Isolated and purified nucleic acids comprising a gene and a...

Details Isolated and purified nucleic acids comprising a gene and a... Isolated and purified nucleic acids comprising a gene and a...

1999-02-19

2001-07-24

Benzion, Gary (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C435S320100, C435S419000, C435S468000, C536S023600, C800S298000

Reexamination Certificate

active

06265633

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to nucleic acids comprising a gene specifically expressed in lupulin glands of hops and to regulatory sequences thereof.
2. Discussion of the Background
Plants produce and store a wide variety of low molecular weight organic compounds including terpenoids, alkaloids, phenolics, saponins, etc. Since, formerly, these compounds were not considered to be directly involved in supporting living matter having only minor biological functions, they were conventionally called “secondary metabolic products”.
Now, however, it has been elucidated that these secondary metabolic products function for promoting cellular differentiation and protecting cells from external harmful factors, and, furthermore, these secondary metabolic products formed by plants have been utilized and applied in a wide field of popular foods, medicaments, dyes, etc.
These secondary metabolic products have been paid so much attention with respect to their usefulness that the production pathways thereof in plant cells have been successively elucidated, indicating that these compounds are produced via a complicated biosynthetic cascade involving a number of enzymes. Most of these compounds biosynthesized via a cascade of enzymatic reactions can be isolated by directly extracting plant materials, but such a direct extraction from plants not only does not meet the demand for production on a large scale, but also is generally expensive. Therefore, the development of methods for synthesizing these compound in vitro using cultured cells, etc. has been under way.
On the one hand, it has been elucidated that hops, a major material for rendering a refreshing bitterness and flavor to beer, secrete a variety of secondary metabolic products in lupulin glands on the cones, contributing a great deal to the bitterness and flavor of beer.
Based on these circumstances, hops have been subjected to various breeding attempts focused on secondary metabolic products accumulated in lupulin glands such as bitter substance, essential oils, etc. in addition to the improvement of their agricultural properties.
However, hops are a dioecious plant, and especially the male plant bears no cones, materials for beer, is not commercially appreciated, and accordingly has not been actively studied so that its genetic properties useful for beer brewing have hardly been elucidated at all. Therefore, at present, hop breeding by conventional crossing relies largely on breeders' experience and intuition, and no prediction can be made especially on the quality of fermentation products at all till the time of the actual bearing of cones.
On the other hand, these days, breeding methods using genetic engineering such as a transformation technique and marker assisted selection have become available for various plants. In these methods, a more objective breeding can be performed compared with those conventional breeding methods that largely depend on breeders' experience and intuition. The transformation technique is a technique for directly introducing a desired character by transferring and expressing a foreign gene in plant cells. The expression of a foreign gene can be performed by linking a desired structural gene and a terminator capable of functioning in plant cells to a gene expression regulating promoter which is capable of functioning also in plant cells, and transferring the resulting transformed promoter into plant cells. Promoters frequently used in experiments are exemplified by CaMV 35S capable of expressing a transferred gene in regardless of any tissues of relatively numerous varieties of plants, and the promoter for the nopaline synthetase gene (Sanders, P. R., et al., Nucleic Acid Res., 15 (1987) 1543-1558). Furthermore, in the practical aspect of genetic transformation, the transferred gene might be harmful for the plant growth, etc. Therefore, there has been also a demand for promoters capable of expressing a foreign gene in a desired tissue, desired period, and desired quantity. The advantage of the breeding method using the transformation technique over the conventional traditional breeding method is that the former method is capable of transducing a desired character to plants regardless of their species with a relatively high accuracy in a short time. Also in the case of hops, since they can be proliferated by root-planting, the procedure for fixing the transduced character is not required. Therefore, the breeding method using the transformation technique is especially effective for hops.
Marker assisted selection is an example of a breeding method using the DNA marker such as RFLP (Restriction Fragment Length Polymorphism) marker, and have been put into practical use, especially for rice and wheat. It has been generally agreed that transformation techniques are capable of transducing a character regulated by a single gene, but incapable of transducing a character regulated by multiple genes. Marker assisted selection is capable of compensating for these defects of transformation techniques.
A prerequisite for such a breeding method using gene technology is to elucidate genes related with the desired character and those regulating those genes. Especially, from the viewpoint of hops as the beer material as well as the source of effective drug ingredient, if genes related to the biosynthesis of secondary metabolic products secreted from lupulin glands contained in the cones of female plants are elucidated, these genes can be applied to the hop breeding method using the gene technology, and, furthermore, also to the field of medical treatment.
SUMMARY OF THE INVENTION
Therefore, in order to elucidate genes specifically expressed in lupulin glands and facilitate their practical application, it is an object of the present invention to isolate, purify and provide such genes, as well as regulatory sequences thereof, such as promoters, for these genes.
As described above, nucleic acids isolated and purified in the present invention comprise genes specifically expressed in the lupulin glands of hops, promoters specifically functioning in lupulin glands, and portions thereof.
Using these nucleic acids, the conventional method for breeding hops wholly dependent on the breeders experience and intuition can be converted to a more objective method using genetic engineering. As described above, since important secondary metabolic products, such as beer materials and effective drug ingredients, are secreted exclusively in lupulin glands on the hop cones, genes specifically expressed to function in lupulin glands are likely related to the biosynthesis of these secondary metabolic products. Thus, by utilizing genes capable of participating in the biosynthesis of secondary metabolic products as a marker, an improved marker assisted selection can be developed for the breeding of hops which will contribute significantly to the food and drug industries. In addition, by transferring the above-described genes to hops using a transformation technique, breeding of industrially useful cultivar can be accomplished. That is, by breeding hops using a genetic engineering technique with these nucleic acids, the composition of secondary metabolic products accumulating in lupulin glands can be regulated. Furthermore, the nucleic acids of the present invention enable the maintenance and improvement of hop quality for beer brewing, and the use of hops for drug production.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings.


REFERENCES:
Sive et al, Nucl. Acids Res., vol. 16, No. 22, p. 10937, 1988.*
Likens et al, Crop Sci., vol. 18, pp. 380-386, 1978.*
Speirs et al, Meth. Plant Biochem., vol. 10, pp. 1-32, 1993.*
Sparvoli et al, Plant Mol. Biol., vol. 24, pp. 743-755, 1994.*
Suen-Ying Fung, et al., Phytochemistry, vol. 44, No. 6, pp. 1047-1053, “Conversion of Deoxyhumulo

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