Gene combinations, multi-gene expression cassettes and their applications for improving cadmium accumulation and cadmium tolerance in plants

Abstract

The present invention provides a gene combination and multi-gene expression cassette for improving cadmium accumulation and cadmium tolerance in plants, relating to the field of biological genetic engineering technology. The present invention combines the PPKS gene, the MT1ES gene, the MT2S gene, the MT3S gene, and the MT4S gene, and constructs gene expression cassettes based on the codon preference of the target plant after codon optimization. The five gene expression cassettes are then serially incorporated into a plant transformation vector to produce a recombinant plant transformation vector. The recombinant plant transformation vector is then used to transform the target plant, yielding an engineered plant capable of efficiently expressing the exogenous gene. Experimental measurements confirm that the introduction of the PPKS, MT1ES, MT2S, MT3S, and MT4S genes improves the plant's cadmium accumulation and tolerance, resulting in an engineered plant with a more ideal cadmium-contaminated soil remediation effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a gene combination, a multi-gene expression cassette and an application thereof for improving the cadmium accumulation and cadmium resistance of plants. Background Art

[0002] Cadmium (Cd) is a highly toxic heavy metal pollutant, ranking first among inorganic soil pollutants. Cadmium is primarily produced during the processing and smelting of non-ferrous metals and is widely present in environmental media such as water, soil, and food. Due to its high mobility and solubility, Cd is easily absorbed by plants. This not only affects plant metabolism and reduces crop quality, but can also accumulate and harm the human skeleton, lungs, kidneys, and central nervous system through its transport and accumulation within the food chain, potentially threatening life.

[0003] Currently, the main remediation technologies for heavy metal contaminated soil include physical remediation, chemical remediation, and bioremediation. Compared to traditional physical and chemical remediation techniques, bioremediation technology can be used to repair heavy metal-contaminated soil. The products produced do not harm the plant growth environment. Furthermore, bioremediation technology offers advantages such as low cost, high efficiency, no secondary pollution, simple operation, and wide applicability. Therefore, bioremediation technology has become a research hotspot in recent years.

[0004] Plants detoxify heavy metals through processes such as acidification, compartmentalization, chelation, free radical scavenging, and transport. Among these, metallothioneins (MTs), metal-binding proteins rich in cysteine ​​residues, can bind free Cd in the cytoplasm, mitigating its effects on organelle damage and metabolic regulation. Furthermore, reports indicate that polyphosphate kinase (PPK) can enhance plant accumulation of metal ions and reduce environmental metal ion concentrations. However, there are currently no reports on the combined effects of these two genes on plant accumulation and tolerance to Cd. Summary of the Invention

[0005] The purpose of the present invention is to provide a gene combination, a multi-gene expression cassette and its application for improving the cadmium tolerance of plants. The tandem expression of the gene combination in plants not only improves the plant's ability to absorb cadmium near the rhizosphere, but also avoids the toxicity to the plant caused by excessive absorption of cadmium ions, thereby improving the plant's tolerance to cadmium and enabling the engineered plants to have a more ideal cadmium-contaminated soil remediation effect.

[0006] The invention provides a gene combination for improving the cadmium accumulation and cadmium tolerance of plants. The gene combination comprises a PPKS gene, an MT1ES gene, an MT2S gene, an MT3S gene and an MT4S gene. The nucleotide sequence of the PPKS gene is shown in SEQ ID NO.1, the nucleotide sequence of the MT1ES gene is shown in SEQ ID NO.2, the nucleotide sequence of the MT2S gene is shown in SEQ ID NO.3, the nucleotide sequence of the MT3S gene is shown in SEQ ID NO.4, and the nucleotide sequence of the MT4S gene is shown in SEQ ID NO.5.

[0007] In the present invention, the PPKS gene, MT1ES gene, MT2S gene, MT3S gene and MT4S gene in the gene combination are sequentially connected in order to obtain the gene combination.

[0008] The present invention also provides a multi-gene expression cassette for improving the cadmium accumulation and cadmium resistance of plants. The multi-gene expression cassette is obtained by serially connecting a PPKS gene expression cassette formed by fusing the PPKS gene with a 35S promoter and a NOS terminator, a MT1ES gene expression cassette formed by fusing the MT1ES gene with a 35S promoter and a NOS terminator, a MT2S gene expression cassette formed by fusing the MT2S gene with a 35S promoter and a NOS terminator, a MT3S gene expression cassette formed by fusing the MT3S gene with a 35S promoter and a NOS terminator, and a MT4S gene expression cassette formed by fusing the MT4S gene with a 35S promoter and a NOS terminator.

[0009] In the present invention, the 35S promoter is derived from cauliflower mosaic virus; and the NOS terminator is derived from Agrobacterium tumefaciens.

[0010] In the present invention, the sequence of the multi-gene expression cassette is shown as SEQ ID NO.6.

[0011] The present invention also provides a recombinant plant transformation vector comprising the above gene combination or the above multi-gene expression cassette.

[0012] In the present invention, the backbone vector of the recombinant plant transformation vector is the pCAMBIA1301 vector.

[0013] In the present invention, the recombinant plant transformation vector is obtained by inserting the multi-gene expression cassette between the EcoR I and Hind III restriction sites of the pCAMBIA1301 vector.

[0014] The present invention also provides an application of the above gene combination, the above multi-gene expression cassette or the above recombinant plant transformation vector, wherein the application comprises at least one of the following:

[0015] S1. Application in the preparation of cadmium-enriched and / or cadmium-tolerant plants;

[0016] S2. Application in remediation of cadmium-contaminated soil.

[0017] In the present invention, the plant is a monocotyledonous plant.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a gene combination for improving plant cadmium tolerance. The combination combines the PPKS gene, the MT1ES gene, the MT2S gene, the MT3S gene, and the MT4S gene, and expresses them in tandem within the plant genome. After codon optimization based on the codon preference of the target plant, gene expression cassettes are constructed. The five gene expression cassettes are then serially incorporated into a plant transformation vector to produce a recombinant plant transformation vector. The recombinant plant transformation vector is then transformed into the target plant to obtain an engineered plant capable of efficiently expressing the exogenous gene. The engineered plants constructed by the present invention, which incorporate the PPKS gene, the MT1ES gene, the MT2S gene, the MT3S gene, and the MT4S gene combination, exhibit enhanced cadmium accumulation and tolerance, resulting in more ideal cadmium-contaminated soil remediation effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the structure of the pCAMBIA1301:PPKS:MT1ES:MT2S:MT3S:MT4S plant transformation vector.

[0021] Figure 2 PCR electrophoresis diagram of transgenic lines.

[0022] Figure 3 Figure 2 shows the phenotypes of engineered rice and wild-type rice plants after treatment with Cd ion solution.

[0023] Figure 4 These are the results of Cd content determination in soil after 30 days of treatment with Cd ion solution. DETAILED DESCRIPTION

[0024] The invention provides a gene combination for improving the cadmium accumulation and cadmium tolerance of plants. The gene combination comprises a PPKS gene, an MT1ES gene, an MT2S gene, an MT3S gene and an MT4S gene. The nucleotide sequence of the PPKS gene is shown in SEQ ID NO.1, the nucleotide sequence of the MT1ES gene is shown in SEQ ID NO.2, the nucleotide sequence of the MT2S gene is shown in SEQ ID NO.3, the nucleotide sequence of the MT3S gene is shown in SEQ ID NO.4, and the nucleotide sequence of the MT4S gene is shown in SEQ ID NO.5. In the present invention, the PPKS gene was optimized and synthesized based on the PPK gene of Escherichia coli (SEQ ID NO. 7), the MT1ES gene was optimized and synthesized based on the MT1E gene of Homo sapiens (SEQ ID NO. 8), the MT2S gene was optimized and synthesized based on the MT2 gene of Homo sapiens (SEQ ID NO. 9), the MT3S gene was optimized and synthesized based on the MT3 gene of Homo sapiens (SEQ ID NO. 10), and the MT4S gene was optimized and synthesized based on the MT4 gene of Homo sapiens (SEQ ID NO. 11). In the present invention, based on the PPK gene of Escherichia coli, the structures of the MT1E gene, MT2 gene, MT3 gene, and MT4 gene of Homo sapiens were optimized according to the following principles: (1) Optimizing gene codons, taking into account the codon preferences of E. coli and plants, and improving gene translation efficiency. (2) Eliminate the recognition sites of commonly used restriction endonucleases within the gene to facilitate the construction of the expression cassette. (3) Eliminate inverted repeat sequences, stem-loop structures, and transcription termination signals to balance the GC / AT ratio within the gene and improve the stability of the RNA. (4) Make the gene-encoded protein conform to the N-terminal principle to improve the stability of the translated protein. (5) Optimize the free energy of the mRNA secondary structure to improve the efficiency of gene expression. As an embodiment, the PPKS gene, MT1ES gene, MT2S gene, MT3S gene, and MT4S gene in the gene combination are sequentially connected to obtain the gene combination. In the present invention, the PPKS gene is used to improve the plant's enrichment of cadmium ions, and the MT1ES gene, MT2S gene, MT3S gene, and MT4S gene can bind to free cadmium ions in the cytoplasm, thereby alleviating the damage of cadmium ions to plant organelles and the effects of metabolic regulation, thereby improving the plant's resistance and tolerance to cadmium ions. In the present invention, the gene combination for improving the cadmium accumulation and cadmium tolerance of plants is introduced into plants and can be efficiently expressed in plants, thereby improving the cadmium tolerance of transgenic plants while improving the ability of plants to accumulate cadmium, so that transgenic plants have a more ideal cadmium-contaminated soil remediation effect.

[0025] The present invention also provides a multi-gene expression cassette for improving plant cadmium accumulation and tolerance. The multi-gene expression cassette is a cassette consisting of a PPKS gene expression cassette fused with the 35S promoter and NOS terminator, an MT1ES gene expression cassette fused with the 35S promoter and NOS terminator, an MT2S gene expression cassette fused with the 35S promoter and NOS terminator, an MT3S gene expression cassette fused with the 35S promoter and NOS terminator, and an MT4S gene expression cassette fused with the 35S promoter and NOS terminator. In the present invention, the fusion of the above genes with the 35S promoter and NOS terminator utilizes a modified overlap extension PCR technique. In the present invention, the 35S promoter is derived from cauliflower mosaic virus, and the NOS terminator is derived from Agrobacterium rhizogenes. As an embodiment, the PPKS gene expression cassette, MT1ES gene expression cassette, MT2S gene expression cassette, MT3S gene expression cassette, and MT4S gene expression cassette constructed above are sequentially connected in the order of PPKS-MT1ES-MT2S-MT3S-MT4S to obtain the multi-gene expression cassette. Preferably, the sequence of the multi-gene expression cassette is shown in SEQ ID NO. 6.

[0026] The present invention also provides a recombinant plant transformation vector comprising the aforementioned gene combination or multi-gene expression cassette. In the present invention, the backbone vector of the recombinant plant transformation vector is the pCAMBIA1301 vector. In the present invention, the recombinant plant transformation vector is obtained by inserting the multi-gene expression cassette between the EcoR I and Hind III restriction sites of the pCAMBIA1301 vector. In one embodiment, EcoR I and Hind III restriction sites are introduced at both ends of the sequence of the multi-gene expression cassette, respectively.

[0027] The present invention also provides applications of the above gene combination, the above multi-gene expression cassette or the above recombinant plant transformation vector, wherein the application comprises at least one of the following:

[0028] S1. Application in the preparation of cadmium-enriched and / or cadmium-tolerant plants;

[0029] S2. Application in remediation of cadmium-contaminated soil.

[0030] The gene combination, multi-gene expression cassette, and recombinant plant transformation vector of the present invention improve the plant's ability to accumulate and tolerate cadmium, enabling the engineered plants to have a more ideal cadmium-contaminated soil remediation effect.

[0031] The plant in the above application is preferably a monocotyledonous plant. The monocotyledonous plant is preferably a grass plant. The grass plant is preferably a plant of the genus Oryza. The plant of the genus Oryza is preferably rice, such as Oryza sativa L.

[0032] To further illustrate the present invention, the gene combination, multi-gene expression cassette and recombinant plant transformation vector for improving plant cadmium tolerance provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional molecular biological methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0034] The test materials used in the present invention and their sources include:

[0035] Wild-type rice Zhonghua 11 was cultured in an artificial climate chamber at 26°C with a 16-hour photoperiod. Escherichia coli DH5α was maintained by the Plant Genetic Engineering Laboratory, Institute of Biotechnology, Shanghai Academy of Agricultural Sciences.

[0036] Restriction enzymes and ligases were purchased from Shanghai Haojia Co., Ltd. Chemicals not specified were of analytical grade and purchased from Sangon Biotechnology (Shanghai) Co., Ltd. or Shanghai Sinopharm Group Co., Ltd.

[0037] For details of conventional molecular biological operations in the present invention, please refer to Molecular Cloning Experimental Guide, 3rd Edition (translated by Huang Peitang et al., Science Press, China, 2002).

[0038] Example 1 Construction of a multigene plant expression vector

[0039] 1. Optimization design and synthesis of five genes

[0040] Based on the PPK gene (SEQ ID NO.7) of Escherichia coli, the MT1E gene (SEQ ID NO.8), MT2 gene (SEQ ID NO.9), MT3 gene (SEQ ID NO.10) and MT4 gene (SEQID NO.11) of Homo sapiens, the structures were optimized according to the following principles: (1) Optimize gene codons, taking into account the codon preferences of E. coli and plants, and improve gene translation efficiency. (2) Eliminate the recognition sites of commonly used restriction endonucleases within the gene to facilitate the construction of expression cassettes. (3) Eliminate inverted repeat sequences, stem-loop structures and transcription termination signals to balance the GC / AT ratio within the gene and improve the stability of RNA. (4) Make the gene-encoded protein conform to the N-terminal principle to improve the stability of the translated protein. (5) Optimize the free energy of the mRNA secondary structure to improve gene expression efficiency. The nucleotide sequence PPKS shown in SEQ ID NO.1 and the nucleotide sequence MT1ES shown in SEQ ID NO.2 were obtained by optimization and synthesis, the nucleotide sequence MT2S shown in SEQ ID NO.3 was obtained by synthesis, the nucleotide sequence MT3S shown in SEQ ID NO.4 was obtained by synthesis, and the nucleotide sequence MT4S shown in SEQ ID NO.5 was obtained by synthesis, and their sequences were respectively sequenced to confirm.

[0041] 2. Construction of multi-gene plant transformation vector:

[0042] The five optimized genes were fused to the 35S promoter and NOS terminator, respectively. These gene fusions were amplified using a modified overlap extension PCR technique, as described in the literature (Rihe Peng, Aisheng Xiong, Quanhong Yao; "A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mulagenesis," Applied Microbiology Biotechnology, 2006, 73:234-240). Phanta Max Super-Fidelity DNA Polymerase, suitable for high-fidelity amplification of long genes, was used (Vazyme Biotech Co., Ltd., Nanjing). The PCR amplification program was as follows: initial denaturation at 95°C for 30 s; denaturation at 95°C for 45 s, annealing at 56-72°C for 45 s, extension at 72°C for 5-20 min (1000 bp / min), for 25-35 cycles; and final extension at 72°C for 10 min. Five gene expression cassettes were constructed separately; the five gene expression cassettes were then linked in sequence in the order of PPKS-MT1ES-MT2S-MT3S-MT4S using the ClonExpress MultiS multi-fragment one-step seamless rapid cloning kit (Novozymes) to form a complete sequence containing a multi-gene expression cassette; EcoR I and Hind III restriction sites were introduced at both ends of the complete sequence, and the complete sequence was analyzed and determined by Sangon Biotech (Shanghai) Co., Ltd. for nucleotide sequence analysis. Finally, the correctly sequenced complete synthetic fragment was double-digested with EcoR I and Hind III and then connected to the same restriction enzyme-digested vector pCAMBIA1301, thus obtaining a multi-gene plant transformation vector pCAMBIA1301:PPKS:MT1ES:MT2S:MT3S:MT4S containing five genes. The schematic diagram of the vector structure is shown in the figure. Figure 1 shown.

[0043] The multi-gene plant transformation vector pCAMBIA1301:PPKS:MT1ES:MT2S:MT3S:MT4S was efficiently transformed into Escherichia coli DH5α competent cells, and plasmid extraction and sequencing were performed. After successful plasmid sequencing, the plasmid was deposited.

[0044] Example 2 Transformation of rice

[0045] 1. Acquisition and identification of engineered rice

[0046] 1.1 Preparation and electroporation of Agrobacterium

[0047] (1) Agrobacterium tumefaciens strain EHA105 was selected and inoculated into 5 mL of LB liquid medium (rifampicin 50 μg / mL, chloramphenicol 100 μg / mL) and cultured at 28°C and 250 rpm for 20 h.

[0048] (2) Take 1 mL of bacterial solution and transfer it into 20-30 mL of LB liquid medium (rifampicin 50 μg / mL, chloramphenicol 100 μg / mL), culture at 28°C and 250 rpm for about 12 h, and measure the OD 600 =1.5.

[0049] (3) Centrifuge at 8000 rpm, 4°C for 10 min to collect the cells, resuspend them in Agrobacterium transformation solution (5 wt% sucrose, 0.05 wt% Silwet L-77) and dilute to OD 600 =0.8.

[0050] (4) Resuspend the cells thoroughly in 0.5 times the volume of sterile water pre-cooled at 4°C. Centrifuge at 3500g for 10 min and carefully discard the supernatant. Resuspend the cells thoroughly in 1-2 mL of sterile 10% glycerol pre-cooled at 4°C. The culture solution can be used immediately or stored at -70°C.

[0051] (5) Add 40 μL of bacterial suspension and 1-2 μL of plasmid DNA (a constructed plant transformation vector containing five genes) to a 1.5 mL eppendorf tube. The plasmid DNA concentration is 0.4 pg-0.3 μg. Mix well in a 0.2 cm diameter electroporation cuvette and place on ice for about 1 minute. Adjust the electroporation parameters to 25 μF, 2.5 kV / cm, and 400 Ω. Electroporation is performed with a discharge time of 4-5 msec.

[0052] (6) After electroporation, 1.0 mL of expression culture medium was added to the cells, incubated at 29°C for 1 hour, and plated onto YEB plates supplemented with antibiotics (rifampicin (50 μg / mL) + kanamycin (50 μg / mL)). Using the above transformation procedure, the plant expression vector pCAMBIA1301:PPKS:MT1ES:MT2S:MT3S:MT4S prepared in Example 1 was electroporated into Agrobacterium tumefaciens EHA105 to obtain Agrobacterium tumefaciens strain EHA105 (pCAMBIA1301:PPKS:MT1ES:MT2S:MT3S:MT4S).

[0053] 1.2 Agrobacterium infection and co-cultivation with rice callus

[0054] (1) Rice callus induction: Select mature seeds of Zhonghua 11 rice (shelled), soak them in 70% ethanol for 1 min, rinse them with sterile water, soak them in 2% sodium hypochlorite for 20 min for disinfection, and rinse them repeatedly with sterile water. Place them in a sterile culture dish on a clean bench, dry them with sterilized filter paper, and then use a knife to peel off the mature embryos. Inoculate them on the culture medium with the scutellum facing up. Place the embryos on callus induction medium (including MS 4.4 g / L, 2,4-D 2.5 mg / L, casein 600 mg / L, sucrose 30 g / L and plant gel 5 g / L, adjust the pH to 5.8 with KOH, and sterilize them by high pressure) for callus culture. Induce callus at 25-28 ° C for 8-10 days. When the embryos grow to 1 cm, strictly select the scutellum callus with good quality and inoculate them on NBD2 (including NB and 2,4-D 2 mg / L) culture medium, culture in the dark at 25-28°C for 4-7 days for subculture.

[0055] (2) Infection with Agrobacterium tumefaciens culture

[0056] Agrobacterium EHA105 carrying the plant expression vector (pCAMBIA1301:PPKS:MT1ES:MT2S:MT3S:MT4S) was picked from the YEB plate and inoculated into 5 mL of YEB liquid medium containing 50 mg / L kanamycin. The culture was shaken at 200 rpm at 28°C until the OD 600 = 0.5, and then re-inoculated into fresh YEB medium at a ratio of 1:100 and cultured with shaking. The antibiotics and culture conditions in YEB medium were the same as before. 600 = 0.5, centrifuge the bacterial solution at a relative centrifugal force of 6000g at 4°C for 10 min to collect the Agrobacterium cells, and resuspend the Agrobacterium in 2 / 3MS + 1 / 3YEB to an OD of 600 =0.5, reserve.

[0057] (3) Co-cultivation of Agrobacterium tumefaciens:

[0058] Take the embryonic callus of rice with vigorous growth, cut it into 2mm small pieces, put it into sterile culture dish, add OD 600 = 0.5 Agrobacterium tumefaciens liquid, soak for 25 minutes, dry the surface liquid with sterile filter paper, and then inoculate the rice callus on the co-cultivation medium and culture in the dark at 28°C for 2-3 days.

[0059] (4) Rice callus differentiation and plant regeneration

[0060] The rice callus tissue after co-cultivation was collected, washed three times with sterile water containing 500 mg / L cephalosporin, and excess water was removed by aspiration.

[0061] The callus tissue was transferred to the screening medium (including NB medium, 0.5 mg / L 2,4-D, 600 mg / L cefotaxime and 25 mg / L hygromycin) and cultured for 12-15 days, of which the first 6-7 days were cultured in the dark and the next 8-9 days were cultured in the light with a light intensity of 45-55 mmoL·m -2 ·s -1 The callus that had differentiated into adventitious buds was then transferred to a differentiation medium RE2-H and cultured for 15 days, allowing the adventitious buds to grow into 2-4 cm seedlings, thereby obtaining transgenic rice seedlings. The differentiation medium RE2-H contained MS, 30 g / L sucrose, 10-20 g / L sorbitol, 500 mg / L hydrolyzed casein, 1 mg / L 6-benzylaminopurine, 0.5 mg / L naphthaleneacetic acid, 0.5 mg / L kinetin, 0.2 mg / L zeatin, 50 mg / L hygromycin, and 4.5 g / L phytagel, with a pH of 5.8.

[0062] Finally, these seedlings were transferred to rooting medium, took root after 2 weeks, and were transplanted into long test tubes for culture for 20-30 days. The larger genetically modified rice plants were transplanted into the fields of the Baihe Transgenic Base of the Shanghai Academy of Agricultural Sciences.

[0063] 2. Identification of genetically modified rice

[0064] 2.1 PCR detection of genomic DNA of transformed plants

[0065] Genomic DNA was extracted from leaves of transgenic rice transplanted into the field using the SDS method and used as a template. PCR amplification was performed to detect the exogenous genes PPKS, MT1ES, MT2S, MT3S, and MT4S. The primers used were as follows:

[0066] PPKS:

[0067] F(SEQ ID No.12):5'-GATGTAACGTGCCTTCACAGTGTC-3';

[0068] R (SEQ ID No. 13): 5'-GCAATCTCAATCGTGGATCGTTAC-3'.

[0069] MT1ES:

[0070] F(SEQ ID No.14):5'-AGCACAGCAAGAGCACTTCTC-3';

[0071] R (SEQ ID No. 15): 5'-ATGGACCCAAACTGCTCTTGT-3'.

[0072] MT2S:

[0073] F(SEQ ID No.16):5'-TTAAGCACAACAAGAGCACTTG-3';

[0074] R (SEQ ID No. 17): 5'-ATGGACCCAAACTGCTCCTGT-3'.

[0075] MT3S:

[0076] F(SEQ ID No.18):5'-TTACTGACAGCAAGAGCACTTC-3';

[0077] R (SEQ ID No. 19): 5'-ATGGACCCTGAGACCTGCCCA-3'.

[0078] MT4S:

[0079] F(SEQ ID No.20):5'-TGGACAGCAAGAACACTTGTC-3';

[0080] R (SEQ ID No. 21): 5'-ATGGACCCACGTGAATGTGTC-3'.

[0081] The amplification program used was: 95°C initial denaturation for 30 seconds; 95°C denaturation for 45 seconds, 56-72°C annealing for 45 seconds, 72°C extension for 5-20 minutes (1000 bp / min), amplification for 25-35 cycles; final extension at 72°C for 10 minutes. Figure 2 PCR electrophoresis diagram.

[0082] Figure 2 In the PCR electrophoresis diagram, M: 2000bp DNA marker; WT is wild-type rice; PPKS, MT1ES, MT2S, MT3S and MT4S are the corresponding detection results in the transgenic engineering lines. Figure 2 It can be seen that the transgenic lines can amplify the above five genes, indicating that the exogenous genes PPKS, MT1ES, MT2S, MT3S and MT4S are completely integrated into the rice genome.

[0083] 3. Identification of cadmium resistance in transgenic rice plants

[0084] The transgenic rice seeds harvested above were self-pollinated for three generations to obtain homozygous transformants, and the seeds were harvested. After sowing, the plants were grown in a 25°C incubator for three weeks and then irrigated with a 20mM cadmium solution. Three transgenic lines were selected and named OE1, OE2, and OE3. Wild-type rice (WT) was used as a control. After three weeks of irrigating, the cadmium resistance of the transgenic and wild-type rice was observed ( Figure 3 ). Figure 3 WT in the figure is wild-type rice; OE1, OE2, and OE3 are transgenic rice (i.e., plants of homozygous transformed strains after sowing), of which OE1, OE2, and OE3 are all transgenic lines that have successfully been transformed with the exogenous genes PPKS, MT1ES, MT2S, MT3S, and MT4S. Figure 3 It can be seen that after three weeks of irrigation, the overall growth of WT wild-type rice was weaker than that of the transgenic rice lines OE1, OE2, and OE3.

[0085] The cadmium content of the soil matrix in which the transgenic rice plants were grown was determined 30 days after treatment with Cd ion solution according to GB / T 5750.6-2006 and GB 5009.268-2016 ( Figure 4 ), Figure 4 The results showed that under the same concentration treatment, the average residual Cd ion concentration in the soil of transgenic rice was 60 mg / kg, while the average residual Cd ion concentration in the soil of wild rice was 110 mg / kg. The former was about half of the latter. The significant difference analysis (t-test, *P<0.05, **P<0.01) showed that transgenic plants have a more ideal effect in remediating cadmium-contaminated soil.

[0086] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of a multi-gene expression cassette or a recombinant plant transformation vector comprising the multi-gene expression cassette in at least one of the following applications, characterized in that: S1. Application in the preparation of cadmium-enriched and / or cadmium-tolerant rice; S2. Application in remediation of cadmium-contaminated soil; The complete sequence of the multi-gene expression cassette is shown in SEQ ID NO.

6.

2. The application according to claim 1, characterized in that The backbone vector of the recombinant plant transformation vector is the pCAMBIA1301 vector.

3. The application according to claim 1, characterized in that The recombinant plant transformation vector is obtained by inserting the multi-gene expression box between the EcoR I and HindIII restriction sites of the pCAMBIA1301 vector.

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