|Author:||Lee, Wang-fat Fred|
|Title:||Proteomic study of harmful algal blooming causative agents : nitrogen-induced growth and identification of dinoflagellates|
|Subject:||Hong Kong Polytechnic University -- Dissertations.|
Algal blooms -- Control.
Dinoflagellate blooms -- Control.
Red tide -- Environmental aspects.
|Department:||Department of Applied Biology and Chemical Technology|
|Pages:||xxvi, 326 leaves : ill. (some col.) ; 30 cm.|
|Abstract:||Harmful Algal Blooms (HABs) are an unavoidable worldwide problem and there is an apparent global increase in the occurrence of HABs. Consequently, there is an ever increasing risk of occurrence of HABs which represents expanding threats to human health, fishery resources and tourism industries. A huge and massive red tide occurred in the coastal waters of South China Sea, including Hong Kong from mid-March through mid-April in 1998. In Hong Kong alone, the blooms inflicted an estimated direct economic loss of around HK$ 250 million. There is an urgent need to understand the blooming mechanism. Although it is still not sure the extent to which the increase in red tides can be attributed to the increase of nutrient level, there is a strong relationship between algal blooming and the nitrogen load of coastal waters. Therefore, nitrogen is believed to be an important factor in the initiation and maintenance of phytoplankton blooms. Dinoflagellates are the major HABs causative agents. Although there are many studies on the effects of nitrogen on the growth of dinoflagellates, very little is known about the changes in the cells at molecular level. In addition, dissection into the proteome of dinoflagellate was not reported. Proteomic studies using 2D gels with or without nitrogen supplements on dinoflagellates have the potential to uncover the cellular pathways and mechanisms involved in blooming at the molecular level. Given that no effective method for controlling blooming is available yet, the best strategy for control is prevention. On top of it, a fore-warning system will be very useful. Therefore, rapid identification of HABs species is another important issue of the study of HABs. Traditional HABs species identification method is based on the morphological features. However, this type of taxonomic identification method not only is time-consuming but also requires a high level of expertise. Because of the nature of the technique, taxonomic confusion and arguments on similar looking HAB species are common. To resolve the taxonomic debate, different types of identification methods are being introduced, including the molecular probes using lectins, antibody and oligonucleotide. However, none of them are entirely satisfactory. Therefore, a fast, simple, accurate and systematic identification method is required for field applications. Studies described in this thesis were divided into two parts. The first part was aimed to study protein expression profiles in the model dinoflagellate Alexandrium affine under nitrogen stress. By investigating protein expressions in response to nitrogen availability, we may understand more about the mechanism of growth in this organism. Methodologies required to study protein expression profiles in dinoflagellates were derived and streamlined to allow production of high-quality two-dimensional gel electrophoretograms (2DE).When comparing cells grown under nitrogen depletion conditions with that under depletions, more than 15 differentially expressed proteins (>= 5-fold differences) were annotated by 2-DE. Two of them were found to be highly down-regulated (>= 16-fold) upon N-depletion; where one of them was 55 kDa with pI 5-6 and was successfully identified as ribulose-1,5 bisphosphate carboxylase/oxygenase form II (Rubisco II). Another one with 50 kDa, pI 5-6 and it was completely de novel and named as NAP50. Using a combination of N-terminal amino acid sequencing, tandem-mass pectrometry, PCR and molecular cloning technologies, the entire putative amino acid sequences of NAP50 were successfully obtained. When searched against the NCBI non-redundant database, no homology was found. Down-regulation of these two nitrogen-associated proteins (NAPs) was further confined to occur upon 30 hours after N-depletion through the immunoblotting experiments. However, their mRNA expression levels remained unchanged during N-depletion and repletion. Therefore, the down-regulation of these two NAPs seems to be controlled at protein level rather than at transcriptional level. Down-regulation of these NAP proteins under N-depletion could be prevented either by the replenishment of N sources or the addition of protease inhibitors. Specifically addition of serine protease inhibitors (AEBSF and benzamidine) could prevent the proteins became down-regulated in vivo and in vitro. Therefore, it was speculated that specific serine protease(s) was involved in the degradation of both proteins under the N-depletion condition. This specific serine protease(s) was partially fractionated using benzamidine-bound affinity column chromatography and the active fractions further suggested that there could be a NAPs-specific serine protease(s) being activated in response to nitrogen stress. To the best of our knowledge, this is the first study of dinoflagellate proteome in response to nitrogen stress. The novel protein NAP50 and the involvement of a serine protease(s) in the NAPs degradation under nitrogen stress had never been reported as well. Results from the present study not only provide new insights on how dinoflagellates react with nitrogen (one of the main factors thought to trigger HABs) at molecular level, but also act as the first step to dissect proteomes of dinoflagellates under optimal growth conditions. In the second part, it was aimed to design and validate a fast and accurate method for identification of dinoflagellates that is not dependent on morphological aspects. Different dinoflagellates were identified using their respective protein/peptide mass fingerprint profiles obtained with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The peptide mass fingerprint spectral patterns found for each species of dinoflagellates are unique and are easily distinguishable by visual inspection. In addition to the whole mass spectra, several specific biomarkers were identified from the mass spectrum of different species. These mass spectral patterns and the biomarker ions form an unambiguous basis for species discrimination. Results of our investigations clearly demonstrated the capability of this method in rapid identification of different species of dinoflagellates (namely Alexandrium affine, Prorocentrum minimum, Scrippsiella rotunda, Karenia brevis and a yet to be identified species), In addition, identification and differentiation of closely related species such as Alexandrium affine, Alexandrium catenella, Alexandrium tamarense, Alexandrium minutum, can be accomplished easily by the MALDI-MS analysis. The method is simple, fast and reproducible. Lastly, I had demonstrated that it was possible to identify individual dinoflagellate species in a mixed population of different dinoflagellate species based on characteristic peak mass spectral patterns or species-specific signature biomarkers in the spectrum. Although the identification process would become very complicated when more than two species are presented in a mixed culture, it may still be possible to identify the species compositions with the aid of computer software based on the analysis of the specific signature biomarker peak ions. The workflow of HAB species protein/peptide peak mass fingerprint spectral profiling is a straightforward approach. It represents an excellent alternative to classical microscopic-based identification techniques. Furthermore, this approach shows great potentials to be used for the continuous monitoring of water samples for the possible occurrence of HABs.|
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