CN114989274A

CN114989274A - Phaeodactylum tricornutum Myb transcription factor PtMYB3 gene and encoding protein and application thereof

Info

Publication number: CN114989274A
Application number: CN202210598993.1A
Authority: CN
Inventors: 范思哲; 严小军; 徐继林; 周成旭; 李政; 李小辉; 张金荣
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2022-05-30
Filing date: 2022-05-30
Publication date: 2022-09-02
Anticipated expiration: 2042-05-30
Also published as: CN114989274B

Abstract

The invention discloses a Phaeodactylum tricornutum Myb transcription factor PtMYB3 gene and its coding protein and application, characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO: 2, the construction method of the gene overexpression recombinant algae comprises the step of designing a primer according to the gene sequence of PtMYB3 and pPha-T1 carrier information; obtaining a PtMYB3 homologous recombination product through PCR amplification, and then constructing the PtMYB3 gene into a pPha-T1 vector through a homologous recombination method to obtain a recombinant plasmid; finally, the recombinant plasmid is transformed into phaeodactylum tricornutum through an electrotransformation method, and recombinant algae with positive over-expression PtMYB3 is obtained through antibiotic screening and expression quantity detection screening; has the advantage of obviously improving the fucoxanthin content in the phaeodactylum tricornutum.

Description

Phaeodactylum tricornutum Myb transcription factor PtMYB3 gene and encoding protein and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a phaeodactylum tricornutum Myb transcription factor PtMYB3 gene and a coding protein and application thereof.

Background

Marine diatoms play an important role in the global carbon cycle. They may account for 40% of the primary productivity of the ocean. In addition, there are many valuable substances in diatoms, such as fucoxanthin. Fucoxanthin is a marine carotene found in diatoms, forms a complex of light capture and chlorophyll, and captures, transfers and converts light energy for photosynthesis. As an important component of this light-harvesting complex, fucoxanthin helps diatoms living in the deep ocean floor to capture enough blue and green light to survive. In addition to its light trapping function, fucoxanthin has been shown to possess a range of other biological activities, including antioxidant, anti-inflammatory, anti-obesity, anti-diabetic and anti-cancer activities. Therefore, the fucoxanthin has wide application prospect.

Fucoxanthin synthesis starts from glyceraldehyde 3-phosphate (G3P) and produces lycopene by 1-deoxy-d-xylose 5-phosphate synthase (DXS), Phytoene Synthase (PSY), Phytoene Desaturase (PDS), zeta-carotene desaturase (ZDS) and carotenoid isomerase (CRTISO). Lycopene is converted into beta-carotene under the catalysis of lycopene beta-cyclase (LCYB), and is finally converted into beta-cryptoxanthin and zeaxanthin under the action of beta-carotene hydroxylase (BCH) or other isoenzymes. Zeaxanthin is converted to anthracenes and then to violaxanthins under catalysis of Zeaxanthin Epoxidase (ZEP). These conversions are reversible under intense light in the presence of ultraviolet xanthine deoxygenase (VDE). Finally, violaxanthin produces fucoxanthin through a series of unknown enzymatic reactions.

Myb transcription factors are ubiquitous in microalgae and play an important role, such as CZMYB1 in Zuofu green algae, which mediates the synthesis of triacylglycerol by regulating the expression of triacylglycerol synthase genes through combining with triacylglycerol synthesis key enzyme gene promoters; in phaeodactylum tricornutum, the Myb transcription factor PtPSR is induced by phosphorus stress and can be combined with a phosphorus metabolism related gene promoter, so that the adaptation of microalgae to phosphorus stress is regulated. However, the role of Myb class transcription factors in the synthesis of microalgal fucoxanthin has not been reported.

Disclosure of Invention

The invention aims to solve the technical problem of providing a Myb transcription factor PtMYB3 capable of obviously improving the fucoxanthin content of Phaeodactylum tricornutum, a coding protein, a cloning method thereof, a construction method of an over-expression recombinant alga and application thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows:

1. a Phaeodactylum tricornutum Myb transcription factor PtMYB3 gene has a nucleotide sequence shown as SEQ ID NO: 1 is shown.

2. A protein coded by a Myb transcription factor PtMYB3 of Phaeodactylum tricornutum has an amino acid sequence shown as SEQ ID NO: 2, respectively.

3. The cloning method of the Phaeodactylum tricornutum Myb transcription factor PtMYB3 gene is characterized by comprising the following steps:

(1) extracting the total RNA of the phaeodactylum tricornutum and performing reverse transcription to obtain cDNA serving as a template;

(2) primers were designed according to the gene sequence of PtMYB 3:

the sequence of the upstream primer is as follows: 5'-ATGCCTTGGACCGCCGACGAAG-3', and the adhesive tape is used for adhering the film to a substrate,

the sequence of the downstream primer is as follows: 5'-TCATGCCAGGTACTTTTCAATT-3', respectively;

(3) and (3) PCR amplification: the amplification product of the PtMYB3 gene is obtained by PCR amplification.

4. The construction method of the phaeodactylum tricornutum Myb transcription factor PtMYB3 overexpression recombinant algae comprises the following steps:

(1) according to the gene sequence of PtMYB3 and pPha-T1 carrier informationCounting primers: the sequence of the upstream primer is as follows: 5' -CGG GGATCCTCTAGAGTCGACATGCCTTGGACCGCCGACGAAG-3', downstream primer sequence: 5' -GATAGCACGCTTCTG AAGCTTTCATGCCAGGTACTTTTCAATT-3', both ends of the primer respectively comprise the sequence of the vector as the homologous arm (the underlined part);

(2) and (3) PCR amplification: obtaining a PtMYB3 gene vector construction amplification product through PCR amplification;

(3) obtaining of recombinant plasmid: constructing the PtMYB3 gene into a pPha-T1 vector by a homologous recombination method to obtain a recombinant plasmid;

(4) obtaining of recombinant alga overexpressing PtMYB 3: and (3) transforming the recombinant plasmid into phaeodactylum tricornutum through an electrotransformation method, and screening through an antibiotic screening and expression quantity detection mode to obtain the recombinant alga with positive over-expression of PtMYB 3.

Preferably, the reaction system for the PCR amplification in step (2) is: mu.L cDNA, 10. mu.L 2 XPrimeSTAR Max Premix, 0.5. mu.L upstream and downstream primers, 8.5. mu.L ddH ₂ O; the PCR amplification procedure was: denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 1min, and 30 cycles; and purifying and recovering the PCR amplification product by using 1wt% agarose gel electrophoresis to obtain a PtMYB3 homologous recombination product.

Preferably, the step (3) is to perform homologous recombination reaction on the pPha-T1 double-enzyme digestion product and the PtMYB3 homologous recombination product, wherein the reaction system is as follows: 2. mu.L of pPha-T1 double cleavage product, 2. mu.L of PtMYB3 PCR homologous recombination product, 2. mu.L of Exnase II, 4. mu.L of 5 × CE II Buffer, 10. mu.L of ddH ₂ O, reacting for 30min at 37 ℃; and E.coli transformation is carried out on the homologous product, and a plasmid containing pPha-T1-PtMYB3 positive bacteria is obtained through identification.

Preferably, in the step (4), the pPha-T1-PtMYB3 plasmid is subjected to plasmid linearization treatment by ScaI, wherein the enzyme cutting system is as follows: 26 mu L of pPha-T1-PtMYB3, 1 mu L of ScaI, 3 mu L of 10 XFastdigest buffer, and enzyme digestion at 37 ℃ for 3 hours, wherein the enzyme digestion product is cleaned and recovered by a glue recovery kit; the density is 2x 10 ⁹ 100 mu l of cell/ml resuspended Phaeodactylum tricornutum cells, 4 mu g linearized pPha-T1-PtMYB3 plasmid and 40 mu g salmon sperm DNA, and after incubation on ice for at least 10min, electroporation is carried out, and cells are immediately transferred to cells containing the linearized pPha-T1-PtMYB3 plasmid and 40 mu g salmon sperm DNAIncubating in a test tube with 10ml of f/2 culture solution for 24h under low light intensity, transferring to normal light intensity for culturing for 24h, centrifuging at 3000g for 10min to collect cells, resuspending the cells with 0.6ml of f/2 culture solution, and screening by bleomycin to obtain primary positive algae; and screening by an expression quantity detection mode to obtain the recombinant alga positively over-expressing PtHSF 1.

5. The application of the Phaeodactylum tricornutum Myb transcription factor PtMYB3 in increasing the content of fucoxanthin in Phaeodactylum tricornutum is provided.

Compared with the prior art, the invention has the advantages that:

1. the fact that the Myb transcription factor PtMYB3 in the phaeodactylum tricornutum is over-expressed can promote the content of fucoxanthin is proved for the first time;

2. the over-expression PtMYB3 can provide a new algae species for the industrial utilization of the phaeodactylum tricornutum to produce fucoxanthin.

Drawings

FIG. 1 is a diagram of the structure of the linearized pPha-T1-PtMYB3 vector, note:PtHSF1is expressed byfcpAPromoter driven bleomycin resistance genesh bleIs expressed byfcpBDriven by a promoter, the screening antibiotic of the vector in the escherichia coli is ampicillin (Amp), and the enzyme used for vector linearization is ScaI;

FIG. 2 shows the antibiotic gene in recombinant algae overexpressing PtMYB3sh bleIdentification, note: T1-T5 are five different strains of PtMYB3 over-expressed recombinant algae, vector represents transgenic phaeodactylum tricornutum transferred into pPha-T1 vector, WT is wild type strain, five over-expressed strains and empty vector are insh bleThe size and position of the fragment are all provided with strips;

FIG. 3 is an analysis of the expression of PtMYB3 gene in PtMYB3 overexpressed recombinant algae, note: asterisks on the columns indicate significant differences in statistical analysis: (tAnd (4) checking the test result,p<0.05), WT represents wild type phaeodactylum tricornutum, vector represents transgenic phaeodactylum tricornutum transferred into pPha-T1 vector, 1# to 5# represent different strains of transgenic phaeodactylum tricornutum transferred into pPha-T1-PtMYB3 vector, wherein 3# and 5# have higher expression level than other transgenic algae strains;

fig. 4 is a growth curve of recombinant algae overexpressing PtMYB3, note: WT represents wild type phaeodactylum tricornutum, vector represents transgenic phaeodactylum tricornutum transferred into pPha-T1 vector, 3# and 5# represent different strains of transgenic phaeodactylum tricornutum transferred into pPha-T1-PtMYB3 vector, the total measurement is 8 days, and the vertical coordinate counting unit is cells/ml; vector plants, WT plants, 5# plants and 3# plants are arranged on the rightmost side of the line in the figure from top to bottom in sequence;

FIG. 5 is a chromatogram analysis of fucoxanthin content in recombinant algae overexpressing PtMYB3, noting that: the peak value of fucoxanthin is 14 min, and the peak area represents the content. WT represents wild type Phaeodactylum tricornutum, and 3# and 5# represent different strains of transgenic Phaeodactylum tricornutum transformed with pPha-T1-PtMYB3 vector;

FIG. 6 is an analysis of fucoxanthin content in recombinant algae overexpressing PtMYB3, note: asterisks on the columns indicate statistically significant differences (tAnd (4) checking the test result,p<0.05), WT represents wild type phaeodactylum tricornutum, 3# and 5# represent different strains of transgenic phaeodactylum tricornutum transferred into pPha-T1-PtMYB3 vector, and the fucoxanthin content of both 3# and 5# over-expression strains is obviously higher than that of wild type strains;

FIG. 7 shows the expression of fucoxanthin synthesis-related genes in recombinant algae overexpressing PtMYB3, note: analysis of fucoxanthin synthesis-related gene expression in PtMYB3 overexpressed recombinant algae: psy: hytoene synthsase; zds: ζ -Carotene desaturase; and (3) Lycb: lycopene-beta-cyclase; criptioso, Carotenoid isomerase; asterisks on the columns indicate statistically significant differences (tAnd (4) checking the test result,p<0.05), WT represents wild type phaeodactylum tricornutum, and # 3 and # 5 represent different lines of transgenic phaeodactylum tricornutum transformed into the ppa-T1-PtMYB 3 vector.

Detailed Description

The invention is described in further detail below with reference to the accompanying examples.

Detailed description of the preferred embodiment

Cloning and sequence analysis of Phaeodactylum tricornutum heat shock transcription factor PtMYB3 gene

1. Extracting Phaeodactylum tricornutum (with algae concentration of 1 × 10) ⁶ cells/ml) and reverse transcribed into cDNA as template.

2. Primers were designed based on the gene sequence of PtMYB3 in Genebank:

the sequence of the upstream primer is as follows: 5'-ATGCCTTGGACCGCCGACGAAG-3', respectively;

the sequence of the downstream primer is as follows: 5'-TCATGCCAGGTACTTTTCAATT-3' is added.

3. And (3) PCR amplification: obtaining an amplification product of the PtMYB3 gene through PCR amplification, wherein the reaction system of the PCR amplification is as follows: mu.L of cDNA, 10. mu.L of 2 XPrimeSTAR Max Premix (PCR amplification kit: 2 XPrimeSTAR Max Premix from Baozhi Bio Inc.), 0.5. mu.L of upstream and downstream primers, 8.5. mu.L of ddH ₂ O; the PCR amplification procedure was: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min, and 30 cycles.

4. And purifying and recovering the PCR amplification product by using 1wt% agarose gel electrophoresis, connecting the PCR amplification product with a pMD19-T vector, further verifying by PCR, and sequencing to obtain the PtMYB3 gene, wherein the nucleotide sequence of the PtMYB3 gene is shown as SEQ ID NO: 1, and the following components: ATGCCTTGGACCGCCGACGAAGACGACCTCATCCGTCGGCACGTACAAAGACACGGACTCAAGGGCTGGACCTTGCTGGCTAAAGAACGCATGCCGGGCCGTCGAGGAAAACAGTGCCGCGAACGATGGATCAATCAGTTGGATCCGGACATTGATCGGACGGGCTGGAGGTTCCCGGAAGATCTCTTTCTATTGCAAGGTCTGACCCGATGGGGTCCGAAATGGGCTTTGATTGCCAAACAGTTGCCCGGACGCCCGGAAAATAACGCCAAGAATCGCTTCAACGCATACCTGCATCCCAAAATTGAAAAGTACCTGGCATGA are provided.

The amino acid sequence of the protein coded by the Phaeodactylum tricornutum heat shock transcription factor PtMYB3 is shown as SEQ ID NO: 2, as shown in the figure: MPWTADEDDLIRRHVQRHGLKGWTLLAKERMPGRRGKQCRERWINQLDPDIDRTGWRFPEDLFLLQGLTRWGPKWALIAKQLPGRPENNAKNRFNAYLHPKIEKYLA are provided.

Detailed description of the invention

PtMYB3 overexpression recombinant algae

1. Designing primers according to the gene sequence of PtMYB3 and pPha-T1 vector information: the sequence of the upstream primer is as follows: 5' -CGGG GATCCTCTAGAGTCGACATGCCTTGGACCGCCGACGAAG-3', downstream primer sequence: 5' -GATAGCACGCTTCTGA AGCTTTCATGCCAGGTACTTTTCAATT-3', the two ends of the primer respectively comprise the sequence of the vector as the homologous arms (the underlined positions).

2. And (3) PCR amplification: obtaining a PtMYB3 homologous recombination product through PCR amplification, wherein the reaction system of the PCR amplification is as follows: 0.5. mu.L of cDNA, 10. mu.L of 2 XPrimeSTAR Max Premix (PCR amplification kit: 2 XPrim)eSTATAR Max Premix from Baozchen Bio Inc.), upstream and downstream primers were 0.5. mu.L, 8.5. mu.L ddH ₂ O; the PCR amplification procedure was: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min, and 30 cycles; the PCR amplification product was purified and recovered by 1wt% agarose gel electrophoresis.

3. Vector construction: pPha-T1 was double digested with SalI and HindIII in the following system: mu.L of pPha-T1, 1. mu.L of SalI, 1. mu.L of HindIII, 3. mu.L of 10 XFastdigest buffer (Thermo Scientific), digested at 37 ℃ for 3 hours, and the digested products were recovered by washing with a gel recovery kit.

Carrying out homologous recombination reaction on the pPha-T1 double-enzyme digestion product and the PtMYB3 homologous recombination product, wherein the reaction system is as follows: 2. mu.L of pPha-T1 double cleavage product, 2. mu.L of PtMYB3 PCR homologous recombination product, 2. mu.L of Exnase II, 4. mu.L of 5 × CE II Buffer, 10. mu.L of ddH ₂ O, reacting at 37 ℃ for 30 min. And transforming the homologous product into escherichia coli, and identifying to obtain a plasmid containing pPha-T1-PtMYB3 positive bacteria.

4. Converting phaeodactylum tricornutum: the pPha-T1-PtMYB3 plasmid was extracted and linearized with ScaI (FIG. 1), the enzyme cut was: 26. mu.L of pPha-T1-PtMYB3, 1. mu.L of ScaI, 3. mu.L of 10 XFastdigest buffer (Thermo Scientific), digested at 37 ℃ for 3 hours, and the digested products were recovered by washing with a gel recovery kit.

Phaeodactylum tricornutum grows to logarithmic phase density of 4 × 10 ⁶ -5×10 ⁶ cell/ml, collected at 4 ℃ for 10min at 3000g centrifugation for a total of 2X 10 ⁸ The cells were resuspended 3 times in 1ml of 375mM sorbitol (sterile frozen) and finally in 100. mu.l of 375mM sorbitol to a final density of 2X 10 ⁹ cell/ml, put on ice for use. 100 μ l of resuspended algal cells were incubated with 4 μ g (0.2 μ g/μ l) of linearized pPha-T1-PtMYB3 plasmid and 40 μ g (10 μ g/μ l) salmon sperm DNA (1 min water bath boiling above 95 ℃) on ice for at least 10min, then transferred into a 2mm electroporation cuvette and electroporated using a bio-rad electroporator. The electroporation system was adjusted to exponential decay, 0.5kv field strength, 25 muf capacitance and 400 ohm parallel resistance. Cells were transferred to a 15ml assay containing 10ml f/2 medium immediately after electroporationIn tubes, at low light intensity (30. mu. mol. m) ^-2 .s ^-1 ) And (4) incubating for 24h, culturing for 24h under the condition of transferring to normal light intensity, then centrifuging at 3000g for 10min to collect cells, resuspending the cells by using 0.6ml f/2 culture solution, averagely coating the cells on 3 solid culture media containing 75 mug/ml bleomycin, and carrying out algae shedding after 15-25d, wherein the next step of treatment can be carried out. FIG. 2 shows the antibiotic gene in recombinant algae overexpressing PtMYB3sh bleIdentification, note: T1-T5 are five different strains of PtMYB3 over-expressed recombinant algae, vector represents transgenic phaeodactylum tricornutum transferred into pPha-T1 vector, WT is wild type strain, and it can be known from FIG. 2 that five over-expressed strains and empty vector are in the recombinant algaesh bleThe size and position of the fragment show a band, which indicates the antibiotic gene in the transgenic algash bleSuccessful expression was achieved, which initially demonstrated the success of the transgene.

5. PtMYB3 overexpression positive algae identification

Transgenic algal cells were cultured with normal shaking (150 r/min) in selective liquid f/2 medium containing 50 μ g/mL bleomycin as a pre-culture to reach log phase. 50ml of logarithmic phase algal solution was centrifuged at 4000 Xg for 5min at 4 ℃ and the supernatant was discarded, and the resulting precipitate was snap-frozen in liquid nitrogen. Ground to a frozen powder in liquid nitrogen, and Total RNA was extracted using RNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA, USA)/Total RNA Kit I/miRNA Isolation Kit. 500ng of RNA was reverse transcribed into cDNA using PrimeScript RT Reagent Kit (Perfect Reagent Time) (Takara Bio, Otsu, Japan). The cDNA samples were diluted to 80 ng/. mu.L with TE buffer and stored at-20 ℃. qRT-PCR was performed on a LightCycler 96 Real-Time PCR system (Roche, Basel, Switzerland) using a 2X SYBR Green I PCR Master Mix (Applied Biosystems, Calif., USA). The forward and reverse primers used in (table 1) thermocycling conditions were as follows: denaturation at 95 ℃ for 2min, 40 cycles of 95 ℃ for 5s, 60 ℃ for 30 s; and 95 ℃ for 5s, 65 ℃ for 5s, 95 ℃ for 5 s. Each qRT-PCR sample has three repeats, actin gene is used for internal reference, and 2 is adopted for gene expression analysis ^-ΔΔCt The method of (4).

TABLE 1 quantitative PCR primers used in this patent

As shown in the expression situation of PtMYB3 gene in the PtMYB3 overexpression recombinant algae in FIG. 3, the expression level of different strains of transgenic phaeodactylum tricornutum transferred into pPha-T1-PtMYB3 vector is obviously increased compared with that of a control group, wherein 3# and 5# are higher than those of other transgenic algae strains.

FIG. 4 is a growth curve of recombinant algae overexpressing PtMYB3, and it can be seen from FIG. 4 that the growth of the strain overexpressing PtMYB3 is similar to that of the control group, indicating that PtMYB3 does not regulate the growth of microalgae.

Detailed description of the preferred embodiment

Fucoxanthin content in PtMYB3 over-expressed algae

According to the method of Kwon et al and modified. 10mL of crude liquid of the transgenic alga at the early stage of the logarithm was collected and the density of each transgenic alga was recorded on a hemocytometer, centrifuged at 4000 Xg for 10min at 4 ℃ and the supernatant was discarded. Adding 10mL ethanol, mixing, and extracting fucoxanthin at 24-30 deg.C by ultrasonic (New Zhi brand ultrasonic instrument) for 1 h. The supernatant (0.22 μm) was filtered for HPLC analysis. The whole process is carried out in the dark. An Agilent 1200 HPLC system (Agilent Technologies, usa) 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, methanol and water, was run at 0.7 mL min ^-1 The flow rate of (2) is eluted at 35 ℃. Columns of YMC carotenoids (250 mm long X4.6 mm inner diameter; 5 μm particle size; Waters, USA) were used for separation under the following gradient program: methanol was increased from 90% to 100% for 20 minutes, held at 100% for the next 5 minutes, decreased to 90% for 5 minutes, and then held at 90% for 5 minutes. A sample solution (10. mu.L) was injected and the chromatogram recorded at 445 nm. Based on the concentration range of 0.5-50 mug mL ^-1 The fucoxanthin was quantified by the calibration curve of (2).

The results in fig. 5 and 6 show that the fucoxanthin content of both PtMYB3 overexpression lines 3# and 5# are significantly higher than that of the wild type strain.

Quantitative PCR primers of the genes related to fucoxanthin synthesis are designed (see the primers in Table 1), and the gene expression is detected by quantitative PCR, and the gene expression result in FIG. 7 shows that the expression of the genes related to fucoxanthin synthesis in the PtMYB3 overexpression strain is also obviously higher than that of a control group (Psy: hytoene synthsase; Zds: zeta-Carotene desaturase; Lyccb: Lyccopene-beta-cyclase; Crotiso: Carotenoid isomerase). These results indicate that PtMYB3 overexpression can increase fucoxanthin content in phaeodactylum tricornutum.

The above description is not intended to limit the invention, nor is the invention limited to the examples set forth above. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.

Sequence listing

<110> Ningbo university

<120> Phaeodactylum tricornutum Myb transcription factor PtMYB3 gene and encoding protein and application thereof

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 324

<212> DNA

<213> Phaeodactylum tricornutum heat shock transcription factor PtMYB3 gene (ATGCCTTGGACCGCCGACGAAGACGACCTCATCCGTCGGCACGTACAAAGACACGGACTCAAGGGCTGGACCTTGCTGGCTAAAGAACGCATGCCGGGCCGTCGAGGAAAACAGTGCCGCGAACGATGGATCAATCAGTTGGATCCGGACATTGATCGGACGGGCTGGAGGTTCCCGGAAGATCTCTTTCTATTGCAAGGTCTGACCCGATGGGGTCCGAAATGGGCTTTGATTGCCAAACAGTTGCCCGGACGCCCGGAAAATAACGCCAAGAATCGCTTCAACGCATACCTGCATCCCAAAATTGAAAAGTACCTGGCATGA)

<400> 1

<210> 2

<211> 107

<212> RNA

<213> protein encoded by the Heat shock transcription factor PtMYB3 of Phaeodactylum tricornutum (MPWTADEDDLIRRHVQRHGLKGWTLLAKERMPGRRGKQCRERWINQLDPDIDRTGWRFPEDLFLLQGLTRWGPKWALIAKQLPGRPENNAKNRFNAYLHPKIEKYLA)

<400> 2

<210> 3

<211> 22

<212> DNA

<213> gene upstream amplification primer of PtMYB3 (5'-ATGCCTTGGACCGCCGACGAAG-3')

<400> 3

<210> 4

<211> 22

<212> DNA

<213> gene downstream amplification primer of PtMYB3 (5'-TCATGCCAGGTACTTTTCAATT-3')

<400> 4

<210> 5

<211> 43

<212> DNA

<213> Gene sequence of PtMYB3 and upstream primer of pPha-T1-GFP vector (5'-CGGGGATCCTCTAGAGTCGACATGCCTTGGACCGCCGACGAAG-3')

<400> 5

<210> 6

<211> 43

<212> DNA

<213> Gene sequence of PtMYB3 and downstream primer of pPha-T1-GFP vector (5'-GATAGCACGCTTCTGAAGCTTTCATGCCAGGTACTTTTCAATT-3')

<400> 6

<210> 7

<211> 21

<212> DNA

<213> Forward amplification primer for Actin Gene quantitative PCR (AGGCAAAGCGTGGTGTTCTTA)

<400> 7

<210> 8

<211> 21

<212> DNA

<213> reverse amplification primer for Actin Gene quantitative PCR (TCTGGGGAGCCTCAGTCAATA)

<400> 8

<210> 9

<211> 22

<212> DNA

<213> forward amplification primer for quantitative PCR of Lycb gene (GTACGCAATGCCAATAAAGGAT)

<400> 9

<210> 10

<211> 22

<212> DNA

<213> reverse amplification primer for Lycb Gene quantitative PCR (AAACAACGATCTTTGCATTCCT)

<400> 10

<210> 11

<211> 22

<212> DNA

<213> Forward amplification primer for Psy Gene quantitative PCR (GTCTATGTTTGGTGTCGACGAA)

<400> 11

<210> 12

<211> 22

<212> DNA

<213> reverse amplification primer for Psy Gene quantitative PCR (AAGCACAGGTCAAAGACATCCT)

<400> 12

<210> 13

<211> 22

<212> DNA

<213> forward amplification primer for quantitative PCR of Zds Gene (GTTTACAGCAATGGGAGGTAGC)

<400> 13

<210> 14

<211> 22

<212> DNA

<213> reverse amplification primer for quantitative PCR of Zds Gene (CATGCATCGGGCACTTATACTA)

<400> 14

<210> 15

<211> 22

<212> DNA

<213> Forward amplification primer for Crtiso Gene quantitative PCR (GAGGATCGGCTCATACATTCTC)

<400> 15

<210> 16

<211> 22

<212> DNA

<213> reverse amplification primer for Crtiso Gene quantitative PCR (GCATCTCTTCTTCCAGGACATC)

<400> 16

<210> 17

<211> 21

<212> DNA

<213> forward amplification primer for PtMYB3 gene quantitative PCR (CTTTGATTGCCAAACAGTTGC)

<400> 17

<210> 18

<211> 22

<212> DNA

<213> reverse amplification primer of PtMYB3 gene quantitative PCR (ACTTTTCAATTTTGGGATGCAG)

<400> 18

Claims

1. A Phaeodactylum tricornutum Myb transcription factor PtMYB3 gene is characterized in that: the nucleotide sequence of the gene is shown as SEQ ID NO: 1 is shown.

2. A protein coded by a Myb transcription factor PtMYB3 of Phaeodactylum tricornutum is characterized in that: the amino acid sequence of the protein is shown as SEQ ID NO: 2, respectively.

3. The cloning method of the phaeodactylum tricornutum Myb transcription factor PtMYB3 as defined in claim 1, comprising the following steps:

(2) primers were designed according to the gene sequence of PtMYB 3:

the sequence of the upstream primer is as follows: 5'-ATGCCTTGGACCGCCGACGAAG-3' the flow of the air in the air conditioner,

4. The construction method of the phaeodactylum tricornutum Myb transcription factor PtMYB3 overexpression recombinant algae of claim 1, which is characterized by comprising the following steps:

(1) designing primers according to the gene sequence of PtMYB3 and pPha-T1 vector information: the sequence of the upstream primer is as follows: 5'-CGGGGATCCTCTAGAGTCGACATGCCTTGGACCGCCGACGAAG-3', downstream primer sequence: 5'-GATAGCACGCTTCTGAAGCTTTCATGCCAGGTACTTTTCAATT-3', the two ends of the primer respectively comprise the sequences of the vector as homologous arms;

5. The method for constructing phaeodactylum tricornutum Myb transcription factor PtMYB3 overexpressed recombinant algae according to claim 4, wherein the PCR amplification reaction system in the step (2) is as follows: mu.L cDNA, 10. mu.L 2 XPrimeSTAR Max Premix, 0.5. mu.L upstream and downstream primers, 8.5. mu.L ddH ₂ O; the PCR amplification procedure was: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min, and 30 cycles; and purifying and recovering the PCR amplification product by using 1wt% agarose gel electrophoresis to obtain a PtMYB3 homologous recombination product.

6. The method for constructing phaeodactylum tricornutum Myb transcription factor PtMYB3 overexpressed recombinant algae according to claim 4, wherein the step (3) is specifically to perform homologous recombination reaction on the pPha-T1 double enzyme digestion product and the Ptmyb3 homologous recombination product, and the reaction system is as follows: 2. mu.L of pPha-T1 double cleavage product, 2. mu.L of PtMYB3 PCR homologous recombination product, 2. mu.L of Exnase II, 4. mu.L of 5 × CE II Buffer, 10. mu.L of ddH ₂ O, reacting for 30min at 37 ℃; and E.coli transformation is carried out on the homologous product, and a plasmid containing pPha-T1-PtMYB3 positive bacteria is obtained through identification.

7. The method for constructing phaeodactylum tricornutum Myb transcription factor PtMYB3 overexpressed recombinant algae according to claim 4, wherein the method comprises the step (4)Specifically, the pPha-T1-PtMYB3 plasmid was linearized with ScaI, and the digestion system was: 26 mu L of pPha-T1-PtMYB3, 1 mu L of ScaI, 3 mu L of 10 XFastdigest buffer, and performing enzyme digestion at 37 ℃ for 3 hours, wherein the enzyme digestion product is cleaned and recovered by using a gel recovery kit; the density is 2x 10 ⁹ carrying out electroporation after 100 mu l of cell/ml heavy suspension on Phaeodactylum tricornutum cells, 4 mu g of linearized pPha-T1-PtMYB3 plasmid and 40 mu g of salmon sperm DNA are incubated on ice for at least 10min, immediately transferring the cells after electroporation into a test tube containing 10ml f/2 culture solution, incubating for 24h under low light intensity, then transferring to culture for 24h under normal light intensity, then centrifuging for 10min at 3000g, collecting the cells, carrying out heavy suspension on the cells by using 0.6ml f/2 culture solution, and screening by using bleomycin to obtain primary positive algae; and screening by an expression quantity detection mode to obtain the recombinant alga positively overexpressing PtHSF 1.

8. The use of the Phaeodactylum tricornutum Myb transcription factor PtMYB3 of claims 1-7 for increasing the content of fucoxanthin in Phaeodactylum tricornutum.