Phaeodactylum tricornutum high-temperature-resistant engineered strain, and construction method and application thereof

Abstract

This invention discloses a heat-resistant engineered strain of *Phaeodactylum tricornutum*, its construction method, and its applications. The key feature is that the heat-resistant engineered strain is an overexpression of... bd1311 The gene-bearing brown finger algae, bd1311 The nucleotide sequence of the gene is shown in SEQ ID NO.1. The construction method of this algal strain includes using pPha-T1 as a vector and employing seamless cloning based on homologous recombination. bd1311 The full-length sequences of the genes are ligated and fused to obtain bd1311 Steps for gene overexpression vector design; bd1311 The steps include: amplifying the target fragment using primers for gene overexpression and amplifying it with cDNA from wild-type *Phaeodactylum tricornutum* as a template; recombinantly cloning the target fragment and the enzyme-digested linearized vector; transforming the recombinant vector into *Phaeodactylum tricornutum* and then electroporating it into *Phaeodactylum tricornutum* to obtain a heat-resistant engineered strain of *Phaeodactylum tricornutum*; the advantages are that it is resistant to high summer temperatures and positively regulates the fucoxanthin synthesis gene.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, and in particular relates to a heat-resistant engineered algal strain of *Phaeodactylum tricornutum*, its construction method, and its application. Background Technology

[0002] Diatoms are an important group of eukaryotic photosynthetic organisms. Despite their tiny size, they are widely distributed and diverse in species. *Phaeodactylum tricornutum*, as one of the representative species of marine diatoms, is widely known for its unique triangular or spindle-shaped cell morphology. This species was the first to complete whole-genome sequencing of diatoms, becoming an important model in microalgae research, particularly in areas such as photosynthesis, lipid metabolism, environmental remediation, and biofuel production. Its high lipid content and rapid growth characteristics make it an ideal candidate for the development of biofuels and bioproducts.

[0003] Heat shock proteins (HSPs) are a highly conserved family of molecular chaperone proteins, widely distributed in life forms from prokaryotes to higher eukaryotes. They mainly participate in intracellular protein synthesis, folding, transport, and degradation, playing a crucial role in maintaining protein homeostasis. Heat shock transcription factors (HSFs) are core regulators of cellular stress responses, responsible for activating and protecting the expression of related genes. *Phaeodactylum tricornutum*, a microalga of significant economic value, has an optimal growth temperature of 22°C, but exhibits significant temperature sensitivity above 25°C, limiting its large-scale application in high-temperature environments. Therefore, developing thermotolerant algal strains is key to solving this problem. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a heat-resistant engineered algal strain of *Phaeodactylum tricornutum* that can withstand high summer temperatures and positively regulate the fucoxanthin synthesis gene, as well as its construction method and application.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0006] This invention provides a heat-resistant engineered strain of *Phaeodactylum tricornutum*, wherein the heat-resistant engineered strain is an overexpression... bd1311 The gene of the brown finger algae, as described bd1311 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Furthermore, the amino acid sequence of the protein encoded by bd1311 is shown in SEQ ID NO.2.

[0008] Furthermore, the aforementioned bd1311 The nucleotide sequence of the primers for forward gene amplification is shown in SEQ ID NO.3: ATGACGTTCCCACCGACGGA. bd1311The nucleotide sequence of the gene reverse amplification primer is shown in SEQ ID NO.4: ATCGACCACAACTGCCCTAG.

[0009] Furthermore, the aforementioned bd1311 The nucleotide sequence of the gene overexpression positive amplification is shown in SEQ ID NO.5: TGTCTGCCGTTTCGAGAATTCATGACGTTCCCACCGACGG. bd1311 The nucleotide sequence of the reverse amplification primer for gene overexpression is shown in SEQ ID NO.6: CAGGTCGACTCTAGAGGATCCCTAGGGCAGTTGTGGTCGATAAA.

[0010] The present invention also provides a method for constructing the above-mentioned heat-resistant engineered strain of *Phaeodactylum tricornutum*, comprising the following steps:

[0011] Step 1: Construct an overexpression vector using pPha-T1 as the vector, and employ seamless cloning based on homologous recombination. bd1311 The full-length sequences of the genes are ligated and fused to obtain bd1311 Gene overexpression vectors;

[0012] Step 2, Design bd1311 Gene overexpression amplification primers, among which bd1311 The nucleotide sequence of the gene overexpression positive amplification is shown in SEQ ID NO.5: TGTCTGCCGTTTCGAGAATTCATGACGTTCCCACCGACGG. bd1311 The nucleotide sequence of the reverse amplification primer for gene overexpression is shown in SEQ ID NO.6: CAGGTCGACTCTAGAGGATCCCTAGGGCAGTTGTGGTCGATAAA;

[0013] Step 3: Use the design from Step 1 bd1311 Gene overexpression amplification primers were used to amplify the target fragment using wild-type *Phaeodactylum tricornutum* cDNA as a template; pPha-T1 was linearized using EcoRI and BamHI to obtain the enzyme-digested linearized vector.

[0014] Step 4: The target fragment obtained in Step 3 and the enzyme-digested linearized vector are recombinantly cloned to obtain the recombinant vector;

[0015] Step 5: Transform the recombinant vector obtained in Step 4 into E. coli, take bacterial culture samples with positive bands for amplification, and extract plasmids using a plasmid extraction kit to obtain... bd1311 Gene overexpression vectors;

[0016] Step 6: Obtain the result from Step 5 bd1311 The gene overexpression vector was linearized by restriction endonuclease digestion. bd1311 Gene overexpression vectors will linearize bd1311 Gene overexpression vectors were electroporated into *Phaeodactylum tricornutum*, and the transformed *Phaeodactylum tricornutum* cells were plated on f / 2 plates for culture. Positive algal strains were screened to obtain heat-resistant engineered *Phaeodactylum tricornutum* strains.

[0017] The present invention also provides the application of the above-mentioned heat-resistant engineered algal strain of *Phaeodactylum tricornutum* in the preparation of heat-resistant algal strains.

[0018] The present invention also provides the application of the above-mentioned heat-resistant engineered algal strain of *Phaeodactylum tricornutum* in the preparation of algal strains with high fucoxanthin content.

[0019] This invention also provides the above-mentioned heat shock transcription factor. bd1311 The application of genes in increasing fucoxanthin content in *Phaeodactylum tricornutum*, as described above bd1311 The nucleotide sequence of the gene is shown in SEQ ID NO.1. This was achieved through overexpression of *Phaeodactylum tricornutum*. bd1311 The gene causes it to synthesize more fucoxanthin.

[0020] Compared with the prior art, the advantages of the present invention are as follows:

[0021] 1. For the first time, it was confirmed that the heat shock transcription factor bd1311 was found by screening the gene library of *Phaeodactylum tricornutum*, and that overexpression of bd1311 resulted in... bd1311 The overexpression mutant strain showed phenotypic changes in its tolerance to high temperatures and fucoxanthin synthesis, demonstrating the positive role of the Browniana triangularis transcription factor bd1311 in Browniana triangularis's response to high temperatures.

[0022] 2. This study is the first to confirm the heat shock transcription factor of *Phaeodactylum tricornutum*. bd1311 The increased content of fucoxanthin synthesis-related genes in the overexpressed algal strains suggests that the increased expression of transcription factors under heat stress may facilitate the accumulation of fucoxanthin and lipids to resist high temperatures. Attached Figure Description

[0023] Figure 1 The results of PCR electrophoresis identification of bd1311 overexpression transformants in Example 4 are shown. Note: T3 and T6 transformants are highlighted in red.

[0024] Figure 2 The expression of fucoxanthin synthesis-related genes in the bd1311 overexpressing transgenic algae in Specific Example 5 is shown. Note: * indicates statistically significant differences, and empty vector 1 and empty vector 2 represent the pPha-T1 empty vector algae.

[0025] Figure 3The expression level of HSP-related genes in the bd1311 overexpressing transgenic algae in Specific Example 5 is shown. Note: The control group represents the expression level of HSP protein-related genes in the pPha-T1 empty vector algae. Different letters indicate statistically significant differences.

[0026] Figure 4 This shows the growth of the bd1311 overexpressing algal strain in Example 6 on a solid culture medium at high temperature. Note: The algal strains from top to bottom are, respectively, overexpressing algal strains bd-3 and bd-6, empty vector algal strains pp-2, pp-5 and pp-6, and wild type, with three replicates for each group;

[0027] Figure 5 This is a specific example of the growth of the bd1311 overexpressing algal strain in a plate at high temperature. The algal strains, from left to right, are the overexpressing algal strains bd-3 and bd-6 and the empty vector algal strains pp-2 and pp-5, with three replicates for each strain. The plate contains 2 mL of algal solution. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0029] Specific Implementation Example 1: Heat Shock Transcription Factor bd1311 Gene cloning and sequence analysis includes the following steps:

[0030] Step 1: Extract algae at a concentration of 1×10⁻⁶. 6 Total RNA from *Phaeodactylum tricornutum* in logarithmic phase (cells / mL) was used as a reactant for reverse transcription to obtain cDNA templates.

[0031] Step 2, PCR amplification: Obtain the desired result through PCR amplification. bd1311 The gene amplification product was amplified using a PCR reaction system consisting of: 0.5 μL cDNA and 10 μL 2×PrimeSTAR Max Premix. bd1311 0.5 μL each of the forward and reverse gene amplification primers, and 8.5 μL of ddH2O; the PCR amplification program was: denaturation at 98℃ for 10 s, annealing at 55℃ for 15 s, extension at 72℃ for 8 s, for 30 cycles; bd1311 The nucleotide sequence of the primers for forward gene amplification is shown in SEQ ID NO.3: ATGACGTTCCCACCGACGGA. bd1311 The nucleotide sequence of the reverse gene amplification primer is shown in SEQ ID NO.4: ATCGACCACAACTGCCCTAG;

[0032] Step 3: The PCR amplification product was purified and recovered by 1% agarose gel electrophoresis, ligated into the pMD19-T vector, further verified by PCR, and then sequenced to obtain... bd1311 The gene, whose nucleotide sequence is shown in SEQ ID NO.1:

[0033] The amino acid sequence of the protein encoded by the heat shock transcription factor bd1311 is shown in SEQ ID NO.2:

[0034] Mtfpptesskdeqpvaakdvahepkhqspilpaesptagpdeawsvplapyafpysmyhppallpmhlsvppsstlgstypdlsgvpdpqslndwrhrqnhggvaaafpeklhk mlaycareklqdvasfyphgrafaihqphrfvtevmpqffrqskltsfqrqlnlygfqriphgpdhggyyhehflrgrpglaaalkrvtvkgkakaalqlerfpeyvyrpqlp.

[0035] Specific Example 2: Heat Shock Transcription Factor bd1311 The construction of the overexpression vector includes the following steps:

[0036] An overexpression vector was constructed using pPha-T1 as the vector, and then ligated and fused with the full-length sequence of bd1311 using a seamless clone based on homologous recombination. The specific steps are as follows:

[0037] Step 1: Select the target gene bd1311 to be overexpressed. Using the online primer design tool provided by Vazyme, and through bioinformatics analysis, design primers based on a double enzyme digestion strategy (BamHI and EcoRI). bd1311 Gene overexpression amplification primers, among which bd1311 The nucleotide sequence of the gene overexpression positive amplification is shown in SEQ ID NO.5: TGTCTGCCGTTTCGAGAATTCATGACGTTCCCACCGACGG. bd1311 The nucleotide sequence of the reverse amplification primer for gene overexpression is shown in SEQ ID NO.6: CAGGTCGACTCTAGAGGATCCCTAGGGCAGTTGTGGTCGATAAA.

[0038] Step 2: Use the design from Step 1 bd1311 Gene overexpression amplification primers were used to amplify the target fragment using wild-type *Phaeodactylum tricornutum* cDNA as a template. The amplification reaction system consisted of: 0.5 μL cDNA template and 10 μL 2×PrimeSTAR Max DNAPolymerase. bd13111 μL each of the forward and reverse amplification primers for gene overexpression, and 7.5 μL of ddH2O were used. The PCR amplification program was as follows: pre-denaturation at 95℃ for 2 min, denaturation at 98℃ for 10 s, annealing at 55℃ for 5 s, extension at 72℃ for 5-10 s / kb, for 30 cycles, to obtain the target fragment. pPha-T1 was linearized using EcoRI and BamHI. The enzyme digestion system was as follows: 1 μL restriction enzyme, 5 μL 10× Buffer, 2 μL pPha-T1 plasmid, and 42 μL ddH2O to obtain the linearized vector.

[0039] Step 3: Using the target fragment obtained in Step 2 and the enzyme-digested linearized vector, calculate the optimal molar ratio of the insert fragment to the linearized vector according to the ClonExpress II OneStep Cloning Kit instructions for recombinant cloning. The reaction mixture consists of: 2 μL linear plasmid, 4 μL 5× CE II buffer, 1 μL target fragment, 2 μL Exnase II enzyme, and 11 μL ddH2O. The reaction is carried out in a PCR instrument at 37℃ for 30 min to obtain the recombinant vector. After the reaction is terminated, immediately transfer the mixture to an ice box for rapid cooling.

[0040] Step 4: Transform the recombinant vector obtained in Step 3 into E. coli. The specific steps are as follows:

[0041] (1) Take a 100 μL DH5α competent cell from -80℃ and place it in ice to thaw naturally for 4-8 minutes;

[0042] (2) Under a sterile clean bench environment, take 10 μL of the recombinant vector prepared in step 3 and add it to the cell suspension that has been thawed. Place the mixture in an ice bath and let it stand for 30 min.

[0043] (3) After gently shaking the bacterial culture, immediately place it in a 42℃ constant temperature water bath for heat shock treatment (45-60s), and then quickly transfer it to an ice bath and let it stand for 2-3 minutes to terminate the reaction;

[0044] (4) Take 400 μL of antibiotic-free LB medium into a centrifuge tube in the ultra-clean bench, shake to mix, and then incubate at 37°C and 200 × g for 60 minutes with constant temperature shaking. Maintain a sterile operating environment throughout the process.

[0045] (5) Under aseptic conditions, accurately measure 100 μL of the bacterial suspension prepared in step (4) using a pipette and spread it evenly on the surface of an LB agar plate containing 50 μg / mL ampicillin. After the inoculum is completely absorbed, invert the petri dish and incubate it in a 37°C constant temperature incubator.

[0046] (6) Use sterile and breathable sealing film to seal the culture dish, and mark the experimental parameters in detail (including key information such as culture date, strain number, antibiotic type and concentration), and then transfer it to a 37℃ constant temperature incubator for 12-16 h of aerobic culture;

[0047] (7) Observe the growth. If single colonies are found, pick 8 morphologically regular independent single colonies in a sterile environment under a laminar flow hood and inoculate them into 300 μL LB liquid medium (1.5 mL centrifuge tubes) containing 50 μg / mL ampicillin. Place the centrifuge tubes in a 37℃ constant temperature shaker and incubate at 200 rpm for 4 h to allow the cells to reach the logarithmic growth phase. Take 2 μL of the bacterial solution as a template and use... bd1311 PCR mixture was prepared by adding 1 μL each of forward and reverse primers for gene overexpression, 10 μL of 2×Flash Hot StartMaster, 2 μL of bacterial culture, and 6 μL of ddH2O. The reaction program was as follows: 95℃ for 5 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 5-10 s / kb, for 30 cycles; then cooled to 4℃ for bacterial culture identification and amplification.

[0048] The obtained PCR products were subjected to gel electrophoresis to check the results. A bacterial culture sample with a positive band was taken, and 100 μL of the bacterial culture was added to 4 mL of ampicillin-containing solution. + The culture was expanded in LB liquid medium containing antibiotics, and plasmids were extracted using the Plasmid MiniKit II plasmid extraction kit to obtain two plasmids. bd1311 Gene overexpression vector.

[0049] Specific Implementation Example 3: *Phaeodactylum tricornutum* and Linearization bd1311 Gene overexpression vectors are electroporated.

[0050] The target plasmid was linearized using restriction endonucleases, and specific restriction sites (pPha-T1: NdeⅠ) were selected in the non-coding functional region of the vector backbone. 8 μg of the target plasmid was linearized using restriction endonucleases. bd1311 The gene overexpression vector was digested with enzymes, and then purified using a Gel Extraction Kit to obtain linearized plasmid DNA. The specific steps are as follows:

[0051] (1) Culture the brown finger algae to the logarithmic phase of 5×10 6 Centrifuge at 1500×g for 10 min at 4℃ and discard the supernatant. Note that the operation should be carried out at low temperature.

[0052] (2) 1 mL of sterile 375 mM sorbitol was pre-cooled in an ice bath, and the algal cells were washed three times. After each wash, the cells were centrifuged at 1500 × g at 4°C for 1–2 minutes, and transferred to a 1.5 mL centrifuge tube before the third wash. After centrifugation, the supernatant was discarded, and the cell pellet was resuspended in 100 μL of pre-cooled 375 mM sorbitol solution, resulting in an algal cell density of approximately 2 × 10⁻⁶ cells / mL. 9 cells·mL -1 Cell resuspension;

[0053] (3) The 0.2 cm electroporation reaction vessel was pre-cooled on ice, and then the following components were mixed: 100 μL cell resuspension, 4 μg linearized plasmid DNA (concentration of 0.2 μg / μL), and 4 μL salmon sperm DNA solution (10 μg / μL) that had been denatured at 100℃. After the mixture was allowed to stand on ice for 10 minutes, it was immediately transferred to the pre-cooled electroporation vessel; the electroporation transformation parameters were optimized as follows: field strength 500 V / cm, capacitance 25 μF, parallel resistance 400 Ω, and pulse time 4-5 s.

[0054] (4) After electroporation, the transformed cell suspension was quickly transferred to a 15 mL sterile centrifuge tube containing 10 mL of f / 2 medium for resuscitation culture, and placed under low light conditions (light intensity approximately 30 μmol·m⁻¹). -2 ·s -1 After 24 hours of resuscitation, the cells were transferred to a standard culture environment and cultured for another 24 hours.

[0055] (5) After the algal cells are centrifuged at 4℃ and 1500 × g for 10 minutes, the supernatant is removed. The cell suspension is then resuspended in 600 μL of f / 2 liquid culture medium. 200 μL of the suspension is evenly spread on the surface of f / 2 solid culture medium containing bleomycin. After the spread layer is completely dry, it is cultured under standard culture conditions for 12-14 days.

[0056] Specific Implementation Example 4 bd1311 The specific steps for screening and identifying gene-overexpressing algal strains are as follows:

[0057] Step 1: Pick a single algal colony from the bleomycin-resistant plate obtained in Specific Example 3, and streak it 3-5 mm long on f / 2 solid medium containing 50 μg / mL bleomycin. Incubate at 18℃ under light for 7 days. Wait until a 2 mm new algal colony forms at the end of the streak, ensuring sterility during the process. Then, take half of the algal colony sample and add 20 μL of algal lysis buffer for thermal lysis. The resulting lysis product is used as a PCR amplification template. The algal lysis buffer formulation is: 1% Nonidet P40 (NP40), 10.00 mM Tris, 0.14 mM NaCl, 5.00 mM KCl;

[0058] Step 2: Design the nucleotide sequence of the forward primer for algal identification as shown in SEQ ID NO.7: TGTTTGGACCTGGATAAGA, and the nucleotide sequence of the reverse primer for algal identification as shown in SEQ ID NO.8: ACACGACCTCCGACCACT. The algal PCR reaction system is as follows: 10 μL 2×Flash Hot Start Master, 1 μL forward primer for algal identification, 1 μL reverse primer for algal identification, 1 μL algal solution, and ddH2O to a total volume of 20 μL. The algal PCR reaction program is as follows: 95℃ for 5 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 5-10 s / kb, 30 cycles; cool to 4℃.

[0059] The PCR-identified products were analyzed by electrophoresis, such as... Figure 1 As shown, the results indicated that all transformants amplified the expected 500 bp target band, while the corresponding fragment was not detected in the wild-type algae. Further sequencing confirmed that this fragment was completely identical to the bleomycin resistance gene sequence (100% similarity). Based on this, two transformants, T3 and T6, were randomly selected for further research. After homozygous identification, homozygous algae bd-3 and bd-6, which stably overexpress bd1311, were finally obtained. According to the above results, the bd-3 and bd-6 algal colonies were inoculated into f / 2 liquid medium for expansion culture, and after appropriate dilution, they were spread onto fresh plates for further culture.

[0060] Specific Implementation Example 5 bd1311 RNA extraction from overexpressed transgenic algae and quantitative real-time PCR of genes related to fucoxanthin synthesis and HSP protein-related transcription factor genes.

[0061] Step 1: RNA extraction from the bd1311 overexpressing transgenic algae. The specific steps are as follows:

[0062] (1) Collect 50 mL of algae in the logarithmic growth phase (algal cell concentration 2×10⁻⁶).6 cells·mL -1 The culture medium of the bd1311 overexpressing transgenic algae was centrifuged at 3000×g for 5 min in a pre-cooled centrifuge at 4℃. After removing the supernatant, the algal cell pellet was immediately transferred to liquid nitrogen for quick-freezing and preservation.

[0063] (2) Pre-cool the mortar and grinding rod in liquid nitrogen, then quickly transfer the frozen algal mud sample into the pre-cooled mortar. Continuously replenish liquid nitrogen during grinding to maintain the low-temperature environment, and use the mortar and grinding rod for intermittent grinding (3-5 min) until the sample reaches a uniform powder state. Avoid sample splashing and ensure the algae are always submerged in liquid nitrogen to prevent RNA degradation;

[0064] (3) Total RNA extraction was performed using the RNeasy Plant Mini Kit, strictly following the manufacturer's operating procedures;

[0065] (4) The concentration and purity of RNA samples were quantitatively detected using a micro-volume nucleic acid analyzer. RNA samples that met the quality standards were then used for cDNA first-strand synthesis, and the remaining RNA solutions were aliquoted and stored at -80℃ for long-term preservation.

[0066] (5) Purified high-integrity RNA was used as the starting template. The reverse transcription reaction was performed according to the standard procedure in the PrimeScript™ RTReagent Kit manual to synthesize cDNA template. The entire experiment used dedicated pipette tips and centrifuge tubes that were inactivated by RNase. The reaction system was as follows: PrimeScript™RT Enzyme Mix Ⅰ 0.5 μL, PrimeScript™ Buffer (for Real Time) 2 μL, Random 6 mers (100 μM) 0.5 μL, OligodT primer (50 μM) 0.5 μL, total RNA 2 μL, and RNase-free ddH2O was added to 10 μL. The amplification program was: 37℃ for 15 min, 85℃ for 5 s, 4℃ for ∞, and then the cDNA template was obtained.

[0067] Step 2: Real-time PCR (RT-PCR) of fucoxanthin synthesis-related genes and HSP protein-related transcription factor genes in bd1311-overexpressed transgenic algae:

[0068] Genes related to fucoxanthin synthesis include lycb, psy, zds, and crtiso5; HSP protein-related transcription factor genes include PHATRDRAFT_47062, PHATRDRAFT_35158, PHATRDRAFT_54150, PHATRDRAFT_36981, PHATRDRAFT_54656, PHATRDRAFT_54019, PHATRDRAFT_55215, PHATRDRAFT_55890, and PHATRDRAFT_17633. Full-length genes were obtained from NCBI, and quantitative PCR primers were designed using Primer 3.

[0069] The nucleotide sequence of the forward primer for psy fluorescence quantitative PCR is shown in SEQ ID NO.9: GTCTATGTTTGGTGTCGACGAA, and the nucleotide sequence of the reverse primer for psy fluorescence quantitative PCR is shown in SEQ ID NO.10: AAGCACAGGTCAAAGACATCCT.

[0070] The nucleotide sequence of the lycb fluorescence quantitative forward primer is shown in SEQ ID NO.11: GCCTTTAATGACGGTCCTACTG, and the nucleotide sequence of the lycb fluorescence quantitative reverse primer is shown in SEQ ID NO.12: AATTTCGTCACCCTCGAAAGTA.

[0071] The nucleotide sequence of the forward primer for CRTiso5 quantitative PCR is shown in SEQ ID NO.13: GAGGATCGGCTCATACATTCTC, and the nucleotide sequence of the reverse primer for CRTiso5 quantitative PCR is shown in SEQ ID NO.14: GCATCTCTTCTTCCAGGACATC.

[0072] The nucleotide sequence of the ZDS quantitative PCR forward primer is shown in SEQ ID NO.15: CGGCATACCAGGTAGTCTTTTC, and the nucleotide sequence of the ZDS quantitative PCR reverse primer is shown in SEQ ID NO.16: TTCGGATCTTTCAACCTTGTCT.

[0073] The nucleotide sequence of the forward primer for the internal control Actin fluorescence quantitative PCR is shown in SEQ ID NO.17: GACTCCACCTTCCAGACCATTA, and the nucleotide sequence of the reverse primer for the internal control Actin fluorescence quantitative PCR is shown in SEQ ID NO.18: GACCCTCCAATCCAAACAGAG.

[0074] The nucleotide sequence of the forward primer for quantitative fluorescence detection of PHATRDRAFT_47062 is shown in SEQ ID NO.19: CATGCTGAAGACTTGCTACAGG, and the nucleotide sequence of the reverse primer for quantitative fluorescence detection is shown in SEQ ID NO.20: GTCCTTTCATTGAGCAAAAAGC.

[0075] The nucleotide sequence of the forward primer for quantitative fluorescence detection of PHATRDRAFT_35158 is shown in SEQ ID NO.21: GAGAAAATGACGGCTAACCTTG, and the nucleotide sequence of the reverse primer for quantitative fluorescence detection is shown in SEQ ID NO.22: CGCAATCTTTTTGAGAGGTTTC;

[0076] The nucleotide sequence of the PHATRDRAFT_54150 fluorescence quantitative forward primer is shown in SEQ ID NO.23: ACAATCCGTCATTCCTGAAATC, and the nucleotide sequence of its fluorescence quantitative reverse primer is shown in SEQ ID NO.24: AAGCTTTTGACTTCTTCGTCAGG;

[0077] The nucleotide sequence of the forward primer for quantitative fluorescence detection of PHATRDRAFT_36981 is shown in SEQ ID NO.25: CGGACAATGATGAGAAGTTTGA, and the nucleotide sequence of the reverse primer for quantitative fluorescence detection is shown in SEQ ID NO.26: AGCTATACGTCCCACTCTTGGA.

[0078] The nucleotide sequence of the PHATRDRAFT_54656 fluorescence quantitative forward primer is shown in SEQ ID NO.27: GAAGCGAGCTTGATGGTAGAGT, and the nucleotide sequence of its fluorescence quantitative reverse primer is shown in SEQ ID NO.28: TCTAATGGCTCCTTTGTCCAGT.

[0079] The nucleotide sequence of the forward primer for quantitative fluorescence detection of PHATRDRAFT_54019 is shown in SEQ ID NO.29: CGATTGGGTACCACCTACAGT, and the nucleotide sequence of the reverse primer for quantitative fluorescence detection is shown in SEQ ID NO.30: TAAAGGCAACGTACGAAGGAGT.

[0080] The nucleotide sequence of the forward primer for quantitative fluorescence detection of PHATRDRAFT_55215 is shown in SEQ ID NO.31: GTTGCTAATCCCATTCTCAAGC, and the nucleotide sequence of the reverse primer for quantitative fluorescence detection is shown in SEQ ID NO.32: CCGTCCAAATCTTCACCTAAAA;

[0081] The nucleotide sequence of the PHATRDRAFT_55890 fluorescence quantitative forward primer is shown in SEQ ID NO.33: TTCCGATTTCTATCTGGAAGGA, and the nucleotide sequence of its fluorescence quantitative reverse primer is shown in SEQ ID NO.34: CTGTCTTCATTTTCGGTTCCTC.

[0082] The nucleotide sequence of the forward primer for quantitative fluorescence detection of PHATRDRAFT_17633 is shown in SEQ ID NO.35: ACTGAGAAATCGCTCACAGACA, and the nucleotide sequence of the reverse primer for quantitative fluorescence detection is shown in SEQ ID NO.36: GTCACCGTCCTTAACTTCCTTG.

[0083] The PCR reaction system consisted of: 0.4 μL of forward primer, 0.4 μL of reverse primer, 2 μL of cDNA, 10 μL of 2×Taq Pro UniversalSYBR qPCR Master Mix, and ddH2O to a final volume of 20 μL. The quantitative PCR program was as follows: pre-denaturation at 95℃ for 30 s, followed by 40 cycles of 95℃ for 10 s, then 60℃ for 30 s, and finally melting at 95℃ for 15 s, 60℃ for 60 s, and 95℃ for 15 s.

[0084] Based on the above qRT-PCR experimental results, as follows: Figure 2 The results showed that among the genes known to be related to fucoxanthin synthesis, four were differentially expressed between the overexpressing algal strain BD-3 and the empty vector control: psy, zds, lycb, and crtiso5. Compared with the empty vector control, the expression levels of these four fucoxanthin synthesis-related genes were significantly increased in the overexpressing algal strain BD-3, but there was no significant difference in the overexpressing algal strain BD-6. These results indicate that BD1311 may affect the expression of fucoxanthin synthesis-related genes through a differential regulatory mechanism, thereby effectively regulating the fucoxanthin biosynthesis pathway.

[0085] To determine whether transcription factor bd1311 affects the expression levels of other HSP protein-related genes, qRT-PCR was performed on homozygous algal strains overexpressing transcription factor bd1311. Figure 3As shown, the results indicate that, compared with the control, the expression levels of HSP protein-related genes were increased in the overexpression strain bd-3, while there was no significant difference in bd-6. These results suggest that the transcription factor bd1311 may regulate the thermotolerant response mechanism of *Phaeodactylum tricornutum* by affecting the expression of HSP proteins.

[0086] Specific Example 6: High-temperature experiment on BD1311 overexpression transcribed algae.

[0087] Step 1: The homozygous bd1311 overexpressing algal strain was inoculated into f / 2 liquid medium containing 50 μg / mL bleomycin and cultured under standard conditions until the logarithmic growth phase. Algal cells were then collected by aseptic centrifugation and washed three times with sterile f / 2 medium to thoroughly remove antibiotic residues. Finally, the algal cells were resuspended in fresh f / 2 medium, and the initial cell density was adjusted to 3 × 10⁻⁶ cells / mL using a hemocytometer. 5 cells·mL -1 To prepare an algal suspension for the experiment.

[0088] Step 2: Spot the algal solution evenly onto f / 2 solid medium, using wild-type and algal strains containing the pPha-T1 empty vector as controls. Apply 5 μL per spot, with three replicates per group. Transfer to a 27℃ constant temperature and light incubator, using a white cold light lamp and 50 μmol·m² light intensity. -2 ·s -1 They were cultured under conditions of a 12-hour light-dark cycle: 12-hour dark cycle.

[0089] Step 3: Transfer 2 mL of liquid algal culture to a 12-well plate, using algal strains containing the pPha-T1 empty vector as controls (pp-5, pp-2), with three replicates per group. Incubate at 27℃ under constant temperature and light, using a white cold light lamp and 50 μmol·m⁻² light intensity. -2 ·s -1 Cultured under a 12 h:12 h light-dark cycle. If growth is not possible at 27℃, the temperature should be lowered and the experiment repeated.

[0090] Through high temperature test Figure 4 It can be seen that, compared with the control, the overexpressing algal strains bd-3 and bd-6 showed a deeper color on the solid medium at high temperatures, indicating better growth. bd-3 had the deepest color, while bd-6 was slightly lighter but still significantly darker than the control. This indicates that the overexpressing algal strains can survive on solid medium at 26℃, while the wild-type and empty vector algal strains could not grow. Figure 5 As shown, neither the overexpressed algal strain nor the wild-type could survive in the well plate, while the overexpressed algal strain grew better than the empty vector algal strain at 26℃, and its color was significantly darker than the control.

[0091] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A heat-resistant engineered strain of *Phaeodactylum tricornutum*, characterized in that: The high-temperature resistant engineered algal strain described above is an overexpression bd1311 The gene of the brown finger algae, as described bd1311 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The heat-resistant engineered strain of *Phaeodactylum tricornutum* according to claim 1, characterized in that: The amino acid sequence of the protein encoded by bd1311 is shown in SEQ ID NO.

2.

3. The heat-resistant engineered strain of *Phaeodactylum tricornutum* according to claim 1, characterized in that: The aforementioned bd1311 The nucleotide sequence of the primers for forward amplification of gene overexpression is shown in SEQ ID NO.5: TGTCTGCCGTTTCGAGAATTCATGACGTTCCCACCGACGG. bd1311 The nucleotide sequence of the reverse amplification primer for gene overexpression is shown in SEQ ID NO.6: CAGGTCGACTCTAGAGGATCCCTAGGGCAGTTGTGGTCGATAAA.

4. A method for constructing a heat-resistant engineered algal strain of *Phaeodactylum tricornutum* as described in claim 1, characterized in that... Includes the following steps: Step 1: Construct an overexpression vector using pPha-T1 as the vector, and employ seamless cloning based on homologous recombination. bd1311 The full-length sequences of the genes are ligated and fused to obtain bd1311 Gene overexpression vectors; Step 2, Design bd1311 Gene overexpression amplification primers, among which bd1311 The nucleotide sequence of the primers for forward amplification of gene overexpression is shown in SEQ ID NO.5: TGTCTGCCGTTTCGAGAATTCATGACGTTCCCACCGACGG. bd1311 The nucleotide sequence of the reverse amplification primer for gene overexpression is shown in SEQ ID NO.6: CAGGTCGACTCTAGAGGATCCCTAGGGCAGTTGTGGTCGATAAA; Step 3: Use the design from Step 1 bd1311 Gene overexpression amplification primers were used to amplify the target fragment using wild-type brown finger algae cDNA as a template; pPha-T1 was linearized using EcoRI and BamHI to obtain the enzyme-digested linearized vector; Step 4: The target fragment obtained in Step 3 and the enzyme-digested linearized vector are recombinantly cloned to obtain the recombinant vector; Step 5: Transform the recombinant vector obtained in Step 4 into E. coli, take bacterial culture samples with positive bands for amplification, and extract plasmids using a plasmid extraction kit to obtain... bd1311 Gene overexpression vectors; Step 6: Obtain the result from Step 5 bd1311 The gene overexpression vector was linearized by restriction endonuclease digestion. bd1311 Gene overexpression vectors will linearize bd1311 Gene overexpression vectors were electroporated into *Phaeodactylum tricornutum*, and the transformed *Phaeodactylum tricornutum* cells were plated on f / 2 plates for culture. Positive algal strains were screened to obtain heat-resistant engineered *Phaeodactylum tricornutum* strains.

5. The application of the heat-resistant engineered algal strain of *Phaeodactylum tricornutum* as described in claim 1 in the preparation of heat-resistant algal strains.

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