Cloning And Gene Expression Analysis Of Phosphate-stress Gene And Cell-lysis Gene Of Prorocentrum Donghaiense

Prorocentrum donghaiense, one of dinoflagellates, has caused most frequent large-scale red tides at Changjiang River Estuary and Zhoushan Mountain Archipelago in recent years, bringing us tremendous economic losses and local environments serious impacts. Currently, there are much more knowledge concerning P. donghaiense's life history, ecology and taxonomy. However, we know little about how the bloom forming and declineA cDNA library was constructed using P. donghaiense at exponential growth phase. Poly(A)+ RNA was extracted and purified using mRNA Purification Kit MagExtractor? - mRNA- and cDNAs were synthesized and amplified by Super SMART? PCR cDNA Synthesis Kit. Finally, the ligated cDNAs with pMD 18-T vector were transformed into E. coli JM109. Results showed that transform efficiency high above 109 and almost all the insert cDNA were between 500bp and 1500bp. Using this method, it is possible to constructed a cDNA library from micro-quantities of P. donghaiense (ca. <105 cells).The cDNA-amplified fragment length polymorphism (cDNA-AFLP) profiling technique was used to analysis early gene expression associated with inorganic phosphate-stress of dinoflagellate P. donghaiense. And the resulting difference cDNA-AFLP fragments were sequenced. Homology searches revealed that 8 of 43 transcript-derived fragments (TDFs) induced by phosphate stress were significantly homologous to genes in GenBank. We subsequently analyzed 3 of those TDFs and the results indicated that all of them are up-regulated when subjected to phosphate-stress, and the differences are significant compare to the control: of TDF72 (antioxidant, AhpC/Tsa family), TDF68(dual-specificity protein phosphatase) and TDF85(Phosphoglycerate/bisphosphoglycerate mutase), it is 1.24(p=0.04), 1.60(p=0.002) and 2.30(p=0.0001) folds higher than that of the control respectively.Usually, phytoplankton are considered immortal unless the cells are eaten by heterotrophic zooplankton or infected by viruses, or sink irreversibly into the deep ocean. However over the past decade, it has become clear that these organisms can undergo an autogenic control process in response to environmental stress, which shows similarity with programmed cell death (PCD) as commonly known in multicellular organisms. We have observed cell-lysis of senescence P. donghaiense and we have retrieved partial capsase encoding gene sequences in aging P. donghaiense EST analysis. In this study, we cloned the full-length cDNA sequence of caspase encoding gene of P. donghaiense which was 520bp in length, containing a 258bp open reading frame encoding 85 amino acids and two stretches of 135 and 127bp in length for the 5′and 3′untranslated regions, respectively. We also detected the expression level of caspase encoding gene usingβ-actin as a reference during culture senescence and found that, an increase at beginning and then a decrease of expression level was observed. On Day 6, the value reached the maximum, 0.689 (19 folds higher than that of Day 0). The transcripts of both genes were not detectable on Day 9. Our results suggested that caspase may play a role in the molecular mechanism of self-lysis in aging P. donghaiense. This may provide us some clues of how the bloom of Prorocentrum donghaiense crashed.