Zanthoxylum zanthoxyloides is an endangered African tree producing numerous bioactive substances including antileukemic and antisickling agents. Here, the potential of Z. zanthoxyloides hairy root cultures was tested for the production of bioactive substances with limited natural resources. The efficiency of Agrobacterium rhizogenes LBA9402‐mediated transformation of leaf material was evaluated using different techniques. An optimal transformation frequency of 77% was obtained after 11 days by inoculating A. rhizogenes directly onto the central vein of 14‐week‐old leaves followed by a co‐cultivation period of 3 days. Different treatments in immersion mode (manual wounding, acetosyringone, CaCl 2 , ultrasonication) never exceeded these results. A maximum growth rate of 0.37 cm/day was determined during the exponential phase. Liquid chromatography‐diode array detection analysis showed the presence of skimmiamine, sesamine, chelerythrine, and chelerythrine derivatives in Z. zanthoxyloides hairy root lines. The maximum production of skimmiamine and chelerythrine in 28‐day‐old hairy root cultures was 45 ± 2 and 107 ± 4 mg/100 g dry weight, respectively. The present results highlight the potential of Z. zanthoxyloides hairy root cultures for the sustainable production of skimmiamine and chelerythrine.
We designed an efficient transformation system for Candida guilliermondii wild-type strains. We demonstrated that the Staphylococcus aureus MRSA 252 ble coding sequence placed under the control of the yeast phosphoglycerate kinase gene transcription-regulating regions confers phleomycin resistance to transformed C. guilliermondii cells. To illustrate the potential of this drug-resistant cassette, we carried out the disruption of the C. guilliermondii ADE2 gene. This new dominant selectable marker represents a powerful tool to study the function of various genes in this yeast of clinical and biotechnological interest.
RésuméLe transfert d'une souche chlorophyllienne de C. ternata à l'obscurité provoque une diminution de la croissance et des teneurs en acides aminés libres. Les concentrations en proline, en glutamine et en arginine diminuent alors que celle d'asparagine augmente. L'accumulation séquentielle d'acides aminés observée dans les tissus à la lumière est modifiée à l'obscurité. Les teneurs en platydesminium diminuent, les chloroplastes se transforment en étioplastes. Certaines de ces modifications pourraient être provoquées par la disparition des apports énergétiques quand la photosynthèse ne peut avoir lieu.
Abstract It is now accepted that some signal transduction systems in plants are built of modules that are homologous to the autophosphorylating histidine kinases, the histidine-containing phosphotransfer (HPt) domains and the response regulators that compose two-component systems (TCS) as well as multistep phosphorelay systems (MPS) firstly uncovered in Prokaryotes. There are now good evidences that TCS and MPS play crucial roles in the signaling pathways for ethylene and cytokinin, respectively. In the present study, a full length cDNA (designated CrHPt1) was isolated by PCR amplification of a cDNA library from periwinkle cell cultures. The deduced protein (151 amino acids) is 68% identical to the HPt protein AHP3 of Arabidopsis thaliana. Involvement of a HPt protein in the cytokinin signaling pathway leading to alkaloid biosynthesis enhancement in Madagascar periwinkle cell cultures is discussed.
Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the fungal CTG clade. This yeast remains actively studied as a result of its moderate clinical importance and most of all for its potential uses in biotechnology. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both a methionine auxotroph recipient strain and a functional MET gene as selection marker. We first disrupted the MET2 and MET15 genes encoding homoserine-O-acetyltransferase and O-acetylserine O-acetylhomoserine sulphydrylase, respectively. The met2 mutant was shown to be a methionine auxotroph in contrast to met15 which was not. Interestingly, met2 and met15 mutants formed brown colonies when cultured on lead-containing medium, contrary to the wild-type strain, which develop as white colonies on this medium. The MET2 wild-type allele was successfully used to transfer a yellow fluorescent protein (YFP) gene-expressing vector into the met2 recipient strain. In addition, we showed that the loss of the MET2-containing YFP-expressing plasmid can be easily observed on lead-containing medium. The MET2 wild-type allele, flanked by two short repeated sequences, was then used to disrupt the LYS2 gene (encoding the α-aminoadipate reductase) in the C. guilliermondii met2 recipient strain. The resulting lys2 mutants displayed, as expected, auxotrophy for lysine. Unfortunately, all our attempts to pop-out the MET2 marker (following the recombination of the bordering repeat sequences) from a target lys2 locus were unsuccessful using white/brown colony colour screening. Nevertheless, this MET2 transformation/disruption system represents a new versatile genetic tool for C. guilliermondii.
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