Heat shock transcription factor 49557 gene for increasing fucoxanthin content in Phaeodactylum tricornutum and its application

Abstract

The invention discloses a heat shock transcription factor for increasing the content of fucoxanthin in Phaeodactylum tricornutum 49557 Genes and their applications are characterized by 49557 The nucleotide sequence of the gene is shown in SEQ ID NO: 3, and the amino acid sequence of the protein encoded by 49557 is shown in SEQ ID NO: 4. 49557 The gene increases the content of fucoxanthin synthesized by it, which has the advantage of significantly increasing the content of fucoxanthin in Phaeochromis triangularis.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a heat shock transcription factor 49557 gene for increasing the content of fucoxanthin in Phaeodactylum tricornutum and an application thereof. Background Art

[0002] As an important biological resource, microalgae has broad application prospects in the fields of food, medicine, energy, etc. Fucoxanthin is a natural carotenoid with multiple biological activities in microalgae, showing good application prospects in anti-oxidation, anti-inflammatory, and anti-tumor. Microalgae is one of the important biological sources for synthesizing fucoxanthin, but the content of fucoxanthin in wild microalgae is limited, and the methods for regulating microalgae growth and fucoxanthin accumulation still have certain limitations. Therefore, it is of great significance to use genetic engineering technology to increase the content of fucoxanthin in microalgae and improve the production efficiency of microalgae growth.

[0003] Heat shock transcription factors (HSFs) are an important class of transcription factors that initially attracted widespread attention because of their key role in responding to heat stress. Subsequent studies have found that HSFs are also involved in the regulation of many other physiological processes and metabolic pathways in cells, involving different stress responses, growth and development, and material metabolism. However, research on the role of specific HSFs in regulating the content of fucoxanthin in P. tricornutum is still relatively scarce. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a heat shock transcription factor for increasing the content of fucoxanthin in Phaeodactylum triangularis. 49557 Genes and their applications, 49557 Gene knockout algae strains can positively regulate the expression of fucoxanthin synthesis genes.

[0005] The technical solution adopted by the present invention to solve the above technical problems is: a heat shock transcription factor for increasing the content of fucoxanthin in Phaeodactylum triangularis 49557 Gene, described 49557 The nucleotide sequence of the gene is shown in SEQ ID NO:3.

[0006] Furthermore, the amino acid sequence of the protein encoded by 49557 is shown in SEQ ID NO:4.

[0007] The present invention also provides the heat shock transcription factor 49557 Application of genes in increasing the content of fucoxanthin in Phaeodactylum triangularis, the 49557 The nucleotide sequence of the gene is shown in SEQ ID NO: 3.49557 The gene causes an increase in the amount of fucoxanthin it synthesizes.

[0008] Compared with the prior art, the advantages of the present invention are:

[0009] 1. For the first time, we found the heat shock transcription factor 49557 by screening the gene library of P. tricornutum. After knocking out the CRISPR gene, we found 49557 The mutant strain had phenotypic changes in fucoxanthin synthesis;

[0010] 2. The heat shock transcription factor of P. tricornutum was confirmed for the first time 49557 Genes can promote the increase of fucoxanthin content. Through genetic modification and optimization of culture conditions, the fucoxanthin content of genetically modified microalgae strains is significantly higher than that of wild-type, providing a better source of raw materials for the industrial production of fucoxanthin. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 For two 49557 The knockout results of the gene knockout algae strain and the sequence chromatogram generated by sequencing the WT wild type;

[0012] Figure 2 For two 49557 Analysis of fucoxanthin content in gene knockout algae strains and WT. Note: WT means wild type. 49557-1 and 49557-10 Indicates two strains 49557 Gene knockout Phaeochromis tricornutum strains;

[0013] Figure 3 For two 49557 Fucoxanthin synthesis of gene-edited algal strains and WT CRTISO5 Relative gene expression;

[0014] Figure 4 For two 49557 Fucoxanthin synthesis of gene-edited algal strains and WT ZEP1 Relative gene expression;

[0015] Figure 5 For two 49557 The changes in the number of algae between the knockout algae strain and the WT at different temperatures and culture times, i.e., the growth curve;

[0016] Figure 6 For two 49557 Heat shock proteins in knockout strains and WT HSP20 The relative expression of genes. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below with reference to the accompanying drawings.

[0018] Specific Example 1: Heat shock transcription factor 49557 Gene cloning and sequence analysis include the following steps:

[0019] Step 1: Extraction concentration is 1×10 6 cells / mL of total RNA from the logarithmic phase of Phaeodactylum triangularis, and the extracted RNA was used as a reactant for reverse transcription to obtain a cDNA template.

[0020] Step 2: PCR amplification: 49557 Gene amplification products, PCR amplification reaction system: 0.5 μL cDNA template, 10 μL 2× PrimeSTAR Max Premix, 49557 0.5 μL of upstream and downstream amplification primers, 8.5 μL of ddHO 2 O; PCR amplification program was: denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 1 min, 30 cycles; 49557 The nucleotide sequence of the upstream amplification primer of the gene is shown in SEQ ID NO: 1: ATGGTATTCTACAGAGCTTCCACGT, 49557 The nucleotide sequence of the primer for downstream amplification of the gene is shown in SEQ ID NO: 2: TCAAAAGTCATCTACAGTTGCCACC.

[0021] Step 3: The PCR amplification product was purified by 1% agarose gel electrophoresis and then connected to the pMD19-T vector. After further verification by PCR, sequencing was performed to obtain 49557

[0022] The amino acid sequence of the protein encoded by heat shock transcription factor 49557 is shown in SEQ ID NO: 4 shown: MVFYRASTFAPESKERHTEGYKVTPPFKNELQDGFPNNQYFRHSFQLDDLPSPDSLLQRSNISNTGDCRIASFRHPVHANIVNPTHFRSVSNNGH TIANQVCLGDYGIDMMSTQRLNEQICFSEIPPRMVVNHKYFDHYRDAVNFNVQPSKPESTSNPKYPFRGGVAVHFPERLFEMLDRVEELGMSHIVSWQPHGR SFLVHKPKEFVSAIMPHFFRQSKFTSFQRQLNLYGFVRLTVGKDSGSYYHELFLRGCPMLCRCIVRRRIKGNGVKPAPSPSTEPDFYSMERCESTGPKTENDTLDTQKNEPGPHQDCRHLNEDIHFSCTPARIEDQMMPVPICPTEPENWMQPNRRLFELLAKATSMDTHIYDELDSVYRELDIPVPDPLFEIDLTVATVDDF.

[0023] Specific embodiment 2: Construction 49557 Gene knockout vectors.

[0024] Using the pPtPuc3-Cas9-sgRNA (Addgene ID: 109219) vector as the CRISPR / Cas9 expression cassette, two 49557 The knockout vector (CRISPR-49557-1 / 2) is as follows:

[0025] Step 1: Select the 49557For the target gene, two groups of sgRNA sequences were obtained using online sgRNA design software, wherein the first group of sgRNA sequences included the first sgRNA1 and the first sgRNA2, and the second group of sgRNA sequences included the second sgRNA1 and the second sgRNA2. The nucleotide sequence of the first sgRNA1 was shown in SEQ ID NO:5: 5'-AGCAAAGAGCGTCACACGG-3', the nucleotide sequence of the first sgRNA2 was shown in SEQ ID NO:6: 5'-ACACGCAAAAAAATGAACC-3', the nucleotide sequence of the second sgRNA1 was shown in SEQ ID NO:7: 5'-GAGCAAAGAGCGTCACACGG-3', and the nucleotide sequence of the second sgRNA2 was shown in SEQ ID NO:8: 5'-TCTCCGGATTCTCTTCTCCA-3'.

[0026] Step 2. Add restriction sites to the sgRNA sequence obtained in step 1 and synthesize two pairs of single-stranded primers. The nucleotide sequence of the first sgRNA1 single-stranded upstream primer is shown in SEQ ID NO: 9: 5'-AGGTCTCGTCGAGAGCAAAGAGCGTCACACGGGTTTTAGAGCTAGAAAT-3', and the nucleotide sequence of the first sgRNA2 single-stranded downstream primer is shown in SEQ ID NO: 10: 5'-GGGTCTCCAAACGGTTCATTTTTTTGCGTGTCTCGACTTTGAAGGTGTT-3'.

[0027] The nucleotide sequence of the second sgRNA1 single-stranded upstream primer is shown in SEQ ID NO: 11: 5'-AGGTCTCGTCGAGAGAGAGCAAAGAGCGTCACAGTTTTAGAGCTAGAAAT-3', and the nucleotide sequence of the second sgRNA2 single-stranded downstream primer is shown in SEQ ID NO: 12: 5'-GGGTCTCCAAACTCTCCGGATTCTCTTCTCCACTCGACTTTGAAGGTGTT-3'.

[0028] Step 3. The pPtPuc3-Cas9-sgRNA vector contains U6 Promoter, sgRNA scaffold and U6 3' region elements. The whole gene is synthesized in the order of sgRNA scaffold-U6 3' region-U6 Promoter. The synthesized product is used as a template for PCR amplification. The PCR amplification reaction system is 2× PrimeSTAR Max DNA Polymeras (high-fidelity enzyme), 25 μL; 1 μL of 10μM first sgRNA1 single-stranded upstream primer, 1 μL of 10μM first sgRNA2 single-stranded downstream primer, and 1 μL of template. Use ddH 2 O was added to 22 μL and the reaction system was mixed; PCR amplification program: 98°C 10 s, 55°C 15 s, 72°C 7 s, 30 cycles, cooled to 4°C, and the first double-stranded DNA fragment was amplified.

[0029] The above amplification primers were replaced with the second sgRNA1 single-stranded upstream primer and the second sgRNA2 single-stranded downstream primer, and the second double-stranded DNA fragment was amplified using the same method.

[0030] Step 4: The first double-stranded DNA fragment, the second double-stranded DNA fragment and the pPtPuc3-Cas9-sgRNA plasmid obtained in step 3 were digested with BsaⅠ-HF respectively. The digestion system was as follows: 10×Buffer 5 μL, restriction endonuclease BsaⅠ-HF 1 μL, 1 μg / μL target 2 μg, ddH 2 O to a total volume of 50 μL, react in a 37°C constant temperature incubator for 2 h, and then recover and detect the concentration.

[0031] The double-stranded DNA fragments recovered by enzyme digestion and the linear plasmids recovered by enzyme digestion were connected with T4 ligase at 16°C for 1h. The connection system was 1 μL of T4 10×Buffer, 150 ng of pPtPuc3-Cas9-sgRNA linear plasmid, 26.5 ng of the first double-stranded DNA fragment, 0.5 μL of T4 DNA ligase, and water was used to fill up to 10 μL to obtain the first recombinant vector. The first double-stranded DNA fragment was replaced with the second double-stranded DNA fragment and connected with T4 ligase to obtain the second recombinant vector.

[0032] Step 5: Transform the first recombinant vector and the second recombinant vector into Escherichia coli respectively. The specific steps are as follows:

[0033] (1) Take 10 μL of the two recombinant vectors obtained in step 4 and add them to the E. coli DH5α competent cells respectively. Place them on ice for 30 min. Gently shake the cells, heat shock them in a 42°C water bath for 90 s, and then quickly place them on ice for 3-5 min.

[0034] (2) Then, add 400 μL of LB liquid culture medium without antibiotics to each tube and mix gently, and then shake and culture at 220 rpm in a shaker at 37°C for 1 h; the LB liquid culture medium consists of 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, and the solvent is water;

[0035] (3) Take 100 μL of the mixture obtained in step (2) and add it to the + ) on the LB solid medium containing antibiotics, spread evenly until the bacterial solution is completely dry on the plate, seal it with a sealing film, mark it, and culture it in a 37°C constant temperature incubator overnight for 12-16 hours;

[0036] Take out the overnight cultured plate from the incubator and observe the colonies on the plate. If there are single colonies, pick 8 single colonies on the clean bench and place them in 1 mL of agar containing ampicillin (Amp + ) antibiotics in LB liquid medium, shaken at 220 rpm in a 37°C constant temperature incubator for 3 h, and then PCR identification of the bacterial solution was performed. A sequence on the pPtPuc3-Cas9-sgRNA vector was used as the reverse primer for identification. The nucleotide sequence of the reverse primer for bacterial solution identification is shown in SEQ ID NO: 13: CAGGAAACAGCTATGACC. The single-stranded upstream primer in step 2 was used as the forward primer for bacterial solution identification (the first recombinant vector used the first sgRNA1 single-stranded upstream primer as the forward primer for bacterial solution identification, and the second recombinant vector used the second sgRNA1 single-stranded upstream primer as the forward primer for bacterial solution identification). PCR identification was performed. The bacterial solution PCR reaction system was: 10 μL 2×Flash Hot Start Master, 1 μL bacterial solution identification forward primer, 1 μL bacterial solution identification reverse primer, 2 μL bacterial solution, and ddH 2 O to a total volume of 20 μL. The PCR reaction program of the bacterial solution was: 95℃ for 5 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 7 s, 30 cycles; and cooled to 4℃.

[0037] The obtained PCR products were subjected to gel electrophoresis to check the results, and samples with positive bands were taken for expansion culture (100 μL bacterial solution and 4 mL of ampicillin (Amp +) LB liquid medium containing antibiotics) and the plasmid was extracted using the Plasmid Mini Kit Ⅱ plasmid extraction kit to obtain two 49557 Gene knockout vectors.

[0038] Specific embodiment three: 49557 The specific steps for preparing gene knockout algae strains are as follows:

[0039] Step 1: Use EcoNI to monitor the two 49557 The gene knockout vector was linearized and digested with the following enzyme system: 5 μL 10× Buffer, 3 μL EcoNI, 6 μg 49557 The knockout vector was filled with water to 50 μL. After enzyme digestion for 3 hours, it was purified using a gel extraction kit and the product was recovered by enzyme digestion to obtain two 49557 Gene knockout vector linear plasmid.

[0040] Step 2: Mix the triangular brown finger algae with 49557 The gene knockout vector linear plasmid is electrotransformed, and the specific steps are as follows:

[0041] (1) Cultivate the triangular algae to the logarithmic stage 1×10 6 cells / mL, centrifuged at 1500×g at 4°C for 10 min, and discarded the supernatant; the precipitate was washed three times with 1 mL of 375 mM sorbitol, and the washed cells were resuspended with 100 μL of 375 mM sorbitol to obtain a final algal density of 2×10 9 cells / mL resuspension solution;

[0042] (2) Add 100 μL of resuspension solution and 4 μg of the purified protein in step 1 at a concentration of 0.2 μg / μL. 49557 The knockout vector linear plasmid and 40 μg salmon sperm DNA (boiled at high temperature for 10 min) at a concentration of 10 μg / μL were mixed together, incubated on ice for 10 min, and then transferred to a 0.2 cm ice-precooled electroporation cuvette. The parameters were set as follows: 500 V field strength, 25 μF capacitance, and 400 Ω parallel resistance for electroporation transformation;

[0043] (3) After electroporation, the cells were immediately transferred to 10 mL of f / 2 liquid culture medium and placed under low light (about 30 μmol / m 2·s) for 24 h to recover, and then transferred to normal light conditions for 24 h. Centrifuge the algae solution at 1500×g, 4°C for 10 min, discard the supernatant, resuspend in 600 μL f / 2 liquid medium, take 200 μL and add it to the f / 2 medium solid plate containing bleomycin, and culture under normal conditions for 12-14 d.

[0044] Step 3 49557 The specific steps for screening and identification of gene knockout algae strains are as follows:

[0045] (1) Pick the monoclonal algae colony grown on the bleomycin resistance plate in step 2, draw a short line of 3-5 mm on the f / 2 solid medium containing bleomycin, and culture under normal conditions until algae colonies grow. Take half of the algae colony, add 20 μL algae lysis buffer, and place it in an ice bath for 15 min, then in a 100℃ water bath for 10 min. Take the algae lysis product as a template for algae liquid PCR identification. The formula of the algae lysis buffer is: 1% NP40, 10.00 mM Tris, 0.14 mM NaCl, 5.00 mM KCl;

[0046] (2) Designing primers for identifying algae liquid, wherein the nucleotide sequence of the forward primer for identifying the first recombinant algae is shown in SEQ ID NO:14: 5'-TGCACTATGTAAGCCGGTACTACAG-3', and the nucleotide sequence of the reverse primer for identifying the first recombinant algae is shown in SEQ ID NO:15: 5'-ATCAATCTCAAAAAGAGGATCGGG-3'; the nucleotide sequence of the forward primer for identifying the second recombinant algae is shown in SEQ ID NO:16: 5'-TACTACAGAGCTAACATGACTTCGGA-3', and the nucleotide sequence of the reverse primer for identifying the second recombinant algae is shown in SEQ ID NO:17: 5'-TTTCGCTGAAACAAATTTGCTC-3'.

[0047] The algae liquid PCR reaction system is as follows: 10 μL 2×Flash Hot Start Master, 1 μL algae liquid identification forward primer, 1 μL algae liquid identification reverse primer, 1 μL algae liquid, ddH 2 O to a total volume of 20 μL. The PCR reaction program for algae solution was as follows: 95℃ for 5 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 5-10 s / kb, 30 cycles; and cooled to 4℃.

[0048] The PCR amplification products were sequenced and analyzed, and the successful knockout was selected 49557The positive transformants of the gene were selected, and the knockout heterozygous algae strains were selected and cultured in f / 2 liquid medium, and then diluted and spread for a new round of screening. The PCR and sequencing steps were repeated until two homozygous transformants were screened out. The first recombinant algae was named 49557-1 The second recombinant algae was named 49557-10 Gene knockout strains.

[0049] Wild type (native P. triangularis without genetic modification), 49557-1 and 49557-10 The sequencing results of homozygous transformants are as follows Figure 1 As shown by Figure 1 It can be seen that 49557-1 The homozygous transformant had a deletion of 868 bp relative to the wild type; 49557-10 The homozygous transformant had a deletion of 107 bp relative to the wild type.

[0050] Specific embodiment 4: 49557 Extraction and determination of fucoxanthin from gene knockout algae strains

[0051] In the logarithmic period 49557 After freeze-drying the knockout strains and wild-type P. tricornutum for 48 h, 90% ethanol was added at a solid-liquid ratio of 1 g:10 mL, and fucoxanthin was extracted by ultrasound at 40 °C for 1 h. After filtration, the supernatant was filtered through a 0.22 μm filter membrane for HPLC analysis, and the whole process was carried out under dark conditions. An Agilent 1200 HPLC system (Agilent Technologies, American) consisting of a G1312A binary pump, a G1367B autosampler, a G1315D PDA detector, and a G1316A column oven was used for fucoxanthin quantification. The mobile phase, namely methanol and water, was eluted at 0.7 mL min -1 The YMC carotenoid column (250 mm length × 4.6 mm inner diameter; 5 μm particle size; Waters, American) was used for separation at 35 °C under the following gradient program: methanol increased from 90% to 100% for 20 min, maintained at 100% for 5 min, decreased to 90% within 5 min, and then maintained at 90% for 5 min. 10 μL of sample solution was injected and the chromatogram was recorded at 445 nm. Fucoxanthin was quantified based on a calibration curve with a concentration range of 0.5-50 μg / mL.

[0052] Figure 2 The cell density was 1.5×10 6 cells / mL 49557 The fucoxanthin content of the knockout strains and wild type was Figure 2 It can be seen that 49557-1Knockout strains and 49557-10 The fucoxanthin content of the gene knockout strain increased by 0.2-0.25 times. Experimental data showed that 49557 Gene knockout resulted in an increase in fucoxanthin content. 49557 The gene knockout algae plays a positive regulatory role in the synthesis of fucoxanthin. Note: WT represents wild-type P. triangularis.

[0053] Specific embodiment 5: 49557 Detection of expression levels of key genes for fucoxanthin synthesis in gene knockout algal strains.

[0054] Select and synthesize fucoxanthin synthesis genes CRTISO5 , ZEP1 Fluorescence quantitative primers,

[0055] CRTISO5 The nucleotide sequence of the fluorescent quantitative upstream primer is shown in SEQ ID NO: 18: GAGGATCGGCTCATACATTCTC, CRTISO5 The nucleotide sequence of the fluorescence quantitative downstream primer is shown in SEQ ID NO: 19: GCATCTCTTCTTCCAGGACATC.

[0056] ZEP1 The nucleotide sequence of the fluorescent quantitative upstream primer is shown in SEQ ID NO: 20: GGTACGCTTCGATACCCTACAG, ZEP1 The nucleotide sequence of the fluorescence quantitative downstream primer is shown in SEQ ID NO: 21: GTAATTGGCAATACGGGACTTG.

[0057] Internal reference Actin The nucleotide sequence of the fluorescent quantitative upstream primer is shown in SEQ ID NO: 22: AGGCAAAGCGTGGTGTTCTTA, the internal reference Actin The nucleotide sequence of the fluorescence quantitative downstream primer is shown in SEQ ID NO: 23: TCTGGGGAGCCTCAGTCAATA.

[0058] Use respectively 49557-1 Gene knockout strains 49557-10cDNA extracted from RNA reverse transcribed from gene knockout algae and wild-type P. tricornutum was used as template for real-time PCR quantification. The reaction system of Taq Pro UniversalSYBR qPCR Master Mix (Vazyme #Q712) used as quantitative reagent is as follows: 10 μL 2×Taq Pro UniversalSYBR qPCR Master Mix, 0.4 μL fluorescent quantitative upstream primer, 0.4 μL fluorescent quantitative downstream primer, 1 μL cDNA, ddH 2 O to a total volume of 20 μL. The reaction program was as follows: 95°C for 2 min; 95°C for 5 s, 60°C for 30 s, for a total of 39 cycles; 95°C for 15 s, 60°C for 60 s, 95°C for 15 s; and 4°C until the end.

[0059] The results are as follows Figure 3 and Figure 4 As shown, compared with WT, 49557 Two key genes of fucoxanthin in gene knockout algae strains ZEP1 and CRTISO5 The expression level of 49557 Gene knockout algae strains can regulate the expression of fucoxanthin synthesis genes, and the regulation is positive.

[0060] Specific embodiment six: 49557 Determination of the growth curve of gene knockout algae strains under high temperature conditions.

[0061] The obtained product prepared in the specific embodiment 3 49557 The knockout algae strain was cultured in f / 2 liquid medium containing bleomycin under normal conditions until the logarithmic phase, then washed three times with f / 2 liquid medium without bleomycin and inoculated into 50 mL f / 2 liquid medium. The wild type was used as the control, and the initial concentration was adjusted to 3×10 5 cells / mL, initially cultured at 30°C for 2 days, then transferred to 22°C for 4 days, at 30°C for the first time, 30°C for 2 days, 22°C for 2 days, and 22°C for 4 days, the number of algal cells was counted under an optical microscope at a fixed time every day to determine the algal cell density, and this was repeated three times until the culture reached the logarithmic phase. HSP20 Gene expression level.

[0062] Figure 5 for 49557 The growth curves of the knockout algae strains were measured at 30°C initially, 30°C for 2 days, 22°C for 2 days, and 22°C for 4 days. 49557-1 Gene knockout strains 49557-10The cell concentrations of the knockout strain and the wild type (WT) were used to obtain the growth curve of the knockout strain. Figure 5 It can be concluded that 49557-1 Knockout strains and 49557-10 The cell number of the knockout strain was lower than that of the wild type, and this phenomenon was most significant on the 8th day. 49557 After the gene was added, the growth of the triangular brown finger algae decreased.

[0063] like Figure 6 As shown, 49557-1 Knockout strains and 49557-10 Knockout strains and wild-type P. tricornutum HSP20 The gene expression level is consistent with the trend of algae quantity change, that is, HSP20 Gene expression in 49557 The knockout algae had a lower level than the wild type, which showed that 49557 After the gene, HSP20 Gene expression is also suppressed.

[0064] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. An application of a heat shock transcription factor 49557 gene in increasing the fucoxanthin content in Phaeodactylum triangularis, characterized in that: The nucleotide sequence of the 49557 gene is shown in SEQ ID NO:

3. By knocking out the 49557 gene in Phaeodactylum tricornutum, the content of synthesized fucoxanthin is increased.

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