Idetification Of The Biosynthesis Gene Cluster Of Deacetylmycoepoxydiene(DAM) By RNA Interference

The hot spot for searching novel drugs has been focused on mangrove plants and the endophytic fungi of mangrove plants for the decrease of medicinal plant resources and land resources.The strain Phomopsis sp. A123is one of the endophytic fungus isolated from the leaves of mangrove plant Kandelia candel previously, which produces deacetyl mycoepoxydiene (DAM), a natural product that contains a rare oxygen bridge ring skeleton, with strong antitumor activity and the AMPK activation activity. A mutant strain (G818) of the fungus that produces a high yield of deacylmycoepoxydiene was obtain through many runs of protoplast mutagenesis and genome shuffling. A PKS fragment was obtained from the Suppression subtractive hybridization (SSH) library based on the transcription differences between DAM high-producing strain G818and low-producing strain A123and identified a50Kb cluster containing two PKS genes (PKS1and PKS2) and other aligned genes.It need more functional evidences to identify whether the hypothetical gene cluster be associated with the DAM biosynthesis. But that two PKS cluster (PKSl and PKS2) in Phomopsis sp. G818may be associated with the DAM biosynthesis.Gene knockout and heterologous expression of this gene cluster have been unsuccessful due to their multinuclei in Phomopsis sp. cells and the large cluster size. Herein, the present study adopted RNA interference, a gene knockdown strategy to construct the PKSl and PKS2gene scilence plasmid and obatin the PKS1and PKS2gene scilence mutants. The results showed that the6PKS1or PKS2gene scilence mutants (1-4,1-8,1-9and2-2,2-11,2-13) losing the ability to produce DAM and their mRNA level of pksl and pks2significantly decreased in all mutants, ranging from approximately20%to100%of the control in reverse transcription PCR (RT-PCR) and Quantitative real-time PCR (qRT-PCR). This means that two PKS genes (PKS1and PKS2) are associated with the DAM biosynthesis. Meanwhile the DAM high-yield strain Phomopsis sp. G818and transformed an empty silencing plasmid strain have the high mRNA level of pksl and pks2gene expression and DAM biosynthesis ability. These results indicated that the pksl and pks2gene as well as the linkage gene beside them are responsible for DAM biosynthesis.