Abstract
The uses and value of plant minor components such as carotenoids, flavonoid and sterols have recently been discovered. Carotenoids, in particular lycopene, have been shown to be potential for its anticancer and antioxidant activities. Extensive studies have been conducted to elucidate the plant carotenoid pathway. Oil palm is known to be the richest natural source for carotene. However, to date, there has been lack of work carried out to elucidate the pathway in this species. This work is the first effort towards the understanding of the oil palm carotenoid biosynthesis pathway. More importantly, the work has also provides the necessary genetic materials that could be used to genetically engineer oil palm for lycopene accumulation. This paper is the first report on the isolation of cDNA clones coding for lycopene â-cyclase (lcyb) and lycopene -cyclase (lcye) from oil palm. Fragments containing partial sequences of these genes were successfully generated through RT-PCR amplification using degenerated primers. The complete DNA sequence of these fragments was determined. Primers were then designed based on these sequences to facilitate the amplification of the 3' and 5' end regions of the genes. Both ends were successfully obtained for both cyclases. A consensus sequence of 1962 bp and 1759 bp was generated for oil palm lcye and lcyb, respectively. An open reading frame (ORF) of 1617 bp encoding 539 amino acid (AA) residues was identified for lcye. Similarly, an ORF of 1509 bp encoding for 503 AA residues was also identified for lcyb. Deduced AA sequences were shown to be highly identical to their respective counterparts from other plants at about 80% identity. Although the enzymes were functionally equivalent, they were shown to share a little resemblance at about 30 percent identity. However, oil palm lcyb was shown to share a relatively high identity to plant neoxanthin and capxanthin-capsorubin synthases, suggesting the common ancestor of the cyclases and synthases. In addition, there are also other conserved regions present within the sequences. The regulation and expression of these two carotenogenic genes were studied in developing mesocarp tissues using real-time PCR analysis. The results indicated that all of the carotenogenic genes were expressed at a low level in the tissues tested. Both genes were shown to be expressed at a relatively high level in young and late developing mesocarp tissues, as well as in leaves. Nevertheless, their expression was seen to be correlated to the accumulation of carotenoids in the tissues both for developmental process and storage. At present, work is being carried out to prepare the transformation gene constructs that are to be used in a model plant and oil palm.Incluye referencias bibliográficas.The uses and value of plant minor components such as carotenoids, flavonoid and sterols have recently been discovered. Carotenoids, in particular lycopene, have been shown to be potential for its anticancer and antioxidant activities. Extensive studies have been conducted to elucidate the plant carotenoid pathway. Oil palm is known to be the richest natural source for carotene. However, to date, there has been lack of work carried out to elucidate the pathway in this species. This work is the first effort towards the understanding of the oil palm carotenoid biosynthesis pathway. More importantly, the work has also provides the necessary genetic materials that could be used to genetically engineer oil palm for lycopene accumulation. This paper is the first report on the isolation of cDNA clones coding for lycopene â-cyclase (lcyb) and lycopene -cyclase (lcye) from oil palm. Fragments containing partial sequences of these genes were successfully generated through RT-PCR amplification using degenerated primers. The complete DNA sequence of these fragments was determined. Primers were then designed based on these sequences to facilitate the amplification of the 3' and 5' end regions of the genes. Both ends were successfully obtained for both cyclases. A consensus sequence of 1962 bp and 1759 bp was generated for oil palm lcye and lcyb, respectively. An open reading frame (ORF) of 1617 bp encoding 539 amino acid (AA) residues was identified for lcye. Similarly, an ORF of 1509 bp encoding for 503 AA residues was also identified for lcyb. Deduced AA sequences were shown to be highly identical to their respective counterparts from other plants at about 80% identity. Although the enzymes were functionally equivalent, they were shown to share a little resemblance at about 30 percent identity. However, oil palm lcyb was shown to share a relatively high identity to plant neoxanthin and capxanthin-capsorubin synthases, suggesting the common ancestor of the cyclases and synthases. In addition, there are also other conserved regions present within the sequences. The regulation and expression of these two carotenogenic genes were studied in developing mesocarp tissues using real-time PCR analysis. The results indicated that all of the carotenogenic genes were expressed at a low level in the tissues tested. Both genes were shown to be expressed at a relatively high level in young and late developing mesocarp tissues, as well as in leaves. Nevertheless, their expression was seen to be correlated to the accumulation of carotenoids in the tissues both for developmental process and storage. At present, work is being carried out to prepare the transformation gene constructs that are to be used in a model plant and oil palm.