Application of two ciliate desert-grass AsIII reverse transport proteins in improvement of arsenic hyperaccumulation capability of plants

Abstract

The invention discloses an application of two ciliate desert-grass AsIII reverse transport proteins in improving the arsenic hyperaccumulation capability of plants. The application is as follows. According to the invention, the PvACR3 of a cell membrane and the PvACR3 positioned in a vacuolar membrane are simultaneously subjected to heterologous overexpression in a plant through a hybridization technology; 1, the biomass of the plant is obviously improved, the hyperaccumulation capability of the plant on arsenic is enhanced, and the absorption and transport efficiency of the plant on arsenic is promoted; the invention provides an efficient, environment-friendly and low-cost new technical choice for the bioremediation technology of the arsenic-polluted soil.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to applications of two AsIII antiporter proteins of Scolopendra suber in improving the arsenic hyperaccumulation ability of plants. Background Art

[0002] With the acceleration of industrialization and mineral mining, arsenic pollution has become a global environmental chemical pollution problem in modern society. Currently, agricultural soils are contaminated with arsenic over a large area, with low and medium levels of contamination being prominent.

[0003] Arsenic hyperaccumulators have a far greater capacity to absorb arsenic (As) from soil and other environments than ordinary plants. They can accumulate large amounts of arsenic in their tissues (such as roots, stems, and leaves) without becoming seriously toxic due to the accumulated arsenic. Phytoremediation is a key approach to remediating heavy metal contamination in soils. It utilizes hyperaccumulators to absorb and transport heavy metals from contaminated soils, which are then removed to reduce heavy metal concentrations in the soil. While phytoremediation offers the advantages of being green and low-carbon, its efficiency is limited by the physiological characteristics of the hyperaccumulators used. Centipede grass is currently the primary plant used for phytoremediation of arsenic-contaminated soils. However, its arsenic extraction efficiency is limited by its natural growth and arsenic uptake and transport capacity. Therefore, there is an urgent need to improve the arsenic accumulation capacity of plants other than centipede grass, overcome the limitations of hyperaccumulator germplasm, and develop a new generation of engineered plants for soil arsenic remediation.

[0004] The AsIII antiporter (ACR3) family of key arsenic metabolism genes is a key protein for arsenic accumulation in centipede grass. These proteins, including PvACR3, PvACR3;1, PvACR3;2, PvACR3;3, and PvACR3;4, are divided into two groups based on their localization. PvACR3 is located at the cell membrane, while PvACR3;1 is localized to the vacuole membrane. These two proteins have distinct functions and differ significantly in their applications. In particular, PvACR3;1 reduces arsenic accumulation in the shoots and instead fixes arsenic in the roots, preventing its absorption, transport, and accumulation, making it difficult to remediate arsenic contaminated soil (DOI: 10.1021 / acs.est.7b03369). Therefore, effectively utilizing these two ACR3 proteins to enhance plant arsenic accumulation remains a challenging technical challenge for those skilled in the art. Summary of the Invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides the use of two AsIII antiporter proteins from Centipede sphaerocarpus in improving the arsenic hyperaccumulation ability of plants.

[0006] The first object of the present invention is to provide an application of PvACR3 protein and / or PvACR3 gene in combination with PvACR3;1 protein and / or PvACR3;1 gene in improving the arsenic hyperaccumulation ability of plants.

[0007] The second object of the present invention is to provide a use of PvACR3 protein and / or PvACR3 gene in combination with PvACR3;1 protein and / or PvACR3;1 gene in cultivating plants for repairing arsenic-contaminated soil.

[0008] The third object of the present invention is to provide a use of a biomaterial that promotes the expression of PvACR3 protein and / or PvACR3 gene and promotes the expression of PvACR3;1 protein and / or PvACR3;1 gene in improving the arsenic hyperaccumulation ability of plants.

[0009] A fourth object of the present invention is to provide a use of a biomaterial that promotes the expression of PvACR3 protein and / or PvACR3 gene and promotes the expression of PvACR3;1 protein and / or PvACR3;1 gene in cultivating plants for repairing arsenic-contaminated soil.

[0010] The fifth object of the present invention is to provide a method for creating plants with arsenic hyperaccumulation ability.

[0011] The sixth object of the present invention is to provide the use of arsenic hyperaccumulator plants created according to the method in remediating arsenic-contaminated soil.

[0012] In order to achieve the above object, the present invention is implemented through the following scheme: The present invention significantly improves the arsenic hyperaccumulation ability of the plant, enhances the arsenic accumulation ability of its aboveground part, and increases the total arsenic content by simultaneously heterologously expressing two types of centipede grass AsIII antiporter proteins (PvACR3 and PvACR3;1) in the plant.

[0013] Therefore, the present invention seeks protection for the following: The invention relates to an application of PvACR3 protein and / or PvACR3 gene in combination with PvACR3;1 protein and / or PvACR3;1 gene in improving the arsenic hyperaccumulation ability of plants, wherein the plants are plants other than centipede grass.

[0014] The invention relates to an application of PvACR3 protein and / or PvACR3 gene in combination with PvACR3;1 protein and / or PvACR3;1 gene in cultivating plants for repairing arsenic-contaminated soil, wherein the plants are plants other than centipede grass.

[0015] The invention relates to an application of a combined use of PvACR3 protein and PvACR3;1 protein in improving the arsenic hyperaccumulation ability of plants, wherein the plants are plants other than centipede grass.

[0016] The invention relates to the application of PvACR3 protein and PvACR3;1 protein in combination in cultivating plants for repairing arsenic-contaminated soil, wherein the plants are plants other than centipede grass.

[0017] The invention discloses an application of the combined use of the PvACR3 gene and the PvACR3;1 gene in improving the arsenic hyperaccumulation ability of plants, wherein the plants are plants other than centipede grass, and the plants are plants other than centipede grass.

[0018] The invention relates to an application of the combined use of the PvACR3 gene and the PvACR3;1 gene in cultivating plants for repairing arsenic-contaminated soil, wherein the plants are plants other than centipede grass, and the plants are plants other than centipede grass.

[0019] Application of a biological material that promotes the expression of PvACR3 protein and / or PvACR3 gene and promotes the expression of PvACR3;1 protein and / or PvACR3;1 gene in improving the arsenic hyperaccumulation ability of plants, wherein the plants are plants other than centipede grass.

[0020] Use of a biological material that promotes the expression of PvACR3 protein and / or PvACR3 gene and promotes the expression of PvACR3;1 protein and / or PvACR3;1 gene in cultivating plants for repairing arsenic-contaminated soil, wherein the plants are plants other than centipede grass.

[0021] Application of a biological material that promotes the expression of PvACR3 protein and PvACR3;1 protein in improving the arsenic hyperaccumulation ability of plants, wherein the plants are plants other than centipede grass.

[0022] Application of biomaterials that promote the expression of PvACR3 protein and PvACR3;1 protein in cultivating plants for repairing arsenic-contaminated soil.

[0023] Application of biomaterials that promote PvACR3 gene expression and PvACR3;1 gene expression in improving the arsenic hyperaccumulation ability of plants.

[0024] Application of biomaterials that promote PvACR3 gene expression and PvACR3;1 gene expression in cultivating plants for repairing arsenic-contaminated soil.

[0025] Preferably, the amino acid sequence of the PvACR3 protein is shown in SEQ ID NO: 2.

[0026] Preferably, the amino acid sequence of the PvACR3;1 protein is shown in SEQ ID NO: 4.

[0027] Preferably, the nucleotide sequence of the PvACR3 gene is shown in SEQ ID NO: 1.

[0028] Preferably, the nucleotide sequence of the PvACR3;1 gene is shown in SEQ ID NO: 3.

[0029] Preferably, the plant is Arabidopsis thaliana.

[0030] More preferably, the expression of the endogenous rhodamine / cell cycle control phosphatase superfamily protein in Arabidopsis thaliana is inhibited, or the endogenous rhodamine / cell cycle control phosphatase superfamily protein in Arabidopsis thaliana is knocked out.

[0031] Further preferably, the Arabidopsis thaliana is hac The mutant Arabidopsis thaliana was accessioned as SALK_058016C.

[0032] Preferably, the biomaterial is any one of the following (1) to (4): (1) a nucleic acid molecule 1 encoding a PvACR3 protein, and a nucleic acid molecule 2 encoding the PvACR3;1 protein; (2) an expression cassette containing the nucleic acid molecule 1 and / or the nucleic acid molecule 2 described in (1); (3) a recombinant expression vector containing the nucleic acid molecule 1 and / or the nucleic acid molecule 2 described in (1); (4) A microorganism containing the recombinant expression vector described in (3).

[0033] More preferably, in (1), the nucleotide sequence of the nucleic acid molecule 1 is as shown in SEQ ID NO: 1 or a completely complementary sequence of the sequence shown in SEQ ID NO: 1, and the nucleotide sequence of the nucleic acid molecule 2 is as shown in SEQ ID NO: 3 or a completely complementary sequence of the sequence shown in SEQ ID NO: 3.

[0034] A method for creating a plant with arsenic hyperaccumulation ability, wherein the PvACR3 gene and the PvACR3;1 gene are heterologously expressed in the plant.

[0035] Preferably, the plant is Arabidopsis thaliana.

[0036] More preferably, the expression of the endogenous rhodamine / cell cycle control phosphatase superfamily protein in Arabidopsis thaliana is inhibited, or the endogenous rhodamine / cell cycle control phosphatase superfamily protein in Arabidopsis thaliana is knocked out.

[0037] Further preferably, the Arabidopsis thaliana is hac The mutant Arabidopsis thaliana was accessioned as SALK_058016C.

[0038] Preferably, the screening method comprises hygromycin screening.

[0039] Preferably, a plant heterologously expressing the PvACR3 gene is used as parent 1, and a plant heterologously expressing the PvACR3;1 gene is used as parent 2, and parent 1 and parent 2 are hybridized; after screening, the progeny heterologously expressing both the PvACR3 gene and the PvACR3;1 gene are retained to obtain.

[0040] More preferably, a plant heterologously expressing the PvACR3 gene is used as the female parent, and a plant heterologously expressing the PvACR3;1 gene is used as the female parent, and the female and male parents are hybridized; after screening, the offspring heterologously expressing both the PvACR3 gene and the PvACR3;1 gene are retained to obtain the product.

[0041] In some specific embodiments, the PvACR3 gene is heterologously expressed in hac The mutant Arabidopsis thaliana was used as parent 1 to heterologously express the PvACR3;1 gene. hac The mutant Arabidopsis thaliana was used as parent 2, and parent 1 and parent 2 were crossed; after screening, the mutants that heterologously expressed both the PvACR3 gene and the PvACR3;1 gene were retained. hac The mutant Arabidopsis offspring is obtained; hac The mutant Arabidopsis thaliana is accessioned as SALK_058016C.

[0042] In some specific embodiments, the PvACR3 gene is heterologously expressed in hac The mutant Arabidopsis thaliana was used as the maternal parent to heterologously express the PvACR3;1 gene. hac The mutant Arabidopsis thaliana was used as the male parent, and the female and male parents were crossed; after screening, the plants expressing both the PvACR3 gene and the PvACR3;1 gene were retained. hac The mutant Arabidopsis offspring is obtained; hac The mutant Arabidopsis thaliana is accessioned as SALK_058016C.

[0043] Arsenic hyperaccumulator plants produced according to any of the methods.

[0044] The use of arsenic hyperaccumulator plants created according to any of the methods in remediating arsenic-contaminated soil.

[0045] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses hybridization technology to simultaneously overexpress the cell membrane PvACR3 and the tonoplast-localized PvACR3;1 in plants, significantly increasing plant biomass, enhancing the plant's ability to hyperaccumulate arsenic, and promoting the plant's absorption and transport efficiency of arsenic. Specific advantages are as follows: 1. Promote plant growth: Increasing biomass not only enhances the plant's own repair ability, but also provides more biomass resources for subsequent plant harvesting, processing and resource utilization, significantly improving the efficiency and economic benefits of the overall repair system.

[0046] 2. Enhance the plant's ability to accumulate arsenic: The total amount of arsenic in the aboveground part of the plant increases significantly, especially under high concentration AsV conditions. It can absorb more arsenic from the soil, significantly reducing the residual arsenic concentration in the soil, thereby accelerating the remediation process of arsenic-contaminated soil.

[0047] 3. High efficiency, environmental protection and low cost: The present invention uses plant resources for bioremediation, avoiding the secondary environmental pollution problems that may be caused by traditional physical or chemical remediation methods, reducing dependence on energy and chemical reagents, and has significant environmental advantages and economic benefits.

[0048] 4. Strong sustainability: Based on the self-repair mechanism of natural ecosystems, this method has sustainable development potential and can maintain stable remediation effects in long-term applications. This method does not require large-scale external investment and can maintain stable remediation effects in long-term applications, making it suitable for large-scale application and contributing to the long-term and effective treatment of soil pollution.

[0049] In summary, the present invention provides a new technology option for bioremediation of arsenic-contaminated soil that is efficient, environmentally friendly and low-cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 30 strains of PvACR3-PvACR3;1 hybrid hac Relative expression levels of PvACR3 and PvACR3;1 genes in Arabidopsis thaliana.

[0051] Figure 2 The growth of Arabidopsis plants after 14 days of treatment with different culture media; A is 1 / 2MS culture medium; B is 1 / 2MS culture medium containing 10 μM AsIII; C is 1 / 2MS culture medium containing 150 μM AsV; WT is wild-type Arabidopsis; hac is hac Mutant Arabidopsis thaliana; PvACR3 is PvACR3 Arabidopsis thaliana; PvACR3;1 is PvACR3;1 Arabidopsis thaliana; L9, L25, and L26 are PvACR3-PvACR3;1 hybrids hac Mutant Arabidopsis thaliana.

[0052] Figure 3The aboveground biomass of each Arabidopsis plant after 14 days of treatment with different culture media; A is 1 / 2MS medium; B is 1 / 2MS medium containing 10 μM AsIII; C is 1 / 2MS medium containing 150 μM AsV; WT is wild-type Arabidopsis; hac is hac Mutant Arabidopsis thaliana; PvACR3 is PvACR3 Arabidopsis thaliana; PvACR3;1 is PvACR3;1 Arabidopsis thaliana; L9, L25, and L26 are PvACR3-PvACR3;1 hybrids hac Mutant Arabidopsis thaliana.

[0053] Figure 4 are the arsenic concentrations of each Arabidopsis plant after 14 days of treatment with different culture media; A is the arsenic concentration in the aboveground part of each Arabidopsis plant treated with 1 / 2MS culture medium containing 10 μM AsIII; B is the arsenic concentration in the root system of each Arabidopsis plant treated with 1 / 2MS culture medium containing 10 μM AsIII; C is the arsenic concentration in the aboveground part of each Arabidopsis plant treated with 1 / 2MS culture medium containing 150 μM AsV; D is the arsenic concentration in the root system of each Arabidopsis plant treated with 1 / 2MS culture medium containing 150 μM AsV.

[0054] Figure 5 The total amount of arsenic in the aerial parts of each Arabidopsis plant after 14 days of treatment with different culture media; A is 1 / 2MS medium containing 10 μM AsIII; B is 1 / 2MS medium containing 150 μM AsV. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0056] Example 1 Construction of PvACR3 recombinant expression vector and PvACR3;1 recombinant expression vector 1. Construction of PvACR3 recombinant expression vector (1) Obtaining the fragment sequence used to construct the PvACR3 recombinant expression vector The coding region of the PvACR3 gene of Centipedegrass was obtained from the NCBI database. The nucleotide sequence is as follows:

[0057] (2) Amplification of target fragment PCR amplification was performed using cDNA from the leaves of P. truncatula as a template using the upstream primer PvACR3-F (5'-gaacgataggagctcggtaccATGGAGAACTCAAGCGCGG-3') and the downstream primer PvACR3-R (5'-caggtcgactctagaggatccCTAAACAGAAGGCCCCTTCCTC-3'). The PCR reaction system consisted of 1 μL template, 2 μL PvACR3-F, 2 μL PvACR3-R, 25 μL high-fidelity enzyme, and 20 μL ddH2O. The PCR program was as follows: 95°C for 3 min for initial denaturation; at the beginning of each cycle, 35 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 15 s, and 72°C for 2 min; and exhaustive extension at 72°C for 5 min.

[0058] (3) Connection conversion The PvACR3 amplified product was purified and then introduced into the pCAMBIA 1305 vector, which had been double-digested with BamHI and KpnI, via homologous recombination. Following recombination, the resulting vector was transformed into competent E. coli cells (Trelief5α) and cultured on solid medium.

[0059] (4) Vector identification After colony PCR identification and sequencing analysis, the vector with the sequence consistent with SEQ ID NO: 1 was retained, and the PvACR3 recombinant expression vector was obtained, which was recorded as 1305::PvACR3.

[0060] 2. Construction of PvACR3;1 recombinant expression vector (1) Obtaining the fragment sequence used to construct the PvACR3;1 recombinant expression vector The coding region of the PvACR3;1 gene of Centipedegrass was obtained from the NCBI database. The nucleotide sequence is as follows:

[0061] (2) Amplification of target fragment PCR amplification was performed using cDNA from the leaves of P. truncatula as a template using the upstream primer PvACR3;1-F (5'-gaacgataggagctcggtaccATGGCCGACAGCACTCGTT-3') and the downstream primer PvACR3;1-R (5'-caggtcgactctagaggatccCTAGTAGGGTTCTGCAGGCCAC-3'). The PCR reaction system consisted of 1 μL template, 2 μL PvACR3;1-F, 2 μL PvAC3;1-R, 25 μL high-fidelity enzyme, and 20 μL ddH2O. The PCR program was as follows: 95°C for 3 min for initial denaturation; 35 cycles of 95°C for 15 s, denaturation at 56°C for 15 s, annealing at 72°C for 2 min, and complete extension at 72°C for 5 min.

[0062] (3) Connection conversion The PvACR3;1 amplified product was purified and then introduced into the pCAMBIA 1305 vector, which had been double-digested with BamHI and KpnI, via homologous recombination. Following recombination, the resulting vector was transformed into competent E. coli cells (Trelief 5α) and cultured on solid medium.

[0063] (4) Vector identification After colony PCR identification and sequencing analysis, the vector with the sequence consistent with SEQ ID NO: 3 was retained, and the PvACR3;1 recombinant expression vector was obtained and recorded as 1305::PvACR3;1.

[0064] Example 2 PvACR3-PvACR3;1 hybridization hac Construction of Arabidopsis 1. Preparation of Agrobacterium The 1305::PvACR3 obtained in Example 1 was transformed into Agrobacterium competent cells (GV3101), and after transformation into Escherichia coli (Trelief 5α) and sequencing, the Agrobacterium with the sequence consistent with SEQ ID NO: 1 was obtained, namely the Agrobacterium transformed with 1305::PvACR3, which was recorded as Agrobacterium 1.

[0065] The 1305::PvACR3;1 obtained in Example 1 was transformed into Agrobacterium competent cells (GV3101), and after transformation into Escherichia coli (Trelief 5α) and sequencing, the Agrobacterium with the sequence consistent with SEQ ID NO: 3 was obtained, namely the Agrobacterium transformed with 1305::PvACR3;1, which was recorded as Agrobacterium 2.

[0066] 2. Cultivation of Arabidopsis Will hac After sterilizing, T1 seeds of the Arabidopsis thaliana mutant (Cat. No. SALK_058016C, Gene ID: At2g21045, Gene name: Rhodanese / cell cycle control phosphatase superfamily protein) were evenly spread on 1 / 2 MS medium and incubated in a lighted incubator for 5 days. When the plants had three leaves, they were transplanted to nutrient soil. During this time, the first 4 cm long primary inflorescence was trimmed to promote the growth of more lateral inflorescences.

[0067] Planting hac The mutant Arabidopsis thaliana plants were cultured in an artificial climate chamber under the following greenhouse conditions: 14 h light, 10 h dark; daytime temperature of 26°C, nighttime temperature of 20°C; relative humidity of 60%; light intensity of 210 μmol / m 2 / s(PAR).

[0068] 3. Infection by sticking flowers (1) Preparation of flower dipping solution Agrobacterium 1 bacterial solution (OD 600 0.8) and Agrobacterium tumefaciens 2 bacterial solution (OD 600 0.8) was added to LB medium containing kanamycin and rifampicin, and cultured at 28℃ for 24 h. The bacterial solution was centrifuged and the bacteria were then 600 Resuspend the culture medium at a concentration of 0.8 in 120 mL of infiltration solution (ultrapure water containing 10% w / v sucrose and 400 μL / L Silwet-77) to obtain the floral infiltration solution containing Agrobacterium 1 and the floral infiltration solution containing Agrobacterium 2, respectively. Mix thoroughly before infiltration.

[0069] (2) Infection hac The mutant Arabidopsis thaliana was cultured until it bolted and flowered, and the fruit pods and pollinated flowers were removed before transformation. hac The aboveground part of the mutant Arabidopsis thaliana was immersed in the floral solution containing Agrobacterium tumefaciens 1 for 45 seconds with slight shaking; the infection was repeated three times with a week interval between each infection; the culture was continued until the plant matured, and the seeds were collected and placed in a dry environment to obtain the plants infected with Agrobacterium tumefaciens 1. hac The seeds of the mutant Arabidopsis thaliana were designated as seeds 1.

[0070] According to the same method, the Agrobacterium tumefaciens 2-infected hac The seeds of the mutant Arabidopsis thaliana were designated as seeds 2.

[0071] 3. Screening and identification of infected seeds Seeds 1 and 2 were screened using 1 / 2MS medium supplemented with 30 mg / L hygromycin, and the seeds 1 and 2 that continued to grow were retained and transplanted into nutrient soil for further cultivation, and the heterologous expression of PvACR3 was obtained. hac Mutant Arabidopsis (denoted as PvACR3 Arabidopsis) and heterologously expressing PvACR3;1 hac mutant Arabidopsis thaliana (denoted as PvACR3;1 Arabidopsis thaliana).

[0072] 4. Arabidopsis hybridization Once both plants have grown to bolting and flowering, prepare for hybridization, using the PvACR3 Arabidopsis thaliana as the female parent and the PvACR3;1 Arabidopsis thaliana as the male parent. Under a dissecting microscope, use tweezers to remove all sepals, petals, and stamens from the female parent about to flower, retaining only the stigma. Simultaneously, use finely pointed tweezers to carefully remove the stamens from the male parent. Apply pollen from these stamens to the stigma of the female parent under a dissecting microscope. Mark the hybrid plant with a marker. Continue culturing until the plant matures, then harvest the T0 generation seeds and store them in a dry environment.

[0073] 5. Hybrid seed screening and identification (1) Hybrid seed screening The T0 generation seeds were screened using 1 / 2MS medium containing 30 mg / L hygromycin, and the T0 generation seeds that continued to grow were retained to obtain the PvACR3-PvACR3;1 hybrid. hac The T0 generation seeds of Arabidopsis thaliana were transplanted into nutrient soil for further cultivation. After the plants matured, the hybrids PvACR3-PvACR3;1 were obtained. hac T1 generation seeds of Arabidopsis thaliana, set aside.

[0074] (2) Hybrid seed identification 30 T1 generation PvACR3-PvACR3;1 hybrids were extracted hac Total RNA from Arabidopsis thaliana was reverse transcribed to obtain cDNA, which was used as a template for qRT-PCR analysis using the quantitative PCR primers shown in Table 1, Taq Pro Universal SYBR qPCR Master Mix, and QuantStudio 3 Real-time Fluorescence Quantitative PCR Detection System. The actin gene (AtActin) from Arabidopsis thaliana was used as an internal control. The relative quantification of 2 -ΔΔCt Methods The relative expression levels of PvACR3 and PvACR3;1 genes in each plant were calculated.

[0075] Table 1 Quantitative PCR primers

[0076] like Figure 1 As shown, 30 T1 generation PvACR3-PvACR3;1 hybrids hac Both PvACR3 and PvACR3;1 genes were expressed in Arabidopsis thaliana (L1-L30). The relative expression levels of PvACR3 and PvACR3;1 genes were comparable in L9, the relative expression level of PvACR3;1 gene in L25 was significantly higher than that of PvACR3 gene, and the relative expression level of PvACR3 gene in L26 was significantly higher than that of PvACR3 gene. In other words, the PvACR3 and PvACR3;1 genes had different expression levels in these three lines. Therefore, L9, L25 and L26 were selected for subsequent experiments.

[0077] Example 3 Identification of the Arsenic Accumulation Ability of PvACR3-PvACR3;1 Hybrid Arabidopsis thaliana 1. Arabidopsis thaliana arsenic treatment plate experiment The T1 seeds of wild-type Arabidopsis thaliana (denoted as WT) were hac The T1 generation seeds of the mutant Arabidopsis thaliana (No.: SALK_058016C, denoted as hac), the T1 generation seeds of the PvACR3 Arabidopsis thaliana prepared in Example 2 (denoted as PvACR3), the T1 generation seeds of the PvACR3;1 Arabidopsis thaliana (denoted as PvACR3;1), and three PvACR3-PvACR3;1 hybrids were obtained. hac After disinfection, the T1 generation seeds of mutant Arabidopsis thaliana (L9, L25 and L26) were evenly spread on 1 / 2 MS medium. After the plants sprouted at the same time, they were transplanted into 1 / 2 MS medium (denoted as CK), 1 / 2 MS medium containing 10 μM AsIII (denoted as 10 μM AsIII) and 1 / 2 MS medium containing 150 μM AsV (denoted as 150 μM AsV) respectively. Three seedlings were transplanted into each medium for each plant.

[0078] The culture conditions were as follows: 14 hours of light, 10 hours of darkness; daytime temperature of 26°C, nighttime temperature of 20°C; relative humidity of 60%; light intensity of 210 μmol / m 2 / s(PAR).

[0079] After 14 days of culture, the phenotype of each plant in each culture medium was observed, and the aboveground and root tissues were collected for subsequent testing.

[0080] 2. Determination of Arabidopsis plant biomass Each plant was cut at the root-stem junction with a razor blade to ensure complete separation of the aboveground portion from the root system. Root samples were immersed in pre-chilled arsenic desorption solution (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 minutes to remove arsenic adsorbed in the root apoplast. After rinsing the roots with water, they were spread flat on paper and gently pressed to remove surface moisture. The fresh weight of the aboveground portion and root system were weighed separately using an analytical balance and recorded. The aboveground fresh weight was used as the plant biomass.

[0081] 3. Determination of arsenic concentration in Arabidopsis plants The aerial parts and roots of each plant grown in different culture media were weighed and placed in digestion tubes. The plant tissues were digested in a graphite furnace digester at 105°C using a concentrated nitric acid-hydrogen peroxide system (3 mL 1:1 (w / v) HNO3 and 1 mL 30% (w / v) H2O2) from USEPA3050B. The arsenic concentration (μg kg -1 The total amount of arsenic in each plant was obtained by multiplying the arsenic concentration by the biomass.

[0082] 4. Measurement results like Figure 2 A and Figure 3 As shown in A, under arsenic-free treatment, three strains of PvACR3-PvACR3;1 hybrid hac The growth of mutant Arabidopsis thaliana was basically the same as that of other Arabidopsis thaliana plants, and there was no significant difference in aboveground biomass.

[0083] like Figure 2 B and Figure 3 As shown in B, under 10 μM AsIII treatment, three PvACR3-PvACR3;1 hybrids hac The growth of mutant Arabidopsis, PvACR3 Arabidopsis and PvACR3;1 Arabidopsis is basically the same, while that of wild-type Arabidopsis and hac The growth of mutant Arabidopsis thaliana was poor; 3 hybrids of PvACR3-PvACR3;1 hac There was no significant difference in the aboveground biomass of the mutant Arabidopsis thaliana compared with that of other Arabidopsis thaliana plants.

[0084] like Figure 2 C and Figure 3 As shown in C, under 150 μM AsV treatment, three PvACR3-PvACR3;1 hybrids hac The mutant Arabidopsis thaliana grew better than other plants, and the hybrid PvACR3-PvACR3;1 hacThe aboveground biomass of mutant Arabidopsis thaliana was significantly higher than that of wild-type Arabidopsis thaliana, hac The aboveground biomass of mutant Arabidopsis thaliana, PvACR3 Arabidopsis and PvACR3;1 Arabidopsis thaliana increased significantly, with an increase of 49% to 100% compared with wild-type Arabidopsis. hac The expression of ACR3 in Arabidopsis thaliana increased by 95% to 166% compared with the mutant, by 12% to 54% compared with the PvACR3;1 Arabidopsis, and by 38% to 89% compared with the PvACR3;1 Arabidopsis.

[0085] like Figure 4 As shown in A, under 10 μM AsIII treatment, three strains of PvACR3-PvACR3;1 hybrid hac There was no significant difference in the aboveground arsenic concentration of the mutant Arabidopsis plants compared with other Arabidopsis plants. Figure 4 As shown in B, under 10 μM AsIII treatment, three strains of PvACR3-PvACR3;1 hybrid hac The arsenic concentration in the roots of mutant Arabidopsis was significantly lower than that in wild-type Arabidopsis. hac mutant Arabidopsis and PvACR3;1 Arabidopsis, and was significantly lower than PvACR3 Arabidopsis. Figure 4 As shown in C, under 150 μM AsV treatment, three PvACR3-PvACR3;1 hybrids hac The arsenic concentration in the aboveground part of mutant Arabidopsis was significantly higher than that in wild-type Arabidopsis. hac The mutant Arabidopsis thaliana, PvACR3 Arabidopsis and PvACR3;1 Arabidopsis showed an increase of 41% to 73% and 44% to 77% compared with PvACR3 Arabidopsis and PvACR3;1 Arabidopsis. Figure 4 As shown in D, under 150 μM AsV treatment, three PvACR3-PvACR3;1 hybrids hac The arsenic concentrations in the roots of mutant Arabidopsis were lower than hac The mutant, PvACR3 Arabidopsis and PvACR3;1 Arabidopsis, among which the root arsenic concentration of L25 was significantly reduced by 53% compared with that of PvACR3;1 Arabidopsis.

[0086] like Figure 5 As shown in A, under the condition of 10μM AsIII treatment, the three strains PvACR3-PvACR3;1 hybrid hac The total arsenic content in the aboveground parts of mutant Arabidopsis thaliana was significantly different from that in wild-type Arabidopsis thaliana and hac There was no significant difference between the mutant Arabidopsis thaliana. Figure 5 As shown in B, under the condition of 150μM AsV treatment, the three PvACR3-PvACR3;1 hybrids hac The total amount of arsenic in the aboveground parts of mutant Arabidopsis was significantly higher than that in wild-type Arabidopsis,hac The mutant Arabidopsis, PvACR3 Arabidopsis and PvACR3;1 Arabidopsis increased by 89% to 127% and 137% to 185% compared with PvACR3 Arabidopsis and PvACR3;1 Arabidopsis.

[0087] The above results show that the simultaneous heterologous expression of PvACR3 and PvACR3;1 in Arabidopsis and the deletion of hac can significantly improve the growth of plants under high AsV concentration conditions, enhance arsenic hyperaccumulation ability, and increase arsenic accumulation.

[0088] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Use of PvACR3 protein and / or PvACR3 gene in combination with PvACR3;1 protein and / or PvACR3;1 gene in improving the arsenic hyperaccumulation ability of plants, characterized in that: The plants are plants other than centipede grass.

2. Use of PvACR3 protein and / or PvACR3 gene in combination with PvACR3;1 protein and / or PvACR3;1 gene in cultivating plants for remediating arsenic-contaminated soil, characterized in that: The plants are plants other than centipede grass.

3. Use of a biomaterial that promotes the expression of PvACR3 protein and / or PvACR3 gene and promotes the expression of PvACR3;1 protein and / or PvACR3;1 gene in improving the arsenic hyperaccumulation ability of plants, characterized in that: The plants are plants other than centipede grass.

4. Use of a biomaterial that promotes the expression of PvACR3 protein and / or PvACR3 gene and promotes the expression of PvACR3;1 protein and / or PvACR3;1 gene in cultivating plants for repairing arsenic-contaminated soil, characterized in that: The plants are plants other than centipede grass.

5. The use according to any one of claims 1 to 4, characterized in that: The plant is Arabidopsis thaliana.

6. The use according to claim 3 or 4, characterized in that: The biomaterial is any one of the following (1) to (4): (1) a nucleic acid molecule 1 encoding a PvACR3 protein, and a nucleic acid molecule 2 encoding the PvACR3;1 protein; (2) an expression cassette containing the nucleic acid molecule 1 and / or the nucleic acid molecule 2 described in (1); (3) a recombinant expression vector containing the nucleic acid molecule 1 and / or the nucleic acid molecule 2 described in (1); (4) A microorganism containing the recombinant expression vector described in (3).

7. A method for creating plants with arsenic hyperaccumulation ability, characterized in that: The PvACR3 gene and the PvACR3;1 gene are heterologously expressed in the plant.

8. The method according to claim 7, characterized in that The plant is Arabidopsis thaliana.

9. The method according to claim 7, characterized in that The plant heterologously expressing the PvACR3 gene is used as parent 1, and the plant heterologously expressing the PvACR3;1 gene is used as parent 2. Parent 1 and parent 2 are hybridized; after screening, the progeny heterologously expressing both the PvACR3 gene and the PvACR3;1 gene are retained to obtain the product.

10. Use of the arsenic hyperaccumulator plant produced according to the method according to any one of claims 7 to 9 in remediation of arsenic-contaminated soil.

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