Application of the rice OsFWL2 gene in increasing the content of trace metal elements in rice grains

Abstract

This invention discloses the application of the rice OsFWL2 gene in increasing the content of trace metal elements in rice grains, belonging to the field of plant genetic engineering technology. This invention designs gene knockout target sites in the coding region of the rice OsFWL2 gene and constructs gene knockout mutants using CRISPR / Cas9 technology to knock out the OsFWL2 gene in rice. Through progeny isolation and detection, homozygous mutants without transgene insertion were obtained. The grain weight of different mutants was not significantly different from the wild type, but the content of trace metal elements Fe, Mn, and Cu in brown rice was significantly increased compared to the control, indicating better nutritional quality. Therefore, the method of knocking out the rice OsFWL2 gene to enhance the content of trace metal elements in grains provided by this invention has significant application value and can generate good economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to the application of the rice OsFWL2 gene in increasing the content of trace metal elements in rice grains. Background Technology

[0002] Rice is one of the world's most important food crops, with nearly half of the global population relying on it as a staple food. As people's living standards improve, the demands for rice quality are also increasing. Developing rice varieties with high accumulation of trace metals and low accumulation of cadmium is crucial for improving rice quality and ensuring safe rice production. However, traditional breeding methods are time-consuming, inefficient, and slow. Utilizing biotechnology to improve rice varieties is a feasible strategy to accelerate the development of safe and high-quality rice varieties.

[0003] FW2.2-like (FWL) proteins are a newly discovered class of metal transporters involved in the transport of various metal ions. The Arabidopsis FWL gene AtPCR2 encodes a Zn export transporter involved in long-distance Zn transport. Overexpression of the common wheat FWL genes TaCNR2 and TaCNR5, and the diploid wheat FWL gene TuCNR10 in rice significantly increased the Zn and Mn content in brown rice. Rice OsFWL5 gene knockdown plants showed a significant increase in Zn and Mg content in brown rice, but a decrease in Mn content. OsFWL4 knockdown plants showed a decrease in Mn content in brown rice. OsFWL7 knockout mutants showed an increase in Mn and Cu content in brown rice. The OsFWL2 gene regulates rice plant height, seed size, and tiller number.

[0004] The expression of FWL family genes varies across different species. Functional studies of FWL genes in many species have been reported, demonstrating their crucial role in plant growth and development. Some studies have reported that the OsFWL2 gene regulates the number of secondary branches, grains per panicle, and yield per plant in rice plants; however, whether the OsFWL2 gene has other biological functions in rice remains largely unreported.

[0005] The CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-Associated Protein 9) system is a guide RNA-mediated genome editing system widely used in genome editing of various organisms. Because the T-DNA insertion site and mutation site are located at different positions, the T-DNA can be separated and removed from the gene-edited offspring, obtaining mutants without transgene insertion, thus avoiding the potential safety risks associated with transgenes. Summary of the Invention

[0006] In view of the above-mentioned problems in the existing technology, the technical problem to be solved by the present invention is to provide the application of the rice OsFWL2 gene in increasing the content of trace metal elements in rice grains.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] The application of the OsFWL2 gene, with a nucleotide sequence as shown in SEQ ID NO.1, in increasing the content of trace metal elements in rice grains, wherein the trace metal elements are Fe and / or Mn and / or Cu and / or Zn.

[0009] The application described uses CRISPR / Cas9 technology to target the OsFWL2 gene in mutant rice.

[0010] The specific steps of the application are as follows:

[0011] 1) Select target sequences in the coding region of the rice OsFWL2 gene;

[0012] 2) Synthesize adapter primers with sticky ends according to the target sequence to construct a CRISPR / Cas9 recombinant vector for targeting the OsFWL2 gene; the recombinant vector includes an sgRNA expression cassette with the target sequence and a Cas9 nuclease expression cassette that can be expressed in rice cells;

[0013] 3) The recombinant vector was transferred into an engineered strain of Agrobacterium tumefaciens, and conventional rice varieties were transformed using the Agrobacterium-mediated transformation method to obtain transgenic rice plants;

[0014] 4) Genomic primers covering the target site are used to amplify and sequence the genomic DNA of transgenic plants, thereby identifying the genotype of T0 generation transgenic plants and obtaining homozygous or biallelic mutants.

[0015] 5) By planting homozygous mutants in the T1 generation and using primers to perform PCR detection on HPT, sgRNA and Cas9 transgenes in the T1 generation plants, rice with no transgene insertion and significantly increased Fe and / or Mn and / or Cu and / or Zn content in the grains can be obtained.

[0016] In step 1), the target sequence is as shown in SEQ ID NO.6 or SEQ ID NO.7.

[0017] In step 2), the nucleotide sequence of the sgRNA expression cassette having the target sequence is shown in SEQ ID NO.3 and SEQ ID NO.4; the nucleotide sequence of the Cas9 nuclease expression cassette is shown in SEQ ID NO.5.

[0018] In step 5), the primer sequences used for HPT, sgRNA, and Cas9 transgene amplification are as follows:

[0019] HPTF: 5'-GGGTTGTCACGTTGCAAGACC-3',

[0020] HPTR: 5'-ATGCCTCCGCTCGAAGTAGC-3',

[0021] sgRNAF: 5'-TCCCAGTCACGACGTTGTAA-3',

[0022] sgRNAR: 5'-GGCCATTTGTCTGCAGAAT-3',

[0023] Cas9F: 5'-CACCATCTACCACCTGAGAA-3',

[0024] Cas9R: 5'-CGAAGTTGCTCTTGAAGTTG-3'.

[0025] In the aforementioned application, step 1), the primer sequences for the target sequence are as follows:

[0026] OsFWL2a-P1:5'- TGTG GCGCTGGTGATGCTCCTCAC-3',

[0027] OsFWL2a-P2:5'- AAAC GTGAGGAGCATCACCAGCGC-3';

[0028] OsFWL2b-P1:5'- TGTG CATCTTGGCGCGGTAGAAGC-3',

[0029] OsFWL2b-P2:5'- AAAC GCTTCTACCGCGCCAAGATG-3'.

[0030] Application of knockout vectors OsFWL2a-CRISPR / Cas9 or OsFWL2b-CRISPR / Cas9 in increasing Fe and / or Mn and / or Cu and / or Zn content in rice grains, and / or decreasing Cd content.

[0031] The application includes the following steps:

[0032] 1) Design two CRISPR / Cas9 gene knockout target sites, OsFWL2a and OsFWL2b, in the coding region of the rice OsFWL2 gene;

[0033] 2) The maize ZmUBI promoter was linked to the hSpCas9 gene and inserted into the pCAMBIA1300 binary vector to obtain the intermediate vector;

[0034] 3) After removing the original BsaI restriction site from the pCAMBIA1300 vector, the sequences of the ccdB negative selection marker gene containing the OsU6 promoter, the ccdB negative selection marker gene flanked by BsaI restriction sites, the OsFWL2a sgRNA expression cassette, and the OsFWL2b sgRNA expression cassette were inserted into the intermediate vector to construct a CRISPR / Cas9 binary vector.

[0035] 3) OsFWL2a-P1 and OsFWL2a-P2, as well as OsFWL2b-P1 and OsFWL2b-P2, were annealed to form double-stranded DNA with sticky ends, which were then used as insert fragments for constructing CRISPR / Cas9 gene knockout vectors.

[0036] 4) The CRISPR / Cas9 binary vector was digested with BsaI restriction endonuclease at 37°C to obtain the vector backbone fragment;

[0037] 5) The insert fragment and the vector backbone were ligated using T4 ligase to obtain the recombinant expression vectors OsFWL2a-CRISPR / Cas9 and OsFWL2b-CRISPR / Cas9 targeting the target site.

[0038] 6) The recombinant expression vector was transferred into an engineered strain of Agrobacterium tumefaciens, and conventional rice varieties were transformed using the Agrobacterium-mediated transformation method to obtain transgenic rice plants;

[0039] 4) Genomic primers covering the target site are used to amplify and sequence the genomic DNA of transgenic plants, thereby identifying the genotype of T0 generation transgenic plants and obtaining homozygous or biallelic mutants.

[0040] 5) Plant the above T0 generation homozygous or biallelic mutants in the T1 generation, and use primers to perform PCR detection on HPT, sgRNA and Cas9 transgenes in the T1 generation plants to obtain rice without transgene insertion, with significantly increased Fe and / or Mn and / or Cu and / or Zn content and / or significantly decreased Cd content.

[0041] The HPT primer sequence is as follows:

[0042] HPT-F: 5'-GGGTTGTCACGTTGCAAGACC-3',

[0043] HPT-R: 5'-ATGCCTCCGCTCGAAGTAGC-3';

[0044] The sgRNA primer sequence is as follows:

[0045] sgRNA-F: 5'-TCCCAGTCACGACGTTGTAA-3',

[0046] sgRNA-R: 5'-GGCCATTTGTCTGCAGAAT-3';

[0047] The Cas9 primer sequence is as follows:

[0048] Cas9-F: 5'-CACCATCTACCACCTGAGAA-3',

[0049] Cas9-R: 5'-CGAAGTTGCTCTTGAAGTTG-3'.

[0050] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0051] 1) This invention utilizes CRISPR / Cas9 technology to target and mutate the OsFWL2 gene to obtain transgene-free mutants. By using three pairs of primers in the T1 generation for detection, it ensures that the obtained mutants are transgene-free and equivalent to naturally occurring mutants, thus avoiding the safety risks associated with transgenic technology. This method improves rice quality while enhancing the safety of rice production, increases the content of trace elements in rice grains, and generates higher commercial value. It allows for rapid and targeted improvement of existing rice varieties, significantly shortening the breeding cycle and saving time and costs. Because the mutation is targeted at the target gene, it does not alter the genetic background of the edited material and therefore does not change the original beneficial traits of the recipient variety.

[0052] 2) When the T2 generation homozygous mutant without transgene insertion obtained in this invention was planted under normal field conditions, the grain weight of the mutant plants was not significantly different from that of the wild type.

[0053] 3) The contents of Fe, Mn, Cu, and Zn in the OsFWL2a target mutant brown rice obtained in this invention increased by 99.2%, 32.9%, 31.0%, and 10.2% respectively compared with the wild-type control, while the content of heavy metal Cd decreased by 9.9%. The Zn content of the OsFWL2b target homozygous mutant brown rice was not significantly different from the control, while the contents of Fe, Mn, and Cu increased by 5.2%, 35.3%, and 29.0% respectively. Although the Cd content of the OsFWL2b mutant brown rice was slightly higher than that of the control, its content was only 6.1 μg / kg, which is far below the national standard of 0.2 mg / kg (GB 2762-2017) for Cd content in rice. Attached Figure Description

[0054] Figure 1 The structure of the T-DNA segment in the gene knockout vector in Example 1;

[0055] Figure 2 This is a graph showing the sequencing identification results of different mutants in Example 1;

[0056] Figure 3 The image shows the detection results of the T1 generation non-transgenic insertion mutant in Example 1 (where 1-10 represent randomly selected T1 generation plants, VC represents the vector control, WT represents the wild type, and M represents the DNA molecular weight marker).

[0057] Figure 4 The image shows the thousand-grain weight of the mutant and wild-type seeds in Example 1 (n=5);

[0058] Figure 5 The graph shows the content of trace metal elements in the seeds of mutant and wild-type plants in Example 1 (n=6, *P<0.05, ***P<0.001);

[0059] Figure 6 The graph shows the Cd content of seeds from mutant and wild-type plants in Example 1 (n=6). Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0061] Example 1

[0062] 1. Constructing a rice OsFWL2 gene knockout recombinant expression vector

[0063] The nucleotide sequence of the rice OsFWL2 gene is shown in SEQ ID NO.1. Sequence analysis shows that the gene includes three exons, namely positions 1-121 (exon 1), positions 224-508 (exon 2), and positions 603-685 (exon 3) of the sequence in SEQ ID NO.1.

[0064] Two CRISPR / Cas9 gene knockout target sites, OsFWL2a and OsFWL2b, were designed in the coding region of the rice OsFWL2 gene to target the antisense and sense strands of exon 2, respectively. The target sequence of OsFWL2a is shown in SEQ ID NO.6, and the target sequence of OsFWL2b is shown in SEQ ID NO.7. CRISPR / Cas9-based primers for the target sequences were synthesized, and the primer sequences are shown below:

[0065] OsFWL2a-P1:5'- TGTG GCGCTGGTGATGCTCCTCAC-3',

[0066] OsFWL2a-P2:5'- AAAC GTGAGGAGCATCACCAGCGC-3';

[0067] OsFWL2b-P1:5'- TGTG CATCTTGGCGCGGTAGAAGC-3',

[0068] OsFWL2b-P2:5'- AAAC GCTTCTACCGCGCCAAGATG-3'.

[0069] The underlined portion represents the adhesive ends used for carrier connection, while the ununderlined portion represents the target sequence or its complementary sequence.

[0070] The maize ZmUBI promoter (SEQ ID NO.8) was ligated to the hSpCas9 gene (DOI: 10.1126 / science.1231143, SEQ ID NO.5) and inserted into the pCAMBIA1300 binary vector to obtain an intermediate vector. The original BsaI restriction site in the pCAMBIA1300 vector was removed using a point mutation kit (TransGen Biotech). Then, a fragment containing the OsU6 promoter (SEQ ID NO.9), a ccdB negative selection marker gene flanked by BsaI restriction sites (SEQ ID NO.10), and an sgRNA backbone fragment (SEQ ID NO.11) was inserted into the intermediate vector to construct a CRISPR / Cas9 binary vector (vector structure shown in Figure 1). Figure 1 (As shown). The binary vector was preserved in Escherichia coli strain DB3.1.

[0071] OsFWL2a-P1 and OsFWL2a-P2, as well as OsFWL2b-P1 and OsFWL2b-P2, were annealed to form double-stranded DNA with sticky ends, which served as the insert fragments for constructing CRISPR / Cas9 gene knockout vectors. The CRISPR / Cas9 binary vectors were digested with BsaI restriction endonuclease (NEB) at 37°C to obtain the vector backbone fragment. The insert fragment and the vector backbone were ligated using T4 ligase (NEB) to obtain the recombinant expression vectors OsFWL2a-CRISPR / Cas9 and OsFWL2b-CRISPR / Cas9 targeting the target site. The recombinant expression vectors OsFWL2a-CRISPR / Cas9 and OsFWL2b-CRISPR / Cas9 were transformed into Escherichia coli for amplification. After the vector plasmids were extracted and verified to be correct by sequencing (Shanghai Sangon Biotech Co., Ltd.), they were transformed into Agrobacterium tumefaciens strain EHA105 for Agrobacterium-mediated rice genetic transformation.

[0072] 2. Obtaining and identifying OsFWL2 gene knockout rice

[0073] Following the method of Nishimura et al. (Nishimura A, Aichi I, Matsuoka MA protocol for Agrobacterium-mediated transformation in rice. Nature Protocols, 2006, 1(6): 2796-2802), the recombinant expression vectors targeting the above-mentioned target sites were transformed into the conventional japonica rice variety Zhonghua 11. The two recombinant vectors yielded 16 and 14 T0 generation transgenic rice plants, respectively.

[0074] DNA was extracted from two T0 generation transgenic rice plants. DNA fragments containing target sites were amplified using KOD DNA polymerase (TOYOBO) and genomic primers. The amplified products were sequenced at Shanghai Sangon Biotech Co., Ltd. The primer sequences are shown below:

[0075] OsFWL2-F: 5'-CTCGATCGATCAGCGTGTCA-3'

[0076] OsFWL2-R: 5'-TGATTGATCGTTGTGCGTGC-3'.

[0077] This primer can cover two target sites simultaneously.

[0078] Sequencing results showed that 15 transgenic plants carrying the target mutation at the OsFWL2a target site were 1 homozygous mutant and 14 biallelic mutants; 11 transgenic plants carrying the target mutation at the OsFWL2b target site were 3 homozygous mutants and 8 biallelic mutants.

[0079] 3. Obtaining and phenotypic analysis of transgenic homozygous mutants

[0080] One homozygous T0 generation mutant was selected for each target site for subsequent analysis, named osfwl2a and osfwl2b, respectively. The mutant genotypes are shown below. Figure 2 .

[0081] Simultaneously, in the T1 generation, genomic DNA of mutant plants was amplified using primers for HPT (HPT-F: 5'-GGGTGTCACGTTGCAAGACC-3', HPT-R: 5'-ATGCCTCCGCTCGAAGTAGC-3'), sgRNA (sgRNA-F: 5'-TCCCAGTCACGACGTTGTAA-3', sgRNA-R: 5'-GGCCATTTGTCTGCAGAAT-3') and Cas9 (Cas9-F: 5'-CACCATCTACCACCTGAGAA-3', Cas9-R: 5'-CGAAGTTGCTCTTGAAGTTG-3'). Mutants without transgene insertion were identified in 10 randomly selected individual plants from both mutant systems. Figure 3 This indicates that the number of T-DNA insertion sites in the transgenic plants obtained using this method is extremely small, and mutants without transgenic insertion can be obtained in the T1 generation.

[0082] 1) Results are as follows Figure 4 As shown, when the homozygous mutants of the T2 generation without transgene insertion were planted under normal field conditions, the grain weight of the mutant plants was not significantly different from that of the wild type.

[0083] 2) The mutant and wild-type brown rice were ground into a fine powder using a Cole-Parmer sample mill. 0.4 g of powder was accurately weighed for each replicate of each sample and digested with HNO3 and H2O2 in a microwave digester (MARS 5). After dilution, the metal ion content was determined using inductively coupled plasma atomic emission spectrometry (Inductively Coupled Plasma Optical Emission Spectrometry) (Thermo Scientific iCAP 6300).

[0084] The results are as follows Figure 5-6As shown, the contents of Fe, Mn, Cu, and Zn in the OsFWL2a target mutant brown rice increased by 99.2%, 32.9%, 31.0%, and 10.2%, respectively, compared to the wild-type control, while the content of heavy metal Cd decreased by 9.9%. The Zn content in the OsFWL2b target homozygous mutant brown rice was not significantly different from the control, while the contents of Fe, Mn, and Cu increased by 5.2%, 35.3%, and 29.0%, respectively. Although the Cd content in the OsFWL2b mutant brown rice was slightly higher than the control, its content was only 6.1 μg / kg, far below the national standard of 0.2 mg / kg (GB 2762—2017) for Cd content in rice.

[0085] In summary, mutant grains exhibit superior nutritional quality, enhancing essential micronutrients in the diet while reducing harmful metal elements. The rice material with increased Fe, Mn, Cu, and Zn content obtained in this invention does not contain any genetically modified components, thus avoiding the potential safety risks associated with genetic modification. Furthermore, since only one gene is altered, it will not affect other agronomic traits of the rice variety.

[0086] The above description is illustrative only and not restrictive of this invention. Those skilled in the art will understand that many modifications, variations, or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations, or equivalents will fall within the protection scope of this invention.

Claims

1. The application of knocking out the OsFWL2 gene with the nucleotide sequence shown in SEQ ID NO.1 in increasing the content of trace metal elements in rice grains. This application involves knocking out the OsFWL2 gene at two target sites, OsFWL2a and OsFWL2b, using CRISPR / Cas9 technology. The OsFWL2a mutant rice grains show increased contents of Fe, Mn, Cu, and Zn, while the OsFWL2b mutant rice grains show increased contents of Mn and Cu. The OsFWL2a target sequence is shown in SEQ ID NO. 6, and the OsFWL2b target sequence is shown in SEQ ID NO.

7.

2. The application according to claim 1, characterized in that, The specific steps are as follows: 1) Select target sequences in the coding region of the rice OsFWL2 gene; 2) Synthesize adapter primers with sticky ends according to the target sequence to construct a CRISPR / Cas9 recombinant vector for targeting the OsFWL2 gene; the recombinant vector includes an sgRNA expression cassette with the target sequence and a Cas9 nuclease expression cassette that can be expressed in rice cells; 3) The recombinant vector was transferred into an engineered strain of Agrobacterium tumefaciens, and conventional rice varieties were transformed using the Agrobacterium-mediated transformation method to obtain transgenic rice plants; 4) Genomic primers covering the target site are used to amplify and sequence the genomic DNA of transgenic plants, thereby identifying the genotype of T0 generation transgenic plants and obtaining homozygous or biallelic mutants. 5) Homozygous mutants were planted in the T1 generation, and primers were used to detect HPT, sgRNA and Cas9 transgene in the T1 generation plants by PCR.

3. The application according to claim 2, characterized in that, In step 2), the nucleotide sequence of the sgRNA expression cassette having the target sequence is shown in SEQ ID NO.3 and SEQ ID NO.4; the nucleotide sequence of the Cas9 nuclease expression cassette is shown in SEQ ID NO.

5.

4. The application according to claim 2, characterized in that, In step 5), the primer sequences used for HPT, sgRNA, and Cas9 transgene amplification are as follows: HPTF: 5'-GGGTTGTCACGTTGCAAGACC-3', HPTR: 5'-ATGCCTCCGCTCGAAGTAGC-3', sgRNAF: 5'-TCCCAGTCACGACGTTGTAA-3', sgRNAR: 5'-GGCCATTTGTCTGCAGAAT-3', Cas9F: 5'-CACCATCTACCACCTGAGAA-3', Cas9R: 5'-CGAAGTTGCTCTTGAAGTTG-3'.

5. The application according to claim 2, characterized in that, In step 1), the primer sequences for the target sequence are as follows: OsFWL2a-P1: 5'-TGTGGCGCTGGTGATGCTCCTCAC-3', OsFWL2a-P2: 5'-AAACGTGAGGAGCATCACCAGCGC-3'; OsFWL2b-P1: 5'-TGTGCATCTTGGCGCGGTAGAAGC-3', OsFWL2b-P2: 5'-AAACGCTTCTACCGCGCCAAGATG-3'.

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