Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide contains a tissue – organ – or cell...
Reexamination Certificate
1999-11-29
2004-01-20
Mehta, Ashwin (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide contains a tissue, organ, or cell...
C435S320100, C435S419000, C536S023600, C536S024100
Reexamination Certificate
active
06680425
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel plant chimeric promoters that are responsive to sugar depletion conditions in a plant cell.
BACKGROUND OF THE INVENTION
The Amy3D and Amy3E genes are two sugar-regulated genes in the rice &agr;-amylase family (Huang, N. et al.,
Nucleic Acids Res
., 18(23):7077 (1990)). Amy3D is expressed in seedlings in response to sugar depletion and is not expressed when sugar, such as glucose, concentrations are elevated. Further, Amy3D is upregulated strongly in cell culture under conditions of sugar depletion or sugar deprivation. Unlike many other &agr;-amylase genes, Amy3D is not induced in response to gibberellic acid.
Metabolic regulation of Amy3D expression provides a signal mechanism to help control sugar production in the source tissues of germinating cereal,seedling. Thus, the rate of starch breakdown is modulated in response to the rate at which the embryo axis can utilize sugar for its growth.
SUMMARY OF THE INVENTION
In one aspect, the invention includes a chimeric plant promoter for upregulating expression of a coding sequence operatively linked to the promoter, under conditions of sugar depletion or deprivation in a plant cell. The promoter includes a promoter element effective to express the coding sequence, under selected conditions and, carried in the promoter element, one or more of the following heterologous sequences which are responsive to (upregulated by) sugar depletion in plant cells: SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, and combinations of these sequences. An exemplary combination is produced from sequences from SEQ ID NOS: 2 and 3, identified as SEQ ID NO:5.
In one embodiment the heterologous sequences is duplicated. In another embodiment, the promoter element is a monocot amylase-gene promoter, and the heterologous sequence is inserted in the promoter element at a position homologous to the position of the heterologous sequence in the rice Amy3D gene. Exemplary promoter elements are from the RAmy1A, RAmy1B, RAmy2A, RAmy3A, RAmy3B, RAmy3C, pM/C, gKAmy141, gKAmy155, Amy32b, and HV18 genes.
Also forming part of the invention is a method of enhancing the inducibility by sugar depletion or deprivation of a plant or plant-virus promoter in plant cells. The method includes introducing into a plant promoter or plant-virus promoter, one or more of the above heterologous sequences, to achieve at least a 2-fold greater sugar-depletion induction level over that of the unmodified promoter.
The promoter may be, for example, an &agr;-amylase promoter from a monocot &agr;-amylase gene, such as the RAmy1A, RAmy1B, RAmy2A, RAmy3A, RAmy3B, RAmy3C, pM/C, gKAmy141, gKAmy155, Amy32b, or HV18 genes. The promoter may be duplicated in the chimeric promoter. The plant may be a monocot plant.
In another aspect, the invention includes a vector for use in transforming a plant. The vector includes a chimeric gene having, operatively linked in sequence in a 5′ to 3′ direction, (i) the above chimeric plant promoter, (ii) a gene encoding a protein to be expressed, and (iii) a 3′ untranslated terminator region. Also disclosed are plant cells transfected with the vector.
REFERENCES:
patent: 5460952 (1995-10-01), Yu et al.
patent: WO 89/09267 (1989-10-01), None
patent: WO 95/03690 (1995-02-01), None
patent: WO 95/14099 (1995-05-01), None
Lu, et al., “Sugar Response Sequence in the Promoter of a Rice &agr;-Amylase Gene Serves as a Transcriptional Enhancer,”J. Biol. Chem., 273(17):10120-10131 (Apr. 24, 1998).
Chan, Ming-Tsair, et al., :Novel Gene Expression System for Plant Cells Based on Induction of&agr;-Amylase Promoter by Carbohydrate Starvation:,The Journal of Biological Chemistry 269(26):17635-17641, (1994).
* Chun-An L., et al., “Sugar Response Sequence in the Promoter of a Rice&agr;-amylase Gene Serves as a Transcriptional Enhancer,”J Biol Chem 273(17):10120-10131, (1998).
Huang, N., et al., “Structural Organization and differential expression of rice&agr;-amylase genes”,Nuc. Acids Res. 18(23):7007-7014, (1990).
Huang, N., et al., “Classification and characterization of the rice&agr;-amylase multigene family”,Plant Molecular Biology 14:655-668, (1991).
Huang, N., et al., “Structural organization and differential expression of rice&agr;-amylase genes”,Nucleic Acids Research 18: (23):7007-7014, (1991).
Huang, N., et al., “Metabolic regrlation of a&agr;-amylase Gene Expression in Transgenic Cell Cultures of Rice,”Plant Mol Bio 23:737-747, (1993).
Hwang, Y.S., et al., “Three cis-elements Required for rice&agr;-amylase Amy3D Expression During Sugar Starvation,”Plant Mol Biol 36:331-341, (1998).
Mitsunaga, Shin-Ichiro, et al., “Sequence-specific Interactions of a Nuculear Protein Factor with the Pormoter Region of a Rice Gene for&agr;-amylase RAmy3D,”Nuc Acids Res 22(11):1948-1953, (1994).
Thomas, Bruice, R. et al., “Gene Regulation and Protein Secretion from Plant Cell Cultures: the Rice&agr;-amylase System,”Advances in Plant Biotechnology 4:37-55, (1994).
Mehta Ashwin
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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