Gene combination for improving cadmium enrichment and cadmium tolerance of plants, multi-gene expression cassette and application

Abstract

The invention provides a gene combination and a polygene expression cassette for improving cadmium enrichment and cadmium resistance of plants, and relates to the technical field of biological gene engineering. A PPKS gene, an MT1ES gene, an MT2S gene, an MT3S gene and an MT4S gene are combined, gene expression cassettes are constructed respectively after codon optimization is performed according to codon preference of a target plant, and the five gene expression cassettes are connected in series into a plant transformation vector to obtain a recombinant plant transformation vector. And transforming the recombinant plant transformation vector into a target plant to obtain an engineering plant capable of efficiently expressing an exogenous gene in the target plant. Experimental determination results prove that transfer of the PPKS gene, the MT1ES gene, the MT2S gene, the MT3S gene and the MT4S gene improves the enrichment capacity and tolerance of the plant to cadmium, so that the engineering plant has a more ideal cadmium-contaminated soil remediation effect.

Description

Technical Field

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

[0002] Cadmium (Cd) is a highly toxic heavy metal pollutant, ranking first among inorganic soil pollutants. Cadmium is mainly produced during the processing and smelting of non-ferrous metals and is widely present in environmental media such as water sources, soil, and food. Cd is easily absorbed by plants due to its strong fluidity and solubility. It not only affects the normal physiological metabolism of plants and reduces the quality of crops, but also can cause harm to human bones, lungs, kidneys, and central nervous system through transmission and enrichment in the food chain, and even threaten life.

[0003] At present, the main remediation technologies for heavy metal pollution in soil include physical remediation, chemical remediation and biological remediation. Compared with traditional physical and chemical remediation technologies, the use of biological remediation technology to repair heavy metal polluted soil will not damage the growth environment of plants. At the same time, biological remediation technology also has the characteristics of low cost, high efficiency, no secondary pollution, simple operation and wide application range. Therefore, biological remediation technology has become a research hotspot in recent years.

[0004] The detoxification of heavy metals in plants includes acidification, compartmentalization, chelation, free radical scavenging and transport. Among them, metallothionein (MT), a metal binding protein rich in cysteine ​​residues, can bind to free Cd in the cytoplasm, thereby alleviating the damage of Cd to organelles and metabolic regulation. In addition, there are reports that polyphosphate kinase PPK can increase the enrichment of metal ions in plants and reduce the concentration of metal ions in the environment. However, there are no reports on the combined effect of these two genes on plant enrichment and cadmium tolerance. Summary of the invention

[0005] The purpose of the present invention is to provide a gene combination, a multi-gene expression box and an application for improving the cadmium resistance of plants. The gene combination is expressed in series in plants, which 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 plant to have a more ideal cadmium-contaminated soil remediation effect.

[0006] The invention provides a gene combination for improving the cadmium accumulation and cadmium resistance of plants, comprising a PPKS gene, an MT1ES gene, an MT2S gene, an MT3S gene and an MT4S gene, wherein 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 connecting in series 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 box.

[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, the application comprising 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 the cadmium resistance of plants, combining PPKS gene, MT1ES gene, MT2S gene, MT3S gene and MT4S gene, and expressing them in series in the plant genome. Gene expression cassettes are respectively constructed after codon optimization according to the codon preference of the target plant, and five gene expression cassettes are connected in series into a plant transformation vector to obtain a recombinant plant transformation vector. The recombinant plant transformation vector is transformed into the target plant to obtain an engineered plant that can efficiently express exogenous genes in the target plant. The engineered plant constructed by the present invention, which is transferred with a combination of PPKS gene, MT1ES gene, MT2S gene, MT3S gene and MT4S gene, has better cadmium enrichment ability and tolerance, so that the engineered plant has a more ideal cadmium-contaminated soil remediation effect. 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 This is the PCR electrophoresis diagram of the transgenic strain.

[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 resistance of plants, comprising a PPKS gene, an MT1ES gene, an MT2S gene, an MT3S gene and an MT4S gene, wherein 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 is optimized and synthesized based on the PPK gene (SEQ ID NO.7) of Escherichia coli, the MT1ES gene is optimized and synthesized based on the MT1E gene (SEQ ID NO.8) of Homo sapiens, the MT2S gene is optimized and synthesized based on the MT2 gene (SEQ ID NO.9) of Homo sapiens, the MT3S gene is optimized and synthesized based on the MT3 gene (SEQ ID NO.10) of Homo sapiens, and the MT4S gene is optimized and synthesized based on the MT4 gene (SEQ ID NO.11) of Homo sapiens. In the present invention, based on the PPK gene of Escherichia coli, the MT1E gene, MT2 gene, MT3 gene and MT4 gene of Homo sapiens are structurally optimized according to the following principles: (i) Gene codons are optimized, taking into account the codon preferences of Escherichia coli and plants, and improving gene translation efficiency. (ii) Eliminate the recognition sites of commonly used restriction endonucleases within the gene to facilitate the construction of the expression cassette. (iii) Eliminate reverse repeat sequences, stem-loop structures and transcription termination signals to balance the GC / AT within the gene and improve the stability of RNA. (iv) Make the gene-encoded protein conform to the N-terminal principle to improve the stability of the translated protein. (v) Optimize the free energy of the mRNA secondary structure to improve the efficiency of gene expression. As an implementable 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 enrichment of cadmium ions in plants, 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, and improving the resistance and tolerance of plants to cadmium ions. In the present invention, the gene combination for improving the cadmium accumulation and cadmium resistance of plants is transferred into plants and can be efficiently expressed in plants, thereby improving the cadmium accumulation ability of plants while improving the cadmium resistance of transgenic plants, 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 the cadmium accumulation and cadmium tolerance of plants, wherein the multi-gene expression cassette is a multi-gene expression cassette obtained by connecting in series the PPKS gene expression cassette formed by fusing the PPKS gene with the 35S promoter and the NOS terminator, the MT1ES gene expression cassette formed by fusing the MT1ES gene with the 35S promoter and the NOS terminator, the MT2S gene expression cassette formed by fusing the MT2S gene with the 35S promoter and the NOS terminator, the MT3S gene expression cassette formed by fusing the MT3S gene with the 35S promoter and the NOS terminator, and the MT4S gene expression cassette formed by fusing the MT4S gene with the 35S promoter and the NOS terminator. In the present invention, the fusion of the above-mentioned gene with the 35S promoter and the NOS terminator adopts the improved overlap extension PCR technology. In the present invention, the 35S promoter is derived from cauliflower mosaic virus; and the NOS terminator is derived from Agrobacterium rhizogenes. As an implementation method, 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 above gene combination or the above multi-gene expression cassette. In the present invention, the backbone vector of the recombinant plant transformation vector is a 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. As an implementable embodiment, EcoR I and Hind III restriction sites are introduced at both ends of the sequence of the multi-gene expression cassette.

[0027] 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:

[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, so that the engineered plants have a more ideal cadmium-contaminated soil restoration effect.

[0031] The plant in the above application is preferably a monocotyledonous plant. The monocotyledonous plant is preferably a Gramineae plant. The Gramineae plant is preferably a Rice plant. The Rice plant is preferably Rice, such as Rice Zhonghua 11.

[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 construed as limiting the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are all conventional molecular biological methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

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

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

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

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

[0038] Example 1 Construction of a multi-gene 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 structure was optimized according to the following principles: (i) Optimize gene codons, taking into account the codon preferences of Escherichia coli and plants, and improve gene translation efficiency. (ii) Eliminate the recognition sites of commonly used restriction endonucleases inside the gene to facilitate the construction of expression cassettes. (iii) Eliminate reverse repeat sequences, stem-loop structures and transcription termination signals to balance the GC / AT inside the gene and improve the stability of RNA. (iv) Make the gene-encoded protein conform to the N-terminal principle to improve the stability of the translated protein. (v) Optimize the free energy of the mRNA secondary structure to improve the efficiency of gene expression. The nucleotide sequence PPKS as shown in SEQ ID NO.1 was obtained by optimization and synthesis, the nucleotide sequence MT1ES as shown in SEQ ID NO.2 was obtained by synthesis, the nucleotide sequence MT2S as shown in SEQ ID NO.3 was obtained by synthesis, the nucleotide sequence MT3S as shown in SEQ ID NO.4 was obtained by synthesis, and the nucleotide sequence MT4S as shown in SEQ ID NO.5 was obtained by synthesis, and their sequences were determined respectively.

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

[0042] The optimized five genes were fused to the 35S promoter and NOS terminator, respectively. The above genes were fused to the 35S promoter and NOS terminator using the improved overlap extension PCR technology. The specific reference of the improved overlap extension PCR technology is (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), and the Phanta Max Super-Fidelity DNA Polymerase suitable for high-fidelity amplification of long genes from Nanjing Vazyme Biotech Co., Ltd. was used. The PCR amplification program was: 95°C pre-denaturation for 30s; 95°C denaturation for 45s, 56-72°C annealing for 45s, 72°C extension for 5-20min (1000bp / min), amplification for 25-35 cycles; 72°C final extension for 10min. Five gene expression cassettes were constructed respectively; the five gene expression cassettes were then connected in sequence in the order of PPK S-MT1ES-MT2S-MT3S-MT4S using the ClonExpress MultiS multi-fragment one-step seamless rapid cloning kit (Novozyme) to form a complete sequence containing a multi-gene expression cassette; EcoR I and HindⅢ restriction sites were introduced at both ends of the complete sequence, and the complete sequence was analyzed and determined by the nucleotide sequence analysis of Sangon Biotech (Shanghai) Co., Ltd. Finally, the complete synthetic fragment with correct sequencing was double-digested with EcoR I and HindⅢ, and then connected to the same restriction 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 sequence determination were performed. After the plasmid sequencing was successful, it was preserved.

[0044] Example 2 Transformation of rice

[0045] 1. Acquisition and identification of engineered rice

[0046] 1.1 Preparation and electroporation of Agrobacterium

[0047] (1) The Agrobacterium tumefaciens strain EHA105 was selected and inoculated into 5 mL of LB liquid culture 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 infiltration solution (5 wt% sucrose, 0.05 wt% Silwet L-77) and dilute to OD 600 =0.8.

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

[0051] (5) Add 40 μL of bacterial solution and 1-2 μL of plasmid DNA (a plant transformation vector containing five genes that has been constructed) to a 1.5 mL eppendorf tube, where the mass concentration of the plasmid DNA is 0.4 pg-0.3 μg. Mix well in a 0.2 cm diameter electric shock cup and place on ice for about 1 minute. Adjust the electric shock parameters to 25 μF, 2.5 kV / cm, and 400 Ω. Electric shock, discharge time is 4-5 msec.

[0052] (6) After electroporation, 1.0 mL of expression culture medium was added to the bacteria, cultured at 29° C. for 1 hour, and then applied to a YEB plate containing 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 (hulled), 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 scalpel 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 phytogels 5 g / L, adjust the pH to 5.8 with KOH, and sterilize them by high pressure) for callus culture. Induce callus by dark culture 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) medium and culture in the dark at 25-28°C for 4-7 days for subculture.

[0055] (2) Infection with Agrobacterium tumefaciens

[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 28°C on a shaker at 200 rpm until the OD 600 =0.5, and then re-inoculated into fresh YEB medium at a ratio of 1:100 and cultured under shaking. The antibiotics and culture conditions in the 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, collect the Agrobacterium cells, and resuspend the Agrobacterium in 2 / 3MS+1 / 3YEB to OD 600 =0.5, for standby use.

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

[0058] Take the vigorously growing rice embryonic callus, cut it into 2 mm small pieces, put it into a sterile culture dish, add OD 600 = 0.5 of Agrobacterium tumefaciens culture medium, soak for 25 minutes, dry the surface culture medium with sterile filter paper, and then inoculate the rice callus on the co-cultivation medium and culture it 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 the 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 has differentiated into adventitious buds is then transferred to a differentiation medium RE2-H for 15 days, which allows the adventitious buds to grow into 2-4 cm seedlings, thereby obtaining transgenic rice seedlings; wherein the differentiation medium RE2-H includes 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, pH 5.8.

[0062] Finally, the 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 transgenic rice plants were transplanted into the fields of the White Crane 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] The transgenic rice leaves transplanted to the field were taken, and the genomic DNA was extracted by SDS method as a template. The exogenous genes PPKS, MT1ES, MT2S, MT3S and MT4S were detected by PCR amplification method. The primers used are 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℃ pre-denaturation for 30s; 95℃ denaturation for 45s, 56-72℃ annealing for 45s, 72℃ extension for 5-20min (1000bp / min), amplification for 25-35 cycles; 72℃ final extension for 10min. 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 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 transformed plants and harvested seeds. After sowing, the plants were grown in a 25°C culture room for three weeks and then irrigated with a 20 mM cadmium solution. Three transgenic lines were selected and named OE1, OE2, and OE3. At the same time, wild-type rice (WT) was used as a control. After three weeks of irrigation, the results of the resistance of transgenic rice and wild-type rice to cadmium were 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 strains successfully introduced with 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 in accordance with GB / T 5750.6-2006 and GB 5009.268-2016 after 30 days of treatment with Cd ion solution ( Figure 4 ), Figure 4 The results showed that under the same concentration treatment, the average residual Cd ion concentration in the soil planted with genetically modified rice was 60 mg / kg, while the average residual Cd ion concentration in the soil planted with wild-type rice was 110 mg / kg. The former was about half less than the latter. The significant difference analysis (t-test, *P<0.05, **P<0.01) showed that genetically modified plants have a more ideal effect on the remediation of cadmium-contaminated soil.

[0086] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. A gene combination that improves plant cadmium accumulation and cadmium tolerance, It is characterized in that The gene combination includes PPKS gene, MT1ES gene, MT2S gene, MT3S gene and 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.

2. The gene combination according to claim 1, It is characterized in that The PPKS gene, the MT1ES gene, the MT2S gene, the MT3S gene and the MT4S gene are sequentially connected to obtain the gene combination.

3. A multi-gene expression cassette for improving cadmium accumulation and cadmium tolerance in plants, It is characterized in that The multi-gene expression cassette is a multi-gene expression cassette obtained by connecting in series a PPKS gene expression cassette formed by fusing the PPKS gene described in claim 1 with a 35S promoter and a NOS terminator, a MT1ES gene expression cassette formed by fusing the MT1ES gene described in claim 1 with a 35S promoter and a NOS terminator, a MT2S gene expression cassette formed by fusing the MT2S gene described in claim 1 with a 35S promoter and a NOS terminator, a MT3S gene expression cassette formed by fusing the MT3S gene described in claim 1 with a 35S promoter and a NOS terminator, and a MT4S gene expression cassette formed by fusing the MT4S gene described in claim 1 with a 35S promoter and a NOS terminator.

4. The multi-gene expression cassette according to claim 3, It is characterized in that The 35S promoter is derived from cauliflower mosaic virus; the NOS terminator is derived from Agrobacterium tumefaciens.

5. The multi-gene expression cassette according to claim 3 or 4, It is characterized in that The complete sequence of the multi-gene expression cassette is shown in SEQ ID NO.

6.

6. A recombinant plant transformation vector comprising the gene combination according to claim 1 or 2 or the multi-gene expression cassette according to any one of claims 3 to 5.

7. The recombinant plant transformation vector according to claim 6, It is characterized in that The backbone vector of the recombinant plant transformation vector is the pCAMBIA1301 vector.

8. The recombinant plant transformation vector according to claim 7, It is characterized in that The recombinant plant transformation vector is obtained by inserting the multi-gene expression box between the EcoR I and Hind III restriction sites of the pCAMBIA1301 vector.

9. Use of the gene combination according to claim 1 or 2, the multi-gene expression cassette according to any one of claims 3 to 5, or the recombinant plant transformation vector according to any one of claims 6 to 8, It is characterized in that The application includes at least one of the following: S1. Application in the preparation of cadmium-enriched and / or cadmium-tolerant plants; S2. Application in remediation of cadmium contaminated soil.

10. The use according to claim 9, It is characterized in that The plant is a monocotyledonous plant.

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