Maria Michela Spiriti

Comparative determination of some phytohormones in wild-type and genetically modified plants by gas chromatography–mass spectrometry and high-performance liquid chromatography–tandem mass spectrometry

The analytical performances of two optimized analytical methodologies used for the determination of auxins, cytokinins, and abscisic acid in plant samples were critically compared. Phytohormones were extracted from Nicotiana glauca samples using a modified Bieleski solvent and determined both by gas chromatography-mass spectrometry (GC-MS), after derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) on the Bieleski extract without any further treatment. HPLC-MS/MS gave better results in terms of higher coefficients of determination of the calibration curves, higher and more reproducible recoveries, lower limits of detection, faster sample preparation, and higher sample throughput. Thus, two sets of N. glauca and N. langsdorffii samples, both wild-type and genetically modified by inserting the glucocorticoid receptor (GR) gene encoding for the rat glucocorticoid receptor, were first characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and then analyzed by HPLC-MS/MS. Significant differences in the phytohormone content between the two sample sets were found and are very important in terms of understanding the mechanisms and effects on growth processes and the development of transgenic plants.

A DNA-based piezoelectric biosensor: strategies for coupling nucleic acids to piezoelectric devices.

A DNA-based piezoelectric biosensor has been here studied in terms of probe immobilisation and DNA sample pre-treatment. The biosensor is specific for the detection of the mecA gene of methicillin-resistant Staphylococcus aureus (MRSA). Methicillin-resistant S. aureus is responsible of several infections in humans, like pneumonia, meningitis and endocarditic. MRSA is also a major cause of hospital-acquired infections worldwide. The antibiotics resistance is conferred by the gene mecA, codifying for an anomalous protein. Two different immobilisation procedures of the probe specific for mecA gene are reported: immobilisation via streptavidin-biotin interaction and direct immobilisation of thiolated probes. After the study with synthetic oligonucleotides, the system has been applied to the analysis of bacterial DNA from MRSA, amplified by polymerase chain reaction. These samples were pre-treated with two different denaturation procedures and the performances of the sensor in the two cases were compared. The two immobilisation methods and denaturation protocols were here used to study the influences of these parameters on the performances of the sensor, applied here to the detection of the mecA gene. Better results in terms of sensitivity and reproducibility were obtained when using the biotinylated probe and the PCR-amplified samples treated by a denaturation procedures involving the use of high temperature and blocking oligonucleotides.

Response to metal stress of Nicotiana langsdorffii plants wild-type and transgenic for the rat glucocorticoid receptor gene

Recently our findings have shown that the integration of the gene coding for the rat gluco-corticoid receptor (GR receptor) in Nicotiana langsdorffii plants induced morphophysiological effects in transgenic plants through the modification of their hormonal pattern. Phytohormones play a key role in plant responses to many different biotic and abiotic stresses since a modified hormonal profile up-regulates the activation of secondary metabolites involved in the response to stress. In this work transgenic GR plants and isogenic wild type genotypes were exposed to metal stress by treating them with 30ppm cadmium(II) or 50ppm chromium(VI). Hormonal patterns along with changes in key response related metabolites were then monitored and compared. Heavy metal up-take was found to be lower in the GR plants. The transgenic plants exhibited higher values of S-abscisic acid (S-ABA) and 3-indole acetic acid (IAA), salicylic acid and total polyphenols, chlorogenic acid and antiradical activity, compared to the untransformed wild type plants. Both Cd and Cr treatments led to an increase in hormone concentrations and secondary metabolites only in wild type plants. Analysis of the results suggests that the stress responses due to changes in the plants hormonal system may derive from the interaction between the GR receptor and phytosteroids, which are known to play a key role in plant physiology and development.

Simultaneous detection of transgenic DNA by surface plasmon resonance imaging with potential application to gene doping detection.

Surface plasmon resonance imaging (SPRi) was used as the transduction principle for the development of optical-based sensing for transgenes detection in human cell lines. The objective was to develop a multianalyte, label-free, and real-time approach for DNA sequences that are identified as markers of transgenosis events. The strategy exploits SPRi sensing to detect the transgenic event by targeting selected marker sequences, which are present on shuttle vector backbone used to carry out the transfection of human embryonic kidney (HEK) cell lines. Here, we identified DNA sequences belonging to the Cytomegalovirus promoter and the Enhanced Green Fluorescent Protein gene. System development is discussed in terms of probe efficiency and influence of secondary structures on biorecognition reaction on sensor; moreover, optimization of PCR samples pretreatment was carried out to allow hybridization on biosensor, together with an approach to increase SPRi signals by in situ mass enhancement. Real-time PCR was also employed as reference technique for marker sequences detection on human HEK cells. We can foresee that the developed system may have potential applications in the field of antidoping research focused on the so-called gene doping.