Screening Of Environmental Factors Responsive Genes From Alexandrium Catenella And The Isolation And Identification Of Nanoplanktonic Algae From The North Yellow Sea

(Ⅰ)Abstract:With the development of worldwide industrialization and environmental deterioration,red tides occurred frequently.Some research shows that water eutrophication and trace elements is the factors of red tides organisms burst growing. At present,there is no relative research on environment factors which connect with molecular biology,reveals its burst growing mechanism from molecular biology. Generally,it is considered that the higher concentration of P and Mn would promote growth of Alexandruim catenella,We contacted the favorable conditions of red tides with molecular techniques,constructed suppressive subtractive hybridization(SSH) library of high phosphor cultivating condition of Alexandrium catenella The expression of the clones from the library were further demonstrated by the cells cultivated under the medium with manganese additive.The expression of the clones from the library were further demonstrated by the cells cultivated under the medium with manganese additive.The result will contribute to the obtaining of burst-growth related genes of HAB species and their applications,it reduced the false positive of the response of single factor.It established the foundation of red tides burstgrowing as well as provided data for its prediction and forecast.Three hundred and thirty-eight clones were obtained from the SSH library, involving 348 positive clones with the posotive ratio of 90.9%.The PCR products from 348 clones were purified,and then screened by two round of dot hybridization with the first round done by the probes produced from the cDNA of cells cultivated under low and high phosphor condition respectively.The clones showing a signal ratio higher than 1.4 between these two kind of hybridization were selected and undergone a second round of dot hybridization which operated on the probes produced from the cDNA of cells cultivated under with and without manganese additive condition respectively.Twelve clones were obtained with a signal ration higher than 1.4 between these two kind of hybridization and sequenced.Six gene fragmnent were identified with four of them showing the predicted function of growth, the initial of translation and energy delivering,respectively.Other two had unknown function and the other one was weakly similar to transforming protein.Then analysis the expression of different genes using Real Time PCR,the expression of eIF-4A and SSH298 is similar to Northern blot.It was suggested that the burst growth of this HAB species may relate to the influence or promoting of the initial of translation,thus initiating or accelerate the fast growth and division of the cells.(II)Nanoplankton is the most important primary producer in the marine environment, Identification and classification are the basic research as well as one of the difficult subject of alga.Distribution density of nanoplankton is high in the waters,also a main component with marine plankton.There is little research aimed at taxonomy of nanoplankton,so without unified catalogue of taxonomy,it including many un-identification species,it is different to identification,and the research progress shows slowly.In order to enrich the recorded vatiety of nanoplanktonic algal as well for the demand of scientific research.A nanoplanktonic species was observed during water sample analysis,described as a unknown species after SEM analysis,belonging to Prorocentrum Ehrenberg.The alga was round to ellipse,with the cell length of 15-23μm,width of 8-15μm.The cell surface was rich with granule knobs,about 7-10 each squareμm.A metacytic zone was in the middle of the cell with width of 0.60-1.50μm, became narrow and ultimately a slim slot when elongated to the other side.The same kind of knobs also existed within the metacytic zone.There were shallow depressions beside metacytic zone from the valve view,and 2-3 assistant holes around surface trichocyst pore.Compared with Prorocentrum donghaiense Lu and Prorocentrum minimum,we found that they had some similar structures,but they have some obvious differences.Deficiency of dinoflagellate taxonomy,especially the disadvantage of nanoplanktonic algae taxonomy,on the other hand,we didn't cultured the nanoplanktonic algae after collecting of water samples,so we can't do further research using molecular techniques,we considered that this nanoplanktonic algae as a unknown species Prorocentrum Ehrenberg.We also cultured a no fixation samples in F/2 medium by filtration,then obtained a new purification nanoplanktonic algae using limiting dilution.It is similar to Minutocellus polymorphus by the observation under SEM and TEM,but it still has some differences in morphology,So we designed 18SrDNA primers from NCBI and amplified its 18Sr DNA,after sequencing we found that it has high similarity sequence to Minutocellus polymorphus.Due to the highly conservation of 18sr DNA, it just classed the species in genus,the interspecific classification need the 5.8srDNA and ITS sequences,but there is no relative reports on it,we cannot do further analysis. Combining with cell morphology with molecular biology,we considered that it maybe a newly recorded diatoms belongs to Minutocellus Halse,can also a deformation about Minutocellus Halse,we should do further research to denominating.