Abstract
The potential development of potato (Solanum tuberosum) as a low-cost eukaryotic system for the production of a commercially valuable enzyme feed supplement was examined. AFibrobacter succinogenes 1,3-1,4-β-glucanase [1,3-1,4-β-D-glucan 4-glucanohydro-lase] gene under the control of the constitutive cauliflower mosaic virus 35S promoter was transferred into the potato cultivar, Desiree. The presence of the β-glucanase cDNA in the plant genome of independent transgenic potato lines was confirmed by PCR and Southern analysis. Northern analysis identified the presence of the β-glucanase mRNA in the leaf tissue of transgenic plants. Furthermore, western analysis showedF. succinogenes β-glucanase accumulations of 0.1% and 0.05% of total soluble protein in the leaves and tubers, respectively. Specific activities of the enzyme in leaves (1693 units mg-1 β-glucanase) and tubers (2978 units mg-1 β-glucanase) were comparable to that previously reported for the enzyme produced in bacteria. Lyophilization of leaves had no effect on the specific activity of the β-glucanase, and only marginally influenced the specific activity of the enzyme expressed in tubers. Relative to the control line (cv. Desiree), tuber yields were significantly reduced by 28%-72% in all lines expressing theF. succinogenes β-glucanase, and microscopy showed that expression of the β-glucanase caused changes in cell wall structure. Results of this study demonstrate that a 1,3-1,4-β-glucanase can be expressed in potato tissues, and that potato plants have the potential to be used for the commercial production of heterologous enzymes.
Resumen
Se examinó el desarrollo potencial de la papa (Solanum tuberosum) como un sistema eucarióntico de bajo costo para la producción de enzimas alimenticias suplementarias con valor comercial. Con ese fin se transfirió al cultivar de papa Desiree un gen 1,3-1,4-β-glucanasa [l,3-l,4-$-D-glucana 4-glucanahidrolasa] deFibrobacter succinogenes bajo el control del promotor constitutivo del mosaico del virus 35S de la coliflor. La presencia del cADN de β-glucanasa en el genoma de la planta de las líneas de papa transgénicas independientes fue confirmada por la reacción en cadena de la polimerasa (PCR) y el análisis meridional. El análisis septentrional identificó la presencia de mRNA de ²-glucanasa en el tejido de las hojas de las plantas transgénicas. Además, el análisis occidental mostró acumulaciones de 0.1% y 0.05% de las proteínas totales solubles deF. succinogenes β-glucanasa en las hojas y tubérculos, respectivamente. Las actividades específicas de la enzima en las hojas (1693 unidades mg-1 β-glucanasa) y en los tubérculos (2978 unidades mg-1 β-glucanasa) fueron comparables a las reportadas previamente para la enzima producida en la bacteria. La liofilización de las hojas no tuvo efecto en la actividad especifica de la β-glucanasa y sólo influyó marginalmente en la actividad especifica de la enzima expresada en los tubérculos. En relación a la línea de control (cv. Desiree), los rendimientos del tubérculo se redujeron significativamente entre 28 a 72% en todas las líneas, expresando laF. succinogenes β-glucanasa, y el microscopio mostró que la expresión de la β-glucanasa causó cambios en las estructuras de la pared de la célula. Los resultados de este estudio demuestran que la 1,3-1,4-β-glucanasa puede expresarse en tejidos de papa y que las plantas de papa tienen potencial para ser usadas en la producción comercial de enzimas heterólogas.
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Armstrong, J.D., Inglis, G.D., Kawchuk, L.M. et al. Expression of afibrobacter succinogenes 1,3-1,4-β-glucanase in Potato (Solanum tuberosum). Am. J. Pot Res 79, 39–48 (2002). https://doi.org/10.1007/BF02883522
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DOI: https://doi.org/10.1007/BF02883522