Apple promoters for expression of transgenes in plants

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

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C536S024100, C435S320100, C435S419000, C435S468000, C800S295000

Reexamination Certificate

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06392122

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to novel apple fruit-associated and Thi 1.3:actin fusion promoters, and to heterologous nucleic acid constructs, vectors, kits, and transformation methods employing such promoters. The invention further relates to transgenic plant cells and plants transformed with heterologous nucleic acid constructs comprising an apple fruit-associated or Thi 1.3:actin fusion promoter.
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BACKGROUND OF THE INVENTION
Transcriptional regulatory sequences or promoters that regulate gene expression in plants are essential elements of plant genetic engineering. Several examples of promoters useful for the expression of heterologous genes in plants are now available (Zhu, et al., 1995; Ni, et al., 1995).
Most promoters are from about 500-1500 bases. Promoters for expressing a heterologous gene sequence in plants can be derived from plant DNA, e.g., the cauliflower heat shock protein 80 (hsp80, Brunke and Wilson, 1993; U.S. Pat. No. 5,612,472), or from other sources, for example, plant viruses e.g., the 35S cauliflower mosaic virus promoter, or bacteria which infect plants, e.g., the nopaline synthase (nos) promoter (Rogers, 1991), the octopine synthase (ocs) promoter (Leisner and Gelvin, 1988) and the mannopine synthase (mas) promoter from Agrobacterium.
Expression of heterologous genes or selected sequences of genes in transgenic plants has typically involved the use of constitutive promoters, which drive the expression of a product throughout the plant at all times and in most tissues (e.g., hsp80), the tomato ubiquitin promoter (Picton, et al., 1993), and the raspberry E4 promoter (U.S. Pat. Nos. 5,783,393; and 5,783,394).
A limited number of inducible and/or tissue specific promoters are known. Promoters that provide fruit-specific expression include the E4 and E8 promoter from tomato (Cordes, et al., 1989; Bestwick, et al., 1995; U.S. Pat. No. 5, 859,330). Another fruit-specific promoter is the tomato 2AII gene promoter. It has been demonstrated that nucleic acid sequences placed under the regulatory control of the 5′ non-coding region of the tomato 2AII gene (Van Haaren, 1993) are preferentially transcribed in developing fruit tissue. Fruit specific regulation of the kiwifruit actinidin promoter has been reported to be conserved in transgenic petunia plants (Lin, et al., 1993).
Differential screening has been used to identify abundant transcripts in developing and ripening fruit. In banana, for example, a cDNA clone encoding a putative thaumatin-like protein is identified as among the most abundant transcripts in ripening fruit, and in kiwifruit a metallothionein-like transcript is identified as very abundant in ripening fruit (Clendennen and May, 1997; Ledger and Gardner, 1994). Abundant transcripts have also been identified in the fruit of grape, cherry, and apple (Fils-Lycaon et al., 1996; Lee et al., 1993).
A transcript was previously identified in Golden Delicious apple (GenBank L15194; Lee, Gardner, and Lay-Yee, 1993
, Plant Physiol
. 103: 1017) that is abundant in fruit and shows sequence similarity to an auxin-repressed protein (ARP) of unknown function from strawberry.
Apple is a fruit which has been the subject of a great deal of study over the past several decades (Knee, 1993). Ethylene reduction is desired by packers and shippers in order to maintain apples from over-ripening and rotting. Refrigeration, high concentrations of CO
2
and low concentrations of O
2
are currently being employed to reduce the harmful effects of ethylene during storage. Such methods suffer from the disadvantages that fruits picked at preclimacteric stages (prior to full ripening) respond better to controlled atmosphere conditions than those at mature stages and many varieties of apple suffer from chilling injury and physiological disorders due to controlled atmosphere conditions, rendering them unmarketable.
Ethylene is a plant hormone influencing many aspects of plant growth and development, and is known to play a major role in the ripening process in fruits and vegetables. A large amount of ethylene is also produced following trauma caused by chemicals, temperatur

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