Development of a transient promoter assay system for oil palm.
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Optimization of the physical parameters was carried out to produce transient Beta glucuronidase (GUS) and green fluorescence protein (GFP) activities in oil palm tissues (leaf mesocarp and root) for promoter analysis. The optimum conditions for DNA delivery into mesocarp slices and root tissue were 1550 psi helium pressure, 9 cm distance from macrocarrier to the sample and using 1 um gold particles as microcarrier to penetrate the tissues. For leaf explants, a lower pressure of 1350 psi sufficed with the same size gold particles and distance. The specificity of the Type 3 metallothionein-like gene (MT3-B) promoter was determined using the optimized system developed. The MT3-B promoter was cloned into an expression cassette (pBI221) containing GUS as the reporter gene with the removal of the existing Cauliflower Mosaic Virus (CaMV) 35S promoter to form a pBI-4C vector. The original pBI221 plasmid containing the CaMV 355 promoter was used as a control. In our study, express ion of the MT3-B promoter was only observed in the root tissue at a low level but not in the other tissues (mesocarp and leaf). In contrast, with the constitutive promoter (CaMV 355), expression was observed in all the tissues tested. These results correlated with the expression profile obtained for the MT3-B gene using northern blot analysis.
Incluye 20 referencias bibliográficas.
Optimization of the physical parameters was carried out to produce transient Beta glucuronidase (GUS) and green fluorescence protein (GFP) activities in oil palm tissues (leaf mesocarp and root) for promoter analysis. The optimum conditions for DNA delivery into mesocarp slices and root tissue were 1550 psi helium pressure, 9 cm distance from macrocarrier to the sample and using 1 um gold particles as microcarrier to penetrate the tissues. For leaf explants, a lower pressure of 1350 psi sufficed with the same size gold particles and distance. The specificity of the Type 3 metallothionein-like gene (MT3-B) promoter was determined using the optimized system developed. The MT3-B promoter was cloned into an expression cassette (pBI221) containing GUS as the reporter gene with the removal of the existing Cauliflower Mosaic Virus (CaMV) 35S promoter to form a pBI-4C vector. The original pBI221 plasmid containing the CaMV 355 promoter was used as a control. In our study, express ion of the MT3-B promoter was only observed in the root tissue at a low level but not in the other tissues (mesocarp and leaf). In contrast, with the constitutive promoter (CaMV 355), expression was observed in all the tissues tested. These results correlated with the expression profile obtained for the MT3-B gene using northern blot analysis.
Incluye 20 referencias bibliográficas.
Optimization of the physical parameters was carried out to produce transient Beta glucuronidase (GUS) and green fluorescence protein (GFP) activities in oil palm tissues (leaf mesocarp and root) for promoter analysis. The optimum conditions for DNA delivery into mesocarp slices and root tissue were 1550 psi helium pressure, 9 cm distance from macrocarrier to the sample and using 1 um gold particles as microcarrier to penetrate the tissues. For leaf explants, a lower pressure of 1350 psi sufficed with the same size gold particles and distance. The specificity of the Type 3 metallothionein-like gene (MT3-B) promoter was determined using the optimized system developed. The MT3-B promoter was cloned into an expression cassette (pBI221) containing GUS as the reporter gene with the removal of the existing Cauliflower Mosaic Virus (CaMV) 35S promoter to form a pBI-4C vector. The original pBI221 plasmid containing the CaMV 355 promoter was used as a control. In our study, express ion of the MT3-B promoter was only observed in the root tissue at a low level but not in the other tissues (mesocarp and leaf). In contrast, with the constitutive promoter (CaMV 355), expression was observed in all the tissues tested. These results correlated with the expression profile obtained for the MT3-B gene using northern blot analysis.
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Aceite de palma, Adn.