Molecular Identification Of HAB Species And Detecting Them Using Fluorescence In Situ Hybridization Technique

During the last two decades, serious economic and public health impacts caused by Harmful Algal Blooms (HABs)have increased in intensity, frequency and geographic distribution. It had great effect on national economy, public health, and oceanic ecosystem as well as. Thus, HABs is receiving increased international attention。 Studying HAB events , then predict and govern HAB, The basic biological concern of HAB was identify and distinguish its species. The speedy progress of molecular techniques offers a new way to identify phytoplankton. At present More and more studies using LSU/SSU rRNA gene or internal transcribed spacers(ITS) as molecular criteria to study dinoflagellate phylogeny have been proved to be powerful tools. PCR amplification, cloning and sequencing the ITS and 5.8S rDNA or LSU rDNA from some strains of dinoflagellates and diatoms. Blast searches of these sequences had been done on NCBI(the website: http:// www.ncbi.nlm.nih.gov). Softwares such as Mega 2.0, DNAStar,BioEdit and Phlip3.5 were used to compare the three sequences studied in this paper with other ITS sequences of diatoms retrieved from GenBank. The Phylogenetic tree was constructed with NJ method. The aims of this paper were hoping that verify their phylogeneny by molecular data , and more nucleotide sequences obtained could be offer more choice for designing species-specific probes as well as. The results were as follows: 1.Studing 2 strains of dinoflagellate and 3 strains of diatoms according to ITS and 5.8S rDNA sequences (1) The ITS length of P.micans APBM was 631 base pairs, and P. donghaiense's ITS sequence was 552 bp. In the ITS sequence, P.donghaiense has very high level of similarity with P.minimum (showing 88% identity), but low level similarity with other species of P.triestinum and P.micans (about 60%-70%), while P.micans APBM show the lowest level similarity with other P.micans according to ITS sequence, it was about 30% only. The results from phylogenetic tree of ITS2 sequence was the same as that showed by phylogenetic tree of ITS and 5.8S sequence. The results from phylogenetic tree constructed with ITS and 5.8S sequence accorded with that of sequence similarity analysis. The results of the phylogenetic tree constructed with 5.8S sequence were inaccurate; therefore, the use of 5.8S rDNA as a classification standard between species is not reasonable. (2) The ITS sequence of P. pungens was 693 bp, and the ITS sequence of SK-1 was 715 bp, while the ITS sequence of SK-2 was 331bp. Suquence similarity and phylogenetic tree showed that biogeographic groups of P.pungens have little difference in the ITS sequence, showing 100% identity, and classifications of SK-2 and SK-1 based on morphological features maybe incongruous. SK-2 seems to belong to species of S.pseudocostatum. (3) To overcome the shortage of fresh materials demanded by molecular biological experiments, and to get complete target sequence when sequencing as well as, an improved cloning method was applied. This cloning method is simple, feasible and efficient able to meet the need for regular cloning in lab. This study can provide target DNA on call. 2.Obtaining more nucleotide sequences could be offer more choice for designing species-specific probes (1)Complete LSU rDNA sequence of two strains of dinoflagellate were obtained by PCR amplifying three DNA fragments. The complete LSU rDNA sequence of P.micans was 3332 bp,and that of P.donghaiense was 3376 bp. The D1-D2 region of P.micans was 659 bp while the same region of P.donghaiense was713 bp. The D9-D10 region of these species were both 330bp。 (2)analyzing the sequences of complete LSU rDNA, D1-D2 and D9-D10 regions showed that the D1-D2 of P.micans was hypervariable between this strain of P.micans and other species of dinoflagellate, but the same region of p.donghaiense was relatively conserve compared with that of other species of dinoflagellate, especially with P.minimun. The D1-D2 region and D9-D10 region betweenp.donghaiense and P.minimun all have above 96% identity, so was the complete LSU rDNA sequ...