Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2002-05-31
2004-07-06
Yucel, Remy (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S419000, C800S278000
Reexamination Certificate
active
06759526
ABSTRACT:
TECHNICAL FIELD
This invention relates to a DNA fragment having a promoter activity in plants. More particularly, this invention relates to a novel DNA fragment having a promoter activity derived from a gene encoding metallothionein and a vector containing the same and a plant cell, a plant and a seed transformed thereby.
BACKGROUND ART
Improvement of plants by using a genetic engineering method has become practical recently. Particularly, a mechanism of tumor formation in plant by Ti plasmid of Agrobacterium tumefaciens which is a soil microorganism is solved in molecular level, and taking this opportunity, since a transformation system using a Ti plasmid vector has been established, research on plants in molecular level has been making a significant progress. In these days, the transformation system using the Ti plasmid vector can be applied to a main agricultural products including rice, soy been, etc., as well as to the model plants such as tobacco and
Arabidopsis thaliana
, etc.
It has been generally known that a 5′ upstream region of a structural gene called promoter is involved in a transcription of a gene. The promoter is a DNA sequence located in an upstream region of a structural gene, containing a signal (TATA region) for an RNA polymerase to start transcription, thereby enabling a following protein synthesis. Therefore, the promoter is an important and essential gene to produce a genetically recombinant plant.
In a 5′ upstream of the TATA region, there exists a specific nucleotide sequence called cis element, and this region interacts with a DNA binding protein (trans element) to determine a strength of promoter activity and control of the transcription.
For example, a promoter of
Arabidopsis thaliana
gene (rd29A) whose expression is induced by drought [Koizumi et al., Gene 129:175-182 (1993)] was isolated, and after that, from a research in which a promoter region is mutated by deletion or a research in which various kinds of DNA fragments are linked, it has been reported that the cis element of rd29A gene controlling an induction by drought is a 9 base sequence comprising TACCGACAT [Yamaguchi-Shinozaki et al., J. Plant Res. 108:127-136 (1995)], and it has been elucidated that the above mentioned cis element comprising 9 bases is essential for serving as a promoter induced by drought.
Currently, as a promoter for a gene transduction, 35S promoter of a cauliflower mosaic virus [Guilley et al., Cell 30:763-773 (1982)] has been frequently used. From analyses using tobacco [Morell et al., Nature 315:200-204 (1985)] on petunia [Sander, Nucl. Acid Res. 15:1543-1558 (1987)], 35S promoter has been shown to have an activity 30 times or more as strong as that of nopaline synthetase promoter. As shown above, since the promoter activity of 35S promoter is strong in dycotyledons, this promoter is widely used in order to transfer a structural gene and have it highly expressed in dycotyledons.
However, 35S promoter shows only relatively low promoter activity in a rice family plant which is an important monocotyledon in agriculture [Hauptmann et al., Plant Cell Rep. 6:265-270 (1987)].
On the contrary, a promoter derived from alcohol dehydrogenase (Adh) of corns merely gives an extremely low expression in protoplast of
Nicotiana plumbaginifolia
which is a dicotyledon [Ellis et al., EMBO J. 6:11-16 (1987)].
In order to produce practically useful novel plant strain in a wide range of plants by recombinant DNA technology in the future, it is thought to be an important technology, how to carry out efficiently an expression of a structural gene to be transferred or how to control an expression. However, with the 35S promoter or the Adh promoter, it is not easy to control a tissue-specific expression, or to control an expression by chemical substances.
Therefore, a promoter having a promoter activity not only in monocotyledons but also in dicotyledons, and being able to control a tissue specific and part specific expression has been sought.
Incidentally, metallothionein (Mt) has been known as a protein with a low molecular weight, playing an important role in metabolism of metals in vivo in animals, microorganisms and plants [Yu et al., Gene 206:29-35 (1998)].
Mt was initially isolated from the kidneys of horses and research on its structure and its function has made progress by using organisms such as cyanobacteria or fungi as a sample [Yu et al., Plant Biotechnology 15:167-172 (1998)]. When Mt originated from Chinese hamster is expressed in a metallo-sensitive yeast, the yeast becomes resistant to cadmium. This suggests Mt takes a part in detoxification of metals.
Regarding rice Mt gene, cDNA has been isolated from YAMAHOUSHI, and a promoter region has been isolated from Sasanishiki, whose base sequences have been reported [Japanese Provisional Patent Publication No. 10-248570].
The present invention has been aimed to obtain and provide a novel DNA fragment having a promoter activity which makes a linked exogenous structural gene express in the plant or plant cells, and enabling an induction of expression or control of a tissue-specific and part-specific expression by substances.
The present inventors have made intensive studies to solve the above-mentioned problems and they have isolated a promoter region of Mt gene from rice plant NNipponbare. As a result of comparison of nucleotide sequences with a promoter region isolated from Sasanishiki, the both sequences are totally different and a novel promoter has been identified. Further, they have found that this promoter has a promoter activity in a different plant strain, regardless of monocotyledons or dicotyledons, and that it enables an expression of an exogenous gene and control of the expression, and thus, the present invention has been completed.
SUMMARY OF THE INVENTION
The present invention comprises promoters shown below, which is derived from a gene encoding a rice metallothionein. That is, it is a DNA fragment having a promoter activity comprising a whole or a part of a base sequence shown by SEQ ID NO:1 wherein one or two or more bases maybe deleted, inserted or substituted provided that the sequence has a function in plants or in plant cells to control expression of a structural gene which can be expressed in plants, for example, a DNA of about 2.7 Kbp shown by SEQ ID NO:2.
Further, the present invention is an expression vector into which a DNA fragment shown by SEQ ID NO:1 or 2 is transferred.
In addition, the present invention relates to an expression vector wherein the DNA sequence shown by SEQ ID NO:1 or 2 is transferred to an exogenous structural gene.
Yet further, the present invention relates to a transformed plant cell obtainable by introducing the above-mentioned vector into a host plant cell, a transformed plant regenerated from the plant cells and a seed obtainable from the plant.
BEST MODE FOR CARRYING OUT THE INVENTION
In the description below, unless otherwise specified, recombinant DNA technology used as a conventional method can be carried out using techniques described in the following reference: “Molecular Cloning” (Fristch et al., Cold Spring Harbour Press (1989)).
The DNA fragment having a promoter activity of the present invention can be isolated from a plant genomic library by a plaque hybridization, etc. using cDNA of rice metallothionein as a probe.
Genomic library can be obtained by extracting genomic DNA from plant such as a rice plant, partially digesting the isolated genomic DNA by an appropriate restriction enzyme such as EcoRI, fractionating longer DNA fragments, for example, DNA fragments of 9 to 23 kb by sucrose density-gradient centrifugation or by agarose gel electrophoresis, incorporating this into an appropriate vector such as &lgr; phage and packaging, followed by infecting
Escherichia coli
such as XL-1 Blue strain with this recombinant phage, and culturing them on a flat culture medium such as an LB plate (1% Bactotryptone, 0.5% yeast-extract, 1% NaCl and 1.3% agarose)
Fukuzawa Hiromitsu
Tagawa Michito
Uchimiya Hirofumi
Birch & Stewart Kolasch & Birch, LLP
Katcheves Konstantina
Nissan Chemical Industries Ltd.
Yucel Remy
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