Method for constructing recombinant genetic transformation vector of abcg22 gene to improve plant absorption and accumulation of pfoa

Abstract

The present application relates to the technical field of agricultural environmental pollutant control, more particularly, it relates to a method for constructing a recombinant genetic transformation vector of ABCG22 gene, thereby increasing the absorption and accumulation of emerging persistent organic pollutants PFOA by plants.The nucleotide sequence of the ABCG22 gene is shown as SEQ ID NO.1, and the ABCG22 gene can promote the absorption of PFOA by plants.The AtABCG22 gene of the plant is amplified to obtain a complete gene sequence of ABCG22 protein, and the complete gene sequence of the ABCG22 protein is used for absorbing and transporting PFOA.These results clarify the mechanism of the key gene in the process of absorbing and transporting PFOA by plants from the cell level, and realize the remediation and removal of PFOA.

Description

Technical Field

[0001] This invention relates to the field of agricultural environmental pollutant control technology, and more specifically, to a method for constructing a recombinant genetic transformation vector of the ABCG22 gene to enhance the absorption and accumulation of PFOA in plants. Background Technology

[0002] Perfluoroalkyl acids (PFAAs), due to their unique hydrophobic and oleophobic properties and extreme chemical stability, are widely used in various commercial products and industrial fields. However, this also leads to their large-scale release into the environment, making them a globally significant emerging persistent organic pollutant. PFAAs have half-lives in the human body that can last for years or even more than 10 years, causing various forms of toxicity and even inducing cancer. PFAAs can enter farmland soil in large quantities through various pathways, including atmospheric deposition (both dry and wet), agricultural use of sewage sludge, surface runoff, and groundwater infiltration. It is important to note that PFAAs, especially their representative compound perfluorooctanoic acid (PFOA), have a unique anionic structure and high water solubility, making them easily absorbed and accumulated by crops, particularly leafy vegetables. Consuming agricultural products has become one of the important pathways for human exposure to ionic PFOA and other compounds.

[0003] Perfluorooctanoic acid (PFOA), a typical perfluorinated compound, has extremely high carbon-fluorine bond energies in its molecular structure, making it difficult to decompose effectively through biological or chemical pathways in the natural environment. Therefore, it persists in soil (and is even considered a permanent compound), posing a long-term threat to ecosystems and human health. Currently, remediation technologies for PFOA-contaminated soil still face significant challenges: while traditional microbial remediation methods are widely used in the treatment of organic pollutants, there is a lack of microbial strains capable of efficiently degrading PFOA; the few reported anaerobic degrading bacteria are not only demanding in terms of conditions and limited in efficiency, but also difficult to adapt to actual soil environments, failing to meet the needs of engineered remediation. Meanwhile, although phytoremediation technology has achieved success in the remediation of heavy metal pollution, the lack of plant resources in nature with a significant capacity to accumulate PFOA has prevented this technology from achieving a breakthrough in the treatment of such organic pollutants. Therefore, developing a phytoremediation system capable of actively and efficiently absorbing and accumulating PFOA has become an urgent technological need to solve this soil pollution problem. Summary of the Invention

[0004] This invention provides a method for constructing a recombinant genetic transformation vector of the ABCG22 gene (a member of the ATP-binding box protein G family gene, which has functions such as resisting biotic and abiotic stresses and controlling nutrient intake) to absorb perfluorooctanoic acid (PFOA). The method utilizes genetic engineering techniques to regulate the absorption and transport of PFOA by protoplast separation and transient gene expression in lettuce, thereby increasing its accumulation in lettuce.

[0005] In a first aspect, the present invention provides an application of the ABCG22 gene in the absorption and enrichment of PFOA in plants through multiple transmembrane structures. The nucleotide sequence of the ABCG22 gene is shown in SEQ ID NO.1, and the ABCG22 gene has the function of improving the enrichment of PFOA in plants.

[0006] Preferably, the amino acid sequence of the protein encoded by the ABCG22 gene is shown in SEQ ID NO.2.

[0007] Preferably, the AtABCG22 gene of the plant is amplified to obtain the complete gene sequence of the synthesized ABCG22 protein, and the complete gene sequence of the ABCG22 protein is used to absorb and enrich PFOA.

[0008] Preferably, the ABCG22 protein interacts with the PFOA, enabling the protein to transport PFOA across the membrane.

[0009] Preferably, the plant is a dicotyledonous plant, preferably lettuce or Arabidopsis thaliana.

[0010] Secondly, the present invention provides a method for constructing a recombinant genetic transformation vector for the ABCG22 gene to absorb PFOA, comprising:

[0011] Obtain the gene sequence of the ABCG22 gene;

[0012] The gene sequence of the ABCG22 gene was identified and arranged to obtain candidate sgRNA target sites;

[0013] The off-target sites of the predicted candidate sgRNA target sites are calculated to obtain target sgRNA-1 and target sgRNA-2;

[0014] The target sgRNA-1 and target sgRNA-2 were amplified respectively to obtain the amplified products;

[0015] The amplified product was used to construct a vector to obtain a plasmid for the ABCG22 overexpression vector.

[0016] Preferably, the sequences of target sgRNA-1 and target sgRNA-2 are as follows:

[0017] Target sgRNA-1: 5'-ATGTCAATGGAGAAGCCACCTT-3';

[0018] Target sgRNA-2: 5'-TTATGTTACGATCTTCATTTGCCTTA-3'.

[0019] Preferably, the plasmid of the ABCG22 overexpression vector is transformed into protoplasts and incubated with different concentrations of PFOA, wherein the concentrations of PFOA are 1, 2.5, 5, 10, 15, and 20 mg / L.

[0020] Preferably, the competitive agent ABA reduces the absorption and enrichment of PFOA by ABCG22 protein through competitive action, further confirming the absorption and transport function of PFOA by ABCG22 protein.

[0021] Thirdly, the present invention provides a plant that has been constructed with high PFOA enrichment, in which the ABCG22 gene is expressed for the purpose of remediating environmental pollution.

[0022] In summary, the present invention has the following beneficial effects:

[0023] This invention constructs a highly efficient protoplast isolation and transient expression system by improving key conditions in the protoplast isolation and transfection process of lettuce. This system provides a powerful and multifunctional technique for the analysis of plant gene and protein functions. By optimizing the conditions of the polyethylene glycol (PEG)-mediated transient expression system in plants, highly efficient transfection of the target gene into lettuce protoplasts was achieved, with a transfection rate of up to 85%. Using the constructed lettuce protoplast isolation and transient expression cells, the key gene ABCG22 was found to be located in the cell membrane. Overexpression of this gene in lettuce cells or Arabidopsis thaliana cells increased the uptake and accumulation of PFOA in both. These results elucidate the mechanism of the ABCG22 gene in the process of PFOA uptake and translocation in plants at the cellular and plant levels, and may serve as a feasible means to improve crop uptake and accumulation of PFOA for soil remediation.

[0024] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit the scope of protection of the present invention. Attached Figure Description

[0025] Figure 1 The protein structure prediction of ABCG22 in this embodiment of the invention is shown in (a) Arabidopsis thaliana and (b) lettuce.

[0026] Figure 2 This is a diagram showing the sequence alignment results of Arabidopsis thaliana and lettuce ABCG22 protein in an embodiment of the present invention.

[0027] Figure 3 This is a clustering analysis diagram of the ABCG22 amino acid sequences among different species in an embodiment of the present invention.

[0028] Figure 4 This is a graph showing the accumulation of PFOA after overexpression of the ABCG22 gene in lettuce protoplasts in an embodiment of the present invention.

[0029] Figure 5 This is a diagram showing the growth of Arabidopsis thaliana plants in MS medium at different PFOA concentrations in this embodiment of the invention.

[0030] Figure 6 This is a biomass diagram of ABCG22 overexpressing Arabidopsis thaliana plants treated with different concentrations of PFOA in this embodiment of the invention.

[0031] Figure 7 This is a graph showing the PFOA content of Arabidopsis thaliana plants overexpressing ABCG22 under different concentrations of PFOA treatment in this embodiment of the invention.

[0032] Figure 8 This is a correlation analysis diagram of biomass and PFOA content in ABCG22 overexpressing plants in this embodiment of the invention.

[0033] Figure 9 This is a graph showing the gene expression levels of Arabidopsis ABCG22 overexpressing plants under different PFOA concentration treatments in this embodiment of the invention.

[0034] Figure 10 This is a graph showing the PFOA uptake content in the aboveground parts of Arabidopsis thaliana overexpressed with ABCG22 in an embodiment of the present invention.

[0035] Figure 11 This is a graph showing the PFOA uptake in the roots of Arabidopsis thaliana overexpressed with ABCG22 in an embodiment of the present invention.

[0036] Figure 12 This invention describes the change in PFOA content in Arabidopsis thaliana plants when ABA is present (Col-0 is the wild type, and ABCG22 is the overexpression type of this gene). Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the embodiments. It should be noted that: unless otherwise specified, the conditions in the following embodiments are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from commercially available sources.

[0038] The amino acid sequence of the ABCG22 protein was obtained from NCBI, and its transmembrane domains were predicted using the NCBI website, BioEdit, GeneDoc, and other software for sequence alignment.

[0039] Example

[0040] Example 1

[0041] Construction of a recombinant genetic transformation vector for the wild-type Arabidopsis ABCG22 gene

[0042] Using wild-type Arabidopsis thaliana as material, Arabidopsis thaliana plants were obtained by disinfecting and planting seeds. RNA was extracted and reverse transcribed to synthesize cDNA, which was then temporarily stored in a -20°C freezer.

[0043] Download AtABCG22 from the NCBI website. Based on the restriction endonuclease sites in the sequence and the multiple cloning site information in the expression vector pET-28a(+), add 15 bp homologous arms of restriction enzyme sites to the 5' ends of the upstream and downstream primers. The restriction enzyme sites are determined as follows: AtABCG22-ORF-1-EcoRI-F / AtABCG22-ORF-2253-HindIII-R. Using Arabidopsis cDNA as a template, amplify the ORF sequence of the ABCG22 gene. Primers are shown in the table below. Take 15 μL of PCR amplification product for electrophoresis to detect whether the band size meets the expectations. Use the Tiangen gel extraction kit to extract the PCR product of the determined target fragment.

[0044] Primers for fragment amplification of target expression

[0045] ABCG22-FatgggtcgcggatccgaattcATGTCAATGGAGAAGCCACCTT

[0046] ABCG22-RctcgagtgcggccgcaagcttTTATGTTACGATCTTCATTTGCCTTA

[0047] Note: F is the upstream primer, and R is the downstream primer.

[0048] Example 2

[0049] ABCG22 protein function and structure prediction:

[0050] The presence of transmembrane structures in proteins is often closely related to their function. Structural predictions of ABCG22 proteins from lettuce and Arabidopsis thaliana show that AtABCG22 and LsABCG22 proteins possess ABC-type transporter activity and are located on the cell membrane. AtABCG22 protein has a four-transmembrane structure, while LsABCG22 protein has a five-transmembrane structure. Figure 1 As shown.

[0051] The results of the sequence alignment of the ABCG22 protein between Arabidopsis thaliana and lettuce are as follows: Figure 2 As shown, the similarity of ABCG22 between the two is 70.2%, indicating that the two proteins have high homology and strong functional conservation.

[0052] Cluster analysis of ABCG22 amino acid sequences among the same species, such as Figure 3As shown in the phylogenetic tree, the key proteins in Arabidopsis and lettuce have high homology and may have the same biological functions. This provides a feasible guarantee for subsequent research on Arabidopsis using model plants to reveal the functional mechanism of key genes mediating the absorption and transport of PFOA in plants (lettuce).

[0053] Example 3

[0054] Analysis of key gene expression levels and PFOA content

[0055] Using lettuce cDNA as a template and pCHF1 as a vector, the full-length coding sequence of the ABCG22 gene was cloned using a high-fidelity enzyme. Based on this, a recombinant genetic transformation vector for this gene, pCHF1-KpnI-1-LsABCG22ORF-2220-BamHI, was constructed. Protoplasts were extracted from lettuce shoot tips, and the plasmid of the overexpression vector was transfected into cells using a protoplast transient transfection system. Untransfected cells served as a control group. After incubation at 25 °C for 14 h, RNA was extracted, and cDNA was obtained through reverse transcription. The gene expression level was detected using quantitative real-time PCR.

[0056] To investigate the mechanism of PFOA uptake mediated by key genes in protoplasts, lettuce protoplast cells were obtained using the isolation method described above. The plasmid of the ABCG22 overexpression vector was transformed into protoplasts and incubated for 14 h. After treatment with different concentrations of PFOA, the PFOA content in the supernatant and cells was measured. Seven PFOA concentration gradients were set up: blank control group (no PFOA), 1 mg / L PFOA group, 2.5 mg / L PFOA group, 5 mg / L PFOA group, 10 mg / L PFOA group, 15 mg / L PFOA group, and 20 mg / L PFOA group. All concentrations were final PFOA concentrations. Each experimental group had three replicates. After culturing at 25 ℃ for 24 h, PFOA was extracted from cells and supernatant using the method described above. Origin 2021 software was used to fit a curve, with the PFOA content in the supernatant as the x-axis and the PFOA content in the cells as the y-axis.

[0057] Experimental results:

[0058] After transfecting the plasmid of the LsABCG22 gene into protoplasts, the results of quantitative real-time PCR are as follows: Figure 4As shown, compared with the control group, the expression levels of key genes in the transfection group (overexpression group) were significantly increased (p < 0.05). The detection of PFOA content directly reflects the level of PFOA uptake by cells, showing that the PFOA content in the transfection group cells was higher than that in the control group, and the uptake process conformed to the Michaelis-Menten equation. The Km value in the Michaelis-Menten equation is mainly related to the plant's affinity for the substance and its uptake efficiency. The smaller the Km value, the higher the uptake rate at a lower substrate concentration, which usually means that the plant has high uptake efficiency and selectivity. The Vmax value mainly reflects the plant's maximum uptake rate. The Km values ​​of PFOA uptake in the transfection group cells were significantly lower than those in the untransfected protoplasts (p < 0.05), indicating that the overexpressing protoplasts could achieve a higher uptake rate at a lower PFOA concentration, exhibiting high affinity and selectivity for PFOA. The Vmax of the LsABCG22 gene was significantly higher than that of the untransfected protoplasts (p < 0.05), indicating that this gene can promote the accumulation of PFOA in cells and can act as a protein carrier in the uptake and transport of PFOA. This result reveals the functional mechanism of this gene in transporting PFOA at the cellular level.

[0059] Example 4

[0060] Obtaining and cultivating transgenic Arabidopsis thaliana:

[0061] Using the LsABCG22 gene cDNA from lettuce plants as a template and pCHF1 as a vector, the full-length coding sequence of the gene was cloned using a high-fidelity enzyme. Based on this, a recombinant genetic transformation vector was constructed. This vector was transformed into Agrobacterium, and after infecting wild-type Arabidopsis plants, transgenic plants were obtained. Homozygous plants were screened and identified, ultimately yielding homozygous Arabidopsis plants overexpressing OE-ABCG22. Homozygous seeds were then collected for further plant experiments.

[0062] Select an appropriate amount of Arabidopsis thaliana seeds (wild-type Col-0, OE-ABCG22) into 1.5 mL centrifuge tubes and sterilize them with 10% sodium hypochlorite (NaClO, v / v) for 10 min, continuously shaking the tubes during this period to ensure thorough sterilization. Then sterilize twice with 75% ethanol for 1 min each time, followed by washing 4-5 times with sterile water for 1 min each time. Vernalize the seeds at 4℃ for 2-3 days. Prepare 1 / 2 MS solid medium, sterilize at high temperature, and pour plates in a laminar flow hood, ensuring uniform medium thickness. Suspend the vernalized seeds in sterile water in a laminar flow hood and evenly place them on 1 / 2 MS medium. Blow until watermarks disappear, then seal the plates and incubate for 15 days to obtain Arabidopsis thaliana seedlings. The incubator temperature was 25℃, the light conditions were 18 h light and 6 h darkness, and the humidity was 60%. Select healthy seedlings and transplant them into vermiculite for two weeks, watering them with a 1 / 2 nutrient solution during this period. When the roots reach 3-5 cm in length and are relatively robust, transplant them into hydroponic pots and cultivate them in a greenhouse at 25 ℃. Change the nutrient solution every 7 days, with the formula shown in Table 1.

[0063] Table 1 Formulation Composition

[0064]

[0065] Phenotypic analysis experiments:

[0066] To investigate the changes in the physiological and biochemical properties of Arabidopsis thaliana overexpressing plants (OE-ABCG22) under PFOA treatment, Arabidopsis thaliana seeds were grown in disposable sterile culture dishes, and seed germination and plant growth under PFOA stress were observed. Five treatment groups were set up: a control group, and 0.2, 1, 5, and 10 mg / L PFOA treatment groups. Seeds of both overexpressing and wild-type plants were sterilized using the above-mentioned sterilization method and vernalized at 4 ℃ for 2-3 days. The PFOA stock solution (100 mg / L) was filtered into sterile 50 mL centrifuge tubes using a disposable sterile filter in a laminar flow hood. MS medium was prepared, sterilized at high temperature, and then, after cooling to approximately 60 ℃, sterilized PFOA solution was added to prepare MS medium with the corresponding PFOA concentration. Quickly pour the mixture into petri dishes. After the plates solidify and dry, evenly spot 8 seeds from both overexpression and wild-type plants onto the sides of the petri dish, ensuring the seeds are in a straight line. After the moisture has evaporated, seal the petri dishes and place them in a 25°C incubator to allow the seeds to grow upright for approximately 10 days. Once the Arabidopsis plants have grown three leaves, photograph and observe the growth of the overexpression and wild-type plants under different concentrations of PFOA treatment. Collect Arabidopsis plants of the same strain from the same plate, weigh them, and measure the plant biomass. Extract PFOA from the samples using ultrasonic extraction. Simultaneously, extract RNA from different strains of Arabidopsis plants to detect the expression levels of related genes, using the same experimental methods as described above. Set up three replicates for each gene and each concentration. As shown in Table 2.

[0067] Table 2 Primer Table

[0068]

[0069] Example 5

[0070] Substrate competition experiment:

[0071] ABCG22 transporter is a specific protein for abscisic acid (ABA). In this experiment, 100 μg / L ABA was used as a substrate competitor for ABCG22 transporter to treat OE-ABCG22-overexpressing Arabidopsis thaliana to detect PFOA uptake. Wild-type (Col-0) Arabidopsis thaliana plants of uniform size and growth were selected and transferred to hydroponic pots containing fresh nutrient solution. Inhibitors were added to the treatment groups, and PFOA was added to the hydroponic pots to a concentration of 1 mg / L after 30 min. The control group was treated with PFOA only. After 6 h of exposure, samples were taken and washed with tap water and deionized water, respectively. The aboveground and underground parts were then separated using a scalpel. 1 g of aboveground and underground tissues were weighed separately, and the fresh samples were ground with a steel ball. PFOA was extracted using the above-mentioned method. Each treatment was performed in triplicate.

[0072] Main results:

[0073] (1) Phenotypic analysis:

[0074] Arabidopsis seeds grown in media with different concentrations of PFOA showed varying phenotypes. For example... Figure 5 As shown, root growth in both wild-type and overexpressing plants was inhibited with increasing PFOA concentration. Wild-type plants showed less impact from PFOA, with only lateral root growth inhibited; taproot growth remained largely unchanged compared to the control group. However, the ABCG22 overexpressing plants exhibited more significant root changes. Compared to both the control and wild-type plants, both taproot and lateral root growth were significantly inhibited, and the germination rate of the overexpressing plants was also affected. At a PFOA concentration of 10 mg / L, the average germination rate was only 50%. This result may be due to the overexpressing plants absorbing more PFOA, thus affecting seed germination and root growth.

[0075] The biomass of overexpressing plants and wild-type plants was measured separately, and the results are as follows: Figure 6 As shown, with the increase of PFOA concentration, the biomass of overexpressing plants decreased significantly compared with wild-type plants (p < 0.05). This indicates that overexpression of the ABCG22 gene can enable plants to absorb more PFOA, thereby inhibiting the plant's own growth. This confirms the functional mechanism of the above key genes mediating the absorption and transport of PFOA in plants at the seedling level.

[0076] The enrichment coefficients of Arabidopsis thaliana plants are shown in the table below. For the same Arabidopsis thaliana variety, the enrichment coefficients decreased with increasing PFOA concentration. Under PFOA stress, the enrichment coefficients of overexpressing plants were significantly higher than those of wild-type plants (p < 0.05), indicating that the ABCG22 transporter protein plays an important role in the absorption and accumulation of PFOA in Arabidopsis thaliana. With increasing PFOA concentration, the enrichment coefficients decreased, indicating saturation of the transport process and a decline in the ability of Arabidopsis thaliana to transport PFOA. The results are shown in Table 3.

[0077] Table 3. PFOA enrichment coefficient (BCF) of ABCG22 overexpression lines

[0078]

[0079] Note: Uppercase letters indicate differences between different strains of Arabidopsis thaliana, while lowercase letters indicate differences between Arabidopsis thaliana strains at different concentrations.

[0080] To verify the above experimental results, the PFOA content absorbed by the plants was detected using ultrasonic extraction. The results are as follows: Figure 7As shown, under different concentrations of PFOA treatment, the PFOA uptake of overexpressing plants was significantly higher than that of wild-type plants (p < 0.05).

[0081] Correlation analysis was performed on plant biomass and PFOA content, and the results are as follows: Figure 8 As shown, the biomass of ABCG22 overexpressing plants was negatively correlated with the PFOA uptake (p < 0.05), meaning that the lower the biomass, the higher the PFOA content in the plant, indicating that the overexpressing plants absorbed more PFOA during growth, which led to the inhibition of Arabidopsis thaliana growth.

[0082] Simultaneously, the relative expression levels of key genes in overexpressed and wild-type plants were detected, and the results were as follows: Figure 9 As shown, the gene expression levels in ABCG22-overexpressing plants were significantly higher than those in wild-type plants, but these levels were not correlated with PFOA concentration. These results indicate that the ABCG22 gene plays a crucial role in the uptake and accumulation of PFOA in crops, providing a basis for further research on gene action mechanisms.

[0083] Example 6

[0084] Analysis of PFOA content and physiological and biochemical indicators in mature Arabidopsis thaliana plants overexpressing PFOA:

[0085] Arabidopsis thaliana plants overexpressing PFOA were treated with 0.2, 0.5, and 1 mg / L PFOA, and the results were as follows: Figure 10 and 11 As shown in Table 4, compared with wild-type Arabidopsis (Col-0) plants, the PFOA uptake in both the aboveground and underground parts of the overexpressing Arabidopsis plants was increased, and the results were significantly different in the 0.5 and 1 mg / L PFOA treatment groups (p < 0.05). The PFOA translocation coefficients in Arabidopsis are shown in Table 4. Under different PFOA concentrations, the translocation coefficients of the ABCG22 overexpressing plants were significantly increased (p < 0.05), indicating that this gene plays an important role in the uptake and translocation of PFOA. Note: Different letters in the figure indicate significant differences (p < 0.05).

[0086] Table 4. PFOA transport coefficient (TF) in Arabidopsis thaliana overexpressed with ABCG22

[0087]

[0088] Note: Uppercase letters indicate significance analysis of translocation coefficients in overexpression lines of different genes, while lowercase letters indicate significance analysis of translocation coefficients in Arabidopsis thaliana at different concentrations in the same line.

[0089] Example 7

[0090] Analysis of the effect of the competing agent (ABA) on the absorption of PFOA in Arabidopsis thaliana

[0091] ABA is a specific substrate of ABCG22. For example... Figure 12 As shown, the presence of ABA significantly reduced PFOA accumulation in both wild-type and ABCG22 gene-overexpressing plants (p < 0.05), indicating that ABA can competitively reduce the uptake and transport of PFOA by the ABCG22 protein. This result further demonstrates the important role of the ABCG22 gene in the uptake and transport of PFOA in plants.

[0092] The above description is merely an exemplary embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The application of the ABCG22 gene in the absorption and enrichment of PFOA in plants through multiple transmembrane structures, characterized in that... The nucleotide sequence of the ABCG22 gene is shown in SEQ ID NO.

1. The ABCG22 gene has the function of increasing the accumulation of PFOA in plants.

2. The application of the ABCG22 gene according to claim 1 in the absorption and enrichment of PFOA in plants through multiple transmembrane structures, characterized in that, The amino acid sequence of the protein encoded by the ABCG22 gene is shown in SEQ ID NO.

2.

3. The application of the ABCG22 gene according to claim 1 in the absorption and enrichment of PFOA in plants through multiple transmembrane structures, characterized in that, The AtABCG22 gene of a plant was amplified to obtain the complete gene sequence of the synthesized ABCG22 protein, which was used to absorb and enrich PFOA.

4. The application of the ABCG22 gene according to claim 1 in the absorption and enrichment of PFOA in plants through multiple transmembrane structures, characterized in that, The ABCG22 protein interacts with the PFOA, enabling the protein to transport PFOA across the membrane.

5. The application of the ABCG22 gene according to claim 1 in the absorption and enrichment of PFOA in plants through multiple transmembrane structures, characterized in that, The plant is a dicotyledonous plant, preferably lettuce or Arabidopsis thaliana.

6. A method for constructing a recombinant genetic transformation vector for the ABCG22 gene to absorb PFOA, characterized in that, include: Obtain the gene sequence of the ABCG22 gene; The gene sequence of the ABCG22 gene was identified and arranged to obtain candidate sgRNA target sites; The off-target sites of the predicted candidate sgRNA target sites are calculated to obtain target sgRNA-1 and target sgRNA-2; The target sgRNA-1 and target sgRNA-2 were amplified respectively to obtain the amplified products; The amplified product was used to construct a vector to obtain a plasmid for the ABCG22 overexpression vector.

7. The method for constructing a recombinant genetic transformation vector for the ABCG22 gene to absorb PFOA according to claim 6, characterized in that, The sequences of the target sgRNA-1 and target sgRNA-2 are as follows: Target sgRNA-1: 5'-ATGTCAATGGAGAAGCCACCTT-3'; Target sgRNA-2: 5'-TTATGTTACGATCTTCATTTGCCTTA-3'.

8. The method for constructing a recombinant genetic transformation vector for the ABCG22 gene to absorb PFOA according to claim 6, characterized in that, The plasmid of the ABCG22 overexpression vector was transformed into protoplasts and Arabidopsis plants and incubated with different concentrations of PFOA, namely 1, 2.5, 5, 10, 15, and 20 mg / L.

9. The method for constructing a recombinant genetic transformation vector for the ABCG22 gene to absorb PFOA according to claim 6, characterized in that, The inhibitor and the competitor ABA reduce the uptake and accumulation of PFOA by ABCG22 protein through competitive action.

10. A plant that has been constructed to highly enrich PFOA, characterized in that, The ABCG22 gene of claim 1 is expressed in plants to enhance the absorption and accumulation of PFOA, thereby achieving environmental pollution remediation.

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