Method for improving arsenic enrichment capacity of plants and application of method in repairing arsenic-polluted soil

Abstract

The invention discloses a method for improving the arsenic enrichment capacity of plants and application of the method in repairing arsenic-polluted soil. The application is as follows. The key amino acid rare mutation of the pteris vittata phosphorus transporter is applied to other non-arsenic hyperaccumulator plants, so that the arsenic absorption and transport efficiency of the plants is remarkably improved, the arsenic enrichment capacity is enhanced, the remediation process of arsenic-polluted soil is accelerated, the stable remediation effect can be kept in long-term application, and the method has a wide application prospect. The method is suitable for large-scale popularization and application, and an efficient, environment-friendly and sustainable new solution is provided for phytoremediation of the arsenic-polluted soil.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for enhancing the arsenic accumulation capacity of plants and its application in the remediation of arsenic-contaminated soil. Background Technology

[0002] Arsenic (As) is a highly toxic metallic element, and along with lead, chromium, cadmium, and mercury, it is considered one of the five major toxins in environmental pollution. Traditional arsenic pollution remediation technologies are not only costly but also prone to causing secondary pollution. Phytoremediation technology, due to its low-carbon and environmentally friendly advantages, holds promise for widespread application.

[0003] *Pteris vittata* L. was the first discovered arsenic hyperaccumulator, exhibiting highly efficient arsenic absorption and translocation capabilities and extremely strong arsenic tolerance. It can accumulate up to 22,600 mg / kg (dry weight) of arsenic and can grow normally in arsenic-contaminated soils with concentrations as high as 1,500 mg / kg, making it a primary material for phytoremediation technologies. Currently, several other *Pteris* species have also been identified as arsenic hyperaccumulators. However, these existing arsenic hyperaccumulators have low biomass, slow growth rates, and weak stress resistance, making large-scale application difficult.

[0004] Arsenic and phosphorus are chemical analogs, and they interact extensively during migration from soil to plants. In arsenic-contaminated soil, arsenic and phosphorus compete for absorption and translocation in plants. Plants can absorb and translocate arsenate [As(V)] from soil through a high-affinity Phosphate transporter 1st family (Pht1). The Pht1 family proteins of different plants exhibit remarkably similar structures, with interspecies homology reaching 76%, demonstrating high conservation. Studies show that compared to other plants, members of the *Pht1* family exhibit significantly stronger arsenic translocation capabilities and higher affinity for As(V). Currently, how to selectively modify fast-growing crops such as *Arabidopsis thaliana* and rice based on the *Pht1* protein family to cultivate novel plant lines with both high biomass and arsenic hyperaccumulation capacity has become a hot topic in the field of phytoremediation of arsenic-contaminated soil. Summary of the Invention

[0005] To address the problems in the prior art, this invention provides a method for improving the arsenic accumulation capacity of plants and its application in the remediation of arsenic-contaminated soil.

[0006] The first objective of this invention is to provide a method for improving the arsenic accumulation capacity of plants.

[0007] A second objective of this invention is to provide the application of plants obtained by the method in the remediation of arsenic-contaminated soil.

[0008] A third objective of this invention is to provide a biomaterial.

[0009] A fourth objective of this invention is to provide the application of the aforementioned biomaterials in enhancing the arsenic accumulation capacity of plants and / or remediating arsenic-contaminated soils.

[0010] To achieve the above objectives, the present invention is implemented through the following solution: Arsenic enrichment refers to the process by which plants absorb arsenic from arsenic-contaminated soil through their roots and then transport and accumulate it in their above-ground parts (stems, leaves, etc.). The stronger a plant's arsenic enrichment capacity, the higher its efficiency in extracting arsenic from the soil, and the more suitable it is for soil remediation.

[0011] This invention, through amino acid sequence alignment analysis of Pht1 protein from multiple species, identified a core amino acid site (G324) involved in arsenic absorption, transport, and recognition in the Pht1;5 protein sequence of the arsenic hyperaccumulator *Pteris vittata*. This site is homologous to D in many higher plants, with no sequence variation observed, except for Pht1;5, where it is G. This site has great potential for constructing arsenic hyperaccumulating engineered plants.

[0012] This invention uses phosphorus-absorbing mutant yeast as a heterologous expression system to clarify the effect of the rare G324 site of the Pht1;5 protein mutation in *Physalis alkekengi* on the arsenic transport capacity of the Pht1;4 protein in *Arabidopsis thaliana* and rice. Subsequently, point mutant plants of the model plant (*Arabidopsis thaliana*) and the important crop (rice) were constructed using genetic engineering strategies such as homologous gene substitution expression and single-base directed editing, and the changes in arsenic absorption, transport, and accumulation in these plants were evaluated. Rice, due to its flood adaptability, high biomass, and high degree of germplasm mechanization, holds promise as an important biological material for large-scale remediation of arsenic pollution in farmland, paddy fields, and drylands. Therefore, this invention seeks protection for the following: A method for improving the arsenic accumulation capacity of plants involves mutating a key amino acid site of the Pht1;4 protein of the plant from aspartic acid to glycine; if the plant is Arabidopsis thaliana, then the key amino acid site is the 309th amino acid of the Pht1;4 protein; if the plant is rice, then the key amino acid site is the 306th amino acid of the Pht1;4 protein.

[0013] Preferably, the plant is Arabidopsis thaliana, then the Pht1;4 protein is AtPht1;4 protein, and its amino acid sequence is shown in SEQ ID NO.2.

[0014] More preferably, if the plant is Arabidopsis thaliana, then the Pht1;4 protein after mutation will be a point mutation. AtPht1; 4 D309G Its amino acid sequence is shown in SEQ ID NO.11.

[0015] More preferably, the expression of the Arabidopsis Pht1;4 protein is first knocked out or inhibited, and then the expression of the Arabidopsis Pht1;4 protein D309Q mutant is reintroduced. The Arabidopsis Pht1;4 protein D309Q mutant is the Arabidopsis Pht1;4 protein in which the amino acid at position 309 is mutated from aspartic to glycine.

[0016] Preferably, the plant is rice, then the Pht1;4 protein is the OsPht1;4 protein, and its amino acid sequence is shown in SEQ ID NO.3.

[0017] More preferably, if the plant is rice, then the Pht1;4 protein after mutation is a point mutation. OsPht1;4 D306G Its amino acid sequence is shown in SEQ ID NO.12.

[0018] More preferably, the plant is rice, and the 306th amino acid of the OsPht1;4 protein is mutated from aspartic acid to glycine using single-base editing technology.

[0019] Application of plants obtained by any of the methods described in the remediation of arsenic-contaminated soil.

[0020] A biological material, wherein the biological material is any one of the following (1) to (5): (1) a Pht1;4 protein mutant, which is a D309Q mutant of Arabidopsis Pht1;4 protein or a D306Q mutant of rice Pht1;4 protein; (2) a nucleic acid molecule encoding the Pht1;4 protein mutant of (1); (3) an expression cassette containing the nucleic acid molecule of (2); (4) a recombinant expression vector containing the expression cassette of (3); (5) a microorganism containing the recombinant expression vector of (4).

[0021] Preferably, the amino acid sequence of the Arabidopsis Pht1;4 protein described in (1) is as shown in SEQ ID NO.2. More preferably, the amino acid sequence of the D309Q mutant of the Arabidopsis Pht1;4 protein described in (1) is as shown in SEQ ID NO.11. Even more preferably, the nucleotide sequence of the nucleic acid molecule described in (2) is as shown in SEQ ID NO.8.

[0022] Preferably, the amino acid sequence of the rice Pht1;4 protein described in (1) is shown in SEQ ID NO.3. More preferably, the amino acid sequence of the D306Q mutant of the rice Pht1;4 protein described in (1) is shown in SEQ ID NO.12. Even more preferably, the nucleotide sequence of the nucleic acid molecule described in (2) is shown in SEQ ID NO.9.

[0023] The present invention does not have any particular limitation on the source and type of the recombinant expression vector in (4) of the biological material. Recombinant expression vectors that can express the Pht1;4 protein mutant, such as yeast expression vectors and Agrobacterium expression vectors, can achieve the purpose of the present invention.

[0024] Preferably, the recombinant expression vector in (4) includes a yeast expression vector or an Agrobacterium expression vector. More preferably, the yeast expression vector uses the pAG426GPD-ccdB vector as its backbone. More preferably, the Agrobacterium expression vector uses the pCAMBIA1305 vector as its backbone.

[0025] The present invention does not specifically limit the source and type of microorganisms in (5) of the biological material, including but not limited to yeast, Escherichia coli, Agrobacterium, and other conventional microorganisms that can carry the recombinant expression vector to achieve the purpose of the present invention. Preferably, the microorganisms in (5) include yeast or Agrobacterium. More preferably, the yeast is the EY917 yeast mutant strain. More preferably, the Agrobacterium is Agrobacterium GV3101.

[0026] Application of any of the described biomaterials in enhancing the arsenic accumulation capacity of plants.

[0027] In some specific embodiments, the plant is rice, and the 306th amino acid of its Pht1;4 protein is mutated from aspartic to glycine using single-base editing technology. Preferably, the nucleotide sequence of the sgRNA used in the single-base editing technology is shown in SEQ ID NO.13.

[0028] Application of any of the described biomaterials in the remediation of arsenic-contaminated soil.

[0029] Application of any of the described biomaterials in enhancing the arsenic accumulation capacity of plants and remediating arsenic-contaminated soil.

[0030] Compared with the prior art, the present invention has the following beneficial effects: This invention applies a rare mutation of a key amino acid in the phosphotransporter protein of Centipede Grass to other non-arsenic hyperaccumulators, significantly improving the plant's absorption and translocation efficiency of arsenic, enhancing its arsenic accumulation capacity, helping to accelerate the remediation process of arsenic-contaminated soil, maintaining stable remediation effects in long-term application, and is suitable for large-scale promotion and application, providing a new, efficient, environmentally friendly, and sustainable solution for the phytoremediation of arsenic-contaminated soil. Attached Figure Description

[0031] Figure 1 The results of cohort analysis of key amino acid sites of Pht1 protein in centipede grass, rice, and Arabidopsis thaliana.

[0032] Figure 2 for PvPht1;5 yeast and PvPht1;5 G324D Yeast growth curve and arsenic accumulation; a is... PvPht1;5 Yeast growth curve; b is PvPht1;5 G324D Yeast growth curves; c represents arsenic accumulation in the two yeasts; -As represents yeast liquid culture medium containing 3 mM Pi; +As represents SD-U liquid culture medium containing 3 mM Pi and 1.5 mM As (V).

[0033] Figure 3 for AtPht1;4 yeast and AtPht1;4 D309G Yeast growth curve and arsenic accumulation; a is... AtPht1;4 Yeast growth curve; b is AtPht1;4 D309G Yeast growth curves; c represents the arsenic tolerance of the two yeast species; -As represents yeast liquid culture medium containing 3 mM Pi; +As represents SD-U liquid culture medium containing 3 mM Pi and 1.5 mM As (V).

[0034] Figure 4 for OsPht1;4 yeast and OsPht1;4 D306G Yeast growth curve and arsenic accumulation; a is... OsPht1;4 Yeast growth curve; b is OsPht1;4 D306G Yeast growth curves; c represents arsenic accumulation in the two yeasts; -As represents yeast liquid culture medium containing 3 mM Pi; +As represents SD-U liquid culture medium containing 3 mM Pi and 1.5 mM As (V).

[0035] Figure 5 Results of homologous recombination expression identification from different Arabidopsis thaliana plants; a is Δatpht1;4 Arabidopsis thaliana, WT Arabidopsis thaliana and 3 strains AtPht1;4 -Ex Arabidopsis thaliana ( AtPht1;4 -Ex1~ AtPht1;4 -Ex3) identification results; b is Δatpht1;4 Arabidopsis thaliana, WT Arabidopsis thaliana and 3 strains AtPht1;4 D309G -Ex Arabidopsis thaliana ( AtPht1;4 D309G -Ex1~ AtPht1;4 D309G -Ex3) identification results; ND indicates not detected.

[0036] Figure 6Arsenic accumulation in different Arabidopsis thaliana plants; a represents WT Arabidopsis thaliana, Δatpht1;4 Arabidopsis thaliana, 3 strains AtPht1;4 -Ex Arabidopsis thaliana ( AtPht1;4 -Ex1~ AtPht1;4 -Ex3) and 3 plants AtPht1;4 D309G -Ex Arabidopsis thaliana ( AtPht1;4 D309G -Ex1~ AtPht1;4 D309G Arsenic accumulation in the aboveground parts of Arabidopsis thaliana (-Ex3); b represents WT Arabidopsis thaliana, Δatpht1; 4 Arabidopsis thaliana, 3 strains AtPht1;4 -Ex Arabidopsis thaliana ( AtPht1;4 -Ex1~ AtPht1;4 -Ex3) and 3 plants AtPht1;4 D309G -Ex Arabidopsis thaliana ( AtPht1;4 D309G -Ex1~ AtPht1;4 D309G Arsenic accumulation in the roots of -Ex3);* p <0.05.

[0037] Figure 7 A map of the adenine base editing vector pH-ABE8e-NG.

[0038] Figure 8 For WT rice and 3 plants osPht1;4 D306G rice (os) pht1;4 D306G -1~os pht1;4 D306G Arsenic accumulation status (-3). Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0040] Example 1: Determination of key amino acid sites and biochemical function study of Pht1 family proteins from Centipede Grass 1. Determination of key amino acid sites in Pht1 family proteins of Centipede Grass This example uses fungi with analyzed crystal structures. Piriformospora indicaThe key amino acid residues of the phosphate transporter (PiPT) of *P. tumefaciens* are referenced (DOI: https: / / doi.org / 10.1038 / nature12042). The amino acid sequences of *PvPht1;1* (NCBI: KM192135.1), *PvPht1;2* (NCBI: KM192136.1), *PvPht1;3* (NCBI: KM192137.1), *PvPht1;4* (NCBI: MN260326.1), and *PvPht1;5* (amino acid sequences as shown in SEQ ID) are also described. As shown in NO.1), the following OsPht1;1 (NCBI: NM_001402044.1), OsPht1;2 (NCBI: NM_001402045.1), OsPht1;3 (NCBI: NM_001423046.1), OsPht1;4 (NCBI: NM_001402342.1), OsPht1;5 (NCBI: NM_001419081.1), and OsPht1;6 (N CBI: NM_001422695.1), OsPht1;7 (NCBI: NM_001402038.1), OsPht1;8 (NCBI: NM_001423048.1), OsPht1 ;9 (NCBI: OsPht1;12 (NCBI: NM_001417461.1) and OsPht1;13 (NCBI: NM_001419103.1), and AtPht1;1 (NCBI: NM_123701.4), AtPht1;2 (NCBI: NM_001203533.1), AtPht1;3 (NCBI: NM_123702.1), and AtPht1;4 (NCBI: NM_12945) of Arabidopsis thaliana. 2.4) AtPht1;5 (NCBI: NM_128843.4), AtPht1;6 (NCBI: NM_123700.1), AtPht1;7 (NCBI: NM_001339687.1), AtPht1;8 (NCBI: NM_001332472.1), and AtPht1;9 (NCBI: NM_106293.4) ​​were subjected to cohort analysis, which involved aligning the amino acid sequences according to their similarity.

[0041] like Figure 1As shown, Pht1 family proteins are highly conserved in the three plants, with only a few amino acids differing. Unlike Arabidopsis and rice, the 324th amino acid of the PvPht1;5 protein (SEQ ID NO.1) of Centipede Grass is glycine (Gly, G) instead of aspartic acid (Asp, D). Therefore, it is inferred that this site (i.e., the D324 site) is a unique mutant amino acid site and is a key amino acid site of the Pht1 family protein of Centipede Grass.

[0042] Cohort analysis revealed that the D324 site of the Centipede Grass PvPht1;5 protein (SEQ ID NO.1) corresponds to amino acid 309 (i.e., D309) in the Arabidopsis thaliana AtPht1;4 protein (amino acid sequence shown in SEQ ID NO.2), and to amino acid 3306 (i.e., D306) in the rice OsPht1;4 protein (amino acid sequence shown in SEQ ID NO.3).

[0043] 2. Biochemical Functional Study of Key Amino Acid Sites in Pht1 Family Proteins from Centipede Grass (1) Primer design Based on the gene coding region sequences of PvPht1;5 (SEQ ID NO.1), AtPht1;4 (SEQ ID NO.2), and OsPht1;4 (SEQ ID NO.3) of rice, gene cloning primers as shown in Table 1 and gene point mutation primers as shown in Table 2 were designed using the NCBI online website.

[0044] Table 1. Primers for gene cloning

[0045] Table 2 Primers for gene point mutations

[0046] Note: Lowercase letters in the sequence indicate the bases corresponding to mutation sites.

[0047] (2) Construction and transformation of recombinant expression vectors encoding Pht1 family proteins High-quality total RNA was extracted from *Pteris vittata*, *Arabidopsis thaliana*, and rice (varieties: Zhonghua 11 and ZhongHua 11), and cDNA was obtained from each plant by reverse transcription. These cDNAs were then used as templates for PCR amplification using the three sets of gene cloning primers shown in Table 1. PvPht1;5 The cloned product of the gene (nucleotide sequence as shown in SEQ ID NO.4). AtPht1;4 The cloned product of the gene (nucleotide sequence as shown in SEQ ID NO.5) and OsPht1;4The cloned gene products (nucleotide sequences as shown in SEQ ID NO. 6) were purified and ligated into the pBM23 universal cloning vector (pBM23 Toposmart Cloning Kit (T vector TOPO cloning vector)). This yielded recombinant expression vectors encoding PvPht1;5 (SEQ ID NO. 1), AtPht1;4 (SEQ ID NO. 2), and OsPht1;4 (SEQ ID NO. 3), respectively denoted as pBM23-PvPht1;5 vector, pBM23-AtPht1;4 vector, and pBM23-OsPht1;4 vector.

[0048] The three recombinant expression vectors were then transformed into competent *E. coli* cells (DH5α strain). Positive single clones were screened using ampicillin-resistant medium, and colony PCR and sequencing were performed to confirm that the sequences of the cloned products of each gene were consistent with those in the database, indicating that the pBM23-PvPht1;5, pBM23-AtPht1;4, and pBM23-OsPht1;4 vectors were successfully constructed. The *E. coli* strains transformed with each recombinant expression vector were stored at -80°C.

[0049] (3) Construction and transformation of recombinant expression vectors for point mutations in Pht1 family proteins Using pBM23-PvPht1;5, pBM23-AtPht1;4, and pBM23-OsPht1;4 vectors as templates, three sets of gene point mutation primers as shown in Table 2 were used sequentially, combined with a site-directed mutagenesis kit (Mut Express II Fast Mutagenesis Kit V2, manufacturer: Nanjing Novizan Biotechnology Co., Ltd., catalog number: C214) for site-directed mutagenesis PCR amplification, yielding the corresponding results. PvPht1;5 Point mutation products of genes (nucleotide sequences as shown in SEQ ID NO.7), AtPht1;4 Point mutation products of the gene (nucleotide sequence as shown in SEQ ID NO. 8) and OsPht1;4 The point mutation product of the gene (nucleotide sequence as shown in SEQ ID NO.9) was digested with DpnI enzyme and purified by gel extraction, and then subjected to recombination to obtain the gene encoding the point mutation. PvPht1;5 G324D The recombinant expression vector of (SEQ ID NO.10), and the point mutation AtPht1;4 D309G Recombinant expression vector of (SEQ ID NO.11) and point mutation OsPht1;4 D306GThe recombinant expression vectors of (SEQ ID NO.12) are designated as pBM23-PvPht1;5-D324, pBM23-AtPht1;4-D309, and pBM23-OsPht1;4-D306.

[0050] The three recombinant expression vectors were then transformed into competent *E. coli* cells (DH5α strain). Positive single clones were screened using ampicillin-resistant medium, and colony PCR and sequencing confirmed that the point mutations in each gene were as expected, indicating successful construction of the pBM23-PvPht1;5-D324, pBM23-AtPht1;4-D309, and pBM23-OsPht1;4-D306 vectors. The *E. coli* strains transformed with each recombinant expression vector were stored at -80°C.

[0051] (4) Construction of yeast expression vector Designed using CE Design software (Nanjing Novizan Biotechnology Co., Ltd.) PvPht1;5、 AtPht1;4 and OsPht1;4 The homologous recombination primers for the yeast expression vector are shown in Table 3.

[0052] Table 3. Primers for homologous recombination of yeast expression vectors

[0053] Using pBM23-PvPht1;5, pBM23-AtPht1;4, pBM23-OsPht1;4, pBM23-PvPht1;5-D324, pBM23-AtPht1;4-D309, and pBM23-OsPht1;4-D306 vectors as templates, PCR amplification was performed. Homologous arms were ligated to both ends of the CDS of each gene to obtain homologous recombination products of each gene and its point mutation.

[0054] The yeast expression vector pAG426GPD-ccdB (Addgene:#14156) was digested with restriction endonucleases SpeⅠ and HindⅢ to obtain the linearized pAG426GPD-ccdB vector. The homologous recombination products and the linearized pAG426GPD-ccdB vector were purified by gel extraction and then subjected to homologous recombination using a one-step rapid cloning kit (ClonExpress II OneStep Cloning Kit, manufacturer: Nanjing Novizan Biotechnology Co., Ltd., catalog number: C112-01). Yeast expression vectors that met the expected sequencing results were retained. The expression vectors... PvPht1;5The yeast expression vector of (SEQ ID NO.1) is designated pAG426GPD- PvPht1;5- G vector, expression point mutation PvPht1;5 G324D The yeast expression vector of (SEQ ID NO.10) is designated pAG426GPD- PvPht1;5- D-carrier, expression AtPht1;4 The yeast expression vector of (SEQ ID NO.2) is designated pAG426GPD- AtPht1;4 -D vector, expressing point mutations AtPht1;4 D309G The yeast expression vector of (SEQ ID NO.11) is designated pAG426GPD- AtPht1;4 -G carrier, expression OsPht1;4 The yeast expression vector of (SEQ ID NO.3) is designated pAG426GPD- OsPht1;4 -D vector, expressing point mutations OsPht1;4 D306G The yeast expression vector of (SEQ ID NO.12) is designated pAG426GPD- OsPht1;4 -G carrier.

[0055] (5) Construction of recombinant yeast To prepare competent yeast cells of the phosphorus-transporting yeast mutant strain EY917, the six yeast expression vectors were transformed into competent EY917 cells using a classic yeast transformation kit (manufacturer: Beijing Coollab Technology Co., Ltd., catalog number: SK2400-200T). The cells were then plated onto SD-U yeast culture medium (containing no Ura amino acids) plates and incubated at 30°C for one week. Positive single clones were picked and inoculated into SD-U liquid medium, and cultured in a shaker at 200 rpm and 30°C to obtain recombinant yeast cells transformed with the six yeast expression vectors.

[0056] Take the culture broth of the above 6 recombinant yeasts, streak off single colonies and culture them in SD-U liquid medium until OD. 600 To achieve a cell enrichment rate of 1.0, yeast cells were enriched. Yeast expression vectors were extracted using a yeast plasmid extraction kit (EZNA Yeast Plasmid Kit D3376-01, manufacturer: Zhejiang Yuxiang Biotechnology Co., Ltd., catalog number: D3376-01). These vectors were then transferred back into competent *E. coli* cells (DH5α strain) for secondary sequencing identification. Recombinant yeast cells that met the expected sequencing results were retained. Among them, the pAG426GPD-... PvPht1;5Recombinant yeast with the -G vector is denoted as PvPht1;5 Yeast, transferred to pAG426GPD- PvPht1;5 Recombinant yeast with the -D vector is denoted as PvPht1;5 G324D Yeast, transferred to pAG426GPD- AtPht1;4 Recombinant yeast with the -D vector is denoted as AtPht1;4 Yeast, transferred to pAG426GPD- AtPht1;4 Recombinant yeast with the -G vector is denoted as AtPht1;4 D309G Yeast, transferred to pAG426GPD- OsPht1;4 Recombinant yeast with the -D vector is denoted as OsPht1;4 Yeast, transferred to pAG426GPD- OsPht1;4 Recombinant yeast with the -G vector is denoted as OsPht1;4 D306G yeast.

[0057] (6) Yeast growth experiment To investigate the effect of the key amino acid D324 site of PvPht1;5 on the transmembrane transport biochemical function of Pht1 protein As(V), yeast growth curves were first determined. The six recombinant yeast strains were inoculated into SD-U liquid medium with a phosphorus concentration of 3 mM for expansion. When the recombinant yeasts reached OD... 600 When the OD value is 1.0, collect fresh bacterial culture, centrifuge at 1000×g for 5 min, discard the supernatant, resuspend the precipitate in sterile water, and aliquot into 25 mL of SD-U liquid medium containing 3 mM Pi and 25 mL of SD-U liquid medium containing 3 mM Pi and 1.5 mM As(V), respectively, and adjust the initial OD value. 600 The value was uniformly set to 0.2, and three parallel replicates were set up for each experimental group.

[0058] The conical flasks containing each culture medium were placed in a shaker (200 rpm, 30℃) for incubation. Periodically, 1 mL of bacterial suspension was taken from each flask, and its OD value was measured using a UV-Vis ratio dual-beam spectrophotometer. 600 The OD value was used to plot the yeast growth curve. 600 The value is y, and the time is x, using the exponential function y(x) = a·e kx The growth curves of each yeast transformant were fitted, where k is the yeast growth rate constant.

[0059] like Figure 2 As shown in a and b, compared with no As(V), under the condition of 1.5 mM As(V) PvPht1;5 yeast and PvPht1;5 G324D Yeast growth was inhibited, and PvPht1;5The yeast was more significantly inhibited. (Explanation) PvPht1;5 yeast and PvPht1;5 G324D Yeasts all possess a certain capacity for As(V) absorption and transport, while PvPht1;5 Yeast has a stronger ability to absorb and transport As(V). For example... Figure 3 As shown in a and b, compared with no As(V), under the condition of 1.5 mM As(V) AtPht1;4 yeast and AtPht1;4 D309G Yeast growth was inhibited in all cases. For example... Figure 3 As shown in c, AtPht1;4 D309G yeast ratio AtPht1;4 Decreased arsenic tolerance in yeast indicates AtPht1;4 yeast and AtPht1;4 D309G Yeasts all possess a certain capacity for As(V) absorption and transport, while AtPht1;4 D309G Yeast has a stronger ability to absorb and transport As(V). For example... Figure 4 As shown in a and b, compared with no As(V), under the condition of 1.5 mM As(V) OsPht1;4 yeast and OsPht1;4 D306G Yeast growth was inhibited by arsenic stress, and OsPht1;4 D306G The yeast was more significantly inhibited. (Explanation) OsPht1;4 yeast and OsPht1;4 D306G Yeasts all possess a certain capacity for As(V) absorption and transport, while OsPht1;4 D306G Yeast has a stronger ability to absorb and transport As(V).

[0060] (7) Yeast arsenic absorption experiment To investigate the effect of the key amino acid D324 site of PvPht1;5 on the transmembrane transport biochemical function of Pht1 protein As(V), an arsenic uptake experiment was also conducted. The initial OD... 600 Each transformed yeast with a value of 1 was cultured in SD-U yeast liquid medium containing 1.5 mM P and 150 μM As(V) in a constant temperature shaker (30℃, 200 rpm) for 2 hours. After washing the bacterial solution three times with ice water, the cells were enriched by centrifugation, dried and digested, and the yeast arsenic concentration was determined. The effects of mutations at key amino acid sites on the arsenic transport capacity of Pht1 protein were compared.

[0061] like Figure 2 As shown in c, PvPht1;5 yeast ratio PvPht1;5 G324DYeast accumulated arsenic at a significantly higher level (p<0.01), with a 43.5% higher accumulation rate. This indicates that the mutation of the amino acid residue at position D324 of PvPht1;5 (SEQ ID NO.1) from G to D significantly weakens the arsenic transport capacity of the centipede phosphate transporter PvPht1;4. Figure 4 As shown in c, OsPht1;4 D306G The arsenic accumulation in yeast is significantly higher than that in other organisms. OsPht1;4 In yeast, the arsenic transport capacity was increased by 80%. This indicates that when the amino acid residue at position D309 of OsPht1;4 (SEQ ID NO.3) is mutated from D to G, the arsenic transport capacity of the rice phosphorus transporter OsPht1;4 is significantly enhanced.

[0062] Example 2: Functional study of key amino acid sites in the As(V) absorption process in Arabidopsis thaliana 1. Construction of transgenic Arabidopsis plants (1) Mutation of key amino acid sites at AtPht1;4 in Arabidopsis thaliana and construction of expression vector Designed using CE Design software (Nanjing Novizan Biotechnology Co., Ltd.) AtPht1;4 The homologous recombination primers for the plant expression vector are shown in Table 4.

[0063] Table 4. Primers for homologous recombination of plant expression vectors

[0064] Using the pBM23-AtPht1;4 vector and the pBM23-AtPht1;4-D309 vector from Example 1 as templates, PCR amplification was performed, and homologous arms were ligated to both ends of the CDS, respectively, to obtain the following results. AtPht1;4 and AtPht1;4 D309G Homologous recombination products.

[0065] The plant expression vector pCAMBIA1305 was double-digested with restriction endonucleases BamHI and KpnI, resulting in a linearized pCAMBIA1305 vector. After gel purification, the homologous recombination products and the linearized pCAMBIA1305 vector were subjected to homologous recombination using a one-step rapid cloning kit (ClonExpress II One Step Cloning Kit, Nanjing Novizan Biotechnology Co., Ltd., catalog number: C112-01). Plant expression vectors that met the expected sequencing results were retained. Among them, the expression vector for Arabidopsis thaliana was... AtPht1;4 The plant expression vector of (SEQ ID NO.2) is designated pCAMBIA1305- AtPht1;4 -D vector, expressing point mutations AtPht1;4D309G The plant expression vector of (SEQ ID NO.11) is designated pCAMBIA1305- AtPht1;4 -G carrier.

[0066] (2) Construction of Agrobacterium pCAMBIA1305- AtPht1;4 The -D vector was transformed into Agrobacterium competent cells GV3101, and Agrobacterium that had been transformed back into E. coli and identified by sequencing as meeting the expected Agrobacterium expression was obtained, yielding Arabidopsis thaliana expression. AtPht1;4 Agrobacterium, denoted as AtPht1;4 -D Agrobacterium. pCAMBIA1305- AtPht1;4 -G vector was transformed into Agrobacterium competent cells GV3101, and Agrobacterium that had been transformed back into E. coli and identified by sequencing as meeting the expected Agrobacterium strain was obtained, resulting in point-mutated Arabidopsis thaliana. AtPht1;4 Agrobacterium, denoted as AtPht1;4 -G Agrobacterium.

[0067] (3) Infection The bacterial cultures of the above-mentioned Agrobacterium species (initial OD) 600 Add 0.8 g of the solution to LB medium containing kanamycin and rifampin, respectively, and incubate at 28°C with shaking for 24 h. Centrifuge the bacterial culture and then adjust the OD value of the culture. 600 The concentration was 0.8, and the solution was resuspended in 120 mL of permeate (ultrapure water containing 10% w / v sucrose and 400 μL / L Silwet-77). The corresponding concentration was 0.8. AtPht1;4 -D Agrobacterium tumefaciens flower dipping solution and containing AtPht1;4 The Agrobacterium tumefaciens dipping solution should be thoroughly mixed before dipping the flowers.

[0068] Take healthy Arabidopsis thaliana AtPht1;4 mutant plants (denoted as) Δatpht1;4 Arabidopsis thaliana (Arabidopsis no. N680086) was cultured until bolting and flowering, and the pods and pollinated flowers were removed before transformation. The inflorescence infection method was used to inoculate the plants... Δ atpht1;4 The above-ground parts of Arabidopsis thaliana are submerged AtPht1;4 -D Agrobacterium tumefaciens flower dipping solution and containing AtPht1;4 Infect the plants with Agrobacterium tumefaciens in a flower-dipping solution for 45 seconds, followed by gentle shaking; repeat this process three times, with one week between each infection; continue culturing until the plants mature, then collect the seeds to obtain the final product. AtPht1;4 -G Agrobacterium Δatpht1;4 The seeds of Arabidopsis thaliana (denoted as) AtPht1;4 seeds) and AtPht1;4 - Agrobacterium tumefaciens infection Δatpht1;4 The seeds of Arabidopsis thaliana (denoted as) AtPht1;4 D309G Seeds should be stored in a dry environment.

[0069] (4) Screening of infected seeds Screening was performed using 1 / 2 MS medium containing 30 mg / L hygromycin. AtPht1;4 Seeds and AtPht1;4 D309G Seeds, retaining those that continue to grow, are transplanted into nutrient soil for further cultivation; the resulting plants are sequentially denoted as... AtPht1; 4 -Ex Arabidopsis thaliana and AtPht1;4 D309G -Ex Arabidopsis thaliana. Continue culturing until the plants mature, harvest T1 generation seeds for later use.

[0070] (5) Expression identification for Δatpht1;4 Arabidopsis thaliana, wild-type Arabidopsis thaliana (designated as WT Arabidopsis thaliana) AtPht1;4 -Ex Arabidopsis thaliana and AtPht1;4 D309G -Ex Arabidopsis thaliana, total RNA was extracted from 10 T1 generation seeds, reverse transcribed to obtain cDNA, used as templates, and designed and used as shown in Table 5. AtPht1;4 and AtPht1;4 D309G Gene-specific quantitative PCR primers, Taq Pro UniversalSYBR qPCR Master Mix, and QuantStudio 3 real-time quantitative PCR detection system were used for qRT-PCR analysis of the actin gene in Arabidopsis thaliana. AtActin As an internal control (the sequences of its quantitative PCR primers are shown in Table 5), relative quantification was performed using 2... -ΔΔCt Method to calculate the number of plants AtPht1;4 Genes and AtPht1;4 D309G The relative expression level of genes.

[0071] Table 5 Quantitative PCR Primers

[0072] like Figure 5 As shown in a and b, retain AtPht1;4 The three T1 generations with the highest relative gene expression AtPht1;4 -Ex Arabidopsis thaliana (denoted as AtPht1;4 -Ex1~ AtPht1;4 -Ex3), and AtPht1;4 D309G The three T1 generations with the highest relative gene expression levels AtPht1;4 D309G -Ex Arabidopsis thaliana (denoted as AtPht1;4 D309G -Ex1~ AtPht1;4D309G -Ex3), used for subsequent experiments.

[0073] 2. Effect of the mutation at site D309 of AtPht1;4 on As(V) uptake in Arabidopsis thaliana. (1) Arsenic-treated plate experiment To investigate the effect of the AtPht1;4 D309 mutation on the ability of Arabidopsis thaliana to absorb As(V), healthy T1 generation WT Arabidopsis thaliana with uniform growth were selected. Δatpht1;4 Arabidopsis thaliana, 3 strains AtPht1;4 -Ex Arabidopsis thaliana ( AtPht1;4 -Ex1~ AtPht1;4 -Ex3) and 3 plants AtPht1;4 D309G -Ex Arabidopsis thaliana ( AtPht1;4 D309G -Ex1~ AtPht1;4 D309G -Ex3), in 1 / 2 MS medium containing 100 μM As(V), were cultured in an artificial climate chamber under the following conditions: 14 hours light, 10 hours dark; daytime temperature 26°C, nighttime temperature 20°C; relative humidity 60%, and light intensity 210 μmol / m². 2 / s(PAR).

[0074] After 14 days of culture, the phenotype of each plant in each culture medium was observed, and the aboveground parts and root tissues were collected for subsequent testing.

[0075] (2) Determination of plant biomass Cut each Arabidopsis plant at the root-stem junction with scissors to ensure complete separation of the shoot and root. Immerse the root sample in pre-cooled arsenic desorption solution (prepared by uniformly mixing 5 mL of 100 mM K₂HPO₄, 5 mL of 50 mM Ca(NO₃)₂, 5 mL of 500 mM MES, and 485 mL of ultrapure water, pH 6.0) for 10 min to remove arsenic adsorbed by the root apoplast. After rinsing the roots with water, lay them flat on paper and gently press to absorb surface moisture. Weigh the fresh weight of the shoot and roots separately using an analytical balance and record the data. The fresh weight of the shoot represents the biomass of each Arabidopsis plant.

[0076] (3) Determination of arsenic concentration in plants The aerial parts and roots of Arabidopsis thaliana plants grown in different culture media were weighed into digestion tubes. The plant tissues were digested at 105 °C using a USEPA 3050B system of concentrated nitric acid-hydrogen peroxide (3 mL 1:1 (w / v) HNO3 and 1 mL 30% (w / v) H2O2). The arsenic concentration (μg / kg) was then determined using inductively coupled plasma mass spectrometry (ICP-MS). -1 Arsenic accumulation in each Arabidopsis plant was obtained by multiplying the arsenic concentration by the biomass. The differences in arsenic accumulation in the aboveground parts (Shoot) and roots (Root) of each Arabidopsis plant were compared.

[0077] (4) Results Analysis like Figure 6 As shown in a and b in the figure, with Δatpht1;4 Compared to Arabidopsis thaliana, 3 strains AtPht1;4 -Ex Arabidopsis thaliana ( AtPht1; 4 -Ex1~ AtPht1;4 Arsenic accumulation in the aboveground parts of plants with the arsenic-ex-3 group increased by only 78.3%–83.8%, and there was no significant difference in arsenic accumulation in the roots; while compared with... Δatpht1;4 Compared to Arabidopsis thaliana, 3 strains AtPht1;4 D309G -Ex Arabidopsis thaliana ( AtPht1;4 D309G -Ex1~ AtPht1;4 D309G Arsenic accumulation in the aboveground parts and roots of the *Ex3* group increased by 331.4%–500.2% and 5.4%–15.1%, respectively, and compared with the WT group, the arsenic accumulation in the three plants was significantly higher. AtPht1;4 D309G -Ex Arabidopsis thaliana ( AtPht1;4 D309G -Ex1~ AtPht1;4 D309G Arsenic accumulation in the aboveground parts of Arabidopsis thaliana (-Ex3) also increased by 170.1%–275.4%. This indicates that the mutation of the amino acid at the D309 site of AtPht1;4 from D to G can significantly enhance the arsenic absorption and translocation capacity of Arabidopsis thaliana.

[0078] Table 6 This embodiment AtPht1;4 D309G -Comparison of the effects of Arabidopsis thaliana and existing transgenic Arabidopsis thaliana plants on arsenic absorption.

[0079] Note: The percentage increase in arsenic accumulation in the aboveground parts refers to: AtPht1;4 D309G -Ex Arabidopsis thaliana or each transgenic Arabidopsis thaliana plant showed a higher proportion of arsenic concentration in the aboveground parts compared to their corresponding wild-type Arabidopsis thaliana plants.

[0080] Furthermore, as shown in Table 6, compared with the Arabidopsis transgenic plants in the prior art, the transgenic plants constructed in this embodiment... AtPht1;4 D309G -Ex Arabidopsis thaliana exhibits significantly stronger arsenic absorption and accumulation capabilities.

[0081] Example 3: Functional study of key amino acid sites in the absorption of As(V) in rice 1. Construction of single-base edited rice plants (1) Target site design and construction of single-base editing vectors First, based on the codon bias of cereal plants, the sequences of tRNA editing deaminase (ABE8e) and nCas9-NG fusion protein (D10A) were codon optimized. These were then synthesized by GENEWIZ (Suzhou, China) to form fragments of each element. Finally, the two were fused to form... Figure 7 The adenine base editing vector pH-ABE8e-NG is shown.

[0082] The single-base editing requirement at the D306 site of OsPht1;4 (SEQ ID NO.3) led to the design of a target site and the synthesis of sgRNA, denoted as OsPht1;4-sgRNA, with the nucleotide sequence 3'-GCTGGACATCGCCTTCTACTCGT-5' (SEQ ID NO.13). Subsequently, the annealed OsPht1;4-sgRNA was constructed into the aforementioned pH-ABE8e-NG vector using Golden Gate technology, driven by OsU3, and designated as the ABE8e-OsPht1;4 plasmid (DOI: 10.1016 / j.cell.2023.06.023).

[0083] The ABE8e-OsPht1;4 plasmid was introduced into rice protoplasts using the polyethylene glycol (PEG) method, and the editing efficiency of the target sites was evaluated using a transient assay. After incubation for 48 hours, genomic DNA was prepared from each sample, and DNA fragments containing each target site were amplified by PCR. The PCR products were digested with restriction endonucleases, and the PCR amplicon was cloned into a TA cloning vector. DNA sequencing analysis was then used to verify the efficient editing of the target sites.

[0084] (2) Induction of rice callus Wild-type rice (i.e., WT rice, japonica rice, variety: Zhonghua 11) seeds were deglunded, removing the inner and outer glumes. The seeds were washed three times with distilled water, then soaked in 75% ethanol for 2 minutes, followed by three more washes with distilled water, and finally washed with 0.15% HgCl2 for 20 minutes. Afterward, the HgCl2 was poured out from the centrifuge tube containing the seeds on a sterile workbench, and the seeds were washed 4-5 times with distilled water. The seeds were then poured onto sterilized filter paper and dried (1 hour). They were inoculated onto hormone-free induction medium and cultured at 28°C in the dark. After 30 days, the induced callus tissue was observed. The endosperm and plumule were removed, and the callus was peeled off and transferred to fresh N6D medium for subculture. Subculture was then performed every 7-8 days.

[0085] (3) Agrobacterium-mediated transformation of rice callus cells The *Agrobacterium* strain EHA105 was selected and competent cells were prepared. These cells were then transformed into the plasmid ABE8e-OsPht1;4 and plated onto LB agar plates containing kanamycin and rifampin. Single colonies appeared after incubation at 28°C for 2 days. Single colonies of *Agrobacterium* were picked and inoculated onto YEP liquid medium (containing kanamycin and rifampin) and cultured with shaking at 28°C until OD200. 600 Approximately 0.8. Collect bacterial cells by centrifugation and resuspend in AAM medium (containing 100 μM acetylsyl syringone). Immerse callus tissue in Agrobacterium suspension for 10–15 minutes, aspirate excess bacterial solution, and then spread onto co-culture medium (N6D medium containing 100 μM As) and incubate in the dark at 25°C for 3 days.

[0086] Wash the callus three times with sterile water containing cephalosporin (250 mg / L) and transfer it to selection medium (N6D medium containing 50 mg / L hygromycin). Subculture every 2 weeks for 4–6 weeks to obtain resistant callus.

[0087] (4) Plant regeneration The resistant callus was transferred to regeneration medium (MS medium containing 2 mg / L 6-BA and 0.2 mg / L NAA) and cultured under light (16 h light / 8 h dark). When the seedlings grew to 3 cm–5 cm, they were transferred to rooting medium (1 / 2 MS medium containing 0.5 mg / L NAA). The selected plantlets were collected and recorded as follows: ospht1;4 D306G Rice.

[0088] (5) Identification of mutation sites Cut a 2cm piece with scissors ospht1;4 D306GRice leaves were placed in a grinding tube containing zirconia grinding beads and pulverized using a grinder with liquid nitrogen. Extraction was then performed using the magnetic bead method with the YZMG Fast Plant Total DNA kit (manufacturer: Jizhang Technology Investment Hainan Co., Ltd., catalog number: YZ0046-S). ospht1;4 D306G The DNA of rice.

[0089] The first round of PCR was performed using the specific primers shown in Table 7, amplifying... ospht1;4 The target fragment is located near the base region. A second round of PCR is then performed, adding a specific barcode sequence and sequencing adapter to both ends of the target fragment to construct a HiDecode mixed amplicon library. The mixed library sample is then sent to a sequencing company for sequencing using the Illumina platform. The obtained next-generation sequencing results are decoded and analyzed using HiDecode software to obtain... ospht1;4 D306G Mutations in rice, as well as heterozygosity and homozygosity.

[0090] Kimura B nutrient solution was prepared according to the instructions using 5000×Calcium salt solution and Kimura B nutrient salts. Three single-base edited strains were retained. ospht1;4 D306G homozygous rice plants, i.e., OS pht1; 4 D306G -1~os pht1;4 D306G -3), continue greenhouse cultivation in Kimura B nutrient solution, with a light cycle of 16 hours (30°C) / 8 hours (22°C) and a relative humidity of 70%.

[0091] Table 7 OsPht1;4 Specific primers for mutation sites

[0092] 2. Rice OsPht1;4 The effect of the D306 site mutation on As(V) uptake in rice (1) Arsenic absorption hydroponic experiment To investigate the effect of the D306 mutation in rice OsPht1;4 on the rice's ability to absorb As(V), a hydroponic experiment on As(V) absorption was conducted. WT rice and os rice of similar growth were compared. pht1;4 D306G -1~os pht1;4 D306G -3, pretreated for 7 days in phosphorus-deficient Kimura B nutrient solution, and cultured for 3 days in Kimura B nutrient solution containing 20 μM As(V).

[0093] (2) Sample collection and processing Use scissors to cut WT rice and os pht1;4 D306G -1~os pht1;4 D306G Cut the rice plant at the root-stem junction (-3), ensuring complete separation of the shoot and root. Immerse the roots of each rice plant in a pre-cooled arsenic desorption solution (prepared by uniformly mixing 5 mL of 100 mM K₂HPO₄, 5 mL of 50 mM Ca(NO₃)₂, 5 mL of 500 mM MES, and 485 mL of ultrapure water, pH 5.5) for 10 min to remove arsenic adsorbed by the root apoplast. After rinsing with water, lay the roots flat on paper and gently press to absorb surface moisture. Place the shoot and the treated roots of each rice plant separately in envelopes and dry them in a forced-air drying oven at 60°C for three days. After chopping, weigh 0.025 g of shoot and 0.025 g of root from each rice plant and place them in a digestion tube, recording the actual dry weight.

[0094] (3) Determination of arsenic concentration The aboveground parts and roots of the rice plants obtained in the previous step were digested in a graphite furnace digester at 105 °C using a concentrated nitric acid-hydrogen peroxide system (6 mL 1:1 (w / v) HNO3 and 2 mL 30% (w / v) H2O2) prepared by USEPA 3050B. The arsenic concentration (μg kg) was then determined using inductively coupled plasma mass spectrometry (ICP-MS). -1 The arsenic concentration was multiplied by the biomass to obtain the arsenic accumulation of each rice plant. The differences in arsenic accumulation in the aboveground parts (shoot) and roots of each rice plant were compared.

[0095] (4) Results Analysis like Figure 8 As shown, compared to WT rice, os pht1;4 D306G -1~os pht1;4 D306G The arsenic accumulation in the aboveground parts and roots of rice with the Ospht1;4 protein was increased by 76.8%–145.3% and 28.9%–48.8%, respectively, demonstrating a stronger arsenic absorption capacity. This indicates that the mutation of the amino acid from D to G at the D306 site of Ospht1;4 can enhance the arsenic absorption and translocation capacity of rice.

[0096] Table 8 This embodiment ospht1;4 D306G Comparison of the effects of transgenic rice on arsenic absorption with existing transgenic rice plants

[0097] Note: The percentage increase in arsenic accumulation in the aboveground parts refers to: ospht1;4 D306G The proportion of arsenic accumulation in the aboveground parts of rice or various transgenic rice plants was increased compared to their corresponding wild-type rice plants.

[0098] Furthermore, as shown in Table 8, compared with the transgenic rice plants in the prior art, the transgenic rice plants constructed in this embodiment... OsPht1; 4 -G rice exhibits significantly stronger arsenic absorption and accumulation capabilities.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for improving the arsenic accumulation capacity of plants, characterized in that, The key amino acid site of the Pht1;4 protein in the plant was mutated from aspartic acid to glycine. If the plant is Arabidopsis thaliana, then the key amino acid site is amino acid 309 of the Pht1;4 protein; If the plant is rice, then the key amino acid site is the 306th amino acid of the Pht1;4 protein.

2. The method according to claim 1, characterized in that, If the plant is Arabidopsis thaliana, then the amino acid sequence of the Pht1;4 protein is shown in SEQ ID NO.

2.

3. The method according to claim 2, characterized in that, If the plant is rice, then the amino acid sequence of the Pht1;4 protein is shown in SEQ ID NO.

3.

4. The application of plants obtained by the method according to any one of claims 1 to 3 in the remediation of arsenic-contaminated soil.

5. A biomaterial, characterized in that, The biomaterial is any one of the following (1) to (5): (1) Pht1;4 protein mutant, which is the D309Q mutant of Arabidopsis Pht1;4 protein or the D306Q mutant of rice Pht1;4 protein; (2) The nucleic acid molecule encoding the Pht1;4 protein mutant described in (1); (3) An expression cassette containing the nucleic acid molecules described in (2); (4) A recombinant expression vector containing the expression cassette described in (3); (5) Microorganisms containing the recombinant expression vector described in (4).

6. The biomaterial according to claim 5, characterized in that, The amino acid sequence of the Arabidopsis Pht1;4 protein described in (1) is shown in SEQ ID NO.

2.

7. The biomaterial according to claim 6, characterized in that, The amino acid sequence of the D309Q mutant of the Arabidopsis Pht1;4 protein described in (1) is shown in SEQ ID NO.

11.

8. The biomaterial according to claim 5, characterized in that, The amino acid sequence of the rice Pht1;4 protein described in (2) is shown in SEQ ID NO.

3.

9. The biomaterial according to claim 8, characterized in that, The amino acid sequence of the D306Q mutant of the rice Pht1;4 protein described in (2) is shown in SEQ ID NO.

12.

10. The application of the biomaterial according to any one of claims 5 to 9 in improving the arsenic accumulation capacity of plants and / or remediating arsenic-contaminated soil.

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