Combinatorial genetic transformation of cereals and the creation of metabolic libraries for the carotenoid pathway.
ABSTRACT Combinatorial nuclear transformation is used to generate populations of transgenic plants containing random selections from a collection of input transgenes. This is a useful approach because it provides the means to test different combinations of genes without the need for separate transformation experiments, allowing the comprehensive analysis of metabolic pathways and other genetic systems requiring the coordinated expression of multiple genes. The principle of combinatorial nuclear transformation is demonstrated in this chapter through protocols developed in our laboratory that allow combinations of genes encoding enzymes in the carotenoid biosynthesis pathway to be introduced into rice and a white-endosperm variety of corn. These allow the accumulation of carotenoids to be screened initially by the colour of the endosperm, which ranges from white through various shades of yellow and orange depending on the types and quantities of carotenoids present. The protocols cover the preparation of DNA-coated metal particles, the transformation of corn and rice plants by particle bombardment, the regeneration of transgenic plants, the extraction of carotenoids from plant tissues, and their analysis by high-performance liquid chromatography.
- SourceAvailable from: Hikaru Seki
Dataset: Combi, PCP,54,740, 2013
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ABSTRACT: Triterpenoid saponins are a diverse group of specialized (secondary) metabolites with many biological properties. The model legume Medicago truncatula has an interesting profile of triterpenoid saponins from which sapogenins are differentiated into hemolytic and nonhemolytic types according to the position of their functional groups and hemolytic properties. Gene co-expression analysis confirmed the presence of candidate P450s whose gene expressions correlated highly with that of β-amyrin synthase (bAS). Among these, we identified CYP716A12 and CYP93E2 as key enzymes in hemolytic and nonhemolytic sapogenin biosynthetic pathways. The other candidate P450s showed no β-amyrin oxidation activity. However, among the remaining candidate P450s, CYP72A61v2 expression highly correlated with that of CYP93E2, and CYP72A68v2 expression highly correlated with that of CYP716A12. These correlation values were higher than occurred with bAS expression. We generated yeast strains expressing bAS, CPR, CYP93E2, and CYP72A61v2, and bAS, CPR, CYP716A12, and CYP72A68v2. These transgenic yeast strains produced soyasapogenol B and gypsogenic acid, respectively. We were therefore able to identify two CYP72A subfamily enzymes: CYP72A61v2, which modifies 24-OH-β-amyrin, and CYP72A68v2, which modifies oleanolic acid. Additionally, P450s that seemed not to work together in planta were combinatorially expressed in transgenic yeast. The yeast strains (expressing bAS, CPR, CYP72A63, and CYP93E2 or CYP716A12) produced rare triterpenoids that do not occur in M. truncatula. These results show the potential for combinatorial synthesis of diverse triterpenoid structures and enable identification of the enzymes involved in their biosynthesis.Plant and Cell Physiology 01/2013; · 4.13 Impact Factor
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ABSTRACT: Plant Synthetic Biology aims to apply engineering principles to plant genetic design. One strategic requirement of Plant Synthetic Biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here we describe GoldenBraid2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for Plant Synthetic Biology. GB2.0 relies on the use of TypeIIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an ever-growing open collection of DNA parts, including a group of functionally-tested, pre-made genetic modules to build frequently-used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of web resources which include a publicly available database, tutorials and a software package that provides in silico simulations and lab protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement Plant Synthetic Biology projects.Plant physiology 05/2013; · 6.56 Impact Factor