Carbon Allocation Function Of UDP-glucose Pyrophosphorylase In Phaeodactylum Tricornutum

Phaeodactylum tricornutum has a fully sequenced genome so that, together with a perfect transgenic platform and the feasibility of exogenously triggered RNA-mediated silencing, it is an appealing model system for further study to explore the functions of genes,β-1,3 glucan (chrysolaminarin) is the main storage polysaccharide of P. tricornutum, and UDP-glucose (UDPG) serves as the substrate for chrysolaminaran synthesis. In higher plants, UDP-glucose pyrophosphorylase (UGPase) is a key enzyme in carbohydrate metabolism, catalyzing the reversible production of UDPG and pyrophosphate (PPi) from glucose-1-phosphate (G-1-P) and UTP. Based upon its strategic positioning at the cross roads of several pathways for carbohydrate synthesis, UGPase might also play an important role in carbon partitioning. In order to study in vivo the functionality of UGPase in synthesizing chrysolaminaran and regulating carbon partitioning, the design of experimental technology route is as the following two parts:The first part, according to the existing research of abiotic stress on UGPase, we selected the phosphorus concentration and temperature to regulate the metabolism of UGPase. we designed 5 kind phosphorus concentration and 4 kind temperature to culture P. tricornutum to the late-exponential phase, then analysed the transcription level of the UGP by real-time quantitative PCR (QRT-PCR) technology. We also measured UGPase activity, the content of chrysolaminarin and the amount of total lipids. We attempt to inhibit the synthesis of UGP, which encodes UDP-glucose pyrophosphorylase, by changing the phosphorus concentration and temperature, decrease the content of chrysolaminarin, increase the amount of total lipids.The second part, based on the whole genome sequencing results of P. tricornutum. UGP was analyzed by bioinformatics method; according to the information of UGP which analyzed by bioinformatics method, we used theexpression vector pha-T1 to construct successfully one inverted repeat RNAi vector (pUGP-IR) and two antisense interference vectors (pUGP-AS516, pUGP-AS237), then all of them were introduced into P. tricornutum using the biolistic transformation. Bombarded cells were cultivation with ZeocinTm for the preliminary screening of transformants. We isolated the total genomic DNA of transformants to check the integration of Sh ble gene by PCR. To further verify the integration of interference vectors, we locate the integration sites of interference vectors by LA PCR technology. The transcript abundance, enzyme activity, specific growth rate, chrysolaminaran content, and total lipid content were quantified in both transgenic and wild-type strains. We expect to shunt photosynthetic carbon fluxes from chrysolaminarin synthesis pathway into total lipids pathway, constructing high-yield oil engineering strain.At the first part of the experiment, the results of the study show that the transcriptional level of UGP is being down-regulated by decreased phosphorus concentration and increased temperature, respectively; together with declined UGPase activity, decreased chrysolaminarin content and added total lipids. Through this part of the experiment, we firstly prove that UGPase is being regulated positively by the phosphorus concentration and being regulated negatively by temperature. We also find that declines of UGPase activity in P. tricornutum result in decreases in chrysolaminaran content and increases in lipid synthesis. It suggest that the UGPase is a rate-limiting enzyme and may play an important role in chrysolaminarin biosynthesis and carbon allocation.The second part of the experiment, we found that the 69% decrease in UGPase activity was accompanied by a 4.89 fold reduction in UGP transcript abundance. Accumulation of total lipid reflected the reduction in chrysolaminaran content in transgenic strains. Inactivation of UGPase in P. tricornutum led to a significant decrease in chrysolaminaran content and an increase in lipid synthesis. These findings suggest that UGPase is a rate-limiting enzyme and may play an important role in chrysolaminarin biosynthesis and carbon allocation, especially in lipid biosynthesis of P. tricornutum. Our results support a theoretical deduction that UGP is a good candidate for improving lipid synthesis in diatoms.