| The artificial culture of turbot(Scophthalmus maximus)is one of the most important pillar industries in China’s mariculture.However,with the rapid development of the scale of turbot culture,various diseases followed,suticociliatosis caused by scuticociliates is one of the most serious diseases.In order to prevent and control suticociliatosis,the development of new biological insecticides has a very broad application prospect.Marine microorganisms can produce metabolites with unique structure and diversity of biological activity,they are different from terrestrial sources and some of them can be used as insecticidal agents.In this study,a bacterium with the effect of killing ciliates was screened from offshore microbial resources bank,and its insecticidal ability and genetic background were studied.The main research results are as follows:1.Eleven turbots infected with ciliates were analyzed by pathological observation.The results of histological sections showed that the skin,fin,gills,liver,intestine and brain of diseased fishes were all infected with ciliates.We isolated a ciliate from the brain tissue of an infected turbot and identified it as Uronema marinum based on morphological and molecular evidences.The infection experiments confirmed that U.marinum can infect turbots and cause suticociliatosis.2.We identified YCSC6 as Salinivibrio costicola using morphological and molecular biological methods.We conducted co-culture pharmacodynamics experiments in which YCSC6 killed pathogenic scuticociliates.We characterized the insecticidal properties of YCSC6 by optical and scanning electron microscopy.The results showed that the fermentation supernatant of YCSC6 could lead to membrane perforation in scuticociliates,after which the scuticociliates losed their membrane integrity and cracked.After a certain period of time,the cracking ability strengthened with time.3.In order to further study the insecticidal mechanism and secondary metabolites biosynthetic gene clusters,The genome was sequenced using the Illumina Hiseq platform,and then analyzed using relevant software for genome assembly,gene prediction and functional annotation and total secondary metabolite biosynthesis gene clusters prediction,it laid the foundation for the analysis of late transcriptional and metabolic groups.The best genome assembly results were 3.87 Mb with 3896 genes.It includes two circular chromosomes,the long sequence(scf7180000000004)is the genome sequence with an average GC content of 50.03%,the short one(scf7180000000005)is a plasmid with an average GC content of 48.80%.According to genome sequences analysis,21 secondary metabolite biosynthetic gene clusters were predicted.This Whole Genome Shotgun project has been deposited at GenBank under the accession NHNL00000000.The version described in this paper is version NHNL01000000.4.On the basis of the whole genome of YCSC6,we analyzed the YCSC6 transcriptional and metabolic groups of marine bacteria at different incubation times.By comparing transcriptional and metabolic groups between different groups,we predicted a large number of differentially expressed genes and metabolites.The number of substances identified in the metabonomics was 1129,and the number of substances that could not be identified was 16814.After comparison,84 kinds of significant up regulated metabolites were screened.Later,we will verify the insecticidal and membrane cracking activities of these substances by experiments.Sample expression pattern analysis shows that,In the transcriptional group and the metabolic group,a group of module expression patterns coincide with the effect pattern of the target product,and all of them are highly expressed after 48 h,and the target product may exist in it.In the next job,we will focus on the analysis of differential genes and differential metabolites to find the insecticidal gene and its metabolic pathway,it can be used to develop a safe and efficient insecticide for killing ciliates. |
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