Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing a carotene nucleus
Reexamination Certificate
1999-06-04
2003-04-22
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing a carotene nucleus
C435S468000, C435S471000, C435S419000, C435S320100, C536S023100, C536S023200, C536S023600, C536S024100
Reexamination Certificate
active
06551793
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of Invention
This invention concerns a method for production of high amounts of carotenoids in plants. In particular, the invention concerns carotenoid-associated proteins CHRC and CHRD isolated from corollas of
Cucumis sativus
, CHRC deduced sequence, the molecular cloning, cDNA, RNA and a homologous gene involved in expression of CHRC. The
Cucumis sativus
corollas proteins were found to be involved and to regulate carotenoid accumulation and sequestration in chromoplasts. Invention additionally concerns a new CHRC promoter able to direct expression of foreign genes. The cloned promoter comprises approximately 3.5 kb of the CHRC upstream region.
2. Background Art and Related Art Disclosures
Carotenoids are very important dietary components in animals and are also important for plants as they are essential for plant photosynthesis. In humans, they are essential dietary components, which possess anticancer activity and protect against heart and age related diseases. Commercially, they are used-to color food products. In flowers and plants they are responsible for their pigmentation. In photosynthetically active organisms, carotenoids are essential to the proper functioning of the light-harvesting apparatus and they determine color of fruits and flowers. Moreover, carotenoid synthetic pathway leads to the synthesis of key growth regulators.
In plants, carotenoids are accumulated in high amounts chromoplasts, that are carotenoid-containing plastids responsible for the yellow, orange and red colors of petals, fruits and some roots in various plant species. Information on the structural organization of chromoplasts comes mainly from studies of ripening processes in fruits [Plant Sci. 94:1-17 (1983)]. The disappearance of chlorophyll and accumulation of carotenoids have been shown to parallel fruit maturation. Following the chloroplast to chromoplast conversion thylakoid membranes disintegrate, most of the components of the photosynthetic machine disappear, and a new set of proteins accumulates instead. In pepper fruits containing fibrillar chromoplasts, two very abundant proteins with approximate molecular masses of 35 kDa (ChrB) and 58 kDa (ChrA) have been identified as chromoplast-specific [Plant Mol. Biol. 21:549-554 (1993)]. When their pattern of expression was studied, the former was found to accumulate early and remain throughout ripening, whereas the latter appeared only at the final stage of fruit-color development and was found to be a carotenoid-associated protein [Plant Physiology 91:455-458 (1989)].
Identification of the protein fibrillin in bell peppers as an essential structure component of chromoplast-specific, carotenoid-accumulating lipoprotein structures, termed fibrils, and the isolation of its cDNA and genomic clones were very important steps towards understanding the internal structure of chromoplasts [Plant Cell 6:119-133 (1994)]. Analysis of this gene's expression in bell pepper revealed that both the protein and the transcript accumulate in parallel to fruit ripening. The bell pepper clone, however, did not reveal homologous transcripts in other tissues or plants, ibid.
To date, a number of genes from the carotenoid biosynthetic pathway in fruits have been cloned [Plant Mol. Biol. 27:425-428 (1995); Plant Mol. Biol. 27:1153-1162 (1995)]. Of these, however, only one, encoding capsanthin-capsorubin synthase [Plant Cell 7:1027-1038 (1995); Plant J. 6:45-54 (1994); Curr. Genet. 26:524-527 (1994)], shows fruit-chromroplast-specific expression.
Flower pigmentation due to carotenoid accumulation has also been studied. In
Tropaeolum majus
corollas, a 30 kDa protein accumulated in parallel to flower development and appeared to be a major and obligatory component of chromoplast fibrils [Protoplasma 157:128-135 (1990)]. Proteins of 30 and 68 kDa were characterized as the main proteins of
Viola tricolor
chromoplast globules [Plant Cell Res. 1:111-114 (1982)]. In nasturtium flowers, an immunocomplex band of 32 kDa was revealed with fibrillin antibodies [Plant Cell 6:119-133 (1994)]. With respect to genes of the carotenoid biosynthetic pathway, expression of phytoene synthase (PSY) and phytoene desaturase (PDS) in tomato flowers was shown to peak just before anthesis [Plant Cell 5:379-384 (1993)]. The petals and anthers of mature flowers accumulated the highest levels of these transcripts, as compared with other organs. Nevertheless, expression of both PSY and PDS was found not to be flower-specific.
Young green flower buds of
Cucumis sativus
contain chloroplasts, which are converted to fibrillar chromoplasts as the flower matures. Only chromoplasts are found in the mature yellow corollas [Physiol. Plant 104:321-326 (1994)]. An isolated 35 kDa chromoplast-specific protein (CHRC) from cucumber corollas was shown to be associated with carotenoids. While the above-findings are interesting from the botanical point of view, they do not have a practical utility.
Since carotenoids are so important for both plants and animals it would be of a great advantage to utilize in some way the above findings to achieve and control a higher production or accumulation of carotenoids in plants or cells, or their easy production by bacterial cells.
It is, therefore a primary object of this invention to provide a method for genetic control of production and/or accumulation of high levels of carotenoids in cells of plants, bacteria or other organisms.
SUMMARY
One aspect of the current invention is a method for production, accumulation and sequestration of high amounts of carotenoids in plant, bacteria or other cells by molecular and other type manipulations of carotenoid-associated proteins.
Another aspect of the current invention is a gene of which expression produces a protein which controls and is involved in a production, accumulation and sequestration of carotenoids in plant, bacteria or other cells.
Another aspect of the current invention is a method for molecular cloning of chrc gene encoding for the carotenoid-associated protein CHRC from
Cucumis sativus.
Still another aspect of the current invention is a gene containing domain which is homologous to domains present in a variety of chromoplasts containing plants.
Still another aspect of the current invention is a nucleotide sequence (SEQ ID NO:1) of the chrc gene encoding the
Cucumis sativus
protein CHRC.
Still another aspect of the current invention is an amino acid sequence (SEQ ID NO:2) comprising 322 amino acid of the CHRC protein of
Cucumis sativus.
Still yet another aspect of the current invention is a partly sequenced CHRC promoter having a nucleotide sequence (SEQ ID NO:10).
Another aspect of the current invention is a method for high production of carotenoids using bacterial cells having introduced CHRC gene intracellularly together with genes encoding enzymes for carotenoid biosynthesis.
DEFINITIONS
As used herein:
“CHRC” means chromoplast-specific carotenoid-associated protein.
“CHRD” means a minor chromoplast-specific protein of about 14 kD isolated from cucumber corolla chromoplasts.
“Anthesis” means the flowering period in flowers and plants.
“Corolla” means collectively the petals and flowers.
“Thylakoid” means a membranous lamella of protein and lipid implant chloroplasts where the photochemical reactions of photosynthesis take place.
“Gibberellins” or “GA” means plant hormones that regulate various aspects of plant growth and development, such as germination, cell growth, stem elongation, flower and fruit development and pigmentation.
“Gibberellin A
3
” or “GA
3
” means a hormone which up-regulates production of CHRC protein expression.
REFERENCES:
patent: 5429939 (1995-07-01), Misawa et al.
Vishnevetsky, M. GenBank Accession No. X95593 (1996), Sep. 1996.*
Deruere et al. Structure and Expression of Two Plant Genes Encoding Chromoplast-Specific Proteins. Biochem. Biophys. Res. Comm. (1994) 199(3): 1144-1150, Mar. 1994.*
Schantz, R. GenBank Accession No.
Itzhaki Hanan
Ovadis Marianna
Vainstein Alexander
Vishnevetsky Michael
Achutamurthy Ponnathapu
Cooper & Dunham LLP
Ken Kathleen
White John P.
Yissum Research Development Company of the Hebrew University of
LandOfFree
Carotenoid-associated proteins useful for high carotenoid... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Carotenoid-associated proteins useful for high carotenoid..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Carotenoid-associated proteins useful for high carotenoid... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3105374