Team:CHINA-FAFU/Results

Result

Overview

Due to the impact of COVID-19, our time in the laboratory has been reduced to a certain extent. As of October 2021, we have completed part of the experimental design part. We explored the effect of exogenous AsA on the growth and lipid accumulation of Phaeodactylum tricornutum. We found that when the amount of AsA added to Phaeodactylum tricornutum was 250mg/L, its lipid content and growth rate were the highest relative to the control group and other treatment groups, and its fucoxanthin content and the value of Fv/Fm increased significantly. At the same time, we detected by qPCR that APX1, DHAR, UGP and VTC2 gene expression levels were significantly up-regulated.
Using the cDNA of Phaeodactylum tricornutum as a template, we successfully cloned the VTC2 gene by Nested PCR, and successfully synthesized the APX1, DHAR and UGP genes in Ailurus vec^TM company. Afterwards, these four genes were successfully recombined into a plasmid vector to construct an overexpression system.
Now we are trying to transfer the overexpression system to the chassis organism-Pichia pastoris for functional verification. Next, we will transfer the expression system to Phaeodactylum tricornutum to continue the detection and verification of the transformation results. We expect to complete the unfinished experimental design and conduct a complete display before 2021 Giant Jamboree.

The effect of AsA on the growth and metabolism of microalgae

We first added different concentrations of AsA to the Phaeodactylum tricornutum culture medium, VC-CK: control group, no AsA added; VC-80: AsA added at 80mg/L; VC-160: AsA added at 160mg/L; VC-200: the addition amount of AsA is 200mg/L; VC-250: the addition amount of AsA is 250mg/L; VC-300: the addition amount of AsA is 300mg/L. During the cultivation process, we carried out the following physiological and biochemical indicators and gene expression tests:

Table 1 Measurement indicators and tools used

The test results are as follows

Cell density

According to the changes in the density of Phaeodactylum tricornutum, we speculate that the addition of 250mg/L of AsA may be the most conducive to the growth of Phaeodactylum tricornutum .

Fig.1 Density of Phaeodactylum tricornutum under different concentrations of AsA

Chlorophyll fluorescence parameters

Fig.2 Fv/Fm values of Phaeodactylum tricornutum under different concentrations of AsA.Asterisks (*) indicates significant difference by t test from no AsA treatment ( P < 0.05; Student’s t test)

According to the Fv/Fm value, we judge that 250mg/L of AsA may be the most beneficial for the photosynthesis of Phaeodactylum tricornutum

Fucoxanthin content
We extracted the Phaeodactylum tricornutum fucoxanthin in the VC-250 treatment group and tested the content with a microplate reader. The results showed that the content of Phaeodactylum tricornutum fucoxanthin in the VC-250 treatment group was higher than that of the control group. About 1.5 times.

Fig.3 Fucoxanthin content of Phaeodactylum tricornutum in the control group and the VC-250 treatment group.Asterisks (*) indicates significant difference by t test from no AsA treatment (P < 0.05; Student’s t test)

Lipid content
According to the changes in the intracellular lipid content of Phaeodactylum tricornutum, we judged that the addition of 250mg/L of AsA may be the most conducive to the accumulation of lipids of Phaeodactylum tricornutum.

Fig.4 Lipid content of Phaeodactylum tricornutum under different concentrations of AsA Asterisks (*) indicates significant difference by t test from no AsA treatment after 9 days. (P < 0.05; t test)

Fig.5 LSM observation of the fat body of Phaeodactylum tricornutum stained with BODIPY dye on the day of AsA culture

Fig.6 The percentage of lipid content of Phaeodactylum tricornutum in the control group and the VC-250 treatment group (the percentage of the dry weight of the lipid in the dry cell weight).Asterisks (*) indicates significant difference by t test from no AsA treatment (P < 0.05; Student’s t test).

According to the changes in the intracellular lipid content of Phaeodactylum tricornutum, we judged that the addition of 250mg/L of AsA may be the most conducive to the accumulation of lipids of Phaeodactylum tricornutum.

Lipid components

We used GC-MS to analyze the composition of lipids produced by Phaeodactylum tricornutum in the control group and the VC-250 treatment group. The results showed that the content of monounsaturated fatty acids in the VC-250 treatment group increased by about 9% compared to the control group. The content is reduced by about 15%, the polyunsaturated fatty acid content is increased by about 12%, and the total oil content is increased by about 25%.

Table 2 Lipid composition of Phaeodactylum tricornutum in VC-250 treatment group(% of total FAs ± SD, n = 3) Three groups were taken for the determination of fatty acid composition.FA, fatty acid; DW, dry weight; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; ND, not detected.

Gene expression

The results showed that the expressions of APX1, UGP, VTC2, and DHAR genes in the VC-250 treatment group were up-regulated by about 9, 7.5, 3.5, and 7.5 times, respectively.

Fig.7 qPCR analysis of APX1, UGP, VTC2, DHAR gene expression in the VC-250 treatment group

Get gene

Using the cDNA obtained by reverse transcription of Phaeodactylum tricornutum RNA as a template, the VTC2 gene was cloned by Nested PCR. These three genes were synthesized according to the APX1, DHAR and UGP gene sequences obtained from NCBI.

Fig.8 Principle of Nested PCR

Nested PCR has a very high specificity, which can guarantee the accuracy of the genes cloned by our PCR.

Fig.9 Electrophoresis image of the target gene

APX1 and DHAR are the key genes in the AsA-GSH cycle and participate in the regeneration of AsA. VTC2 encodes GGPase, which catalyzes the reaction between GDP-L-galactose and GTP to produce L-galactose-1-phosphate. Studies have proved that VTC2 is the seventh rate-limiting gene in the process of ascorbic acid synthesis. UGPase encoded by UGP catalyzes UTP reaction to produce UDPG, which is an important substance in the synthesis and metabolism of plant organisms.

Ligation

The vector we use is pPink-HC-FHG-cp. pPink-HC-FHG-cp is 8678bp in length, contains Paox1 promoter and CYC1 terminator, and is pre-linked with sfGFP gene.We use T4 ligase to connect APX1, DHAR, UGP and VTC2 to the vector.

Fig.10 pPink-HC-FHG-cp plasmid map.

Overexpression system: We inserted APX1, DHAR, UGP, and VTC2 between the promoter and sfGFP, respectively.

Fig.11 A: APX1 overexpression system. B: DHAR overexpression system. C: UGP overexpression system. D: VCT2 overexpression system.

Transform

The ligated plasmid is transferred to competent cells and cultured. The following is the growth on the medium containing ampicillin.

Fig.12 A: APX1 overexpression system. B: DHAR overexpression system. C: UGP overexpression system. D: VCT2 overexpression system.

Positive colony PCR

Pick a single positive colony for culture, and perform PCR detection, and then send the colony with the correct electrophoresis band to the company for sequencing.

Fig.13 A:VTC2. B:UGP. C:DHAR. D:APX1.

Get the plasmid

According to the sequencing results, the bacterial solution was expanded and cultured and the plasmid was extracted, and the plasmid was verified by PCR detection. 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.

Fig.14 Plasmid PCR results

Electric shock conversion and functional verification

The plasmid was transferred into Pichia pastoris and Phaeodactylum tricornutum using an electroporator. As of October 2021, the system we have built is still being tested and verified. Please go to our Engineering section for detailed plans. We will continue to complete subsequent experiments in accordance with Engineering's plan.Here are some of the test results we have achieved:

Fig.15 PCR detection of DNA

After electroporation transformation, we expanded Phaeodactylum tricornutum and then extracted its DNA using the CTAB method, and used the DNA as a template to detect whether the recombinant plasmid gene 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.

Fig.16 LSM photographs of Phaeodactylum tricornutum containing an overexpression system stained with BODIPY.Images were acquired randomly from at least 20 cells per sample, and typical images are presented here. Bar was shown in the fifigure.

We added bleomycin to screen Phaeodactylum tricornutum containing UGP overexpression system and VTC2 overexpression system. After screening, Phaeodactylum tricornutum was dyed with BODIPY 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.