Hemicellulose and cellulose are essential polysaccharides for plant development and major components of cell wall. They are also an important energy source for the production of ethanol from plant biomass, but their conversion to fermentable sugars is hindered by the complex structure of cell walls. The glucuronic acid substitution of xylan (GUX) enzymes attach glucuronic acid to xylan, a major component of hemicellulose, decreasing the efficiency of enzymes used for ethanol production. Since loss-of-function gux mutants of Arabidopsis thaliana enhance enzyme accessibility and cell wall digestion without adverse phenotypes, GUX genes are potential targets for genetically improving energy crops. However, comprehensive identification of GUX in important species and their evolutionary history are largely lacking. Here, we identified putative GUX proteins using hidden Markov model searches with the GT8 domain and a GUX-specific motif, and inferred the phylogenetic relationship of 18 species with Maximum likelihood and Bayesian approaches. Each species presented a variable number of GUX, and their evolution can be explained by a mixture of divergent, concerted and birth-and-death evolutionary models. This is the first broad insight into the evolution of GUX gene family in plants and will potentially guide genetic and functional studies in species used for biofuel production.
The successful development of genetically engineered monocots using
Agrobacterium-mediated transformation has created an
increasing demand for compatible vectors. We have developed a new expression
vector, pGVG, for efficient transformation and expression of different
constructs for gene overexpression and silencing in sugarcane. The pCAMBIA2300
binary vector was modified by adding Gateway recombination sites for fast gene
transfer between vectors and the maize polyubiquitin promoter Ubi-1
(ZmUbi1), which is known to drive high gene expression
levels in monocots. Transformation efficiency using the pGVG vector reached up
to 14 transgenic events per gram of transformed callus. Transgenic plants
expressing the β-glucuronidase (GUS) reporter gene from pGVG
showed high levels of GUS activity. qRT-PCR evaluations demonstrated success for
both overexpression and hairpin-based silencing cassettes. Therefore, pGVG is
suitable for plant transformation and subsequent applications for
high-throughput production of stable transgenic sugarcane. The use of an
expression cassette based on the ZmUbi1 promoter opens the
possibility of using pGVG in other monocot species.
There are several published works about the success in sugarcane transformation, with a wide range of possible methodologies. However, there are still difficulties in establishing an efficient and reproducible tissue culture techniques, being of great importance the establishment of an effective protocol for the transformation of sugarcane.
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