[PKS gene screening based on metagenome of Halichondria rugosa]

Metagenome DNA was extracted from Halichondria rugosa which was collected from South China Sea and kept in -4 degrees C. PKS gene fragment was amplified using PCR with KS domain primers in PKS gene. A DNA fragment about 671bp in length was obtained by PCR. The PCR product was measured by agrose gel electrophoresis. Then the product was recovered from gel and cloned into pUCm-T vector. After that vectors were transformed into competent cells (DH5alpha). PKS gene fragment in positive clones was sequenced. Consequently, the corresponding amino acid sequence was deduced based on nucleotide sequence. BLAST analysis showed that the homology of this amino acid sequence with that deduced from KS domain of PKS gene in Rhodobacterales bacterium was up to 96%. Phylogenetic analysis indicated that the obtained PKS gene belongs to trans-AT KS domains. Meanwhile the result demonstrated the diversity and differences of microorganisms associated with and around sponge in different sea area. It is the first time to find bacterial PKS gene in sponge Halichondria rugosa, which provide powerful proof to the microbial origin hypothesis of sponge active compounds. At the same time, this study lay basis for the utilization of uncultured microorganisms associated with sponge from the aspect of genes.