Fluorescent quantum dots (QDs) have shown great promise for use as biolabels in cell and animal biology and more recently in plant sciences. An important use of QDs is for monitoring the dynamics, intracellular trafficking, and fate of carrier-DNA nanocomplexes in cell transfection and potentially in plant transformation. In this study, a low cost aqueous procedure has been developed to efficiently prepare biocompatible QDs for monitoring nanoparticle-mediated gene transfer in conjunction with molecular breeding of Jatropha curcas. Water-soluble CdSe nanoparticles were synthesized by self-assembly using L-Cysteine as stabilizer and optimal synthesis scheme established by fluorescence spectroscopy. The QDs were used to label chitosan-DNA nanoparticles via electrostatic interaction and the resultant QD-labeled chitosan-DNA complexes were shown to have superior fluorescence properties with red shift of emission and absorption spectra relative to the CdSe QDs alone. This system is being explored as a superior alternative to Agrobacterium-mediated genetic transformation of Jatropha curcas cells. PCR amplification of the full length of the carried reporter gene (GFP) suggests that the DNA was not digested in Jatropha curcas cells transfected with CdSe/CS-DNA complexes. Furthermore, GFP gene expression in the transfected callus cells, as evidenced by fluorescence detection, suggests that the target DNA was integrated into the plant genome.
Phosphoenolpyruvate carboxykinase (PEPCK), a critical gluconeogenic enzyme, catalyzes the first committed step in the diversion of tricarboxylic acid cycle intermediates toward gluconeogenesis. According to the relative conservation of homologous gene, a bioinformatics strategy was applied to clone Fusarium oxysporum phosphoenolpyruvate carboxykinase gene (PEPCK) by blasting search of EST database with homologous gene cDNA of Neurospora crassa and identified. Some characters of the PEPCK that were analyzed and predicted by the tools of bioinformatics in the following aspects include the composition of amino acid sequences, physical and chemical properties, O-glycosylation site, hydrophobicity or hydrophilicity, secondary and tertiary structure of the protein and function. These results showed that the full-length of PEPCK was 1771 bp and it contained a complete ORF (1575 bp), encoded 524 amino acids, which is much conserved in ascomycetes. The calculated molecular weight of PEPCK was 58358.2 Da, theoretical pI of 6.84. It has 20 α-helices, 37 sheets, and 12 glycosylation sites. It was a hydrophilic and stable protein with active site, ATP-binding site, metalbinding site and substrate-binding site.
The fatty acid compositions of Jatropha oil and Vernicia oil are strikingly different, which leads to a great difference in combustion performance, low temperature performance and oxidation stability. A comparative transcriptomic study was made in Vernicia and Jatropha, with a focus on the gene regulation of differential oil accumulation process. Transcriptome sequencing was conducted with seeds at the initial-and fast-stage of oil accumulation from both. More than 24 billion bases of cDNA sequence were obtained, with 49,583 and 45,414 high-quality unigenes identified for Vernicia and Jatropha seeds, respectively. Multiple comparative transcriptome approaches revealed a number of species-specific fatty acid desaturases (FAD2, FADX, FAH12 etc.) contributing to their differentiated fatty acid compositions in seeds of Vernicia and Jatropha. Meanwhile, the results suggested that DGAT majorly regulates TAG synthesis than PDAT in Vernicia seed, and PDAT may have more important role regulating TAG synthesis in Jatropha seed than in Vernicia seed. It was also implied that specific oleosins involving in oil bodies may have member bias and may affect lipid contents in seeds of Vernicia and Jatropha, as some of which were 30-50 fold up-regulated (with their RPKM values over 10,000 at fast-stage). Some important factors were identified and can differentially regulate lipid pathways in seeds of Vernicia and Jatropha.
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