Microbial Diversity In The Rhizosphere Soil Of Mangrove Kandelia Candel And The Mechanism Of Anti-marine Biofouling Mechanism Of 3,3'-diindolylmethane

This thesis mainly includes three points: first,study on the microbial diversity and novelty of rhizosphere soil of a mangrove plant Kandelia candel in Mai Po Nature Reserve,Hong Kong,and the classification and identification of a new strain J95T;second,study on antifouling action of 3,3'-diindolylmethane(DIM)against the fouling alga Nitzschia closterium;third,study on the transcriptomics profile of bryozoan Bugula neritina larvae with the treatment of DIM.1.Study on the microbial diversity a nd novelty of rhizosphere soil of a mangrove plant Kandelia candel in Mai Po Nature Reserve,Hong Kong,and the classification and identification of a new strain J95TFocusing on the study of the microbial diversity of the rhizosphere soil of Kandelia candel in Mai Po Nature Reserve,Hong Kong,separation,purification and identification(based on 16 S r DNA sequences)of microorganisms was applied Totally 103 bacterial strains belonging to 27 genus and 3 suspected novel bacteria.In this part,the novel strain J95 T would be identified using the method of polyphasictaxonomy.The above demonstrates the diversity and novelty of culturable microbes from the rhizosphere soil of mangrove plants in Mai Po Nature Reserve,Hong Kong,and also the potential bioavailability of these microorganisms.In this part,we describe the exact taxonomic position of strain J95 T using a polyphasic approach.A Gram-negative,rod-shaped and motile bacterium,designated strain J95 T,was isolated from the rhizosphere soil of a mangrove plant Kandelia candel(L.)Druce in Mai Po Nature Reserve,Hong Kong.Phylogenetic analysis based on 16 S r RNA gene sequences indicated that strain J95 T belongs to the genus Ruegeria with highest sequence similarity(96.8 %)to the type strain Ruegeria marina ZH17 T.The genomic contained a circular chromosome of 5.48 Mb with a DNA G+C content of 65.7 mol%.The genome included 5,299 genes.Growth of strain J95 T was observed at p H 5.0–8.5(optimum,6.0–7.0),between 10–40 ?(30–37?)and in the presence of 0–9%(w/v)Na Cl(0.5–3%).Chemotaxonomic analysis showed ubiquinone-10 as the predominant respiratory quinone and C18 : 1?7c and C19 : 0 cyclo?8c as the major fatty acids.The major polar lipids were lipid,aminolipid,phospholipid,phosphatidylcholine,phosphatidylglycerol and phosphatidylethanolamine.The combined phenotypic,chemotaxonomic and phylogenetic data suggested that strain J95 T represents a novel species of the genus Ruegeria,for which the name Ruegeria kandeliae sp.nov.is proposed.The type strain is J95T(=MCCC 1K03284T=DSM 104293T).2.Study on antifouling action of 3,3'-diindolylmethane(DIM)against the fouling alga Nitzschia closteriumThe compound 3,3'-diindolylmethane(DIM)showec excellent antifouling activity against the fouling alga Nitzschia closterium.With the EC50 and EC90 values(after 96 h)of 0.3713 and the 1.00 ?g/m L,respectively.Observe that the NOEC value is 0.03 ?g/m L.Based on non-invasive micro-test-NMT,atomic force microscopy-AFM and X-ray photoelectron spectroscopy-XPS,the preliminary investigation of antifouling action of DIM against the fouling alga Nitzschia crassifolia was carried out.In situ measurement of hydrogen ion flux on the cell surface using NMT technique revealed that the hydrogen ion influx velocity of cells treatmented with a certain concentration of DIM decreased or even effluxed relative to the control group,which may be due to the blance of ionic absorption of cell wall.Excess cations cause the cell wall to require efflux of H+ to reach equilibrium,and also may be related to the accumulation of cations in the cell vacuole and the accumulation of internally synthesized organic acid anions.DIM causes Ca2+ efflux at high concentration,the ability of cell wall to adsorb Ca2+ is weakened and a certain amount of Ca2+ is absent in the cell,which may cause cell wall synthesis to be blocked.Through the roughness measurement of AFM,it was found that DIM could reduce the integrity of the cell wall of N.crassifolia by increasing the roughness of its cell wall,which would lead to the decrease for the type and quantity of trace metals-adsorption groups on the surface.Meanwhile,Young's modulus measurements showed that DIM affected the adsorption capacity of the surface of cell wall by weakening its mechanical strength.The results of XPS suggested that DIM changed the type and quantity of organic ligand groups dominant on the cell wall,which provided data support for the difference in adsorption capacity of different metal ions on different cell surface groups.The anti-fouling compound DIM was used to study the anti-fouling mechanism of fouling Buluga neritina larvae.The B.neritina larvae were treated with DIM low concentration group(2 ?g/m L)and DIM high concentration group(4 ?g/m L)for 3 hours,and bryozoa treated with RNA-Seq technique for the control group,DIM low concentration group and high concentration group.The larvae were sequenced and transcriptome techniques were used to analyze how different co ncentrations of DIM affect the attachment and metamorphosis of B.neritina larvae.The results showed that 686,759,362 original sequences were obtained from 13 groups of 3 samples.After screening,670,566,046 high-quality sequences were obtained.After splicing,37,159 unigene sequences were obtained,with a total size of 43,932,439 bp,the shortest length was 301 bp,and the maximum length was 27,362 bp.The average length is 1,182 bp.Gene function annotations were performed in seven databases,and a total of 19,893(53.53%)of the 37,159 genes were annotated.Gene differential expression analysis showed that compared with the control group,888 genes were up-regulated and 855 genes were down-regulated in the DIM low-concentration group;2,148 genes were up-regulated and 1,876 genes were down-regulated in the DIM high-concentration group;DIM high concentration Compared with the low concentration group of DIM,990 genes were up-regulated and 591 genes were down-regulated.The above results showed that DIM exerts its anti-fouling function by up-regulating the fatty acid metabolic genes,stress indicators and protein degradation genes,protein translation machinery genes and apoptotic genes,down-regulation and expression of genes related to developmental pathways and collagen.