Phaeodactvlum Tricornutum MiRNA Identification, Regulatory Mechanism And Metabolic Flux Analysis

Diatoms are important planktons that are believed to produce around one-fifth ofthe primary productivity on earth, and are vital for cycling of minerals such as silica.Diatoms has an unusual evolutionary position of secondary endosymbiotic origin,thus are good species for research on evolutionary. Phaeodactylum tricornutum is anatypical diatom with three convertible morphotypes （i.e. oval, fusiform and triradiate）,and silicification essentially restricted to one valve of the oval cells. With itscharacteristics of short life-cycle, small genome size and ease of transformation, P.tricornutum has become an attractive photosynthetic model. EST analysis indicatedthat some of its genes were more similar to those of animals than of photosyntheticcounterparts. P. tricornutum has a mosaic genome that contains ‘animal-like’,‘plant-like’ and ‘bacteria-like’ genes. P. tricornutum has a complete urea cycle, and itsresponse to nitrogen starvation is different from that of green algae and higher plants.In this study we do the following research on P. tricornutum.（1） Using a homology search and RNA structure analysis,13,3and7predictedmicroRNA （miRNA） genes were found in genomes from P. tricornutum, T.pseudonana and C. merolae, respectively. Of the23miRNA genes,18had sequencesimilarity with animal miRNAs, implying that those unicellular algae may adopt amiRNA regulation pattern similar to that of animals’. A phylogenetic tree based oncommon miRNA families shared by these three unicellular algae, higher plants andanimals showed that P. tricornutum shared most miRNAs with animals. Thephylogenetic tree also showed that C. merolae shared more common miRNAs withplants than with the two diatoms, and most of its miRNAs were shared with the twodiatoms. Our results indicated the origin of diatoms was from a secondaryendosymbiosis. Potential targets of P. tricornutum miRNAs involved fatty acidmetabolism, cell cycle and so on, indicated that miRNAs might play important rolesin P. tricornutum. （2） Using Solexa high throughput sequencing technology, we constructed andsequenced small RNA （sRNA） libraries from P. tricornutum under normal （PT1）,nitrogen-limited （PT2） and silicon-limited （PT3） conditions. A total of13miRNAswere identified. They were probable P. tricornutum-specific novel miRNAs. MoremiRNAs were sequenced from P. tricornutum under nitrogen-limited and/orsilicon-limited conditions, and their potential targets were involved in variousprocesses, such as signal transduction, fatty acid biosynthetic process. Our resultsindicated miRNA might play an important role in P. tricornutum nitrogen and siliconmetabolism.（3） Based on13C metabolism flux analysis, the centre carbon metabolism of P.tricornutum was studied. Results indicated that P. tricornutum can used glycerol ascarbon source. There was little malic enzyme activity in P. tricornutum, and theactivity of Calvin cycle was low. The exchange fluxes between3C and4C compoundswere high, indicating the presence of a C₄-like photosynthesis mechanism in P.tricornutum.（4） The expression level of C₄key enzymes were analyzed by real-time quantificationPCR and P. tricornutum cells were stained to analyze organelle partitioning requiredfor single-cell C₄photosynthesis. The results showed that the expression of ca underlow inorganic carbon condition was higher than that of normal condition, indicatedthat ca might play an important role in P. tricornutum CO2concentration mechanism,and there might be organelle partitioning required for single-cell C₄photosynthesis inP. tricornutum cells.