Plasmid for pyrimidine base mutation in phaeodactylum tricornutum and application thereof

Abstract

The invention provides a plasmid for pyrimidine base mutation in phaeodactylum tricornutum and application of the plasmid, and belongs to the field of bioengineering. The plasmid provided by the invention is named as pPtCBE; the plasmid comprises an evoAPOBEC1-nCas9-UGI expression box, a targeting sequence spacer fragment and two BsaI sites, the nucleotide sequence of the evoAPOBEC1-nCas9-UGI expression box is as shown in SEQ ID No.2, and nuclear localization signal peptides of phaeodactylum tricornutum are fused at the two ends of the evoAPOBEC1-nCas9-UGI expression box respectively; the target sequence spacer fragment is derived from phaeodactylum tricornutum; the BsaI site is used for inserting a target sequence spacer fragment. The plasmid provided by the invention can realize pyrimidine editing on genes so as to realize gene editing, and has great application potential.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to a plasmid for pyrimidine base mutation in Phaeodactylum tricornutum and application thereof. Background Art

[0002] Large-scale industrial production and synthetic biology transformation of P. triangularis require efficient and simple genetic manipulation tools. However, gene editing technology in P. triangularis is relatively backward, and only CRISPR-Cas9 has been used for gene knockout. The CRISPR-Cas9 system has the problems of large randomness of editing results and high off-target probability, resulting in low editing efficiency and potential biosafety risks. Therefore, the development of more accurate, efficient and safer gene editing tools is an important link in promoting the industrialization of gene editing in P. triangularis. Base editors can achieve precise single-base replacement (such as C→T) without causing double-strand breaks by fusing deaminase with inactivated Cas9 protein (such as nCas9D10A). However, existing base editors have not been optimized for the genomic characteristics of P. triangularis (such as codon preference, nuclear localization signal requirements, etc.), resulting in insufficient editing efficiency and cannot be applied in actual production. Summary of the invention

[0003] The technical problem to be solved by the present invention is: how to improve the directional editing efficiency of base editors.

[0004] To solve the above technical problems, the first aspect of the present invention provides a plasmid for pyrimidine base mutation in Phaeodactylum triangularis, the plasmid is named pPtCBE; the plasmid comprises an evoAPOBEC1-nCas9-UGI expression cassette, a targeting sequence spacer fragment, and two BsaⅠ sites, the nucleotide sequence of the evoAPOBEC1-nCas9-UGI expression cassette is shown in SEQ ID No. 2, and the two ends of the evoAPOBEC1-nCas9-UGI expression cassette are respectively fused with nuclear localization signal peptides of Phaeodactylum triangularis; the targeting sequence spacer fragment is derived from Phaeodactylum triangularis; the BsaⅠ site is used to insert the targeting sequence spacer fragment.

[0005] The plasmid for pyrimidine base mutation in Phaeodactylum tricornutum provided by the present invention can express evoAPOBEC1-nCas9-UGI fusion protein, wherein the evoAPOBEC1-nCas9-UGI fusion protein comprises the following expression units: codon-optimized evoAPOBEC1 deaminase (NCBI accession number: Q9UBU7), nickase nCas9D10A (RefSeq: NP_001394100.1), and uracil glycosidase inhibitor UGI (RefSeq: NP_001073816.1).

[0006] The plasmid for pyrimidine base mutation in Phaeodactylum tricornutum provided by the present invention further comprises two BsaⅠ sites, and the BsaⅠ sites are used for inserting a targeting sequence spacer fragment.

[0007] The targeting sequence spacer fragment includes the selected mutation site of the whole genome of Phaeodactylum tricornutum, and the spacer fragment includes at least 20 bases. The selection of the spacer fragment should follow the following rules: a DNA fragment of at least 20 base sequences before any NGG (N is any base) sequence in the whole genome of Phaeodactylum tricornutum is selected as the targeting sequence spacer fragment, and the NGG sequence is not in the targeting sequence spacer fragment, wherein positions 4 to 8 of the targeting sequence spacer fragment should have at least one cytosine base as the target base for base editing.

[0008] Preferably, the Phaeodactylum triangularis nuclear localization signal peptide at the N-terminus of the evoAPOBEC1-nCas9-UGI expression cassette is SV40 NLS, and the Phaeodactylum triangularis nuclear localization signal peptide at the C-terminus of the evoAPOBEC1-nCas9-UGI expression cassette is nucleoplasmin NLS.

[0009] Preferably, the plasmid further comprises an sgRNA expression unit, which is driven by a U6 promoter, and the DNA sequence of the sgRNA expression unit is shown in SEQ ID No. 3. In the present invention, the principle of base-directed editing by the evoAPOBEC1-nCas9-UGI fusion protein is as follows: under the guidance of nCas9-sgRNA, the evoAPOBEC1 protein specifically targets a specific site of the genome through the principle of base complementary pairing, and the evoAPOBEC1 protein can catalyze the deamination reaction of cytosine (C) at the site to generate uracil (U), while nCas9 cuts the non-edited chain, allowing the cell to perform DNA repair using the edited chain as a template. During the repair process, uracil (U) will be recognized as thymine (T), thereby achieving the directed mutation of cytosine to thymine at a specific site.

[0010] Preferably, the plasmid further comprises a construction screening gene, and the construction screening gene is selected from at least one of kanamycin resistance gene, chloramphenicol resistance gene, ampicillin resistance gene, tetracycline resistance gene, erythromycin resistance gene, gentamicin resistance gene and bleomycin resistance gene.

[0011] Preferably, the plasmid further comprises a transformation screening gene, and the transformation screening gene is selected from at least one of the bleomycin resistance gene, the neomycin resistance gene, the hygromycin B resistance gene and the puromycin resistance gene.

[0012] Preferably, the plasmid further comprises a CEN / ARS autonomously replicating element.

[0013] Furthermore, the second aspect of the present invention provides an application of the plasmid described in the first aspect, wherein the application is to use the plasmid with pyrimidine base mutation in the triangular phycocyanin to direct the mutation of cytosine at any site on the triangular phycocyanin genome to thymine.

[0014] Preferably, the active window for directed mutation of cytosine to thymine at any site on the genome of Phaeodactylum tricornutum is position 4 to 8 of the spacer fragment of the targeting sequence, from 5' to 3'.

[0015] Furthermore, the third aspect of the present invention provides an expression cell strain, which contains the plasmid described in the first aspect, and the expression cell strain is selected from any one or more of bacteria, fungi, and algae.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The plasmid for pyrimidine base mutation in triangular phytosporium provided by the present invention is designed based on the CRISPR-Cas9 system and APOBEC1 cytosine deaminase. The plasmid provided by the present invention can directly mutate cytosine at any site in triangular phytosporium to thymine, realize base editing, and further realize amino acid-directed mutation or gene inactivation in triangular phytosporium. The plasmid provided by the present invention has the advantages of high editing efficiency and low off-target rate, and is of great significance to the research on physiological characteristics and industrial application of triangular phytosporium. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The plasmid map of the pPtCBE plasmid provided in the embodiment of the present invention; Figure 2 A schematic diagram of the technical principle of mutating cytosine into thymine by the pPtCBE plasmid provided by the present invention; Figure 3 The electrophoresis result in Example 3 of the present invention; Figure 4 This is the sequencing result in Example 3 of the present invention. DETAILED DESCRIPTION

[0018] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the scope of protection of the claims of the present invention.

[0019] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0020] Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventionally understood meanings are defined herein for the purpose of clarification or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters given by the manufacturer.

[0021] As described in the background technology, base editing technology can achieve directional changes in DNA bases without relying on double-strand breaks of genomic DNA and homologous recombination. However, currently, due to the genomic characteristics of P. tricornutum, base editors cannot be directly applied to gene editing of P. tricornutum.

[0022] Based on this, a specific embodiment of the present invention provides a plasmid for pyrimidine base mutation in P. triangularis, the plasmid is named pPtCBE, the pPtCBE plasmid includes an evoAPOBEC1-nCas9-UGI expression cassette, a targeting sequence spacer fragment, and two BsaⅠ sites. The nucleotide sequence of the evoAPOBEC1-nCas9-UGI expression cassette is shown in SEQ ID No. 2, and the nuclear localization signal peptide of P. triangularis is fused at both ends of the evoAPOBEC1-nCas9-UGI expression cassette; the targeting sequence spacer fragment is derived from P. triangularis; and the BsaⅠ site is used to insert the targeting sequence spacer fragment.

[0023] In some embodiments, the nucleotide sequence of the pPtCBE plasmid is shown in SEQ ID No. 1, and the plasmid map of the pPtCBE plasmid is shown in Figure 1 shown.

[0024] In some embodiments, the Phaeodactylum tricornutum nuclear localization signal peptide at the N-terminus of the evoAPOBEC1-nCas9-UGI expression cassette is SV40 NLS, and the Phaeodactylum tricornutum nuclear localization signal peptide at the C-terminus of the evoAPOBEC1-nCas9-UGI expression cassette is nucleoplasmin NLS.

[0025] In some embodiments, the plasmid further comprises an sgRNA expression unit, wherein the sgRNA expression unit is driven by a U6 promoter, and the DNA sequence of the sgRNA expression unit is shown in SEQ ID No.3.

[0026] In some embodiments, the plasmid further comprises a construction screening gene, wherein the construction screening gene is selected from at least one of kanamycin resistance gene, chloramphenicol resistance gene, ampicillin resistance gene, tetracycline resistance gene, erythromycin resistance gene, gentamicin resistance gene, and bleomycin resistance gene. The function of the construction screening gene is to enable the plasmid to be transformed and screened in Escherichia coli.

[0027] In some embodiments, the plasmid further comprises a transformation screening gene, wherein the transformation screening gene is selected from at least one of a bleomycin resistance gene, a neomycin resistance gene, a hygromycin B resistance gene, and a puromycin resistance gene. The function of the transformation screening gene is to enable the plasmid to be transformed and screened in Phaeodactylum tricornutum.

[0028] In some embodiments, the plasmid further comprises a CEN / ARS autonomous replication element. The function of the CEN / ARS autonomous replication element is to enable the plasmid to stably replicate and propagate in Phaeodactylum tricornutum.

[0029] More specifically, the aforementioned plasmid for pyrimidine base mutation in Phaeodactylum tricornutum is capable of expressing evoAPOBEC1-nCas9-UGI fusion protein and sgRNA for targeted editing of specific cytosine bases to thymine bases in Phaeodactylum tricornutum.

[0030] More specifically, the aforementioned plasmid-directed editing for pyrimidine base mutation in Phaeodactylum tricornutum works as follows: Figure 2It should be noted that the targeting sequence spacer fragment in the pPtCBE plasmid is derived from Phaeodactylum tricornutum, and the specific selection rule is: a DNA fragment of at least 20 base sequences before any NGG (N is any base) sequence in the whole genome of Phaeodactylum tricornutum is selected as the targeting sequence spacer fragment, and the NGG sequence is not in the targeting sequence spacer fragment, wherein positions 4 to 8 of the targeting sequence spacer fragment should have at least one cytosine base as the target base for base editing.

[0031] Since the targeting sequence spacer fragment is inserted into the pPtCBE sequence through the BsaⅠ site, the 5' segment of the selected targeting sequence usually needs to be connected to the base segment of TCGA, and the 5' segment of the anti-complement sequence of the targeting sequence spacer fragment usually needs to be connected to the base segment of AAAC.

[0032] The technical solution of the present invention is further described below by means of specific examples. Unless otherwise specified, all the strains of Phaeodactylum triangularis in this article are Phaeodactylum tricornutum CCMP2561 algal strain.

[0033] Example 1

[0034] pPtCBE plasmid construction In this example, the pPtCBE plasmid has the following fragments: (1) an evoAPOBEC1-nCas9-UGI expression cassette, whose sequence is shown in SEQ ID No. 2; (2) an sgRNA expression cassette regulated by the U6 promoter, whose sequence is shown in SEQ ID No. 3; (3) a kanamycin resistance gene; (4) a bleomycin resistance gene; and (5) a CEN / ARS replication element for stable genetic replication of the plasmid in Phaeodactylum tricornutum, whose nucleotide sequence is shown in SEQ ID No. 4.

[0035] The construction method of pPtCBE plasmid is as follows: S1: First, synthesize the evoAPOBEC1-nCas9-UGI expression cassette shown in SEQ ID No.2 and the sgRNA expression cassette shown in SEQ ID No.3; S2: Using PtPuc3_diaCas9_sgRNA plasmid as template, amplify the plasmid backbone, which contains kanamycin resistance gene fragment, bleomycin resistance gene fragment and CEN / ARS replication element. Use 2×Phanta MaxMaster Mix for PCR amplification. The reaction system is: 23μl ddH 2O, 0.75μl 5' Primer (10μM), 0.75μl 3' Primer (10μM), 0.5μl template DNA (100 ng / μl), 25μl 2× Phanta Max Master Mix. After the system was prepared, polymerase chain reaction (PCR) was performed with the following cycles: 98 o C 2 min; then 98 o C 30 s, 55 o C 30 s, 72 o C 5min, 30 cycles in total; the final 72 o C for 10 min, extract the amplified DNA fragments, and the specific steps are carried out according to the kit operation manual; S3: Assemble the three DNA fragments synthesized in step S1 and step S2 to form a circular plasmid. The reaction system is: 2 μl 2×CE Mix, 200 ng of each of the three DNA fragments, and finally add appropriate amount of ddH 2 O to a total volume of 10 μl, and perform the reaction in a PCR instrument: 50 o C for 15 min; then cool to 4°C or immediately store on ice.

[0036] The reaction product of step S3 was transformed into E. coli DH5α strain and plated on LB solid medium containing 50 μg / ml kanamycin. After the transformation solution was absorbed by the solid medium, incubate at 37 o Incubate the cells in an inverted position at C incubator overnight. Transfer the transformed strain grown on the culture medium to a plasmid extraction kit to extract the pPtCBE plasmid for subsequent experiments. The specific steps are carried out according to the kit manual. At the same time, the plasmid is sequenced to confirm the accuracy of the pPtCBE plasmid sequence.

[0037] The plasmid map of pPtCBE plasmid was finally determined as follows Figure 1 As shown, the sequence of the pPtCBE plasmid is shown in SEQ ID No.1. The pPtCBE plasmid is a shuttle plasmid that can replicate and propagate in Escherichia coli and P. triangularis; it is kanamycin-resistant in Escherichia coli and bleomycin-resistant in P. triangularis; the plasmid can perform genome gene editing in P. triangularis to achieve a mutation of a specific base on the genome from cytosine to thymine; and it contains two BsaI sites that can be used to insert a spacer fragment.

[0038] Example 2

[0039] Construction of pPtCBE plasmid for gene editing of CRTISO5 in Phaeodactylum tricornutum Phaeodactylum tricornutum Phaeodactylum tricornutumEfficient base editing of different genes was achieved in the CCMP2561 algae strain. CRTISO5 Gene as an example, construct a targeted CRTISO5 pPtCBE editing plasmid for gene editing.

[0040] First, in the triangular algae CRTISO5 A DNA fragment of 20 bases before a certain NGG (N is an arbitrary base) sequence is selected on the gene. This 20-base DNA fragment is the targeted sequence spacer fragment, NGG is not included in it, and the cytosine C that needs to be mutated should be located at positions 4 to 8 of the 20 bases on the spacer (counted in the order of 5'→3').

[0041] For example, in this embodiment, select CRTISO5 The DNA sequence of the gene spacer is: 5'-gtgatCgaggtacgtatggaa-3' (SEQ ID NO.5), in which the cytosine C (uppercase bold) that needs to undergo base editing is at position 6.

[0042] In order to insert the targeted sequence spacer fragment into the pPtCBE plasmid, TCGA needs to be added to the 5' end of the single-stranded DNA sequence of the selected spacer. At the same time, the anti-complement sequence of the spacer needs to be synthesized, and AAAC needs to be added to the 5' end of the anti-complement sequence. The specific sequence design is as follows: CRTISO5spF: 5'-TCGAgtgatcgaggtacgtatggaa-3' (SEQ ID NO. 6); CRTISO5spR: 5'-AAACttccatacgtacctcgatcac-3' (SEQ ID NO. 7).

[0043] Primers were designed according to SEQ ID No.6 and SEQ ID No.7, and a double-stranded DNA was synthesized from SEQ ID No.6 and SEQ ID No.7. The specific reaction system was as follows: 5 μl 10×T4 DNA ligase Buffer (NEB), 10 μl CRTISO5spF (10 μM), 10 μl CRTISO5spR (10 μM), 25 μl ddH 2 O. In 95 o C for 5 min, then slowly cool to room temperature within 1 to 2 hours to allow the two single-stranded primers to form double-stranded DNA through base pairing. 2 The obtained product was diluted 20 times.

[0044] The double-stranded DNA obtained above was inserted into the BsaI site of the pCyCBE plasmid. The reaction system was: 1 μl 10×T4 DNA ligase Buffer, 1 μl of the above 20-fold diluted phosphorylated double-stranded DNA, 20 fmol of the pCyCBE plasmid obtained in Example 1, 0.5 μl T4 DNA ligase (400 units / μl), 0.5 μl BsaI-HF (20 units / μl), and finally an appropriate amount of ddH 2 O to a total volume of 10 μl. The buffer and enzymes used in the reaction were produced by NEB. The reaction was carried out in a PCR instrument with the following cycles: 37 o C 2 min; 16 o C for 3 min, for 25 cycles; then 50 o C 5 min, 80 o C for 15 min.

[0045] The 10 μl reaction product was transformed into competent E. coli DH5α strain and plated on a LB solid culture plate containing 50 μg / ml kanamycin. o C incubator overnight. The transformed strain grown on the culture medium was transferred and stored, and the plasmid was extracted using a plasmid extraction kit and sequenced for verification, and finally the successfully constructed pPtCBE-CRTISO5 was obtained.

[0046] Example 3

[0047] pPtCBE-CRTISO5 plasmid for efficient base editing in Phaeodactylum tricornutum The specific steps are as follows: (1) Linearize 9 μg of pPtCBE-CRTISO5 plasmid using restriction endonuclease EcoNI, recover and purify it, and store it at -20°C for later use; (2) Use f / 2 medium to culture a certain volume of Phaeodactylum tricornutum cells to the logarithmic phase (approximately 5×10 6 cells mL -1 ). Take 50 mL of algae cells and centrifuge at 4°C and 1500 × g for 10 min. Discard the supernatant, resuspend with 1 mL of 375 mM sorbitol, and centrifuge again at 4°C and 1500 × g for 10 min; repeat the wash twice, discard the supernatant, and resuspend with 100 μL of sorbitol. At this time, the cell density is 2×10 9 cells mL -1 ; (3) Mix 100 μL of resuspended algae with 4 μL of salmon sperm (1 μg / μL), which needs to be boiled at 100°C for 1 min. Incubate the mixture on ice for 10 min and transfer it to a 1 mm electroporation cup. Use an electroporator (Bio-Rad, Gene Pulser Xcell) for electroporation, with the parameters of 500 V, 25 μF, and 400 Ω. (4) Transfer the electroporated algae to 10 mL f / 2 medium and incubate under low light for 24 h, then incubate under normal light for 24 h. Centrifuge the incubated algae solution at 1500 × g for 10 min, discard the supernatant, resuspend with 600 μL of fresh medium, take 200 μL and spread on f / 2 solid medium containing bleomycin, and culture for 10-12 days for antibiotic selection; (5) Pick a single algae colony grown on the bleomycin-resistant plate and inoculate it into a 5 mL centrifuge tube containing f / 2 medium. Incubate for 5 to 7 days until the color of the algae solution is observed; take 200 μL of the algae solution and extract genomic DNA; (6) PCR was performed using the bleomycin resistance gene primers shble-F and shble-R to identify whether the plasmid pPtCBE-CRTISO5 was successfully transferred into the algae cells. The PCR reaction procedure was: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 30 cycles, and finally extension at 72°C for 5 min. The primer sequences are as follows: shble-F: 5'-ATGAAAACGTTTAATATCTCGC-3' (SEQ ID NO. 8); shble-R: 5'-TCAATTTCGCGTGTATTTAAG-3' (SEQ ID NO.9), The PCR products were subjected to DNA gel electrophoresis, and the results showed that the algae cell samples into which the plasmid was transferred had a single bright DNA band, while the wild-type (WT) sample as the control group had no band (e.g. Figure 3 ), indicating that the editing plasmid has been successfully transferred into the P. tricornutum cells.

[0048] (7) In order to identify whether the knockout transformants are homozygous, for the samples with bands shown in the above electrophoresis results and the wild type (WT), a pair of specific fragment amplification primers JD-F and JD-R were designed using DNA as a template to amplify the CRTISO5 gene location. The PCR procedure was the same as step (6). The PCR primers are as follows: JD-F: 5'-CCCGCTCATTTAGTAGTTCA-3' (SEQ ID NO. 10); JD-R: 5'-GGGGTTTCATTGTGTTCGCTG-3' (SEQ ID NO. 11); (8) Perform nucleic acid electrophoresis on the PCR products and compare them with the wild-type bands. Select samples with normal bands for testing and use Snapgene software to analyze the sequencing results.

[0049] (9) Repeat steps (7) and (8) until homozygous transformants are screened. The sequencing results show that CRTISO5 The cytosine (C) on the gene has completely mutated to thymine (T), indicating that the pPtCBE plasmid has successfully edited the gene in P. tricornutum. The specific results are as follows: Figure 4 shown.

[0050] The results of analyzing the sequencing map using EditR software (http: / / baseeditr.com / ) showed that more than 99% of the cytosine (C) at this gene site were mutated to thymine (T).

[0051] It can be seen from the experimental results of the above examples that the pPtCBE plasmid provided by the present invention has extremely high efficiency and accuracy in gene editing and has great application potential.

[0052] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A plasmid for pyrimidine base mutation in Phaeodactylum tricornutum, characterized in that: The plasmid is named pPtCBE; the plasmid includes an evoAPOBEC1-nCas9-UGI expression cassette, a targeting sequence spacer fragment, and two BsaⅠ sites. The nucleotide sequence of the evoAPOBEC1-nCas9-UGI expression cassette is shown in SEQ ID No.2, and the two ends of the evoAPOBEC1-nCas9-UGI expression cassette are respectively fused with nuclear localization signal peptides of triangular phytoplankton; the targeting sequence spacer fragment is derived from triangular phytoplankton; and the BsaⅠ site is used to insert the targeting sequence spacer fragment.

2. The plasmid according to claim 1, characterized in that The triangular phytosporum nuclear localization signal peptide at the N-terminus of the evoAPOBEC1-nCas9-UGI expression cassette is SV40 NLS, and the triangular phytosporum nuclear localization signal peptide at the C-terminus of the evoAPOBEC1-nCas9-UGI expression cassette is nucleoplasmin NLS.

3. The plasmid according to claim 2, characterized in that The plasmid also includes a sgRNA expression unit, which is driven by a U6 promoter, and the DNA sequence of the sgRNA expression unit is shown in SEQ ID No.

3.

4. The plasmid according to claim 1, characterized in that The plasmid also includes a construction screening gene, which is selected from at least one of kanamycin resistance gene, chloramphenicol resistance gene, ampicillin resistance gene, tetracycline resistance gene, erythromycin resistance gene, gentamicin resistance gene and bleomycin resistance gene.

5. The plasmid according to claim 1, characterized in that The plasmid also includes a transformation screening gene, which is selected from at least one of a bleomycin resistance gene, a neomycin resistance gene, a hygromycin B resistance gene, and a puromycin resistance gene.

6. The plasmid according to claim 1, characterized in that The plasmid also includes a CEN / ARS autonomously replicating element.

7. A use of the plasmid according to any one of claims 1 to 6, characterized in that: The application is to use the plasmid for pyrimidine base mutation in the triangular phycodactylum to direct mutation of cytosine at any site on the triangular phycodactylum genome into thymine.

8. The use according to claim 7, characterized in that From 5' to 3', the active window for directed mutation of cytosine to thymine at any site on the genome of P. tricornutum is position 4 to 8 of the spacer fragment of the targeting sequence.

9. An expression cell line, characterized in that The expression cell strain contains the plasmid according to any one of claims 1 to 6, and the expression cell strain is selected from any one or more of bacteria, fungi, and algae.

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