Genetically engineered algae with controllable flocculation as well as construction method and application of genetically engineered algae

Abstract

The invention discloses a flocculation-controllable genetic engineering alga and a construction method and application thereof, and is characterized in that the genetic engineering alga is phaeodactylum tricornutum into which an enabling plasmid pPha-J47869-EG01992-eGFP-FSP is introduced, the FSP is directionally expressed on the outer side of a phaeodactylum tricornutum cell, and the construction method comprises the following steps: constructing an induced expression plasmid pPha-J47869-eGFP and a directional expression plasmid pPha-J47869-EG01992-eGFP; artificially synthesizing a codon-optimized FSP coding sequence, inserting the codon-optimized FSP coding sequence into the downstream of the eGFP of the directional expression vector, and constructing an enabling plasmid; finally, the enabling plasmids are electrically transformed into phaeodactylum tricornutum, an f / 2 plate culture medium is coated with the phaeodactylum tricornutum for culture, positive algae strains are screened and identified, and the gene engineering algae with controllable flocculation are constructed and have the advantages that the gene engineering algae can be efficiently combined with white carbon black, and the enabling process of microalgae is efficiently coupled with the culture and collection process of the microalgae.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and in particular relates to a controllable flocculation genetic engineering algae and a construction method and application thereof. Background Art

[0002] Microalgae have a much higher photosynthetic carbon fixation efficiency than higher plants, and can synthesize large amounts of high value-added substances such as polyunsaturated fatty acids, astaxanthin, and fucoxanthin, and have huge economic development potential. Microalgae represented by diatoms have a fucoxanthin content that is tens or even hundreds of times higher than that of large brown algae, and have attracted widespread attention in the field of large-scale production of fucoxanthin. However, the mass density of microalgae cells is close to that of water, and the individual cells are tiny; after achieving high-density culture, it is difficult to separate them from the water body. Therefore, how to efficiently collect microalgae cells in the culture water body has become a bottleneck restricting the development of the microalgae industry.

[0003] Although there are currently a variety of cell collection methods, such as flocculation, microfiltration, plate and frame filtration, flotation and high-speed centrifugation, these existing methods cannot achieve efficient collection of microalgae cells while saving energy. Take high-speed centrifugation, the most mainstream cell collection method in the microalgae industry, as an example: centrifugal collection relies on expensive large centrifuges, which not only require huge initial investment, but also have high maintenance costs; in addition, centrifugal energy consumption is extremely high and operating costs are very high. It is estimated that the cost of centrifugal collection even reaches more than one-third of the total cost of microalgae production. Therefore, the development of a new collection system that is efficient, safe and low-energy is a key issue that needs to be urgently addressed in the microalgae industry.

[0004] Cell-enabling technology refers to the process of introducing certain biological molecules with special functions into target cells for expression through genetic engineering and other means, thereby giving the target cells specific new functions. In addition, by introducing an induced expression system, the enabling process of microalgae can be efficiently coupled with its cultivation and collection process, thereby significantly improving its overall performance in biotechnology applications. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a controllable flocculation genetically engineered algae capable of efficiently coupling the enabling process of microalgae with its cultivation and collection process, a construction method thereof and an application thereof.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a genetically engineered algae with controllable flocculation, wherein the genetically engineered algae is a triangular shaped algae into which an enabling plasmid pPha-J47869-EG01992-eGFP-FSP is introduced, and the FSP ( Functional Silica-binding peptide, functional silicon-binding peptide) has the ability to efficiently and specifically bind to white carbon black, and can be directed to be expressed on the outside of the triangular brown algae cells through fusion expression with EG01992, and the J47869 promoter can show high transcriptional activity under low phosphate concentration conditions. The nucleotide sequence of the enabling plasmid pPha-J47869-EG01992-eGFP-FSP is shown in SEQ ID NO.1.

[0007] The present invention also provides a method for constructing the above-mentioned genetically engineered algae capable of controllable flocculation, comprising the following steps: Step 1: Using the genomic DNA of Phaeodactylum triangularis as a template, amplify the Phatr3_J47869 promoter and replace the FcpA promoter of the basic expression plasmid pPha-T1-eGFP to construct the inducible expression plasmid pPha-J47869-eGFP; Step 2: Using the genomic DNA of Phaeodactylum tricornutum as a template, amplify the coding sequence of Phatr3_EG01992, and insert it into the upstream of eGFP of the inducible expression vector to construct the directional expression plasmid pPha-J47869-EG01992-eGFP; Step 3, artificially synthesize the codon-optimized FSP coding sequence and insert it into the downstream of eGFP in the directional expression vector to construct the enabling plasmid pPha-J47869-EG01992-eGFP-FSP; Step 4: electro-transform the enabling plasmid pPha-J47869-EG01992-eGFP-FSP into P. tricornutum, and spread the transformed P. tricornutum cells on f / 2 plate culture medium for culture, and screen positive algae strains to obtain genetically engineered algae with controllable flocculation.

[0008] Furthermore, step 1 is as follows: (1) According to the Phatr3_J47869 promoter sequence, a forward amplification primer containing an NdeI restriction site and a reverse amplification primer containing an EcoRI restriction site were designed, and PCR amplification was performed using the DNA of Phaeodactylum tricornutum as a template to obtain the amplification product of the Phatr3_J47869 promoter, wherein the nucleotide sequence of the forward amplification primer of the Phatr3_J47869 promoter is shown in SEQ ID NO.3: 5'-GTACTGAGAGTGCACCATATGTGGTGGTGAATCAACACTTAATGTG-3', and the nucleotide sequence of the reverse amplification primer of the Phatr3_J47869 promoter is shown in SEQ ID NO.4: 5'-TTAGTCGATGATATCGAATTCCGAAGAATTCTTTTCACCAGGG-3'; (2) The original FcpA promoter of pPha-T1-eGFP was removed by using NdeI and EcoRI restriction endonucleases. The amplified product of Phatr3_J47869 promoter was connected with the restriction product of pPha-T1-eGFP to construct the inducible expression plasmid pPha-J47869-eGFP.

[0009] Furthermore, step 2 is as follows: (1) Forward and reverse primers containing KpnI restriction site were designed based on the coding sequence of Phatr3_EG01992, and PCR amplification was performed using the DNA of Phaeodactylum triangularis as a template to obtain the amplification product of the coding sequence of Phatr3_EG01992, where The nucleotide sequence of the primer for forward amplification of the Phatr3_EG01992 coding sequence is shown in SEQ ID NO.6: 5'-GACTAATTCGAGCTCGGTACCATGAGGCTACGTTCATCCATTACC-3', and the nucleotide sequence of the primer for reverse amplification of the Phatr3_EG01992 coding sequence is shown in SEQ ID NO.7: 5'-TCTAGAGGATCCCCGGGTACCACGGCAAAAGATTCCAAAACG-3'; (2) The pPha-J47869-eGFP plasmid was digested with KpnI restriction endonuclease, and the amplified product of the Phatr3_EG01992 coding sequence was ligated with the digested pPha-J47869-eGFP to construct the directional expression plasmid pPha-J47869-EG01992-eGFP.

[0010] Furthermore, step 3 is as follows: (1) Synthesis of double-stranded template: The coding sequence of the FSP short peptide was codon optimized to obtain the nucleotide sequence of the FSP coding gene as shown in SEQ ID NO.9: 5'-ATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3'. FSP double-stranded forward and reverse synthetic primers were designed according to the FSP coding sequence and the flexible linker coding sequence, and the two synthetic primers were synthesized into double-stranded nucleotides as PCR amplification templates by annealing reaction, wherein the nucleotide sequence of the FSP double-stranded forward synthetic primer is shown in SEQ ID NO.10: 5'-GGCGGCGGTGGTAGCGGTGGCGGCGGTTCCATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3', and the nucleotide sequence of the FSP double-stranded reverse synthetic primer is shown in SEQ ID NO.11: 5'-TTACCGCACAGCAGCCGACATGAGGTCGCTGCGGTGCATGGAACCGCCGCCACCGCTACCACCGCCGCC-3'; (2) Designing forward and reverse primers for FSP amplification according to the XbaI restriction site sequence and the FSP coding sequence, and using the double-stranded nucleotide obtained in step (1) as a template for PCR amplification to obtain an amplification product containing the FSP coding sequence, the corresponding restriction site and the linker coding sequence, wherein the nucleotide sequence of the FSP forward amplification primer is as shown in SEQ ID NO.12: 5'-GACGAGCTGTACAAGTCTAGGGCGGCGGTGGTAGCGGT-3', and the nucleotide sequence of the FSP reverse amplification primer is as shown in SEQ ID NO.13: 5'-TGCCTGCAGGTCGACTCTAGATTACCGCACAGCAGCCGA-3'; (3) The pPha-J47869-EG01992-eGFP plasmid was digested with XbaI restriction endonuclease, and the FSP amplification product was ligated with the digested pPha-J47869-EG01992-eGFP to construct the enabling plasmid pPha-J47869-EG01992-eGFP-FSP, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0011] Furthermore, step 4 is as follows: (1) Transformation of Phaeodactylum triangularis: The enabling plasmid pPha-J47869-EG01992-eGFP-FSP was transformed into competent cells, and after incubation, the cells were spread onto LB solid culture medium; after visible colonies were formed, the colonies were picked up and transferred to LB liquid culture medium for shaking culture; the transformed competent cells were collected, the enabling plasmid pPha-J47869-EG01992-eGFP-FSP was extracted, and the cells were transformed into Phaeodactylum triangularis by electroporation; (2) Screening of positive transformants: The transformed Phaeodactylum tricornutum cells were spread on a solid f / 2 plate medium containing bleomycin and placed under a light intensity of 50-70 µmol photons m -2 s -1 , culture under the condition of temperature of 20-24 ℃, screen the positive transformation column, and obtain the genetically engineered algae with controllable flocculation.

[0012] The present invention also provides the use of the above-mentioned controllable flocculation genetically engineered algae in preparing a microalgae flocculation system, wherein the microalgae flocculation system is an algae liquid added with white carbon black, and the amount of white carbon black added is 0.5-1.5 g per liter of P. triangularis culture liquid.

[0013] Compared with the prior art, the advantages of the present invention are: the present invention is a genetically engineered algae with controllable flocculation, a construction method and an application thereof, the amino acid sequence MHRSDLMSAAVR (FSP for short) used in the present invention shows high affinity to white carbon black, and the fragment is expressed on the outside of the microalgae cell, which can give the microalgae the ability to efficiently bind white carbon black. The Phatr3_EG01992 gene encodes a protein located on the outside of the microalgae cell. The protein is fused with FSP for expression, and the directional expression of FSP on the outside of the microalgae cell can be achieved. During the cultivation process of microalgae, as the cell density increases, the nutrient salt content in the culture system will decrease rapidly. When the cell density reaches a level that can be collected, the nutrient salt in the culture system is usually exhausted. By placing Phatr3_EG01992 and FSP under the control of a promoter induced by a low nutrient salt concentration, the timing of the directional expression of FSP on the outside of the microalgae cell and the optimal collection time of the microalgae can be synchronized. Placing the Phatr3_EG01992-FSP fusion gene under the regulation of the Phatr3_J47869 promoter can efficiently couple the FSP enabling process with the cultivation and collection of microalgae. Adding silica to the culture system, since the density of silica is much greater than that of water, the microalgae cells combined with silica can quickly sink to the bottom of the water, achieving efficient collection of microalgae cells.

[0014] In summary, the present invention provides a genetically engineered algae with controllable flocculation, a construction method and an application thereof, and genetically improves microalgae species by utilizing the ability of FSP to specifically and efficiently bind to white carbon black, the property of Phatr3_EG01992 being located on the outside of the cell, and the characteristic that the promoter activity of Phatr3_J47869 is regulated by phosphate concentration. The microalgae germplasm transformed by this strategy can use white carbon black as a flocculant to construct an efficient and low-energy flocculation system, thereby providing a new idea for breaking through the pain points in the microalgae industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 To show the changes in fluorescence intensity of green fluorescent protein (eGFP) under different phosphate concentration conditions after the green fluorescent protein (eGFP) coding sequence was placed under the control of the Phatr3_J47869 promoter; Figure 2 This is the electrophoresis diagram of the amplified Phatr3_J47869 promoter, where lane 1: 5000 bp Marker, lanes 2-3: Phatr3_J47869 promoter amplification results; Figure 3 This is the electrophoresis diagram of the amplified Phatr3_EG01992 coding sequence, where lane 1: 2000 bp Marker, lanes 2-3: Phatr3_EG01992 coding sequence amplification results; Figure 4 The results of screening positive transformants of Phaeodactylum tricornutum by using the plate method with the help of antibiotics; Figure 5 The results are the sequencing results after the DNA of the transformed algae strain was amplified by the specific primers based on the eGFP coding sequence; Figure 6 To compare the transcription of Phatr3_EG01992 in wild-type strains, inducible plasmid pPha-J47869-eGFP transformants, and enabling plasmid pPha-J47869-EG01992-eGFP-FSP transformants based on real-time quantitative PCR; Figure 7 To compare and analyze the eGFP signals in wild-type strains, induction plasmid pPha-J47869-eGFP transformants, and enabling plasmid pPha-J47869-EG01992-eGFP-FSP transformants based on laser confocal microscopy. Figure 8 This is a comparative analysis of the binding of Phaeodactylum triangularis cells and wild strains with white carbon black based on the enabling plasmid pPha-J47869-EG01992-eGFP-FSP. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below with reference to the accompanying drawings.

[0017] Microalgae: Phaeochromis triangularis; basic plasmid: pPha-T1-eGFP; amplification kit: 2×Phanta FlashMaster Mix (Dye Plus) (Vazyme, P520-01); competent cells: DH5α; ligation kit: ClonExpress® II One Step Cloning Kit (Vazyme, C112-02); DNA extraction: 2×CTAB (Coolaber, SL2071); plasmid extraction kit: FastPure Plasmid Mini Kit (Vazyme, DC201-01), etc.; the culture medium used was according to the literature (Guillard RRL, Ryther JH. Studies of marine planktonic diatoms:I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran[J].Canadian Journal of Microbiology, 1962, 8(2): 229–239) f / 2 medium; microalgae culture equipment: Zhichu ZQZY-88CGES full-temperature shaking incubator; microalgae culture conditions: light intensity 60 µmol photons m -2 s -1 , temperature 22°C, shaker speed 90 rpm.

[0018] Different genetic transformation systems, different promoters, different positioning tags, different functional peptides and corresponding affinity materials of each microalgae can all be used for the microalgae germplasm transformation based on the cell enabling strategy mentioned in this patent and the subsequent flocculation system construction. This specific embodiment only lists the use of triangular brown finger algae as research materials, the use of electrotransformation method, FSP as enabling factor, the use of Phatr3_J47869 promoter to achieve induced enabling, and Phatr3_EG01992 as a positioning tag to construct a genetically engineered triangular brown finger algae introduced with the enabling plasmid pPha-J47869-EG01992-eGFP-FSP, and white carbon black as a flocculation medium to establish a corresponding flocculation collection system, wherein the nucleotide sequence of the enabling plasmid pPha-J47869-EG01992-eGFP-FSP is shown in SEQ ID NO.1.

[0019] Specific Example 1: Construction of inducible expression plasmid pPha-J47869-eGFP.

[0020] The promoter activity of Phatr3_J47869 gene is regulated by phosphate concentration and shows high transcriptional activity under low phosphate conditions, e.g. Figure 1 As shown. Therefore, by controlling the expression of Phatr3_EG01992 and the enabling factor FSP through the Phatr3_J47869 promoter, the directional expression of FSP on the outside of the microalgae cells is triggered when the phosphate concentration in the culture system decreases. The construction method of the inducible expression plasmid pPha-J47869-eGFP includes the following steps: Step 1: Extract genomic DNA of Phaeodactylum triangularis: Collect cells in the logarithmic phase (cell density 5×10 6 cells / mL) of Phaeochromis triangularis algae liquid, and genomic DNA was extracted; Step 2, PCR amplification: According to the Ensembl Protists database, the upstream sequence of the open reading frame of Phatr3_J47869 was searched, and its promoter sequence was predicted with the help of Promoter2.0, and the nucleotide sequence of the Phatr3_J47869 promoter was obtained as shown in SEQ ID NO.2. According to the Phatr3_J47869 promoter sequence, a forward amplification primer containing an NdeI restriction site and a reverse amplification primer containing an EcoRI restriction site were designed, wherein the nucleotide sequence of the forward amplification primer of the Phatr3_J47869 promoter is shown in SEQ ID NO.3: 5'-GTACTGAGAGTGCACCATATGTGGTGGTGAATCAACACTTAATGTG-3', and the nucleotide sequence of the reverse amplification primer of the Phatr3_J47869 promoter is shown in SEQ ID NO.4: 5'-TTAGTCGATGATATCGAATTCCGAAGAATTCTTTTCACCAGGG-3'; The PCR amplification system was as follows: 0.5 μL of P. tricornutum DNA, 10 μL of 2×Phanta Flash Master Mix, 0.5 μL each of the forward and reverse amplification primers of the Phatr3_J47869 promoter, and 8.5 μL of ddH2O; The PCR amplification program was as follows: 94°C for 5 min; 94°C for 30 sec, 68°C for 30 sec, 72°C for 1 min, 35 cycles, and stored at 4°C; Step 3: Verification of PCR amplification products: After amplification, the PCR products were separated and verified by agarose gel electrophoresis. Figure 2 As shown, lane 1 is a DNA marker; lanes 2 and 3 are amplified Phatr3_J47869 promoter sequences, which were subsequently excised and recovered and sequenced, and the length of the amplified fragments was consistent with the theoretical length; Step 4: Use NdeI and EcoRI restriction endonucleases to remove the original FcpA promoter of pPha-T1-eGFP; connect the Phatr3_J47869 promoter amplification product obtained in step 3 with the restriction product of pPha-T1-eGFP, react at 37°C for 30 min, and construct the inducible expression plasmid pPha-J47869-eGFP.

[0021] Specific Example 2: Construction of directional expression plasmid pPha-J47869-EG01992-eGFP.

[0022] The protein encoded by Phatr3_EG01992 is localized outside the microalgae cells. The protein is fused with FSP to express the directional expression of FSP outside the microalgae cells. The construction method of the directional expression plasmid pPha-J47869-EG01992-eGFP includes the following steps: Step 1. Extraction of genomic DNA from P. triangularis: Collect the cells in the logarithmic phase (cell density of 5×10 6 cells / mL) of Phaeochromis triangularis algae liquid, and genomic DNA was extracted; Step 2, PCR amplification: According to the Ensembl Protists database, the open reading frame sequence of Phatr3_EG01992 was searched to obtain the nucleotide sequence encoded by Phatr3_EG01992 as shown in SEQ ID NO.5, and forward and reverse amplification primers containing KpnI restriction site were designed according to the Phatr3_EG01992 coding sequence, wherein the nucleotide sequence of the forward amplification primer of the Phatr3_EG01992 coding sequence is shown in SEQ ID NO.6: 5'-GACTAATTCGAGCTCGGTACCATGAGGCTACGTTCATCCATTACC-3', and the nucleotide sequence of the reverse amplification primer of the Phatr3_EG01992 coding sequence is shown in SEQ ID NO.7: 5'-TCTAGAGGATCCCCGGGTACCACGGCAAAAGATTCCAAAACG-3'; The PCR amplification system was as follows: 0.5 μL of P. tricornutum DNA, 10 μL of 2×Phanta Flash Master Mix, 0.5 μL each of the forward and reverse amplification primers of the Phatr3_EG01992 coding sequence, and 8.5 μL of ddH2O; The PCR amplification program was as follows: 94°C for 5 min; 94°C for 30 sec, 68°C for 30 sec, 72°C for 1 min, 35 cycles, and stored at 4°C; Step 3, PCR amplification product identification: After amplification, the PCR products were separated and verified by agarose gel electrophoresis. Figure 3 As shown, lane 1 is a DNA marker, and lanes 2 and 3 are amplified Phatr3_EG01992 coding regions, which are then subjected to gel excision, recovery, and sequencing; the length of the amplified fragment is consistent with the theoretical length.

[0023] Step 4, construction of directional expression vector: pPha-J47869-eGFP plasmid was digested with KpnI restriction endonuclease, and the amplified product of Phatr3_EG01992 coding sequence obtained in step 3 was connected with the digested pPha-J47869-eGFP, and the reaction was carried out at 37°C for 30 min to construct the directional expression plasmid pPha-J47869-EG01992-eGFP.

[0024] Specific example three: Construction of enabling plasmid pPha-J47869-EG01992-eGFP-FSP.

[0025] The amino acid sequence of the FSP short peptide is shown in SEQ ID NO.8: MHRSDLMSAAVR, which has an efficient and specific affinity for silica. If the short peptide is directed to be expressed on the outside of the microalgae cells, it can give the microalgae cells the ability to efficiently bind to silica. Since the density of silica is much greater than that of water, the microalgae cells bound to silica can quickly settle to the bottom of the water. The microalgae germplasm transformed by this strategy can use silica as a flocculant to construct an efficient flocculation system, providing a new solution for the collection of microalgae cells. The steps of the method for constructing the enabling plasmid pPha-J47869-EG01992-eGFP-FSP are as follows: Step 1, synthesizing a double-stranded template: codon optimization is performed on the coding sequence of the FSP short peptide to obtain a nucleotide sequence encoding FSP as shown in SEQ ID NO.9: 5'-ATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3', and FSP double-stranded forward and reverse synthesis primers are designed according to the FSP coding sequence and the flexible linker (the nucleotide sequence of the flexible linker is shown in SEQ ID NO.18: GGGGSGGGGS) coding sequence, and the two synthesis primers are synthesized into double-stranded nucleotides as a PCR amplification template by annealing reaction, wherein the nucleotide sequence of the FSP double-stranded forward synthesis primer is shown in SEQ ID NO.10: 5'-GGCGGCGGTGGTAGCGGTGGCGGCGGTTCCATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3', and the nucleotide sequence of the FSP double-stranded reverse synthesis primer is shown in SEQ ID NO. Shown in NO.11: 5'-TTACCGCACAGCAGCCGACATGAGGTCGCTGCGGTGCATGGAACCGCCGCCACCGCTACCACCGCCGCC-3'; Step 2, designing FSP forward and reverse amplification primers according to the XbaI restriction site sequence and the FSP coding sequence, wherein the nucleotide sequence of the FSP forward amplification primer is as shown in SEQ ID NO.12: 5'-GACGAGCTGTACAAGTCTAGGGCGGCGGTGGTAGCGGT-3', and the nucleotide sequence of the FSP reverse amplification primer is as shown in SEQ ID NO.13: 5'-TGCCTGCAGGTCGACTCTAGATTACCGCACAGCAGCCGA-3'; The PCR amplification system was as follows: 0.5 μL double-stranded nucleotide, 10 μL 2× PrimeSTAR Max Premix, 0.5 μL each of the forward and reverse amplification primers of the FSP gene, and 8.5 μL ddH2O; The PCR program was as follows: 94°C for 5 min; 94°C for 30 sec, 62°C for 30 sec, 72°C for 1 min, for 35 cycles, and stored at 4°C.

[0026] Step 3, PCR amplification product verification: After amplification, the PCR products were separated by agarose gel electrophoresis, followed by gel excision, recovery and sequencing. The sequencing results were consistent with the FSP theoretical coding sequence; Step 4, construction of enabling plasmid: pPha-J47869-EG01992-eGFP plasmid was digested with XbaI restriction endonuclease, and the FSP amplification product obtained in step 3 was ligated with the digested pPha-J47869-EG01992-eGFP, and the ligation was carried out at 37°C for 30 min to construct the enabling plasmid pPha-J47869-EG01992-eGFP-FSP, whose nucleotide sequence is shown in SEQ ID NO.1.

[0027] Specific Example 4: The enabling plasmid pPha-J47869-EG01992-eGFP-FSP constructed in Specific Example 3 was electroporated into Phaeodactylum tricornutum, and the specific steps are as follows: The pPha-J47869-EG01992-eGFP-FSP plasmid constructed in the specific example 3 was transformed into the triangular flavonoids by electroporation, and the transformed triangular flavonoids cells were spread on f / 2 plate culture medium, and positive algae strains were screened and identified to obtain genetically engineered algae with controllable flocculation. The specific steps are: Step 1, transformation of P. triangularis: transform the enabling plasmid pPha-J47869-EG01992-eGFP-FSP into competent cells, incubate, and spread on LB solid culture medium. When visible colonies are formed, pick the colonies into LB liquid culture medium, and place them in a constant temperature shaking incubator at 37°C to culture the transformed competent cells. Collect the transformed competent cells, extract the pPha-J47869-EG01992-eGFP-FSP plasmid, and transform them into P. triangularis by electrotransformation. The electrotransformation parameters are: 500 V, 25 μF, 400 Ω; Step 2, screening of positive transformants: The transformed P. tricornutum cells were spread on f / 2 plate medium containing 1.0% agar and a bleomycin concentration of 75 μg / mL, and placed under a light intensity of 60 μmol photons m -2 s -1 , cultured at 22°C. After about 3 weeks, the successfully transformed strains formed algal colonies on the f / 2 plate. Figure 4 As shown; pick visible algae colonies and culture them in liquid f / 2 medium with a bleomycin concentration of 75 μg / mL for 1 week to obtain the algal liquid of P. triangularis, and extract the algal liquid DNA as an identification template; Step 3, identification of positive transformants: design identification primers based on the eGFP coding sequence, wherein the nucleotide sequence of the algae liquid forward identification primer is shown in SEQ ID NO.14: 5'-GACGACGGCAACTACAAGAC-3', and the nucleotide sequence of the algae liquid reverse identification primer is shown in SEQ ID NO.15: 5'-CGAACTCCAGCAGGACCAT-3'; The PCR amplification system was as follows: 0.5 μL of Phaeodactylum triangularis DNA, 10 μL of 2×Phanta Flash Master Mix, 0.5 μL of each of the forward and reverse identification primers designed based on the eGFP coding sequence, and 8.5 μL of ddH2O; The PCR amplification program was: 94°C for 5 min; 94°C for 30 sec, 58°C for 30 sec, 72°C for 1 min, 35 cycles, and stored at 4°C. The PCR products were separated by agarose gel electrophoresis and then sequenced. Figure 5 As shown, the Sanger sequencing results showed that the amplified fragment originated from the eGFP coding segment of the enabling plasmid, indicating that the enabling plasmid had been successfully transformed into P. tricornutum.

[0028] Specific example 5: Verification of positive transformants of Phaeodactylum tricornutum.

[0029] 1. Verification of transcription level: Total RNA of each strain of P. tricornutum was extracted and reverse transcribed to obtain cDNA. Using cDNA as a template, real-time quantitative PCR was used to compare and analyze the transcription of Phatr3_EG01992 in the wild strain and the positive transformed strain.

[0030] Based on the Phatr3_EG01992 sequence, the forward and reverse primers for real-time quantitative PCR were designed, wherein the nucleotide sequence of the forward primer for real-time quantitative PCR is shown in SEQ ID NO.16: 5'-CGAGAACCGAAGTGACCG-3'; the nucleotide sequence of the reverse primer for real-time quantitative PCR is shown in SEQ ID NO.17: 5'-ACCAGCACAACCAGGGAC-3'; Real-time quantitative PCR system: 0.5 μL of Phaeodactylum triangularis cDNA, 10 μL of 2×PhantaQuantiNovaSYBRGreen PCR Master Mix, 0.5 μL of each of the forward and reverse primers for real-time quantitative PCR, and 8.5 μL of ddH2O; The real-time quantitative PCR program was: 94°C for 5 min; 94°C for 15 sec, 60°C for 30 sec, and cycled 40 times. Figure 6As shown, in the enabling plasmid transformant, the transcription abundance of Phatr3_EG01992 was several times higher than that of the wild-type strain (negative control 1) and the inducible expression plasmid pPha-J47869-eGFP transformant (negative control 2), indicating that the target nucleotide sequence was successfully transcribed in the enabling plasmid transformant.

[0031] 2. Protein level verification: Each strain of P. triangularis was cultured to the logarithmic growth phase (cell density was about 5×10 6 cells / mL), the position and intensity of the eGFP signal of each strain of P. tricornutum were detected and compared with the laser confocal microscope under the conditions of excitation and emission wavelengths of 488 nm and 507 nm, respectively. Figure 7 As shown, the green fluorescence signal of the P. tricornutum strain transformed with the enabling plasmid was accurately localized to the outside of the cell; no eGFP signal was observed in the cells of the wild strain (negative control 1); and the eGFP signal of the strain transformed with the inducible expression plasmid pPha-J47869-eGFP (negative control 2) was localized in the cytoplasm.

[0032] In summary, after identification of transcription and protein expression levels, it can be determined that the FSP enabling factor carried by the positive transformant can be correctly transcribed, translated, and accurately located on the outside of the P. tricornutum cells.

[0033] Specific Example 6: Verification of the efficient flocculation effect based on the positive transformation strain of Phaeodactylum triangularis.

[0034] Cultivation of P. triangularis: The positive transformed strains of P. triangularis and the wild strain (negative control) were inoculated into liquid f / 2 medium at the same starting density (cell density was about 1×10 6 cells / mL) and cultured in a constant temperature and light shaker; the culture conditions were a light intensity of 60 µmol photons m -2 s -1 , temperature 22°C, rotation speed 90 rpm.

[0035] Culture to the stationary phase (cell density is about 4×10 7 After culturing the algae solution in a constant temperature and light shaker for 24 hours, the algae solution was left to stand for 5 minutes. Figure 8 As shown, the positive transformed strains of P. triangularis will quickly settle to the bottom of the culture container due to the binding of a large amount of silica, achieving efficient and low-energy collection; however, the cells of the wild strain of P. triangularis lack the ability to bind silica, and their cells are still evenly dispersed in the algae liquid and cannot be collected.

[0036] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A genetically engineered algae capable of controlled flocculation, characterized in that: The genetically engineered algae is a triangular shaped algae into which an enabling plasmid pPha-J47869-EG01992-eGFP-FSP is introduced. The FSP is directionally expressed outside the triangular shaped algae cells. The nucleotide sequence of the enabling plasmid pPha-J47869-EG01992-eGFP-FSP is shown in SEQ ID NO.

1.

2. A method for constructing a genetically engineered algae capable of controlled flocculation according to claim 1, characterized in that The following steps are involved: Step 1: Using the genomic DNA of Phaeodactylum triangularis as a template, amplify the Phatr3_J47869 promoter and replace the FcpA promoter of the basic expression plasmid pPha-T1-eGFP to construct the inducible expression plasmid pPha-J47869-eGFP; Step 2: Using the genomic DNA of Phaeodactylum tricornutum as a template, amplify the coding sequence of Phatr3_EG01992, and insert it into the upstream of eGFP of the inducible expression vector to construct the directional expression plasmid pPha-J47869-EG01992-eGFP; Step 3, artificially synthesize the codon-optimized FSP coding sequence and insert it into the downstream of eGFP in the directional expression vector to construct the enabling plasmid pPha-J47869-EG01992-eGFP-FSP; Step 4: electro-transform the enabling plasmid pPha-J47869-EG01992-eGFP-FSP into P. tricornutum, and spread the transformed P. tricornutum cells on f / 2 plate culture medium for culture, and screen positive algae strains to obtain genetically engineered algae with controllable flocculation.

3. The method for constructing a genetically engineered algae capable of controlled flocculation according to claim 2, characterized in that Step 1 is as follows: (1) According to the Phatr3_J47869 promoter sequence, a forward amplification primer containing an NdeI restriction site and a reverse amplification primer containing an EcoRI restriction site were designed, and PCR amplification was performed using the DNA of Phaerotactum tricornutum as a template to obtain the amplification product of the Phatr3_J47869 promoter, wherein the nucleotide sequence of the Phatr3_J47869 promoter forward amplification primer is shown in SEQ ID NO.3: 5'-GTACTGAGAGTGCACCATATGTGGTGGTGAATCAACACTTAATGTG-3', and the nucleotide sequence of the Phatr3_J47869 promoter reverse amplification primer is shown in SEQ ID NO.4: 5'-TTAGTCGATGATATCGAATTCCGAAGAATTCTTTTCACCAGGG-3'; (2) The original FcpA promoter of pPha-T1-eGFP was removed by using NdeI and EcoRI restriction endonucleases. The amplified product of Phatr3_J47869 promoter was connected with the restriction product of pPha-T1-eGFP to construct the inducible expression plasmid pPha-J47869-eGFP.

4. The method for constructing a genetically engineered algae capable of controlled flocculation according to claim 2, characterized in that Step 2 is as follows: (1) Forward and reverse primers containing KpnI restriction site were designed based on the coding sequence of Phatr3_EG01992, and PCR amplification was performed using the DNA of Phaeodactylum triangularis as a template to obtain the amplification product of the coding sequence of Phatr3_EG01992, where The nucleotide sequence of the primer for forward amplification of the Phatr3_EG01992 coding sequence is shown in SEQ ID NO.6: 5'-GACTAATTCGAGCTCGGTACCATGAGGCTACGTTCATCCATTACC-3', and the nucleotide sequence of the primer for reverse amplification of the Phatr3_EG01992 coding sequence is shown in SEQ ID NO.7: 5'-TCTAGAGGATCCCCGGGTACCACGGCAAAAGATTCCAAAACG-3'; (2) The pPha-J47869-eGFP plasmid was digested with KpnI restriction endonuclease, and the amplified product of the Phatr3_EG01992 coding sequence was ligated with the digested pPha-J47869-eGFP to construct the directional expression plasmid pPha-J47869-EG01992-eGFP.

5. The method for constructing a genetically engineered algae capable of controlled flocculation according to claim 2, characterized in that Step 3 is as follows: (1) Synthesis of double-stranded template: The coding sequence of the FSP short peptide was codon optimized to obtain the nucleotide sequence of the FSP coding gene as shown in SEQ ID NO.9: 5'-ATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3'. FSP double-stranded forward and reverse synthetic primers were designed according to the FSP coding sequence and the flexible linker coding sequence, and the two synthetic primers were synthesized into double-stranded nucleotides as PCR amplification templates by annealing reaction. The nucleotide sequence of the FSP double-stranded forward synthetic primer was shown in SEQ ID NO.10: 5'-GGCGGCGGTGGTAGCGGTGGCGGCGGTTCCATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3', and the nucleotide sequence of the FSP double-stranded reverse synthetic primer was shown in SEQ ID NO.11: 5'-TTACCGCACAGCAGCCGACATGAGGTCGCTGCGGTGCATGGAACCGCCGCCACCGCTACCACCGCCGCC-3'; (2) Designing forward and reverse primers for FSP amplification according to the XbaI restriction site sequence and the FSP coding sequence, and using the double-stranded nucleotide obtained in step (1) as a template for PCR amplification to obtain an amplification product containing the FSP coding sequence, the corresponding restriction site and the linker coding sequence, wherein the nucleotide sequence of the FSP forward amplification primer is as shown in SEQ ID NO.12: 5'-GACGAGCTGTACAAGTCTAGGGCGGCGGTGGTAGCGGT-3', and the nucleotide sequence of the FSP reverse amplification primer is as shown in SEQ ID NO.13: 5'-TGCCTGCAGGTCGACTCTAGATTACCGCACAGCAGCCGA-3'; (3) The pPha-J47869-EG01992-eGFP plasmid was digested with XbaI restriction endonuclease, and the FSP amplification product was ligated with the digested pPha-J47869-EG01992-eGFP to construct the enabling plasmid pPha-J47869-EG01992-eGFP-FSP, the nucleotide sequence of which is shown in SEQ ID NO.

1.

6. The method for constructing a genetically engineered algae capable of controlled flocculation according to claim 2, characterized in that Step 4 is as follows: (1) Transformation of Phaeodactylum triangularis: The enabling plasmid pPha-J47869-EG01992-eGFP-FSP was transformed into competent cells, and after incubation, the cells were spread onto LB solid culture medium; after visible colonies were formed, the colonies were picked up and transferred to LB liquid culture medium for shaking culture; the transformed competent cells were collected, the enabling plasmid pPha-J47869-EG01992-eGFP-FSP was extracted, and the cells were transformed into Phaeodactylum triangularis by electroporation; (2) Screening of positive transformants: The transformed P. tricornutum cells were spread on a solid f / 2 plate medium containing bleomycin and placed under a light intensity of 50-70 µmol photons m -2 s -1 , culture under the condition of temperature of 20-24°C, screen the positive transformation columns, and obtain the genetically engineered algae with controllable flocculation.

7. A use of the controllable flocculation genetically engineered algae according to claim 1 in preparing a microalgae flocculation system, characterized in that The microalgae flocculation system is an algae liquid added with white carbon black, and the amount of white carbon black added is 0.5-1.5 g per liter of triangular brown finger algae culture liquid.

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