Metagenomic Study Of Symbiontic Microbes Of Marine Sponge From Southern China

Marine sponges， usually attached to the seabed or reef， are among the oldestmetazoans. Their bodies changed when environmental conditions are altered. Usingonly morphological features to classify marine sponges is controversial. Spongespicules are useful feature in traditional classification. It is easier to classify the kindsof marine sponge with their spicules unlikeness. However， some times spicules aresimilar in shape but different in spicule curve and length. In this case， theclassification of marine sponges becomes unreliable just only depending on the spiculeshapes. Application of the barcoding technology (DNA barcoding)， the classificationof marine sponges is beco分钟g more reliable.The16S-rRNA genes are cloned through polymerase chain reaction (PCR) viaprimer27F and1492R. The symbiotic microbial libraries of six different marinesponges are constructed with16S-rDNA clone library technology via plasmidPMD-19T. The name of the six sponges clone library is as follow: PBS2， PBS4，PBS5， PBS7， PBS8and PBS10. And analyzed clones are240，203，288，288，288and288respectively. After constructing the clone library， the inserts whichcontain the target16S rRNA gene fragment by universal primer of plasmid (M13F andRV-47) is amplified to produce enough copies. Then the restriction enzyme Mbo I andBstU I are used to digest the fragments and different operational taxonomic units (OTU)are identified. The OTUs of PBS2and PBS4are29and21. The representative clonesof different OTUs are sequenced and the types of bacteria are identified by comparingthe sequences with NCBI and RDP database. PBS2library comprises five categories ofbacteria: Actinobacteria (23clones，9.58%)， Bacteroidetes (23clones，5.42%)， Firmicutes (1clones，0.42%)，Proteobacteria (62clones，25.83%)，Cyanobacteria(137clones，57.08%)， and unclassified bacteria (4clones，1.67%). PBS4librarycontain203clones， and bacteria categories are: Actinobacteria (3clones，1.48%)Bacteroidetes (3clones，1.48%)， Proteobacteria (164clones，80.79%)，Cyanobacteria (9clone，4.43%)， unclassified bacteria(24clones，11.82%).Metagenomic approach based on next generation sequence and16S-rDNA clonedlibrary have been applied in a marine sponge sample analyses. The16S-rDNA libraryof sponge Jing1was analysed and244clones were classified into fifteen types OTU.Actinobacteria(1clones，0.41%)， Planctomycetes(1clones，0.41%)，Proteobacteria(16clones，6.56%)， Cyanobacteria(222clones，91.39%)， andunclassified bacteria(3clones，1.23%) were composed of the categories of bacteria ofjing1. The percentage of bacteria Cyanobacteria covers up to91.39%whilePlanctomycetes and Actinobacteria only occupy0.41%. Proteobacteria bacteria is6.56%， and unclassified bacteria is1.23%.From the sponge sample of Jing1about20GB data were collected through Illu分钟asequencer，GAⅡx. After using software to assemble，about346，749contigs，73.52MB non-redundant data were acquired. The average fragment length is212bp whilethe longest fragment is27.73kb， the shortest is100bp. Among the data，65.09%ofcontigs were distribute in the length100-149bp，14.69%of the contigs in150-199bp，over200bp length contigs is20.22%. Those contigs accord to RDP16S classifieranalysis， a total of176contigs belong to Archaea， accounting for0.05%of allcontigs;227，021contigs belong to Bacteria，accounting for65.47%; the contigs withno clear classification are about119，553， accounting for34.48%. The176contigsbelonging to Archaea， there are32contigs are Crenarchaeota; the remaining has noclear classification.227021bacteria contigs，15contigs belong to Actinobacteria，1fall within the category Bacteroidetes，937fall within the category Firmicutes，5fallwithin the category Planctomycetes，10belong to Proteobacteria，9fall in thecategory Cyanobacteria.