DNA encoding fruit-ripening-related proteins, DNA constructs, ce

Details DNA encoding fruit-ripening-related proteins, DNA constructs, ce DNA encoding fruit-ripening-related proteins, DNA constructs, ce

1996-01-11

1999-06-01

Robinson, Douglas W.

Multicellular living organisms and unmodified parts thereof and

Plant, seedling, plant seed, or plant part, per se

4353201, 435419, 435468, 536 232, 536 236, 800260, 800278, A01H 102, A01H 500, A01H 510, C12N 514

Patent

active

059089733

DESCRIPTION:

BRIEF SUMMARY
The modification of plant gene expression has been achieved by several methods. The molecular biologist can choose from a range of known methods to decrease or increase gene expression or to alter the spatial or temporal expression of a particular gene. For example, the expression of either specific antisense RNA or partial sense RNA has been utilised to reduce the expression of various target genes in plants (as reviewed by Bird and Ray, 1991, Biotechnology and Genetic Engineering Reviews 9:207-227). These techniques involve the incorporation into the genome of the plant of a synthetic gene designed to express either antisense or sense RNA. They have been successfully used to down-regulate the expression of a range of individual genes involved in the development and ripening of tomato fruit (Gray et al, 1992, Plant Molecular Biology, 19:69-87). Methods to increase the expression of a target gene have also been developed. For example, additional genes designed to express RNA containing the complete coding region of the target gene may be incorporated into the genome of the plant to "over-express" the gene product. Various other methods to modify gene expression are known; for example, the use of alternative regulatory sequences.
In work leading to the present invention, we have identified genes which encode proteins involved in ripening-related processes and which show novel expression patterns in normal and ripening inhibitor (rin) mutant tomatoes. DNA sequences encoding these proteins have been cloned and characterised. The DNA sequences may be used to modify plant characteristics, particularly the ripening characteristics of fruit, including tomatoes. The sequences in question are encoded (almost completely) in the following clones: ERXT1b, ERT10, ERT13, ER14, ERT15, ERT16b, ERT17, ERTD1, ERTR1 and ERTS2 (herein referred to as the "ERT clones" or "ERT sequences"). The clones were isolated as part of a research programme to identify genes expressed at the onset of tomato fruit ripening (Picton et al, 1993, Plant Molecular Biology, in press).


BACKGROUND TO THE ISOLATION OF THE ERT CLONES

Considering the large number of complex pathways involved in the fruit ripening process, and despite construction and screening of many tomato fruit cDNA libraries in the past decade, surprisingly few fruit- or ripening-specific genes and their functions have been identified. In order to address this specific area a new cDNA library was constructed (pERT clone series, Early Ripening Tomato) from the pericarp of a very early ripening stage of wild-type tomato fruit and differentially screened against mRNA obtained from the pericarp of ripening inhibitor (rin) mutant fruit of a similar developmental stage.
The rin mutation, first reported in 1968, is recessive, maps to chromosome 5 and is closely linked to the macrocalyx locus. It has pleiotropic effects on ripening, resulting in an extremely retarded ripening phenotype. Fruit demonstrate an increased resistance to many common post harvest pathogens and have been maintained for years without further signs of normal ripening or deterioration. Following extensive storage of fruit seeds may germinate and grow precociously within the fruit. Other aspects of plant growth and early fruit development appear unaffected by the rin mutation. However, the rin fruit fail to attain a normal level of pigmentation as a result of decreased accumulation of carotenoids, particularly lycopene, and there is a decreased rate of chlorophyll loss so that rin fruit remain green when wild-type fruit are fully red. The rin fruit eventually "ripen" to a lemon yellow colour after several weeks. These mutant fruit also fail to achieve normal flavour or aroma which has been correlated with the reduced production of a number of aromatic compounds.
Despite these considerable shortfalls the rin mutation is used in the heterozygous state in commercial tomato production, as in such a genetic state the deficiencies of the homozygous rin mutation regarding fruit quality are at least partially overcome. The

REFERENCES:
patent: 5328999 (1994-07-01), Bennett et al.
Gray J, et al. "Molecular biology of fruit ripening and its manipulation with antisense genes." Plant Mol. Biol. 19: 69-87, 1992.
Iusem ND, et al. Tomato (Lycopersicon esculentum) transcript induced by water deficit and ripening. Plant Physiol. 102: 1353-1354, Aug. 1993.
Iusem ND, et al. GenBank accession No.L08255, 1993.

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