DXS expression in Morinda citrifolia cell suspension cultures to increase antrhaquinones production

QUEVEDO CV; PERASSOLO M; BUSTO, V.; CARDILLO, AB; MARTÍNEZ CA; GIULIETTI AM; RODRÍGUEZ TALOU J

Carlos Paz, Cordoba

Congreso; XLIV Congreso de SAIB; 2008

Plant cell suspension cultures are attractive alternatives for large scale production of plant have a potential therapeutic use. AQs are anthracene derivatives whose basal structure, B rings of AQs are synthetized from shikimic and α-ketoglutaric acids via the ischorismate phosphate pathway (MEP). 1-deoxy-D-Xylulose-5-phosphate synthase (DXS), the first deoxy-D-Xylulose-5-phosphate. The aim of this work was to overexpress the DXS in M. citrifolia phosphate pathway (MEP). 1-deoxy-D-Xylulose-5-phosphate synthase (DXS), the first deoxy-D-Xylulose-5-phosphate. The aim of this work was to overexpress the DXS in M. citrifolia α-ketoglutaric acids via the ischorismate phosphate pathway (MEP). 1-deoxy-D-Xylulose-5-phosphate synthase (DXS), the first deoxy-D-Xylulose-5-phosphate. The aim of this work was to overexpress the DXS in M. citrifoliaM. citrifolia binary vector pMOG22-GUS, with hygromycin resistance, GUS reporter gene and the left by biolistic and A. tumefaciens strain LBA4404 methods. Transformation was confirmed levels of AQs (21% and 30% after 3 and 6 days of culture; p>0.01) and higher DXS activity Los cultivos de suspensiones celulares vegetales son una alternativa para la síntesis de productos naturales con gran potencial terapéutico. M. citrifolia produce metabolitos secundarios tales como las AQs [1] las cuales son derivados del antraceno. El componente basal de las AQs (9,10- dioxoantraceno) puede ser sustituido, dando lugar a una gran diversidad de estructuras. La figura 1 muestra la biosíntesis de las AQs por las vías metabólicas del Shikimato y de los Terpenos [2]. En esta ultima vía esta involucrada la DXS representando uno de los pasos importantes en dicha vía [3]. metabolitos secundarios tales como las AQs [1] las cuales son derivados del antraceno. El componente basal de las AQs (9,10- dioxoantraceno) puede ser sustituido, dando lugar a una gran diversidad de estructuras. La figura 1 muestra la biosíntesis de las AQs por las vías metabólicas del Shikimato y de los Terpenos [2]. En esta ultima vía esta involucrada la DXS representando uno de los pasos importantes en dicha vía [3]. levels of AQs (21% and 30% after 3 and 6 days of culture; p>0.01) and higher DXS activity Los cultivos de suspensiones celulares vegetales son una alternativa para la síntesis de productos naturales con gran potencial terapéutico. M. citrifolia produce metabolitos secundarios tales como las AQs [1] las cuales son derivados del antraceno. El componente basal de las AQs (9,10- dioxoantraceno) puede ser sustituido, dando lugar a una gran diversidad de estructuras. La figura 1 muestra la biosíntesis de las AQs por las vías metabólicas del Shikimato y de los Terpenos [2]. En esta ultima vía esta involucrada la DXS representando uno de los pasos importantes en dicha vía [3]. metabolitos secundarios tales como las AQs [1] las cuales son derivados del antraceno. El componente basal de las AQs (9,10- dioxoantraceno) puede ser sustituido, dando lugar a una gran diversidad de estructuras. La figura 1 muestra la biosíntesis de las AQs por las vías metabólicas del Shikimato y de los Terpenos [2]. En esta ultima vía esta involucrada la DXS representando uno de los pasos importantes en dicha vía [3]. A. tumefaciens strain LBA4404 methods. Transformation was confirmed levels of AQs (21% and 30% after 3 and 6 days of culture; p>0.01) and higher DXS activity Los cultivos de suspensiones celulares vegetales son una alternativa para la síntesis de productos naturales con gran potencial terapéutico. M. citrifolia produce metabolitos secundarios tales como las AQs [1] las cuales son derivados del antraceno. El componente basal de las AQs (9,10- dioxoantraceno) puede ser sustituido, dando lugar a una gran diversidad de estructuras. La figura 1 muestra la biosíntesis de las AQs por las vías metabólicas del Shikimato y de los Terpenos [2]. En esta ultima vía esta involucrada la DXS representando uno de los pasos importantes en dicha vía [3]. metabolitos secundarios tales como las AQs [1] las cuales son derivados del antraceno. El componente basal de las AQs (9,10- dioxoantraceno) puede ser sustituido, dando lugar a una gran diversidad de estructuras. La figura 1 muestra la biosíntesis de las AQs por las vías metabólicas del Shikimato y de los Terpenos [2]. En esta ultima vía esta involucrada la DXS representando uno de los pasos importantes en dicha vía [3]. M. citrifolia produce metabolitos secundarios tales como las AQs [1] las cuales son derivados del antraceno. El componente basal de las AQs (9,10- dioxoantraceno) puede ser sustituido, dando lugar a una gran diversidad de estructuras. La figura 1 muestra la biosíntesis de las AQs por las vías metabólicas del Shikimato y de los Terpenos [2]. En esta ultima vía esta involucrada la DXS representando uno de los pasos importantes en dicha vía [3].. El objetivo del presente estudio es la obtención de líneas transgénicas estables de M. citrifolia que sobreexpresen la enzima DXS, para aumentar la producción de AQs. citrifolia que sobreexpresen la enzima DXS, para aumentar la producción de AQs. objetivo del presente estudio es la obtención de líneas transgénicas estables de M. citrifolia que sobreexpresen la enzima DXS, para aumentar la producción de AQs.que sobreexpresen la enzima DXS, para aumentar la producción de AQs. DXS Figura 1: Ruta biosintética de las AQs en Rubiceae.Rubiceae. INTRODUCCIÓN RESULTADOS Fig 3: Células no transformadas Fig 4: Células transformadas, clon 1 Fig. 5: Células transformadas, clon 4 Obtención de líneas transgénicas estables CONCLUSIONES Se obtuvieron suspensiones celulares transgénicas de M. citrifolia que sobreexpresan el gen dxs mostrando ser positivas para el test de GUS. el gen dxs mostrando ser positivas para el test de GUS. Se obtuvieron suspensiones celulares transgénicas de M. citrifolia que sobreexpresan el gen dxs mostrando ser positivas para el test de GUS.dxs mostrando ser positivas para el test de GUS. La actividad DXS fue un 62% mayor en las líneas transgénicas con respectos a las líneas salvajes luego de 6 días de cultivo (p<0.05),no hubo diferencias a los 3 y 9 días. líneas salvajes luego de 6 días de cultivo (p<0.05),no hubo diferencias a los 3 y 9 días. La actividad DXS fue un 62% mayor en las líneas transgénicas con respectos a las líneas salvajes luego de 6 días de cultivo (p<0.05),no hubo diferencias a los 3 y 9 días. Los niveles de AQs en las líneas transgénicas fueron mayores en un 21 % a los 3 días y un 24 % a los 6 días de cultivo comparados con las líneas salvajes (p<0.01). Se puede concluir que las líneas transgénicas obtenidas exhiben una clara sobreexpresión de la enzima DXS. días y un 24 % a los 6 días de cultivo comparados con las líneas salvajes (p<0.01). Se puede concluir que las líneas transgénicas obtenidas exhiben una clara sobreexpresión de la enzima DXS. Los niveles de AQs en las líneas transgénicas fueron mayores en un 21 % a los 3 días y un 24 % a los 6 días de cultivo comparados con las líneas salvajes (p<0.01). Se puede concluir que las líneas transgénicas obtenidas exhiben una clara sobreexpresión de la enzima DXS. CELL SUSPENSION CULTURES TO Giulietti AM and Rodríguez Talou J. plant-derived natural products. Morinda citrifolia is able to produce Anthraquinones (AQs) that 9, 10-dioxoanthracene, can be substituted resulting in a diversity of structures. The A and ischorismate/o-succinylbenzoate pathway. The C ring is originated from the 2-C-methyl-D-erythritol 4- enzyme in the MEP pathway converts pyruvate and glyceraldehyde 3-phosphate into 1- suspension cultures. The dxs cDNA from Catharantus roseus was inserted into the and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. enzyme in the MEP pathway converts pyruvate and glyceraldehyde 3-phosphate into 1- suspension cultures. The dxs cDNA from Catharantus roseus was inserted into the and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. 9, 10-dioxoanthracene, can be substituted resulting in a diversity of structures. The A and ischorismate/o-succinylbenzoate pathway. The C ring is originated from the 2-C-methyl-D-erythritol 4- enzyme in the MEP pathway converts pyruvate and glyceraldehyde 3-phosphate into 1- suspension cultures. The dxs cDNA from Catharantus roseus was inserted into the and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. enzyme in the MEP pathway converts pyruvate and glyceraldehyde 3-phosphate into 1- suspension cultures. The dxs cDNA from Catharantus roseus was inserted into the and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. Morinda citrifolia is able to produce Anthraquinones (AQs) that 9, 10-dioxoanthracene, can be substituted resulting in a diversity of structures. The A and ischorismate/o-succinylbenzoate pathway. The C ring is originated from the 2-C-methyl-D-erythritol 4- enzyme in the MEP pathway converts pyruvate and glyceraldehyde 3-phosphate into 1- suspension cultures. The dxs cDNA from Catharantus roseus was inserted into the and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. enzyme in the MEP pathway converts pyruvate and glyceraldehyde 3-phosphate into 1- suspension cultures. The dxs cDNA from Catharantus roseus was inserted into the and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. o-succinylbenzoate pathway. The C ring is originated from the 2-C-methyl-D-erythritol 4- enzyme in the MEP pathway converts pyruvate and glyceraldehyde 3-phosphate into 1- suspension cultures. The dxs cDNA from Catharantus roseus was inserted into the and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. dxs cDNA from Catharantus roseus was inserted into the and right T-DNA borders from A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. A. tumefaciens. M citrifolia cell cultures were transformed by analysis of the GUS reporter gene. Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. (60% in a 6 day-culture; p>0.05) compared to wild type cell lines. . Transgenic cells lines showed significantly higher (60% in a 6 day-culture; p>0.05) compared to wild type cell lines.