Mutated sequence of rice osabci13 gene and method and application thereof in regulating cadmium accumulation in rice
By editing the rice OsABCI13 gene and designing a CRISPR/Cas9 vector mutant nucleotide sequence to alter the protein structure, the problem of excessive cadmium content in rice was solved, and rice varieties with reduced cadmium absorption were cultivated, thus improving food security.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-02
- Publication Date
- 2026-03-31
AI Technical Summary
Existing technologies have shown that excessive cadmium content in rice leads to environmental pollution and harms human health, making it difficult to effectively reduce the accumulation of cadmium in rice.
By editing the rice OsABCI13 gene, a CRISPR/Cas9 editing vector was designed to mutate the nucleotide sequence and change the protein structure, thereby reducing cadmium accumulation.
Significantly reduce the cadmium content in rice grains, cultivate rice varieties with reduced cadmium absorption, and improve food security.
Smart Images
Figure CN118895279B_ABST
Abstract
Description
Technical Field
[0001] This disclosure relates to the field of rice genetic engineering technology, specifically to a mutant sequence of the rice OsABCI13 gene, a protein encoded by the mutant sequence, a vector, a transformant, a method for reducing cadmium accumulation in rice plants, and its application. Background Technology
[0002] The indiscriminate discharge of industrial waste and the excessive application of pesticides and fertilizers have led to an increase in the accumulation of heavy metals, resulting in increasingly serious environmental pollution. Cadmium (Cd), due to its long half-life, high toxicity, difficulty in degradation, and tendency to accumulate in plants, can harm human health through the food chain. Studies have found that cadmium accumulation can cause serious diseases such as osteoporosis, kidney failure, and immune system cancers.
[0003] Rice, as an energy source for half the world's population, is not only an important food crop but also an ideal plant for the remediation of heavy metal pollution. Because cadmium shares similar physicochemical properties with elements such as zinc, manganese, and iron, it can "hitch a ride" into plants via transport proteins of essential metals, leading to cadmium accumulation, especially in edible parts. This accumulation can then enter the human body through the food chain, posing a health risk. Simultaneously, the accumulation of cadmium in plants can affect their absorption and utilization of essential elements such as nitrogen, phosphorus, potassium, zinc, iron, and manganese, harming normal plant growth and development.
[0004] Therefore, exploring the functions of more genes that regulate cadmium absorption, transport, and accumulation, elucidating the molecular mechanisms of cadmium accumulation in rice grains, identifying target genes that can reduce cadmium content in rice, and cultivating low-cadmium-accumulation rice are important ways to solve the problem of cadmium harm to human health and are of great significance to solving the food security problem. Summary of the Invention
[0005] In order to address the problem of excessive cadmium in rice in existing technologies, the purpose of this disclosure is to screen genes and methods that can reduce cadmium in rice, and to cultivate rice varieties with reduced cadmium absorption.
[0006] To achieve the above objectives, the first aspect of this disclosure provides a mutant sequence of the rice OsABCI13 gene, said mutant sequence being obtained by mutation of the nucleotide sequence of the rice OsABCI13 gene; said mutation includes substitution, deletion and / or addition of one or more nucleotides in the rice OsABCI13 gene.
[0007] The nucleotide sequence of the rice OsABCI13 gene is shown in SEQ ID NO: 1; the amino acid sequence of the protein encoded by the rice OsABCI13 gene is shown in SEQ ID NO: 3.
[0008] Optionally, the mutant sequence is selected from at least one of the following sequences:
[0009] (1) The nucleotide sequence shown in SEQ ID NO:4 obtained by deleting the TGAT fragment from position 6 to position 9 of the target site I sequence of the rice OsABCI13 gene.
[0010] (2) The nucleotide sequence shown in SEQ ID NO: 5 was obtained by inserting a base T between the 6th and 7th bases of the target site I sequence of the rice OsABCI13 gene;
[0011] The nucleotide sequence of target site I is shown in SEQ ID NO: 8.
[0012] The second aspect of this disclosure provides a protein encoded by the mutant sequence described in the first aspect, the amino acid sequence of which is shown in SEQ ID NO: 6 and / or 7;
[0013] The nucleotide sequence of target site I is shown in SEQ ID NO: 8.
[0014] A third aspect of this disclosure provides a vector, which is a CRISPR / Cas9 editing vector, wherein the vector is inserted with a target site I and produces a mutant sequence as described in the first aspect;
[0015] The nucleotide sequence of target site I is shown in SEQ ID NO: 8.
[0016] The fourth aspect of this disclosure provides a transformant, wherein the host of the transformant is a genetically engineered bacterium; the transformant contains a target site I, or the transformant contains the vector described in the third aspect.
[0017] The fifth aspect of this disclosure provides a method for reducing cadmium accumulation in rice plants, the method comprising:
[0018] Gene editing of the rice OsABCI13 gene mutated the nucleotide sequence of the OsABCI13 gene and altered the protein encoded by the OsABCI13 gene.
[0019] The mutations include substitutions, deletions, and / or additions of one or more nucleotides in the rice OsABCI13 gene.
[0020] Optionally, the gene editing method includes:
[0021] S1. Design a CRISPR / Cas9 editing vector based on target site I of the rice OsABCI13 gene;
[0022] S2. The CRISPR / Cas9 editing vector was introduced into rice plants by Agrobacterium tumefaciens. Mutations were randomly performed at the target site I in the rice plants. The T0 generation of positive plants with functional defect mutations were then screened according to the designed primers.
[0023] S3. Self-pollinate the T0 generation positive plants and screen to obtain T1 generation homozygous mutant plants, thus obtaining rice plants with reduced cadmium content.
[0024] The nucleotide sequence of target site I is shown in SEQ ID NO: 8;
[0025] The sequences of the primers are shown in SEQ ID NO: 9 and SEQ ID NO: 10.
[0026] Optionally, in step S2, the mutation includes one of the following:
[0027] (a) TGAT deletion of the 6th to 9th segments of the target site I of the rice OsABCI13 gene.
[0028] (b) Insert a T base between the 6th and 7th bases of the target site I sequence of the rice OsABCI13 gene.
[0029] The sixth aspect of this disclosure provides the application of the rice OsABCI13 gene and / or the protein encoded by the rice OsABCI13 gene in regulating cadmium accumulation in rice and / or breeding rice varieties with reduced cadmium uptake.
[0030] The seventh aspect of this disclosure provides the application of the mutant sequence of the rice OsABCI13 gene described in the first aspect, the protein described in the second aspect, the vector described in the third aspect, or the transformant described in the fourth aspect in regulating cadmium accumulation in rice and / or cultivating rice varieties with reduced cadmium uptake.
[0031] Through the above-described technical solution, this disclosure reduces the accumulation of cadmium in rice by causing gene mutations in the nucleotide sequence of the OsABCI13 gene, thereby eliminating the gene's function. This provides genetic resources and technical support for cultivating rice varieties with reduced cadmium absorption, and offers new methods and ideas for breeding low-cadmium varieties and producing safe food. This disclosure can effectively address the problem of excessive cadmium content in rice and is of great significance for solving food security issues.
[0032] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description
[0033] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:
[0034] Figure 1 This refers to the OsABCI13 gene structure and mutant type in the examples.
[0035] Figure 2 Gene-edited rice osabci13-1 and osabci13-2 Cadmium content test results for mutants and wild-type (WT). Detailed Implementation
[0036] The specific embodiments of this disclosure will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this disclosure.
[0037] The first aspect of this disclosure provides a mutant sequence of the rice OsABCI13 gene, which is obtained by mutating the nucleotide sequence of the rice OsABCI13 gene; the mutation includes substitution, deletion and / or addition of one or more nucleotides in the rice OsABCI13 gene.
[0038] The nucleotide sequence of the rice OsABCI13 gene is shown in SEQ ID NO: 1, the nucleotide sequence of the coding region of the rice OsABCI13 gene is shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by the rice OsABCI13 gene is shown in SEQ ID NO: 3.
[0039] In this disclosure, the inventors have made a surprising discovery that the OsABCI13 gene (accession number LOC_Os01g42830) can regulate the accumulation of cadmium in rice. This disclosure reduces the accumulation of cadmium in rice by causing gene mutations targeting the nucleotide sequence of the rice OsABCI13 gene to lose the function of the gene.
[0040] In one specific embodiment of this disclosure, the mutant sequence is selected from at least one of the following sequences:
[0041] (1) The nucleotide sequence shown in SEQ ID NO:4 obtained by deleting the TGAT fragment from position 6 to position 9 of the target site I sequence of the rice OsABCI13 gene.
[0042] (2) The nucleotide sequence shown in SEQ ID NO: 5 was obtained by inserting a base T between the 6th and 7th bases of the target site I sequence of the rice OsABCI13 gene;
[0043] The nucleotide sequence of target site I is shown in SEQ ID NO: 8.
[0044] In the above embodiments, the inventors of this disclosure designed and edited a vector based on target site I, and infected rice plants with Agrobacterium. Mutations were randomly performed at the target site within the rice plants. Then, the mutant sequences were detected using designed primers, and mutant lines with T base insertion and TGAT deletion were selected. The TGAT fragment was deleted or the T base was inserted into the nucleotide sequence of the rice OsABCI13 gene, resulting in corresponding changes in the codon sequence and composition after the insertion site. The stop codon appeared prematurely, causing the encoded protein to terminate prematurely. The structure and function of the protein encoded by the mutant sequence were changed. In addition, the inventors of this disclosure have also discovered that homozygous mutant plants with functional defective mutations of the OsABCI13 gene obtained by transgenic technology have a cadmium content in rice grains that is about 60% lower than that of wild-type plants. Therefore, analyzing the biological function of the OsABCI13 gene in rice has potential application value in agricultural development.
[0045] The second aspect of this disclosure provides a protein encoded by the mutant sequence described in the first aspect, the amino acid sequence of which is shown in SEQ ID NO: 6 and / or 7.
[0046] According to this disclosure, the nucleotide sequence shown in SEQ ID NO:4 encodes the protein shown in SEQ ID NO:6; the nucleotide sequence shown in SEQ ID NO:5 encodes the protein shown in SEQ ID NO:7.
[0047] A third aspect of this disclosure provides a vector, which is a CRISPR / Cas9 editing vector, wherein the vector is inserted with a target site I and produces a mutant sequence as described in the first aspect;
[0048] The nucleotide sequence of target site I is shown in SEQ ID NO: 8.
[0049] The fourth aspect of this disclosure provides a transformant, the host of which is a genetically engineered bacterium; the transformant contains a target site I, or the transformant contains the vector described in the third aspect;
[0050] The nucleotide sequence of target site I is shown in SEQ ID NO: 8.
[0051] In this disclosure, the genetically engineered bacterium can be Agrobacterium EHA105.
[0052] The fifth aspect of this disclosure provides a method for reducing cadmium accumulation in rice plants, the method comprising:
[0053] Gene editing of the rice OsABCI13 gene mutated the nucleotide sequence of the OsABCI13 gene and altered the protein encoded by the OsABCI13 gene.
[0054] The mutations include substitutions, deletions, and / or additions of one or more nucleotides in the rice OsABCI13 gene.
[0055] In this disclosure, gene editing of the rice OsABCI13 gene alters the protein encoded by the OsABCI13 gene, thereby reducing or eliminating the expression level of the OsABCI13 gene and decreasing the activity of the protein encoded by the rice OsABCI13 gene. This is beneficial for cultivating rice varieties with reduced cadmium absorption and effectively solves the problem of excessive cadmium content in rice.
[0056] In one specific embodiment of this disclosure, the gene editing method includes:
[0057] S1. Design a CRISPR / Cas9 editing vector based on target site I of the rice OsABCI13 gene;
[0058] S2. The CRISPR / Cas9 editing vector was infected into rice plants by Agrobacterium tumefaciens. Mutations were randomly performed on target site I in the rice plants. The T0 generation of positive plants with functional defect mutations were then screened according to the designed primers.
[0059] S3. Self-pollinate the T0 generation positive plants and screen to obtain T1 generation homozygous mutant plants, thus obtaining rice plants with reduced cadmium content.
[0060] The nucleotide sequence of target site I is shown in SEQ ID NO: 8.
[0061] In a preferred embodiment of this disclosure, in step S2, the mutation includes one of the following:
[0062] (a) TGAT deletion of the 6th to 9th segments of the target site I of the rice OsABCI13 gene.
[0063] (b) Insert a T base between the 6th and 7th bases of the target site I sequence of the rice OsABCI13 gene.
[0064] The sixth aspect of this disclosure provides the application of the rice OsABCI13 gene and / or the protein encoded by the rice OsABCI13 gene in regulating cadmium accumulation in rice and / or breeding rice varieties with reduced cadmium uptake.
[0065] The seventh aspect of this disclosure provides the application of the mutant sequence of the rice OsABCI13 gene described in the first aspect, the protein described in the second aspect, the vector described in the third aspect, or the transformant described in the fourth aspect in regulating cadmium accumulation in rice and / or cultivating rice varieties with reduced cadmium uptake.
[0066] In this disclosure, by mutating the rice OsABCI13 gene and altering the structure and function of the protein encoded by the OsABCI13 gene, the cadmium content in the rice grains of the resulting mutant plants was significantly reduced. This demonstrates that the mutation of the rice OsABCI13 gene can effectively reduce the cadmium content in rice grains. Developing rice varieties with reduced cadmium uptake using mutants of the rice OsABCI13 gene has potential application value in agriculture.
[0067] The present disclosure is further described in detail below through examples.
[0068] All raw materials used in the examples are commercially available. Unless otherwise specified, the experimental conditions used in the examples were performed under conventional conditions known to those skilled in the art.
[0069] Example 1
[0070] This example illustrates how the OsABCI13 mutant in rice was obtained.
[0071] To study the function of the rice OsABCI13 gene, the nucleotide sequence of the rice OsABCI13 gene was edited at specific sites using CRISPR / Cas9 technology, as shown in SEQ ID NO.1.
[0072] Specific methods include:
[0073] 1. Obtaining rice ABC transporter gene OsABCI13 mutant plants:
[0074] like Figure 1 As shown, A is the structure diagram of the OsABCI13 gene, B is the nucleotide type of the OsABCI13 mutant plant, where black lines indicate target sequences and red lines indicate adjacent motifs of candidate recognition sites.
[0075] The OsABCI13 gene is 4053 bp in length and has 8 exons and 7 introns.
[0076] A mutant family of the OsABCI13 gene was constructed using CRISPR-Cas9 technology. First, target site I was selected: GTTATGATAGCAGCAGG (SEQ ID NO: 8), and a pCRISPR-OsABCI13 plasmid containing the OsABCI13-specific target site I was constructed. Then, mutant plants were constructed by infecting wild-type Nipponbare bacteria with Agrobacterium strains. Finally, specific primers were designed:
[0077] Forward primer F: 5'-ACTCCATTGTTGTACTTGTTCTGTTCT-3' (SEQ ID NO: 9); Reverse primer R: 5'-TGCTTACCACCTCAACACTATGATC-3' (SEQ ID NO: 10). PCR amplification was performed at the target site to screen for mutation types, ultimately identifying two independent homozygous mutant families: osabci13-1 and osabci13-2 Its mutation types are as follows Figure 1 As shown, the phenotypic characteristics of these two mutant families were investigated.
[0078] 2. Analysis of cadmium content in grains of the OsABCI13 mutant family:
[0079] To investigate the effect of the OsABCI13 mutant family on cadmium content in rice grains, wild-type Nipponbare seeds and mutant seeds were compared. osabci13-1 and osabci13-2 The T1 generation homozygous seeds were placed in petri dishes and germinated in the dark at 37°C for three days. The germinated rice seedlings were then evenly scattered onto prepared rice paddies. Three weeks later, the uniformly growing wild-type Nipponbare and mutant seeds were transplanted. osabci13-1 and osabci13-2 The seedlings were transplanted into cadmium-contaminated rice fields, with eight seedlings per family, spaced 15cm apart and 15cm apart in rows. Water and fertilizer management and pest and disease control were carried out in the same way as in ordinary paddy fields throughout the rice growth period.
[0080] Harvest wild-type Nipponbare and mutants at maturity. osabci13-1 and osabci13-2 Mature rice seeds from the main panicle were dried in an oven at 65℃ for 20 hours, then the husks were removed, the seeds were weighed, and the mixture was digested with nitric acid. The volume was then adjusted to 10 mL with 1% nitric acid. Finally, the Cd content of the solution was determined by ICP-MS, and the cadmium concentration per gram of seed was calculated.
[0081] The results are as follows Figure 2 As shown, "*" indicates a comparison with the wild type. p <0.05. This example shows that after mutation of the rice ABC transporter gene OsABCI13, the cadmium content of mature rice grains under field conditions is significantly reduced by about 60%.
[0082] The results above demonstrate that mutating the rice OsABCI13 gene using gene editing methods can reduce cadmium content in grains. This provides insights for breeding rice varieties with low cadmium accumulation.
[0083] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.
[0084] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.
[0085] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.
Claims
1. A method of reducing cadmium accumulation in rice grains, characterized by, The method comprises: The method comprises: The method comprises: The method comprises: S1, designing a CRISPR / Cas9 editing vector according to a target site I of the rice OsABCI13 gene; S2, infecting the CRISPR / Cas9 editing vector into the rice plant through agrobacterium, mutating randomly at the position of the target site I in the rice plant body, and then detecting and screening a T0 generation positive plant with a functional defect type mutation according to the designed primer; S3, selfing the T0 generation positive plant, screening a T1 generation homozygous mutant plant, and obtaining a rice plant with reduced cadmium content; The nucleotide sequence of the target site I is shown as SEQ ID NO:
8. The sequence of the primer is shown as SEQ ID NO: 9 and SEQ ID NO:
10. The nucleotide sequence of the rice OsABCI13 gene is shown as SEQ ID NO:
1.
2. The method of claim 1, wherein, In step S2, the mutation is one of the following: (a) deleting a fragment TGAT of positions 6 to 9 of the sequence of the target site I of the rice OsABCI13 gene; (b) inserting a base T between the 6th and 7th bases of the sequence of the target site I of the rice OsABCI13 gene.
3. Use of a knockout of the OsABCI13 gene or the protein encoded thereby for breeding rice varieties with reduced cadmium uptake in the grains of rice, wherein, The nucleotide sequence of the OsABCI13 gene is shown as SEQ ID NO: 1.