Expression of strictosidine β-D-glucosidase cDNA from Catharanthus roseus, involved in the monoterpene indole alkaloid pathway, in a transgenic suspension culture of Nicotiana tabacum

• Published in: Plant physiology and biochemistry 2001, Vol.39 (9), p.763-769
• Main Authors: Zárate, Rafael, Bonavia, Marc, Geerlings, Arjan, van der Heijden, Robert, Verpoorte, Robert
• Format: Article
• Language: English
• Subjects: AGROBACTERIUM TUMEFACIENS; Agronomy. Soil science and plant productions; BIOCHEMICAL PATHWAYS; Biological and medical sciences; CATHARANTHUS ROSEUS; CELL CULTURE; CULTIVO DE CELULAS; CULTURE DE CELLULE; Fundamental and applied biological sciences. Psychology; Genetic engineering applications; genetic transformation; Genetics and breeding of economic plants; GLUCOSIDASA; GLUCOSIDASE; GLUCOSIDASES; INDOL; INDOLES; Metabolism; Metabolism. Physicochemical requirements; NICOTIANA TABACUM; Plant breeding: fundamental aspects and methodology; Plant physiology and development; PLANTAS TRANSGENICAS; PLANTE TRANSGENIQUE; strictosidine β-D-glucosidase; terpenoid indole alkaloid; TRANSGENIC PLANTS; VIA BIOQUIMICA DEL METABOLISMO; VOIE BIOCHIMIQUE DU METABOLISME
• ISSN: 0981-9428; 1873-2690
• DOI: 10.1016/S0981-9428(01)01295-5

Different transgenic cell lines of Nicotiana tabacum expressing strictosidine β-D-glucosidase cDNA from Catharanthus roseus were established following Agrobacterium tumefaciens infection. The fresh weight and protein levels did not appear to be affected by the gene insertion performed, and in several instances these seemed to be boosted. Regarding strictosidine β-D-glucosidase activity (SGD, EC 3.2.1.105) in some of the cell lines, this seemed growth associated but in others it appeared steady during growth, suggesting a continuous activation of the inserted gene cassette triggered by the CaM35S promoter, showing maximum values of ca. 170 pkat·mg –1 protein following incubation of crude protein extracts. Liquid nutrient medium failed to show any enzyme activity and lacked proteins, indicating that the gene product was not released into the medium. Molecular data showed, as seen in C. roseus cells, that SGD activity was associated with a protein aggregate of a size of 650 kDa, and this was absent in control and samples of the transgenic lines which failed to show SGD activity.