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  • Author or Editor: Sungil Kim x
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Carotenoids are plant compounds that serve a variety of essential functions in the plant and have also been found to have several health-promoting activities in humans. Carotenoids found in watermelon (Citrullus lanatus) flesh are responsible for the various colors such as red, yellow and orange. Previous inheritance studies of flesh color revealed that six genes were involved in color determination. The relationship and interaction of these genes suggests that some color-determining genes may be the result of mutations on the structural genes encoding enzymes in the carotenoid biosynthesis pathway. In this study we were able to isolate and sequence six genes encoding enzymes involved in the carotenoid biosynthetic pathway, and determine their expression in different colored watermelon fruit. The cDNA was synthesized from total RNA using RACE (Rapid Amplification of cDNA ends) kit (SMART RACE cDNA Amplification Kit; Clontech, Palo Alto, Calif.). Degenerate primers were designed based on published homologous genes from other species and were used to isolate gene fragments and full-length cDNAs of phytoene synthase, phytoene desaturase, _-carotene desaturase, β-cyclase, β-carotene hydroxylase and zeaxanthin expoxidase. RT-PCR was carried out to examine any differential expression of cloned genes in white, yellow, orange and red-fleshed watermelon. All cloned enzyme-encoding genes were expressed regardless of flesh colors. These results indicate that carotenoid biosynthesis may be regulated at the post-transcriptional level. One interesting feature supports this hypothesis. In case of β-cyclase, a 229-bp leader intron was identified, and an unspliced mRNA with this leader intron existed dominantly in cDNA pool of all samples.

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RNA isolation from ripe fruit can be complicated by high concentrations of sugar and water. These sugars interfere with RNA extraction often resulting in low RNA quality and quantities, and high water concentrations dilute the RNA, making isolation difficult. We report a simple but novel method by which the majority of the excess sugar and water in mature fruit of tomato (Lycopersicon esculentum Mill.), watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai], and muskmelon (Cucumis melo L.) can be easily removed from tissue before RNA extraction. This method produced quality RNA in a shorter time than the currently accepted method for fruit tissue RNA isolation and does not require liquid nitrogen or a freeze dryer.

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