Recombinant vectors for creating highly arsenic-accumulating engineered rice and their applications in arsenic-contaminated water and soil remediation.

Abstract

To provide a recombinant vector for creating high-arsenic-accumulation engineering rice and application of the recombinant vector in arsenic-polluted water body and soil remediation.SOLUTION: By selecting a rice OsLsi1 gene promoter pLsi1 to drive expression of a Pteris vittata PvACR3 gene of ciliate in rice, under such driving the PvACR3 protein can transfer arsenic absorbed by rice roots to an above-ground part, and since an expression amount is lower than that of a common constitutive Ubiquitin promoter pUbi, a poisoning phenomenon caused by excessive accumulation of heavy metals can be avoided, a balance between an accumulation amount and tolerance is achieved, and not only an increase in arsenic accumulated in the above-ground part of rice is ensured but also biomass is ensured.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a recombinant vector for creating high-arsenic-accumulating engineered rice and its application to the purification of arsenic-polluted water areas and soil.

Background Art

[0002] The problem of arsenic pollution in water areas and soil has become a global concern. Arsenic in the environment may have toxic and carcinogenic effects on humans through the food chain. The purification of water areas and soil contaminated with arsenic has become an urgent task. Existing arsenic-polluted water treatment technologies mainly include physical and chemical methods such as ultrafiltration, precipitation, coagulation, ion exchange, and adsorption technologies, but they have high economic costs and certain dependencies. Arsenic-polluted soil treatment technologies mainly include technologies such as soil leaching, in-situ chemical oxidation-reduction, solidification-stabilization, heat treatment, and electrochemical purification. However, these methods have large investment amounts and high costs, so it is not only difficult to promote large-scale application, but also cause damage to soil structure, reduction of microbial activity, and reduction of soil fertility, having a profound impact on soil health, so they cannot be applied to the purification of large areas of paddy fields slightly contaminated with arsenic. Plant purification technology that uses hyperaccumulator plants to absorb, transform, and concentrate pollutants has become a new type of environmental pollution management technology. The desert grass of ciliates is the most effective plant among the arsenic hyperaccumulator plants discovered so far, but it has the disadvantages of a long growth and reproduction cycle and being easily affected by environmental conditions, so there are limitations in its application to arsenic pollution purification.

Summary of the Invention

[0003] In order to solve the above problems, the present invention provides a recombinant vector for creating high-arsenic-accumulating engineered rice​​​​​​​​​​​​ The recombinant vector described in this invention provides applications for culling and remediation of arsenic-contaminated waters and soils. The above-ground parts of the engineered rice plants created by this method have high arsenic accumulation levels, strong arsenic tolerance, and biomass It is large, has a short growth cycle, mature planting techniques, is suitable for hydroponics, and is suitable for both paddy and dry fields. Furthermore, it has the advantage of being economical, fast, simple, and effective for reducing and purifying heavy metals in water bodies and soil. Furthermore, harvesting before the rice hulling period when biomass is at its maximum shortens the purification cycle. Not only does this help, but it also helps avoid the risks associated with rice consumption. To achieve the above objectives, the present invention provides the following technical solutions: This invention provides a recombinant vector for creating high arsenic-accumulating engineered rice, and recombinant genetics The plant expression vector contains the recombinant gene and the pLsi1 promoter and PvACR It contains 3 genes, and the nucleotide sequence of the pLsi1 promoter is SEQ ID NO As shown in .1, the nucleotide sequence of the PvACR3 gene is SEQ ID NO.2 It will be shown. Preferably, the plant expression vector includes the pSN1301 vector. The present invention provides a primer set for constructing the recombinant vector described in the above technical solution. It includes a first primer pair and a second primer pair, and the sequence of the first primer pair is SEQ The sequence of the second primer pair is shown in ID NO.7 and SEQ ID NO.8. These are shown in SEQ ID NO.9 and SEQ ID NO.10. The present invention provides a method for constructing recombinant vectors as described in the above technical solution, and the following steps Includes: Using the entire rice DNA as a template, the first PCR amplification was performed to obtain the pLsi1 promoter. , We targeted the ciliate desert grass cDNA and performed a second PCR amplification to obtain the PvACR3 gene. The pLsi1 promoter is inserted into the plant expression vector by double enzyme digestion. A transforming vector containing the si1 promoter was obtained. The PvACR3 gene is transformed by homologous recombination into the pLsi1 in the transformation vector. The recombinant vector is obtained by inserting it downstream of the motor. Preferably, the primer pair for the first PCR amplification is the primer described in the technical solution above. The set includes the first primer pair, and the primer pair for the second PCR amplification is the above technical solution Includes the second primer pair in the primer set described in the solution. The present invention relates to the recombinant vector described in the above technical solution, and the ply described in the above technical solution. Recombinant vectors constructed by marset or the construction method described in the above technical solution This provides an application in the creation of high arsenic-accumulating engineered rice. This invention provides a highly arsenic-accumulating engineered rice, and the recombinant vector described in the above technical solution and Contains transgenic recipeoin. Preferably, the variety of the transgenic recipe rice is Zhonghua11. include. The present invention relates to the recombinant vector described in the above technical solution, and the ply described in the above technical solution. Recombinant vectors constructed by marset or the construction method described in the above technical solution or the high arsenic-accumulating engineered rice described in the above technical solutions, in arsenic-contaminated waters and / or soil It offers applications in soil purification. The present invention provides a method for remediating arsenic-contaminated water and / or soil, comprising the following steps: : Plant highly arsenic-accumulating engineered rice in water bodies and / or soils that need to be purified, and before the milling stage, the rice plants are treated in the soil. Harvest the upper plants. The present invention has the following beneficial effects: The present invention provides a recombinant vector for creating high-arsenic-accumulating engineered rice, including a recombinant gene and a plant expression vector. The recombinant gene contains a pLsi1 promoter and a PvACR3 gene. The nucleotide sequence of the pLsi1 promoter is shown in SEQ ID NO.1, and the nucleotide sequence of the PvACR3 gene is shown in SEQ ID NO.2. The recombinant vector provided by the present invention selects the rice root-specific promoter pLsi1 to drive the expression of the PvACR3 gene of the ciliate desert grass in rice. Under this drive, the PvACR3 protein can move the arsenic absorbed by the rice roots to the above-ground parts. Since the expression level is lower than that of the general constitutive promoter pUbi, it can avoid the poisoning phenomenon caused by excessive accumulation of heavy metals, achieve the balance of accumulation amount and tolerance, ensure the increase of arsenic accumulation in the above-ground parts of rice, and also ensure the biomass. The engineered rice plants created using the recombinant vector described in the present invention have high arsenic accumulation in the above-ground parts, strong arsenic tolerance, high effective arsenic removal rates from water arsenic and soil, large biomass, short growth cycle, mature cultivation technology, are suitable for water areas, paddy fields and dry fields, and have the advantages of being economical, rapid, simple and effective for reducing and purifying heavy metals. 子と植物発現ベクターを含み、前記組換え遺伝子はpLsi1プロモーターとPvACR 3遺伝子を含み、前記pLsi1プロモーターのヌクレオチド配列はSEQ ID NO .1に示され、前記PvACR3遺伝子のヌクレオチド配列はSEQ ID NO.2に 示される。本発明によって提供される組換えベクターは、イネ根部の特異的プロモーター pLsi1を選択してイネでの繊毛虫の砂漠草PvACR3遺伝子の発現を駆動し、その 駆動下でPvACR3タンパク質はイネ根部によって吸収されたヒ素を地上部に移動させ ることができ、発現量が一般的な構成的プロモーターpUbiよりも低いため、重金属の 過剰蓄積による中毒現象を回避することができ、蓄積量と耐性のバランスを達成し、イネ の地上部の蓄積ヒ素の増加が保証され、バイオマスも確保される。本発明に記載の組換え ベクターを用いて作成された工学イネ植物の地上部のヒ素蓄積量が高く、ヒ素耐性が強く 、水域ヒ素と土壌の有効なヒ素除去率が高く、バイオマスが大きく、生育周期が短く、栽 培技術が成熟しており、水域·水田と乾田に適しており、重金属の減少と浄化に対して経 済的で迅速かつ簡単で有効である利点がある。 Furthermore, the present invention selects a simple rice harvesting method. Since soil arsenic is transported and accumulated in the above-ground parts of rice, arsenic removal can be achieved by harvesting only the above-ground parts without removing the rootstock, and it has good operability. され、根株を除去することなく、地上部のみを収穫すればヒ素除去を達成でき、良好な作 業性を有する。

Brief Description of the Drawings

[0004] To more clearly explain the technical solutions in the embodiments of the present invention or the prior art, the following The attached drawings that need to be used in the examples are briefly described below. [Figure 1] This is a flowchart for creating pLsi1::PvACR3 transgenerated high arsenic-accumulating engineered rice. [Figure 2] This is an electrophoresis diagram showing the amplification of the pLsi1 promoter and the PvACR3 gene. [Figure 3] This is a schematic diagram of the pLsi1::PvACR3 binary expression vector construction. [Figure 4] This is a quantitative expression diagram of the PvACR3 gene in transgenerated plants of pUbi::PvACR3 and pLsi1::PvACR3. [Figure 5] This shows the growth map and biomass measurement results of the pLsi1::PvACR3 transgenerated plant in contaminated water and soil. [Figure 6] This figure shows the accumulated arsenic content of the pLsi1::PvACR3 transgenetic plant in contaminated water and soil. [Figure 7] This figure shows the effective arsenic residue levels in contaminated soil after remediation of the pLsi1::PvACR3 transgenerated plant. [Figure 8] This is an electrophoresis diagram amplified by the pUbi promoter. [Figure 9] This is a schematic diagram of the pUbi::PvACR3 binary expression vector construction. [Figure 10] This figure shows the growth results of the pUbi::PvACR3 transgenerated plant in contaminated water. [Figure 11] This figure shows the results after culturing pUbi::PvACR3 transgenerated plants in contaminated soil for two weeks. [Modes for carrying out the invention]

[0005] This invention provides a recombinant vector for creating high arsenic-accumulating engineered rice, and recombinant genetics The plant expression vector contains the recombinant gene and the pLsi1 promoter and PvACR It contains 3 genes, and the nucleotide sequence of the pLsi1 promoter is SEQ ID NO As shown in .1, the nucleotide sequence of the PvACR3 gene is SEQ ID NO.2 This was shown, and specifically as follows: SEQ ID NO.1:5'-aagtaaaaccatggttattctgaat ctaaacgatgcttcttcagagaatggttcagggcaccata tcaacttagcagatatcggatggtacacctgacctcaaca tggggtgcttttgctactgcttgatcacagggtagggcct agcattatgccaggaaatgtagttagattcatacacacaa aagtgattaacaagaactacactagtggcctgaggagcgc agacaaaagaagatcaataatcagcgaaggtaattatgcc agcttctggatggatggtgcatctgtgattgattcactga tctgatcgctcactcgtcagctatcttggttccatgcctc tcatgaaaagctaaggggttgcagagaagcgtggattttc ccctcttgtggcctggcctcatgccaatgcgctagtctcat ctcaggcagcacaagctgtcttttcatcctgtagatcgtg caaaataaggtgctggttctaaacgtgccccagaaagcgc tctctgtttgcacatgtgtgtatttagtggtatttttcag taccaatatccatttattttcatttaatttgctttgctcg taaagtagttcttgattctacatgtatacatctacaaagt attgatgagtgctcattacagaaggcatctcaaatcaata aattatctgcatttttgcgaaagaaacctgattgaaacac ctcgtgaacgaaatacctagcaaactctgtaaggcctgag attttcaccaagtcgagtggctgatctgcaacgagctgta ccgatcaaaatatgggttctttcattctttgtgatgtgtg ctgattttccaatcgaaaatcattgtggcaagattttgtc agggcatcgccgtccacactctgctcccccaccggggatg cctaccaagggaagaagaggcgtcataactgccatacact tgtgctgtctacggccatcagagcattgaccatacgggcc tacttcacagaacatgattgacctgtaaaaatcagcttca gactttgagttccgaatcctgttgatttttcattgagttt aattaggagtaggtggcattgctcttcagatgatatgtcg atttctggcattgctctttttaatacaaggtgatgaaaat tcagctgcctgaattggagttttgttttcctgaactgtag tatctgaactctgaagacagttactgatagtggtagtaca agatagtactccctccgttttgaaatgtttgacgccgttg actttttatcacatgtttgatcattcgtcttattcaaaaa atttaagtaattattaattattttcctatcatttgattca ttattaaatatatttttatgtagacatataattttacata tctcacaaaagtttttgaataagacgaacagttaaacatg tgctaaaaagtcaacggtgtcaaacatttcgaactggagg gagtatcctacaggtacagtacggcaaaaaaagaaaaact gaatgtgagctaagctcaatgagagaagctaggattgcaa attgctgaagtactccaactgacatgagatttttcaatag tagcaggtcagttttgacagtgaccatccaagtgcaacgt cctctgctctgacattgcttagcattgctaaccgaagcat gcacactgcgtaatagagtggttaggataaccccttattg taatgtcacctttgcaaatccttaactgctcggatatttc aatttggtcaccagagatggcaatcctacaattgaaaatt tgttcagttgccacggatccatcattaatctggcaatggc ggcaacctctgacagggacaatggcaaattcggccaatag taaatttcggtacggtttatcctagttggcattggcacac atggttcgtctcttctacgagtatagattatgaaaaatgt caacttacaacaggtgacgaatttcgcaaaaaaaacgtat taacattcggcatggaaaacgtacgtagaatgaccaaaaa tatccatccctatagtatcatttctttcaggggagccccc aatctacaaaagaaaaagaatttgttcgtcacccatatat cggcgtcatgacctcgacgtcgcgctttatccaggcatat agtttacaacaccttgtgaattgaaaacccacaattattt cagtctaacagcagacagaggcaacgttgctctcgttgtc gttcacggggggatgacgcgcggttttatgccctcgacga gaatacaaaatcaagtatgcgtttctgtttctcggccaat gctgatccgacaacgtgtttgaacggattaaacaaaatct gaatccccgtcgaaaaattagaccagaaacaatgatctta tgctgattaattagggctaatgagctatgcatgcaagcac tgtacccagtggtgctccgacaagtaggcctgcctaatca aaaggcagtgaggactgtaactactagtacctgcc-3’、 SEQ ID NO.2:5’-atggagaactcaagcgcggagcgga agcagcaactggccctggacattgctgatgggaacgaccc gtccgatgcggcaaaaaaccctgacggcagaactaaactg caagggcttttcaagcagctttcgctgctcgatagatatc tctatgtctggattttcatagtcatggcagtgtccatcat ctttgggtactatgtcaagggtgtgaagaaggcgttccaa gtagcggagataacatccgtttcactacccatcgcaattg ggctgtgggtaatgatgtaccctgtcctatgcaaggtgca gtatgaaattctgggtggggtcttaaggcaagcagggtca ctcaagacaatctcactgagtgttgtgctgaactgggtag taggacccgcactcatgacaggtttagcgtgggcgaccct cccagatttgccagatttccgcactggtgtgatcctggtt ggcatagcacgatgcatagcgatggttctgatttggaatg atctcgcaaaaggagacgctgactattgcgccattctggt ggccatcaattcgattttgcagattatcctgtttactccg gtggcactcttatacctcaaagtggtctcccgaggcaagg gatttcatgtgagctcatggacagtggcaaagagtgtgct ccttttccttggggttccgcttgcggcaggtgttcttaca cgactcatcttgatgaatgcttttgggcggaagtggtacg agtcgaagtttctgcgctttatcggaccttgggctctcat tggtttgctgtacaccatttttgtcatgttctcaattcaa gctcatcagattgttgacaacatagggcatgtggtaagag ttgcagtaccacttcttctgtactttggcattcttttctt tgggtcattgggcatatgtaggtggctgaaggtgccatac ccattgatggtcacacaatgctttacggctgcgagcaaca atttcgagcttgcgattgcagttgcagttggtagctttgg cattgattccacgcaggctcttgctgccacaattggccct cttattgaagtaccggtgctgttgctgttcgtatacatcg ttggcttctttcagaggaaggggccttctgtttag-3'. In the present invention, the plant expression vector preferably includes the pSN1301 vector. This invention provides a target gene constructor recombinant vector for the desert grass of a naturally occurring arsenic-accumulating plant ciliate. We selected the gene PvACR3 which encodes an arsenite transporter protein, and The encoded protein has high transporter efficiency for arsenic, and the target gene The rice root-specific promoter pLsi1 was used as a regulatory element to adjust expression. Selected, this promoter specifically expresses the target gene in the rice root, and the expression level is typical. It employs a weaker promoter than the constitutive promoter pUbi to target downstream genes By initiating this process, it is possible to avoid poisoning caused by the excessive accumulation of heavy metals. The present invention provides a primer set for constructing the recombinant vector described in the above technical solution. It includes a first primer pair and a second primer pair, and the sequence of the first primer pair is SEQ The sequence of the second primer pair is shown in ID NO.7 and SEQ ID NO.8. These are shown in SEQ ID NO.9 and SEQ ID NO.10. The first primer pair specifically amplifies the pLsi1 promoter sequence, and the second primer The pair can specifically amplify the PvACR3 gene.

[0006] The present invention provides a method for constructing recombinant vectors as described in the above technical solution, and the following steps Includes: Using the entire rice DNA as a template, the first PCR amplification was performed to obtain the pLsi1 promoter. , We targeted the ciliate desert grass cDNA and performed a second PCR amplification to obtain the PvACR3 gene. The pLsi1 promoter is inserted into the plant expression vector by double enzyme digestion, A transforming vector containing the si1 promoter was obtained. The PvACR3 gene is transformed by homologous recombination into the pLsi1 in the transformation vector. The recombinant vector is obtained by inserting it downstream of the motor. In the present invention, the primer pair for the first PCR amplification is preferably the same as the above technical solution. The primer set includes the first primer pair from the set of primers described, and the primers for the second PCR amplification. The pair preferably includes the second primer pair in the primer set described in the technical solution above. . The present invention relates to the recombinant vector described in the above technical solution, and the ply described in the above technical solution. Recombinant vectors constructed by marset or the construction method described in the above technical solution This provides an application in the creation of high arsenic-accumulating engineered rice. This invention provides a highly arsenic-accumulating engineered rice, and the recombinant vector described in the above technical solution and The present invention includes transgenic recipeintoine. The rice varieties selected in this invention include Zhonghua11. The infectious recipient plant is the rice variety Zhonghua11, which is stable. In addition to being genetically transformed, they have a large biomass, a short growth cycle, and cultivation techniques It is mature, highly adaptable, and has the advantage of being able to be planted in both paddy and dry fields, China It can be widely used and applied to paddy fields and dry fields in the southern and northern regions, and therefore, soil purification It has potential for a wide range of applications in the field of chemistry. The present invention relates to the recombinant vector described in the above technical solution, and the ply described in the above technical solution. Recombinant vectors constructed by marset or the construction method described in the above technical solution or the high arsenic-accumulating engineered rice described in the above technical solutions, in arsenic-contaminated waters and / or soil It offers applications in soil purification. The present invention provides a method for remediating arsenic-contaminated water and / or soil, comprising the following steps: : Plant highly arsenic-accumulating engineered rice in water bodies and / or soils that need to be purified, and before the milling stage, the rice plants are treated in the soil. Harvest the upper plants. In this invention, a simple rice harvesting method is selected, and arsenic in the soil is transported and accumulated in the above-ground parts of the rice plant. By harvesting only the above-ground parts without removing the rootstock, arsenic removal can be achieved. It can be harvested before the rice hulling period when biomass is at its maximum, and it has good workability. This not only shortens the purification cycle but also helps avoid the risk associated with rice consumption. To further illustrate the present invention, the present invention is described below in conjunction with the accompanying drawings and examples. Recombinant vectors for creating highly arsenic-accumulating engineered rice and for remediation of arsenic-contaminated soil. The application to the present invention will be described in detail, but should be interpreted as limiting the scope of protection of the present invention. There isn't one.

[0007] Example 1 Cloning of the regulatory element pLsi1 promoter and the arsenic hyperaccumulation gene PvACR3 : The root system of rice Zhonghua11 seedlings was pulverized into a powder using liquid nitrogen, and Novozy The entire genome DNA was extracted using the mes plant whole DNA extraction kit, and the rice OsLsi1 The upstream 2500bp sequence of the gene (SEQ ID NO.1) is used as a template for amplification via pLsi. One promoter was selected, and the upstream and downstream primers were synthesized by the Beijing Genome Institute. The nucleotide sequence is as follows: Upstream primer: 5'-aagtaaaaccatggttattctgaat-3', SEQ ID NO.3, Downstream primer: 5'-ggcaggtactagtagttacagtcct-3', SEQ ID NO.4. Ciliated desert grass seedlings were pulverized into a powder using liquid nitrogen, and the whole Novozymes plant RN The total RNA was extracted using the A extraction kit, and the Novozymes reverse transcription extraction kit was used. Then, reverse transcribe the PvACR3 gene cDNA sequence provided by NCBI ( According to SEQ ID NO.2), the upstream and downstream primers were designed at the Beijing Genome Research Institute. It was synthesized by the laboratory, and its nucleotide sequence is as follows: Upstream primer: 5'-atggagaactcaagcgcggagc-3', SEQ ID NO.5, Downstream primer: 5'-ctaaacagaaggccccttcctctga-3', SEQ ID NO.6. After primer synthesis, KOD high-fidelity enzyme was used to extract total DNA from rice roots and ciliate sand, respectively. Using the cDNA of the common grass as a template, PCR amplification was performed to obtain pLsi1 and PvACR3. The PCR amplification reaction system is as follows: 2× PCR Buffer for KO D 25 μl, dNTP 10 μl, E (KOD×NEO high fidelity enzyme) 1.25 μl, upstream primer 1.25 μl, downstream primer 1.25 μl, template 2 0.5 μl and ddH 2 O 8.75 μl, The reaction procedure for the PCR amplification is as follows: the law of nature: Pre-denaturation at 94°C for 2 minutes, denaturation at 98°C for 30 seconds, and annealing at 60°C for 10 seconds. The reaction was carried out at 72°C for 3 minutes / kb, for a total of 32 cycles, with each cycle lasting 10 minutes at 72°C. 。 The amplified product is separated into target bands by 1% agarose gel electrophoresis, and then all-in-one go The pLsi1 and PvACR3 amplification products were recovered using a Ludger recovery and extraction kit, and then electrophoresed. The diagram is shown in Figure 2. The recovered product was then extracted using the pEASY-Blunt Cloning Kit. The pEASYR- Blunt vector was ligated to the E. coli receptor Top1 After transformation to 0, monoclonal strains are picked for sequencing and correctly sequenced The quenched monoclones were inoculated into LB medium containing ampicillin and incubated at 37°C. After evening shaking culture, plamid was extracted using a plamid mini extraction kit, and pLsi1- Prepare by obtaining 19B and PvACR3-19B. Example 2 Construction of heterogeneous fusion expression binary vectors: pSN1301 binary vector containing chaotropic screening genes back We used bone vectors and further constructed heterogeneous fusion expression binary vectors. (1) Acquisition of the pLsi1 promoter: including the Hind III and Sal I digestive junctions We designed PCR upstream and downstream primers for the pLsi1 promoter, and the Beijing Genome Institute... It is synthesized, and its nucleotide sequence is as follows: Upstream primer: 5'-aagcttaagtaaaaccatggttattctga atctaa-3', SEQ ID NO.7, Downstream primer: 5'-gtcgacggcaggtactagtagttacagtc ctca-3', SEQ ID NO.8. After primer synthesis, pLsi1-19B was used as a template with KOD high-fidelity enzyme. PCR amplification was performed (the reaction system and procedure were the same as in Example 1), and 1% agarose was used. Electrophoresis was performed, and approximately 2.5kb was extracted using the All-in-One Gold Gel Recovery and Extraction Kit. A band of a certain size was collected and recovered using the Hind III and Sal I enzymes via PCR. The material was digested, and the digestion products were collected. Construction of the pSN1301-pLsi1 backbone vector: Hind III and Sal The pSN1301 vector was digested using I, and electrophoresis was performed using 1% agarose. Next, the large band digestion vector was collected, and the prepared Hind III and Sal I digestion vectors were used. The pLsi1 digestion product including the ligation site and the recovered digestion vector product were fermented overnight with T4 ligase. Gated, transformed with the Top 10 E. coli receptors, and selected monoclonal strains. Sequence and correctly sequenced monoclones containing kanamycin The sample was inoculated into LB medium and incubated overnight at 37°C with shaking, and then plamisdomini extraction was performed using a plamisdomini extraction kit. The donuts were extracted and prepared, and the resulting vector-plated donut pSN1301-pLsi1 was shaped This will be used as a backbone vector for qualitative transformation. (3) Construction of pSN1301-pLsi1::PvACR3 transformation vector:Xba PCR upstream and downstream of the PvACR3 gene, including the homologous recombination digestive junction of I and Kpn I. The lymer was designed and synthesized by the Beijing Genome Institute, and its nucleotide sequence is as follows: It is: Upstream primer: 5'-gagaacacgggggactctagaatggagaa ctcaagcgcgc-3', SEQ ID NO.9, Downstream primer: 5'-gggaaattcgagctcggtaccctaaacag aaggccccttcctc-3',SEQ ID NO.10. After primer synthesis, PvACR3-19B was used as a template with KOD high fidelity enzyme. PCR amplification was performed (the reaction system and procedure were the same as in Example 1), and 1% agarose was added. Electrophoresis was performed using the All-in-One Gold Gel Recovery and Extraction Kit, yielding approximately 1.2k Collect and prepare a band of size b. The constructed pSN1301-pLsi1 plasma Sudo was double-digested with Xba I and Kpn I, and the digested products were electrophoresed with 1% agarose. Then, the larger fragments are collected and prepared using the All-in-One Gold Gel Recovery Kit. PvACR3 gene containing homologous recombination junction and pSN13 with Xba I and Kpn I 01-pLsi1 plamised homologous recombination is double digested, and the extraction kit for the homologous recombination is T OROIVD R One Step Fusion Cloning Mix, and the opposite The application is as follows: One step fusion cloning mix 5 μl, double digested linear vector 200 ng, target gene 200 ng, dd H2 Add 10 μl of O, prepare the reaction system, then gently wipe several times to mix the components, and set the reaction system to 6 After allowing the reaction to proceed at 0°C for 60 minutes, the reaction solution is cooled on ice and the transformation is performed directly, or It was stored at -20°C. Homologous recombination products were transformed into E. coli receptor Top10 by thermal excitation, and monochromatic Select a strain and sequence it, and obtain a correctly sequenced monoclone. Inoculate into LB medium containing kanamycin and culture overnight at 37°C with shaking, then extract pramisdomini. Using the kit, plamisd is extracted and prepared, and the resulting recombinant plamisd is processed using pSN130. The recombinant plamised profile is represented as 1-pLsi1::PvACR3 and is shown in Figure 3. . Correctly sequenced pSN1301-pLsi1::PvACR3 recombinant Ramid was transformed into Agrobacterium rhisogenes EHA105 by thermal excitation. Plates containing kanamycin and rifampicin were coated, and positive monoclonal absorpti were used. Select and save the Globacterium to prepare it. Example 3 pLsi1::PvACR3 rice gene transformation and transgenosis-positive seedlings Fixed: The positive monoclonal Agrobacterium selected in Example 2 was administered at 50 mg / L rifampic acid. The cells were inoculated into 5 ml of LB liquid medium containing 100 mg / L kanamycin and incubated overnight. (28℃, 200rpm). Take 2 mL of Agrobacterium cultured overnight and add 40 mL Add to fresh culture medium and adjust the OD of the bacterial suspension. 600 Repeat under the same conditions until the value reaches 0.8. Eye activation was performed. Cultured Agrobacterium was centrifuged at 4000 g for 10 minutes. Then, the supernatant was removed, the precipitate was collected, and resuspended in 5 mL MS liquid medium. Contains 0 μM acetosyringone, which induces gene activation in the Agrobacterium Vir region. This promoted the entry and integration of Agrobacterium T-DNA into the plant genome. The solution will be used in subsequent gene transformation experiments. At the same time, select uniform and well-developed Zhonghua11 rice seeds and ferment them in 75% alcohol for 3 minutes. Disinfect for 0 seconds, then disinfect with 20% sodium hypochlorite for 20 minutes, and then use a healing tissue medium (MS medium). +2.0 mg / L (2,4-D) is used to induce healing tissue. After one week, the above activation is performed. EHA105 Agrobacterium rhizogenes was infiltrated into the healing tissue. 0.1 mg / M contains L 6-BA, 1.0 mg / L 2, 4-D and 100 μM acetosyringone. After inoculating into S medium and co-culturing in the dark for 3 days, a culture containing 300 mg / L cephalosporin was performed. Wash it with sterile water, and then use a screening medium (MS medium + 30 mg / L thaumarane). Chin + 500 mg / L Cephalosporin + 1.0 mg / L (2,4-D) After culturing and subculturing every two weeks, the untransformed organisms died after several weeks. The transformed organisms were destroyed, and the transformed organisms were able to continue in the Western Regions. Subsequently, the transformed resistant healing tissue was Transplant into differentiation medium (MS medium + 1.0 mg / L 6-BA + 0.2 mg / L NAA) and seedlings. The culture was continued until the seedlings grew. After seedling growth, the growth induction medium (MS medium + 30 mg / L thaumatin) was added. (+600mg / L cephalosporin) was transplanted and root culture was performed, and resistant transplants with good rooting were selected. We transplanted plants into soil and cultivated them to obtain transgenerated plants. The resulting transgenerated plants were screened for three generations by self-crossing with a segregation ratio of 3:1. We obtained the T3 generation (denoted as L1 and L2), which consists of single-copy pure lineage material. We sowed the seeds of the T3 generation. After sowing, the rice seedlings (wild type is denoted as WT) are pulverized into a powder using liquid nitrogen, and the rice Total RNA is extracted from the upper and root portions and mixed into a HiScript premix specifically for RT-qPCR. Reverse transcription is performed using Q Select RT SuperMix for qPCR. cDNA was obtained using this method, and PvAC of transgenosis-positive plants was used with the SYBR method. The expression level of the R3 gene was identified, and the internal reference gene was OsActin. The plastic used for identification was The imager sequence is as follows: PvACR3-F:5′-atggagaactcaagcgcggagcgga-3′ SEQ ID NO.17, PvACR3-R:5′-ctaaacagaaggccccttcctctga-3′ SEQ ID NO.18, OsActin-F:5′-gaagatcactgccttgctcc-3′, SEQ ID NO.19, OsActin-R:5′-cgataacagctcctcttggc-3′, SEQ ID NO.20, The identification results are shown in Figure 4. The PvACR3 gene is not expressed in the above-ground tissues of rice. It was expressed only in the roots of rice plants. Example 4 Arsenic removal amount from contaminated waters by pLsi1::PvACR3 transgenosis-positive rice measurement: The obtained T3 generation positive transgenerated plants were transplanted to arsenic-contaminated water areas and hydroponic experiments were conducted. The arsenic-containing test area was treated with 1 / 2 Hoagland nutrient solution, to which 5 μM NaAsO2 was added. The solution was prepared. After transplanting the rice plants, they were hydroponically cultured for 15 days. Rice growth at harvest time is shown in Figure 5a and Table 1. It will be shown. Table 1. Biomass (g) of wild-type and trans-genetic rice in contaminated waters. As can be seen from Figure 5a and Table 1, trans-genosis rice is superior in the contaminated water area. It demonstrated resistance to arsenic. Plant samples were collected, thoroughly washed alternately with tap water and deionized water, and then incubated at 105°C for 30 minutes. The material was insecticided and dried at 65°C until it reached a certain temperature. After crushing, it was processed to 0.0500±0.00 Weigh out 0.5g of sample and use 5ml of HNO3 and 2ml of H2O2 in a graphite digester. After complete digestion at 120°C until clarified, the volume was reduced to 50 mL. ICP-MS was performed. The arsenic accumulation concentrations in the roots and above-ground parts of wild-type rice and trans-genotic rice were detected, respectively. The amount of arsenic removed from the contaminated water was analyzed. The results are shown in Figure 6a and Table 2. Table 2 Arsenic Removal Amounts from Contaminated Waters by Wild-Type and Trans-Genosis Rice As can be seen from Figure 6a and Table 2, the amount of arsenic accumulation in the root tissue of trans-genosis rice is low. However, the accumulation of arsenic in the above-ground tissues was significantly higher than in the wild control group, indicating that a large amount of arsenic was present in the rice. It is transported to the above-ground parts of the plant, and at the same time, low accumulation in the roots reduces arsenic poisoning, improving the arsenic tolerance of rice plants. Furthermore, among them, L2 rice has a higher arsenic removal capacity in its above-ground parts. Example 5 pLsi1::PvACR3 transgenosis-positive rice and the amount of arsenic removed from contaminated soil and soil Measurement of effective arsenic residue: The resulting T3 generation positive transgenetic plants were transplanted to arsenic-contaminated soil and potted. An experiment was conducted. The test soil was collected from rice soil in Xuzhou City, Jiangsu Province (pH 5.15, total arsenic content). (Amount: 38.51 mg / kg). After transplanting rice plants, potting culture was performed for 90 days, before the flowering and hulling stage. The rice was harvested. The rice growth at the time of harvest is shown in Figure 5b and Table 3. Table 3 Biomass (g) of wild-type and trans-genetic rice As can be seen from Figure 5b and Table 3, trans-genosis rice is superior in the contaminated soil. It demonstrated resistance to arsenic. Plant samples were collected, thoroughly washed alternately with tap water and deionized water, and then incubated at 105°C for 30 minutes. The material was treated with insecticide and dried at 65°C until it reached a certain weight. After crushing, it was processed to 0.5000±0.00 Weigh out 0.5g of sample and use 5ml of HNO3 and 2ml of H2O2 in a graphite digester. After thoroughly digesting at 120°C until clarified, the volume was reduced to 50 mL. ICP-MS was performed. The arsenic accumulation concentrations in the roots and above-ground parts of wild-type rice and trans-genotic rice were detected, respectively. The amount of arsenic removed from contaminated soil was analyzed. The results are shown in Figure 6b and Table 4. Table 4. Amount of contaminated soil arsenic removed by wild-type and trans-genotic rice. TIFF0007842389000004.tif40135 As can be seen from Figure 6b and Table 4, the amount of arsenic accumulation in the root tissue of transgenic rice is Although low, the accumulation of arsenic in the above-ground tissues was significantly higher than in the wild control group, indicating that a large amount of arsenic was present in rice. It is transported to the above-ground parts of the plant, and at the same time, arsenic poisoning is reduced due to low accumulation in the roots, thus improving the arsenic tolerance of rice. The process has improved, and among them, the arsenic removal capacity of the above-ground parts of L1 rice is higher. After purification, potting soil was collected and ICP-MS was used to determine the amount of residual DGT active arsenic and B in the soil. The CR step extractive arsenic content was detected, and the DGT active arsenic extraction method was [Sun, Q., Chen, J., Zhang, H., Ding, SM, Li, Z., Wil. Liams, P.N., Cheng, H., Han, C., Wu, L.H., & Zhang, CS Improved diffusive gradient s in thin films (DGT) measurement of tota l dissolved inorganic arsenic in waters and soils using a hydrous zirconium oxide e binding layer. Analytical Chemistry 86 See 3060-3067 (2014) for the BCR step extraction method for arsenic extraction, Pueyo, M., Mateu, J., Rigol, A., Vidal, M., Lopez-Sanchez, JF, & Rauret, G. Use of t he modified BCR three-step sequential ex traction procedure for the study of trac e element dynamics in contaminated soils Environmental Pollution 152, 330-341 (2 (008) was referenced. The results are shown in Figure 7 and Table 5. Table 5 Results of DGT available arsenic and BCR step-extracted arsenic content in soils after different plant remediation processes. TIFF0007842389000005.tif95168 Note: The original soil refers to untreated soil. As can be seen from Figure 7 and Table 5, the available arsenic concentration in the purified soil is higher compared to the control soil. It decreased significantly. As can be seen from the above results, the present invention relates to the rice pLsi1 promoter and the ciliate desert grass High arsenic-accumulating engineered rice, created through heterologous fusion of the PvACR3 gene, exhibits high arsenic tolerance. In addition to possessing [certain properties], it removes a large amount of arsenic from the above-ground portion and has a high effective arsenic removal effect on the soil. By harvesting the above-ground parts of the rice plant, the arsenic-contaminated soil can be effectively treated without removing the rootstock. This method can achieve purification and is an effective solution for remediating arsenic-contaminated soil. Comparative Example 1 Cloning of the regulatory element pUbi promoter: Corn seedlings were pulverized into a powder using liquid nitrogen, and the whole genome of Novozymes plants was obtained. Using a DNA extraction kit, the entire DNA was extracted from corn Ub provided by NCBI. We designed pUbi upstream and downstream primers according to the iqutin promoter sequence, and Beijing G Synthesized by Nomu Laboratories, its nucleotide sequence is as follows: Upstream primer: 5'-ctgcagtgcagcgtgacccggtcgt-3', SEQ ID NO.11, Downstream primer: 5'-ctgcagaagtaacaccaaacaacag-3', SEQ ID NO.12. After primer synthesis, the entire maize DNA was used as a template with KOD high fidelity enzyme. pUbi was amplified (the reaction system and procedure were the same as in Example 1). The amplified product was divided into 1% The target band was separated by agarose gel electrophoresis, and the all-in-one gold gel was recovered and extracted. The product was recovered using the kit, and the electrophoresis of the pUbi amplification product is shown in Figure 8. Further recovery was then performed. The product was ligated into the pEASYR-Blunt vector and then into the E. coli receptor T Transformed into op10, selected monoclonal strains and sequenced correctly. The encapsulated monoclones were inoculated into LB medium containing ampicillin and shaken overnight at 37°C. The culture was performed, and plamis was extracted using a plamis mini extraction kit to obtain pUbi-19B Prepare it by obtaining it. Binary vector construction: (1) Acquisition of pUbi promoter: p including Hind III and BamHI digestive junction We designed the PCR upstream and downstream primers for the Ubi promoter and collaborated with the Beijing Genome Institute. It was created, and its nucleotide sequence is as follows: Upstream primer: 5'-aagcttctgcagtgcagcgtgaccc-3', SEQ ID NO.13, Downstream primer: 5'-ggatccctgcagaagtaacaccaaacaac ag-3', SEQ ID NO.14. After primer synthesis, pUbi-19B was used as a template with KOD high-fidelity enzyme. PCR amplification was performed (the reaction system and procedure were the same as in Example 1), and electrolysis was performed with 1% agarose. Electrophoresis was performed, and a sample size of approximately 2.0 kb was extracted using the All-in-One Gold Gel Recovery and Extraction Kit. The band was recovered, and the PCR-recovered product was digested using Hind III and BamHI. The digestion products described above were recovered. Construction of the pSN1301-pUbi backbone vector: Hind III and BamHI The pSN1301 vector was digested using [method / tool ​​name], and electrophoresis was performed using 1% agarose. The large band digestion vector was recovered, and the prepared Hind III and BamHI digestion junction was located. The pUbi digestion products and the recovered digestion vector products were ligated overnight with T4 ligase. The cells were then transformed with the Top 10 E. coli receptors, and monoclonal strains were selected for the next stage. Ensuring and correctly sequenced monoclones are cultured in LB culture medium containing kanamycin. The sample was inoculated into soil and incubated overnight at 37°C with shaking, and then plamised using a plamised mini extraction kit. Extract and prepare the resulting vector plamised pSN1301-pUbi, transform This will be used as the backbone vector. (3) Construction of pSN1301-pUbi::PvACR3 transformation vector:BamHI PCR upstream and downstream primers of the PvACR3 gene, including the KpnI homologous recombination digestion junction. It was designed and synthesized by the Beijing Genome Institute, and its nucleotide sequence is as follows: the law of nature: Upstream primer: 5'-gtgttacttctgcagggatccatggagaa ctcaagcgcgg-3',SEQ ID NO.15, Downstream primer: 5'-gggaaattcgagctcggtaccctaaacag aaggccccttcctc-3',SEQ ID NO.16. After primer synthesis, PvACR3-19B was used as a template with KOD high fidelity enzyme. PCR amplification was performed (the reaction system and procedure were the same as in Example 1), and 1% agarose was added. Electrophoresis was performed using the All-in-One Gold Gel Recovery and Extraction Kit, yielding approximately 1.2k Collect and prepare a band of size b. The constructed pSN1301-pUbi plasmid The product was double-digested with BamHI and KpnI, and the digested product was electrophoresed with 1% agarose. The fragments are recovered and prepared using the All-in-One Gold Gel Recovery Kit. Homologous recombination junction. The PvACR3 gene, which includes the part, and BamHI and KpnI are pSN1301-pUbi The ramisud homologous recombination was double digested (the method was the same as in Example 2). The homologous recombination product was heated. By transforming with the Top10 receptors of E. coli using the method, monoclonal strains were selected and screened. Quensing and correctly sequenced monoclones containing kanamycin LB The culture medium is inoculated and incubated overnight at 37°C with shaking, and the pramis are extracted using a pramisdomini extraction kit. The 'do' is extracted and prepared, and the resulting recombinant 'plamisdo' is pSN1301-pUbi::PvA It is CR3, and the recombinant pramised profile is shown in Figure 9. The sequencing is correct. The modified pSN1301-pUbi::PvACR3 recombinant plasma was obtained by thermal excitation. Agrobacterium rhizogenes EHA105 was transformed and treated with kanamycin and rifampic acid. Prepare the plates by coating them with the synth, selecting and storing the positive monoclones. Comparative Example 2 Identification of pUbi::PvACR3 rice seedlings positive for gene transformation and transgenosis. The method is the same as in Example 3, except for the following point, and the positive monoclonal selected in Example 2. Luagrobacterium was placed in the positive monoclonal agrobacterium selected in Comparative Example 1. The identification results are shown in Figure 4. The PvACR3 gene was found in both the above-ground and root parts of rice. It was expressed in the roots of rice plants in the pUbi::PvACR3 transgenosis. PvACR3 expression levels in pLsi::PvACR3 transgenosis in rice roots It was significantly higher than the PvACR3 expression level. Comparative Example 3 Growth status of pUbi::PvACR3 transgenosis-positive rice in arsenic-contaminated waters: The obtained T3 generation pUbi::PvACR3-positive transgenerated plants were germinated for 3 days. After that, transfer to a 1 / 2 Hoagland nutrient solution containing 5 μM and 20 μM NaAsO2. The plants were treated hydroponically for 5 days. After 15 days, photographs were taken and the phenotype was recorded. The results are shown in Figure 10. The transgenic rice species pUbi::PvACR3 showed significant growth inhibition in arsenic-containing waters. It was done. Comparative Example 4 pUbi::PvACR3 transgenosis-positive rice proliferating in arsenic-contaminated soil: The T3 generation positive transgenetic plants obtained in Comparative Example 2 were transplanted into actual arsenic-contaminated soil. A potting experiment was conducted. The test soil was collected from rice soil in Xuzhou City, Jiangsu Province (pH 5 0.15, total arsenic content 38.51 mg / kg). Transgenic rice was found in the contaminated soil. After two weeks of growth, the plants showed signs of wilting due to poisoning (Figure 11). As can be seen from the results of Comparative Examples 2-4, the ubiquitous expression promoter pUbi is used in rice P The vACR3 gene is overexpressed, causing rice plants to accumulate large amounts of arsenic, and the arsenic levels exceed the standard limit. In soil conditions, it was evident that clear signs of poisoning and death were already present at the seedling stage. The rice material constructed by driving the PvACR3 gene with the TAS expression promoter pUbi is It cannot be applied to the remediation of contaminated waters and soil. This invention selects the specific weak promoter pLsi1 in rice roots to inherit PvACR3 genes. The offspring is expressed in rice, and under its influence, arsenic absorbed by the rice roots is moved to the above-ground parts. It is possible. However, the PvACR3 gene expression level is strongly driven by the promoter pUbi. The amount is also low, balancing accumulation and tolerance. The increase in arsenic accumulation in the above-ground parts of rice and biomass Both increases and decreases are ensured. The created high arsenic-accumulating engineered rice reduces and purifies heavy metals in the soil. It can be used for economical, quick, simple, and effective purposes. . Although the present invention has been described in detail in the above examples, these are only a part of the present invention. Not all embodiments, but those skilled in the art can follow these embodiments without performing any creative work. Examples can be obtained, and all of these examples are included within the scope of protection of the present invention. do.

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INSDQualifier_value> < / insdqualifier> <insdqualifier id="q7"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> aagtaaaaccatggttattctgaat< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="4"> <insdseq> <INSDSeq_length>25< / INSDSeq_length> <INSDSeq_moltype>RNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..25< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q9"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>ggcaggtactagtagttacagtcct< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="5"> <insdseq> <INSDSeq_length> 22< / INSDSeq_length> <INSDSeq_moltype> RNA< / INSDSeq_moltype> <INSDSeq_division> PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..22< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q11"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> atggagaactcaagcgcggagc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="6"> <insdseq> <INSDSeq_length> 25< / INSDSeq_length> <INSDSeq_moltype> RNA< / INSDSeq_moltype> <INSDSeq_division> PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..25< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q13"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> ctaaacagaaggccccttcctctga< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="7"> <insdseq> <INSDSeq_length> 35< / INSDSeq_length> <INSDSeq_moltype> RNA< / INSDSeq_moltype> <INSDSeq_division> PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..35< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q15"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> aagcttaagtaaaaccatggttattctgaatctaa< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="8"> <insdseq> <INSDSeq_length>33< / INSDSeq_length> <INSDSeq_moltype>RNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..33< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q17"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gtcgacggcaggtactagtagttacagtcctca< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="9"> <insdseq> <INSDSeq_length> 40< / INSDSeq_length> <INSDSeq_moltype> RNA< / INSDSeq_moltype> <INSDSeq_division> PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..40< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q19"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> gagaacacggggactctagaatggagaactcaagcgcgc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="10"> <insdseq> <INSDSeq_length> 43< / INSDSeq_length> <INSDSeq_moltype> RNA< / INSDSeq_moltype> <INSDSeq_division> PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..43< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q21"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> gggaaattcgagctcggtaccctaaacagaaggccccttc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="11"> <insdseq> <INSDSeq_length> 25< / INSDSeq_length> <INSDSeq_moltype> RNA< / INSDSeq_moltype> <INSDSeq_division> PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..25< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q23"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> ctgcagtgcagcgtgacccggtcgt< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="12"> <insdseq> <INSDSeq_length> 25< / INSDSeq_length> <INSDSeq_moltype> RNA< / INSDSeq_moltype> <INSDSeq_division> PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..25< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q25"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> ctgfrequencyxfrequency< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="13"> <insdseq> <INSDSeq_length> 25< / INSDSeq_length> <INSDSeq_moltype> RNA< / INSDSeq_moltype> <INSDSeq_division> PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..25< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q27"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> aagcttctgcagtgcagcgtgaccc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="14"> <insdseq> <INSDSeq_length> 31< / INSDSeq_length> <INSDSeq_moltype> RNA< / INSDSeq_moltype> <INSDSeq_division> PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..31< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q29"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> ggatccctgcagagtaacaaaacag< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="15"> <insdseq> <INSDSeq_length>40< / INSDSeq_length> <INSDSeq_moltype>RNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..40< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q31"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gtgttacttctgcagggatccatggagaactcaagcgcgg< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="16"> <insdseq> <INSDSeq_length> 43< / INSDSeq_length> <INSDSeq_moltype> RNA< / INSDSeq_moltype> <INSDSeq_division> PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..43< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q33"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> gggaaattcgagctcggtaccctaaacagaaggccccttc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="17"> <insdseq> <INSDSeq_length> 25< / INSDSeq_length> <INSDSeq_moltype> RNA< / INSDSeq_moltype> <INSDSeq_division> PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..25< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q35"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> atggaactcaagcgcggagcgga< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="18"> <insdseq> <INSDSeq_length> 25< / INSDSeq_length> <INSDSeq_moltype> RNA< / INSDSeq_moltype> <INSDSeq_division> PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..25< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q37"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> ctaaacagaaggccccttcctctga< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="19"> <insdseq> <INSDSeq_length>20< / INSDSeq_length> <INSDSeq_moltype>RNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..20< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q39"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gaagatcactgccttgctcc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="20"> <insdseq> <INSDSeq_length>20< / INSDSeq_length> <INSDSeq_moltype>RNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..20< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q41"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>cgataacagctcctcttggc< / INSDSeq_sequence> < / insdseq> < / sequencedata> < / st26sequencelisting>

[0009]

Claims

1. The plant expression vector comprises a recombinant gene and a pLsi1 promoter. It contains the PvACR3 gene, and the nucleotide sequence of the pLsi1 promoter is SEQ The nucleotide sequence of the PvACR3 gene is shown in ID NO. 1, and is SEQ ID As shown in NO. 2, The aforementioned plant expression vector includes the pSN1301 vector, The method for constructing recombinant vectors is: Using the entire rice DNA as a template, the first PCR amplification was performed to identify the pLsi1 promoter. Steps to obtain, It includes a first primer pair and a second primer pair, the sequence of the first primer pair being SEQ As shown in ID NO. 7 and SEQ ID NO. 8, 5'-aagcttaagtaaaaccatggttatctgaatctaa -3', SEQ ID NO. 7 5'-gtcgacggcaggtactagtagttacagtcctca-3 ',SEQ ID NO. 8 Using the ciliate desert grass cDNA as a template, a second PCR amplification was performed to obtain the PvACR3 gene. Steps to obtain the gene, The sequences of the second primer pair are SEQ ID NO. 9 and SEQ ID NO. As shown in 10, 5'-gagaacacggggactctagaatggagaactcaag cgcgc-3', SEQ ID NO. 9 5'-gggaaattcgagctcggtaccctaaacagaaggcc ccttcctc-3', SEQ ID NO. 10 The pLsi1 promoter is inserted into the plant expression vector by double enzyme digestion, The steps include obtaining a transforming vector containing the Lsi1 promoter, The PvACR3 gene is transformed by homologous recombination into the pLsi1 in the transformation vector. The steps include: inserting downstream of the promoter to obtain the recombinant vector; A recombinant vector for creating arsenic-accumulating engineered rice, characterized by the following features.

2. The recombinant vector and transgenic recipeine described in claim 1 are included. A distinctive arsenic-accumulating variety of rice.

3. The aforementioned transgenic rice variety includes Zhonghua11. The arsenic-accumulating engineered rice described in claim 2 is characterized by the feature of the arsenic-accumulating engineered rice.

4. A method for remediating arsenic-contaminated water and / or soil based on arsenic-accumulating engineered rice according to claim 2. It is a law, The aforementioned arsenic-accumulating engineered rice is planted in the water body and / or soil to be purified, and before the milling stage the rice A purification method characterized by including a step of harvesting above-ground plants.

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