense direction) has been introduced; on the other hand, the expression of the desired characteristic is inhibited in a plant to which an anti-sense gene (the same gene as the sense gene, which is fused to a promoter in an anti-sense direction) has been introduced. The expression of the desired characteristic is suppressed by the introduction of an anti-sense gene because in a plant cell, RNA synthesized from the anti-sense gene as a template is complementarily bound to mRNA derived from a gene of the plant itself relating to the expression of the desired characteristic to inhibit the subsequent synthesis of protein.
However, many genes of a plant form a multi-gene family and genes belonging to such a family show a high homology in a nucleotide sequence respectively. Even if the expression of such a gene belonging to a multi-gene family is controlled using the anti-sense method, the RNA of the anti-sense gene is inevitably bound at random to the mRNAs of many other genes belonging to the same family to control their expression, which makes it impossible to control the expression of only the desired gene, so that various characteristic suppression patterns is caused. Thus, the results are sometimes quite different from those as expected.
Also, the phenylpropanoid biosynthesis pathway is a complicatedly branched reaction system which exists specifically in plants and it relates to the biosynthesis of components of a cell wall (for example, lignin, suberin), pigments of a flower, antibacterial substances and the like. Phenylpropanoid derivatives available through such a biosynthesis pathway can also be used for UV protecting agents, insecticides or the like. If the expression of a gene relating to this phenylpropanoid biosynthesis pathway can be promoted or suppressed accurately, it becomes possible to control this biosynthesis pathway to produce a tree containing a lignin at a low content or carry out mass production of useful substances. In this case, however, it is difficult to control the expression of the gene by the anti-sense method owing to the above-described problem in homology between genes. For example, it is reported that a transformed plant to which a gene of phenylalanine ammonia lyase (PAL) or peroxidase (PRX) which is an enzyme acting in the phenylpropanoid biosynthesis pathway had been introduced in an anti-sense direction exhibited diversified controlling effects such as growth inhibition (M. M. Campbell and R. R. Sederoff,
Plant Physiol
., 110: 3-13 (1996)) and a change in a lignin content of tobacco to which a caffeic acid O-methyltransferase gene had been introduced in an anti-sense direction was not so large as expected (W. Ni et al.,
Transgen. Res
., 3: 120-126 (1994)).
SUMMARY OF THE INVENTION
In consideration of the above-described problems, an object of the present invention is to provide a DNA and a vector which can accurately promote or suppress the expression of a specific gene relating to the phenylpropanoid biosynthesis pathway of a plant.
With a view toward overcoming the above-described problems, the present inventors have carried out an extensive investigation. As a result, the inventors paid attention to the fact that in the 5′-upstream non-translated region of specific genes relating to the phenylpropanoid biosynthesis pathway, for example, a cynnamyl alcohol dehydrogenase (CAD) gene, chalconic acid synthetase (CHS) gene, 4-coumaric acid CoA ligase (4CL) gene, PAL gene and PRX gene, there exist sequences controlling the expression of these genes, and these sequences have a very high homology between these genes. So the inventors isolated a factor promoting the transcription of these genes by binding to these sequences (hereinafter referred to as a “transcription factor”) and then introduced a DNA encoding the factor into a plant as a sense gene or anti-sense gene so that the expression of the above-described gene can be accurately promoted or suppressed. Thus, the present invention has been completed.
Specifically, the above and other objects of the present invention may be accomplished by an isolated and purified DNA having a nucleotide sequence which comprises SEQ ID NO:1; an isolated and purified DNA which hybridizes to a DNA having a nucleotide sequence which comprises SEQ ID NO:1 under stringent conditions, and encodes a transcription factor controlling a phenylpropanoid biosynthesis pathway; a recombinant vector comprising the DNA; the recombinant vector, further comprising a promoter to which the DNA is operably fused; the recombinant vector, wherein the DNA is operably fused to the promoter in the sense or antisense direction; a plant cell into which the DNA has been introduced; a plant regenerated from the plant cell; a method of producing the plant cell, comprising introducing the DNA into the plant cell; a method of producing the plant, comprising regenerating the plant from the plant cell; an isolated and purified protein encoded by the DNA; and an isolated and purified DNA which encodes a protein having the amino acid sequence of SEQ ID NO:2.
REFERENCES:
Baltz R, et al. “Characterization of a pollen-specific cDNA from sunflower encoding a zinc finger protein” Plant J. 2: 713-721 (Abstract Only). 1992.
Ebinuma Hiroyasu
Kawaoka Akiyoshi
Nelson Amy J.
Nippon Paper Industries Co. Ltd.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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