Method for verifying function of Cre in oil-producing microalgae and application of Cre in enhancing biosynthesis of eicosapentaenoic acid

Abstract

The invention provides a method for verifying functions of Cre in oil-producing microalgae and application of the method in enhancing biosynthesis of eicosapentaenoic acid, and belongs to the technical field of genetic modification. According to the invention, functional verification of Cre in oil-producing microalgae is completed by a method of inserting an intron from an endogenous Phatr3J51169 gene of phaeodactylum tricornutum into a Cre gene, that is, a selection marker located between two loxP sequences can be removed, and a free plasmid transformation method more suitable for Cre removal is further developed. Based on a Cre / loxP system, the expression of EPA synthetic pathway genes in the oil-producing microalgae is enhanced to obtain an engineering algal strain with high yield of EPA, and compared with a wild type, the relative content of EPA can be increased by 24.7%.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic modification, and particularly relates to a method for verifying the function of Cre in oil-producing microalgae and application of the method in enhancing the biosynthesis of eicosapentaenoic acid. BACKGROUND

[0002] Eicosapentaenoic acid (EPA, C20:5Δ5,8,11,14,17) is a kind of omega 3 series very long chain polyunsaturated fatty acids (VLC-PUFAs), which can effectively reduce the content of cholesterol and triglyceride, reduce the viscosity of blood lipid of blood vessels, and significantly promote cardiovascular health. At present, the main source of VLC-PUFAs for human beings is still deep sea fish oil, and marine microorganisms including marine microalgae are the initial producers of VLC-PUFAs. Direct extraction of VLC-PUFAs from marine microalgae not only can effectively avoid the huge loss of VLC-PUFAs in the transmission of food chain, but also has the advantages of simpler process, avoidance of irritating fishy smell and cholesterol in traditional fish oil.

[0003] It is an effective method to construct a transgenic microalgae strain producing EPA by genetic modification method. The genetic modification tools include vector construction, transformation and screening technology. At present, there are still some technical problems in these genetic modification tools, for example, the vector capacity and transformation efficiency are limited during transformation, which causes that the number of genes that can be introduced in a single transformation is limited. In addition, when multiple genes need to be integrated in the transformant, multiple different screening markers need to be used. This causes that it is still difficult to construct a complex metabolic pathway in the metabolic engineering modification of microalgae.

[0004] The Cre / loxP recombination system is the most widely used site-specific recombination system at present, which is composed of Cre recombinase and loxP site that can be specifically recognized by it. Cre recombinase can activate and recombine two loxP in the same sequence in the same direction, so that the sequence between the loxP sites and one of the loxP sequences are deleted. By using this characteristic, the screening marker can be inserted between the two same direction loxP sites, and then the integrated screening marker is deleted by using Cre recombinase. Although the Cre recombinase system has the advantages of simplicity and high efficiency, how to realize the expression of Cre in different species is still a problem to be solved. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a method for verifying the function of Cre enzyme in oil-producing microalgae. By inserting a specific intron into the Cre gene, the expressed Cre enzyme removes the screening marker located between the two loxP sequences, proving that the Cre enzyme in the oil-producing microalgae has normal recombinase activity function.

[0006] The application provides an analysis method of Cre recombinase function in oil-producing microalgae, comprising the following steps:

[0007] constructing an expression cassette containing a Cre-J51169-P2A fusion sequence and a first resistance gene, a second resistance gene expression cassette with loxP sequences at both ends into a backbone vector to obtain a first recombination vector;

[0008] transferring the first recombination vector into oil-producing microalgae, and obtaining a recombinant oil-producing microalgae; culturing the recombinant oil-producing microalgae in a culture medium containing an antibiotic corresponding to the first resistance gene for the first time, and transferring the positive monoclonal to a culture medium containing an antibiotic corresponding to the second resistance gene for the second time, to obtain a recombinant oil-producing microalgae that can grow in the first culture but cannot grow in the second culture, indicating that the Cre recombinase is normally expressed in the recombinant oil-producing microalgae and exerts the function of the recombinase;

[0009] The Cre-J51169-P2A fusion sequence is obtained by fusing a Cre gene and a P2A sequence, and the Cre gene is a Cre gene with a Phatr3_J51169 gene intron embedded in the middle.

[0010] Preferably, the nucleotide sequence of the Phatr3_J51169 gene intron is shown in SEQ ID NO: 7.

[0011] Preferably, the 5' end of the Cre gene in the Cre-J51169-P2A fusion sequence is fused with a 3xFlag tag and a nuclear localization signal peptide sequence, and the 3' end of the Cre gene is further fused with a double-type nuclear localization signal sequence of a nuclear lysosome.

[0012] The application provides a group of recombination vectors, including pPhtJ05 and pPhtJ06.

[0013] The pPhtJ05 is a backbone vector containing the following genetic elements: a double-copy Cre gene expression cassette, a single-copy resistance gene expression cassette and a CEN6-ARSH4-HIS3 fusion fragment.

[0014] The pPhtJ06 is a backbone vector containing the following genetic elements: a double-copy PtDes12 gene expression cassette, a single-copy PtDes9 gene expression cassette and a second resistance gene expression cassette with loxP sequences at both ends.

[0015] Preferably, it further includes pPhtJ07.

[0016] The pPhtJ07 is a backbone vector containing the following genetic elements: a PtDes6 gene expression cassette, a PtELO6B_1 gene expression cassette, a PtDes5A gene expression cassette, and a second resistance gene expression cassette connected with loxP sequences at both ends.

[0017] The application provides application of the recombinant vector in promoting biosynthesis of eicosapentaenoic acid in oil-producing microalgae.

[0018] Preferably, the oil-producing microalgae comprises Phaeodactylum tricornutum.

[0019] The application provides a method for promoting biosynthesis of eicosapentaenoic acid in oil-producing microalgae, comprising the following steps:

[0020] The pPhtJ06 in the recombinant vector is introduced into the oil-producing microalgae, and screening culture is performed, so that a first recombinant algae strain is obtained;

[0021] The pPhtJ05 in the recombinant vector is introduced into the first recombinant algae strain by using bacterial conjugation and transfer technology, and after twice screening culture, a second recombinant algae strain without the second resistance gene expression cassette connected with loxP sequences at both ends is obtained;

[0022] The second recombinant algae strain without the second resistance gene expression cassette connected with loxP sequences at both ends is cultured, and a third recombinant algae strain without pPhtJ05 is selected.

[0023] Preferably, after the third recombinant algae strain without pPhtJ05 is obtained, the pPhtJ07 in the recombinant vector is further introduced into the third recombinant algae strain without pPhtJ05, screening culture is performed, and a fourth recombinant algae strain is obtained.

[0024] The application provides a recombinant algae strain for producing eicosapentaenoic acid, wherein oil-producing microalgae are used as hosts, and the recombinant expression of a PtDes12 gene and a PtDes9 gene is further included.

[0025] Or the recombinant expression of a PtDes12 gene, a PtDes9 gene, a PtDes6 gene, a PtELO6B_1 gene and a PtDes5A gene is further included.

[0026] The application provides an analysis method for Cre recombinase function in oil-producing microalgae, wherein an intron sequence derived from a microalgae endogenous Phatr3_J51169 gene is inserted in a Cre coding sequence, so that the complete Cre can be prevented from removing the screening marker on the plasmid itself during plasmid construction, and the intron can also be effectively recognized and removed in Phaeodactylum tricornutum transformation. The complete Cre protein and the blasticidin resistance protein connected through a P2A peptide are successfully translated, and a transformed algae strain with blasticidin resistance is obtained.

[0027] The application provides a method for promoting biosynthesis of eicosapentaenoic acid by oil-producing microalgae, by developing Cre / loxP recombinase system for application in oil-producing microalgae, removal of integrated screening markers can be realized, and removal of free Cre plasmid by simple antibiotic-free medium transmission is realized, and use in algal strain transformation of the same screening marker is realized. The application provides a promising solution for iterative integration of oil-producing microalgae to strengthen complex pathways. The genetically engineered high-EPA engineering algal strain obtained by the application can increase the relative content of EPA by more than 17.3% compared with the wild type, and the highest can reach 24.7%. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Test results of the vector for being successfully recognized and cut in an intron in Phaeodactylum tricornutum;

[0029] Figure 2 Intron screening results;

[0030] Figure 3 Test results of the vector for Cre function verification in Phaeodactylum tricornutum;

[0031] Figure 4 Detection results (a) of loss of bleomycin resistance and PCR detection results (b) of the bleomycin resistance gene caused by the Cre function;

[0032] Figure 5 Combination transfer experiment process;

[0033] Figure 6 Detection results (a) of loss of bleomycin resistance and PCR detection results (b) of the bleomycin resistance gene caused by the Cre function under the combination transfer form;

[0034] Figure 7 Schematic diagram of the first round of multi-gene overexpression plasmid for strengthening the EPA synthesis pathway;

[0035] Figure 8 Detection results (a) of loss of bleomycin resistance and PCR detection results (b) of the bleomycin resistance gene caused by the Cre function in the pPhtJ06 transformed algal strain;

[0036] Figure 9 Detection results (a) of loss of bleomycin resistance and PCR detection results (b) of the bleomycin resistance gene caused by the Cre function in the pPhtJ06 transformed algal strain;

[0037] Figure 10 Schematic diagram of the second round of multi-gene overexpression plasmid for strengthening the EPA synthesis pathway;

[0038] Figure 11 Fatty acid composition detection results. DETAILED DESCRIPTION

[0039] The application provides an analysis method of Cre recombinase function in oil-producing microalgae, comprising the following steps:

[0040] The expression cassette containing the Cre-J51169-P2A fusion sequence and the first resistance gene, and the second resistance gene expression cassette with loxP sequences at both ends are constructed into a backbone vector to obtain a first recombinant vector;

[0041] The first recombinant vector is transformed into the oil-producing microalgae, and the obtained recombinant oil-producing microalgae is cultured in a culture medium containing an antibiotic corresponding to the first resistance gene for the first time, and the grown monoclonal is transferred to a culture medium containing an antibiotic corresponding to the second resistance gene for the second time, to obtain a recombinant oil-producing microalgae that can only grow in the first culture medium but cannot grow in the second culture medium, indicating that the Cre recombinase is normally expressed in the recombinant oil-producing microalgae and exerts the recombinase function;

[0042] The Cre-J51169-P2A fusion sequence is obtained by fusing a Cre gene and a P2A sequence, and the Cre gene is a Cre gene with a Phatr3_J51169 gene intron embedded in the middle.

[0043] The application constructs the expression cassette containing the Cre-J51169-P2A fusion sequence and the first resistance gene, and the second resistance gene expression cassette with loxP sequences at both ends into a backbone vector to obtain a first recombinant vector.

[0044] The kind of the oil-producing microalgae in the application is not particularly limited, and an oil-producing microalgae known in the art can be used, for example, Phaeodactylum tricornutum.

[0045] In the application, the Cre gene is embedded with the Phatr3_J51169 gene intron, which can prevent the Cre gene from removing the selection marker on the plasmid itself, and can also be specifically recognized and removed by enzymes in the microalgae when transformed into the microalgae, so as to obtain complete Cre enzyme and exert the recombinase function in the algal body. The P2A sequence carried in the Cre-J51169-P2A fusion sequence is fused with the Cre enzyme and the resistance gene, and the P2A sequence exerts self-cleavage activity to cleave the obtained fusion protein, so that the recombinant algal strain has resistance to the antibiotic corresponding to the first resistance gene.

[0046] In one embodiment of the present application, in order to screen the verified inserted intron, the intron of Phatr3_J54983 gene of Phaeodactylum tricornutum (SEQ ID NO: 5), the intron of Phatr3_J18559 gene of Phaeodactylum tricornutum (SEQ ID NO: 6), and the intron of Phatr3_J51169 gene of Phaeodactylum tricornutum (SEQ ID NO: 7) are inserted into the Cre gene, respectively, and the results show that only the intron of Phatr3_J51169 gene of Phaeodactylum tricornutum group realizes the expression of Cre gene and resistance gene, and through functional verification, the expressed Cre enzyme has the purpose of removing the loxP sequence containing the target gene.

[0047] In the present application, the nucleotide sequence of the Cre gene is shown in SEQ ID NO: 3. In the Cre-J51169-P2A fusion sequence, the 5' end of the Cre gene is fused with a 3xFlag tag and a nuclear localization signal peptide sequence, and the 3' end of the Cre gene is further fused with a double-type nuclear localization signal sequence of nuclear lysosome, so as to ensure the recombination enzyme function in the nucleus.

[0048] In the present application, the expression cassette containing the Cre-J51169-P2A fusion sequence and the first resistance gene includes the promoters and terminators distributed at both ends in addition to the tandem Cre-J51169-P2A fusion sequence and the first resistance gene. The present application does not have special restrictions on the types of the promoters and terminators, and the promoters and terminators known in the art can be used, for example, NR promoter, NR terminator, or fcpB promoter, fcpB terminator, or fcpA promoter, fcpA terminator. The present application does not have special restrictions on the types of the first resistance gene, and the resistance genes known in the art can be used, for example, bleomycin resistance gene (Sh ble), nourseothricin resistance gene (nat). The Sh ble gene is amplified from pPha-NR plasmid. The nat sequence is amplified from pPha-nat plasmid. The NR promoter and NR terminator are amplified from pPha-NR plasmid.

[0049] In the present application, the second resistance gene expression cassette with loxP sequence at both ends also includes promoters and terminators distributed at both ends. The promoters and terminators are the same as described above, and will not be described here. The types of the promoters and terminators in the expression cassette containing the Cre-J51169-P2A fusion sequence and the first resistance gene and the second resistance gene expression cassette with loxP sequence at both ends are preferably different. The loxP sequence is amplified from pCfB4586 plasmid, and the nucleotide sequence is shown in SEQ ID NO: 4.

[0050] In the present application, the construction method of the first recombinant vector preferably comprises amplifying a first promoter sequence, a first terminator sequence, two end Cre gene sequences, an intron J51169 sequence, a P2A sequence, a resistance gene, a second promoter sequence, a second terminator sequence, a loxP sequence and a linear backbone vector to assemble a circular first recombinant vector. In the assembly, the reaction system is 2x CE Mix, the DNA fragments are each 100 ng, and ddH2O is added to a total volume of 20 μL. The reaction procedure of the assembly is preferably: 50°C, 60 min, 8°C incubation.

[0051] After obtaining the first recombinant vector, the first recombinant vector is introduced into an oleaginous microalgae to obtain a recombinant oleaginous microalgae. The recombinant oleaginous microalgae is first cultured in a culture medium containing an antibiotic corresponding to the first resistance gene, and the grown monoclonal is transferred to a culture medium containing an antibiotic corresponding to the second resistance gene for second culture. The recombinant oleaginous microalgae that can grow in the first culture but cannot grow in the second culture is obtained, indicating that the Cre recombinase is normally expressed in the recombinant oleaginous microalgae and exerts the function of the recombinase.

[0052] The method for introducing the first recombinant vector into the oleaginous microalgae is not particularly limited in the present application, and a method known in the art can be used, for example, a gene gun shooting. When the first resistance gene is a nat gene, the corresponding antibiotic is nourseothricin. When the second resistance gene is a Shble gene, the corresponding antibiotic is bleomycin. The temperature of the first culture or the second culture is preferably 20-24°C, and can be 22°C. The concentration of the bleomycin is 70-80 mg / L, and can be 75 mg / L. The concentration of the nourseothricin is 140-160 mg / L, and can be 150 mg / L. In the first culture, nourseothricin is preferably used for screening, and the screened recombinant algae strain is an algae strain expressing Cre enzyme. In the second culture, bleomycin is preferably used for screening. When the algae strain cannot grow on a plate containing bleomycin, it indicates that the Sh ble gene has been removed, indicating that the Cre enzyme exerts the activity of cleaving the loxP sequence.

[0053] The present application provides a set of recombinant vectors, including pPhtJ05 and pPhtJ06.

[0054] The pPhtJ05 is a backbone vector containing the following genetic elements: a double copy of a Cre gene expression cassette, a single copy of a resistance gene expression cassette, and a CEN6-ARSH4-HIS3 fusion fragment.

[0055] The pPhtJ06 is a backbone vector containing the following genetic elements: a double copy of a PtDes12 gene expression cassette, a single copy of a PtDes9 gene expression cassette, and a second resistance gene expression cassette connected at both ends with a loxP sequence.

[0056] In the present application, the pPhtJ05 and pPhtJ06 each preferably further comprises a promoter and a terminator distributed at both ends in the expression cassette to realize the expression of the target gene alone. The type of the promoter and the terminator is not particularly limited in the present application, and a promoter and a terminator known in the art can be used. The nucleotide sequence of the PtDes12 gene is shown in SEQ ID NO: 8. The nucleotide sequence of the PtDes9 gene is shown in SEQ ID NO: 9. The nucleotide sequence of the CEN6-ARSH4-HIS3 fusion fragment is shown in SEQ ID NO: 10. The resistance gene in the pPhtJ05 is preferably nourseothricin resistance. The resistance gene in the pPhtJ06 is preferably a bleomycin resistance gene.

[0057] The method for constructing the pPhtJ05 and pPhtJ06 is not particularly limited in the present application, and an assembly method known in the art can be used, for example, the gene elements in the expression cassette are mixed with a linear backbone vector to assemble, and the assembly conditions are as described in the above technical solution.

[0058] In the present application, the recombinant vector preferably further comprises pPhtJ07. The pPhtJ07 is a backbone vector comprising the following gene elements: a PtDes6 gene expression cassette, a PtELO6B_1 gene expression cassette, a PtDes5A gene expression cassette, and a second resistance gene expression cassette connected with a loxP sequence at both ends. The expression cassette in the pPhtJ07 preferably further comprises a promoter and a terminator distributed at both ends in addition to the specific target gene. The type of the promoter and the terminator is not particularly limited in the present application, and a promoter and a terminator known in the art can be used. The nucleotide sequence of the PtDes6 gene is shown in SEQ ID NO: 11. The nucleotide sequence of the PtELO6B_1 gene is shown in SEQ ID NO: 12. The nucleotide sequence of the PtDes5A gene is shown in SEQ ID NO: 13. The method for constructing the pPhtJ07 can use an assembly method known in the art, for example, the gene elements in the expression cassette are mixed with a linear backbone vector to assemble, and the assembly conditions are as described in the above technical solution.

[0059] In the present application, the recombinant vector contains a Cre free plasmid, which can remove the integrated screening marker, so that the algae strain using the screening marker can be used again for overexpression experiment, and the same screening marker is used for two rounds of overexpression of genes in the EPA synthesis pathway, so that higher EPA content can be accumulated. At the same time, in order to facilitate experimental operation and finally realize the removal of Cre, the expression mode of the free Cre plasmid is developed. Under this expression mode, Cre can also successfully remove the screening marker. At the same time, after completing the subsequent continuous passage by antibiotic-free medium to realize the removal of the Cre plasmid.

[0060] The present application provides the use of the recombinant vector in promoting the biosynthesis of eicosapentaenoic acid in oleaginous microalgae.

[0061] In the present application, the oleaginous microalgae preferably include Phaeodactylum tricornutum.

[0062] The present application provides a method for promoting the biosynthesis of eicosapentaenoic acid in oleaginous microalgae, comprising the following steps: transferring pPhtJ06 in the recombinant vector into the oleaginous microalgae, and screening and culturing to obtain a first recombinant algae strain;

[0063] Using bacterial conjugation and transfer technology, pPhtJ05 in the recombinant vector is transferred into the first recombinant algae strain, and after two times of screening and culturing, a second recombinant algae strain without a second resistance gene expression cassette containing two end-connected loxP sequences is obtained.

[0064] The second recombinant algae strain without the second resistance gene expression cassette containing two end-connected loxP sequences is cultured, and a third recombinant algae strain without pPhtJ05 is selected.

[0065] The present application transfers pPhtJ06 in the recombinant vector into the oleaginous microalgae, and screens and cultures to obtain a first recombinant algae strain.

[0066] The method for transferring pPhtJ06 into the oleaginous microalgae is not specially limited in the present application, and a method well known in the art can be used, such as a gene gun method. In the present application, a gene gun is used to transform the wild type of Phaeodactylum tricornutum for the first round of multi-gene overexpression transformation, and a transformed algae strain with bleomycin resistance is obtained.

[0067] After obtaining the recombinant algae strain, the present application uses bacterial conjugation and transfer technology to transfer pPhtJ05 in the recombinant vector into the first recombinant algae strain, and after two times of screening and culturing, a second recombinant algae strain without a second resistance gene expression cassette containing two end-connected loxP sequences is obtained.

[0068] In the present application, the bacterial conjugation transfer technology is used to transform pPhtJ05 into the bacterial competent cell, and the recombinant strain is obtained. The recombinant strain is co-cultured with the first recombinant algal strain, so as to realize the transfer of pPhtJ05 to the first recombinant algal strain and the existence of the free form. The two screening cultures are first screened by the nourseothricin resistance, so as to obtain the recombinant algal strain expressing Cre enzyme. Then, the recombinant algal strain is screened by the bleomycin resistance, so as to select the recombinant algal strain which cannot grow under the culture condition of bleomycin, which indicates that the Cre enzyme can realize the excision of the Sh ble expression cassette.

[0069] In the present application, the free Cre plasmid of the transformed algal strain is cultured on the plate containing nourseothricin, so as to ensure that the free Cre plasmid exists in the algal strain. The single clone with the disappearance of the bleomycin resistance is screened, which proves that the Cre has realized the removal of the bleomycin resistance gene in the first round of transformation. The Cre plasmid is removed by continuous subculture in the medium without the antibiotic, which is convenient for the transformed algal strain to be used for subsequent transformation. The first round of transformed algal strain is transformed by using the gene gun, so as to obtain the second round of transformed algal strain with the bleomycin resistance. The method can realize the repeated use of the same screening marker in the gene integration. Through two rounds of multi-gene overexpression experiments, the content of EPA in the transformed algal strain of Phaeodactylum tricornutum is gradually improved.

[0070] After obtaining the second recombinant algal strain, the second recombinant algal strain without the second resistance gene expression cassette containing the two end connected loxP sequences is cultured, and the third recombinant algal strain without pPhtJ05 is selected.

[0071] In the present application, the culture is preferably the subculture for 1-2 months, and then the resistance screening and identification are performed. The subculture medium is preferably F / 2 medium. The resistance screening is preferably the nourseothricin resistance screening. The identification method is preferably the PCR method for amplifying the Cre sequence. The recombinant algal strain which has no resistance to nourseothricin and cannot amplify the Cre sequence is screened, the free pPhtJ05 plasmid in the recombinant algal strain has been completely removed, and the recombinant algal strain without the resistance marker is obtained.

[0072] In the present application, the third recombinant algal strain takes the oleaginous microalgae as the host, and further includes the recombinantly expressed PtDes12 gene and PtDes9 gene. In the embodiment of the present application, the second recombinant algal strain is subjected to the determination of the fatty acid content, and the results show that the relative contents of 14:0, 16:1, 16:3 and 18:1 fatty acids are lower than those of the wild type, but the content of EPA is increased by 17.3% than that of the wild type. In the present application, after obtaining the third recombinant algal strain without pPhtJ05, the pPhtJ07 in the recombinant vector is preferably transformed into the third recombinant algal strain without pPhtJ05, and the fourth recombinant algal strain is obtained through the screening culture.

[0073] In the present application, the pPhtJ07 is preferably introduced into the third recombinant algal strain by gene gun. The screening culture is preferably carried out in a medium containing bleomycin. The temperature of the screening culture is preferably 20-24℃, which can be 22℃. The light cycle of the screening culture is preferably 16h light: 8h dark, and the light intensity is preferably 75μmol·m -2 ·s -1 .

[0074] The present application provides a recombinant algal strain for producing eicosapentaenoic acid, comprising recombinantly expressed PtDes12 gene, PtDes9 gene, PtDes6 gene, PtELO6B_1 gene and PtDes5A gene.

[0075] In the present application, the recombinant algal strain is subjected to two rounds of metabolic engineering modification, so that the EPA yield is increased by a larger margin, which is increased by 24.7% compared with the wild type.

[0076] The method for verifying the function of Cre in the oil-producing microalgae and the application thereof in enhancing the biosynthesis of eicosapentaenoic acid are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0077] Example 1

[0078] A method for improving the EPA synthesis capacity of Phaeodactylum tricornutum using Cre / loxP recombinase system

[0079] (1) Acquisition of gene

[0080] The Cre gene sequence is obtained from the public database NCBI and codon optimized, and 3×Flag and a nuclear localization signal peptide (SV40 NLS) are fused to the N terminus of the Cre gene sequence, and a nucleolysin double-type nuclear localization signal (nucleoplasmin NLS) is fused to the C terminus. The amino acid sequence of the 3×Flag and SV40 NLS sequence is shown in SEQ ID NO: 1 (MDY KDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAA). The amino acid sequence of the NLS sequence is shown in SEQ ID NO: 2 (KRPAATKKAGQAKKKK). The DNA sequence of the Cre gene with the added nuclear localization signal is shown in SEQ ID NO: 3

[0081] (2) Intron splicing verification

[0082] The Cre sequence synthesized by artificial chemistry was used as a template to amplify the two fragments of Cre, and the amplification primers were Cre-A-F (tcaccacttgtgcgaacggaattatggactataaggaccacgacg, SEQ ID NO: 19), Cre-A-R (catgtcacgggcggctccgac, SEQ ID NO: 20), Cre-B-F (gcccgtgccggagtctcca, SEQ ID NO: 21), and Cre-B-R (gcttcccgtctcgcgcgcctttttcttttttgcctggccg, SEQ ID NO: 22).

[0083] The intron fragments of J54983, J18559, and J51169 were amplified from the genomic DNA of Phaeodactylum tricornutum using primers intronJ54983F (cggagccgcccgtgacatggtaggtcgtcatcgcagtgc, SEQ ID NO: 23), intronJ54983R (gagactccggcacgggcctacggagcgtcaaggatga, SEQ ID NO: 24), intronJ18559F (gagccgcccgtgacatggtgcggataatgtagtgttgacttaagtag, SEQ ID NO: 25), intronJ18559 (gagactccggcacgggcctggaagaggaagcaaacagtaagg, SEQ ID NO: 26), intronJ51169F (ggagccgcccgtgacatggtatgttttagttttgcaatgcatgc, SEQ ID NO: 27), and intronJ51169R (gagactccggcacgggcctagagagacgcagacgggag, SEQ ID NO: 28), respectively.

[0084] The NR promoter was amplified using PNR-HR-F (gagagtgcaccatatggaattggaagtgactgtaaacgagaag, SEQ ID NO: 29) and PNR-HR-R (cgttcgcacaagtggtgact, SEQ ID NO: 30) as primers, the NR terminator was amplified using TNR-HR-F (ggccgaggagcaggactgaaagcttcaatttactttaagactagcgcaat, SEQ ID NO: 31) and TNR-HR-R (gggagaccggcggtacctccggatgcgttcattttagatc, SEQ ID NO: 32) as primers, the BlaR gene (Sh ble) was amplified using BleoR-HR-F (ggacctggactccggcgcatggccaagttgaccagtgc, SEQ ID NO: 33) and BleoR-HR-R (aagctttcagtcctgctcctcggcca, SEQ ID NO: 34) as primers, and the vector backbone was amplified using GJ-HR-F (acgcatccggaggtaccgccggtctccctatagtgag, SEQ ID NO: 35) and GJ-HR-R (acttccggtaccatccgaattctccattgcgtcctgcagcc, SEQ ID NO: 36) as primers, with the pPha-NR plasmid (sequence reference NCBI Accession No: JN180663.1, the plasmid was artificially synthesized) as the template.

[0085] The above PCR amplification was performed using 2x Phanta Max Master Mix high-fidelity enzyme, and the reaction system was as follows: 25 μL 2x Phanta Max Master Mix, 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 1 μL template DNA, 20 μL ddH2O. The prepared system was subjected to PCR amplification, and the program was as follows: 95°C for 5 min; 95°C for 15 s, 56°C for 15 s, 72°C for 3 min, for a total of 35 cycles; 72°C for 5 min; 8°C for incubation. The products obtained by amplification were purified by a gel recovery kit, and the specific steps were performed according to the kit manual.

[0086] 10 μL of each of the forward primer P2A-F (gcgcgcgagacgggaagcggagctactaacttcagcctgctgaagcaggctggcgacgtggaggagaaccctggacctggactccggcgc, SEQ ID NO: 37) (10 μM) and the reverse primer P2A-R (gcgccggagtccaggtccagggttctcctccacgtcgccagcctgcttcagcaggctgaagttagtagctccgcttcccgtctcgcgcgcgc, SEQ ID NO: 38) were mixed and annealed to obtain the P2A product. The reaction program was as follows: 37℃ for 30 min; 95℃ for 5 min; cooling rate 1℃ per minute to 50℃. The nucleotide sequence of P2A is shown in SEQ ID NO: 15 (GCGCGCGAGACGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGCGACGTGGAGGAGAACCCTGGACCTGGACTCCGGCGC).

[0087] according to Figure 1 The products obtained above were assembled using homologous recombinase to match the overlapping sequences of the fragments. The reaction system was: 2×CE Mix, 100 ng of each DNA fragment, and ddH2O added to a total volume of 20 μL. The reaction program was: 50℃, 60 min, and incubation at 8℃, yielding three circular plasmids (pPhtJ01_a, pPhtJ01_b, pPhtJ01_c) with introns J54983, J18559, and J51169 inserted into the Cre sequence, respectively. The Cre sequence (intron J54983 is shown in gray) in plasmid pPhtJ01_a is shown in SEQ ID NO: 16. As shown. The sequence of Cre(intronJ18559) in the pPhtJ01_b plasmid is shown in SEQ ID NO: 17.

[0088] As shown. The sequence of Cre(intronJ51169) in the pPhtJ01_c plasmid is shown in SEQ ID NO: 18. As shown.

[0089] The constructed plasmids pPhtJ01_a, pPhtJ01_b, and pPhtJ01_c are used to... Figure 1 The gene gun was used to transform and introduce the gene into wild-type *Phaeodactylum tricornutum* strains; the bombarded strains were then transferred to F / 2 solid medium containing 75 mg / L bleomycin, and the plates were placed in a 16-hour light-8-hour dark (75 μmol·m⁻¹) environment. -2 ·s -1 Incubate at 22℃. If the intron inserted in Cre can be successfully recognized and cleaved, Cre-P2A-Sh ble will be fully translated, giving the algal strain bleomycin resistance. Bleomycin resistance plate screening results showed that transformation of the pPhtJ01_c plasmid with an intron inserted from the Phatr3_J51169 gene resulted in normal growth of single colonies. Figure 2 This demonstrates that in *Phaeodactylum tricornutum*, the Phatr3_J51169 intron within the Cre sequence can be successfully recognized and cleaved, allowing the Cre-P2A-Shble sequence to be fully translated. Since P2A is a self-cleaving protein, the algal cells possess Shble-resistant proteins.

[0090] (3) Functional verification of Cre in Phaeodactylum tricornutum

[0091] Using pPha-T1 plasmid (sequence reference NCBI accession number: AF219942.1, plasmid was artificially synthesized) as a template, the Shble expression cassette with fcpB as the promoter and fcpA as the terminator was amplified using BleoR-EC-F (gcgcccgttttttcgagacataccttcagcgtcgtctt, SEQ ID NO: 39) and BleoR-EC-R (cgtcccaaaaccttctcctcgagaaaactcatcctgtgc, SEQ ID NO: 40) as primers. The vector backbone was amplified using GJ-2-HR-F (ccttctgccgttctaggaggtaccgccggtctccctatagtgag, SEQ ID NO: 41) and GJ-2-HR-R (agtcacttccaattctccattgcgtcct, SEQ ID NO: 42) as primers.

[0092] The pNR-Cre(intron-J51169)-P2A sequence was amplified from the plasmid pPhtJ01_c using pNR-HR-2-F (aggacgcaatggagaattggaagtgactgtaaacgagaag, SEQ ID NO:43) and Cre-P2A-R (gcgccggagtccaggtccag, SEQ ID NO:44) as primers, and the NR terminator was amplified using tNR-HR-2-F (gtcgacctgcaggcatgcaagcttcaatttactttaagact, SEQ ID NO:45) and tNR-HR-2-R (cacaccaattacggaggatccgaatttccggatgcgttcattttagatcc, SEQ ID NO:46) as primers.

[0093] The pNR-MCS sequence was amplified from the plasmid pPha-NR using pNR-MCS-F (ggctgcaggacgcaatggagaattggaagtgactgtaaacgag aag, SEQ ID NO:47) and pNR-MCS-R (tagtcttaaagtaaattgaagcttgcatgcctgcagg, SEQ ID NO:48) as primers.

[0094] The nat sequence was amplified from the fragment of nat (artificially synthesized fragment) using nat-F (gacctggactccggcgcatgaccactcttgacgacacg, SEQ ID NO:49) and nat-R (tgcatgcctgcaggtcgactcaggggcagggcatgctca, SEQ ID NO:50) as primers.

[0095] The two DNA fragments containing loxP sequence were amplified from the plasmid pCfB4586 (purchased from addgene, #106124) using loxp-1-F (cctccgtaattggtgtgtgt, SEQ ID NO:51) and loxp-1-R (ctcgaaaaaacgggcgccaa, SEQ ID NO:52), loxp-2-F (gagaaggttttgggacgctc, SEQ ID NO:53) and loxp-2-R (tcctagaacggcagaagggt, SEQ ID NO:54) as primers, respectively.

[0096] All PCR amplifications were performed using 2×PhantaMax Master Mix high-fidelity enzyme. The reaction mixture consisted of: 25 μL 2×PhantaMax Master Mix, 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 1 μL template DNA, and 20 μL ddH2O. The prepared mixture was used for PCR amplification according to the following program: 95℃ for 5 min; 95℃ for 15 s, 56℃ for 15 s, 72℃ for 3 min, for a total of 35 cycles; 72℃ for 5 min; and incubation at 8℃. The amplified products were purified using a gel extraction kit, following the instructions in the kit's manual.

[0097] The obtained products pNR-MCS, NR terminator, Shble expression cassette with fcpB as promoter and fcpA as terminator, vector backbone, and two DNA fragments containing the loxP sequence were assembled to obtain a circular plasmid (pPhtJ02); the pNR-Cre(intron-J51169)-P2A sequence, nat sequence, NR terminator, Shble expression cassette with fcpB as promoter and fcpA as terminator, vector backbone, and two DNA fragments containing the loxP sequence were assembled to obtain a circular plasmid (pPhtJ03). The reaction system was: 2×CE Mix, 100 ng each of DNA fragments, and ddH2O added to a total volume of 20 μL. The reaction program was: 50 °C, 60 min, and incubation at 8 °C.

[0098] The constructed plasmids pPhtJ02 and pPhtJ03 were used to construct plasmids ( Figure 3 The gene gun was used to transform and introduce the wild-type *Phaeodactylum tricornutum*. The bombarded algal strain (pPhtJ02) was then transferred to F / 2 solid medium containing 75 mg / L bleomycin, and the bombarded algal strain (pPhtJ03) was transferred to F / 2 solid medium containing 150 mg / L norethindrone. Both were placed under 16 h light / 8 h dark conditions (75 μmol / L). -2 s -1) 22°C incubation. In pPhtJ03 transformed algae, if Cre functions normally after expression, it will excise the Shble expression cassette between the simultaneously integrated loxP sequences, making it not resistant to bleomycin. Further PCR detection using loxp-1-F (cctccgtaattggtgtgtgtct, SEQ ID NO: 55) and loxp-2-R (tcctagaacggcagaagggt, SEQ ID NO: 56) as primers on the 1.3 kb sequence containing the loxP-Sb ble-loxP sequence, if the Cre functions, the algae strain will excise the 1 kb sequence containing the Shble expression cassette, leaving only the 0.3 kb size fragment reserved for detection. The 1.3 kb complete sequence in pPhtJ02 transformed algae serves as a positive control.

[0099] In pPhtJ03 transformed algae, No. 6 and No. 17 cannot grow on plates containing bleomycin, and PCR detection also shows that they completely remove the Shble expression cassette Figure 4 ).

[0100] (4) Cre plasmid transformation platform construction

[0101] The pfcpA-MCS-tfcpA sequence was amplified from the plasmid pPha-T1 using pfcpA-MCS-tfcpA-F (gcaggacgcaatggagaattggacgcaatggagga ttatcac, SEQ ID NO: 57) and pfcpA-MCS-tfcpA-R (caccaattacggaggatccgaatttcgagaaaactcatcct gtgc, SEQ ID NO: 58) as primers, the vector backbone T1-ampR-ori was amplified using T1-ampR-ori-2-F (ccttctgccgttctaggaggtaccgccggtctccctatagtgag, SEQ ID NO: 59) and T1-ampR-ori-2-R (tccattgcgtcctgcagcc, SEQ ID NO: 60) as primers, the fcpA promoter was amplified using T1-pfcpA-F (ggacgcaatggaggattatcac, SEQ ID NO: 61) and T1-pfcpA-R (cgcttcttctttggggccatgaattctcgaaacggc agac, SEQ ID NO: 62) as primers, the tfcpA-pfcpB sequence was amplified using T1-tfcpA-pfcpB-F (caggcaaaaaagaaa aagtaacagaagcgtgctatcgaactca, SEQ ID NO: 63) and T1-tfcpA-pfcpB-R (tgtcgtcaagagtggtcatctt gacatctggcaaccgtg, SEQ ID NO: 64) as primers, and the fcpA terminator was amplified using tfcpA-F (agcatgcc ctgcccctgaccttccttaaaaatttaattttcattagttgc, SEQ ID NO: 65) and tfcpA-R (gtctggacttgacctgagctcct cgagaaaactcatcctgtgc, SEQ ID NO: 66) as primers.

[0102] The loxP-pfcpB-Sh ble-tfc pA-loxP sequence was amplified from the plasmid pPhtJ02 using loxp-1-F (cctccgtaattggtgtgtgt, SEQ ID NO: 67) and / loxp-2-R (tcctagaacggcagaagggt, SEQ ID NO: 68) as primers, the NR promoter was amplified using pNR-HR-2-F (atagggagaccggctctagacggaagtgactgtaaacgagaag, SEQ ID NO: 69) and pNR-HR-2-R (gtggtccttatagtccatgaattccgttcgcacaagtg, SEQ ID NO: 70) as primers, and the NR terminator was amplified using tNR-HR-2-F (caggcaaaaaagaaaaagtaaagcttcaatttactttaagactagcgcaatt, SEQ ID NO: 71) and tNR-HR-2-R (cctccattgcgtcctctagacgttcattttagatcctgatccg, SEQ ID NO: 72) as primers.

[0103] The Cre sequence was amplified from the artificially chemically synthesized Cre fragment using Cre-CAH-F (atggactataaggaccacgacgga, SEQ ID NO: 73) and Cre-CAH-R (ttactttttcttttttgcctggcc, SEQ ID NO: 74) as primers.

[0104] The nat sequence was amplified from the artificially chemically synthesized nat fragment using nat-2-F (atgaccactcttgacgacac, SEQ ID NO: 75) and nat-2-R (tcaggggcagggcatgctca, SEQ ID NO: 76) as primers.

[0105] The CEN6-ARSH4-HIS3 fragment was amplified from the vector PtPuc3_diaCas9_sgRNA plasmid (purchased from addgene, #109219) using CAH-F (cagatcgtcttgccttgctc, SEQ ID NO: 77) and CAH-R (gagctcaggtcaagtccagactc, SEQ ID NO: 78) as primers.

[0106] All PCR amplifications were performed using 2×PhantaMax Master Mix high-fidelity enzyme. The reaction mixture consisted of: 25 μL 2×PhantaMax Master Mix, 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 1 μL template DNA, and 20 μL ddH2O. The prepared mixture was used for PCR amplification according to the following program: 95℃ for 5 min; 95℃ for 15 s, 56℃ for 15 s, 72℃ for 3 min, for a total of 35 cycles; 72℃ for 5 min; and incubation at 8℃. The amplified products were purified using a gel extraction kit, following the instructions in the kit's manual.

[0107] The obtained products, pfcpA-MCS-tfcpA sequence, loxP-pfcpB-Shble-tfcpA-loxP sequence, and vector backbone T1-ampR-ori were assembled to obtain a circular plasmid (pPhtJ04); the NR promoter, Cre fragment, NR terminator, fcpA promoter, Cre fragment, tfcpA-pfcpB fragment, nat fragment, and CEN6-ARSH4-HIS3 fragment, along with the vector backbone T1-ampR-ori, were assembled to obtain a circular plasmid (pPhtJ05). The assembly reaction system consisted of 2×CE Mix, 100 ng of each DNA fragment, and ddH2O added to a total volume of 20 μL. The assembly reaction program was: 50 °C, 60 min, and incubation at 8 °C.

[0108] The constructed pPhtJ04 plasmid was transformed into wild-type *Phaeodactylum tricornutum* using a gene gun; the bombarded algal cells were then transferred to F / 2 solid medium containing 75 mg / L bleomycin and placed under a 16-hour light-8-hour dark (75 μmol / L) environment. -2 s -1 Transformant (pPhtJ04) was obtained by culturing at 22℃. The constructed pPhtJ05 plasmid was introduced into *E. coli* EPI300 via electroporation. After expanding the culture of *E. coli* EPI300, it was incubated with the pPhtJ04 transformed algae to introduce the pPhtJ05 plasmid from *E. coli* EPI300 into the pPhtJ04 transformed algae in a bacterial-binding transfer manner, where it existed in free form. Figure 5 Twenty-four transformed algal strains with bleomycin resistance were randomly selected, of which strains 4 and 17 lacked bleomycin resistance. Further PCR testing using the above method showed that Cre had functioned in strains 4 and 17, effectively cleaving the integrated Shble expression cassette. Figure 6 ).

[0109] (5) Construction of the first round of algal strains for EPA synthesis from *Phaeodactylum tricornutum*

[0110] Three different fcpA promoter sequences with different ends were amplified from the plasmid pPha-T1 using T1-pfcpA-A-HR-F (gccgttctaggaggtaccggacgcaatggagga ttatcacc, SEQ ID NO: 79), T1-pfcpA-A-HR-R (tcgaaacggcagacaaatttgt, SEQ ID NO: 80), T1-pfcpA-B-HR-F (tcagatcctgcccagaccacggacgcaatggaggattatcac, SEQ ID NO: 81), T1-pfcpA-B-HR-R (tcgaaacggcagacaaatttgt, SEQ ID NO: 82) and T1-pfcpA-C-HR-F (caaaggtgcttcgca gcctgggacgcaatggaggattatcac, SEQ ID NO: 83) and T1-pfcpA-C-HR-R (tcgaaacggcagacaaatttgt, SEQ ID NO: 84) as primers, respectively. Three different fcpA terminator sequences with different ends were amplified from the plasmid using T1-tfcpA-A-HR-F (ggatcctctagagtcgacct, SEQ ID NO: 85) and T1-tfcpA-A-HR-R (gtggtctgggcaggatctgactcgagaaaactcatcctgtgcc, SEQ ID NO: 86), T1-tfcpA-B-HR-F (ggatcctctagagtcgacct, SEQ ID NO: 87), T1-tfcpA-B-HR-R (caggctgcgaagcacctttgctcgagaaaactcatcctgtgcc, SEQ ID NO: 88) and T1-tfcpA-C-HR-F (ggatcctctagagtcgacct, SEQ ID NO: 89), T1-tfcpA-C-HR-R (tatagggagaccggcggtaccctcgagaaaactcatcctgtgcc, SEQ ID NO: 90) as primers, respectively.

[0111] The loxP-pfc pB-Sh ble-tfcpA-loxP sequence was amplified from the plasmid pPhtJ04 using shble-HR-F (cggatcctccgtaattggtgtgtgt, SEQ ID NO: 91) and shble-HR-R (ggtacctcctagaacggcagaagggt, SEQ ID NO: 92) as primers, and the vector skeleton T1-ampR-ori was amplified using ampR-ori-HR-F (ggtaccgccggtctccctatagtg, SEQ ID NO: 93) and ampR-ori-HR-R (cacaccaattacggaggatccgaattctccattgcgtcctgcagcc, SEQ ID NO: 94) as primers.

[0112] The desaturase 9 (PtDes9) and desaturase 12 (PtDes12) sequences were amplified from the cDNA of Phaeodactylum tricornutum using PtDes9-HR-F (ttgtctgccgtttcgagaattcatggacttgtcttccgaaatgct, SEQ ID NO: 95) and PtDes9-HR-R (gtcgactctagaggatccttacgcaatcttgcgtgcga, SEQ ID NO: 96) as primers, and PtDes12-HR-F (ctgccgtttcgagaattcatgggtaagggaggtcaacg, SEQ ID NO: 97) and PtDes12-HR-R (gtcgactctagaggatccttatgcggctttgc ttcgcac, SEQ ID NO: 98) as primers. The PCR amplification was performed using 2×Phanta Max Master Mix high-fidelity enzyme, and the reaction system was as follows: 25 μL 2×Phanta Max Master Mix, 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 1 μL template DNA, and 20 μL ddH2O. The prepared system was subjected to PCR amplification, and the reaction program was as follows: 95 °C for 5 min; 95 °C for 15 s, 56 °C for 15 s, 72 °C for 3 min, for a total of 35 cycles; 72 °C for 5 min; and 8 °C for incubation. The amplified products were purified by a gel recovery kit, and the specific steps were performed according to the kit manual.

[0113] The above obtained product, three different end fcpA promoters, three different end fcpA terminators, PtDes9, PtDes12, carrier skeleton T1-ampR-ori and loxP-pfcpB-Shble-tfcpA-loxP sequence are assembled to obtain a circular plasmid pPhtJ06 Figure 7 The reaction system is: 2x CE Mix, 100 ng of each DNA fragment, and ddH2O is added to a total volume of 20 μL. The reaction procedure is: 50°C, 60 min, 8°C incubation.

[0114] The constructed pPhtJ06 plasmid is introduced into a Phaeodactylum tricornutum wild strain by gene gun transformation; the bombarded algal cells are transferred to F / 2 solid medium containing 75 mg / L bleomycin, and are cultured at 22°C under 16h light: 8h dark (75 μmol m -2 s -1 ) to obtain transformants (pPhtJ06). After the Escherichia coli EPI300 is expanded and incubated with the pPhtJ06 transformed algal strain, the pPhtJ05 plasmid in the Escherichia coli EPI300 is introduced into the pPhtJ06 transformed algal strain in the form of bacterial conjugation transfer, and is named as a pPhtJ06-A algal strain. 24 pPhtJ06-A transformed algal strains with nourseothricin resistance are randomly picked, and 3, 4, 5, 8, 10, 12, 13, 18 and 19 do not have bleomycin resistance. Further PCR detection shows that in 3, 4, 5, 8, 10, 12, 13, 18 and 19, Cre has function to remove the integrated Sh ble expression cassette Figure 8

[0115] (6) Removal of free pPhtJ05 plasmid in the pPhtJ06-A algal strain

[0116] After 3 in the pPhtJ06-A algal strain is continuously subcultured in F / 2 liquid medium for 1 to 2 months, it is diluted and plated on F / 2 solid medium. 21 single clones are randomly picked and named as pPhtJ06-B, and are placed on F / 2 + nourseothricin solid plates. 1, 3, 6, 7, 9, 11, 15, 16, 18, 19, 20 and 21 cannot grow on the F / 2 + nourseothricin solid plates. Further PCR detection of 1-20 shows that in 1, 3, 6, 7, 9, 11, 15, 16, 18, 19 and 20, the sequence of Cre cannot be detected Figure 9 ). It is shown that in these pPhtJ06-B single clones, the free pPhtJ05 plasmid removal has been completely removed, and can be used for the next round of transformation.

[0117] ​(7) Triangular brown algae EPA synthesis second round of algal strain construction

[0118] The fcpA promoter sequences with different 3' ends were amplified from the plasmid pPha-T1 using T1-pfcpA-A-HR-F (gccgttctaggaggtaccggacgcaatggagga ttatcacc, SEQ ID NO: 99), T1-pfcpA-A-HR-R (tcgaaacggcagacaaatttgt, SEQ ID NO: 100), T1-pfcpA-B-HR-F (tcagatcctgcccagaccacggacgcaatggaggattatcac, SEQ ID NO: 101), T1-pfcpA-B-HR-R (tcgaaacggcagacaaatttgt, SEQ ID NO: 102), and T1-pfcpA-C-HR-F (caaaggtgcttcgc agcctgggacgcaatggaggattatcac, SEQ ID NO: 103), T1-pfcpA-C-HR-R (tcgaaacggcagacaaatttgt, SEQ ID NO: 104) as primers, respectively. The fcpA terminator with different 3' ends were amplified from the plasmid using T1-tfcpA-A-HR-F (ggatcctctagagtcgacct, SEQ ID NO: 105) and T1-tfcpA-A-HR-R (gtggtctgggca ggatctgactcgagaaaactcatcctgtgcc, SEQ ID NO: 106), T1-tfcpA-B-HR-F (ggatcctctagagtcgacct, SEQ ID NO: 107) and T1-tfcpA-B-HR-R (caggctgcgaagcacctttgctcgagaaaactcatcctgtgcc, SEQ ID NO: 108), and T1-tfcpA-C-HR-F (ggatcctctagagtcgacct, SEQ ID NO: 109) and T1-tfcpA-C-HR-R (tatagggagaccggcggtaccctcgagaaaactcatcctgtgcc, SEQ ID NO: 110) as primers, respectively.

[0119] The loxP-pfcpB-Sh ble-tfcpA-loxP sequence was amplified from the plasmid pPhtJ04 using shble-HR-F (cggatcctccgtaattggtgtgtgt, SEQ ID NO: 111) and shble-HR-R (ggtacctcctagaacggcagaagggt, SEQ ID NO: 112) as primers, and the vector backbone T1-ampR-ori was amplified using ampR-ori-HR-F (ggtaccgccggtctccctatagtg, SEQ ID NO: 113) and ampR-ori-HRR (cacaccaattacggaggatccgaattctccattgcgtcctgcagcc, SEQ ID NO: 114) as primers.

[0120] The desaturase 9 (PtDes6) sequence was amplified from the cDNA of Phaeodactylum tricornutum using ptDes6-HR-F (tctgccgtttcgagaattcatgggcaaaggaggggacgc, SEQ ID NO: 115) and ptDes6-HR-R (gtcgactctagaggatccttacatggccggtccgtcgcgtacaaaatc, SEQ ID NO: 116) as primers, the elongase 6B 1 (PtELO6B 1) was amplified using primers ptELO6B_1-HR-F (ctgccgtttcgagaattcatgagtatgggaatccccgcaattgacccg, SEQ ID NO: 117) and ptELO6B_1-HR-R (gtcgactctagaggatccttatgccgtctttttcttcttcgg, SEQ ID NO: 118), and the desaturase 5A (PtDes5A) sequence was amplified using primers ptDes5A-HR-F (ctgccgtttcgagaattcatggctccggatgcggataagc, SEQ ID NO: 119) and ptDes5A-HR-R (ggtcgactctagaggatccttacgcccgtccggtcaag, SEQ ID NO: 120).

[0121] The above PCR amplification was performed using 2x Phanta Max Master Mix high-fidelity enzyme, and the reaction system was as follows: 25 μL 2x Phanta Max Master Mix, 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 1 μL template DNA, 20 μL ddH2O. The prepared system was subjected to PCR amplification, and the program was as follows: 95°C for 5 min; 95°C for 15 s, 56°C for 15 s, 72°C for 3 min, for a total of 35 cycles; 72°C for 5 min; 8°C for incubation. The product obtained by amplification was purified by a gel recovery kit, and the specific steps were performed according to the kit manual.

[0122] The above obtained product, three different end fcpA promoters, three different end fcpA terminators, PtDes6, PtELO6B_1, PtDes5A, vector skeleton T1-ampR-ori and loxP-pfcpB-Sh ble-tfcpA-loxP sequence were assembled to obtain a circular plasmid pPhtJ07 Figure 10 );The reaction system was as follows: 2x CE Mix, DNA fragments were 100 ng each, and ddH2O was added to a total volume of 20 μL. The reaction program was as follows: 50°C, 60 min, 8°C incubation.

[0123] The constructed pPhtJ07 plasmid was transformed into the Phaeodactylum tricornutum pPhtJ06-B-1 strain by gene gun; the algae cells after bombardment were transferred to F / 2 solid medium containing 75 mg / L bleomycin, and were cultured at 22°C under 16 h light: 8 h dark (75 μmol m -2 s -1 ) to obtain transformants (pPhtJ07).

[0124] (8) Phaeodactylum tricornutum fatty acid composition determination

[0125] Log phase of Phaeodactylum tricornutum wild type, pPhtJ06 strain and pPhtJ07 strain algal cells were collected, centrifuged to remove the culture medium, and then freeze-dried. 5 mg of freeze-dried algal powder was weighed, 30 μL of internal standard (C21:0, 1 mg / mL) and 750 μL of sulfuric acid methanol (v:v=5:95) were added, and then the mixture was placed in a 90 ℃ metal bath for 90 min. After cooling to room temperature, 500 μL of 0.9% NaCl and 200 μL of n-hexane were added, and the mixture was centrifuged at 4000 rpm for 10 min. 100 μL of the upper organic phase was taken to a gas chromatography vial, and the fatty acid composition was determined by gas chromatography. The instrument and its parameters are as follows: 7890A gas chromatograph (Agilent), hydrogen flame ionization detector; chromatographic column: HP-5 flexible quartz, length 30 m, inner diameter 0.32 mm; programmed temperature: initial temperature 210 ℃, holding for 9 min, increasing to 230 ℃ at 20 ℃ / min, holding for 8 min. The temperature of the injection port was 250 ℃, and the temperature of the detector was 280 ℃. The carrier gas was high-purity N2, and the flow rate was 20 mL / min. The constant flow control was used. The flow rate of H2 was 30 mL / min, the flow rate of air was 400 mL / min, and the tail gas was 20 mL / min. The injection amount was 1 μL. The split mode was 30:1.

[0126] The results of the analysis of the fatty acid content of the wild type, pPhtJ06 strain and pPhtJ07 strain showed that the relative contents of 14:0, 16:1, 16:3 and 18:1 of the pPhtJ06 strain and the pPhtJ07 strain were lower than those of the wild type. From the analysis of the EPA content, it can be seen that the EPA content of the pPhtJ06 strain was increased by 17.3% compared with the wild type, and the EPA content of the pPhtJ07 strain was increased by 24.7% compared with the wild type. Figure 11

[0127] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A method for analyzing the function of Cre recombinase in oil-producing microalgae, characterized in that, Includes the following steps: The expression cassette containing the Cre-J51169-P2A fusion sequence and the first resistance gene, and the expression cassette containing the second resistance gene with loxP sequences at both ends were constructed into the backbone vector to obtain the first recombinant vector; The first recombinant vector was transferred into oil-producing microalgae. The resulting recombinant oil-producing microalgae were cultured for the first time in a medium containing an antibiotic corresponding to the first resistance gene. Positive single clones were transferred into a medium containing an antibiotic corresponding to the second resistance gene for the second culture. Recombinant oil-producing microalgae that grew only in the first culture and could not grow in the second culture were obtained, indicating that Cre recombinase was normally expressed and functioned as a recombinase in the recombinant oil-producing microalgae. The Cre-J51169-P2A fusion sequence is obtained by fusing the Cre gene and the P2A sequence. The Cre gene is a Cre gene with an intron of the Phatr3_J51169 gene embedded in its middle.

2. The analytical method according to claim 1, characterized in that, The nucleotide sequence of the intron of the Phatr3_J51169 gene is shown in SEQ ID NO:

7.

3. The analytical method according to claim 1, characterized in that, The Cre-J51169-P2A fusion sequence contains a 3×Flag tag and a nuclear localization signal peptide sequence fused to the 5' end of the Cre gene, and a bigenotyping nuclear localization signal sequence of nucleolysin fused to the 3' end of the Cre gene.

4. A set of recombinant vectors, characterized in that, Including pPhtJ05 and pPhtJ06; The pPhtJ05 is a backbone vector containing the following gene elements: a double-copy Cre gene expression cassette, a single-copy resistance gene expression cassette, and a CEN6-ARSH4-HIS3 fusion fragment. The pPhtJ06 is a backbone vector containing the following gene elements: a double-copy PtDes12 gene expression cassette, a single-copy PtDes9 gene expression cassette, and a second resistance gene expression cassette with loxP sequences at both ends.

5. The recombinant vector according to claim 4, characterized in that, Also includes pPhtJ07; The pPhtJ07 is a backbone vector containing the following gene elements: PtDes6 gene expression cassette, PtELO6B_1 gene expression cassette, PtDes5A gene expression cassette, and a second resistance gene expression cassette with loxP sequences at both ends.

6. The use of the recombinant carrier according to claim 4 or 5 in promoting the biosynthesis of eicosapentaenoic acid by oil-producing microalgae.

7. The application according to claim 6, characterized in that, The oil-producing microalgae include *Phaeodactylum tricornutum*.

8. A method for promoting the biosynthesis of eicosapentaenoic acid by oil-producing microalgae, characterized in that, Includes the following steps: pPhtJ06 in the recombinant vector described in claim 4 was transferred into oil-producing microalgae, screened and cultured, and the first recombinant algal strain was obtained. Using bacterial conjugation transfer technology, pPhtJ05 from the recombinant vector described in claim 4 was transferred into the first recombinant algal strain. After two screening cultures, a second recombinant algal strain without a second resistance gene expression cassette containing loxP sequences at both ends was obtained. The second recombinant algal strain, which does not contain the second resistance gene expression cassette linked at both ends of the loxP sequence, was cultured, and the third recombinant algal strain, which does not contain pPhtJ05, was selected.

9. The method according to claim 8, characterized in that, After obtaining the third recombinant algal strain that does not contain pPhtJ05, the method further includes transferring pPhtJ07 from the recombinant vector described in claim 5 into the third recombinant algal strain that does not contain pPhtJ05, screening and culturing to obtain the fourth recombinant algal strain.

10. A recombinant algal strain for producing eicosapentaenoic acid, characterized in that, Using oil-producing microalgae as the host, it also includes recombinantly expressed PtDes12 and PtDes9 genes; It may also include recombinantly expressed PtDes12, PtDes9, PtDes6, PtELO6B_1 and PtDes5A genes.

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