Phaeodactylum tricornutum strain with high As (V) tolerance and construction method and application of phaeodactylum tricornutum strain

Abstract

The invention discloses a phaeodactylum tricornutum strain with high As (V) tolerance as well as a construction method and application thereof, and belongs to the technical field of genetic engineering. The invention provides a phaeodactylum tricornutum strain with high As (V) tolerance. According to the invention, a PtPHT gene of phaeodactylum tricornutum is knocked out to construct a phaeodactylum tricornutum strain with high As (V) tolerance. The invention provides the application of the phaeodactylum tricornutum strain with high As (V) tolerance in As (V) sewage treatment, the strain has a better growth state under the condition of high-concentration As (V), and a high-quality engineering strain is provided for As water pollution treatment, so that the treatment process is more environment-friendly, and the cost is lower.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a Phaeodactylum tricornutum strain with high tolerance to As (V), a construction method thereof and application. BACKGROUND

[0002] Arsenic (As) is a highly toxic metalloid substance, and high concentration of arsenic or high level of arsenic can cause a series of acute and chronic human health problems, including skin, lung, bladder, liver and kidney cancer, and negative effects on cardiovascular system, nervous system, hematology, kidney, reproduction and respiratory system. Due to overexploitation of arsenic-containing mineral resources, and large use of pesticides and herbicides, a large amount of arsenic enters water bodies, and at present, drinking water in 107 countries and regions in the world is threatened by arsenic pollution, among which the arsenic content of drinking water of about 20 million people in China exceeds the standard, and arsenic pollution has become one of the most serious pollutants that endanger the ecological environment and the health of residents. The biological remediation technology has the advantages of low cost and environmental friendliness. Among them, the purification technology of arsenic contaminated water based on microalgae has become a new research hotspot. Microalgae have good biodegradation and transformation effect on water heavy metal and organic pollutant such as arsenic, and microalgae contain intracellular and extracellular binding sites for metal ions, thereby effectively removing water heavy metal pollutants. However, the natural microalgae has low tolerance to high arsenic and low processing efficiency, and the intracellular arsenate transport mechanism of microalgae is not clear.

[0003] Therefore, how to develop a Phaeodactylum tricornutum mutant strain with high arsenic tolerance so as to make the process of treating arsenic contaminated water more environmentally friendly and lower in cost, and promote the analysis of arsenic absorption and transport mechanism in microalgae, has become an important topic to be solved at present. SUMMARY

[0004] In view of this, the purpose of the present application is to provide a Phaeodactylum tricornutum strain with high tolerance to As (V), a construction method thereof and application in treating As (V) contaminated water.

[0005] In order to achieve the above purpose, the present application provides the following technical scheme: In a first aspect, the present application provides a Phaeodactylum tricornutum strain with high tolerance to As (V), which is a Phaeodactylum tricornutum with PtPHT gene knocked out, and the nucleotide sequence of the PtPHT gene is shown as SEQ ID NO. 1.

[0006] The Phaeodactylum tricornutum is a wild type Phaeodactylum tricornutum strain, CCMP2561, which is purchased from the National Center for Marine Algae and Microbiota, USA.

[0007] In a second aspect, the present application provides a method for constructing a Phaeodactylum tricornutum strain with high tolerance to As (V), wherein the strain is a Phaeodactylum tricornutum strain with high tolerance to As (V), and the method comprises the following steps: S1, designing and synthesizing a gRNA of the PtPHT gene; S2, constructing a pKS diaCas9_sgRNA-PtPHT knockout vector; S3, transforming the knockout vector containing the gRNA into the Phaeodactylum tricornutum; S4, culturing the Phaeodactylum tricornutum; S5, screening the Phaeodactylum tricornutum with complete knockout of the PtPHT gene, obtaining the strain and naming it as PtPHT-KO.

[0008] According to the above technical solution, further, the sequence of the gRNA is 5'-TATCTGGTCGGAACACGAGG-3'.

[0009] According to the above technical solution, further, the knockout vector is constructed by subcloning the gRNA into a pKS diaCas9_sgRNA backbone plasmid through a Bsa I restriction site.

[0010] According to the above technical solution, further, the S3 is realized by a transgenic method of electroporation, and the electroporation refers to an electroporation method.

[0011] According to the above technical solution, further, the culture condition of the S4 is static culture using F / 2 medium in a constant temperature; light and dark cycle culture, light intensity is 80 µmol m -2 s -1 during light period, culture temperature is 20℃; dark light continuous light culture is adopted in a recovery stage after knockout, light intensity is 30 µmol m -2 s -1 .

[0012] According to the above technical solution, further, the screening method comprises microalgae clone PCR, gene fragment recovery, sequencing and TA cloning.

[0013] In a third aspect, the present application provides the use of the above-mentioned Phaeodactylum tricornutum strain with high tolerance to As (V) in treating As (V) sewage.

[0014] Compared with the prior art, the present application has the following beneficial effects: 1. The present application provides a Phaeodactylum tricornutum strain with high tolerance to As (V).

[0015] 2. The application constructs a Phaeodactylum tricornutum strain with high As (V) tolerance by knocking out the PtPHT gene of the Phaeodactylum tricornutum.

[0016] 3. The application provides an application of the Phaeodactylum tricornutum strain with high As (V) tolerance in treating As (V) sewage, and the strain has a better growth state under a high-concentration As (V) condition, thereby providing a high-quality engineering strain for treating As-containing water pollution, so that the treatment process is more environmentally friendly and has a lower cost. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the application, the drawings involved in the embodiments will be briefly introduced below.

[0018] Figure 1 It is a schematic diagram for constructing the Phaeodactylum tricornutum PtPHT knockout vector of the application. Figure 2 It is a specific gene sequence diagram of the Phaeodactylum tricornutum PtPHT-KO strain of the application. Figure 3 It is a specific gene sequence diagram of the Phaeodactylum tricornutum PtPHT-KO strain of the application. Figure 4 It is an absorption test diagram of the PtPHT-KO strain of the application in Example 3 at a concentration gradient of 5 ppb and 50 ppb on arsenic-containing water. Figure 5 It is an absorption test diagram of the PtPHT-KO strain of the application in Example 3 at a concentration gradient of 500 ppb and 5000 ppb on arsenic-containing water. DETAILED DESCRIPTION

[0019] The application will be described in detail below with reference to the embodiments, but the implementation manner of the application is not limited thereto, and obviously, the embodiments described below are only some of the embodiments of the application, and for those skilled in the art, other similar embodiments obtained without creative labor fall within the protection scope of the application.

[0020] Example 1 This embodiment provides a construction process of the PtPHT-KO strain.

[0021] 1. Design and synthesize the gRNA of the PtPHT gene: The PtPHT gene sequence was obtained from the Ensembl Protists database (https: / / protists.ensembl.org) and uploaded to an online website (http: / / www.rgenome.net / cas-designer / ) for gRNA design. The final gRNA sequence was determined to be 5'-TATCTGGTCGGAACACGAGG-3'. The designed gRNA sequence was then synthesized by BioGen (Shanghai Sangon Biotech Co., Ltd.).

[0022] 2. Construct the pKS diaCas9_sgRNA-PtPHT knockout vector: The pKS diaCas9_sgRNA-PtPHT knockout vector was constructed by subcloning the pKS diaCas9_sgRNA backbone plasmid through the Bsa I restriction site.

[0023] The nucleotide sequence of the PtPHT gene is shown in SEQ ID NO.1.

[0024] SEQ ID NO.1 (5'-3'):

[0025] 3. Transform the knockout vector containing gRNA into *Phaeodactylum tricornutum*: Transgenic methods using electroporation were employed in *Phaeodactylum tricornutum* with cell densities of 4-5 × 10⁻⁶. 6 When the concentration is 10 cells / mL, centrifuge at 4℃ and 3000 g for 3 min, and collect 2 × 10⁻⁶ cells / mL. 8 Cells were then washed twice with 1 mL of 375 mM sorbitol (sorbitol must be sterile and pre-frozen). Finally, the cells were resuspended in 100 μL of sorbitol to a final concentration of 2 × 10⁻⁶ cells. 9 Cells / mL. Take 100 μL of the suspension, mix with 4 μg plasmid DNA and 40 μg salmon sperm DNA (denatured by boiling for 1 min), transfer to a 2 mm electroporation cuvette, and incubate on ice for 10 min. Then perform electroporation using the Bio-Rad Gene Pulser Xcell electroporation system. The electroporation system was adjusted to exponential decay, 0.5 kV field strength, 25 μF capacitance, and 400 ohm shunt resistance. Immediately after electroporation, transfer the cells to a 15 mL Erlenmeyer flask containing 10 mL f / 2 medium and culture overnight under low light (~30 μmol photons). -2 s -1 Incubate the cells overnight without shaking. On the second day, centrifuge the cells at 3000 g for 3 min, resuspend them in 300 μL of f / 2 medium, and transfer 150 μL to a solid medium containing 100 μg / mL zeocin. The basal medium for the solid medium is f / 2 medium.

[0026] Table 1 shows the components and preparation method of the f / 2 culture medium: .

[0027] 4. Cultivation of *Phaeodactylum tricornutum*: The constructed pKS diaCas9_sgRNA-PtPHT knockout vector and PUC57 resistance vector were transformed into wild-type *Phaeodactylum tricornutum* strains via electroporation, and then inoculated onto bleomycin (100 μg / mL) plates. The plates were then incubated under continuous low light (30 µmol / mL). -2 s -1 Incubate at 20°C for one week, then transfer to continuous high-intensity light (80 µmol / m²). -2 s -1 Cultured under [a specific environment]. After approximately 21 days, antibiotic-resistant monoclonal algal strains can be observed.

[0028] 5. Screening for *Phododendron triangularis* strains with complete PtPHT gene knockout yielded the PtPHT-KO strain: (1) Clonal PCR analysis and screening: PCR primer names and sequences: PtPHT-cF: 5'-TATCTGGTCGGAACACGAGG-3'; PtPHT-cR: 5'-TATCTGGTCGGAACACGAGG-3'; The obtained resistant monoclonal algal strains were analyzed by clonal PCR, gel recovery and sequencing to obtain possible PtPHT-KO algal strains.

[0029] The clonal PCR analysis specifically involved taking an appropriate amount of *Phaeodactylum tricornutum* (5 × 10⁻⁶). 6 Add 100 cells to a PCR tube containing 50 μL of 10mM EDTA (pH=8.0) solution, vortex to mix (5-10 s), and incubate at 100℃ for 8-10 min. After incubation, immediately remove the tube and place it on ice to cool. After cooling, vortex vigorously again for 5-10 s, and then centrifuge at 14000 rpm for 1 min. Take 1 μL of the supernatant after centrifugation as the PCR template.

[0030] The specific steps for the recovery and purification of PCR target fragments on agar are as follows: After PCR, run the gel at 120V for 25 min in an electrophoresis apparatus. After the gel run, identify the target band to be recovered. Cut off the target band and recover it into a 1.5 mL centrifuge tube. Add 3-6 times the weight of the gel block to the centrifuge tube and incubate at 50℃ for 5-10 min to dissolve the gel. After the gel is completely dissolved, transfer the dissolve to an adsorption column and centrifuge at 8000 ×g for 30 s. Discard the liquid in the collection tube. Add 500 µL of WashSolution and centrifuge at 9000 ×g for 30 s. Discard the liquid in the collection tube. Repeat the previous step. Centrifuge the empty adsorption column at 9000 ×g for 2 min. Place the adsorption column into a clean 1.5 mL centrifuge tube, add 20 µL of deionized water to the center of the adsorption membrane, let it stand at room temperature for 2 min, and then centrifuge for 2 min. Preserve the DNA solution in the tube. Sequencing the recovered product.

[0031] (2) TA clone screening: TA cloning further confirmed the gene sequence of the knockout mutant: the gene fragments of the isolated monoclonal algal strains that may have had gene knockout were recovered, and the recovered gene fragments were ligated with the pGEM-T vector using T4 ligase. Then, they were transformed into DH5α Escherichia coli, and plasmids were extracted from multiple monoclonal strains and sent for sequencing to further determine the knockout status of PtPHT and obtain the specific gene sequence.

[0032] Example 2 This embodiment uses the PtPHT-KO algal strain obtained in Example 1 as the object to verify As (V) tolerance.

[0033] Wild-type *Phaeodactylum tricornutum* WT and PtPHT-KO strains (named 11-4, 11-5, and 11-11) were cultured in F / 2 medium using an arsenic standard solution concentration gradient of 1 ppm, 5 ppm, 10 ppm, 20 ppm, and 40 ppm. The initial cell concentration was set at 2 × 10⁻⁶. 6 The cells / mL concentration was approximately [number] cells / mL, and the cells were cultured until the logarithmic growth phase. For each treatment, three biological replicates and three technical replicates were performed (n=9). Experimental results are as follows Figure 3 As shown, the results indicate that: Under high concentration As(V) culture conditions, the specific growth rate of PtPHT-KO was significantly higher than that of the wild type, and the maximum biomass was also increased compared to the wild type.

[0034] The PtPHT-KO algal strain obtained in this embodiment has higher tolerance to As(V) pollution than its parent strain and can grow better in high-concentration As(V) environments.

[0035] Example 3 This embodiment uses the PtPHT-KO algal strain obtained in Example 1 as the subject to test the absorption of As (V) polluted water.

[0036] PtPHT-KO algal strains (named 11-4, 11-5, and 11-11) were cultured in F / 2 medium using a concentration gradient of arsenic standard solution (As(V)). The concentration gradients were 5 ppb, 50 ppb, 500 ppb, and 5000 ppb. The initial cell concentration was set at 2 × 10⁻⁶ cells / year. 6 The cells / mL concentration was approximately [number], and samples were collected after 10 days of culture. For each treatment, three biological replicates and three technical replicates were performed (n=9).

[0037] Experimental results are as follows Figure 4 and 5 As shown, the results indicate that under high concentration As(V) culture conditions, the PtPHT-KO algal strain exhibits good absorption of As(V) and significant enrichment effect.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A highly As(V)-tolerant strain of *Phaeodactylum tricornutum*, characterized in that, The algal strain is *Phaeodactylum triangularis* with the PtPHT gene knocked out, and the nucleotide sequence of the PtPHT gene is shown in SEQ ID NO.

1.

2. A method for constructing a highly As(V)-tolerant *Phaeodactylum tricornutum* strain, wherein the strain is the highly As(V)-tolerant *Phaeodactylum tricornutum* strain described in claim 1, characterized in that... Includes the following steps: S1, Design and synthesize the gRNA of the PtPHT gene; S2, construct the pKS diaCas9_sgRNA-PtPHT knockout vector; S3, the knockout vector containing the gRNA is transferred into the brown finger algae; S4, culture the aforementioned *Phaeodactylum tricornutum*; S5, screen for the *PtPHT* gene completely knocked out in the *PtPHT* strain, obtain the strain and name it PtPHT-KO.

3. The method for constructing a highly As(V)-tolerant *Phaeodactylum tricornutum* strain according to claim 2, characterized in that, The sequence of the gRNA is 5'-TATCTGGTCGGAACACGAGG-3'.

4. The method for constructing a highly As(V)-tolerant *Phaeodactylum tricornutum* strain according to claim 2, characterized in that, The knockout vector was constructed by subcloning the gRNA into the pKS diaCas9_sgRNA backbone plasmid via the Bsa I restriction site.

5. The method for constructing a highly As(V)-tolerant *Phaeodactylum tricornutum* strain according to claim 2, characterized in that, The S3 is achieved through transgenic transformation via electroporation, where electroporation refers to the electroporation method.

6. The method for constructing a highly As(V)-tolerant *Phaeodactylum tricornutum* strain according to claim 2, characterized in that, The culture conditions for S4 were as follows: static culture in F / 2 medium at a constant temperature; light-dark cycle culture; and a light intensity of 80 µmol / m² during the light period. -2 s -1 The culture temperature was 20℃; during the recovery phase after knockout, culture was conducted under continuous illumination in the dark with a light intensity of 30 µmol / m². -2 s -1 .

7. The method for constructing a highly As(V)-tolerant *Phaeodactylum tricornutum* strain according to claim 2, characterized in that, The screening methods include microalgae cloning PCR, gene fragment recovery, sequencing, and TA cloning.

8. The application of the As(V)-tolerant Brownia delta strain as described in claim 1 in the treatment of As(V) wastewater.

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