CN116926068A - Application of transcription mediator MED31 gene and interaction transcription factor thereof in improving fucoxanthin content in Phaeodactylum tricornutum - Google Patents

Abstract

The invention discloses an application of a transcription mediator MED31 gene and an interaction transcription factor thereof in improving the content of fucoxanthin in Phaeodactylum tricornutum, which is characterized in that the application of the transcription mediator MED31 gene in improving the content of fucoxanthin in Phaeodactylum tricornutum has the nucleotide sequence shown in SEQ ID NO:1, wherein the strong interaction transcription factor of the transcription mediator MED31 gene comprises a MYB2R1 gene, and the application of the strong interaction transcription factor MYB2R1 gene in improving the fucoxanthin content in Phaeodactylum tricornutum is shown in SEQ ID NO:3 is shown in the figure; the application of the strong interaction transcription factor MYB2R1 gene in improving the content of fucoxanthin in Phaeodactylum tricornutum has the advantage of obviously improving the content of fucoxanthin in Phaeodactylum tricornutum.

Description

Application of transcription mediator MED31 gene and interaction transcription factor thereof in improving fucoxanthin content in Phaeodactylum tricornutum

Technical Field

The invention belongs to the field of plant genetic engineering, and in particular relates to a transcription mediator MED31 gene, an interactive transcription factor, a coding protein and application thereof.

Background

In recent years, the use of ocean resources has become increasingly important. Marine diatoms play an important role in the global carbon cycle. Fucoxanthin is a carotenoid, has various biological activities such as antioxidation, anti-inflammatory, anti-obesity, anti-diabetes, anticancer and the like, has wide development prospect in the fields of food, medicine, cosmetology and the like, and has important research significance.

Genetic information of biological organisms is stored in the form of genes on DNA molecules. Transcription is the process by which a DNA sequence is catalyzed by a transcription complex consisting of an RNA polymerase and a transcription cofactor to produce an RNA sequence complementary thereto. Genetic information is transferred from DNA to RNA by transcription. Transcription is one of the most important steps in the central laws of molecular biology, the first step in gene expression. Transcription is therefore a prerequisite for almost every biological process in an organism.

Transcription in prokaryotes is performed in the cytosol and the translation of the protein is performed simultaneously therewith. During the transcription of prokaryotes, the transcription initiation factor sigma factor recognizes the initiation site on the template DNA, where it binds to the RNA polymerase to form a holoenzyme DNA-complex, thereby initiating transcription. The way in which an activator or inhibitor protein acts in the transcriptional regulation of a prokaryote is direct, i.e.directly acts on RNA polymerase. Transcriptional regulation in eukaryotes is more elaborate, but the basic constitution of both are identical, i.e., the presence of RNA polymerase. However, unlike prokaryotes, eukaryotic transcription must be initiated by transcription factors that first recognize the promoter sequence and thus mediate the binding of RNA polymerase to the promoter region. Transcription factors in eukaryotes include gene-specific transcription factors and some common transcription factors (GeneralTranscriptionFactor, GTF). Furthermore, the most prominent difference between eukaryotic and prokaryotic transcription is that this signaling between transcription factors and RNA polymerase is not direct. In eukaryotes, there are a number of intermediate components between them, including chromatin and mediators, which represent a new layer of mediators between maintenance transcriptional regulator proteins and RNA polymerase. Also, more elaborate and complex transcriptional regulation is possible in eukaryotes. During gene expression, RNA polymerase II (Pol II) and the general transcription factor GTFs (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH) are recruited to the promoter of the target gene to form a Pre-initiation complex (Pre-Initiation Complex, PIC) to initiate transcription. Proteins controlling transcription of eukaryotic genes include, in addition to Pol II and the general transcription factors, various activators and inhibitors which bind specifically to the promoter and enhancer sequences of the target gene, and various cofactors which exert a bridging or enzymatic action between the transcription factor and the pre-initiation complex. Mediators as bridges are unique to eukaryotes and are involved in almost all Pol II-mediated transcriptional regulation.

The mediator was originally discovered in yeast by the professor Kornberg, laboratories. One has been looking for a phenomenon in which overexpression of one transcriptional activator instead inhibits transcription downstream of the other transcriptional activator, called squelching, that is, two transcriptional activators may competitively bind to a factor necessary for transcription. It is thought of, of course, that such transcription factors are either general transcription factors or subunits of RNA polymerase II itself. This idea was confirmed in some experimental systems, but it was challenged when in vitro experiments were performed with crude yeast extracts. On the one hand, the addition of either universal transcription factor or RNAPol II itself does not alleviate the squelching; on the other hand, the addition of small portions of crude yeast extract can alleviate squelching. The active substance in such small portions of crude yeast extract is then called an mediator.

Because of the huge molecular weight of the mediator, the internal composition of the mediator is easy to change, the content of the whole complex in the body is low, and the like, the structure of the mediator is not well analyzed. About 80% of the mediator structure has not been resolved, nor is the details of the interaction of the mediator with RNA polymerase known. Therefore, in the future, the structure of the mediator can be better revealed through structural biology to analyze the structure of the mediator, so that the mechanism of gene transcription regulation is deeply known. The mediator serves not only as a bridge linking transcription factors to the RNA polymerase II transcription machinery, but it is involved in a number of biological processes associated with transcription. The mediator may be a transcriptional activator, a transcriptional repressor or a generic class of transcription factors. At present, no related research report on the influence of transcription mediator MED31 gene editing Phaeodactylum tricornutum on fucoxanthin production is reported at home and abroad.

Disclosure of Invention

The invention aims to provide an application of a transcription mediator MED31 gene and an interaction transcription factor thereof in improving the content of fucoxanthin in Phaeodactylum tricornutum, wherein the transcription mediator MED31 gene and the interaction transcription factor thereof can obviously improve the content of fucoxanthin in Phaeodactylum tricornutum.

The technical scheme adopted for solving the technical problems is as follows: an application of a transcription mediator MED31 gene in improving the content of fucoxanthin in Phaeodactylum tricornutum, wherein the nucleotide sequence of the gene is shown in SEQ ID NO: 1.

Application of the transcription mediator MED31 in improving the content of fucoxanthin in Phaeodactylum tricornutum, wherein the amino acid sequence of the protein is shown in SEQ ID NO: 2.

The strong interaction transcription factor of the transcription mediator MED31 gene is MYB2R1 gene.

The application of the strong interaction transcription factor MYB2R1 gene in improving the content of fucoxanthin in Phaeodactylum tricornutum.

Application of the strong interaction transcription factor MYB2R1 gene in improving the content of fucoxanthin in Phaeodactylum tricornutum is provided, and the nucleotide sequence of the gene is shown as SEQ ID NO: 3.

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

1. the first demonstration shows that through screening the gene library of Phaeodactylum tricornutum, the transcription mediator MED31 is found, and after CRISPR gene knockout is carried out on the transcription mediator MED31, the MED31 mutant strain is found to have phenotype change in fucoxanthin synthesis;

2. the research is further conducted, MED31 is taken as a bait to screen interactive transcription factors in the Phaeodactylum tricornutum cDNA library, the transcription factors 14143 (MYB 2R 1) with phenotype in fucoxanthin synthesis are selected, the mechanism of fucoxanthin synthesis is regulated and controlled by the MED31 cooperative transcription factors, a deeper basis is provided for fucoxanthin synthesis of Phaeodactylum tricornutum, and a foundation is provided for industrialized production of fucoxanthin.

Drawings

FIG. 1 is a sequence chromatogram of the knockout results of the gene editing algae strain with WT cells and colonies containing targeted mutant cells. (a) knockdown identification of two genetically engineered algal strains; (b) sequence chromatograms generated by wild-type sequencing; (c) sequence chromatograms generated by MED31-2 sequencing; (d) sequence chromatograms generated by MED31-7 sequencing;

FIG. 2 shows growth curves of two MED31 gene-edited algae strains and wild WT algae;

fig. 3 shows that the fucoxanthin content of two MED31 gene-edited strains and WT shows significant differences between experimental and WT groups (P < 0.05);

FIG. 4 shows the expression levels of fucoxanthin synthesis genes of two MED31 gene-editing algae strains and WT. (a) the amount of ZDS expressed; (b) ZEP3 expression level; (c) CRTISO3 expression level; (d) PDS2 expression level. * Significant differences between experimental and WT groups are indicated (< 0.05: P < 0.01: P < 0.001);

FIG. 5 shows a yeast double hybrid assay in which 300 interacting proteins were co-screened, 15 transcription factors;

FIG. 6 shows the results of yeast double-impurity experiments of MED31-BD and the interaction factor MYB2R 1-AD;

FIG. 7 shows the expression levels of MYB2R1 gene in MYB2R1 overexpressing transgenic algae and WT, showing significant differences between experimental and control groups (P < 0.05; P < 0.0001)

FIG. 8 is a growth curve of MYB2R1 overexpressing transgenic algae versus WT;

fig. 9 is the fucoxanthin content of MYB2R1 overexpressing transgenic algae versus WT, which represents significant differences between experimental and WT groups (P < 0.01).

Detailed Description

The invention is described in further detail below with reference to the embodiments of the drawings.

Detailed description of the preferred embodiments

Transcription mediator MDE31 gene cloning and sequence analysis

1. Using RNA extraction kitExtraction of plant RNAkit (algae concentration 1×10) from Phaeodactylum tricornutum in log phase ⁶ cells/ml) and reverse transcription was performed with reference to the instructions of PrimeScriptTM RTReagent Kit (cat No. RR 037A) using the extracted RNA as a reactant to obtain a cDNA template.

2. Primers were designed based on the gene sequence of MED 31:

upstream primer sequence: 5'-ATGTCGGAAGGGGAACCGAC-3' the number of the individual pieces of the plastic,

downstream primer sequence: 5'-TCAATTGTTCACCGCCGACTC-3';

3. and (3) PCR amplification: obtaining an MDE31 gene amplification 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 2X PrimeSTARMax Premix, 0.5. Mu.L of each of the 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, 72℃for 1min,30 cycles;

4. purifying and recovering the PCR amplified product by using 1% agarose gel electrophoresis, connecting the PCR amplified product with a pMD19-T vector, further verifying by PCR, and sequencing to obtain the MDE31 gene, wherein the nucleotide sequence of the MDE31 gene is shown as SEQ ID NO:1 is shown as follows: ATGTCGGAAGGGGAACCGACCAAGCCCTTGGCGCCGACGAATCCTTTGCCGGAAAACCGGTTCGAGCTGGAACTGGAATTCGTTCAGGCGCTCGCATCGCCGGCGTATCTACACTTTTTGGCCACGTCGAGAGCCGAAGAAGACGGCAAGCTTTTTCTGCAAGATTCTTCCTTCCAACAGTATCTTCGCTATCTTTTCGATACGTGGAGCCGCCCGGAATACGCGAGGTTTTTGTCGTATCCTCACGCTCTGTACTTTCTCGAGTTGCTGATTGAGAAGCCCACTGTCTTGAAGGAATGGAGTTTGCCAGCCTTTCGGAACTTTTGTCACCGGCAACAATTTTTATCCTGGCAACATCGCCACGAGTCTCTCTACGGTAAGGGAACAGTCCCCGCTGCTATGGACCCGAAGATCGACGGTACTACTTCTCCCGTCGTACCTAGAGCAGACGAAGAGTCGGCGGTGAACAATTGA;

MED31 has an amino acid sequence shown in SEQ ID NO:2 is shown as follows:

MSEGEPTKPLAPTNPLPENRFELELEFVQALASPAYLHFLATSRAEEDGKLFLQDSSFQ

QYLRYLFDTWSRPEYARFLSYPHALYFLELLIEKPTVLKEWSLPAFRNFCHRQQFLSW QHRHESLYGKGTVPAAMDPKIDGTTSPVVPRADEESAVNN。

second embodiment

1. Construction of MED31 knockout vector

2MED31 knockout vectors (CRISPR-MED 31-1/2) were constructed using the pPtPuc3-Cas9-sgRNA (Addgene ID: 109219) vector as the CRISPR/Cas9 expression cassette, as follows:

1. the MED31 target gene to be knocked out is selected, and two groups of sgRNA sequences are obtained by using online sgRNA design software, wherein the sequences are shown in table 1:

TABLE 1 two sets of sgRNA sequences

2. Adding enzyme cutting sites to the sgRNA sequence obtained in the step 1 and synthesizing a single-stranded primer, wherein the single-stranded primer sequences are shown in the following table 2, and the two pairs of sgRNA single-stranded primer sequences of the MED31 in the table 2

3. Annealing the paired single-stranded primers in the step 2 to obtain corresponding double-stranded DNA fragments, wherein the reaction system is 10 XBuffer, and 1 mu L;10 μm MED31-fw, 1. Mu.L, 10 μm MED31-rv 1. Mu.L, with ddH ₂ O is added to 10 mu L, the reaction system is mixed uniformly, the reaction is carried out for 10min at 85 ℃ in a program, and the double-stranded DNA is obtained after cooling for 90min at room temperature.

4. The 2 double-stranded DNAs obtained in step 3 were ligated with pPtPuc3-Cas9-sgRNA linear plasmid with T4 ligase, respectively, at 16℃for 1h, with a ligation system of 1. Mu.L of T410 XBuffer, 4. Mu.L of pPtPuc3-Cas9-sgRNA linear plasmid, 3.5. Mu.L of double-stranded DNA, and 0.5. Mu.L of the double-stranded DNAT4 DNA ligase, complemented to 10 mu L with water to obtain 2 recombinant vectors named MED31-sgRNA recombinant vectors; the preparation method of the linear plasmid comprises the following steps: mu.L of 1. Mu.g/. Mu.L of pPtPuc3-Cas9-sgRNA vector (109219) was added to 5. Mu.L of 10 XBuffer, 1. Mu.L of restriction enzyme, and ddH was used ₂ O is complemented to 50 mu L, the reaction system is evenly mixed, the mixture is reacted for 1h in a constant temperature incubator at 37 ℃, and then the pPtPuc3-Cas9-sgRNA linear plasmid is obtained after recovery and concentration detection.

5. E.coli transformation is carried out on the MED31-sgRNA recombinant vector, and the specific steps are as follows

(1) Adding 10 mu L of 2MED 31-sgRNA recombinant vectors obtained in the step 4 into competent cells of escherichia coli DH5 alpha respectively, and placing on ice for 30min; slightly shaking the bacteria, performing heat shock in a water bath at 42 ℃ for 60 seconds, and then rapidly standing on ice for 3-5min;

(2) Then 400. Mu.L of LB liquid medium without antibiotics is added respectively and mixed gently, and then the mixture is cultured for 1h in a shaking table at 37 ℃ and 220 rpm; wherein the LB liquid medium consists of: 5g/L of yeast extract, 10g/L of tryptone and 10g/L of sodium chloride, and water as a solvent;

(3) 100. Mu.L of the mixture obtained in the step (2) was added to a mixture containing ampicillin (Amp ⁺ ) Uniformly coating the LB solid culture medium of the antibiotics until bacterial liquid is dried on a flat plate, sealing by a sealing film, marking, and culturing in a constant temperature incubator at 37 ℃ for 12-16 hours;

(4) Taking the overnight cultured flat plate out of the incubator, and observing the colony condition on the flat plate; if single colonies were present, 8 single colonies were picked on an ultra clean bench and each loaded with 1mL of the sample containing ampicillin (Amp ⁺ ) Culturing in LB liquid medium of antibiotics, shaking culturing at 220rpm in a shaking table at 37deg.C for 3 hr, respectively performing bacterial liquid PCR identification, and taking a segment of sequence on pPtPuc3-Cas9-sgRNA vector as reverse identification primer (CRISPR-rv: CAGGAAACAGCTATGACC), PCR was performed using MED31-fw as a forward identification primer in Table 2. The reaction system and the reaction procedure are shown in tables 3 and 4 below,

TABLE 3 bacterial liquid PCR System

TABLE 4 bacterial liquid PCR procedure

The result was checked by gel electrophoresis of the obtained PCR product, and a sample with a positive band was taken and subjected to amplification culture (100. Mu.L of bacterial liquid and 4mL of ampicillin (Amp) ⁺ ) LB liquid medium for antibiotics) and use thereofPlasmid Mini Kit II Plasmid extraction Kit extracts plasmids to obtain two MED31 gene knockout vectors.

2. Preparation of MED31 gene knockout algae strain

1. The EcoNI is selected to carry out linearization enzyme digestion on two MED31 gene knockout vectors respectively, and the enzyme digestion system is as follows: 5. Mu.L of 10 XBuffer, 3. Mu.L of EcoNI, 6. Mu.g of MED31 gene knockout vector, and after the completion of the digestion treatment for 3 hours, the vector was purified by using a gel recovery extraction kit (Gel Extraction Kit) and the product was recovered by the digestion to obtain two MED31 gene knockout vector linear plasmids.

2. The phaeodactylum tricornutum and the linear plasmid of the MED31 gene knockout vector are subjected to electric transformation, and the specific steps are as follows:

(1) Culturing Phaeodactylum tricornutum to logarithmic phase 1×10 ⁶ centrifuging at 4deg.C for 10min at cells/mL and 1500 Xg, discarding supernatant; washing the pellet with 1mL 37mM sorbitol (sterile, pre-chilled on ice) 3 times, and resuspending the washed cells with 100. Mu.L 375mM sorbitol to give a final density of 2X 10 in the resuspension solution ⁹ cells/mL；

(2) 100. Mu.L of the resuspension solution, 4. Mu.g (0.2. Mu.g/. Mu.L) of the MED31 gene knockout vector linear plasmid purified in step 1, 40. Mu.g (10. Mu.g/. Mu.L) of salmon sperm DNA (boiled at high temperature for 10 min) were mixed together, incubated on ice for 10min, then transferred to a 0.2cm pre-chilled electroporation cuvette, and parameters were set: the electric shock conversion is carried out on the field intensity of 500V, the capacitance of 25 mu F and the parallel resistor of 400 omega;

(3) Electric powerImmediately after transformation, the cells were transferred to an f/2 liquid medium containing 10mL, under low light (about 30. Mu. Mol/m ² S) after incubation for 24h to recover, transfer to culture under normal light conditions for 24h;

(4) The algae liquid obtained by the culture in the step (3) is centrifuged for 10min at 1500 Xg and 4 ℃, the supernatant is discarded, and the algae liquid is resuspended by 600 mu L f/2 liquid culture medium, 200 mu L of the algae liquid is added on a f/2 culture medium solid plate (1.2% agar) containing bleomycin, and the algae liquid is cultured for 12-14d under normal culture conditions.

3. Screening and identification of MED31 gene knockout algae strain

(1) Selecting monoclonal algae colonies growing on a bleomycin resistance plate, drawing a 3-5mm short line on an f/2 solid culture medium containing bleomycin, culturing under normal conditions until algae colonies grow, taking half of the algae colonies, adding 20 mu L of algae lysate, performing PCR identification on the algae lysate as a template after water bath at 100 ℃ for 10min, wherein the formula of the algae lysate is as follows: 1%NP40, 10.00mM Tris,0.14mM NaCl,5.00mM KCl;

(2) The design of the algae liquid identification primer is as follows: MED31-ko-fw: GTCGATGTCGTCATGGTCGGAT; MED31-ko-rw TTCTCAATCAGCAACTCGAGAA,

TABLE 5 reaction system

TABLE 6 reaction conditions

Sequencing and analyzing the PCR amplified product, selecting a positive transformant which is successfully knocked out of the MED31 gene, selecting a strain which is knocked out and heterozygous, diluting and coating the strain after the strain is cultured in the f/2 liquid culture medium, carrying out a new round of screening, repeating the steps of PCR and sequencing until 2 homozygous transformants are screened out, and naming the strain as the strain with the MED31 gene knocked out.

As a result, the sequencing result of the homozygous MED31-2 transformant strain is shown in FIG. 1 (c), the sequencing result of the homozygous MED31-7 transformant strain is shown in FIG. 1 (d), the sequencing result of the wild type (the genetically unmodified Phaeodactylum tricornutum) strain is shown in FIG. 1 (b), and it is clear from FIG. 1 (a) that the homozygous MED31-2 transformant strain lacks 6bp relative to the wild type; FIG. 1 (b) shows that the homozygous MED31-7 transformant lacks 15bp relative to the wild type.

Detailed description of the preferred embodiments

1. Growth curve determination

Culturing the MED31 gene knockout algae strain obtained in the second embodiment under normal conditions in f/2 liquid medium containing bleomycin to logarithmic phase, cleaning with f/2 liquid medium without bleomycin for three times, inoculating into 50mL f/2 liquid medium, and adjusting initial concentration to 3×10 with wild type as control ⁵ cells/mL, cultured under normal conditions. The algal cell density was determined by counting the number of algal cells by light microscopy at a fixed time selected daily, and repeated three times until the cultivation was to the logarithmic phase.

FIG. 2 shows growth curves of MED31 gene knockout algae strain, and MED31 gene knockout algae strain and Wild Type (WT) cell concentrations were measured for 9 consecutive days, respectively, to obtain the growth curves of FIG. 2. It can be seen from the graph that the number of cells of the MED31 knockout algae strain is lower than that of the wild type from the third day on the condition that the initial densities are consistent, and the phenomenon is most remarkable on the ninth day, and the growth amount of phaeodactylum tricornutum after MED31 knockout is reduced.

2. Fucoxanthin extraction and determination

After freeze-drying the MED31 knockout strain and the wild-type Phaeodactylum tricornutum strain in the logarithmic phase for 48 hours, fucoxanthin was extracted by sonication at 40℃for 1 hour using 90% ethanol at a feed-to-liquid ratio of 1:10 (g/mL). After filtration, the supernatant (0.22 μm) was taken for HPLC analysis. The whole process is carried out under dark conditions. An Agilent 1200HPLC system (Agilent Technologies, american) consisting of a G1312A binary pump, a G1367B autosampler, a G1315D PDA detector, and a G1316A column incubator was used for fucoxanthin quantification. The mobile phase, i.e., methanol and water, was at 0.7mL min ^-1 Is eluted at 35℃and YMC carotenoid columns (250 mm long. Times.4.6 mm inside diameter; 5 μm particle size; waters, american) are used for separation under the following gradient procedure: the methanol was increased from 90% to 100% for 20min, kept at 100%% for the next 5min, reduced to 90%5min, and then maintained at 90%5min. 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 Fucoxanthin was quantified by the calibration curve of (c).

FIG. 3 shows that MED31 gene knockout algae strain and wild type strain are respectively measured at cell densities of 1.5x10 ⁶ As can be seen from FIG. 3, the fucoxanthin content of MED31-7 is reduced by 0.13 times compared with that of WT, the fucoxanthin Huang Subi WT of MED31-2 is reduced by 0.14 times, and experimental data show that the reduction of the fucoxanthin content caused by knocking out the MED31 gene in Phaeodactylum tricornutum has an effect of positive regulation in the synthesis of fucoxanthin. And (3) injection: asterisks on the columns indicate significant differences in statistical analysis (t-test, p<0.05 WT represents wild-type phaeodactylum tricornutum.

3. Fucoxanthin synthesis key gene expression quantity detection

Primers were selected which combine to synthesize the fucoxanthin synthesis genes ZDS-like, ZEP3, CRTISO3, PDS2 (primer sequences are shown in Table 4). Real-Time PCR was performed using cDNA reverse transcribed from RNA extracted from MED31 gene knockout algae strains MED31-2, MED31-7 and wild type Phaeodactylum tricornutum as templates, respectively. The quantitative reagents used were Taq Pro Universal SYBR qPCRMaster Mix (Vazyme, Q712), the reaction systems and the reaction procedures are shown in tables 7 and 8

TABLE 7 fluorescent quantitative primer sequences

TABLE 8qPCR reaction System

TABLE 9qPCR reaction procedure

As shown in FIG. 4, in the MED31 knock-out strain, the expression level of the 4 key genes ZDS, ZEP3, PDS2 and CRTISO3 of fucoxanthin was reduced, so that we could prove that MED31 could regulate the expression of the fucoxanthin synthesis gene and was positively regulated.

Detailed description of the preferred embodiments

Confirmation by Yeast two-hybrid System (Y2H)

1. Constructing the full-length sequence of the MED31 on an empty vector by using the pGBKT7 vector as a bait empty vector to obtain a pGBKT7-MED31 recombinant vector;

2. the pGBKT7-MED31 recombinant vector and the Y2H yeast competent cell strain are subjected to heat shock transformation, positive clones are screened, and Y2H-pGBKT7-MED31 recombinant strain is obtained;

3. preparing Y2H-pGBKT7-MED31 recombinant bacteria into Y2H-pGBKT7-MED31 recombinant bacteria competent cells by a yeast transformation kit, transforming a yeast library (purchased from Beijing Living Piao optical biotechnology development Co., ltd.) into Y2H-pGBKT7-MED31 recombinant bacteria competent cells for screening, selecting blue bacterial colonies and culturing to obtain yeast single bacterial colonies;

4. interactive protein determination

Yeast single colonies were picked into the corresponding four-liquid-deficient medium (SD-Ade-His-Leu-Trp medium) and cultured with shaking at 30℃for 1d. After the bacterial liquid is cracked for 10min in a water bath at 100 ℃, bacterial liquid PCR identification and yeast identification forward primer sequence are carried out: 5'-TTTAATACGACTCACTATAGGGCGA-3'; yeast identification reverse primer sequence: 5'-AGATGGTGCACGATGCACAG-3'; yeast sequencing primer sequences: 5'-TTTAATACGACTCACTATAGGGCGA-3'. Sequencing the PCR products, performing blast comparison on NCBI websites to obtain protein information, and screening about 300 interaction proteins, wherein the total number of transcription factors is 15. FIG. 5 shows 15 transcription factors obtained by screening a library in a yeast two-hybrid experiment, and a transcription factor (with higher binding frequency) having strong interaction with MDE31 is selected for research, and MYB family MYB2R1 (locus tag: PHATRDRAFT _14143, 312 bp) is selected.

Further secondary interaction verification is performed on the transcription factor MYB2R1 to be studied, and the result is shown in FIG. 6, which proves that an interaction relationship exists between MED31 and MYB2R1 and a certain channel is possibly regulated together.

Detailed description of the preferred embodiments

The cloning method of the transcription factor MYB2R1 gene comprises the following steps:

1. extracting total RNA of Phaeodactylum tricornutum, and reversely transcribing the total RNA into cDNA as a template;

2. primers were designed based on the gene sequence of MYB2R 1:

upstream primer sequence: 5'-ATGTGGACCAAGGAAGAAGACG-3' the number of the individual pieces of the plastic,

downstream primer sequence: 5'-TCATTCACGTTCGTACTGTCGG-3';

3. and (3) PCR amplification: obtaining a MYB2R1 gene amplification 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 2X PrimeSTAR Max Premix (PCR amplification kit: 2X PrimeSTAR Max Premix available from Takara Bio Inc.), and 0.5. Mu.L of each of the 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, 72℃for 1min,30 cycles;

4. purifying and recovering the PCR amplified product by using 1% agarose gel electrophoresis, connecting the PCR amplified product with a pMD19-T vector, further verifying by PCR, and sequencing to obtain a MYB2R1 gene, wherein the nucleotide sequence of the MYB2R1 gene is shown as SEQ ID NO:3, shown in the following: ATGTGGACCAAGGAAGAAGACGCCATTCTGCTCAAAATTGTGCAAGGGATGCAAATGCCCATGAAGTGGAGTGTTGTCGCACAAAACTTACACGATCGTACGGGAAAGCAGTGTCGCGAGCGCTACGTCAATCATCTCAATCCCCGTCTCAAGGTCACGGACTGGAATCCGGTCGAAGACTCCACCATATTTCACCTTTACAACACTATCGGTAGCCACTGGGCAAAAATGTCCAAGGTCATCCCCGGACGCACGGACAACGGCATCAAGAATCGCTTCCATAATCTCCGCCGACAGTACGAACGTGAATGA.

Description of the preferred embodiments

The construction method of the transcription factor MYB2R1 over-expression recombinant algae comprises the following steps:

1. primers were designed based on the gene sequence of MYB2R1 and pPha-T1 vector information: upstream primer sequence: 5' -TGTC TGCCGTTTCGAGAATTCATGTGGACCAAGGAAGAAGACG-3', downstream primer sequence: 5' -TTAGTCGATGATATCGA ATTCTCATTCACGTTCGTACTGTCGG-3', the primer both ends respectively comprise the sequence of the vector as homology arms (at the scribe line);

2. PCR amplification reactionThe system is as follows: 0.5. Mu.LT vector, 10. Mu.L 2X PrimeSTARMax Premix (PCR amplification kit: 2X PrimeSTAR Max Premix available from Takara Bio Inc.), 0.5. Mu.L each of the 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, 72℃for 1min,30 cycles; purifying and recovering PCR amplified products by using 1% agarose gel electrophoresis;

3. and (3) constructing a carrier: the vector pPha-T1 was digested with the restriction enzyme EcoRI to obtain a linearized plasmid, which was reacted in a constant temperature incubator at 37℃for 3 hours, and after the completion of the reaction, recovered and subjected to concentration detection.

4. Performing homologous recombination reaction on the PCR product purified and recovered in the step (2) and the linearization vector obtained in the step (3) by using a kit ClonExpress II One Step Cloning Kit, and constructing a MYB2R1 gene into the pPha-T1 vector to obtain a pPha-T1-MYB2R1 recombinant plasmid;

5. phaeodactylum tricornutum transformation: the recombinant plasmid pPha-T1-MYB2R1 is transformed into Phaeodactylum tricornutum by electrotransformation method under low light intensity (30 mu mol. M) ^-2 .s ^-1 ) Incubating for 24h, transferring to normal light intensity for culturing for 24h, centrifuging for 10min at 3000g, collecting cells, re-suspending the cells with 0.6ml f/2 culture solution, coating on 3 f/2 solid culture media containing 75 mug/ml bleomycin on average, generating algae fall after 15-25d, picking up preliminary positive algae growing on bleomycin resistant plates, drawing 3-5mm short lines on f/2 solid culture media containing bleomycin, culturing under normal conditions until algae fall grows, taking half of algae fall, adding 20 mug of algae lysate, taking the algae lysate as a template for carrying out algae solution PCR after water bath for 10min at 100 ℃, and initially indicating that transgene is successful after the algae fall appears in a strip, and identifying the primers as follows: MYB2R1-F: GAAGAAGACGCCATTCTGCTC; MYB2R1-R: CACGTTCGTACTGTCGGCGG.

6. MYB2R1 over-expression positive algae identification

Inoculating the algae with successful transgenesis into 50mL f/2 liquid culture medium, culturing to logarithmic phase, centrifuging to collect algae cells, extracting RNA, reverse transcribing into cDNA, qPCR detecting MYB2R1 self expression quantity, MYB2R1 fluorescent quantitative primer sequence is as follows: MYB2R1-qpcr-fw:5'-ATGCCCATGAAGTGGAGTGT-3';

MYB2R1-qpcr-rv：5'-GACGGGGATTGAGATGATTG-3'。

as shown in FIG. 7, the expression level of MYB2R1 over-expressed transgenic algae itself was increased. To study the role of MYB2R1 in fucoxanthin synthesis, transgenic phaeodactylum tricornutum strains overexpressing MYB2R1 were generated and the significant upregulation of MYB2R1 expression levels in three of these strains was confirmed by qRT-PCR.

Detailed description of the preferred embodiments

1. Growth curve measurement of MYB2R1 over-expression transgenic algae

Culturing MYB2R1 over-expressed transgenic algae in f/2 liquid culture medium containing bleomycin under normal condition until logarithmic phase, washing with culture medium without bleomycin for three times, inoculating into 50mL f/2 liquid culture medium, and adjusting initial concentration to 3×10 with wild type as control ⁵ cells/mL, cultured under normal conditions. The algal cell density was determined by counting the number of algal cells by light microscopy at a fixed time selected daily, and repeated three times until the cultivation was to the logarithmic phase.

FIG. 8 shows growth curves of MYB2R1 overexpressing transgenic algae strain, cell concentrations of MYB2R1 overexpressing transgenic algae strain and Wild Type (WT) were measured for 9 consecutive days, respectively, and the growth curves were obtained as shown in FIG. 8. It can be seen from the graph that, in the case of consistent initial density, the number of cells of the MYB2R1 overexpressing transgenic algae strain is higher than that of the wild type from the fifth day, and the phenomenon is most remarkable at the ninth day, and the growth amount of Phaeodactylum tricornutum after the MYB2R1 is overexpressed is increased.

2. Fucoxanthin content determination of MYB2R1 over-expressed transgenic algae

After the MYB2R1 over-expression transgenic algae strain and wild-type phaeodactylum tricornutum strain in logarithmic phase were freeze-dried for 48 hours, fucoxanthin was extracted by sonication at 40 ℃ for 1 hour using 90% ethanol at a feed-to-liquid ratio of 1:10 (g/mL). After filtration, the supernatant (0.22 μm) was taken for HPLC analysis. The whole process is carried out under dark conditions. An Agilent 1200HPLC system (Agilent Technologies, american) consisting of a G1312A binary pump, a G1367B autosampler, a G1315D PDA detector, and a G1316A column incubator was used for fucoxanthin quantification. The mobile phase, i.e., methanol and water, was at 0.7mL min ^-1 Is eluted at 35℃and YMC carotenoid columns (250 mm long. Times.4.6 mm inside diameter; 5 μm particle size; waters, american) are used for separation under the following gradient procedure: the methanol was increased from 90% to 100% for 20min, held at 100% for the next 5min, reduced to 90% for 5min, and then held at 90% for 5min. Sample solution (10. Mu.L) was injected and the chromatogram recorded at 445 nm. Fucoxanthin was quantified based on a calibration curve with a concentration ranging from 0.5 to 50 μg/mL.

As shown in FIG. 9, the fucoxanthin content of MYB2R1 over-expressed transgenic algae and WT was determined, and the fucoxanthin content of MYB2R1 over-expressed transgenic algae was increased by 0.43-0.53 times as compared with that of WT. The expression level of MYB2R1 gene can cause the increase of the content of fucoxanthin, which shows that MYB2R1 plays a role in forward regulation in the synthesis path of fucoxanthin.

The above description is not intended to limit the invention, nor is the invention limited to the examples described above. Variations, modifications, additions, or substitutions will occur to those skilled in the art and are therefore within the spirit and scope of the invention.

Claims (5)

1. An application of a transcription mediator MED31 gene in improving the content of fucoxanthin in Phaeodactylum tricornutum, which is characterized in that: the nucleotide sequence of the gene is shown in SEQ ID NO: 1.

2. Use of the transcriptional mediator MED31 according to claim 1 for increasing the fucoxanthin content of phaeodactylum tricornutum, characterized in that: the amino acid sequence of the protein is shown in SEQ ID NO: 2.

3. A strong interactive transcription factor of the transcription mediator MED31 gene, characterized in that: the strong interaction transcription factor is MYB2R1 gene.

4. Use of the strong interactive transcription factor MYB2R1 of claim 3 for increasing the fucoxanthin content in phaeodactylum tricornutum.

5. The use of the strong interactive transcription factor MYB2R1 according to claim 4 for increasing the fucoxanthin content of phaeodactylum tricornutum, characterized in that: the nucleotide sequence of the gene is shown in SEQ ID NO: 3.