Cloning And Expression Analysis Of PKS Gene In Prorocentrum Lima

Objective:To provied information for the HABs toxin synthesis mechanism and explore the role of polyketide synthase in the synthesis of diarrheic shellfish poisoning(DSP) toxins,potential polyketide synthase(PKS) genes in Prorocentrum lima was amplified,the homology analysis of the PKS gene in related species was conducted and phylogenetic tree was constructed.In addition,the potential relation between the production of DSP and the expression of PKS gene in P.lima was analysed.Methods:Potential polyketide synthase(PKS) gene in Prorocentrum lima was amplified by PCR using degenerate primers.The homology analysis of the PKS gene in related species was conducted and phylogenetic tree was constructed using software of Dnastar and DNAMAN.The expression of PKS in P.lima was determined using reverse transcriptase polymerase chain reaction(RT-PCR).Multiple approaches including amplification of polyadenylate RNA,resistance to methylation-sensitive restriction enzymes,Southern blotting,and isolation of bacteria from cultures were performed to eliminate the possibility of PKS in associate bacteria. The productions of DSP and expressions of PKS gene in different growth phases and under different nutrient conditions in P.lima were observed by ELISA and RT-PCR, respectively.Results:Results showed that type I polyketide synthase may be present in Prorocentrum lima,which grouped strongly with P.mican.High level expression of PKS in P.lima were observed.18S rRNA and PKS gene were amplified successful by RT-PCR using Oligo(T).The analysis of 16S rRNA sequece showed 99% homology between bacteria from P.lima culture and marine actinomycete Pseudonocardia.However,PKS gene was not obtained by PCR and no expression of PKS was detected by RT-PCR in the bacteria.These results suggested that the PKS gene was P.lima originated but not bacteria,the sequence of PKS has been accepted by GenBank(accession NO:EF521601).The production of DSP and expression of PKS gene varied in different growth phases and under different nutrient conditions in P.lima.Significant positive correlations were observed between the production of DSP and expression of PKS gene in different growth phases and under different nutrient conditions with R~2 of 0.9807 and 0.9831,respectively,suggesting that PKS may play an important role in production of DSP toxins.Conclusions:PKS gene was amplified successfully from P.lima.The expression of PKS was significant positively related with the production of DSP,suggeting that PKS gene may play an important role in production of DSP toxins.