Proof Of Concept
In order to prove that we successfully constructed the UGP, VTC2 overexpression system, and functioned in Phaeodactylum tricornutum. We conducted the following experimental verification: PCR to detect whether the recombinant plasmid contains UGP and VTC2 genes; PCR to detect whether P. tricornutum contains our overexpression system after electric shock transformation; LSM to observe the intracellular lipid and chlorophyll content of P. tricornutum. The results showed that the recombinant plasmid contained UGP and VTC2 genes, and was successfully transformed into P. tricornutum, and the intracellular lipid content and chlorophyll content of the successfully transformed P. tricornutum showed very obvious changes.
PCR detection of recombinant plasmid
Before electroporation transformation, we use the recombinant plasmid as a template for PCR detection to verify whether our recombinant plasmid contains the target gene. The results are as follows, the recombinant plasmid contains the target gene.
Figure 1 PCR result of plasmid
Phaeodactylum tricornutum DNA extraction and PCR detection
After electroporation transformation, we expanded the culture of Phaeodactylum tricornutum, and then extracted the DNA of Phaeodactylum tricornutum using the CTAB method. The DNA was used as a template to detect whether the gene of the recombinant plasmid was introduced into the chassis organism by PCR. The results showed that the VTC2 overexpression system and UGP overexpression system we constructed were successfully introduced into Phaeodactylum tricornutum.
Figure 2 PCR detection of DNA
Observation by LSM
The Phaeodactylum tricornutum was dyed with BODIPY 505/515 fluorescent dye and observed under LSM. The excitation wavelength is 488nm and the emission wavelength is 530nm. The results are as follows. The red area represents chlorophyll and the green area represents lipids. The total oil content of Phaeodactylum tricornutum in the VTC2 overexpression system was significantly higher than that of the wild type. The chlorophyll content of Phaeodactylum tricornutum in the UGP overexpression system was significantly higher than that of the wild type.
Figure 3 LSM photographs of Phaeodactylum tricornutum containing overexpression system stained with BODIPY 505/515.Images were acquired randomly from at least 20 cells per sample, and typical images are presented. Bar=10μm
According to the PCR detection results of Phaeodactylum tricornutum DNA, we judged that the VTC2 overexpression system and UGP overexpression system were successfully introduced into Phaeodactylum tricornutum. The results of the LSM observation show that the VTC2 overexpression system and the UGP overexpression system have successfully demonstrated certain functions in the chassis organisms, and have significant effects on the lipid accumulation of Phaeodactylum tricornutum and the chlorophyll synthesis and metabolism, respectively.
In higher plants, UGPase is a key enzyme for carbohydrate metabolism, catalyzing the reversible production of glucose-1 phosphate (Glc 1-P) and UTP to udp-glucose and pyrophosphate (PPi). Studies have found that the overexpression of UGPase in hybrid poplars can make carbon flow to the synthesis of starch and cellulose . Silencing UGPase in Phaeodactylum tricornutum can significantly increase the lipid content of Phaeodactylum tricornutum. According to the results observed by LSM, the oil content of Phaeodactylum tricornutum overexpressing UGP was significantly lower than that of the wild type, and the chlorophyll content showed a very significant increase. Therefore, we infer that the UGP overexpression system changed the carbon flow direction of Phaeodactylum tricornutum, from lipid to carbohydrate, and increased the absorption of CO2 by photosynthesis. Eventually lead to an increase in chlorophyll content.
Studies have found that the levels of VTC2 and AsA in Chlamydomonas reinhardtii are significantly increased during oxidative stress. VTC2 is a highly regulated enzyme involved in the synthesis of AsA in green algae. Together with the ascorbic acid circulation system, it helps cells resist oxidative stress. VTC2 and VTC5 in Arabidopsis are involved in the synthesis of GGP and are regulated by light . According to the results observed by LSM, it was found that the lipid content of Phaeodactylum tricornutum increased to a certain extent. We speculate that the activity of GGP and the content of AsA in the chassis organisms may be increased, and the ability of chassis organisms to eliminate ROS and resist oxidative stress will be improved to a certain extent.
Coleman HD, Canam T, Kang KY, et al. Over-expression of UDP-glucose pyrophosphorylase in hybrid poplar affects carbon allocation. J Exp Bot 2007;58:4257–68.
Zhu B H , Shi H P , Yang G P , et al. Silencing UDP-glucose pyrophosphorylase gene in Phaeodactylum tricornutum affects carbon allocation[J]. New Biotechnology, 2016, 33(1):237.
Urzi Ca E I, Adler L N, MD Page, et al. Impact of Oxidative Stress on Ascorbate Biosynthesis in Chlamydomonas via Regulation of the VTC2 Gene Encoding a GDP-l-galactose Phosphorylase[J]. Journal of Biological Chemistry, 2012, 287(17):14234-45.
Dowdle J, Ishikawa T, Gatzek S, et al. Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability[J]. Plant Journal for Cell & Molecular Biology, 2010, 52(4):673-689.