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, The functional silica-binding peptide) has the ability to efficiently and specifically bind to silica, and can be expressed on the outer side of Phaeodactylum tricornutum cells by fusion expression with EG01992. The J47869 promoter can exhibit 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 genetically engineered algae with controllable flocculation, including the following steps: Step 1: Using the genomic DNA of Phaeodactylum tricornutum as a template, amplify the Phatr3_J47869 promoter, and replace the FcpA promoter of the basic expression plasmid pPha-T1-eGFP to construct an inducible expression plasmid pPha-J47869-eGFP; Step 2: Using the genomic DNA of Phaeodactylum tricornutum as a template, amplify the Phatr3_EG01992 coding sequence, and insert it upstream of eGFP in the inducible expression vector to construct a directional expression plasmid pPha-J47869-EG01992-eGFP; Step 3: Artificially synthesize the codon-optimized FSP coding sequence, and insert it downstream of eGFP in the directional expression vector to construct an enabling plasmid pPha-J47869-EG01992-eGFP-FSP; Step 4: Electrotransform the enabling plasmid pPha-J47869-EG01992-eGFP-FSP into Phaeodactylum tricornutum, coat the transformed Phaeodactylum tricornutum cells on an f / 2 plate medium for culture, and screen for positive algal strains to obtain genetically engineered algae with controllable flocculation.

[0008] Furthermore, step 1 is specifically as follows: (1) Design a forward amplification primer containing an NdeI restriction site and a reverse amplification primer containing an EcoRI restriction site according to the Phatr3_J47869 promoter sequence, perform PCR amplification using the Phaeodactylum tricornutum DNA as a template to obtain the Phatr3_J47869 promoter amplification product. 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)Use NdeI and EcoRI restriction endonucleases to excise the original FcpA promoter of pPha-T1-eGFP; ligate the amplification product of the Phatr3_J47869 promoter with the enzyme-digested product of pPha-T1-eGFP to construct the inducible expression plasmid pPha-J47869-eGFP.

[0009] Furthermore, step 2 is specifically as follows: (1)Design forward and reverse amplification primers containing KpnI restriction sites according to the Phatr3_EG01992 coding sequence, and perform PCR amplification using the DNA of Phaeodactylum tricornutum as a template to obtain the amplification product of the Phatr3_EG01992 coding sequence, where The nucleotide sequence of the forward amplification primer for the Phatr3_EG01992 coding sequence is shown in SEQ ID NO.6: 5’-GACTAATTCGAGCTCGGTACCATGAGGCTACGTTCATCCATTACC-3’, and the nucleotide sequence of the reverse amplification primer for the Phatr3_EG01992 coding sequence is shown in SEQ ID NO.7: 5’-TCTAGAGGATCCCCGGGTACCACGGCAAAAGATTCCAAAACG-3’; (2)Use KpnI restriction endonuclease to digest the pPha-J47869-eGFP plasmid, and ligate the amplification product of the Phatr3_EG01992 coding sequence with the digested pPha-J47869-eGFP to construct the directional expression plasmid pPha-J47869-EG01992-eGFP.

[0010] Furthermore, step 3 is specifically as follows: (1)Synthesize double-stranded template: Optimize the codons of the coding sequence of the FSP short peptide to obtain the nucleotide sequence of the FSP coding gene as shown in SEQ ID NO.9: 5’-ATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3’. Design the forward and reverse synthesis primers of the FSP double-stranded according to the FSP coding sequence and the flexible Linker coding sequence, and synthesize the two synthesis primers into double-stranded nucleotides as the PCR amplification template by means of an annealing reaction. Among them, the nucleotide sequence of the FSP double-stranded forward synthesis primer is as shown in SEQ ID NO.10: 5’-GGCGGCGGTGGTAGCGGTGGCGGCGGTTCCATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3’, and the nucleotide sequence of the FSP double-stranded reverse synthesis primer is as shown in SEQ ID NO.11: 5’-TTACCGCACAGCAGCCGACATGAGGTCGCTGCGGTGCATGGAACCGCCGCCACCGCTACCACCGCCGCC-3’; (2)Design the forward and reverse amplification primers of the FSP according to the XbaI restriction site sequence and the FSP coding sequence, and use the double-stranded nucleotides obtained in step (1) as the template for PCR amplification to obtain the amplification product with the FSP coding sequence, the corresponding restriction site and the Linker coding sequence. Among them, 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)Use the XbaI restriction endonuclease to digest the pPha-J47869-EG01992-eGFP plasmid, and ligate the FSP amplification product with the digested pPha-J47869-EG01992-eGFP to construct the enabling plasmid pPha-J47869-EG01992-eGFP-FSP, whose nucleotide sequence is as shown in SEQ ID NO.1.

[0011] Further, step 4 is specifically as follows: (1)Phaeodactylum tricornutum transformation: Transform the enabling plasmid pPha-J47869-EG01992-eGFP-FSP into competent cells. After incubation, spread them onto LB solid medium. When visible colonies form, pick the colonies and culture them in LB liquid medium on a shaker. Collect the transformed competent cells, extract the enabling plasmid pPha-J47869-EG01992-eGFP-FSP, and transform it into Phaeodactylum tricornutum by electroporation. (2)Screening of positive transformants: Spread the transformed Phaeodactylum tricornutum cells onto a solid f / 2 plate medium containing bleomycin, and culture them under the conditions of light intensity 50 - 70 µmol photons m -2 s -1 -2 and temperature 20 - 24 °C to screen positive transformants, that is, to obtain the genetically engineered algae with controllable flocculation.

[0012] The present invention also provides the application of the above-mentioned genetically engineered algae with controllable flocculation in the preparation of a microalgae flocculation system. The microalgae flocculation system is an algal solution added with silica white, and the addition amount of silica white is 0.5 - 1.5 g per liter of Phaeodactylum tricornutum culture solution.

[0013] Compared with the prior art, the advantages of the present invention are as follows: For the genetically engineered algae with controllable flocculation and its construction method and application of the present invention, the amino acid sequence MHRSDLMSAAVR (abbreviated as FSP) shows high affinity for silica white. Expressing this fragment on the outer side of microalgae cells can endow microalgae with the ability to efficiently bind silica white. The Phatr3_EG01992 gene encodes a protein located on the outer side of microalgae cells. Fusing the expression of this protein with FSP can achieve the directional expression of FSP on the outer side of microalgae cells. During the cultivation of microalgae, as the cell density increases, the nutrient content in the culture system will rapidly decrease. When the cell density reaches the level that can be collected, the nutrients in the culture system are usually exhausted. Placing Phatr3_EG01992 and FSP under the control of a promoter induced by a low nutrient salt concentration can synchronize the timing of the directional expression of FSP on the outer side of microalgae cells with the optimal collection timing of microalgae. Placing the Phatr3_EG01992-FSP fusion gene under the regulation of the Phatr3_J47869 promoter can efficiently couple the enabling process of FSP with the cultivation and collection links of microalgae. Adding silica white to the culture system, since the density of silica white is much greater than that of water, the microalgae cells combined with silica white can quickly settle to the bottom of the water, realizing the efficient collection of microalgae cells.

[0014] In summary, for the genetically engineered microalgae with controllable flocculation, its construction method and application, by utilizing the ability of FSP to specifically and efficiently bind to silica white, the property that Phatr3_EG01992 is located outside the cell, and the characteristic that the promoter activity of Phatr3_J47869 is regulated by phosphate concentration, the microalgae species are genetically improved. The microalgae germplasm modified by this strategy can use silica white as a flocculant to construct an efficient and low-energy-consuming flocculation system, thus providing a new idea for breaking through the pain points in the microalgae industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shows the change in fluorescence intensity of eGFP under different phosphate concentration conditions after placing the green fluorescent protein (eGFP) coding sequence under the control of the Phatr3_J47869 promoter; Figure 2 Is the electrophoresis pattern for amplifying the Phatr3_J47869 promoter, where lane 1: 5000 bp Marker, lanes 2 - 3: Amplification results of the Phatr3_J47869 promoter; Figure 3 Is the electrophoresis pattern for amplifying the Phatr3_EG01992 coding sequence, where lane 1: 2000 bp Marker, lanes 2 - 3: Amplification results of the Phatr3_EG01992 coding sequence; Figure 4 Shows the screening results of positive transformants of Phaeodactylum tricornutum using the plate method with antibiotics; Figure 5 Is the sequencing result after amplifying the DNA of the transformed algal strain using specific primers based on the eGFP coding sequence; Figure 6 Shows the comparative analysis result of the transcription of Phatr3_EG01992 in wild strains, induced plasmid pPha-J47869-eGFP transformed strains, and enabling plasmid pPha-J47869-EG01992-eGFP-FSP transformed strains by real-time quantitative PCR; Figure 7 Is the comparative analysis result of eGFP signals in wild strains, induced plasmid pPha-J47869-eGFP transformed strains, and enabling plasmid pPha-J47869-EG01992-eGFP-FSP transformed strains by laser confocal microscopy; Figure 8 Is the comparative analysis diagram of the binding of Phaeodactylum tricornutum cells with wild strains and silica white based on the enabling plasmid pPha-J47869-EG01992-eGFP-FSP. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] The present invention will be further described in detail below in conjunction with the embodiments with the accompanying drawings.

[0017] Microalgae: *Phaeodactylum tricornutum*; Basic plasmid: pPha-T1-eGFP; Amplification kit: 2×Phanta Flash Master 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 f / 2 medium prepared 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); The microalgae culture equipment was: Zhichu ZQZY-88CGES constant temperature shaking incubator; Microalgae culture conditions: light intensity 60 µmol photons m -2 s -1 , temperature 22 °C, and shaker speed 90 rpm.

[0018] For different genetic transformation systems, different promoters, different localization tags, different functional peptide segments of each microalgae, and the corresponding affinity materials, they can all be used for the microalgae germplasm transformation based on the cell enabling strategy and the subsequent construction of the flocculation system mentioned in this patent. This specific embodiment only lists the construction of the genetically engineered *Phaeodactylum tricornutum* with the *Phaeodactylum tricornutum* as the research material, using the electroporation method, with FSP as the enabling factor, realizing induced enabling with the Phatr3_J47869 promoter, and using Phatr3_EG01992 as the localization tag to introduce the enabling plasmid pPha-J47869-EG01992-eGFP-FSP. With silica white as the flocculation medium, a corresponding flocculation collection system was established. 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 the inducible expression plasmid pPha-J47869-eGFP.

[0020] The activity of the Phatr3_J47869 gene promoter is regulated by phosphate concentration and exhibits high transcriptional activity under low phosphate conditions, as Figure 1 shown. Therefore, by controlling the expression of Phatr3_EG01992 and the enabling factor FSP through the Phatr3_J47869 promoter, the directed expression of FSP outside the microalgae cells can be 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 the genomic DNA of Phaeodactylum tricornutum: Collect the Phaeodactylum tricornutum algal solution in the logarithmic phase (cell density is 5×10 6 cells / mL) and extract the genomic DNA; Step 2: PCR amplification: According to the Ensembl Protists database, search for the upstream sequence of the Phatr3_J47869 open reading frame, and predict its promoter sequence with the help of Promoter2.0 to obtain the nucleotide sequence of the Phatr3_J47869 promoter as shown in SEQ ID NO.2. Design a forward amplification primer containing an NdeI restriction site and a reverse amplification primer containing an EcoRI restriction site according to the Phatr3_J47869 promoter sequence. The nucleotide sequence of the Phatr3_J47869 promoter forward amplification primer is as shown in SEQ ID NO.3: 5’-GTACTGAGAGTGCACCATATGTGGTGGTGAATCAACACTTAATGTG-3’, and the nucleotide sequence of the Phatr3_J47869 promoter reverse amplification primer is as shown in SEQ ID NO.4: 5’-TTAGTCGATGATATCGAATTCCGAAGAATTCTTTTCACCAGGG-3’; The PCR amplification system is: 0.5 μL of Phaeodactylum tricornutum DNA, 10 μL of 2×Phanta Flash Master Mix, 0.5 μL each of the Phatr3_J47869 promoter forward and reverse amplification primers, and 8.5 μL of ddH2O; The PCR amplification program is: 94℃ for 5 min; 94℃ for 30 sec, 68℃ for 30 sec, 72℃ for 1 min, cycle 35 times, and store at 4℃; Step 3: Verification of the PCR amplification product: After the amplification is completed, separate and verify the PCR product by agarose gel electrophoresis. The results are as Figure 2 shown. Lane 1 is the DNA Marker; Lanes 2 and 3 are both the amplified Phatr3_J47869 promoter sequences, and then cut the gel and recover and sequence them. The length of the amplified fragment is consistent with the theoretical length; Step 4: Use NdeI and EcoRI restriction endonucleases to excise the original FcpA promoter of pPha-T1-eGFP; ligate the amplified product of the Phatr3_J47869 promoter obtained in Step 3 with the digested product of pPha-T1-eGFP, and react at 37 °C for 30 min to construct the inducible expression plasmid pPha-J47869-eGFP.

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

[0022] The protein encoded by Phatr3_EG01992 is located outside the microalgae cell. Fusing this protein with FSP can achieve the directional expression of FSP outside the microalgae cell. The construction method of the directional expression plasmid pPha-J47869-EG01992-eGFP includes the following steps: Step 1: Extract the genomic DNA of Phaeodactylum tricornutum: Collect the Phaeodactylum tricornutum algal solution in the logarithmic phase (cell density is 5×10 6 cells / mL), and extract the genomic DNA; Step 2: PCR amplification: According to the Ensembl Protists database, search for the open reading frame sequence of Phatr3_EG01992 to obtain the nucleotide sequence encoding Phatr3_EG01992 as shown in SEQ ID NO.5. Design forward and reverse amplification primers containing KpnI restriction sites according to the Phatr3_EG01992 coding sequence. The nucleotide sequence of the forward amplification primer of the Phatr3_EG01992 coding sequence is as 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 as shown in SEQ ID NO.7: 5’-TCTAGAGGATCCCCGGGTACCACGGCAAAAGATTCCAAAACG-3’; The PCR amplification system is: 0.5 μL of Phaeodactylum 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 is: 94 °C for 5 min; 94 °C for 30 sec, 68 °C for 30 sec, 72 °C for 1 min, cycle 35 times, and store at 4 °C; Step 3, Identification of PCR amplification products: After amplification, the PCR products are separated and verified by agarose gel electrophoresis. The results are as Figure 3 shown. Lane 1 is the DNA Marker, and lanes 2 and 3 are the amplified coding regions of Phatr3_EG01992. Subsequently, gel cutting and recovery are performed followed by sequencing; the length of the amplified fragment is consistent with the theoretical length.

[0023] Step 4, Construction of the directional expression vector: The pPha-J47869-eGFP plasmid is digested with KpnI restriction endonuclease, and the amplified product of the Phatr3_EG01992 coding sequence obtained in Step 3 is ligated to the digested pPha-J47869-eGFP and reacted at 37 °C for 30 min to construct the directional expression plasmid pPha-J47869-EG01992-eGFP.

[0024] Specific Example 3, Construction of the enabling plasmid pPha-J47869-EG01992-eGFP-FSP.

[0025] The amino acid sequence of the FSP peptide is as shown in SEQ ID NO.8: MHRSDLMSAAVR, which has a high and specific affinity for silica. If this peptide is directionally expressed on the outer side of microalgal cells, it can endow microalgal cells with the ability to efficiently bind silica. Since the density of silica is much greater than that of water, microalgal cells bound with silica can quickly settle to the bottom of the water. Through this strategy, the microalgal germplasm can use silica as a flocculant to construct an efficient flocculation system, providing a new solution for the collection of microalgal cells. The construction method steps of the enabling plasmid pPha-J47869-EG01992-eGFP-FSP are as follows: Step 1, Synthesize double-stranded template: Optimize the codons of the coding sequence of the FSP short peptide to obtain the nucleotide sequence encoding FSP as shown in SEQ ID NO.9: 5’-ATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3’. Design the forward and reverse synthesis primers for the FSP double-stranded based on the FSP coding sequence and the coding sequence of the flexible Linker (the nucleotide sequence of the flexible Linker is shown in SEQ ID NO.18: GGGGSGGGGS), and synthesize the two synthesis primers into double-stranded nucleotides as the PCR amplification template by means of annealing reaction. Among them, 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.11: 5’-TTACCGCACAGCAGCCGACATGAGGTCGCTGCGGTGCATGGAACCGCCGCCACCGCTACCACCGCCGCC-3’; Step 2, Design the forward and reverse amplification primers for FSP according to the XbaI restriction site sequence and the FSP coding sequence. Among them, the nucleotide sequence of the FSP forward amplification primer is shown in SEQ ID NO.12: 5’-GACGAGCTGTACAAGTCTAGGGCGGCGGTGGTAGCGGT-3’, and the nucleotide sequence of the FSP reverse amplification primer is shown in SEQ ID NO.13: 5’-TGCCTGCAGGTCGACTCTAGATTACCGCACAGCAGCCGA-3’; The PCR amplification system is: 0.5 μL of double-stranded nucleotides, 10 μL of 2×PrimeSTAR Max Premix, 0.5 μL each of the forward and reverse amplification primers for the FSP gene, and 8.5 μL of ddH2O; The PCR program is: 94°C for 5 min; 94°C for 30 sec, 62°C for 30 sec, 72°C for 1 min, cycle 35 times, and store at 4°C.

[0026] Step 3, Verification of the PCR amplification product: After the amplification is completed, the PCR product is separated by agarose gel electrophoresis, followed by gel cutting and recovery and sequencing. The sequencing result is consistent with the FSP theoretical coding sequence; Step 4. Construction of enabling plasmid: The 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. 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 electrotransformed into Phaeodactylum tricornutum. The specific steps are as follows: The pPha-J47869-EG01992-eGFP-FSP plasmid constructed in Specific Example 3 was transformed into Phaeodactylum tricornutum by electrotransformation. The transformed Phaeodactylum tricornutum cells were spread on an f / 2 plate medium, and positive algal strains were screened and identified to obtain genetically engineered algae with controllable flocculation. The specific steps are as follows: Step 1. Transformation of Phaeodactylum tricornutum: The enabling plasmid pPha-J47869-EG01992-eGFP-FSP was transformed into competent cells. After incubation, the cells were spread on an LB solid medium. When visible colonies formed, the colonies were picked into an LB liquid medium and cultured in a constant temperature shaking incubator at 37°C. The transformed competent cells were collected, and the pPha-J47869-EG01992-eGFP-FSP plasmid was extracted and then transformed into Phaeodactylum tricornutum by electrotransformation. The electrotransformation parameters were: 500 V, 25 μF, 400 Ω; Step 2. Screening of positive transformants: The transformed Phaeodactylum tricornutum cells were spread on an f / 2 plate medium containing 1.0% agar with a bleomycin concentration of 75 μg / mL and cultured under the conditions of a light intensity of 60 µmol photons m -2 s -1 , a temperature of 22°C. After about 3 weeks, the successfully transformed strains formed algal colonies on the f / 2 plate. The results are as Figure 4 shown; Visible algal colonies were picked into a liquid f / 2 medium with a bleomycin concentration of 75 μg / mL and cultured for 1 week to obtain a Phaeodactylum tricornutum algal solution. The DNA of the algal solution was extracted as an identification template; Step 3, Identification of positive transformants: Identification primers were designed based on the eGFP coding sequence. The nucleotide sequence of the forward identification primer for the algal solution is as shown in SEQ ID NO.14: 5’-GACGACGGCAACTACAAGAC-3’, and the nucleotide sequence of the reverse identification primer for the algal solution is as shown in SEQ ID NO.15: 5’-CGAACTCCAGCAGGACCAT-3’; The PCR amplification system was: 0.5 μL of Phaeodactylum tricornutum DNA, 10 μL of 2×Phanta Flash Master Mix, 0.5 μL 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, with 35 cycles, and stored at 4°C. The PCR products were separated by agarose gel electrophoresis and then sequenced. As Figure 5 shown, the Sanger sequencing results showed that the amplified fragment was derived from the eGFP coding region of the enabling plasmid, indicating that the enabling plasmid had been successfully transformed into Phaeodactylum tricornutum.

[0028] Specific Example Five, Verification of positive transformants of Phaeodactylum tricornutum.

[0029] 1. Verification at the transcriptional level: Total RNA of each strain of Phaeodactylum tricornutum was extracted and reverse transcribed to obtain cDNA. Using the cDNA as a template, the transcriptional status of Phatr3_EG01992 in the wild strain and positive transformants was compared and analyzed by real-time quantitative PCR.

[0030] Real-time quantitative PCR forward and reverse primers were designed based on the Phatr3_EG01992 sequence. The nucleotide sequence of the real-time quantitative PCR forward primer is as shown in SEQ ID NO.16: 5’-CGAGAACCGAAGTGACCG-3’; the nucleotide sequence of the real-time quantitative PCR reverse primer is as shown in SEQ ID NO.17: 5’-ACCAGCACAACCAGGGAC-3’; The real-time quantitative PCR system: 0.5 μL of Phaeodactylum tricornutum cDNA, 10 μL of 2×PhantaQuantiNova SYBR Green PCR Master Mix, 0.5 μL each of the real-time quantitative PCR forward and reverse primers, 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, with 40 cycles. As Figure 6As shown, in the enabling plasmid transformant, the transcriptional abundance of Phatr3_EG01992 is several times higher than that of the wild strain (negative control 1) and the induced expression plasmid pPha-J47869-eGFP transformant (negative control 2), indicating that the target nucleotide sequence is successfully transcribed in the enabling plasmid transformant.

[0031] 2. Verification at the protein level: After culturing each strain of Phaeodactylum tricornutum to the logarithmic growth phase (cell density is about 5×10 6 cells / mL), with the aid of a laser confocal microscope, the position and intensity of the eGFP signal in the cells of each strain of Phaeodactylum tricornutum were detected and analyzed by comparison under the conditions of excitation and emission wavelengths of 488 nm and 507 nm respectively. As Figure 7 shown, in the Phaeodactylum tricornutum strain transformed with the enabling plasmid, the green fluorescence signal was accurately located outside the cell; in the wild strain (negative control 1) cells, no eGFP signal was observed; while in the strain transformed with the induced expression plasmid pPha-J47869-eGFP (negative control 2), its eGFP signal was located in the cytoplasm.

[0032] In summary, after identification at the transcriptional 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 outside the cells of Phaeodactylum tricornutum.

[0033] Specific Example Six: Verification of the high-efficiency flocculation effect based on the positive transformant of Phaeodactylum tricornutum.

[0034] Culturing Phaeodactylum tricornutum: Inoculate the positive transformant of Phaeodactylum tricornutum and the wild strain (negative control) at the same initial density into the liquid f / 2 medium (cell density is about 1×10 6 cells / mL), and place them in a constant-temperature light shaker for culturing; the culturing conditions are light intensity of 60 µmol photons m -2 s -1 , temperature of 22°C, and rotation speed of 90 rpm.

[0035] After culturing to the plateau phase (cell density is about 4×10 7 cells / mL), add silica white to the algal solution (the addition ratio of silica white is 1 g of silica white per liter of Phaeodactylum tricornutum culture solution), continue to place the algal solution in a constant-temperature light shaker for culturing for 24 hours, and then let it stand for 5 minutes. As Figure 8 shown, the positive transformant of Phaeodactylum tricornutum will quickly settle to the bottom of the culture container due to binding a large amount of silica white, achieving efficient and low-energy consumption collection; however, the cells of the wild strain of Phaeodactylum tricornutum still uniformly disperse in the algal solution because they lack the ability to bind silica white, and no collection is achieved.

[0036] The above description is not a limitation of the present invention, nor is the present invention limited to the above examples. Changes, modifications, additions or substitutions made by those of ordinary skill in the art within the scope of the essence of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A genetically engineered alga with controllable flocculation, characterized in that: The genetically engineered alga is Phaeodactylum tricornutum into which the enabling plasmid pPha-J47869-EG01992-eGFP-FSP has been introduced. The FSP is directionally expressed outside the Phaeodactylum tricornutum cells. The nucleotide sequence of the enabling plasmid pPha-J47869-EG01992-eGFP-FSP is shown in SEQ ID NO.

1.

2. The construction method of the genetically engineered alga with controllable flocculation according to claim 1, characterized in that It includes the following steps: Step 1: Using the Phaeodactylum tricornutum genomic DNA 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 Phaeodactylum tricornutum genomic DNA as a template, amplify the Phatr3_EG01992 coding sequence, and insert it upstream of eGFP in the inducible expression vector to construct the directionally expressed plasmid pPha-J47869-EG01992-eGFP; Step 3: Artificially synthesize the codon-optimized FSP coding sequence, and insert it downstream of eGFP in the directionally expressed vector to construct the enabling plasmid pPha-J47869-EG01992-eGFP-FSP; Step 4: Electrotransform the enabling plasmid pPha-J47869-EG01992-eGFP-FSP into Phaeodactylum tricornutum, spread the transformed Phaeodactylum tricornutum cells on the f / 2 plate medium for culture, and screen for positive algal strains to obtain the genetically engineered alga with controllable flocculation.

3. The construction method of a genetically engineered alga with controllable flocculation according to claim 2, characterized in that Step 1 is specifically as follows: (1) Design a forward amplification primer containing an NdeI restriction site and a reverse amplification primer containing an EcoRI restriction site according to the Phatr3_J47869 promoter sequence, perform PCR amplification using the Phaeodactylum tricornutum DNA as a template to obtain the Phatr3_J47869 promoter amplification product. 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) Use NdeI and EcoRI restriction endonucleases to excise the original FcpA promoter of pPha-T1-eGFP; ligate the Phatr3_J47869 promoter amplification product with the digested product of pPha-T1-eGFP to construct the inducible expression plasmid pPha-J47869-eGFP.

4. The construction method of a genetically engineered alga with controllable flocculation according to claim 2, characterized in that Step 2 is specifically as follows: (1)Design forward and reverse amplification primers containing KpnI restriction sites according to the Phatr3_EG01992 coding sequence. Using Phaeodactylum tricornutum DNA as a template, perform PCR amplification to obtain the amplification product of the Phatr3_EG01992 coding sequence, where 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’; (2)Use KpnI restriction endonuclease to digest the pPha-J47869-eGFP plasmid, and ligate the amplification product of the Phatr3_EG01992 coding sequence with the digested pPha-J47869-eGFP to construct the directional expression plasmid pPha-J47869-EG01992-eGFP.

5. The construction method of a genetically engineered alga with controllable flocculation according to claim 2, characterized in that Step 3 is specifically as follows: (1)Synthesize double-stranded template: Optimize the codons of the coding sequence of the FSP short peptide to obtain the nucleotide sequence of the FSP coding gene as shown in SEQ ID NO.9: 5’-ATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3’. Design forward and reverse synthesis primers for the FSP double-stranded according to the FSP coding sequence and the flexible Linker coding sequence, and synthesize the two synthesis primers into double-stranded nucleotides as the PCR amplification template through an annealing reaction. Among them, the nucleotide sequence of the forward synthesis primer of the FSP double-stranded is shown in SEQID NO.10: 5’-GGCGGCGGTGGTAGCGGTGGCGGCGGTTCCATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3’, and the nucleotide sequence of the reverse synthesis primer of the FSP double-stranded is shown in SEQ ID NO.11: 5’-TTACCGCACAGCAGCCGACATGAGGTCGCTGCGGTGCATGGAACCGCCGCCACCGCTACCACCGCCGCC-3’; (2) Design forward and reverse amplification primers for FSP according to the XbaI restriction site sequence and the FSP coding sequence, and use the double-stranded nucleotide obtained in step (1) as a template for PCR amplification to obtain an amplification product with the FSP coding sequence, the corresponding restriction site, and the Linker coding sequence. The nucleotide sequence of the FSP forward amplification primer is shown in SEQ ID NO.12: 5’-GACGAGCTGTACAAGTCTAGGGCGGCGGTGGTAGCGGT-3’, and the nucleotide sequence of the FSP reverse amplification primer is shown in SEQ ID NO.13: 5’-TGCCTGCAGGTCGACTCTAGATTACCGCACAGCAGCCGA-3’; (3) Use XbaI restriction endonuclease to digest the plasmid pPha-J47869-EG01992-eGFP, and ligate the FSP amplification product with the digested pPha-J47869-EG01992-eGFP to construct the enabling plasmid pPha-J47869-EG01992-eGFP-FSP, whose nucleotide sequence is shown in SEQ ID NO.

1.

6. The construction method of a genetically engineered alga with controllable flocculation according to claim 2, characterized in that Step 4 is specifically as follows: (1) Transformation of Phaeodactylum tricornutum: Transform the enabling plasmid pPha-J47869-EG01992-eGFP-FSP into competent cells. After incubation, spread them onto the LB solid medium; wait for visible colonies to form, pick the colonies into the LB liquid medium for shaking culture; collect the transformed competent cells, extract the enabling plasmid pPha-J47869-EG01992-eGFP-FSP, and transform it into Phaeodactylum tricornutum by electroporation; (2)Screening of positive transformants: The transformed Phaeodactylum tricornutum cells were spread on a solid f / 2 plate medium containing bleomycin and cultured under the conditions of light intensity of 50 - 70 µmol photons m -2 s -1 -2 and temperature of 20 - 24 °C to screen positive transformants, and thus the genetically engineered algae with controllable flocculation were obtained.

7. Use of the genetically engineered alga with controllable flocculation according to claim 1 in the preparation of a microalga flocculation system, characterized in that The microalgae flocculation system is the algal solution added with silica white, and the addition amount of the silica white is 0.5 - 1.5 g per liter of Phaeodactylum tricornutum culture solution.

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