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
1999-04-28
2002-12-17
Fox, David T. (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, C435S252300, C536S023600, C800S289000
Reexamination Certificate
active
06495742
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a protein which binds to a stress responsive element and regulates the transcription of genes located downstream of the element; a gene coding for the above protein; a recombinant vector comprising the gene; a transformant comprising the recombinant vector; a transgenic plant comprising the gene; a method for producing the above protein using the transformant; and a method for determining a stress level in a plant.
2. Prior Art
Transcription of genes is performed by RNA polymerase. RNA polymerase synthesizes ribonucleoside phosphates in the 5′ to 3′ direction using double-stranded DNA as a template in a primer independent manner. In the case of
Escherichia coli
, for example, its RNA polymerase takes the form of a holoenzyme in which &rgr; factor having promoter recognition ability is bound to the core enzyme &bgr;′&bgr;&agr;
2
. This RNA polymerase initiates transcription and elongates RNA chain; the transcription is terminated by the binding of &rgr; factor. On the other hand, in the case of eucaryotes, RNA polymerase is classified into RNA polymerases I, II and III, any of which has a complicated structure composed of more than 10 subunits. RNA polymerase I selectively transcribes rRNA; RNA polymerase II selectively transcribes mRNA precursor; and RNA polymerase III selectively transcribes tRNA and 5SrRNA. The amount of RNA synthesized by such RNA polymerase varies widely depending on the growth stage of the relevant cells and environmental changes around them. A transcription factor which positively or negatively regulates the transcription initiation of RNA polymerase is deeply involved in the variation in the amount of RNA synthesis.
Generally, living cells are exposed to an external environment composed of a number of factors including temperature, pressure, oxygen, light, radioactive rays, metal ions, organic compounds, etc. When these factors vary, cells perceive such changes as stress and make characteristic responses to them. For example, cells exhibit a response called “heat shock response” to high temperatures. From this response, the expression of a group of heat shock proteins (HSPs) is induced. HSPs prevent the irreversible precipitation of heat-denatured proteins and have the function of molecular chaperone that facilitates the refolding of such proteins, thereby protecting cells from heat stress. It is known that a transcription factor called “heat shock factor (HSF)” plays an important role in the manifestation of the above-described heat shock response in human, Xenopus, Drosophila, etc. [Kazuhiro Nagata, Cell Technology, 10:348-356 (1991)]. When activated by heat shock, HSF binds to heat shock element (HSE) located upstream of a gene coding for HSP (also known as heat shock gene) to thereby promote the transcription of the heat shock gene.
On the other hand, it is also reported that plants induce stress proteins such as LEA proteins, water channel proteins or synthetases for compatible solutes in their cells when they are exposed to stress such as dehydration, low temperature, freezing or salt, thereby protecting their cells from such stress. However, much more research is required to elucidate transcription factors which regulate the transcription of genes encoding those stress proteins.
OBJECTS AND SUMMARY OF THE INVENTION
It is an object of the present invention to provide a protein which regulates the transcription of genes located downstream of a stress responsive element which is essential for controlling stress responsive gene expression; a gene encoding the protein; a recombinant vector comprising the gene; a transformant comprising the recombinant vector; a transgenic plant comprising the gene; a method for producing the above protein using the transformant; and a method for determining a stress level in a plant.
As a result of extensive and intensive researches toward the solution of the above-described problem, the present inventors have succeeded in isolating from a low temperature resistant plant
Arabidopsis thaliana
a gene coding for a transcription factor which binds to a stress responsive element and activates the transcription of genes located downstream of the element. Thus, the present invention has been achieved.
The present invention relates to the following recombinant protein (a) or (b):
(a) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10;
(b) a protein which consists of the amino acid sequence having deletion, substitution or addition of at least one amino acid in the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10 and which regulates the transcription of genes located downstream of a stress responsive element.
Further, the present invention relates to a transcription factor gene coding for the following protein (a) or (b):
(a) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10;
(b) a protein which consists of the amino acid sequence having deletion, substitution or addition of at least one amino acid in the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10 and which regulates the transcription of genes located downstream of a stress responsive element.
Further, the present invention relates to a gene comprising the following DNA (c) or (d):
(c) a DNA consisting of the nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7 or SEQ ID NO: 9;
(d) a DNA which hybridizes with the DNA consisting of the nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7 or SEQ ID NO: 9 under stringent conditions and which codes for a protein that regulates the transcription of genes located downstream of a stress responsive element.
Specific examples of the above-mentioned stress include dehydration stress, low temperature stress and salt stress.
Further, the present invention relates to a recombinant vector comprising the gene of the invention.
Further, the present invention relates to a transformant comprising the recombinant vector.
Further, the present invention relates to a transgenic plant comprising the gene of the invention.
Further, the present invention relates to a method for producing a protein which regulates the transcription of genes located downstream of a stress responsive element, comprising culturing the above transformant in a medium and recovering the protein from the resultant culture.
Further, the present invention relates to a method for determining a stress level in a plant, comprising determining a transcription level of the gene of the invention in the plant.
REFERENCES:
patent: 5891859 (1999-04-01), Thomashow et al.
Liu et al, Two Transcription Factors, DREB1 and DREB2, with an EREBP/AP2 DNA Binding Domain Seperate Two Cell. Signal Transduction Pathways in Drought-and Low-Temp.Respon.Gene Express Respect. in Arabidopsis, Aug. 1998, The Plant Cell, vol. 10, pp 1391-1406.*
Jaglo-Ottosen et al, Arabidopsis CBF1 Overexpression Induces COR Genes and Enhances Freezing Tolerance, Apr. 1998, Science, vol. 280, pp. 104-106.*
Abstract: Lui, Qiang et al., Analysis of DREB Gene Encoding a Protein Binding to the cis-Element DRE which Stimulates Dehydration/Low Temperature Stress-Responsive Gene Expression inArabidopsis Thaliana. 1998 Annual Meeting and the 38thSymposium of the Japanese Society of Plant Physiologists, May 3-5, 1998, F3a-11.
Abstract: Miura, Setsuko, et al., Analysis ofArabidopsis thaliana, in which the Dehydration/Salt/Low Temperature Stress Inducible Transcription Factor DREB1A or DREB2A is Over-expressed. 1998 Annual Meeting and the 38thSymposium of the Japanese Society of Plant Physiologists, May 3-5, 1998, F3a-12.
Abstract: Shirjwari, Zabta K., et al., Identification of the DREB1B Family Encoding Proteins which Bind to the Dehydration/Low Temperature Responsive Element DRE ofArabidopsis thalianaand Analysis of Expression of the Family. 1998 Annual M
Kasuga Mie
Shinozaki Kazuko
Davidson, Davidson & Kappel LLP
Fox David T.
Independent Administrative Institute Japan International Researc
Kruse David H
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