Cloning And Characterization Of Carotenoid Biosynthetic Genes From Sweet Orange (Citrus Sinesis Osbeck) Red-Fleshed Mutants

Citrus red-fleshed mutant are reported with many carotenoids beneficial to human beings. They provide the useful materials not only for the formation of bud sport but also for the carotenoid biosynthesis of citrus fruits. In the present research, two red-fleshed sweet orange mutants, 'Cara Cara' navel orange (C. sinenesis Osbeck) and 'Hong-anliu' sweet orange (C. sinenesis Osbeck), were used to investigate the mechanism of carotenoid metabolism and the expressions of corresponding genes at different ripening stage. A pulp cDNA library from 'Cara Cara' was also constructed .The main results were as follows:1. A modified method for isolating RNA from pulp of ripening citrus fruit was established. This method involved a dehydrating step with absolute ethanol. By this step, most water in citrus pulp was removed, which consequently yielded high quantities of RNA.2. The content and composition of total carotenoids of 'Cara Cara' and 'Washington' navel orange fruits from Augest to November were analyzed. Results showed that total carotenoids, lycopene and phytoene contents in fruits of 'Cara Cara' were higher than those of 'Washington'. Although higher β-carotene content was observed in pulp of 'Cara Cara', less α-content in peel of 'Cara Cara' was present than that of 'Washington'.3. Nine genes involved in carotenoid biosyntheis were cloned and charazterized. Results showed:1) Lcyb was a single allele with two transcripts. Despite differences in these two transcripts, no difference was observed between 'Cara Cara' and 'Washington'. Expression of Lcyb was transcriptionally cotrolled and tissue-specific. No or weak signals were detected in albedo of 'Cara Cara', as revealed by in situ mRNA hybridization and RT-PCR analysis.2) Two copies of Psy were detected in genomes of 'Cara Cara' and 'Washington'. The deduced amino acid sequences were nearly the same, except a single different amino acid. In situ mRNA hybridization and RT-PCR analysis revealed that the Psy expression level in albedo of 'Cara Cara' was higher than that of 'Washington'.3) Lcye was of single copy, but produced two transcripts, of which one was...