Method for creating low-cadmium rice

Abstract

The invention discloses a method for creating low-cadmium rice. The invention relates to the field of biotechnology breeding, in particular to a creation method of low-cadmium rice. The method for preparing the low-cadmium rice comprises the steps that gene editing is used for enabling an OsNramp5 gene of a target rice genome to be subjected to base replacement, the OsNramp5 gene is knocked out, knocked-out rice is obtained, the low-cadmium rice is obtained from the knocked-out rice, and the grain cadmium content of the low-cadmium rice is reduced; gene editing is carried out by adopting a CRISPR / Cas9 system, the CRISPR / Cas9 system contains sgRNA or sgRNA genes, and the target sequence of the sgRNA is the 686 to 705 nucleotides of a sequence 6 and / or the 2683 to 2702 nucleotides of a sequence 8. The rice mutant strain with low cadmium and normal manganese is screened through the method, rice grains are low in cadmium, the yield is not reduced, and the method has important significance on food crop safety and agricultural sustainable development.

Description

Technical Field

[0001] The present invention relates to the field of biotechnological breeding, and particularly to a method for creating low-cadmium rice. Background Art

[0002] Cadmium (Cd) is a non-essential element for the human body and one of the heavy metal elements that are toxic to humans, animals and plants. The World Health Organization has listed cadmium as a highly hazardous toxic substance and a carcinogen and has imposed control measures (WHO). Every year, 20 million tons of grain are contaminated by cadmium worldwide. Therefore, it is urgent to control cadmium pollution in rice. The root cause of cadmium pollution in rice lies in soil pollution. Therefore, solving soil pollution is the long-term solution. There are many methods for repairing cadmium-polluted soil. The most commonly used treatment methods include physical remediation, chemical remediation, bioremediation and agronomic regulation. Using physical remediation methods to remove the surface soil with high cadmium content or covering it with guest soil (Zong and Xu, 2004) can reduce the absorption of cadmium in the soil by plants, but this method has a large workload and high input costs. Chemical remediation methods mainly refer to applying amendments, chelating agents, etc. to cadmium-polluted soil, but this method also affects the absorption of other elements by plants. Bioremediation is considered to be the most promising soil remediation technology. Currently, it mainly refers to phytoremediation and microbial remediation. By using root filtration and biosorption, etc., heavy metals are transferred to the plant body or the bioavailability of heavy metals is reduced to achieve the purpose of remediation, but this method takes too long. Agronomic measures are also an important means to regulate cadmium absorption in rice. The combined application of appropriate amounts of organic fertilizer and chemical fertilizer can not only maintain the balance of trace elements in rice, but also effectively reduce the absorption of cadmium, but the stability is poor. The transformation of tillage methods and the remediation of polluted soil are the fundamental ways to solve the problem of cadmium pollution in rice. Selecting and planting rice varieties with low cadmium accumulation can achieve immediate results and is the best choice to quickly solve the problem of cadmium pollution in rice at low cost currently.

[0003] Currently, the main problem in low-cadmium breeding is the contradiction between yield and cadmium accumulation. Existing editing technologies can easily create loss-of-function mutants. Although such mutants have very low cadmium accumulation, at the same time, because of strong mutations, they do not transport manganese or zinc, seriously affecting the yield and making it difficult to be applied in production. Therefore, the key amino acid sites of proteins can be changed through gene editing technology, so as to change the selectivity of transporter proteins for metal elements, making them not transport cadmium but only transport manganese, in order to quickly cultivate low-cadmium rice varieties. Summary of the Invention

[0004] The main problem to be solved by the present invention is how to solve the problem of cadmium pollution in rice and breed rice varieties with low cadmium accumulation.

[0005] To solve the above problems, the present invention provides a method for preparing low-cadmium rice.

[0006] The method for preparing low-cadmium rice provided by the present invention includes using gene editing to perform a base substitution on the OsNramp5 gene in the genome of the target rice to knockout the OsNramp5 gene to obtain a knockout rice, and obtaining low-cadmium rice from the knockout rice; compared with the target rice, the cadmium content in the grains of the low-cadmium rice is reduced; the gene editing is carried out using the CRISPR / Cas9 system, the CRISPR / Cas9 system contains sgRNA or the sgRNA gene, and the target sequence of the sgRNA is the 686-705th nucleotides of Sequence 6 and / or the 2683-2702nd nucleotides of Sequence 8.

[0007] In the above method, the CRISPR / Cas9 system is any one of the following:

[0008] (b1) includes the sgRNA and Cas9 protein;

[0009] (b2) includes a specific DNA molecule and the coding gene of Cas9 protein, and the specific DNA molecule is the gene of the gRNA;

[0010] (b3) includes a vector with the specific DNA molecule in (b2) and a vector with the coding gene of Cas9 protein;

[0011] (b4) includes a specific recombinant vector, and the specific recombinant vector contains the specific DNA molecule in (b2) and the coding gene of Cas9 protein.

[0012] The specific recombinant vector can express the expression cassette of the specific sgRNA and the expression cassette of the coding gene of the Cas9 protein.

[0013] In the above method, the base substitution of the OsNramp5 gene in the genome of the target rice can be any one of the following:

[0014] 1) Replace 5'-TCCTCGGAGCTCTTGTCATG-3' in the coding gene of the protein in the rice genomic DNA with 5'-TCCTCGGAGCTCTTGCCATG-3', so as to mutate the 234th Val of the OsNramp5 protein into Ala;

[0015] 2) Replace 5'-AGAGAGAGAGCAGTGAGAGA-3' in the OsNramp5 gene in the rice genomic DNA with 5'-AGAGAGGGAGCAGTGAGAGA-3', so as to mutate the 6th Glu of the protein encoded by the OsNramp5 gene into Gly;

[0016] 3) Replace 5'-TCGCCATCGCGCCGAGCCTC-3' in the coding gene of the said protein in the rice genomic DNA with 5'-TCGCCACCGCGCCGAGCCTC-3, so as to mutate Ile at the 371st position of the OsNramp5 protein into Thr;

[0017] 4) Replace 5'-TTCCTGAGCACGAGCTTCGT-3' in the coding gene of the said protein in the rice genomic DNA with 5'-TTCCTGAGCGCGAGCTTCGT-3, so as to mutate Thr at the 450th position of the OsNramp5 protein into Ala;

[0018] 5) Replace 5'-TTCATGCTCGTCTACATCGT-3' in the coding gene of the said protein in the rice genomic DNA with 5'-TTCATGCTCGTCCACATCGT-3, so as to mutate Tyr at the 480th position of the OsNramp5 protein into His.

[0019] The said method further includes self-crossing the knocked-out rice to obtain self-crossed offspring, and screening the knocked-out rice without transgenes from the self-crossed offspring. This knocked-out rice without transgenes is the low-cadmium rice.

[0020] The present invention also provides a method for reducing the cadmium content in rice grains. The said method includes using gene editing to cause a base substitution in the OsNramp5 gene of the target rice genome to knock out the said OsNramp5 gene, and obtaining low-cadmium rice with a reduced cadmium content in the grains; the gene editing is carried out using the CRISPR / Cas9 system, the CRISPR / Cas9 system contains sgRNA or the said sgRNA gene, and the target sequence of the sgRNA is the nucleotide at the 686-705th position of Sequence 6 and / or the nucleotide at the 2683-2702nd position of Sequence 8.

[0021] In the present invention, the said OsNramp5 gene encodes the OsNramp5 protein, and the amino acid sequence of the said OsNramp5 protein is Sequence 7. The coding sequence (CDS) of the said OsNramp5 gene is Sequence 6. The nucleotide sequence of the coding strand of the said OsNramp5 gene (genomic gene) may specifically be Sequence 8.

[0022] The said CRISPR / Cas9 system is a product. The product may specifically be a reagent.

[0023] The vectors described in this article are well-known to those skilled in the art, including but not limited to: plasmids, phages (such as λ phage or M13 filamentous phage, etc.), cosmids (i.e., cosmid plasmids), Ti plasmids or viral vectors. Specifically, it can be vector pH-PABE-7.

[0024] For the convenience of identifying and screening transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) encoding enzymes or luminescent compounds that can produce color changes and are expressible in plants, antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.

[0025] The present invention also provides a DNA molecule, which is a DNA molecule formed by at least one of the following mutations of the DNA molecule with the nucleotide sequence of Sequence 6 in the sequence listing:

[0026] 1) Replace 5'-TCCTCGGAGCTCTTGTCATG-3' in the coding gene of the protein in the rice genomic DNA with 5'-TCCTCGGAGCTCTTGCCATG-3', so as to mutate Val at the 234th position of the encoded OsNramp5 protein into Ala;

[0027] 2) Replace 5'-AGAGAGAGAGCAGTGAGAGA-3' in the coding gene of the protein in the rice genomic DNA with 5'-AGAGAGGGAGCAGTGAGAGA-3', so as to mutate Glu at the 6th position of the encoded OsNramp5 protein into Gly;

[0028] 3) Replace 5'-TCGCCATCGCGCCGAGCCTC-3' in the coding gene of the protein in the rice genomic DNA with 5'-TCGCCACCGCGCCGAGCCTC-3', so as to mutate Ile at the 371st position of the encoded OsNramp5 protein into Thr;

[0029] 4) Replace 5'-TTCCTGAGCACGAGCTTCGT-3' in the coding gene of the protein in the rice genomic DNA with 5'-TTCCTGAGCGCGAGCTTCGT-3', so as to mutate Thr at the 450th position of the encoded OsNramp5 protein into Ala;

[0030] 5) Replace 5'-TTCATGCTCGTCTACATCGT-3' in the coding gene of the said protein in the rice genomic DNA with 5'-TTCATGCTCGTCCACATCGT-3', so as to mutate Tyr at the 480th position of the OsNramp5 protein into His.

[0031] The specific sgRNA or the specific DNA molecule described above also belongs to the scope protected by the present invention.

[0032] The present invention also provides a CRISPR / Cas9 system for gene editing, and the CRISPR / Cas9 system can be the CRISPR / Cas9 system described above.

[0033] The present invention also provides the applications of the method, the DNA molecule, the specific gRNA, the specific DNA molecule and / or the CRISPR / Cas9 system described above, and the applications are any of the following applications:

[0034] (1) Application in reducing the cadmium content in rice;

[0035] (2) Application in cultivating rice varieties with low cadmium accumulation;

[0036] (3) Application in improving rice germplasm resources.

[0037] The present invention uses gene editing technology to saturate and edit the OsNramp5 gene in the rice genome, creates allelic lines with 5 different types of OsNramp5, and uses cadmium-containing hydroponic solution and cadmium-polluted soil for cultivation and screening to obtain low-cadmium and normal-manganese rice varieties. The present invention uses CRISPR / Cas9 technology to saturate and mutate the cadmium and manganese absorption gene OsNramp5 in japonica rice, screens out rice mutants with low cadmium and normal manganese, solves the contradiction between cadmium accumulation and yield in the process of rice breeding, realizes the purpose of low cadmium in rice grains without reducing the yield, provides guarantee for the safety of food crops in China, and has important significance for the sustainable development of agriculture. Description of the Drawings

[0038] Figure 1 It is the homozygous mutant OsNramp5 genotype of the T1 generation.

[0039] Figure 2 It is the contents of Cd and Mn in the stems of homozygous mutant OsNramp5 plants after 2-week-old mutant seedlings and WT seedlings are treated with hydroponic solution containing 0.5um CdCl2 for one week. Among them, A is: the content of cadmium in the seedling stems; B is: the content of manganese in the seedling stems.

[0040] Figure 3Identification of T2 generation OsNramp5 mutant strains without T-DNA insertion. Lanes 1-24 are T2 generation individual plants of OsNramp5 V234A mutant strains, among which lanes 2, 4, 14, and 16 are homozygous mutant individuals without T-DNA insertion; lanes 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23, and 24 are homozygous mutant individuals with T-DNA insertion.

[0041] Figure 4 For mutant OsNramp5 V234A and Cd and Mn contents in brown rice of the control WT; The materials were potted in cadmium soil at 5 mg / kg, with 3 replicates. ** indicates significant difference at the 0.01 level. Among them, A is the cadmium content in rice grains; B is the manganese content in rice grains.

[0042] Figure 5 For mutant OsNramp5 V234A and comparison of yield traits of the control WT; The materials were planted in the field, and 24 individual plants of each strain were planted. Among them, A: yield per plant; B: plant height; C: tiller number; D: 1000-grain weight. Specific implementation manners

[0043] The present invention will be further described in detail below in combination with specific implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0044] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0045] In the following quantitative experiments, unless otherwise specified, three repeated experiments are set.

[0046] The pH-PABE-7 plasmid in the following examples has been recorded in: Li, C. et al. Expanded base editing in rice and wheat using a Cas9-adenosine deaminase fusion. Genome Biol 19, 59 (2018). The public can obtain this biological material from the applicant. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0047] The japonica rice ZH11 in the following embodiments belongs to the conventional japonica rice variety. The public can obtain this biological material from the applicant, and this biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0048] In the following embodiments, SPSS 11.5 statistical software was used to process the data. The experimental results were expressed as mean ± standard deviation, and One-way ANOVA test was used. P < 0.05 (*) indicates significant difference, P < 0.01 (**) indicates extremely significant difference, and P < 0.001 (***) indicates extremely significant difference.

[0049] Example 1. Creation of low-cadmium rice

[0050] The coding sequence (CDS) of the OsNramp5 gene in rice is SEQ ID No. 6, encoding the OsNramp5 protein with the amino acid sequence of SEQ ID No. 7. In the genomic DNA of rice, the genomic sequence encoding the OsNramp5 protein is as shown in SEQ ID No. 8. The 1st to 111th positions of SEQ ID No. 8 are the first exon, the 378th to 456th positions are the second exon, the 585th to 625th positions are the third exon, the 924th to 1002nd positions are the fourth exon, the 1085th to 1189th positions are the fifth exon, the 2189th to 2298th positions are the sixth exon, the 2523rd to 2704th positions are the seventh exon, the 2804th to 2873rd positions are the eighth exon, the 3164th to 3337th positions are the ninth exon, the 3619th to 3723rd positions are the tenth exon, the 4710th to 4841st positions are the eleventh exon, the 5437th to 5532nd positions are the twelfth exon, and the 6542nd to 6874th positions are the thirteenth exon.

[0051] The present invention provides the following method for creating low-cadmium rice, which can effectively improve the trait of cadmium accumulation in grains. The specific steps are as follows:

[0052] 1. Selection of target sequences

[0053] According to the exon sequences of the OsNramp5 gene, 5 target sequences were designed, namely Nramp5-A1 (SEQ ID No. 1) to Nramp5-A5 (SEQ ID No. 5). The specific information is shown in Table 1.

[0054] Table 1. Detailed information of target sequences

[0055]

[0056]

[0057] Note: The sense strand or reverse complementary strand of the above 5 target sequences contains the 5'-(N)X-NGG-3' structure.

[0058] 2. Synthesize double-stranded target sequences with sticky ends as adapter primers

[0059] Synthesize sgRNA (Nramp5-A1 to Nramp5-A5), including synthesizing the sense oligonucleotide strands Nramp5-AF1 to Nramp5-AF5 and their complementary reverse oligonucleotide strands Nramp5-AR1 to Nramp5-AR5. The specific sequence information is shown in Table 2.

[0060] Table 2. Detailed information of primers used for synthesizing target sequences

[0061]

[0062] Among them, the partial target sequences in capital letters are the sequences obtained by removing NGG from Nramp5-A1 (SEQ ID No.1) to Nramp5-A5 (SEQ ID No.5) or their complementary sequences, and the partial sequences in lowercase letters are the sticky ends for connecting vectors.

[0063] 3. Construct the pH-PABE-7 recombinant vector

[0064] 1) Preparation of adapters: Dissolve the adapter primers Nramp5-AF1 to Nramp5-AF5 and their complementary reverse oligonucleotide strands Nramp5-AR1 to Nramp5-AR5 into 100 μM mother solutions respectively. Take 1 μL of each and mix them in pairs to obtain the Nramp5-A1 to Nramp5-A5 mixed solutions. Dilute these 5 mixed solutions to 1 μM. Place them at 95°C for 30 s, then turn off the PCR instrument and cool down slowly to complete annealing.

[0065] 2) Digest the pH-PABE-7 vector: Digest 20000 ng of the pH-PABE-7 vector with 25 U of Bsa I in a 500 μL reaction system for 5 h, and then purify to obtain the digested pH-PABE-7 vector.

[0066] 3) Ligate the adapter obtained in step 1 with the digested vector obtained in step 2: Take 1 μL of 10×T4 DNA ligase buffer, 1 μL of T4 DNA ligase, 0.5 μL of the diluted sgRNA mixture of Nramp5-A1 to Nramp5-A5, 1 μL of the digested linear vector, and finally add ddH2O to a total volume of 10 μL. Ligate overnight at 16°C to obtain recombinant vectors pH-PABE-7-A1, pH-PABE-7-A2, pH-PABE-7-A3, pH-PABE-7-A4, and pH-PABE-7-A5.

[0067] The structure of the pH-PABE-7-A1 recombinant vector is described as follows: The sequence of Nramp5-A1 (i.e., sequence 1) is inserted between the restriction enzyme Bsa I cleavage sites of the starting vector pH-PABE-7, and the other sequences of the empty vector pH-PABE-7 remain unchanged to obtain the recombinant vector. The recombinant expression vector pH-PABE-7-A1 contains an expression cassette that can express the Nramp5-A1 gene with a nucleotide sequence of sequence 1 and a Cas9 expression cassette.

[0068] The structure of the pH-PABE-7-A2 recombinant vector is described as follows: The sequence of Nramp5-A2 (i.e., sequence 2) is inserted between the restriction enzyme Bsa I cleavage sites of the starting vector pH-PABE-7, and the other sequences of the empty vector pH-PABE-7 remain unchanged to obtain the recombinant vector. The recombinant expression vector pH-PABE-7-A2 contains an expression cassette that can express the Nramp5-A2 gene with a nucleotide sequence of sequence 2 and a Cas9 expression cassette.

[0069] The structure of the pH-PABE-7-A3 recombinant vector is described as follows: The sequence of Nramp5-A3 (i.e., sequence 3) is inserted between the restriction enzyme Bsa I cleavage sites of the starting vector pH-PABE-7, and the other sequences of the empty vector pH-PABE-7 remain unchanged to obtain the recombinant vector. The recombinant expression vector pH-PABE-7-A3 contains an expression cassette that can express the Nramp5-A3 gene with a nucleotide sequence of sequence 3 and a Cas9 expression cassette.

[0070] The structure of the pH-PABE-7-A4 recombinant vector is described as follows: The sequence of Nramp5-A4 (i.e., sequence 4) is inserted between the restriction enzyme Bsa I cleavage sites of the starting vector pH-PABE-7, and the other sequences of the empty vector pH-PABE-7 remain unchanged to obtain the recombinant vector. The recombinant expression vector pH-PABE-7-A4 contains an expression cassette that can express the Nramp5-A4 gene with a nucleotide sequence of sequence 4 and a Cas9 expression cassette.

[0071] The structure of the recombinant vector pH-PABE-7-A5 is described as follows: The sequence of Nramp5-A5 (i.e., Sequence 5) was inserted between the restriction enzyme Bsa I cleavage sites of the starting vector pH-PABE-7, and the other sequences of the empty vector pH-PABE-7 were kept unchanged to obtain the recombinant vector. The recombinant expression vector pH-PABE-7-A5 has an expression cassette of the Nramp5-A5 gene with a nucleotide sequence that is Sequence 5 and a Cas9 expression cassette.

[0072] Example 2: Rice genetic transformation and acquisition and screening of positive transgenic plants

[0073] 1. Rice genetic transformation and acquisition of positive transgenic plants

[0074] The recombinant vectors pH-PAVE-7-A1, pH-PAVE-7-A2, pH-PAVE-7-A3, pH-PAVE-7-A4, and pH-PAVE-7-A5 constructed in Example 1 were respectively introduced into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium tumefaciens GV3101 / pH-PAVE-7-A1, GV3101 / pH-PAVE-7-A2, GV3101 / pH-PAVE-7-A3, GV3101 / pH-PAVE-7-A4, and GV3101 / pH-PAVE-7-A5.

[0075] Mature seeds of japonica rice ZH11 were used to induce callus on the induction medium. The specific steps are as follows:

[0076] The above recombinant Agrobacterium tumefaciens GV3101 / pH-PAVE-7-A1, GV3101 / pH-PAVE-7-A2, GV3101 / pH-PAVE-7-A3, GV3101 / pH-PAVE-7-A4, and GV3101 / pH-PAVE-7-A5 were inoculated in LB culture medium containing kanamycin resistance and cultured for 2 days. The collected culture solution was added to NB liquid medium containing 100 μmol / L of acetosyringone, and the OD 600To 0.5 to obtain the bacterial solution. Immerse the rice callus in the above bacterial solution for 30 min, rinse it 3 - 5 times with sterile water, blot dry with sterilized filter paper, air dry, and transfer it to NB agar medium for dark culture at 26°C - 28°C for 3 days. Then transfer the callus to the medium containing 50 mg / L hygromycin for dark culture at 26°C - 28°C, subculture once every 15 days, for a total of 2 subcultures. After resistance screening, transfer it to the differentiation medium for continued culture at 25°C - 28°C, with a light intensity of 14 h / d. When the height of the differentiated seedlings is about 3 - 5 cm, transfer them to the rooting medium to strengthen the seedlings at 25°C - 28°C, with a light intensity of 14 h / d. After 3 - 4 weeks, add an appropriate amount of distilled water, harden the seedlings for 3 days, wash the medium on the roots of the seedlings, hydroponically culture them in an incubator for 1 week, and transplant them to the experimental field. Select the normally growing plants for hygromycin PCR detection. There are 37 transgenic positive seedlings obtained from recombinant Agrobacterium tumefaciens GV3101 / pH - PAVE - 7 - A1, 43 transgenic positive seedlings obtained from recombinant Agrobacterium tumefaciens GV3101 / pH - PAVE - 7 - A2, 24 transgenic positive seedlings obtained from recombinant Agrobacterium tumefaciens GV3101 / pH - PAVE - 7 - A3, 9 transgenic positive seedlings obtained from recombinant Agrobacterium tumefaciens GV3101 / pH - PAVE - 7 - A4, and 32 transgenic positive seedlings obtained from recombinant Agrobacterium tumefaciens GV3101 / pH - PAVE - 7 - A5.

[0077] 2. Identification of Mutation Sites in Transgenic Positive Rice Plants

[0078] Plants to be identified: Wild - type ZH11, transgenic positive rice plants OsNramp5 - A1, OsNramp5 - A2, OsNramp5 - A3, OsNramp5 - A4, and OsNramp5 - A5.

[0079] 1) Extract the genomic DNA of the above - mentioned transgenic positive plants.

[0080] 2) For the DNA fragment within 800 bp containing the target site, design 6 pairs of specific primers on the genome. Using the genomic DNA of the T0 - generation transgenic rice as a template, amplify the DNA fragment containing the target site. The specific steps are as follows:

[0081] Using the DNA extracted in step 1) as a template, and the upstream and downstream primers in Table 3, amplify the DNA fragment containing the target site with 2×KOFU high - fidelity enzyme Mix. The OsNramp5 gene has 13 exons and 12 introns, with a full - length of 6874 bp. Design 6 pairs of primers to amplify the sequences of these 13 exons. The specific primer information is shown in Table 3 below.

[0082] Table 3. Detailed Information of Primers Used for Mutation Identification

[0083]

[0084]

[0085] PCR reaction system: 0.5 μL DNA template, 1 μL N5-F(1-6), 1 μL N5-R(1-6), 20 μL ddH2O, 25 μL 2×kofu high-fidelity enzyme Mix (Beijing Yizhi Biotechnology Co., Ltd., product number Lot#2312ES1). Reaction procedure: Pre-denaturation at 95°C for 3 min, 32 cycles: denaturation at 96°C for 10 s, extension at 68°C for 30 s. Extension at 68°C for 5 min.

[0086] The amplified PCR products are purified and then sent to the company for sequencing. The sequencing results are compared with the sequence of the wild-type plant ZH11. If there is only one kind of PCR amplification product in the regenerated plant and it is consistent with the nucleotide sequence of the PCR amplification product of the wild-type plant ZH11, the regenerated plant is wild-type. If there are two kinds of PCR amplification products in the regenerated plant, one is consistent with the nucleotide sequence of the PCR amplification product of the wild-type plant ZH11, and the other has a mutation compared with the nucleotide sequence of the PCR amplification product of the wild-type plant ZH11 (the mutation includes deletion, insertion or substitution of one or more nucleotides), the regenerated plant is heterozygous. If there are two kinds of PCR amplification products in the regenerated plant, both of which have mutations compared with the nucleotide sequence of the PCR amplification product of the wild-type plant ZH11 (the mutation includes deletion, insertion or substitution of one or more nucleotides), the regenerated plant is double allelic mutant. If there is one kind of PCR amplification product in the regenerated plant and it has a mutation compared with the nucleotide sequence of the PCR amplification product of the wild-type plant ZH11 (the mutation includes deletion, insertion or substitution of one or more nucleotides), the regenerated plant is homozygous mutant. If there are more than three kinds of PCR amplification products in the regenerated plant, the regenerated plant is chimeric. Heterozygous, double allelic mutant, homozygous mutant and chimeric plants are collectively called edited plants.

[0087] The sequencing results are as follows: There are 37 seedlings of the transgenic positive rice plant OsNramp5-A1 in total, among which 31 are unedited and 6 are successfully edited, and these 6 mutant single plants are all heterozygous. Compared with the wild-type rice ZH11, the following mutations occurred in the OsNramp5 gene of the heterozygous mutant plant OsNramp5-A1: The base A at the 17th position of SEQ ID No.6 in the OsNramp5 gene on one chromosome (corresponding to the 17th position of SEQ ID No.8) was replaced by the base G, and the other chromosome sequence is the same as the wild-type.

[0088] There were a total of 43 seedlings of the transgenic positive rice plant OsNramp5-A2. Among them, 37 were unedited, and 6 were successfully edited. Moreover, these 6 mutant individuals were all heterozygous. Compared with the wild-type rice ZH11, the following mutations occurred in the OsNramp5 gene of the heterozygous mutant plant OsNramp5-A2: The base T at position 701 of SEQ ID No.6 in the OsNramp5 gene on one chromosome (corresponding to position 2698 of SEQ ID No.8) was replaced by the base C, and the sequence of the other chromosome was the same as that of the wild type.

[0089] There were a total of 24 seedlings of the transgenic positive rice plant OsNramp5-A3. Among them, 17 were unedited, and 7 were successfully edited. Moreover, these 7 mutant individuals were all heterozygous. Compared with the wild-type rice ZH11, the following mutations occurred in the OsNramp5 gene of the heterozygous mutant plant OsNramp5-A3: The base T at position 1118 of SEQ ID No.6 in the OsNramp5 gene on one chromosome (corresponding to position 4771 of SEQ ID No.8) was replaced by the base C, and the sequence of the other chromosome was the same as that of the wild type.

[0090] There were a total of 9 seedlings of the transgenic positive rice plant OsNramp5-A4. Among them, 7 were unedited, and 2 were successfully edited. Moreover, these 2 mutant individuals were all heterozygous. Compared with the wild-type rice ZH11, the following mutations occurred in the OsNramp5 gene of the heterozygous mutant plant OsNramp5-A4: The base A at position 1348 of SEQ ID No.6 in the OsNramp5 gene on one chromosome (corresponding to position 6605 of SEQ ID No.8) was replaced by the base G, and the sequence of the other chromosome was the same as that of the wild type.

[0091] There were a total of 32 seedlings of the transgenic positive rice plant OsNramp5-A5. Among them, 25 were unedited, and 7 were successfully edited. Moreover, these 2 mutant individuals were all heterozygous. Compared with the wild-type rice ZH11, the following mutations occurred in the OsNramp5 gene of the heterozygous mutant plant OsNramp5-A5: The base T at position 1438 of SEQ ID No.6 in the OsNramp5 gene on one chromosome (corresponding to position 6695 of SEQ ID No.8) was replaced by the base C, and the sequence of the other chromosome was the same as that of the wild type.

[0092] For samples with mutant sequencing results, select the T0 generation mutant single plants that can cause amino acid substitutions for planting, because for proteins with amino acid substitutions, their functions will probably also change; single plants with nonsense mutations are not subjected to further research because they do not change the protein function; strong mutant single plants that cause complete loss of protein function are also not subjected to further research. Plant the corresponding T0 generation plants OsNramp5-A1, OsNramp5-A2, OsNramp5-A3, OsNramp5-A4 and OsNramp5-A5 in the field and harvest the T0 generation seeds.

[0093] Example 3. Phenotypic identification of rice transformed plants

[0094] Plants to be identified: wild type ZH11, T1 generation plants of transgenic positive rice OsNramp5-A1, OsNramp5-A2, OsNramp5-A3, OsNramp5-A4 and OsNramp5-A5.

[0095] Plant 16 seeds of each mutant T0 generation, then extract the genomic DNA of 16 single plants of the T1 generation, and use the upstream primer and downstream primer at the N5-F(1-6) and N5-R(1-6) positions to amplify the DNA fragment containing the target site with KOFU high-fidelity enzyme Mix. Reaction system: 0.5 μL DNA template, 1 μL N5-F(1-6), 1 μL N5-R(1-6), 20 μL ddH2O, 25 μL 2×kofu high-fidelity enzyme Mix. Reaction program: Pre-denature at 95°C for 3 min, 32 cycles: denature at 96°C for 10 s, extend at 68°C for 30 s. Extend at 68°C for 5 min. The amplified PCR products are purified and sent to the company for sequencing. The sequencing results are compared with the wild-type plant sequence to screen samples with homozygous mutations, and the T1 generation seeds of the homozygous mutant plants are harvested later. See Figure 1 。

[0096] Compared with the wild-type rice ZH11, the homozygous mutant plant OsNramp5-A1 has the following mutations in the OsNramp5 gene: The base A at position 17 of SEQ ID No. 6 in the OsNramp5 genes on two homologous chromosomes (corresponding to position 17 of SEQ ID No. 8) are both replaced by the base G, thus knocking out the OsNramp5 gene. This nucleotide substitution causes a change in the function of the OsNramp5 protein, thus obtaining a new OsNramp5 allele.

[0097] Compared with the wild-type rice ZH11, the homozygous mutant plant OsNramp5-A2 has the following mutations in the OsNramp5 gene: The base T at position 701 of SEQ ID No. 6 in the OsNramp5 genes on both chromosomes (corresponding to position 2698 of SEQ ID No. 8) is replaced by the base C, thereby knocking out the OsNramp5 gene. This nucleotide substitution causes a change in the function of the OsNramp5 protein, thus obtaining a new OsNramp5 allele.

[0098] Compared with the wild-type rice ZH11, the homozygous mutant plant OsNramp5-A3 has the following mutations in the OsNramp5 gene: The base T at position 1118 of SEQ ID No. 6 in the OsNramp5 genes on both chromosomes (corresponding to position 4771 of SEQ ID No. 8) is replaced by the base C, thereby knocking out the OsNramp5 gene. This nucleotide substitution causes a change in the function of the OsNramp5 protein, thus obtaining a new OsNramp5 allele.

[0099] Compared with the wild-type rice ZH11, the homozygous mutant plant OsNramp5-A4 has the following mutations in the OsNramp5 gene: The base A at position 1348 of SEQ ID No. 6 in the OsNramp5 genes on both chromosomes (corresponding to position 6605 of SEQ ID No. 8) is replaced by the base G, thereby knocking out the OsNramp5 gene. This nucleotide substitution causes a change in the function of the OsNramp5 protein, thus obtaining a new OsNramp5 allele.

[0100] Compared with the wild-type rice ZH11, the homozygous mutant plant OsNramp5-A5 has the following mutations in the OsNramp5 gene: The base T at position 1438 of SEQ ID No. 6 in the OsNramp5 genes on both chromosomes (corresponding to position 6695 of SEQ ID No. 8) is replaced by the base C, thereby knocking out the OsNramp5 gene. This nucleotide substitution causes a change in the function of the OsNramp5 protein, thus obtaining a new OsNramp5 allele.

[0101] 1. Hydroponic experiment with cadmium-containing hydroponic solution to screen for low cadmium-accumulating rice mutants

[0102] The specific steps of the hydroponic experiment with cadmium-containing hydroponic solution are as follows:

[0103] Select the above 5 different homozygous mutant seeds and wild-type seeds. After germination, grow them in hydroponic solution for 2 weeks, and then transfer them to hydroponic solution containing 0.5 μm CdCl2 for treatment for one week. Take the above-ground stem part, dry it, weigh about 100 mg, digest it with HNO3, and identify the contents of Cd and Mn in the stem by inductively coupled plasma emission spectrometer ICP.

[0104] The results are as Figure 2 shown in A and B below. The Cd content in the stem of the OsNramp5-A2 mutant line decreased significantly compared with the control, and the manganese metal element was not affected.

[0105] Plant 24 homozygous mutant OsNramp5-A2 seeds harvested in the T1 generation in the field, extract rice genomic DNA, and detect the Hpt transgenic element by PCR. The detection primers are: F: 5’-ATGAAAAAGCCTGAACTCACCGCGACG-3’ and R: 5’-ATGGGGATCAGCAATCGCGCATATGAAA-3’. Screen out the edited mutants without transgenic components.

[0106] The results are as Figure 3 shown. By PCR detection of 24 individual plants of the T2 generation of the OsNramp5-A2 mutant line in the field, it was found that no Hpt band was amplified in the 2nd, 4th, 14th, and 16th individual plants, and the Hpt gene band was amplified in the other 20 individual plants. The 2nd, 4th, 14th, and 16th individual plants in the T2 generation are of the same mutant type, and they are named rice line OsNramp5 V234A .

[0107] The homozygous mutant plant OsNramp5 V234A Compared with the wild-type rice ZH11, the following mutations occurred in the OsNramp5 gene: The base T at position 701 of SEQ ID No.6 in the OsNramp5 gene (corresponding to position 2698 of SEQ ID No.8) was replaced by C. This nucleotide substitution caused a change in the function of the OsNramp5 protein, thus creating a new OsNramp5 allele. The sequencing results of this mutation site and its surrounding nucleotides are shown in Figure 1 .

[0108] 2. Pot experiment under cadmium stress

[0109] Conduct a pot experiment under cadmium stress. The soil is flower nutrient soil. Add Cd (CdCl2) to the flower nutrient soil in the form of a solution to make the soil cadmium concentration 5 mg / kg, and adjust the soil pH value to about 5 - 6 with concentrated H2SO4. The 1 low-cadmium-accumulating homozygous mutant OsNramp5 screened in step 1 V234AThe mutant rice and the control rice ZH11 were planted in cadmium-contaminated pots, with 3 plants per pot, 2 of which were mutant plants and 1 was the control, and 5 pots were replicated. Samples were taken at the mature stage of rice and dried to a constant weight at 80 °C. The brown rice was ground, digested with HNO3, and the cadmium content and other related mineral element contents in the brown rice were identified by an inductively coupled plasma optical emission spectrometer ICP.

[0110] The results are as Figure 4 shown in A and B in V234A the cadmium content in the brown rice of the OsNramp5 mutant lines was significantly lower than that of the control, and the manganese content was not affected.

[0111] 3. Study on the agronomic traits at the mature stage of rice

[0112] The low-cadmium-accumulating homozygous mutant OsNramp5 screened in step 1 V234A and the control rice ZH11 were planted in the field, and the agronomic traits were statistically analyzed at the mature stage of rice.

[0113] The results are as Figure 5 shown in A-D in V234A there were no significant differences in plant height, yield and tiller between the OsNramp5 mutant lines and the wild-type lines.

[0114] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including changes made by using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. A method for preparing low-cadmium rice, characterized in that: The method includes using gene editing to cause base substitution in the OsNramp5 gene of the target rice genome to knockout the OsNramp5 gene to obtain knockout rice, and obtaining low-cadmium rice from the knockout rice; compared with the target rice, the cadmium content in the grains of the low-cadmium rice is reduced. The gene editing is carried out using the CRISPR / Cas9 system, the CRISPR / Cas9 system contains sgRNA or the sgRNA gene, and the target sequence of the sgRNA is the 686-705th nucleotides of Sequence 6 and / or the 2683-2702nd nucleotides of Sequence 8.

2. The method according to claim 1, characterized in that: The CRISPR / Cas9 system is any one of the following: (b1) includes the sgRNA and Cas9 protein; (b2) includes the coding gene of the specific DNA molecule and Cas9 protein, and the specific DNA molecule is the gene of the gRNA; (b3) includes the vector with the specific DNA molecule described in (b2) and the vector with the coding gene of Cas9 protein; (b4) includes the specific recombinant vector, and the specific recombinant vector contains the specific DNA molecule described in (b2) and the coding gene of Cas9 protein.

3. The method according to claim 2, wherein: The causing base substitution in the OsNramp5 gene of the target rice genome is any one of the following: 1) Replace 5'-TCCTCGGAGCTCTTGTCATG-3' in the coding gene of the protein in the rice genome DNA with 5'-TCCTCGGAGCTCTTGCCATG-3' 2) Replace 5'-AGAGAGAGAGCAGTGAGAGA-3' in the OsNramp5 gene in the rice genome DNA with 5'-AGAGAGGGAGCAGTGAGAGA-3;; 3) Replace 5'-TCGCCATCGCGCCGAGCCTC-3' in the coding gene of the protein in the rice genome DNA with 5'-TCGCCACCGCGCCGAGCCTC-3'; 4) Replace 5'-TTCCTGAGCACGAGCTTCGT-3' in the coding gene of the protein in the rice genome DNA with 5'-TTCCTGAGCGCGAGCTTCGT-3'; 5) Replace 5'-TTCATGCTCGTCTACATCGT-3' in the coding gene of the protein in the rice genome DNA with 5'-TTCATGCTCGTCCACATCGT-3'.

4. The method according to any one of claims 1 to 3, characterized in that: The method further includes self-crossing the knockout rice to obtain self-crossed offspring, and screening the knockout rice without exogenous gene DNA elements from the self-crossed offspring, and the knockout rice without transgenic elements is the low-cadmium rice.

5. Method for reducing cadmium content in rice grains, characterized in that: The method includes using gene editing to make a base substitution in the OsNramp5 gene of the target rice genome, knocking out the OsNramp5 gene, and obtaining low-cadmium rice with reduced grain cadmium content; the gene editing is carried out using the CRISPR / Cas9 system, the CRISPR / Cas9 system contains sgRNA or the sgRNA gene, and the target sequence of the sgRNA is the 686-705th nucleotides of Sequence 6 and / or the 2683-2702nd nucleotides of Sequence 8.

6. A DNA molecule, characterized in that: The DNA molecule is a DNA molecule formed by at least one of the following mutations in the DNA molecule with the nucleotide sequence of Sequence 5 in the sequence listing: 1) replacing 5'-TCCTCGGAGCTCTTGTCATG-3' in the coding gene of the protein in the rice genome DNA with 5'-TCCTCGGAGCTCTTGCCATG-3'; 2) replacing 5'-AGAGAGAGAGCAGTGAGAGA-3' in the OsNramp5 gene in the rice genome DNA with 5'-AGAGAGGGAGCAGTGAGAGA-3'; 3) replacing 5'-TCGCCATCGCGCCGAGCCTC-3' in the coding gene of the protein in the rice genome DNA with 5'-TCGCCACCGCGCCGAGCCTC-3'; 4) replacing 5'-TTCCTGAGCACGAGCTTCGT-3' in the coding gene of the protein in the rice genome DNA with 5'-TTCCTGAGCGCGAGCTTCGT-3'; 5) replacing 5'-TTCATGCTCGTCTACATCGT-3' in the coding gene of the protein in the rice genome DNA with 5'-TTCATGCTCGTCCACATCGT-3'.

7. The specific sgRNA in the method according to claim 2 or 3 or the specific DNA molecule in the method according to claim 2 or 3.

8. The CRISPR / Cas9 system for reducing the cadmium content in rice grains, characterized in that: The CRISPR / Cas9 system is the CRISPR / Cas9 system as described in any one of claims 1-3.

9. The application of the method according to any one of claims 1-5, the DNA molecule according to claim 6, the specific gRNA according to claim 7, the specific DNA molecule according to claim 7, and / or the CRISPR / Cas9 system according to claim 8 in any one of the following: (1) Application in reducing the cadmium content in rice; (2) Application in cultivating rice varieties with low cadmium accumulation; (3) Application in improving rice germplasm resources.

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