CN114395571B - Phaeodactylum tricornutum ZEP1 gene, protein and application - Google Patents

Abstract

The invention discloses ZEP1 gene, protein and application of Phaeodactylum tricornutum, and the invention discloses a new function of the ZEP1 gene for the first time, which can improve the content of fucoxanthin in Phaeodactylum tricornutum; and, it can be used in photosynthetic organisms (plants, algae, photosynthetic bacteria) to increase the content of fucoxanthin in the photosynthetic organisms, and then increase the light capturing efficiency, and is beneficial to the light capturing efficiency and biomass accumulation, and the protein encoded by the gene can be used for the synthesis of fucoxanthin precursor outside organisms.

Description

Phaeodactylum tricornutum ZEP1 gene, protein and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to Phaeodactylum tricornutum ZEP1 gene, protein and application thereof.

Background

Carotenoids are widely found in photosynthetic organisms and have both antioxidant and light energy capturing properties. Because of the diversity of structures, different carotenoids have different application values. Various carotenoids, such as fucoxanthin, astaxanthin, etc., have high market value. In recent years, fucoxanthin has been reported to have an anticancer function in addition to antioxidation.

In addition, fucoxanthin has important application in photosynthetic light energy capture. Blue-green light has strong penetrability in seawater, so that most of marine algae contain fucoxanthin and are used for capturing blue-green band visible light. Fucoxanthin is transferred to other photosynthetic organisms, and can be used for widening light-capturing spectrum and improving photosynthesis efficiency.

Currently, the synthetic pathway of fucoxanthin has not been completely resolved. There is no report about the synthesis of fucoxanthin gene.

Disclosure of Invention

It is an object of the present invention to provide ZEP1 gene of Phaeodactylum tricornutum (Phaeodactylum tricornutum).

A second object of the present invention is to provide a protein encoded by the above gene.

It is a primary object of the present invention to provide the use of the ZEP1 gene or protein.

In order to achieve the above purpose, the technical scheme adopted by the invention is summarized as follows:

a ZEP1 gene has the nucleotide sequence shown in SEQ ID NO.1, which consists of 1986 bases or DNA molecule hybridized with the DNA sequence defined in SEQ ID NO.1 under strict conditions.

The amino acid sequence of the protein (1) encoded by the gene is shown as SEQ ID No. 2. The sequence consists of 661 amino acid residues.

The protein encoded by the ZEP1 gene can also comprise a protein which is formed by substitution, deletion or addition of one or more ((such as 1-30, preferably 1-20, more preferably 1-10, such as 5, 3)) amino acid residues of the amino acid sequence of SEQ ID NO.2 and has the function of (1) and is derived from (1); or a protein derived from (1) having homology of 80% ((preferably 90% or more, such as 95%,98%,99% or more)) or more with the protein sequence defined in (1) and having the function of the protein of (1).

Recombinant microorganisms containing ZEP1 genes, including algae, fungi or bacteria, are all within the scope of the present invention.

The most important point of the invention is that a novel function of the ZEP1 gene is disclosed, a ZEP mutant is obtained by gene editing, and compared with wild-type Phaeodactylum tricornutum, the mutant has lower fucoxanthin content than the wild-type, but a carotenoid which is not present in the wild-type is accumulated, and the mutant is named as 7',8' -double-dehydrogenated nordinoxanthin according to the similarity with the known compound of the dinoxanthin. It can be seen that the ZEP1 gene or protein is involved in the synthesis of fucoxanthin, that is, the ZEP1 gene has the function of increasing the content of fucoxanthin in phaeodactylum tricornutum, and can be used for increasing the yield of fucoxanthin produced by microorganisms (algae, fungi, bacteria); and, for increasing the content of fucoxanthin in photosynthetic organisms (plants, algae, photosynthetic bacteria) and consequently increasing the light harvesting efficiency, the efficiency of photosynthesis and the accumulation of biomass are advantageous, which can be achieved by means of transgenesis.

In addition, through the identification of the enzymatic function of the ZEP1 protein, it is found that only in the presence of the ZEP1 protein, 7',8' -didehydronordinoflagellate Huang Sucai can be converted into fucoxanthin precursor 7',8' -didehydrofucoxanthin, so that the protein encoded by the ZEP1 gene can be used for in vitro synthesis of fucoxanthin precursor.

It should be noted that the functions of the genes protected by the present invention include not only the ZEP1 gene described above, but also homologous genes having a high homology (e.g., homology of more than 40%, preferably more than 50%, preferably more than 60%, more preferably more than 70%, more preferably more than 80%, more preferably more than 90%, more preferably more than 95%, more preferably more than 98%) with SEQ ID NO. 1.

The invention has the advantages that:

in Phaeodactylum tricornutum, we find a ZEP1 gene for the first time, and the protein encoded by the gene is responsible for the penultimate step of fucoxanthin synthesis, that is, the invention discloses a new function of the ZEP1 gene for the first time, which can improve the fucoxanthin content in Phaeodactylum tricornutum. And, it can be used in photosynthetic organisms (plants, algae, photosynthetic bacteria) to increase the content of fucoxanthin in the photosynthetic organisms, and then increase the light capturing efficiency, and is beneficial to the light capturing efficiency and biomass accumulation, and the protein encoded by the gene can be used for the synthesis of fucoxanthin precursor (7 ',8' -bisdehydrodinoxanthin) outside organisms.

Drawings

FIG. 1 is evidence that the ZEP1 gene was edited in the ZEP1 mutant;

FIG. 2 is evidence of reduced fucoxanthin content of zep1 mutants;

in the figure, the right image is a photograph of the appearance of the cell, and the left image is a High Performance Liquid Chromatography (HPLC) pigment analysis;

FIG. 3 shows the nuclear magnetic resonance identification of the accumulated pigment, i.e.ZEP 1 protein substrate structure, in zepl mutants, i.e.the 7',8' -didehydronoroxyaxanthin molecular structure;

FIG. 4 is a vector for use in the production of ZEP1 protein in E.coli;

FIG. 5 is the results of in vitro synthesis of a fucoxanthin precursor using the ZEP1 protein in the examples;

FIG. 6 is a molecular structure of 7',8' -bisdehydrodinoxanthin;

FIG. 7 is a vector used for producing CRTISO5 protein in E.coli;

FIG. 8 shows the results of in vitro synthesis of fucoxanthin using CRTISO5 protein in the examples.

Detailed Description

The present invention will be described in detail with reference to specific examples. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. The test methods in the following examples are conventional methods unless otherwise specified. The reagents and materials employed, unless otherwise indicated, are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botanicals, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination, and bioinformatics, which will be apparent to one of skill in the art. These techniques are fully explained in the published literature, and the methods of DNA extraction, phylogenetic tree construction, gene editing method, gene editing vector construction, gene editing plant acquisition, etc. used in the present invention can be realized by the methods disclosed in the prior art except the methods used in the examples described below.

The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-or double-stranded structures. Such nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences of non-coding regions. These terms include a gene. "Gene" or "gene sequence" is used broadly to refer to a functional DNA nucleic acid sequence. Thus, a gene may include introns and exons in genomic sequences, and/or coding sequences in cDNA, and/or cDNA and regulatory sequences thereof. In particular embodiments, for example in relation to isolated nucleic acid sequences, it is preferred that they are cDNA.

Examples

1. Acquisition of the ZEP1 Gene

A ZEP1 gene of Phaeodactylum tricornutum is obtained by screening through various technical means such as transcriptome and metabolome, the length of the full-length coding frame nucleotide sequence of the gene is 1698bp, the gene consists of 565 amino acids, the nucleotide sequence is shown as a sequence SEQ ID NO.1, and the protein sequence is shown as a sequence SEQ ID NO. 2.

Phenotypic analysis of Crtiso5 mutants

Previously, no report on the function of the ZEP1 gene in Phaeodactylum tricornutum has been made, and the applicant has conducted the following studies, and has found for the first time that the ZEP1 gene plays an important role in Phaeodactylum tricornutum fucoxanthin synthesis.

Construction of ZEP1 Gene editing vector

The CRTISO5 gene-edited targeting sequence (sgRNA) was selected using the CRISPOR website (http:// CRISPOR. Tefor. Net /), two primers were designed based on the selected sgRNA (Table 1), after annealing, double-stranded DNA containing cohesive ends was obtained, cloned into the target vector containing the blasticidin-S deaminase gene, and the Phaeodactylum tricornutum U6 promoter was responsible for expression of the sgRNA. The Cas9 gene is controlled by the gamma tubulin promoter.

TABLE 1 CRTISO5 sgRNA primers

2. Phaeodactylum tricornutum conjugal transformation and acquisition of zep1 mutant

Coli containing the above vector and pTA-Mob vector required for conjugal transformation was mixed with wild type Phaeodactylum tricornutum at a ratio of 1000:1, concentrated and plated on f/2 medium. Two days later, cells were scraped and plated on f/2 medium plates containing 5. Mu.g/mL blasticidin.

Two weeks later, the clones were picked, resuspended in liquid medium and plated again to obtain subclones. Multiple subclones were picked and colony PCR and sequenced. The CRTISO5 gene in the zepl mutant obtained was missing 15 base pairs from the wild type (fig. 1).

Fucoxanthin phenotyping of zep1 mutants and structural identification of accumulated carotenoids

In constant light (80. Mu. Mol.m) ^-2 ·s ^-1 ) Wild type and mutant were cultured under the conditions. Centrifugal collection 10 ⁷ Cells were added with 250 μl of 90% acetone and mixed sonically under dark conditions. After centrifugation, the supernatant was collected and subjected to a pigment analysis by High Performance Liquid Chromatography (HPLC).

HPLC parameters were as follows:

instrument: thermo Ultimate3000UHPLC;

chromatographic column: the C18 column (5 μm,

250×4.6mm，waters，USA)；

temperature: 20 ℃;

flow rate: 1mL per minute;

sample injection volume: 10. Mu.L;

gradient mobile phase: consists of solvent a (methanol: water=90:10) and solvent B (ethyl acetate). At 0: 100% A,0% B;20-22 minutes: 0% A,100% B;23-28 minutes: 100% A;0% B.

HPLC results showed that the fucoxanthin content of the ZEP mutant was lower than that of the wild type, and that the ZEP1 gene or protein was related to the synthesis of fucoxanthin, ZEP mutant appeared green due to the low fucoxanthin content, but accumulated a carotenoid that was not present in the wild type (FIG. 2).

The accumulated carotenoids were collected during HPLC, and structural analysis was performed using a Bruker AVANCE NEO MHz nmr, the molecular structure of the analysis being shown in fig. 3, and the enzymatic function of ZEP1 protein was further identified in an in vitro system using the accumulated carotenoids as substrates for ZEP1 protein, named 7',8' -didehydronoroxyethyl phycocyanin, according to their similarity to the known compound, dinoxanthin.

Construction of expression vector and protein acquisition

1. Cloning of Phaeodactylum tricornutum ZEP1 Gene cDNA (SEQ ID NO. 1)

Wild Phaeodactylum tricornutum RNA was extracted using RNeasy Plant Minikit kit. Using SuperScript ^TM III Reverse Transcriptase kit reverse transcription was performed. The ZEP1 gene cDNA was cloned by PCR using the obtained total cDNA as a template.

PCR system (table 2):

TABLE 2.20. Mu.L amplification System

PCR cycle:

1)94℃：5min；

2)94℃：30s；

3)55℃：30s；

4)72℃：2min；

steps 2) -4) are cycled 35 times;

6)72℃：5min。

the PCR primers (Table 3) include not only sequences that overlap with the CRTISO5 cDNA sequence (uppercase), but also sequences homologous to the target vector (lowercase).

TABLE 3 ZEP1 cDNA amplification primers

The PCR product was cloned into the pMAL-c5x vector by Information homologous recombination. In the vector, the ZEP1 gene is fused with MBP at the N end, a polyhistidine tag (His-tag) is arranged at the C end, and the expression of the exogenous gene is induced by isopropyl-beta-D-thiogalactoside (IPTG). The constructed plasmid was transformed into the expression BL21 (DE 3) strain and positive clones were screened by PCR. The constructed successful vector is shown in FIG. 4.

2. Expression of Phaeodactylum tricornutum ZEPl gene in Escherichia coli

The strain was cultured at 37℃using LB medium containing 100mg/L of ampicillin until the OD600 was 0.6 to 0.8. Isopropyl- β -D-thiogalactoside (IPTG) was added to a final concentration of 0.4mM and the incubation was continued for 12 hours at 16 ℃.

3. Purification of Phaeodactylum tricornutum ZEP1 protein

After high pressure disruption, the mixture was centrifuged at 13000g at 4℃for 15 minutes. The obtained supernatant was subjected to protein purification using an AKTA system and a Source Q ion exchange column. Protein concentration was performed using a 30kd mwco ultrafiltration tube. Bovine Serum Albumin (BSA) was used as a configuration standard and protein concentration was determined by the bicinchoninic acid method. Split charging to 50 μg per tube, quick freezing with liquid nitrogen, and storing at-80deg.C.

4. Enzymatic functional identification of CRTISO5 protein

7',8' -double dehydrogenation norepoxyfucoxanthin and purified ZEP1 protein are added into an in-vitro system to verify the production of fucoxanthin.

The method comprises the following steps:

1. establishment of enzymatic research system

200. Mu.L of buffer (0.1% TritonX-100, 100mM Tris,10mM MgCl2,1mM DTT,pH7.4) was added to the purified and dried 7',8' -didehydronoroxyaxanthine and the precursor was dissolved by ultrasonic mixing.

Three components were then added to the final concentration in brackets:

flavin adenine dinucleotide (FAD, 100 μm), oxidized form;

nicotinamide adenine dinucleotide phosphate (NADPH; 1 mM);

50 μg of purified polyepoxide.

ZEP1 protein was omitted in the control reaction. After two hours, 200. Mu.L of acetone and 200. Mu.L of ethyl acetate were added and mixed well to terminate the reaction.

Analysis of ZEP1 protein products

The carotenoid product is extracted by centrifugation after termination of the reaction: the organic layer of the supernatant was aspirated, dried with nitrogen and dissolved in 50. Mu.L of aqueous methanol.

The solubilizate was analyzed by High Performance Liquid Chromatography (HPLC) to analyze the production of the fucoxanthin precursor 7',8' -didehydro-dinoxanthin and the residual 7',8' -didehydro-nordinoxanthin. HPLC parameters were as follows:

instrument: waters Acquity UPLC;

chromatographic column: ACQUITYUPLC HSS T31.8.8 μm (3 μm,

2.1×150mm)；

temperature: 45 ℃;

flow rate: 0.3mL per minute;

sample injection volume: 3 μL;

gradient mobile phase: consists of solvent a (acetonitrile: methanol: methyl tert-butyl ether=70:20:10) and solvent B (10 mM ammonium acetate). At 0: 60% A,40% B;4 minutes: 75% A,25% B;12 minutes: 100% A.

As can be seen from FIG. 5, by comparing with the control, after addition of ZEP1 protein, 7',8' -didehydronordinoxanthin is converted into fucoxanthin precursor 7',8' -didehydrofucoxanthin (the substance is subjected to structural analysis by using Bruker AVANCE NEO MHz nuclear magnetic resonance apparatus, the analyzed molecular structure is shown in FIG. 6, and the molecular structure is named as 7',8' -didehydrofucoxanthin according to the similarity with the known compound fucoxanthin), that is, ZEP1 protein has the function of synthesizing fucoxanthin precursor in vitro.

Evidence of penta.7 ',8' -bisdehydromethylfucoxanthin as a precursor to the synthesis of fucoxanthin

1. Cloning of CRTISO5 Gene eDNA (SEQ ID NO. 3) of Phaeodactylum tricornutum

Wild Phaeodactylum tricornutum RNA was extracted using RNeasy Plant Minikit kit. Using SuperScript ^TM III Reverse Transcriptase kit reverse transcription was performed. And cloning the CRTISO5 gene cDNA by using the obtained total cDNA as a template through PCR.

PCR system (table 4):

TABLE 4.20. Mu.L amplification System

PCR cycle:

1)94℃：5min；

2)94℃：30s；

3)55℃：30s；

4)72℃：2min；

steps 2) -4) are cycled 35 times;

6)72℃：5min。

the PCR primers (Table 5) include not only sequences that overlap with the CRTISO5 cDNA sequence (uppercase), but also sequences homologous to the target vector (lowercase).

TABLE 5 CRTISO5 cDNA amplification primers

The PCR product was cloned into the pMAL-c5x vector by Information homologous recombination. In the vector, CRTISO5 gene is fused with MBP at N end, polyhistidine tag (His-tag) is arranged at C end, and exogenous gene expression is induced by isopropyl-beta-D-thiogalactoside (IPTG). The constructed plasmid was transformed into the expression BL21 (DE 3) strain and positive clones were screened by PCR. The constructed successful vector is shown in FIG. 7.

2. Expression of Phaeodactylum tricornutum CRTISO5 Gene in E.coli

The strain was cultured at 37℃using LB medium containing 100mg/L of ampicillin until the OD600 was 0.6 to 0.8. Isopropyl- β -D-thiogalactoside (IPTG) was added to a final concentration of 0.4mM and the incubation was continued for 12 hours at 16 ℃.

3. Purification of Phaeodactylum tricornutum CRTISO5 protein

After high pressure disruption, the mixture was centrifuged at 13000g at 4℃for 15 minutes. The obtained supernatant was subjected to protein purification using an AKTA system and a Source Q ion exchange column. Protein concentration was performed using a 30kd mwco ultrafiltration tube. Bovine Serum Albumin (BSA) was used as a configuration standard and protein concentration was determined by the bicinchoninic acid method. Split charging to 50 μg per tube, quick freezing with liquid nitrogen, and storing at-80deg.C.

Enzymatic functional identification of CRTISO5 protein

7',8' -didehydro-dinoxanthin and purified CRTISO5 protein were added to the in vitro system to verify the production of fucoxanthin. The method comprises the following steps:

4.1 establishment of the enzymatic research System

200. Mu.L of buffer (0.1% TritonX-100, 100mM Tris,10mM MgCl2,1mM DTT,pH 7.4) was added to the purified and dried 7',8' -bisdehydrodinoxanthin and the precursor was dissolved by ultrasonic homogenization.

Three components were then added to the final concentration in brackets:

flavin adenine dinucleotide (FAD, 100 μm), oxidized form;

Na ₂ S ₂ O ₄ (1 mM) to convert FAD to a reduced form;

50 μg of purified CRTISO5 protein.

CRTISO5 protein was omitted in the control reaction. After two hours, 200. Mu.L of acetone and 200. Mu.L of ethyl acetate were added and mixed well to terminate the reaction.

4.2 Analysis of CRTISO5 protein products

The carotenoid product is extracted by centrifugation after termination of the reaction: the organic layer of the supernatant was aspirated, dried with nitrogen and dissolved in 50. Mu.L of aqueous methanol.

The solubilizate was analyzed by High Performance Liquid Chromatography (HPLC) to analyze the production of fucoxanthin and the residual of 7',8' -bisdehydrodinoxanthin. HPLC parameters were as follows:

instrument: waters Acquity UPLC;

chromatographic column: ACQUITY UPLC HSS T31.8 μm (3 μm,

2.1×150mm)；

temperature: 45 ℃;

flow rate: 0.3mL per minute;

sample injection volume: 3 μL;

gradient mobile phase: consists of solvent A (acetonitrile: methanol: methyl tert-butyl ether=70:20:10) and solvent B (10 mM ammonium acetate). At 0: 60% A,40% B;4 minutes: 75% A,25% B;12 minutes: 100% A.

FIG. 8 shows that 7',8' -didehydro-dinoxanthin is converted into fucoxanthin by adding CRTISO5 protein as compared with the control, that is, 7',8' -didehydro-dinoxanthin is a precursor for the synthesis of fucoxanthin.

Sequence listing

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<120> Phaeodactylum tricornutum ZEP1 gene, protein and application

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Asp Asn Asp Val Ile Ser Asn Gln Arg Pro Leu Arg Val Val Ile Ala

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Arg Ile Met His Gly Leu Asp Gln Gly Ala Asp Gly Phe Ala Ala Ser

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Gly Ala Ala Gly Gly Ala Leu Asn Glu Ala Glu Ala Arg Arg Met Ala

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Phe Thr Cys Tyr Ala Ala Leu Thr Glu His Arg Ala Ser Asn Ile Glu

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Lys Leu Thr Arg Leu Leu Gln Glu Phe Asn His Glu Glu Pro Gly Asp

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Gln Asn Gly Asp Val Trp Asp Asp Phe Ala Tyr Glu Leu Phe Lys Ala

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Ala Ala His Pro Met Met Pro Asn Leu Gly Gln Gly Gly Cys Gln Ala

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ttagcgacaa cagctggtag tttgcctttt gtggcaacgc atcctgatgt gtttctcgac 780

ttgtcgctta cgtttgattc tctccacaag acggttgata aaattgtgac ggtccctttc 840

ctccgaaact ttatcgatac catgtgcatt ttctgcggct tcccagccaa gggcgcgatg 900

acggcgcaca tgctttatat cttagagcgc ttctttgaag agtcagcttg ctattctgtt 960

ccgattggag gtacatgcga aatgggaaac acattggtac gcggcttgga aaagtttggt 1020

ggcaaaatcc agttgaatgc tcacgtagac gaaattttgg tcgaaaacgg acgtgccgtg 1080

ggtgttcgtc tcaagaacgg aaatgttgtt aaagcaaaca aagccgtggt gagcaatgcc 1140

acgccttttg ataccgtgaa gatgcttgga gaaaaacaag cacttccaga aggtgtcgcg 1200

aaatggaagg aagagcttgg gaaactccca cgtcacggag cgattatgca tttattttta 1260

gctattgatg cgaaggatct ggacctttcg cacattcaag accccgctca tttagtagtt 1320

caagactggg gacgttcttt acaagactcg cagaacttgt gtagcttctt cattcctagt 1380

ttacttgaca agacgttatg tccggaaggc aagcatgtca ttcatgtata ctcttctgga 1440

ggggaaccgt atgagccgtg ggaaaagctc aagccaggga cacaggagta cgacgattac 1500

aaaaacgaac gcgctaaagt tttgtgggaa gcagtcgaaa ggtgtattcc agatgttcgg 1560

gatcgcttgg aattttccat agtcggatcc cctcttgcac atgaagcctt tcttcgacgt 1620

gatcgaggta cgtatggaat ggcatgggct gctggtacat cagcgcccca ggccggcctt 1680

cttcagaata ttctcccttt cccattccca aaccttaaga caccagtcga tggtctctta 1740

cgatgcggcg actcctgctt tcccggtatc ggaactccaa gtgcggccgc ctcgggagcg 1800

attgcagcga acacaatgaa ccccgtcggc aagcatttag atttgctgaa agaagccagt 1860

caaagagatc ctatgtacaa gtttctggat cctggtgtgt ttggaagtat ttatcgacca 1920

ttcgtcgagt ctttgacgcc aagtaccgaa cttcaggttg aatctatcca aaacactgca 1980

gattag 1986

Claims (1)

1. The application of the protein coded by the Phaeodactylum tricornutum ZEP1 gene in the synthesis of fucoxanthin precursor in vitro is characterized in that the nucleotide sequence of the Phaeodactylum tricornutum ZEP1 gene is shown as SEQ ID NO.1, the fucoxanthin precursor is 7',8' -bisdehydrodinoxanthin, and the molecular structure of the 7',8' -bisdehydrodinoxanthin is as follows:

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