Mutated sequence of rice osabcb2 gene and method and application thereof in regulating cadmium accumulation in rice
By editing the rice OsABCB2 gene and inserting nucleotide bases C or A, resulting in the loss of protein function, the problem of unclear molecular mechanisms of cadmium accumulation in rice grains was solved, and the cadmium content in rice was significantly reduced, thus enabling the cultivation of low-cadmium-accumulation rice varieties.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INSTITUTE OF SUBTROPICAL AGRICULTURE CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2024-08-02
- Publication Date
- 2026-05-29
AI Technical Summary
The molecular mechanism of cadmium accumulation in rice grains is unclear, and excessive cadmium content in rice poses a threat to human health.
By editing the rice OsABCB2 gene and inserting nucleotide bases C or A, protein function is lost, reducing cadmium accumulation.
Significantly reduce the cadmium content in rice grains, cultivate low-cadmium-accumulation rice varieties, and solve the problem of excessive cadmium content in rice.
Smart Images
Figure CN118879724B_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 OsABCB2 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] With industrial development and the irrational use of resources, pollution from metals such as cadmium (Cd), lead (Pb), and chromium (Cr) is becoming increasingly serious. Among them, Cd is the most harmful heavy metal to humans and the environment. Cd in the environment is absorbed and stored by plants through plant roots and atmospheric deposition. Accumulated Cd not only harms plant growth and development but also directly harms human health through the food chain, causing osteoporosis, liver and immune system diseases. Therefore, cultivating low-cadmium-accumulation crops is an important way to solve the cadmium hazard.
[0003] Rice is one of the world's major food crops, providing energy and nutrients for the human body. Because rice has a strong capacity for cadmium absorption and readily transports and accumulates cadmium in its grains, rice is a significant source of cadmium intake for humans. Cadmium accumulation in rice primarily occurs through root absorption, transport, and distribution to various above-ground tissues and grains. Studies have identified multiple families of transporter proteins involved in the absorption, transport, and distribution of cadmium in rice, but the molecular mechanisms of cadmium accumulation in rice grains remain unclear.
[0004] Therefore, elucidating the molecular mechanism 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 harm of cadmium to human health and have significant agricultural application value. 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 OsABCB2 gene, said mutant sequence being obtained by mutation of the nucleotide sequence of the rice OsABCB2 gene; said mutation includes substitution, deletion and / or addition of one or more nucleotides in the rice OsABCB2 gene.
[0007] The nucleotide sequence of the rice OsABCB2 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the rice OsABCB2 gene is shown in SEQ ID NO: 2.
[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:3 was obtained by inserting a base C between the 1100th and 1101st bases of the nucleotide sequence of the rice OsABCB2 gene;
[0010] (2) The nucleotide sequence shown in SEQ ID NO:4 was obtained by inserting base A between the 1100th and 1101st bases of the nucleotide sequence of the rice OsABCB2 gene.
[0011] 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: 5.
[0012] 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 and produces a mutant sequence as described in the first aspect;
[0013] The target site nucleotide sequence is shown in SEQ ID NO: 6.
[0014] The fourth aspect of this disclosure provides a transformant, the host of which is a genetically engineered bacterium; the transformant contains a target site, or the transformant contains the vector described in the third aspect;
[0015] The target site nucleotide sequence is shown in SEQ ID NO: 6.
[0016] The fifth aspect of this disclosure provides a method for reducing cadmium accumulation in rice plants, the method comprising:
[0017] Gene editing of the rice OsABCB2 gene mutated the nucleotide sequence of the rice OsABCB2 gene and altered the protein encoded by the OsABCB2 gene.
[0018] The mutations include substitutions, deletions, and / or additions of one or more nucleotides in the rice OsABCB2 gene.
[0019] Optionally, the gene editing method includes:
[0020] S1. Design a CRISPR / Cas9 editing vector based on the target site of the rice OsABCB2 gene;
[0021] S2. The CRISPR / Cas9 editing vector was infected into rice plants by Agrobacterium, and mutations were performed in the rice plants at the target site. Then, the T0 generation positive plants with functional defect mutations were screened according to the designed primers.
[0022] 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.
[0023] The nucleotide sequence of the target site is shown in SEQ ID NO: 6;
[0024] The sequences of the primers are shown in SEQ ID NO: 7 and SEQ ID NO: 8.
[0025] Optionally, in step S2, the mutation includes one of the following:
[0026] (a) Insert a base C between the 1100th and 1101st bases in the nucleotide sequence of the rice OsABCB2 gene;
[0027] (b) Insert a base A between the 1100th and 1101st bases in the nucleotide sequence of the rice OsABCB2 gene.
[0028] The sixth aspect of this disclosure provides the application of the rice OsABCB2 gene and / or the protein encoded by the rice OsABCB2 gene in regulating cadmium accumulation in rice and / or breeding rice varieties with reduced cadmium uptake.
[0029] The seventh aspect of this disclosure provides the application of the mutant sequence of the rice OsABCB2 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.
[0030] Through the above-described technical solution, this disclosure achieves the loss of function of the OsABCB2 gene by mutating its nucleotide sequence, thereby reducing cadmium accumulation in rice. 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 improve the problem of excessive cadmium content in rice and has significant agricultural application value.
[0031] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description
[0032] 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:
[0033] Figure 1 It uses CRISPR / Cas9 to obtain mutants osabcb2-1 and osabcb2-2Genotypic analysis diagram of wild type.
[0034] Figure 2 Gene-edited rice osabcb2-1 and osabcb2-2 Cadmium content test results for mutants and wild-type (WT) cadmium. Detailed Implementation
[0035] 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.
[0036] The first aspect of this disclosure provides a mutant sequence of the rice OsABCB2 gene, said mutant sequence being obtained by mutation of the nucleotide sequence of the rice OsABCB2 gene; said mutation includes substitution, deletion and / or addition of one or more nucleotides in the rice OsABCB2 gene.
[0037] The nucleotide sequence of the rice OsABCB2 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the rice OsABCB2 gene is shown in SEQ ID NO: 2.
[0038] In this disclosure, the inventors have surprisingly discovered that the OsABCB2 gene (accession number: LOC_Os01g34970) can regulate cadmium accumulation in rice. This disclosure describes a method to reduce cadmium accumulation in rice by causing a loss of function in the OsABCB2 gene through gene mutation targeting its nucleotide sequence. The nucleotide sequence of the rice OsABCB2 gene is shown in SEQ ID NO: 1; the protein encoded by the rice OsABCB2 gene is shown in SEQ ID NO: 2.
[0039] In one specific embodiment of this disclosure, the mutant sequence is selected from at least one of the following sequences:
[0040] (1) The nucleotide sequence shown in SEQ ID NO:3 was obtained by inserting a base C between the 1100th and 1101st bases of the nucleotide sequence of the rice OsABCB2 gene;
[0041] (2) The nucleotide sequence shown in SEQ ID NO:4 was obtained by inserting base A between the 1100th and 1101st bases of the nucleotide sequence of the rice OsABCB2 gene.
[0042] In the above embodiments, the inventors of this disclosure designed and edited a vector based on the target site, and infected rice plants with Agrobacterium. Mutations were randomly performed at the target site within the rice plants. Then, mutation sequence detection was performed using designed primers, selecting mutant lines with C and A base insertions. The insertion of either C or A bases into the nucleotide sequence of the rice OsABCB2 gene caused corresponding changes in the codon sequence and composition after the insertion site, resulting in the premature appearance of the stop codon, producing a shorter peptide chain, and ultimately leading to the loss of protein structure and function. Furthermore, the inventors of this disclosure also discovered that homozygous mutant plants with OsABCB2 gene function defect mutations obtained through transgenics, and rice grains from mutant plants with C or A base insertions in the OsABCB2 gene nucleotide sequence, had significantly lower cadmium content than wild-type rice. This can be used to analyze the biological function of the OsABCB2 gene in rice and has potential application value in agricultural development.
[0043] 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: 5.
[0044] According to this disclosure, the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4 both encode the protein shown in SEQ ID NO:5.
[0045] 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 and produces a mutant sequence as described in the first aspect;
[0046] The target site nucleotide sequence is shown in SEQ ID NO: 6.
[0047] The fourth aspect of this disclosure provides a transformant, the host of which is a genetically engineered bacterium; the transformant contains a target site, or the transformant contains the vector described in the third aspect;
[0048] The target site nucleotide sequence is shown in SEQ ID NO: 6.
[0049] In this disclosure, the genetically engineered bacterium can be Agrobacterium EHA105.
[0050] The fifth aspect of this disclosure provides a method for reducing cadmium accumulation in rice plants, the method comprising:
[0051] Gene editing of the rice OsABCB2 gene mutated the nucleotide sequence of the rice OsABCB2 gene and altered the protein encoded by the OsABCB2 gene.
[0052] The mutations include substitutions, deletions, and / or additions of one or more nucleotides in the rice OsABCB2 gene.
[0053] In this disclosure, gene editing of the rice OsABCB2 gene alters the protein encoded by the OsABCB2 gene, thereby eliminating the activity of the protein encoded by the rice OsABCB2 gene. This is beneficial for cultivating rice varieties with reduced cadmium absorption and effectively solves the problem of excessive cadmium content in rice.
[0054] In one specific embodiment of this disclosure, the gene editing method includes:
[0055] S1. Design a CRISPR / Cas9 editing vector based on the target site of the rice OsABCB2 gene;
[0056] S2. The CRISPR / Cas9 editing vector was infected into rice plants by Agrobacterium tumefaciens. Mutations were randomly performed at the target sites in the rice plants. The results were then detected using the designed primers, and T0 generation positive plants with functional defect mutations were obtained by screening.
[0057] 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.
[0058] The nucleotide sequence of the target site is shown in SEQ ID NO: 6;
[0059] The sequences of the primers are shown in SEQ ID NO: 7 and SEQ ID NO: 8.
[0060] In this disclosure, mutant plants of the rice gene OsABCB2 were obtained using CRISPR-Cas9 technology, and it was verified that the OsABCB2 gene mutation can significantly reduce the cadmium content of rice grains.
[0061] In a preferred embodiment of this disclosure, in step S2, the mutation includes one of the following:
[0062] (a) Insert a base C between the 1100th and 1101st bases in the nucleotide sequence of the rice OsABCB2 gene;
[0063] (b) Insert a base A between the 1100th and 1101st bases in the nucleotide sequence of the rice OsABCB2 gene.
[0064] The sixth aspect of this disclosure provides the application of the rice OsABCB2 gene and / or the protein encoded by the rice OsABCB2 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 OsABCB2 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 OsABCB2 gene and altering the structure and function of the protein encoded by the OsABCB2 gene, the cadmium content in the rice grains of the resulting mutant plants was significantly reduced. This demonstrates that the mutation of the rice OsABCB2 gene can effectively reduce the cadmium content in rice grains. Developing rice varieties with reduced cadmium uptake using mutants of the rice OsABCB2 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 in the examples were performed under conventional conditions known to those skilled in the art.
[0069] Example 1
[0070] This example illustrates the OsABCB rice variety. 2 Obtaining mutants.
[0071] To study the function of the rice OsABCB2 gene, the nucleotide sequence of the rice OsABCB2 gene was edited at specific sites using CRISPR / Cas9 technology, as shown in SEQ ID NO.1.
[0072] Specific methods include:
[0073] 1. Construction of rice ABC transporter gene OsABCB2 mutant plants:
[0074] The target gene sequence was selected as GTGGAATTCAATTGTCCGGAGG. A plasmid with a specific target site of OsABCB2 was constructed. Then, using rice cultivar Nipponbare seeds as transformation material, T0 generation transgenic plants were obtained by Agrobacterium infection transformation.
[0075] Hydroponic identification of T0 generation plants, design of sequencing primers:
[0076] Forward primer mb2F: 5'-TCGCCTATGGGAAGGAGGAT-3' (SEQ ID NO: 7) and reverse primer mb2R: 5'-TCAACCATCTTTCCCTGCTGT-3' (SEQ ID NO: 8) were used to identify whether the OsABCB2 gene had mutated. Homozygous mutants were then selected and T1 generation seeds were harvested.
[0077] This embodiment identified two types of OsABCB2 gene mutations: osabcb2-1 and osabcb2-2 .like Figure 1 As shown, the black solid lines indicate the target sequence, and the red solid lines PAM indicate the adjacent motifs of the candidate recognition sites. osabcb2-1 The C base is inserted in the 5th exon (between positions 1100 and 1101 in the coding region, or between positions 16 and 17 in the target site sequence). osabcb2- 2 involves the insertion of base A in the 5th exon (between positions 1100 and 1101 in the coding region, or between positions 16 and 17 in the target site sequence). All mutations involve frameshift mutations, causing premature termination of the protein encoded by OsABCB2, resulting in a non-functional protein.
[0078] 2. Changes in cadmium content in OsABCB2 mutant grains:
[0079] First, the Nipponbare mutant was... osabcb2-1 and osabcb2-2 T1 generation homozygous seeds were germinated in the dark at 37℃ for 3 days and then sown in rice paddy areas. Twenty days later, uniformly growing seedlings were selected and transplanted into cadmium-contaminated rice paddies, with eight seedlings per row, a row spacing of 15cm, and a plant spacing of 15cm. Water, fertilizer, and pest and disease management throughout the growth period were the same as in ordinary paddy fields. At rice maturity, individual plants of Nipponbare and mutant varieties were harvested. osabcb2-1 and osabcb2-2 Seeds from the main panicle. Harvested homozygous T1 generation mutant families and wild-type Nipponbare mature seeds were first dried at 37℃ and weighed. Then, the husks were removed, and the rice grains were digested with nitric acid to obtain an ionomer solution. The cadmium content of the rice seeds was determined using ICP-OES. Results are as follows: Figure 2 As shown, "**" indicates the difference compared to the wild type. p <0.01; "*" indicates compared to the wild type, p <0.05. (By...) Figure 2 It can be seen that, compared with Nipponbare, the OsABCB2 mutant rice... osabcb2-1 and osabcb2-2 The cadmium content in the grains was significantly reduced, indicating that the OsABCB2 gene plays an important role in regulating cadmium accumulation in rice grains.
[0080] The results above demonstrate that mutating the rice OsABCB2 gene using gene editing methods can reduce cadmium content in grains. This provides insights for breeding rice varieties with low cadmium accumulation.
[0081] 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.
[0082] 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.
[0083] 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 for reducing cadmium accumulation in rice grains, characterized in that, The method includes: By editing the rice OsABCB2 gene, the nucleotide sequence of the rice OsABCB2 gene was mutated, and the protein encoded by the OsABCB2 gene lost its function. The mutations include the substitution, deletion, and / or addition of one or more nucleotides in the rice OsABCB2 gene; The gene editing method includes: S1. Design a CRISPR / Cas9 editing vector based on the target site of the rice OsABCB2 gene; S2. The CRISPR / Cas9 editing vector was infected into rice plants by Agrobacterium tumefaciens. Mutations were randomly performed at the target sites in the rice plants. The results were then detected using the designed primers, and T0 generation positive plants with functional defect mutations were obtained by screening. 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. The nucleotide sequence of the target site is shown in SEQ ID NO: 6; The sequences of the primers are shown in SEQ ID NO: 7 and SEQ ID NO: 8; The nucleotide sequence of the rice OsABCB2 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the rice OsABCB2 gene is shown in SEQ ID NO:
2.
2. The method according to claim 1, wherein, In step S2, the mutation includes one of the following: (a) Insert a base C between the 1100th and 1101st bases in the nucleotide sequence of the rice OsABCB2 gene; (b) Insert a base A between the 1100th and 1101st bases in the nucleotide sequence of the rice OsABCB2 gene.
3. The application of knocking out the rice OsABCB2 gene in breeding rice varieties with reduced cadmium uptake in grains, among which, The nucleotide sequence of the rice OsABCB2 gene is shown in SEQ ID NO: 1.