Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-14
2001-05-22
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024100, C536S023100
Reexamination Certificate
active
06235482
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to plant genetic engineering and more specifically to novel plant genes and selective gene expression in plants. In particular, the present invention relates to novel promoters capable of conferring high levels of transcription of heterologous genes in cells of fruit tissue, novel chimeric genes selectively expressed in cells of fruit, and transformed plants containing said chimeric genes.
BACKGROUND OF THE INVENTION
One of the goals of plant genetic engineering is to obtain plants having improved characteristics or traits. Many different types of characteristics or traits in plants are considered advantageous. Those of particular importance with regard to fruit bearing plants include control of fruit ripening, improvements in the nutritional characteristics of the edible portions thereof, resistance to plant diseases, resistance to insects, cold tolerance and enhanced stability or shelf-life of the ultimate consumer product obtained from the plant.
At least two key components are required to stably engineer a desired trait, or control of such a trait, into a plant. The first key component comprises identifying and isolating the gene(s) which either encode(s) or regulate(s) a particular trait. The second component comprises identifying and isolating the genetic element(s) essential for the actual expression and/or selective control of the newly isolated gene(s) so that the plant will manifest the desired trait and, ideally, manifest the trait in a controlled or controllable manner. This second component, which controls or regulates gene expression, typically comprises transcriptional control elements known as promoters. Although a generic class of promoters which drive the expression of heterologous genes in plants have been identified, a broad variety of promoters active in specific target tissues or cells of plants remain to be described. The identification of such target or tissue-specific promoters is critical to the introduction of the above-mentioned tissue-specific improvements in plants such as fruit bearing plants.
Several promoters useful in expressing heterologous genes in selected fruits have already been identified. For example, the E4 and D8 promoters (Deikman, et al.), the kiwifruit actinidin promoter (Lin, et al.) and promoter for polygalacturonase are known to be fruit specific. U.S. Pat. No. 4,943,674 (Houck et al., Jul. 24, 1990) discloses a 2All promoter as useful in expression of a heterologous gene in tomato fruit. These promoters, however, have been isolated from fruit tissue which comprises mature or maturing ovaries (hereinafter referred to as “traditional fruit”). As such, these traditional fruit promoters would be ineffective in controlling desired traits in such accessory fruit bearing plants as strawberry, apple, pear, quince and the like wherein the major portion of the edible fruit comprises receptacle tissue (see
An Introduction to Plant Biology.
2nd Edition, Braungart & Arnett, eds., C. V. Mosby Co. 1965). Similarly, to date, genes thought to be active in fruit tissue have been isolated from traditional fruit tissue instead of receptacle containing tissue.
There exists a need for receptacle tissue selective promoters in the art. Access to receptacle tissue selective promoters would enable the genetic engineering of fruit tissue from commercially important plants such as strawberry, apple, and pear. Screening of DNA libraries was undertaken as a method for the identification of tissue selective promoters from strawberry. Four such sequences were identified, and the promoters and their associated structural genes sequenced. Expression of reporter genes in tobacco and tomato plants was used as an assay of the tissue specificity of the isolated promoters. Methods for the identification and isolation of analogous promoters and structural genes from other plants is described.
SUMMARY OF THE INVENTION
The present invention provides novel promoters termed “GSRE2, GSRE49, SEL1, and SEL2” herein which cause tissue-specific expression of heterologous DNA in the receptacle tissue of plants.
The present invention also provides novel chimeric genes comprising a receptacle tissue-specific promoter operably coupled to a heterologous DNA sequence.
The present invention further provides novel genes which are highly expressed in ripening receptacle tissue of accessory fruit plants.
The present invention furthermore provides a method for expression of a heterologous gene, the improvement which comprises the use of an accessory fruit plant promoter which causes tissue-specific expression in seed, sink and receptacle tissue of plants, said accessory fruit plant promoter having a sequence selected from the group consisting of those sequences shown in SEQ ID NOS. 1, 2, 3, and 4 and sequences substantially homologous thereto.
The present invention further provides structural DNA sequences transcribed at high levels in the receptacle tissue of fruit bearing plants, the structural DNA sequences having a sequence selected from the group consisting of those shown in SEQ ID NOS. 5, 6, 7, and 8 and sequences substantially homologous thereto.
Novel transformed plant cells and transgenic plants comprising the heterologous genes of the present invention or produced by the methods of the present invention are additionally provided.
DEFINITIONS
The following definitions are provided as an aid to understanding the detailed description of the present invention.
The phrases “coding sequence” and “structural sequence” refer to the region of continuous sequential DNA triplets encoding a protein, polypeptide, or peptide sequence.
The phrase “DNA segment heterologous to the promoter region” means that the coding DNA sequence does not exist in nature in the same gene with the promoter to which it is now attached.
The phrase “expressibly coupled” and “expressibly linked” refer to a promoter or promoter region and a coding or structural sequence in such an orientation and distance that transcription of the coding or structural sequence may be directed by the promoter or promoter region.
The term “expression” refers to the transcription of a gene to produce the corresponding mRNA and translation of this mRNA to produce the corresponding gene product, i.e., a peptide, polypeptide, or protein.
The term “expression of antisense RNA” refers to the transcription of a DNA to produce an first RNA molecule capable of hybridizing to a second RNA molecule encoding a gene product, e.g. a protein. Formation of the RNA-RNA hybrid inhibits translation of the second RNA molecule to produce the gene product.
The term “gene” refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequence involved in the regulation of expression.
“Homology” refers to the level of similarity between nucleic acid or amino acid sequences in terms of percent nucleotide or amino acid positional identity, respectively, i.e., sequence similarity or identity. Note Reeck el al.
Cell
50: 667 (1987) in this regard. Homology also refers to the concept of similar functional properties among different nucleic acids or proteins.
“Hybridization” refers to the ability of a strand of nucleic acid to join with a complementary strand via base pairing. Hybridization occurs when complementary sequences in the two nucleic acid strands bind to one another.
“Overexpression” refers to the expression of a polypeptide or protein encoded by a DNA introduced into a host cell, wherein said polypeptide or protein is either not normally present in the host cell, or wherein said polypeptide or protein is present in said host cell at a higher level than that normally expressed from the endogenous gene encoding said polypeptide or protein.
The term “promoter” or “promoter region” refers to a DNA sequence, usually found upstream (5′) to a coding sequence, that controls expression of the coding sequence by controlling production of messenger RNA (mRNA) by providing the recognition site f
Einsmann Juliet C.
Fredman Jeffrey
Hoerner, Esq. Dennis R.
Howrey Simon Arnold & White , LLP
Monsanto Company
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