Obtaining Of Full-length CDNA Sequence Of Three ABC Transporters In Prorocentrum Lima And Analysis Of Its Expression In Different Environmental Conditions

ATP-binding cassette?ABC?transporters are ubiquitous in all domains of life,which comprise eight subfamilies with prominent functions in secondary metabolism,transmembrane transport of heterologous molecules,metabolic detoxification of external components,and resistance to autotoxicity in plant.As a unicellular marine organism,microalgae are directly exposed to the aquatic environment containing harmful xenobiotics,it should have a similar protection or defense system against harmful or cytotoxic substances.Prorocentrum lima is a cosmopolitan epiphytic-benthic toxic dinoflagellate,which can produce phycotoxins such as okadaic acid?OA?,dinophysistoxin-1?DTX1?,DTX2,and their derivatives.Nevertheless,the biosynthetic and transport mechanism of OA remains elusive,despite their ecological significance.In this study,we identified ABCB1,ABCG2,and ABCC1 genes and characterized their expression patterns under different environmental conditions in order to gain molecular insights into the role of ABC transporters in P.lima.The full length of cDNA sequences of ABCB1,ABCG2,and ABCC1 genes were obtained by using local blast and 5'rapid amplification of cDNA ends?RACE?in P.lima.The full-length cDNA of ABCB1 was 3951 bp,containing an open reading frame?ORF?of length 3834 bp and encoding a transmembrane protein with 1277 amino acid residues.The full-length cDNA of ABCG2 gene was 2404 bp,with 1878 bp ORF and encoding a partial transmembrane protein with 625 amino acid residues.The full-length cDNA of ABCC1 was 4259 bp with 4098 bp of ORF,which encoded a transmembrane protein with 1365amino acid residues.The three ABC transporters all showed a high identity to related ABC proteins from other species and contained some classical features of ABC transport proteins.We found that both nitrogen and phosphorus-limitaiton could promote the prodution of OA in P.lima,and different nitrogen sources and phosphorus sources had different effects on the OA prodution.Cu²⁺in some concentration induced the production of OA,but tributyltin chloride?TBT?inhibited OA synthesis.In the present of artemia,the OA synthesis increased significantly,indicating possibility of OA as an anti-grazing chemicals.The expression of ABCB1,ABCG2 and ABCC1 was greatly regulated by some environmental factors.The expression of ABCB1 and ABCG2 in P.lima were up-regulated under nitrogen and phosphorus-limitaitons.When cultured in different sources of nitrogen and phosphorus,the P.lima cells exhibited different expression profiles of ABCB1,ABCG2 and ABCC1.The exposure of high concentration of Cu²⁺sginificantly up-regulated ABCB1,ABCG2 and ABCC1 in P.lima,suggesting the potential role of ABC transporters in dinoflagellate against metal ion in surrounding waters.Collectively,our results demonstrated intriguing data about the biosynthesis of OA and expression pattern of various ABC transporter genes.However,we could not find any significant correlation between OA production and expression of the three ABC transporters in P.lima,implying the complexity of OA transport in P.lima.