Recombinant plasmid for regulating tolerance of rice to arsenic and cadmium, transgenic rice containing the recombinant plasmid and construction method thereof

Abstract

This invention provides a recombinant plasmid for regulating arsenic and cadmium tolerance in rice. The recombinant plasmid includes a 35S promoter and a gene encoding the TaGlo1 protein. This invention also provides transgenic rice containing the recombinant plasmid and a method for constructing it. This invention verifies that expression of the TaGlo1 gene significantly enhances rice resistance to arsenic and cadmium stress and promotes plant growth. Furthermore, the transgenic rice of this invention exhibits enhanced root accumulation of arsenic and cadmium, but the arsenic and cadmium content in the stem is not significantly different from the wild type. Combined with root-stem translocation coefficient analysis, it shows that TaGlo1 expression reduces the translocation efficiency of arsenic and cadmium from the roots to the aboveground parts by approximately 61.9% and 48.0%, respectively, indicating that it can effectively control the migration of arsenic and cadmium to edible parts.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to recombinant plasmids that regulate the tolerance of rice to arsenic and cadmium, as well as transgenic rice containing said recombinant plasmids and methods for constructing them. Background Technology

[0002] Genetically modified rice has made some progress in research on coping with heavy metal stress. For example, in research on arsenic stress, it was found that it could downregulate... OsLsi1 and OsLsi2 Gene expression can inhibit arsenic absorption and transport in rice; furthermore, knocking out the major arsenic absorption gene... OsNRAMP5 It can block the translocation of arsenic to the aboveground parts. In cadmium stress studies, overexpression of OsHMA3 protein can reduce cadmium accumulation in rice grains, while silencing of OsLCT1 protein can inhibit the translocation of cadmium to the aboveground parts. However, current technological bottlenecks remain: the cross-regulation of arsenic and cadmium metabolic pathways is complex, and single gene editing can easily trigger abnormal absorption of other heavy metals (such as...). OsNRAMP5 Knockout leads to manganese deficiency); low-accumulation varieties may experience reduced yields in uncontaminated fields due to nutrient absorption barriers (zinc and iron deficiency).

[0003] On the other hand, Li, Lijuan et al., in their paper "Metal-Binding Protein TaGlo1 Improves Fungal Resistance to Arsenite (AsIII) and Methylarsenite (MAsIII) in Paddy Soil" (Environmental Science & Technology, Vol. 58, Section 17, pp. 7469-7479), documented the existence of a protein derived from Trichoderma (…). Trichoderma asperellum The metal-binding protein TaGlo1 in SM-12F1 was verified to be present in Escherichia coli (SM-12F1). E. coli The TaGlo1 protein with a His tag and its mutants were overexpressed and purified in BL21, and their growth under different concentrations of arsenic (As(III), As(V), MAs(III), MAs(V)) was investigated. The experiments showed that *E. coli* expressing TaGlo1 had significantly higher biomass than the control group in a medium containing 10 μM As(III). Furthermore, it was revealed that the TaGlo1 protein can bind to As(III) and MAs(III) through conserved cysteine ​​sites, reducing their migration and thus facilitating microbial detoxification, thereby conferring resistance to the main arsenic forms in paddy soil.

[0004] However, the aforementioned literature did not disclose the TaGlo1 protein and only revealed its detoxification effect on arsenic in prokaryotic microorganisms (Escherichia coli) and eukaryotes (fungi). From a technical perspective, on the one hand, it is impossible to predict whether this protein can be efficiently expressed and maintain its activity in higher vascular tissues of rice, i.e., it is impossible to predict whether it will possess the same resistance in higher rice; on the other hand, it is impossible to predict whether this protein can synergistically distribute heavy metals in rice without affecting the absorption of other elements such as iron and manganese, i.e., it is impossible to predict whether it can achieve specific fixation of arsenic and cadmium; furthermore, since arsenic (metalloid) and cadmium (transition metal) are transported through different pathways in plants (e.g., arsenic via OsLsi protein, cadmium via OsNRAMP5 or OsHMA protein), it is also impossible to infer whether there is a difference in the protein's chelating ability for arsenic and cadmium, and it is impossible to predict its effect on reducing arsenic and cadmium accumulation in rice.

[0005] Overall, genetically modified rice has shown potential in reducing heavy metal content in grains through editing key transporters and regulatory factors, but the technical challenges of combined stress still need to be overcome. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a genetic engineering method that can effectively regulate the combined stress of arsenic and cadmium.

[0007] This invention is based on the TaGlo1 protein's specific binding to As(III) and Cd(II) through its conserved cysteine ​​sites. By heterologously expressing the metal-binding protein TaGlo1 derived from Trichoderma, it aims to achieve, for the first time, synergistic resistance to combined arsenic and cadmium stress in rice, a higher plant. Unlike traditional strategies that "shut down" transport proteins (often leading to nutrient deficiencies), this invention constructs an intracellular in-situ fixation mechanism by highly expressing TaGlo1 in the roots or throughout the plant. Root interception effect: Multiple interception barriers are established in the stems and leaves through a strong 35S promoter, further blocking the penetration of heavy metals into the grains. Experiments have shown that this invention not only solves the technical bottleneck of simultaneous arsenic and cadmium remediation in rice but also avoids manganese deficiency and yield reduction similar to that caused by OsNRAMP5 deficiency in uncontaminated fields, achieving a balance between low heavy metal accumulation and high biomass. Based on this, the present invention provides a recombinant plasmid for regulating the tolerance of rice to arsenic and cadmium. The recombinant plasmid includes a 35S promoter and a gene encoding the TaGlo1 protein. The nucleic acid sequence of the TaGlo1 protein is shown in SEQ ID No. 1; the amino acid sequence is shown in SEQ ID No. 2; and the 35S promoter is shown in SEQ ID No. 3.

[0008] In this invention, the TaGlo1 protein is derived from Trichoderma ( Trichoderma asperellumSM-12F1.

[0009] Based on this, the present invention also provides a transgenic rice with tolerance to arsenic and cadmium, characterized in that the transgenic rice expresses the TaGlo1 protein according to claim 1.

[0010] The above-mentioned method for constructing genetically modified rice includes the following steps: (1) Amplification of the target gene according to Taglo1 Gene sequence-specific primers were designed to introduce restriction enzyme sites at the 5' end. Actag and protective bases ggtctc Using the pUC57-TaGlo1 plasmid carrying the TaGlo1 target gene as a template, specific amplification of the target gene was completed. TaGlo1 was amplified by PCR, and the resulting product was confirmed to be correct by 1% agarose gel electrophoresis and recovered. (2) Construction of recombinant plasmids The pEGOEP35S-GFP vector and the amplified target gene fragment TaGlo1 were digested with BsaI-HF via Golden Gate assembly and then ligated under the catalysis of T4 DNA ligase to obtain the pEGOEP35S-H-TaGlo1-GFP vector. (3) Agrobacterium transformation The recombinant vector, verified by sequencing, was transformed into Agrobacterium competent cells and plated on LB solid medium containing hygromycin B. After incubation, positive single colonies were picked and inoculated into LB liquid medium containing hygromycin B and cultured overnight with shaking. Subsequently, the bacterial culture was transferred to AAM liquid medium (Agrobacterium co-culture medium) containing 20 mg / mL acetylsyringone (AS) and cultured for a longer period. When the bacterial culture reached OD600 0.1, it was used as an Agrobacterium suspension for infection. (4) Genetic transformation of rice Take mature rice seeds, thoroughly disinfect and clean them, and inoculate them onto N6D medium (N6 callus induction medium). Select callus and concentrate them in a sterile Erlenmeyer flask, and co-suspend and activate them with Agrobacterium. Then remove the callus, dry it on a laminar flow hood until there is no moisture on the surface, and then place it on a co-culture medium for culture. After co-culture, transfer the callus to an Erlenmeyer flask, thoroughly clean it, and soak the callus in a cephalosporin aqueous solution. Then place it on a sieve medium for continued culture until positive callus appears. Then culture until it differentiates into seedlings. When the seedlings grow to about 1 cm, transfer them to a rooting medium to strengthen the seedlings. Attached Figure Description

[0011] Figure 1Electrophoresis diagram of target gene amplification in Example 1; Marker, nucleic acid molecular weight standard; (a) Taglo1 The amplified target gene product; Figure 2 Here are the electrophoresis images for the PCR verification of the recombinant plasmid in Example 1. (a) 1-8, TaGlo1 was successfully transformed into the pEGOEP35S-GFP vector, and the pEGOEP35S-H-TaGlo1-GFP was verified. Figure 3 Electrophoresis diagram of the vector digested with enzymes in Example 1; Marker, nucleic acid molecular weight standard; (a) pEGOEP35S-H-TaGlo1-GFP plasmid map; (b) Electrophoresis diagram of the digested fragments of the pEGOEP35S-H-TaGlo1-GFP vector digested with EcoRI / HindIII; Figure 4 For Example 2, colocalization of rice protoplast cell nuclei: A, TaGlo1 protein green fluorescence channel; B, nuclear marker autofluorescence channel; C, bright field; D, superposition of bright field and fluorescence channels.

[0012] Figure 5 Example 2 shows the colocalization of rice protoplast cytoplasm: A, TaGlo1 protein green fluorescent channel; B, cytoplasmic marker red fluorescent protein channel; C, bright field; D, superposition of bright field and fluorescent channels.

[0013] Figure 6 Example 2: Induction of mature rice embryos: (a) Inoculation status of rice seeds after dehulling and surface disinfection; (b) Initial callus tissue formed by mature embryos on induction medium.

[0014] Figure 7 Example 2 callus: (a) screening and subculture of embryogenic callus; (b) callus infection and co-culture; (c) resistance screening of transformed callus.

[0015] Figure 8 Example 2: Induction, differentiation and rooting of resistant callus (a) Further differentiation of resistant callus into regenerated buds; (b) Growth of regenerated buds and seedling formation; (c) Rooting and robust seedling culture of transgenic regenerated seedlings.

[0016] Figure 9 This is a fluorescence microscope image of the T1 generation transgenic rice in Example 2.

[0017] Figure 10Gene expression detection of T1 generation rice in Example 2: (a) relative expression level of TaGlo1 in 35S-TaGlo1 line, with UBI as internal reference gene; (b) Western blot verification of TaGlo1 expression level in 35S-TaGlo1 line, with Actin as internal reference gene.

[0018] Figure 11 The changes in rice biomass in Example 3 are as follows: Under arsenic stress, (a) aboveground and (b) underground biomass of rice; under cadmium stress, (c) aboveground and (d) underground biomass of rice; under combined arsenic and cadmium stress, (e) aboveground and (f) underground biomass of rice.

[0019] Figure 12 The changes in heavy metal content in rice in Example 3 are as follows: (a) Arsenic content in the aboveground and (b) underground parts of rice; (c) Cadmium content in the aboveground and (d) underground parts of rice.

[0020] Figure 13 The arsenic-cadmium transfer coefficient in 35S-TaGlo1 in Example 3. Detailed Implementation

[0021] The following examples are used to explain the technical solutions of the present invention in a non-limiting manner.

[0022] In this invention, unless otherwise specified, "%" used to explain concentration refers to mass percentage, and ":" refers to mass ratio.

[0023] The biological materials used in this invention include: the japonica rice variety Nipponbare ( Oryza sativa L. japonica. cv. Nipponbare, NPB), Escherichia coli Escherichia coli DH5α (ToloBio, China) was used for routine transformation. The experimental vectors were pEGOEP35S-GFP (Aidijing, China) and pUC57-TaGlo1.

[0024] The reagents used in this invention include: KOD-FX high-fidelity DNA polymerase (Toyobo, Japan), restriction endonuclease (NEB, USA), T4 DNA ligase (NEB, USA), kanamycin (Solarbio, China), plasmid mini-extraction kit (Tiangen Biotech, China), cellulase R10, macerozyme R10, mannitol, MES, and PEG4000 purchased from BIOSHARP; N6 medium powder, AAM medium powder, MS medium powder, 2,4-D, acid-hydrolyzed casein, L-proline, glucose, sucrose, Phetgel, etc. purchased from Beijing Solarbio Technology Co., Ltd.; and NaCl, KH2PO4, MgCl2, CaCl2, and Fe-EDTA purchased from Sinopharm Chemical Reagent Co., Ltd., etc.

[0025] The solutions used in this invention include: Enzymatic hydrolysate: 0.15 g 1.5% cellulase R10, 0.04 g 0.4% dissociative enzyme R10, 0.728 g 0.4 M mannitol, 1 mL 10 mM MES (pH 5.7), bring to a final volume of 10 mL with ddH2O, and adjust pH to 5.8 with HCl; Polyethylene glycol (PEG4000 solution): 0.728 g 0.4 M mannitol, 0.11 g 100 mM CaCl2, 4 g 40% PEG4000, ddH2O to a final volume of 10 mL, pH adjusted to 5.8 with HCl; W5 buffer: 0.9 g 154 mM NaCl, 1.39 g 125 mM CaCl2, 0.068 g 2 mM KH2PO4, 2 mL 2 mM MES, ddH2O to a final volume of 100 mL, adjust pH to 5.7-5.8 with HCl; sterilize at 110℃, freeze W5 and dispense into 50 mL aliquots. Mannitol–MES–MgCl2 (MMG) solution: 0.72 g 0.4 M mannitol, 0.03 g 15 mM MgCl2·6H2O, 0.4 mL 4 mM MES, ddH2O to a final volume of 10 mL, pH adjusted to 5.7-5.8 with HCl; sterilized at 110 °C, then frozen and dispensed into 1.5 mL tubes.

[0026] The culture medium used in this invention includes: N6D medium: 25 mL of N6D macro-element stock solution (20×), 2.5 mL of N6D micro-element stock solution (200×), 2.5 mL of MS organic stock solution (200×), 2.5 mL of Fe-EDTA (200×) solution, and 5 mL of 2,4-D (200 mg / L) solution. Then, add 0.15 g of acid-hydrolyzed casein, 1.439 g / L proline, 15 g of sucrose, and 2 g of Phetgel sequentially. After thorough dissolution, bring the volume to 0.5 L with distilled water. Adjust the pH to 5.8 with 1 mol / L hydrochloric acid, autoclave at 121℃ for 20 min, and cool before use.

[0027] AAM solution: Weigh 34.25 g sucrose, 18 g glucose and 0.25 g acid-hydrolyzed casein, add 2.5 mL Fe-EDTA solution, 50 mL AAM amino acid solution, 25 mL AAM macro-element stock solution (20×), 2.5 mL AAM micro-element stock solution (200×) and 2.5 mL AAM organic stock solution (200×), dissolve thoroughly and then bring the volume to 0.5 L with distilled water; adjust the pH to 5.8 with 1 mol / L hydrochloric acid, autoclave at 121℃ for 20 min, and cool before use.

[0028] The primers and sequences involved in this invention are shown in Table 1: Table 1

[0029] Example 1: Vector Construction and Identification 1. Amplification and validation of the target gene The TaGlo1 gene sequence was synthesized at Shanghai Sangon Biotech Co., Ltd. and spliced ​​onto the pUC57 plasmid to obtain plasmid pUC57-TaGlo1 for stable preservation. Taglo1 Gene sequence-specific primers were designed to introduce restriction enzyme sites at the 5' end. Actag and protective bases ggtctc Specific primers were designed based on the 35S promoter gene sequence, and restriction enzyme sites were introduced at the 5' end. Actag and protective bases ggtctc ; Specific primers were designed based on TaGlo1, as shown in Table 1.

[0030] Amplification was performed using PCR. Taglo1(1) The PCR system is as follows: 2×PCR buffer 25 μL, 2 mM dNTP 10 μL, primers 1 μL each, KOD-FX 1 μL, template DNA 1 μL, and ddH2O to make up to 50 μL. (2) The PCR reaction program is as follows: 98 ℃ pre-denaturation for 3 min, 98 ℃ denaturation for 10 s, 58 ℃ annealing for 30 s, 68 ℃ extension for 1 min, denaturation to extension for 32 cycles, and finally 68 ℃ final extension for 2 min. The amplified PCR products were detected by 1% agarose gel electrophoresis and the target fragment of the corresponding size was recovered. The target fragment was recovered using the agarose gel DNA recovery kit (DP209-03) from Tiangen Biotech Co., Ltd.

[0031] The results are as follows Figure 1 As shown. The plasmid carrying the target gene was amplified by PCR under high-fidelity DNase KOD-FX conditions. Detection by 1% agarose gel electrophoresis yielded a target band of approximately 438 bp, corresponding to... Taglo1 Gene.

[0032] 2. Plasmid recombination and validation The empty vector pEGOEP35S-GFP and the amplified target gene fragment TaGlo1 were digested with BsaI-HF (NEB, USA) to linearize the empty vector and generate a 4-base nick end. The target gene fragment also generated a 4-base nick end under the action of BsaI. Using the 4-nt sticky ends generated by BsaI-HF, the fragment and the vector were directionally and seamlessly ligated. The ligation was carried out under the catalysis of T4 DNA ligase (NEB, USA) to obtain the pEGOEP35S-H-TaGlo1-GFP vector.

[0033] The reaction system consisted of: 1.5 μL of 10×CutSmart Buffer, 1.5 μL of 10 mM ATP, 100 ng of empty plasmid, and 50 ng of the target fragment. Bsa I-HF 10 U, T4 DNA ligase 35 U, and ddH2O were added to make up to 15 μL. The reaction conditions were: 37 ℃ for 5 min, 20 ℃ for 5 min, for 15 cycles to improve ligation efficiency.

[0034] 3. Escherichia coli transformation, clone screening, and colony identification Take 100 μL of DH5α Escherichia coli competent cells and mix with 2-5 μL of ligation product, and incubate on ice for 30 min; then quickly place in a 42 ℃ constant temperature water bath for 90 s heat shock, followed by an ice bath for 2 min; add 500 μL of LB liquid medium and mix well; incubate at 37 ℃ and 200 rpm for 45 min to allow the cells to recover to normal growth state; then evenly spread the bacterial solution on Kana-resistant LB solid medium plates; after 30 min, incubate overnight at 37 ℃.

[0035] Colony PCR was performed on the recombinant plasmid using 35S-F / eGFP-cx primers. 1% agarose gel electrophoresis yielded a band of approximately 580 bp, indicating successful ligation of the vector pEGOEP35S-GFP and the PCR-amplified fragment via Golden Gate assembly to obtain the pEGOEP35S-H-TaGlo1-GFP vector. Figure 2 As shown.

[0036] The colony PCR amplification system consisted of 10 µL of 2X Taq MIX, 0.5 µL of each primer, a small amount of colonies, and ddH2O to bring the total to 20 µL. The reaction was performed as follows: 95 ℃ for 5 min; 95 ℃ for 30 sec; 55 ℃ for 30 sec; 72 ℃ for 1 min; 25 cycles; 72 ℃ for 2 min; 4 ℃ infinity.

[0037] 4. Plasmid extraction and sequencing (1) Pick single colonies from LB solid medium plates and inoculate them into LB liquid medium with a final concentration of 50 μg / mL for kana resistance, and incubate overnight at 37 °C; (2) Take 4 mL of activated bacterial solution, centrifuge at 10,000 rpm for 2 min at room temperature, and completely discard the supernatant; (3) Take 250 μL of Solution I reagent containing ribonuclease A and thoroughly resuspend the bacterial block; (4) Take 250 μL of Solution II reagent to lyse the bacterial block, and gently invert it several times until the bacterial cells are transparent; (5) Take 350 μL of Solution Ⅲ reagent, invert it several times until a white, firm flocculent substance is formed; (6) Centrifuge at 12,000 rpm for 10 min at room temperature and collect the supernatant; (7) Remove the nucleic acid purification column from the kit and place it on the collection tube; (8) Take the clear supernatant from step 6 above into a nucleic acid purification column, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate; (9) Add 500 μL of Buffer W1 to the nucleic acid purification column, centrifuge at 12000 rpm for 30 s at room temperature, and discard the filtrate; (10) Take 700 μL of Buffer W2 into the nucleic acid purification column, centrifuge at 12000 rpm for 30 s at room temperature, and discard the filtrate; (11) Repeat the above operation step 10; (12) Place the nucleic acid purification column on the collection tube and incubate at 12,000 rpm for 2 min at room temperature to remove as much residual liquid as possible; (13) Discard the collection tube, take the nucleic acid purification column and place it in a 1.5 mL EP tube, and add 50 μL of elution buffer to elute the DNA attached to the nucleic acid purification column membrane (the elution buffer can be preheated in a 65 ℃ constant temperature water bath to help elute the DNA), and let it stand at room temperature for 2 min; (14) Centrifuge at 12,000 rpm for 2 min at room temperature to wash off the DNA attached to the nucleic acid purification column membrane, and store at -40 ℃ for later use; (15) Take a small amount of the recovered product and use 1% agarose gel electrophoresis to detect the plasmid extraction quality.

[0038] The plasmid extracted from the above positive bacterial plaques was subjected to Sanger sequencing. The sequencing primer eGFP-cx was used for one-way sequencing. If the sequencing result sequence matched the target fragment sequence, the overexpression vector was successfully constructed.

[0039] 5. Vector enzyme digestion like Figure 3 Figure a shows the plasmid pattern of the final vector pEGOEP35S-H-TaGlo1-GFP. Verification by enzyme digestion yielded the following results: Figure 3 The enzyme digestion gel image shown in b displays two fragments consistent with the theory, indicating that the enzyme digestion was complete, the reproducibility was good, and the plasmid insertion was successful.

[0040] Example 2 Construction and detection of recombinant rice 1. Preparation of rice protoplasts (1) Cultivate rice seedlings in the dark at about 30 ℃ for about 10 days, take the stems and remove the outermost leaf sheaths; cut the tender parts of the stems into segments about 0.5 mm in length and put them into 50 mL Erlenmeyer flasks; add 5-10 mL of enzymatic hydrolysate to completely soak the tissues, vacuum, wrap with sealing film, protect from light at 30 ℃, and slowly shake (80-100 rpm) for 4 h for enzymatic hydrolysis; (2) Filter the protoplasts into a 2 mL EP tube using 40 μm nylon gauze, and keep the middle part of the filtrate (obtained by gently squeezing). Centrifuge at 600 rpm for 5 min at a centrifugation temperature of 4 ℃ and a speed of rise and fall not exceeding 7. A turbid precipitate will be visible. (3) Aspirate the supernatant from the pipette tip and wash the supernatant twice with 1 mL of pre-cooled W5 solution. Centrifuge at 600 rpm for 5 min each time. The centrifugation temperature is 4 ℃ and the acceleration and deceleration rate should not exceed 7. A turbid precipitate will be visible at the bottom of the tube. Add 1 mL of W5 solution to gently suspend the tube and let it stand on ice for 30 min. (4) Centrifuge the suspension at 600 rpm for 5 min after standing, remove the supernatant, and add MMG solution to suspend as needed (50 μL of protoplast solution is needed for every 10 μL of plasmid), and let stand for 8-10 min (microscopic examination: 20-40 protoplasts per field of view under 40x magnification). (5) Add 50 μL of protoplasts and an equal volume of 40% PEG4000 solution to every 10 μL of plasmid: Common localization: 10 μL target gene plasmid + 50 μL protoplasts + 60 μL 40% PEG4000 solution. Colocalization: 10 μL target gene plasmid + 10 μL marker gene plasmid + 100 μL protoplasts + 120 μL 40% PEG4000 solution; the Bifc experimental method is the same as colocalization; slowly invert to mix, incubate in a 24.5 ℃ water bath for 15-20 min; Step (2) and all operations before the water bath are performed on ice, with gentle movements.

[0041] (6) Dilute the protoplasts with 1 mL of W5 solution, mix well and terminate the reaction; (7) Centrifuge at 600 rpm for 5 min, and discard the supernatant by pipetting. (8) Add 1 mL of W5 solution and wash and centrifuge twice; (9) Add 1 mL of W5 solution, mix slowly, and incubate in the dark at 30 °C overnight; (10) Centrifuge at 600 rpm for 5 min, remove the supernatant with the pipette tip, and observe and photograph with a laser confocal microscope (100× oil immersion).

[0042] TaGlo1 protein is mainly expressed in the cytoplasm and nucleus of rice leaves, as shown in the following results. Figure 4-5 As shown.

[0043] 2. Genetic transformation of rice Callus tissue was induced using mature rice embryos.

[0044] (1) Disinfection: Take mature rice seeds, remove the husks, and soak them in 75% alcohol, 0.15% mercuric chloride, and 30% sodium hypochlorite respectively. After each soaking, rinse them twice with sterile water until they are thoroughly disinfected and cleaned.

[0045] (2) Induction: The sterilized seeds were inoculated onto N6D medium.

[0046] (3) Agrobacterium culture: Prepare callus and activated Agrobacterium plates two days in advance. Select callus (sterile, bright yellow, with an uneven surface) and concentrate them in a sterile Erlenmeyer flask; take 100 ml of sterile Erlenmeyer flask and add 50 mL of AAM suspension (add 125 uL of AS at a concentration of 20 mg / mL to the suspension), scrape the corresponding Agrobacterium 2-3 times into the suspension with an inoculation loop (OD600=0.1, just enough to transmit light when viewed through a fluorescent lamp on a clean bench), shake to suspend and activate the bacteria for 20 min until the bacteria are evenly suspended in the suspension; pour the prepared bacterial solution into the corresponding callus Erlenmeyer flask and soak for about 15 min; then use tweezers to remove the soaked callus and place it on a sterile filter paper; blow dry the callus on a clean bench until there is no moisture on the surface, then invert it on the co-culture medium, making sure that each callus is in contact with the medium, and place it in a co-incubator at 20~22°C for 48-72 h in the dark.

[0047] (4) Cleaning: After co-culturing, transfer the callus to an Erlenmeyer flask and clean it with sterile water until the water is completely clear. Then, add cephalosporin solution (1 g / L) to soak the callus and shake it on a shaker for 30 min. Remove the callus and place it on a clean bench with the lid off to air dry. Shake it every half hour to ensure that the callus is evenly dried. After drying, place the callus corresponding to the resistance on a sieve of culture medium, evenly distributed, with the callus with buds lying flat and the buds of the callus without buds facing down to ensure effective contact between the callus and the culture medium, 15 / 18 callus / plate. Incubate in a 30℃ constant temperature incubator.

[0048] (5) Screening: Change the template every 15 days until positive callus appears.

[0049] (6) Differentiation: Select callus with a hard texture and a bright yellow color and place it in two layers of filter paper. Dry it until the surface is slightly soft when held with tweezers, and then place it on the differentiation medium. Incubate it in a constant temperature incubator at 28 ℃. After 15 days, the buds will emerge. Transfer the buds to the seedling medium. Rooting can be carried out after one week.

[0050] (7) Select resistant callus, transfer it into a culture dish containing differentiation medium, seal it with a sealing film, and place it in a constant temperature culture room to wait for differentiation into seedlings.

[0051] (8) When the seedlings grow to about 1 cm, they are transferred to a rooting medium to strengthen them. They are then transferred to a rooting medium containing screening pressure (hygromycin) for seedling strengthening and resistance screening. After 4 weeks of culture, positive plants with well-developed roots and normal growth are selected. The positive seedlings are then transplanted into greenhouse soil pots and cultivated and managed according to conventional rice cultivation until the grains mature. The T1 generation transgenic rice seeds are then harvested.

[0052] (9) To detect resistance genes, select T1 generation transgenic rice (35S-TaGlo1) grown hydroponically for 4 weeks, slice it, and observe it under a fluorescence microscope. Figure 9 As shown, fluorescent signals were found in both root and stem cells of rice containing 35S-TaGlo1, indicating that TaGlo1 can be expressed in rice.

[0053] Healing Figure 6-8 As shown.

[0054] 3. Detection of TaGlo1 expression level in T1 generation rice T1 generation transgenic rice 35S-TaGlo1, grown hydroponically for 4 weeks, was used. Aboveground (stems and leaves) and underground (roots) tissues were collected, flash-frozen in liquid nitrogen, and stored at -80 °C. Total RNA was extracted from the tissues using the TRIzol method. After removing genomic DNA contamination with DNase, the RNA was synthesized into first-strand cDNA using a reverse transcription kit. Using the rice internal reference gene UBI as a control, quantitative real-time PCR was performed using TaGlo1-specific primers.

[0055] Western blot analysis was used to analyze the expression of TaGlo1 protein, with Actin as an internal control to detect the expression level of the target gene in different tissues. Root and stem tissues from rice of the same age were collected, and plant protein extraction buffer containing protease inhibitors was added. The tissues were ground on ice and centrifuged at 4°C to obtain the protein supernatant. Equal volumes of total protein were separated by SDS-PAGE electrophoresis, and then transferred to a PVDF membrane using a wet transfer technique. Chemiluminescence development was performed using ECL developing solution, and the band intensity was observed and recorded.

[0056] After propagation, seeds of T1 were obtained. After 4 weeks of hydroponics, the above-ground and underground parts were measured. Taglo1 Gene expression levels. For example... Figure 10 As shown in Figure a, at the RNA level, Taglo1 Under the 35S promoter, the expression level of the gene in roots and stems was approximately 21 times higher than that of the internal reference gene UBI. Meanwhile, as... Figure 10 As shown in Figure d, Western blotting experiments revealed that 35S-TaGlo1 was expressed at a higher level in the aerial parts of the plant at the protein level.

[0057] Example 3 Heavy Metal Stress Experiment 1. Biomass changes T1 generation transgenic rice seeds were used as experimental materials. Seeds were surface-sterilized with 30% hydrogen peroxide for 30 min, rinsed five times with sterile water, and then soaked in the dark at 28 °C to break dormancy and germinate for 72 h. After germination, seedlings were transplanted into a light incubator and hydroponically cultured using modified Hoagland nutrient solution. The culture conditions were set as follows: 14 h light / 10 h dark photoperiod, light intensity of 300 μmol·m⁻²·s⁻¹, temperature of 25 °C, and relative humidity of 60%. The nutrient solution was changed every 4 days for a pre-culture period of 2 weeks.

[0058] Two weeks later, the rice seedlings were transferred to modified Hoagland nutrient solution containing the following treatment groups: (1) control group (wild-type WT, without heavy metals); (2) 10 μmol·L⁻¹ arsenic treatment group (As); (3) 10 μmol·L⁻¹ cadmium treatment group (Cd); (4) 10 μmol·L⁻¹ arsenic + 10 μmol·L⁻¹ cadmium combined treatment group (As+Cd). The arsenic source used was sodium arsenite (NaAsO₂, As(III)), and the cadmium source was cadmium chloride (CdCl₂·2.5H₂O).

[0059] Four weeks after stress treatment, rice plant samples were collected. Roots were separated from the aboveground parts, rinsed thoroughly with deionized water, blanched at 105 °C for 30 min, and then dried at 65 °C to constant weight. Dry biomass was measured. After digestion with HNO3-HClO4 (4:1, v / v), the arsenic and cadmium contents in roots and aboveground parts were determined by inductively coupled plasma mass spectrometry (ICP-MS).

[0060] Biomass changes of 35S-TaGlo1 transgenic rice under arsenic stress are as follows: Figure 11 As shown in Figures a and b, arsenic treatment significantly inhibited the growth of wild-type rice compared to the control. Wild-type rice (WT_As) showed a significant decrease in aboveground and underground dry weight of 37.5% and 32.7% respectively compared to the control (WT) (P < 0.01). In contrast, the transgenic line 35S-TaGlo1 exhibited a significant growth advantage under arsenic stress, with aboveground and underground dry weight increases of 77.3% and 39.1% respectively compared to the wild-type control (P < 0.01).

[0061] Biomass changes of 35S-TaGlo1 transgenic rice under cadmium stress as follows Figure 11As shown in Figures c and d, arsenic treatment significantly inhibited the growth of wild-type rice compared to the control. The aboveground and underground dry weights of wild-type rice (WT_As) decreased significantly by 28.6% and 29.9% respectively compared to the control (WT) (P < 0.01). The transgenic line 35S-TaGlo1 performed well under cadmium stress, with significantly increased aboveground and underground biomass of 57.3% and 31.0% respectively compared to WT_Cd (P < 0.05), while there was no significant difference in biomass compared to the control.

[0062] Biomass changes of 35S-TaGlo1 transgenic rice under combined arsenic and cadmium stress are as follows: Figure 11 As shown in e and f, the aboveground and belowground biomass of WT_As+Cd decreased by 25.2% and 22.7% respectively compared to the WT group (P<0.5); in contrast, the aboveground and belowground biomass of 35S-TaGlo1 under arsenic and cadmium stress were significantly increased by 31.4% and 25.0% respectively compared to the WT_As+Cd group (P<0.05).

[0063] 2. Heavy metal content in rice Changes in arsenic content in rice, such as Figure 12 As shown in Figures a and b, compared with wild-type WT_As, the arsenic content in the aboveground parts of 35S-TaGlo1_As showed no significant change (P>0.05), while the arsenic content in the underground parts increased significantly by 154.8% (P<0.001). Under As+Cd co-pollution, the arsenic content in the aboveground and underground parts of 35S-TaGlo1_As+Cd increased by 23.1% and 86.9% respectively compared with wild-type WT_As+Cd (P<0.01).

[0064] Changes in cadmium content in rice, such as Figure 12 As shown in Figures c and d, compared with wild-type WT_Cd, the cadmium content in the aboveground parts of 35S-TaGlo1_Cd showed a significant change (P>0.05), while the cadmium content in the underground parts increased significantly by 49.6%. Similarly, under As+Cd co-pollution, compared with wild-type WT_As+Cd, the aboveground parts of 35S-TaGlo1_As+Cd showed no significant change, while the cadmium content in the underground parts increased significantly by 60.2% (P<0.001).

[0065] Arsenic (or cadmium) transfer coefficient TF=C root / C shoot Among them, C shoot The content of As (or Cd) in the aboveground parts, C root The content of As (or Cd) in the roots is expressed in mg·kg⁻¹. -1 .

[0066] like Figure 13As shown, the transfer coefficients for arsenic and cadmium by wild-type WT were 0.26 and 0.29, respectively. The transfer coefficients for arsenic and cadmium by 35S-TaGlo1 were significantly reduced by 61.9% and 48.0% compared to WT, at 0.11 and 0.15, respectively.

[0067] The above experimental results demonstrate that the expression of the TaGlo1 gene significantly enhances the resistance of rice to arsenic and cadmium stress and promotes plant growth. On the other hand, the transgenic rice of this invention exhibits enhanced root accumulation of arsenic and cadmium, but the arsenic and cadmium content in the stem is not significantly different from the wild type. Combined with root-stem translocation coefficient analysis, it shows that TaGlo1 expression reduces the translocation efficiency of arsenic and cadmium from the roots to the aboveground parts by approximately 61.9% and 48.0%, respectively, indicating that it can effectively control the migration of arsenic and cadmium to edible parts.

Claims

1. A recombinant plasmid for regulating arsenic and cadmium tolerance in rice, the recombinant plasmid comprising a 35S promoter and a gene encoding the TaGlo1 protein, wherein the nucleic acid sequence of the TaGlo1 protein is shown in SEQ ID No. 1; the amino acid sequence is shown in SEQ ID No. 2; and the 35S promoter is shown in SEQ ID No.

3.

2. Transgenic rice containing the recombinant plasmid as described in claim 1.

3. The method for constructing transgenic rice according to claim 2, the method comprising the following steps: (1) Amplification of the target gene Based on the Taglo1 gene sequence, specific primers were designed, and the restriction enzyme site Acta and the protective base ggtctc were introduced at the 5' end. Using the pUC57-TaGlo1 plasmid carrying the TaGlo1 target gene as a template, the specific amplification of the target gene was completed. Taglo1 was amplified by PCR reaction, and the obtained product was confirmed to be correct by 1% agarose gel electrophoresis and recovered. (2) Construction of recombinant plasmids The pEGOEP35S-GFP vector and the amplified target gene fragment Taglo1 were digested with BsaI-HF via Golden Gate assembly and then ligated under the catalysis of T4 DNA ligase to obtain the pEGOEP35S-H-TaGlo1-GFP vector. (3) Agrobacterium transformation The recombinant vector pEGOEP35S-H-TaGlo1-GFP, which was verified by sequencing, was transformed into Agrobacterium competent cells and plated on LB solid medium containing hygromycin. After culturing, positive single colonies were picked and inoculated into LB liquid medium containing hygromycin antibiotic and cultured overnight with shaking. Subsequently, the bacterial culture was transferred to Agrobacterium co-culture medium containing 20 mg / mL acetylsuccinone and cultured for a longer period. When the bacterial culture reached OD600 0.1, it was used as an Agrobacterium suspension for infection. (4) Genetic transformation of rice Take mature rice seeds, thoroughly disinfect and clean them, and inoculate them onto N6 callus induction medium. Select callus and concentrate them in a sterile Erlenmeyer flask, and suspend and activate them together with Agrobacterium. Then remove the callus, dry it on a laminar flow hood until there is no moisture on the surface, and then place it on a co-culture medium for culture. After co-culture, transfer the callus to an Erlenmeyer flask, thoroughly clean it, and soak it in a cephalosporin aqueous solution. Then place it on a sieve medium for continued culture until positive callus appears. Then culture until it differentiates into seedlings. When the seedlings grow to about 1 cm, transfer them to a rooting medium to strengthen the seedlings.

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