Identification Of Two Marine Alexandrium Sp.and Cloning And Analysis Of PCNA Gene Of Alexandrium Catenella

Marine Alexandrium is an important Pyrrophyta species, it have been distributed in the coast of North America, Europe, and Asian extensively. Half amount species of genus Alexandrium can produce paralytic shellfish poisoning. Red tides produced by toxic Alexandrium had caused worldwide aquaculture loss and human poisoning. Thereby, great request is proposed on the growth and identification of Alexandrium.Alexandrium is single cell algae, so morphological character can effect greatly by environment change and different physical phase, some genus have just tiny difference in deck shape.The identification always need special dyeing and deck anatomization,even use electron microscope.It always can't be identified that some genus have similar shape.For example , there are dispute and uncertainness in classify between A. tamarense, A. catenella, A. fundyense.Utilizing molecular biology method can get accurate and reliable results, the most widely application is classify with ribosome 5.8SrDNA and internal transcribed spacers (ITS) sequence . ITS region is credibility dependence in genus classification,and ITS sequence length is suit for primer design and PCR amplification.The method have been in common use of algae identification by biology.Two isolated strains from Chinese qingdao waters are named Alexandrium sp. qd2 andAlexandrium sp. qd3 by morphological describing. The 5.8S ribosomal gene and internal transcribed spacer 1 and spacer 2 (ITS1 and ITS2) from two strain are amplified. By sequenced and phylogenetic analysis ,Alexandrium sp. qd2 and Alexandrium sp. qd3 approach A. catenella which come from South Korea and Japan. So,Alexandrium sp. qd2 and Alexandrium sp. qd3 were identified as A. catenella,which is broadly distributed in Chinese waters, and has different geographical and genotype strains.Red tide is produced by eruptive multiplication of red tide organisms, so it's significant to study in functional gene about red tide organisms multiplication. Proliferation cell nuclear antigen(PCNA)is discovered and named in patient who illed with serum SLE(Systemic Lupus erythematosus)by Miyachi et al.(1978).It is detected that PCNA has great relation with cell DNA synthesize.PCNA is crucial in driving of cell multiplication,and it has been credible index which reflect the cell proliferation. PCNA still concerned with a great deal of important cell event and operated in function switch factor of many protein,thus,researcher have focus on the biologic fuction of PCNA.In our experiment, partial cDNA sequences of A. catenella qd1 and A. catenella qd2 PCNA are obtained, two sequence have 41 bp difference, and there is no different in the amino acid. The gene in A. catenella qd1 possesses 91% and 89% homology to Pyrocystis lunula(Pyrrophyta) and Pfiesteria piscicida(Pyrrophyta) respectively, and shares lower homology to Dunaliella tertiolecta (51%), Aspergillus clavatus (50%),Skeletonema costatum (50%),similar to Pleurochrysis carterae(48%) and Isochrysis galbana(48%), has a lower homology(42%) to plant and animal, too. In general, PCNA gene is highly conserved in Eucaryotes ,it is showed that Pyrrophyta takes a extraordinary position between Eucaryotes and Prokaryota. Accordingly, PCNA gene in Pyrrophyta deserves to study further.The result of Southern blotting showed that PCNA gene in A. catenella qd1 has single copy. Use of Real-time PCR to disscussed the expressing of PCNA gene in various growth phase, the results indicated the expressing of PCNA in either various growth phase or exponential phase were significant different . The expressing of PCNA gene in exponential phase and stationary phase had a positively related to algae cell growth.