ISOLATION AND MOLECULAR CLONING OF GENES FROM Myrciaria dubia “camu-camu” WITH POTENTIAL USE FOR BIOTECHNOLOGICAL PRODUCTION OF VITAMIN C
JUAN C. CASTRO *
Unidad Especializada de Biotecnología, Centro de Investigaciones de Recursos Naturales de la Amazonía (CIRNA), Universidad Nacional de la Amazonía Peruana (UNAP), Pasaje Los Paujiles S/N, Iquitos, Postal Code: 16024, Perú
MARIANELA COBOS
Laboratorio de Biotecnología y Bioenergética, Universidad Científica del Perú (UCP), Av. Abelardo Quiñones km 2.5, Iquitos, Postal Code: 16024, Perú
J. DYLAN MADDOX
The Field Museum of Natural History, Pritzker Laboratory for Molecular Systematics and Evolution, 1400 S. Lake Shore Drive, Chicago, IL, Postal Code: 60605, USA and Environmental Sciences, American Public University System, Charles Town, WV, Postal Code: 25414, USA
SIXTO A. IMÁN
Instituto Nacional de Innovación Agraria (INIA), Estación Experimental San Roque, Área de Conservación de Recursos Fitogenéticos, Calle San Roque # 236, Iquitos, Postal Code: 16024, Perú
JORGE L. MARAPARA
Unidad Especializada de Biotecnología, Centro de Investigaciones de Recursos Naturales de la Amazonía (CIRNA), Universidad Nacional de la Amazonía Peruana (UNAP), Pasaje Los Paujiles S/N, Iquitos, Postal Code: 16024, Perú
*Author to whom correspondence should be addressed.
Abstract
Myrciaria dubia “camu-camu” is a rich source of several bioactive phytochemicals and vitamin C (L-ascorbic acid, AsA). To gain insights about the genes involved in AsA biosynthesis in this plant species and consequently with potential use for its biotechnological production, here we report the isolation and molecular cloning of partial gene sequences of the D-mannose/L-galactose pathway. Degenerate primers designed by the multiple sequence alignment of related plant species were used to isolate in M. dubia the partial sequences of the six D-mannose/L-galactose pathway genes (GMP, GME, GGP, GPP, GDH and GLDH). The deduced protein sequences of the six genes have more than 81% sequence identity to rosids and asterids species, with a closer phylogenetic relationship to Eucalyptus grandis. In conclusion, gene sequences of the D-mannose/L-galactose pathway involved in AsA biosynthesis of M. dubia were successfully isolated and cloned and the phylogenetic analysis indicated that these genes have been relatively well conserved throughout of plant evolution, reflecting the importance of the enzymes of this metabolic pathway for plant growth and survival. Additionally, the isolation and cloning of these genes allow us to implement systems for biotechnological production of AsA.
Keywords: Gene cloning, molecular biotechnology, molecular phylogeny, L-ascorbic acid biosynthesis, tropical fruit