| Over the last several decades, harmful algal blooms (HABs) have emerged as a global concerned environmental problem due to its escalating trend in occurrence and great threat to health of wildlife and human. Multidisciplinary researches ranging from molecular and cell biology to large-scale field surveys, numerical modeling, and remote sensing from space are conducted to study HABs. Due to their virtues of rapid analysis speed and high sensitivity, emerging molecular techniques have been more and more applied in both the qualitative and quantitative detection of toxins and toxic species. On the other hand, understanding the molecular mechanisms of cell proliferation and effects of environmental factors on HABs are important for predicting and managing HABs. In this study, phage display, enzyme-linked immune sorbent assays (ELISA) and quantitative real-time PCR are applied in 1) detection of microcystin 2) exploiting the importance of CaM in dinoflagellate growth and the interaction with the environmental stimuli such as heat stress.Phage display random peptide library was used to select peptides that can bind to microcystin-LR. After five rounds of affinity screening, the recovery of phage clones were increased from 2×10-5 to 3.7×10-2, indicating the effective enrichment of binding phage clones. 70 clones were randomly retrieved from the fourth and fifth round, and then their specificity was evaluated by ELISA. The results show that 63 clones of them can highly positively bind to microcystin-BSA. The genomic DNAs of randomly selected 10 positive clones were sequenced. There is no significant consensus among the amino acids of ten clones. However, a small motif rich in tryptophan (W) was observed in 9 clones, implying that the tryptophan could be the site binding with microcystin. Competitive ELISA was used to test the affinity and specificity of clones 4-8 and 5-2. The results show that the selected clones can bind with free MC, but the affinity is lower than traditional antibodies obtaining from immunizing animals and antibody fragments from phage display antibody library.The distribution of microcystin in the freshwater ecosystems of middle and northern Florida was also investigated in this study. The relation with other environmental factors, including dissolved inorganic nutrients, particle nitrogen (PN), particle phosphorus (PP), chlorophyll a (Chl-a), phycocyanin (PC) were evaluated by using correlation analysis. By implementing multi linear regression analysis, a model was developed to predict the concentration of microcystin from Chl-a, PC, PN and PP. 34.1 % of all the individual samples exceeded the World Health Organization drinking guidance value for microcystin, indicating its potential threat to sources of drinking water. At about 74% of investigated sites, Chl-a concentration was observed to be over the guideline value for algae blooms. In addition, relatively high concentration of PC was observed at most sites, with an average concentration of 370.5μg/L, illustrating the ubiquity of blue-green algae in Florida freshwater ecosystems. Results of correlation analysis show that concentration of microcystin is positively associated with Chl-a, PC,PP, and PN, while there is no significant relation between micocystin and dissolved inorganic nutrients. These results suggest that abundance of microalgae, especially blue-green algae, is the key factor controlling concentration of microcystin. The developed multilinear model can be used to estimate the concentration of microcystin from Chl-a, PC, PN and PP (r2=0.36, p<0.05).Calmodulin gene was isolated and characterized from the HAB species Alexandrium catenella. A full cDNA sequence of cam gene was obtained. It consists of 597 nucleotides (nt), comprising 25 nt of the 5′untranslated region (UTR), 122 nt of the 3′UTR, and an open reading frame (ORF) of 450 nt, encoding 149 amino acid residues. The deduced CaM was found to be highly conserved compared with CaM of other organisms. By implementing quantitative PCR, the relationship between transcription level of cam and growth rate of Alexandrium catenella was investigated. The results show that cam expression level has a similar trend with the cell growth rate through the whole growing stage. The cam transcription was increased more than 8 fold in abundance from lag phase to the exponential phase, and obviously decreased from exponential phase to stationary/decline phase. Additionally, the response of cam gene to heat stress was studied. Relative expression level of cam exhibited a significant decline trend with time during the heat shock experiment. All the results suggest that cam plays an important role in the cell growth of dinoflagellates and their response to environmental stress and still need further study.
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