Method for creating rice suitable for being planted in soil seriously polluted by cadmium

Abstract

The invention discloses a method for creating rice suitable for being planted in soil seriously polluted by cadmium. The invention provides a DNA (deoxyribonucleic acid) molecule. The DNA molecule comprises a promoter and a gene for coding OsNramp5 protein, the promoter is a DNA (deoxyribonucleic acid) fragment formed by connecting two 35S promoters; the OsNramp5 protein is any one of the following proteins: (A1) a protein with an amino acid sequence of SEQ ID No.31; (A2) a protein which is obtained by substitution and / or deletion and / or addition of amino acid residues on the amino acid sequence as shown in SEQ ID No.1 and has the same function as that of the amino acid sequence as shown in SEQ ID No.1; (A3) protein which is derived from rice, has 80% or more of identity with the amino acid sequence in (A1) or (A2) and has the same function with the amino acid sequence in (A1) or (A2); and (A4) any one of the proteins from (A1) to (A3).

Description

Technical Field

[0001] This invention relates to the field of biotechnology breeding, specifically to a method for creating rice suitable for cultivation in heavily cadmium-contaminated soil. Background Technology

[0002] Rice is one of the world's most important food crops, with more than half of the world's population relying on it as their staple food. Cadmium is carcinogenic to humans and has been classified as a Group 1 carcinogen by the International Agency for Research on Cancer. The main sources of cadmium in soil are industrial waste gas, vehicle exhaust deposition, the application of pesticides, fertilizers, and agricultural films, and wastewater irrigation. Rice primarily absorbs cadmium from the soil through its roots. After absorption, cadmium is transported to the vascular bundles via symplasm and apoplastic pathways, then translocated to the stems and leaves, and finally migrated to the grains, ultimately accumulating in the human body through the food chain, thus posing a serious threat to human health. Years of scientific research and practice have proven that breeding low-cadmium rice varieties is the fundamental solution to the problem of excessive cadmium levels in rice.

[0003] Cloning and utilizing genes related to cadmium accumulation in rice is fundamental to the breeding of low-cadmium rice varieties. To date, a series of cadmium accumulation-related genes have been identified in rice. Among them, OsNramp5 is an important transport protein responsible for the absorption of cadmium, manganese, iron, and other metallic minerals by rice root cells. It is mainly expressed in rice roots, exhibiting axial polarity at the distal ends of the outer and inner cortex. Loss of its function can significantly reduce the absorption of cadmium by rice roots. When planted in fields with high cadmium pollution, the cadmium accumulation in both plants and grains is significantly reduced. Previous studies have shown that de-functionalizing the key gene OsNramp5, involved in cadmium absorption and transport, is currently an ideal approach to reduce cadmium content in rice grains. For example, using CRISPR / Cas9 technology to knock out OsNramp5 yielded three low-cadmium mutants. When planted in soil with a total cadmium content of 1.0 mg / kg, the average cadmium content in the grains of the three low-cadmium mutants was 0.024 mg / kg, compared to 0.659 mg / kg for the wild-type control, representing a 96% reduction in cadmium content compared to the control. When planted in soil with a total cadmium content of 5.0 mg / kg, the average cadmium content in the grains of the three low-cadmium mutants was 0.069 mg / kg, compared to 1.537 mg / kg for the wild-type control, representing a 95% reduction in cadmium content compared to the control, all below the national standard limit. However, under conditions of further increases in soil cadmium concentration, i.e., planting in heavily cadmium-contaminated soil, there is a risk that the cadmium content in the grains of OsNramp5-deficient rice may exceed the standard. For example, our previous study found that when the OsNramp5 loss-of-function mutant was planted in soil with a total cadmium content of 10.0 mg / kg, the cadmium content in the grains reached 0.468 mg / kg, which is higher than the national standard limit (0.2 mg / kg). Summary of the Invention

[0004] The technical problem to be solved by this invention is how to grow rice in cadmium-contaminated soil (such as heavily cadmium-contaminated soil) without exceeding the cadmium content in the rice grains.

[0005] Firstly, this invention claims protection for a DNA molecule.

[0006] The DNA molecule claimed in this invention includes a promoter and a gene encoding the OsNramp5 protein; the promoter is a DNA fragment formed by the linkage of two 35S promoters.

[0007] The OsNramp5 protein is any of the following proteins:

[0008] (A1) A protein with the amino acid sequence SEQ ID No. 1;

[0009] (A2) A protein having the same function as the amino acid sequence shown in SEQ ID No. 1, with substitution and / or deletion and / or addition of amino acid residues;

[0010] (A3) is a protein derived from rice that has more than 80% identity with the amino acid sequence described in (A1) or (A2) and has the same function;

[0011] (A4) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of any of the proteins described in (A1)-(A3).

[0012] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0013] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0014] In the aforementioned proteins, the "80% or more identity" can be 80%, 85%, 90%, or 95% or more identity. The "80% or more identity" can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The "85% or more identity" can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 95% or more identity can be at least 95%, 96%, 97%, 98%, or 99% identity.

[0015] In the aforementioned DNA molecule, the promoter is located upstream of the gene.

[0016] In the aforementioned DNA molecules, the 35S promoter may be derived from Scrophularia mosaic virus or cauliflower mosaic virus.

[0017] The promoter may be a DNA fragment of either a) or b) as follows:

[0018] a) The nucleotide sequence is the DNA fragment from position 8784 to 9681 of SEQ ID No. 3;

[0019] b) A DNA fragment that has more than 80% identity with positions 8784 to 9681 of SEQ ID No. 3 and has promoter function.

[0020] In the DNA fragments mentioned above, homology refers to the similarity of nucleotide sequences. The similarity of nucleotide sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI website. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the similarity of a pair of nucleotide sequences, the homology value (%) can be obtained.

[0021] In the aforementioned DNA fragments, the "80% or more identity" can be 80%, 85%, 90%, or 95% or more identity. The "80% or more identity" can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The "85% or more identity" can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 95% or more identity can be at least 95%, 96%, 97%, 98%, or 99% identity.

[0022] In one embodiment of the present invention, the promoter may be a 2×35S promoter, i.e., the DNA fragment of a) above.

[0023] In one embodiment of the present invention, the nucleic acid sequence of the DNA molecule is from SEQ ID No. 3 to 11304.

[0024] Secondly, this invention claims protection for a biological material.

[0025] The biological material claimed in this invention is an expression cassette, recombinant vector, or recombinant bacteria containing the DNA molecule described in claim 1 or 2.

[0026] The expression cassette refers to DNA capable of expressing OsNramp5 in host cells. This DNA may include not only a 2×35S promoter to initiate OsNRramp5 gene transcription, but also a terminator to terminate OsNramp5 transcription. Furthermore, the expression cassette may also include an enhancer sequence. The 35S promoter is a constitutive promoter of cauliflower mosaic virus. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminators (see, for example: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627.

[0027] Construct recombinant expression vectors containing the aforementioned DNA molecules. The plant expression vectors used can be Gateway system vectors or binary Agrobacterium vectors, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. When constructing recombinant expression vectors using the aforementioned DNA molecules, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0028] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants that encode enzymes or luminescent compounds that produce color changes (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes).

[0029] The biological material contains an expression cassette, recombinant vector, or recombinant bacteria containing the DNA molecule described in claim 1 or 2.

[0030] Thirdly, the present invention claims protection for the use of the aforementioned DNA molecules or biological materials in reducing the cadmium content in rice plants to be modified.

[0031] In the above application, the reduction of cadmium content in the rice to be modified means planting modified rice under cadmium-contaminated soil conditions, wherein the cadmium content in the modified rice is less than 0.2 mg / kg.

[0032] Fourthly, the present invention claims protection for the use of the aforementioned DNA molecules or biological materials in the cultivation of rice with low cadmium content.

[0033] Fifthly, the present invention claims a method for reducing the cadmium content in rice.

[0034] The method for reducing cadmium content in rice claimed in this invention includes introducing the aforementioned DNA molecule or the aforementioned biological material into recipient rice to obtain transgenic rice; wherein the cadmium content in the transgenic rice is lower than that in the recipient rice.

[0035] In the above method, genetically modified rice is planted in cadmium-contaminated soil (especially under severe cadmium contamination) and the cadmium content in the genetically modified rice is less than 0.2 mg / kg.

[0036] Sixthly, the present invention claims a method for cultivating rice with low cadmium content.

[0037] The method for cultivating low-cadmium rice claimed in this invention includes introducing the aforementioned DNA molecules or the aforementioned biological materials into recipient rice to obtain low-cadmium rice, wherein the cadmium content of the low-cadmium rice is lower than that of the recipient rice.

[0038] The method for cultivating low-cadmium rice can be achieved through hybridization or genetic modification.

[0039] Seventhly, the present invention also claims protection for a method for cultivating rice suitable for planting in cadmium-contaminated soil.

[0040] The method for cultivating rice suitable for planting in cadmium-contaminated soil, which is claimed in this invention, includes the following steps: introducing the above-mentioned DNA molecules or the above-mentioned biological materials into recipient rice to obtain rice suitable for planting in cadmium-contaminated soil.

[0041] The rice varieties suitable for cultivation in cadmium-contaminated soil are those grown in soils with a total cadmium content ≤ 10.0 mg / kg and a cadmium content in the rice cells below 0.2 mg / kg. This is especially true for rice grown in heavily cadmium-contaminated soils with a cadmium content below 0.2 mg / kg.

[0042] In the above method, the DNA molecule can be introduced into the recipient plant in the form of a recombinant expression vector.

[0043] In this invention, the promoter for initiating transcription of the OsNramp5 gene in the recombinant expression vector is a 2×35S promoter, and the terminator is a Noster poly A terminator.

[0044] In the above methods, the recombinant expression vector is introduced into the recipient plant. Specifically, this can be done by transforming plant cells or tissues using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and then cultivating the transformed plant tissues into plants.

[0045] In the above method, the transgenic plant is understood to include not only first- and second-generation transgenic plants, but also their progeny. For transgenic plants, the gene can be propagated within the species, or it can be transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. The transgenic plant includes seeds, callus tissue, complete plants, and cells.

[0046] In this invention, cadmium accumulation can be reflected by cadmium content.

[0047] The cadmium content mentioned in this invention refers to the cadmium content in rice grains.

[0048] The low-cadmium rice mentioned in this invention refers to rice with a cadmium content of less than 0.2 mg / kg, specifically rice with a cadmium content of less than 0.2 mg / kg in the rice grains.

[0049] The cadmium-contaminated soil described in this invention refers to soil with a total cadmium content ≤ 10.0 mg / kg. Further, the cadmium-contaminated soil can be lightly, moderately, or heavily contaminated. The heavily contaminated soil is soil with a total cadmium content of 10.0 mg / kg.

[0050] The recipient rice or the rice to be modified can be indica rice and / or japonica rice. Indica rice can be varieties such as Huanghuazhan, Wushansimiao, Huazhan, R900, Zhongzao 39, Xiangwanxian 32, 9311, Y58S, and Shuang 1S; japonica rice can be varieties such as Ningxiangjing 9, Nanjing 46, Wuyunjing 7, and Wujing 13. In one specific embodiment of the present invention, the recipient rice is Huanghuazhan.

[0051] The inventors of this invention have demonstrated through experiments that overexpressing the OsNramp5 gene in rice using a dual 35S promoter (2×35S promoter) significantly reduces the cadmium content in rice grains, bringing it below the national standard limit. This enables rice cultivation in cadmium-contaminated soil (such as heavily cadmium-contaminated soil) without exceeding the cadmium content limit in the rice grains. Clearly, by utilizing a dual 35S promoter (2×35S promoter) to activate the OsNramp5 gene, a new cadmium-inhibiting rice material suitable for cultivation in cadmium-contaminated soil conditions can be created. Attached Figure Description

[0052] Figure 1 A schematic diagram of the structure of the recombinant plasmid pEGOEP2×35S-H-LOC_Os07g12900.

[0053] Figure 2 A schematic diagram of the structure of the recombinant plasmid pEGOEP35S-H-LOC_Os07g12900.

[0054] Figure 3 The results of the relative expression levels of the OsNramp5 gene in the OsNramp5 overexpression lines ZDX1, ZDX2 and rice Huanghuazhan; where the horizontal axis represents the rice lines and the vertical axis represents the relative expression level of the OsNramp5 gene. Detailed Implementation

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0056] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0057] The rice variety Huang Huazhan is mentioned in the literature "Tiankang Wang, Yixing Li, et al., Mutation at Different Sites of Metal Transporter Gene OsNramp5 Affects Cd Accumulation and Related Agronomic Traits in Rice (Oryza sativa L.)". Frontiers inPlantScience.The invention is disclosed in 2019 Sep 11; Vol 10. doi:10.3389 / fols.2019.01081. It is available to the public from the Hunan Hybrid Rice Research Center. This biological material is only for repeating the relevant experiments of this invention and cannot be used for other purposes.

[0058] Huang Huazhan is the “Oryza sativa subsp.indica Huanghuazhan” mentioned in the above literature.

[0059] Example 1: Obtaining and Identifying OsNramp5 Transgenic Plants

[0060] This invention relates to a protein derived from rice Huanghuazhan, named OsNramp5 (i.e., LOC_Os07g15370), whose amino acid sequence is shown in SEQ ID No.1, and whose CDS sequence encoding the OsNramp5 protein is shown in SEQ ID No.2.

[0061] 1. Obtaining the OsNramp5 protein-coding gene

[0062] Total RNA was extracted from the indica rice variety Huang Huazhan and reverse transcribed into cDNA. Using this cDNA as a template, the RNA was extracted using primer OsNramp5. CDS -F:5'-actagggtctcGCACCATGGAGATTGAGAGAGAGAGCAGTGAGA-3' and OsNramp5 CDS -R: 5'-actagggtctcTACCGCTACCTTGGGAGCGGGATGTC-3' was used for PCR amplification to obtain the amplification product. The obtained PCR product was detected by 1% agarose gel electrophoresis, and the purified DNA fragment of approximately 1617 bp was recovered and sequenced for verification. The sequencing results showed that the DNA fragment contained the DNA molecule shown in SEQ ID No. 2.

[0063] 2. Construction of the OsNramp5 gene expression vector

[0064] The DNA fragments obtained in step 1 were ligated into the pEGOEP-2×35S vector and the pEGOEP-35S ligation vector using T4 DNA ligase, respectively. The enzyme digestion and ligation reaction systems are as follows:

[0065]

[0066]

[0067] The reaction was carried out at 37℃ for 5 min, followed by at 20℃ for 5 min, yielding ligation product 1 (the product obtained by ligating the DNA fragment obtained in step 1 with the pEGOEP-2×35S vector) and ligation product 2 (the product obtained by ligating the DNA fragment obtained in step 1 with the pEGOEP-35S vector). Ligation product 1 and ligation product 2 were transformed into *E. coli* DH5α competent cells, respectively. After screening with kanamycin-resistant plates, positive clones were picked the next day, and plasmids were extracted and sequenced for verification, naming them pEGOEP2×35S-H-LOC_Os07g15370 and pEGOEP35S-H-LOC_Os07g15370, respectively.

[0068] A schematic diagram of the structure of the recombinant plasmid pEGOEP2×35S-H-LOC_Os07g15370 is shown below. Figure 1 Whole plasmid sequencing revealed the nucleotide sequence of the recombinant plasmid pEGOEP2×35S-H-LOC_Os07g15370 to be SEQ ID No. 3 in the sequence listing. In SEQ ID No. 3, positions 8784 to 9681 represent the 2×35S promoter, positions 9688 to 11304 represent the CDS sequence of OsNramp5, and positions 11318 to 11570 represent the NOS terminator.

[0069] A schematic diagram of the structure of the recombinant plasmid pEGOEP35S-H-LOC_Os07g15370 is shown below. Figure 2 Whole plasmid sequencing revealed that the nucleotide sequence of the recombinant plasmid pEGOEP35S-H-LOC_Os07g15370 is SEQ ID No. 4 in the sequence listing. Positions 131 to 658 of SEQ ID No. 4 in the sequence listing represent the 35S promoter, positions 677 to 2293 represent the CDS sequence of OsNramp5, and positions 2307 to 2559 represent the NOS terminator.

[0070] 3. Construction of recombinant bacteria

[0071] The recombinant pEGOEP2×35S-H-LOC_Os07g15370 obtained in step 2 was introduced into Agrobacterium tumefaciens EHA105 to obtain a recombinant bacterium, which was named EHA105 / pEGOEP2×35S-H-LOC_Os07g15370; the pEGOEP35S-H-LOC_Os07g15370 from step 2 was introduced into Agrobacterium tumefaciens EHA105 to obtain a recombinant bacterium, which was named EHA105 / pEGOEP35S-H-LOC_Os07g15370.

[0072] 4. Perform genetic transformation and obtain regenerated plants.

[0073] The recombinant Agrobacterium EHA105 / pEGOEP2×35S-H-LOC_Os07g15370 and EHA105 / pEGOEP35S-H-LOC_Os07g15370 obtained in step 3 were used to infect embryogenic callus tissue of the rice variety Huang Huazhan. Resistant callus tissue was screened (resistance screening was performed using a medium containing 100 mg / L hygromycin), and then differentiated and regenerated in culture, followed by rooting culture to obtain regenerated plants. The regenerated plant obtained by transforming Huang Huazhan with recombinant Agrobacterium EHA105 / pEGOEP2×35S-H-LOC_Os07g15370 was named Huang Huazhan-Z1, and the regenerated plant obtained by transforming Huang Huazhan with recombinant Agrobacterium EHA105 / pEGOEP35S-H-LOC_Os07g15370 was named Huang Huazhan-Z2.

[0074] 5. Screening and identification of OsNramp5 overexpressing rice

[0075] The regenerated plant Huang Huazhan-Z1 obtained in step 4 was identified as follows: Leaves were taken, genomic DNA was extracted, and PCR amplification was performed using primer pairs consisting of 2×35S-F (5'-CTCAAGCAATCAAGCATTCTAC-3') and NOS-R (5'-ATCATCGCAAGACCGGCAAC-3'). T0 generation OsNramp5 overexpressing plants were screened from the regenerated plant Huang Huazhan-Z1. After harvesting the seeds of the T0 generation plants, they were replanted to obtain T1 generation plants. DNA was extracted and PCR amplification was performed again to identify the T1 generation positive plants (i.e., T2 generation seeds). The seeds of the T1 generation positive plants were screened with hygromycin. The plants with a germination rate higher than 90% were identified as the T2 generation homozygous OsNramp5 overexpressing lines and named ZDX1.

[0076] The regenerated plant Huang Huazhan-Z2 obtained in step 4 was identified as follows: Leaves were taken, genomic DNA was extracted, and PCR amplification was performed using primer pairs consisting of 35S-F (5'-CACGGGGGACTCTTGCCACC-3') and NOS-R (5'-ATCATCGCAAGACCGGCAAC-3'). T0 generation OsNramp5 overexpressing plants were screened from the regenerated plant Huang Huazhan-Z2. After harvesting the seeds of the T0 generation plants, they were replanted to obtain T1 generation plants. DNA was extracted and PCR amplification was performed again to identify the T1 generation positive plants (i.e., T2 generation seeds). The seeds of the T1 generation positive plants were screened with hygromycin. The plants with a germination rate higher than 90% were identified as the T2 generation homozygous OsNramp5 overexpressing lines and named ZDX2.

[0077] To further analyze the expression level of the OsNramp5 gene in ZDX1 and ZDX2, the expression level of the OsNramp5 gene in the overexpression lines ZDX1, ZDX2, and rice Huanghuazhan was detected by RT-PCR. The cDNA of ZDX1, ZDX2, and wild-type Huanghuazhan was used as templates, and primers F (5'-CAGCAGCAGTAAGAGCAAGATG-3') and R (5'-GTGCTCAGGAAGTACATGTTGAT-3') were used for Real-Time PCR identification. The rice OsACTIN1 gene was used as an internal control, and primers F (5'-CCTTCAACACCCCTGCTATG-3') and R (5'-CAATGCCAGGGAACATAGTG-3') were used. Each parallel experiment was performed in triplicate.

[0078] Results (e.g.) Figure 3 The results showed that the expression level of the OsNramp5 gene in the OsNramp5 overexpression lines ZDX1 and ZDX2 was significantly upregulated compared to that in rice Huanghuazhan. Specifically, the expression level of the OsNramp5 gene in ZDX2 was 27 times that in rice Huanghuazhan; while the expression level of the OsNramp5 gene in ZDX1 was 116 times that in rice Huanghuazhan and 4.3 times that in ZDX2.

[0079] Example 2: Phenotypic identification of OsNramp5 overexpression lines

[0080] Test materials: Huang Huazhan, OsNramp5 overexpression lines ZDX1 and ZDX2.

[0081] Pot experiment: Test materials were planted in three soil conditions: slightly contaminated soil (1.0 mg / kg total cadmium), moderately contaminated soil (5.0 mg / kg total cadmium), and heavily contaminated soil (10.0 mg / kg total cadmium). Three plants were planted per pot (one each of Huang Huazhan, ZDX1, and ZDX2), with 40 pots planted for each treatment. Grains were harvested at maturity. The cadmium content of the grains was detected using a Xi'an Jiapu XR700 grain heavy metal rapid analyzer. Quantitative data were obtained from 30 plants. The results are shown in Table 1.

[0082] Table 1. Cadmium content in grains of Huang Huazhan, OsNramp5 overexpression lines ZDX1 and ZDX2

[0083]

[0084] Note: *p<0.05 indicates a significant difference from Huanghuazhan rice, and **p<0.01 indicates a highly significant difference from Huanghuazhan rice.

[0085] As shown in Table 1, regardless of whether the rice Huanghuazhan was planted under slightly, moderately, or severely cadmium-contaminated soil conditions, the cadmium content in Huanghuazhan grains was far higher than the national standard limit (0.2 mg / kg). When the OsNramp5 overexpressing rice line ZDX2 was planted under slightly and moderately cadmium-contaminated soil conditions, the cadmium content in ZDX2 grains was lower than the national standard limit (0.2 mg / kg). However, when ZDX2 was planted under severely cadmium-contaminated soil conditions, the cadmium content in ZDX2 grains was far higher than the national standard limit (0.2 mg / kg). On the other hand, regardless of whether the OsNramp5 overexpressing rice line ZDX1 was planted under slightly, moderately, or severely cadmium-contaminated soil conditions, the cadmium content in ZDX1 grains was far lower than the national standard limit (0.2 mg / kg). It is evident that, compared to Huang Huazhan and ZDX2, the cadmium content in the grains of the OsNramp5-overexpressing rice line ZDX1 decreased under mild, moderate, and severe cadmium pollution conditions. Particularly under severe cadmium-contaminated soil conditions, the cadmium content in ZDX1 grains decreased by 97% and 96% compared to Huang Huazhan and ZDX2, respectively, falling below the national standard limit (0.2 mg / kg). This indicates that overexpression of the OsNramp5 gene in rice via the 2×35S promoter significantly reduces the absorption and translocation of cadmium from the soil by rice, thereby lowering the cadmium content in rice grains. This effectively reduces the cadmium content in rice grains under different levels of cadmium-contaminated soil conditions, solving the problem of excessive cadmium in rice grains grown in cadmium-contaminated soil.

[0086] In addition, the inventors also used the 2×35S promoter to initiate the overexpression of the OsHMA3 gene in rice Huanghuazhan, obtaining the OsHMA3 gene overexpressing plant ZDX3 (the OsHMA3 gene expression level in ZDX3 was higher than that in rice Huanghuazhan). The only difference between the preparation method of the OsHMA3 gene overexpressing plant ZDX3 and the preparation method of ZDX1 is that the OsNramp5 gene in pEGOEP2×35S-H-LOC_Os07g15370 is replaced with the OsHMA3 gene; all other operations are the same.

[0087] Rice varieties Huanghuazhan and ZDX3 were planted in heavily cadmium-contaminated soil with a total cadmium content of 10.0 mg / kg. The results showed that the cadmium content in the grains of ZDX3 was 0.824 mg / kg, while that of Huanghuazhan was 2.26 mg / kg. This indicates that the overexpression of other heavy metal transabsorption and transport genes initiated by the 2×35S promoter in rice cannot effectively reduce the cadmium content in rice grains under heavily cadmium-contaminated soil conditions, and therefore cannot solve the problem of excessive cadmium in rice grains when grown in cadmium-contaminated soil.

[0088] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A DNA molecule comprising a promoter and a gene encoding the OsNramp5 protein; said promoter being a DNA fragment formed by the linkage of two 35S promoters; The OsNramp5 protein is any of the following proteins: (A1) A protein with the amino acid sequence SEQ ID No. 1; (A2) A protein having the same function as the amino acid sequence shown in SEQ ID No. 1, with substitution and / or deletion and / or addition of amino acid residues; (A3) is a protein derived from rice that has more than 80% identity with the amino acid sequence described in (A1) or (A2) and has the same function; (A4) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of any of the proteins described in (A1)-(A3).

2. The DNA molecule according to claim 1, characterized in that, The promoter is a DNA fragment of either a) or b) below: a) The nucleotide sequence is the DNA fragment from position 8784 to 9681 of SEQ ID No. 3; b) A DNA fragment that has more than 80% identity with positions 8784 to 9681 of SEQ ID No. 3 and has promoter function.

3. A biomaterial, characterized in that: The biological material is an expression cassette, recombinant vector, or recombinant bacteria containing the DNA molecule described in claim 1 or 2.

4. The application of the DNA molecule of claim 1 or 2 or the biomaterial of claim 3 in reducing the cadmium content in rice plants to be modified.

5. The application of the DNA molecule of claim 1 or 2, or the expression cassette, recombinant vector, or recombinant bacteria of claim 3, in the cultivation of rice with low cadmium content.

6. A method for reducing cadmium content in rice, comprising the following steps: introducing the DNA molecule of claim 1 or 2 or the biological material of claim 3 into recipient rice to obtain transgenic rice; wherein the cadmium content in the transgenic rice is lower than that in the recipient rice.

7. A method for cultivating rice with low cadmium content, the method comprising introducing the DNA molecule of claim 1 or 2 or the biological material of claim 3 into recipient rice to obtain rice with low cadmium content.

8. The application or method according to claim 5 or 7, characterized in that: The low-cadmium rice refers to rice with a cadmium content of less than 0.2 mg / kg.

9. A method for cultivating rice suitable for planting in cadmium-contaminated soil, comprising the steps of: introducing the DNA molecule of claim 1 or 2 or the biological material of claim 3 into recipient rice to obtain rice suitable for planting in cadmium-contaminated soil.

10. The application or method according to any one of claims 4-9, characterized in that: The cadmium content refers to the cadmium content in rice grains.

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