Heat shock transcription factor bd1650 gene for increasing fucoxanthin content in phaeodactylum tricornutum and application thereof

Abstract

The invention discloses a heat shock transcription factor bd1650 gene for increasing the content of fucoxanthin in phaeodactylum tricornutum and application thereof, and is characterized in that the nucleotide sequence of the bd1650 gene is as shown in SEQ ID NO: 1, the amino acid sequence of protein encoded by the bd1650 gene is as shown in SEQ ID NO: 2, the content of the fucoxanthin synthesized by the bd1650 gene is increased by knocking out the bd1650 gene in the phaeodactylum tricornutum, and the content of the fucoxanthin in the phaeodactylum tricornutum is increased. The method has the advantage that the fucoxanthin content in the phaeodactylum tricornutum can be remarkably increased.

Description

Technical Field

[0001] The invention relates to the field of plant genetic engineering, and in particular to a heat shock transcription factor bd1650 gene for increasing the content of fucoxanthin in Phaeodactylum tricornutum and an application thereof. Background Art

[0002] Marine diatoms play a pivotal role in the global carbon cycle. Their high nutritional value and medicinal potential, as well as their potential as biofuel feedstock, are attracting increasing attention. Among them, P. triangularis stands out for its significant advantages such as rapid growth, strong environmental adaptability, and ease of artificial cultivation. This algae is rich in fucoxanthin, which is several times higher than other algae. Fucoxanthin, as a type of xanthophylls, is an important oxygen-containing derivative of carotene, showing multiple biological activities such as anti-cancer, anti-diabetes, anti-tumor, antioxidant and anti-obesity, and therefore shows broad application prospects in industrial production.

[0003] Heat shock transcription factors are a class of transcription factors that are ubiquitous in higher plants and can respond to a variety of stresses. Heat shock transcription factors may also play an important role in stress responses in microalgae. For example, nitrogen and phosphorus deficiency stresses lead to the expression of many heat shock transcription factors in P. triangularis. Transcriptome analysis showed that heat shock transcription factors were the transcription factors with the greatest expression differences at different growth stages of this species. In fact, heat shock transcription factors accounted for 34.18% of all transcription factors in P. triangularis. However, despite these signs that heat shock transcription factors play an important role in microalgae, the specific functions of heat shock transcription factors in microalgae have been less studied. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a heat shock transcription factor bd1650 gene and its application for increasing the content of fucoxanthin in Phaeodactylum tricornutum. bd1650 Gene knockout algae strains can positively regulate the expression of fucoxanthin synthesis genes.

[0005] The technical solution adopted by the present invention to solve the above technical problems is: a heat shock transcription factor for increasing the content of fucoxanthin in Phaeodactylum triangularis bd1650 Gene, described bd1650 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0006] Furthermore, the amino acid sequence of the protein encoded by bd1650 is shown in SEQ ID NO:2.

[0007] The present invention also provides the use of the heat shock transcription factor bd1650 gene in increasing the fucoxanthin content in Phaeodactylum triangularis. bd1650The nucleotide sequence of the gene is shown in SEQ ID NO: 1. bd1650 The gene causes an increase in the amount of fucoxanthin it synthesizes.

[0008] Compared with the prior art, the advantages of the present invention are: 1. For the first time, we found the heat shock transcription factor bd1650 by screening the gene library of P. tricornutum. After knocking out its CRISPR gene, we found bd1650 The mutant strain had phenotypic changes in fucoxanthin synthesis; 2. For the first time, it was confirmed that the heat shock transcription factor bd1650 gene of P. tricornutum can promote the increase of fucoxanthin content. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 For specific embodiment three bd1650 Sequence chromatograms generated by sequencing the knockout algae strain and the wild-type P. tricornutum; Figure 2 For specific embodiment 4 bd1650 Analysis of fucoxanthin content in gene knockout strains and wild-type P. triangularis. Note: WT represents wild-type P. triangularis. bd1650-1 and bd1650-8 Indicates different transfers bd1650 Transgenic Phaeodactylum tricornutum strains of vector; Figure 3 For the specific embodiment 5 bd1650 Fucoxanthin biosynthesis genes in knockout strains and wild-type Phaeodactylum tricornutum CRTISO5 Expression level; Figure 4 For the specific embodiment 5 bd1650 Fucoxanthin biosynthesis genes in knockout strains and wild-type Phaeodactylum tricornutum ZEP1 Expression amount. DETAILED DESCRIPTION

[0010] The present invention is further described in detail below with reference to the accompanying drawings.

[0011] Specific Example 1: Heat shock transcription factor bd1650 Gene cloning and sequence analysis include the following steps: Step 1: Extract algae at a concentration of 1×10 6 cells / mL of total RNA from the logarithmic phase of Phaeodactylum triangularis, and the extracted RNA was used as a reactant for reverse transcription to obtain a cDNA template.

[0012] Step 2: PCR amplification: bd1650Gene amplification products, PCR amplification reaction system: 0.5 μL cDNA, 10 μL 2× PrimeSTAR Max Premix, bd1650 Gene upstream and downstream amplification primers 0.5 μL each, 8.5 μL ddH2O; PCR amplification program: denaturation at 98℃ for 10s, annealing at 55℃ for 15s, extension at 72℃ for 1min, 30 cycles; bd1650 The nucleotide sequence of the upstream amplification primer of the gene is shown in SEQ ID NO:3: ATGTGCGTTACCAACACTACCCA, bd1650 The nucleotide sequence of the primer for downstream amplification of the gene is shown in SEQ ID NO:4: TTAGTCCTCAAGTAGTCGTTCCAGC.

[0013] Step 3: The PCR amplification product was purified by 1% agarose gel electrophoresis and then connected to the pMD19-T vector. After further verification by PCR, sequencing was performed to obtain bd1650 Heat shock transcription factor bd1650 The amino acid sequence of the encoded protein such as SEQ ID NO:2 shows: MCVTNTTHRRRPQPISLAPSQYGACPSMLSQRLLGSYAAATVSAPASPSPAAPQYYATTIVTEKPRPKRRRKPQKPGKTAKMNERHFVKHNYHDHAMDTVDDMDDHAAEEQQPEDASLRRRGGVSVAFPLKLHAVLDQVEADGLAHVISWQPHGRCFVVHEPKKFVDHVMPKYFRQTKLTSFQRQLN LYGFCRLTRGNDSGGYYHELFLRNKVFLCQKMIRTKVKGTRFKAASSPDQEPDFYTMPVAVTPANTSDEESSFDSGSRESVSSLPTQPTTMPSFDMFATNYAAVPQNISHAVQSFEPRGAFAPLCLPQADDADRVLDEAVEELFLNEAIASDSLDDFVHDWDPSNEGNEAYLDSLQDDCQLGMMLERLLED.

[0014] Specific embodiment 2: Construction bd1650 Gene knockout vectors.

[0015] Using the pPtPuc3-Cas9-sgRNA (Addgene ID: 109219) vector as the CRISPR / Cas9 expression cassette, two bd1650 The knockout vector (CRISPR-bd1650-1 / 2) was constructed. The specific steps are as follows: Step 1: Select the bd1650 For the target gene, two sets of sgRNA sequences were obtained using online sgRNA design software. The nucleotide sequence of sgRNA1 was shown in SEQ ID NO:5: CCCGCAGCCAATCTCTCTGGCAC, and the nucleotide sequence of sgRNA2 was shown in SEQ ID NO:6: CCGTCACAATCGTTGTGGCGTAG.

[0016] Step 2. Add restriction sites to the sgRNA sequence obtained in step 1 and synthesize two pairs of single-stranded primers. The nucleotide sequence of the gRNA1 single-stranded upstream primer is shown in SEQ ID NO: 7: GTGCCAGAGAGATTGGCTGC, and the nucleotide sequence of the gRNA1 single-stranded downstream primer is shown in SEQ ID NO: 8: GCAGCCAATCTCTCTGGCAC; the nucleotide sequence of the gRNA2 single-stranded upstream primer is shown in SEQ ID NO: 9: GCTACGCCACAACGATTGTGA, and the nucleotide sequence of the gRNA2 single-stranded downstream primer is shown in SEQ ID NO: 10: TCACAATCGTTGTGGCGTAGC.

[0017] Step 3. Anneal the two pairs of single-stranded upstream and downstream primers obtained in step 2 to obtain the corresponding double-stranded DNA fragments. The reaction system is 10×Buffer, 1 μL; 10 μM single-stranded upstream primer, 1 μL, 10 μM single-stranded downstream primer, 1 μL, add ddH2O to 10 μL, mix the reaction system, react at 85℃ for 10 min, cool at room temperature for 90 min, and obtain double-stranded DNA.

[0018] Step 4. The two double-stranded DNA fragments obtained in step 3 were connected to the pPtPuc3-Cas9-sgRNA linear plasmid using T4 ligase, and the connection was carried out at 16°C for 1h. The connection system was 1 μL of T4 10×Buffer, 4 μL of pPtPuc3-Cas9-sgRNA linear plasmid, 3.5 μL of double-stranded DNA fragment, 0.5 μL of T4 DNA ligase, and the mixture was made up to 10 μL with water to obtain two recombinant vectors, which were named bd1650-sgRNA1 recombinant vector and bd1650-sgRNA2 recombinant vector respectively. The linear plasmid preparation method was as follows: 1 μL of 1 μg / μL pPtPuc3-Cas9-sgRNA vector (109219) was added with 5 μL of 10×Buffer and 1 μL of restriction endonuclease, and the mixture was made up to 50 μL with ddH2O. The reaction system was mixed and reacted in a 37°C constant temperature incubator for 1 hour. h, followed by recovery and concentration detection to obtain the pPtPuc3-Cas9-sgRNA linear plasmid.

[0019] Step 5: Transform the bd1650-sgRNA1 and bd1650-sgRNA2 recombinant vectors into Escherichia coli respectively. The specific steps are as follows: (1) Take 10 μL of the two recombinant vectors obtained in step 4 and add them to the E. coli DH5α competent cells respectively. Place them on ice for 30 min. Gently shake the cells, heat shock them in a 42°C water bath for 60 s, and then quickly place them on ice for 3-5 min. (2) Then, add 400 μL of LB liquid culture medium without antibiotics to each tube and mix gently, and then shake and culture at 220 rpm in a shaker at 37°C for 1 h; the LB liquid culture medium consists of 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, and the solvent is water; (3) Take 100 μL of the mixture obtained in step (2) and add it to the + ) on LB solid medium containing antibiotics, spread it evenly until the bacterial solution is dry on the plate, seal it with sealing film, mark it, and culture it in a 37℃ constant temperature incubator overnight for 12-16 hours.

[0020] Take out the overnight cultured plate from the incubator and observe the colonies on the plate. If there are single colonies, pick 8 single colonies on the clean bench and place them in 1 mL of agar containing ampicillin (Amp + ) antibiotics in LB liquid medium, and after shaking and culturing at 220 rpm in a constant temperature incubator at 37°C for 3 h, the bacterial solution was taken for bacterial solution PCR identification, and a sequence on the pPtPuc3-Cas9-sgRNA vector was taken as the identification reverse primer. The nucleotide sequence of the bacterial solution identification reverse primer is shown in SEQ ID NO:11: CAGGAAACAGCTATGACC, and the single-stranded upstream primer in step 2 was used as the bacterial solution identification forward primer (gRNA1 single-stranded upstream primer and gRNA2 single-stranded upstream primer) for PCR identification. The bacterial solution PCR reaction system was: 10 μL 2×Flash Hot Start Master, 1 μL bacterial solution identification forward primer, 1 μL bacterial solution identification reverse primer, 1 μL bacterial solution, and ddH2O was added to a total volume of 20 μL. The PCR reaction program of the bacterial solution was: 95°C for 5 min; 98°C for 10 s, 55°C for 5 s, 72°C for 5-10 s / kb, for 30 cycles; and cooled to 4°C.

[0021] The obtained PCR products were subjected to gel electrophoresis to check the results, and samples with positive bands were taken for expansion culture (100 μL bacterial solution and 4 mL of ampicillin (Amp + ) LB liquid medium containing antibiotics) and the plasmid was extracted with Plasmid Mini KitⅡ plasmid extraction kit to obtain two bd1650 Gene knockout vectors.

[0022] Specific embodiment three: bd1650 The specific steps for preparing gene knockout algae strains are as follows: Step 1: Use EcoNI to bd1650The gene knockout vector was linearized and digested with the following enzyme system: 5 μL of 10× Buffer, 3 μL of EcoNI, 6 μg of bd1650 The knockout vector was filled with water to 50 μL. After enzyme digestion for 3 hours, it was purified using a gel extraction kit and the product was recovered by enzyme digestion to obtain two bd1650 Gene knockout vector linear plasmid.

[0023] Step 2: Triangular brown finger algae and bd1650 The gene knockout vector linear plasmid is electrotransformed, and the specific steps are as follows: (1) Cultivate the triangular algae to the logarithmic stage 1×10 6 cells / mL, centrifuged at 1500×g for 10 min at 4°C, and discarded the supernatant; the precipitate was washed three times with 1 mL of 375 mM sorbitol (sterile, pre-cooled on ice) solution, and the washed cells were resuspended with 100 μL of 375 mM sorbitol solution to obtain a final algal density of 2×10 9 cells / mL resuspension solution; (2) Add 100 μL of resuspension solution and 4 μg of the purified protein in step 1 at a concentration of 0.2 μg / μL. bd1650 The knockout vector linear plasmid and 40 μg of salmon sperm DNA (boiled at high temperature for 10 min) solution at a concentration of 10 μg / μL were mixed together, incubated on ice for 10 min, and then transferred to a 0.2 cm ice-precooled electroporation cuvette. The parameters were set as follows: 500 V field strength, 25 μF capacitance, and 400 Ω parallel resistance for electroporation transformation; (3) After electroporation, the cells were immediately transferred to 10 mL of f / 2 liquid culture medium and incubated under low light (about 30 μmol / m 2 ·s) After incubation for 24 h to recover, transfer to normal light conditions and culture for 24 h, centrifuge the algae solution at 1500×g, 4°C for 10 min, discard the supernatant, take the precipitate and resuspend it in 600 μL f / 2 liquid culture medium, take 200 μL and add it to the f / 2 medium solid plate containing bleomycin, and culture it under normal culture conditions for 12-14 days.

[0024] Step 3 bd1650 The specific steps for screening and identification of gene knockout algae strains are as follows: (1) Pick the monoclonal algae colony grown on the bleomycin resistance plate in step 2, draw a short line of 3-5 mm on the f / 2 solid medium containing bleomycin, and culture under normal conditions until algae colonies grow. Take half of the algae colony, add 20 μL algae lysis buffer, and incubate in a 100℃ water bath for 10 min. Take the algae lysis product as a template for algae liquid PCR identification. The formula of the algae lysis buffer is: 1% Nonidet P 40 (NP40), 10.00 mM Tris, 0.14 mM NaCl, 5.00 mM KCl; (2) The nucleotide sequence of the algae liquid identification forward primer is shown in SEQ ID NO:12: ATGTGCGTTACCAACACTACCCA, and the nucleotide sequence of the algae liquid identification reverse primer is shown in SEQ ID NO:13: TGATCGACAAACTTTTTGGGTTC. The algae liquid PCR reaction system is as follows: 10 μL 2×Flash Hot Start Master, 1 μL algae liquid identification forward primer, 1 μL algae liquid identification reverse primer, 1 μL algae liquid, add ddH2O to a total volume of 20 μL. The algae liquid PCR reaction program is as follows: 95℃ 5 min; 98℃ 10 s, 55℃ 5 s, 72℃ 5-10 s / kb, 30 cycles; cool to 4℃.

[0025] The PCR amplification products were sequenced and analyzed, and the successful knockout was selected bd1650 The positive transformants of the gene were selected, and the knockout heterozygous algae strains were selected and cultured in f / 2 liquid medium, then diluted and spread for a new round of screening. The algae liquid PCR identification and sequencing steps were repeated until two homozygous transformants were screened and named bd1650 Gene knockout algae strains.

[0026] Wild type (native Phaeodactylum triangularis without genetic modification), bd1650-1 and bd1650-8 The sequencing results of homozygous transformants are as follows Figure 1 As shown by Figure 1 It can be seen that bd1650-1 The homozygous transformant had a deletion of 135 bp relative to the wild type; bd1650- 8 The homozygous transformant had a deletion of 138 bp relative to the wild type.

[0027] Specific Example 4: Extraction and determination of fucoxanthin.

[0028] In the logarithmic period bd1650After freeze-drying the knockout strains and wild-type P. tricornutum for 48 h, 90% ethanol was added at a solid-liquid ratio of 1 g:10 mL, and fucoxanthin was extracted by ultrasound at 40 °C for 1 h. After filtration, the supernatant was filtered through a 0.22 μm filter membrane for HPLC analysis, and the whole process was carried out under dark conditions. A gilent 1200 HPLC system (Agilent Technologies, American) consisting of a G1312A binary pump, a G1367B autosampler, a G1315D PDA detector, and a G1316A column oven was used for fucoxanthin quantification. The mobile phase, namely methanol and water, was eluted at a flow rate of 0.7 mL / min at 35 °C, and a YMC carotenoid column (250 mm length × 4.6 mm inner diameter; 5 μm particle size; Waters, USA) was used for separation under the following gradient program: methanol increased from 90% to 100% for 20 min, maintained at 100% for 5 min, decreased to 90% for 5 min, and then maintained at 90% for 5 min. 10 μL of the sample solution was injected, and the chromatogram was recorded at 445 nm. Fucoxanthin was quantified based on a calibration curve with a concentration range of 0.5-50 μg / mL.

[0029] Figure 2 The cell density was 1.5×10 6 cells / mL bd1650 The fucoxanthin content of the knockout strain and the wild-type P. triangularis was Figure 2 It can be seen that bd1650-1 and bd1650-8 The fucoxanthin content of the triangular algae was increased by 0.12-0.2 times. The experimental data showed that bd1650 Gene knockout resulted in an increase in fucoxanthin content. bd1650 The gene knockout algae plays a positive regulatory role in the synthesis of fucoxanthin. Note: WT represents wild-type P. triangularis.

[0030] Specific Example 5: Detection of expression level of key genes for fucoxanthin synthesis.

[0031] Select and synthesize fucoxanthin synthesis genes CRTISO5 and ZEP1 Fluorescence quantitative primers, CRTISO5 The nucleotide sequence of the fluorescent quantitative upstream primer is shown in SEQ ID NO: 14: GAGGATCGGCTCATACATTCTC, CRTISO5 The nucleotide sequence of the fluorescence quantitative downstream primer is shown in SEQ ID NO: 15: GCATCTCTTCTTCCAGGACATC. ZEP1The nucleotide sequence of the fluorescent quantitative upstream primer is shown in SEQ ID NO: 16: GGTACGCTTCGATACCCTACAG, ZEP1 The nucleotide sequence of the fluorescence quantitative downstream primer is shown in SEQ ID NO: 17: GTAATTGGCAATACGGGACTTG.

[0032] Use respectively bd1650 Knockout strains bd1650-1 , bd1650-8 The cDNA extracted from RNA extracted from wild-type Phaeodactylum triangularis was used as template for real-time PCR quantification. The reaction system of Taq Pro Universal SYBRqPCR Master Mix (Vazyme #Q712) used as the quantitative reagent is as follows: 10 μL 2×Taq Pro Universal SYBRqPCR Master Mix, 0.4 μL fluorescent quantitative upstream primer, 0.4 μL fluorescent quantitative downstream primer, 2 μL cDNA, add ddH2O to a total volume of 20 μL. The reaction program is as follows: 95℃ 30 s; 95℃ 10 s, 60℃ 30 s, a total of 40 cycles; 95℃ 15 s, 60℃ 60 s, 95℃ 15 s; 4℃ until the end.

[0033] The results are as follows Figure 3 , Figure 4 As shown, compared with the wild-type Phaeodactylum triangularis WT, bd1650 Two key genes of fucoxanthin in gene knockout algae strains CRTISO5 and ZEP1 The expression level of bd1650 Gene knockout algae strains can regulate the expression of fucoxanthin synthesis genes, and the regulation is positive.

[0034] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A heat shock transcription factor for increasing the content of fucoxanthin in Phaeodactylum tricornutum bd1650 A gene characterized by: The bd1650 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. A heat shock transcription factor for increasing the fucoxanthin content in Phaeodactylum tricornutum according to claim 1 bd1650 A gene characterized by: The amino acid sequence of the protein encoded by bd1650 is shown in SEQ ID NO:

2.

3. A heat shock transcription factor according to claim 1 bd1650 The application of the gene in increasing the content of fucoxanthin in Phaeodactylum triangularis is characterized by: The bd1650 The nucleotide sequence of the gene is shown in SEQ ID NO:

1. bd1650 The gene causes an increase in the amount of fucoxanthin it synthesizes.

Images