Aptasensor Detection And Genes Related To Biosynthesis Of Marine Polyether Toxin

Polyether marine toxins,a class compound with complex structure, strong toxicity, good stability, widely distributed in the world. Toxin often produced by toxic dinoflagellates, and enriched through the food chain in shellfish, fish and other seafood, which seriously threat to human health and marine fisheries. Toxin detection methods currently have a variety of disadvantages, so there is an immediate need to develop easy-to-use, rapid detection methods due to the lack of validated protocols for their detection and quantification. In addition, due to the natural marine polyether toxins difficult to extract, and dinoflagellate havelarge and complex genomes, which led polyether toxin biosynthesis poorly understood.This work is focused on biosynthesis and detection of the classic polyether marine toxins, Okadaic acid, Brevetoxin and Palytoxin. The main contents and results are as follows:1. Screening and application of aptamer targeted to BTX. In this study, high affinity ss DNA aptamers binding to BTX were screened by SELEX. On this basis, A5 and B2 aptamer sequences were optimized and mutated. Aptamers secondary structure was analyzed used mfold software in order to remove the sequence of the primer region and non-nucleotide. Finally, we obtained a core sequence of A5-S3G(Kd=72) n M contains only 32 nucleotides with good specificity. Aptamer sensor was prepared by biotin-streptavidin coupling based on SSA chip and BLI technology. We have established a method for detection BTX by BLI aptamer sensors with optimal detection time of 250 s and detection range of 100~2000 n M. The lowest limit of detection was 4.5 n M(4 ng / ml), with lower cross-reactivity and coefficient of variation, which can be used for laboratory testing of BTX-A.2. Screening and optimization of aptamer targeted to PLTX. In this study, high affinity ss DNA aptamers binding to PLTX were screened by SELEX. On this basis, P-5, P-13 and P-18 aptamer sequences were optimized. Aptamers secondary structure were analyzed used mfold software in order to remove the sequence of the primer region an non-nucleotide. Finally, we obtained a core sequence of P-13S2(Kd=0.56 n M)and P-18S2(Kd=0.93 n M). PLTX sensor was prepared by amino-carboxyl coupling based on CM5 chip and SPR technology, which provided the recognition element and technology platform for the preparation of biosensor.3. Transcriptome analysis of Prorocentrum lima to reveal genes related tobiosynthesis of Okadaic Acid. We cultured Prorocentrum lima with different nitrogen concentrations, then sequenced the transcriptome using Illumina Hiseq2000 platform. We used Trinity to de novo assemble the reads so as to obtain transcripts,aligned all the transcripts with Nr database,Uni Prot KB/Swiss-Prot database and COG database to annotate the function and classify using BLASTx algorithm, and assigned the transcript with metabolic pathway by aligning with KEGG database. Then we used RSEM to calculate FPKM value, and used it for preliminary analysis of different gene expression in the related pathways. We used Trinity to assemble these reads into 98594 Unigene with an N50 of 1856 nt and average length of 1319 nt. 73289 Unigene were annotated through BLASTx similarity search, and 333 KEGG pathways were assigned in total. Based on these analyses,we also identified differentially expressed genes of PKS, thioesterase, flavin monooxygenase, epoxidases, cytochrome P450 and epoxidehydrolases under different conditions.In conclusion, high-affinity ss DNA aptamers binding to BTX/PLTX were be screened respectively by SELEX. As a recognition element, it was used to combine bio-layer interferometry, surface plasmon resonance and label-free aptasensor to establish a series of novel, sensitive and rapid detection method. Meanwhile, in the present study, high-throughput de novo transcriptomic sequencing was performed in Prorocentrum lima, which provided the first view of the gene repertoire in this dinoflagellates and we compared the gene expression level under different conditions. This study is important for the understanding of Okadaic acid biosynthesis mechanisms, production and evolution, and also great application prospects for synthesis of Okadaic acid in the future.