Mutant sequence of rice osabcb8 gene and method and application thereof in regulating cadmium accumulation in rice
By editing the rice OsABCB8 gene and designing a CRISPR/Cas9 vector for nucleotide mutation, the problem of excessive cadmium accumulation in rice was solved, resulting in a significant reduction in cadmium content and ensuring food security and human health.
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
- 2026-04-28
AI Technical Summary
Existing technologies have shown that excessive cadmium accumulation in rice leads to reduced grain yields and poses health risks to humans. Furthermore, existing genetic modification methods may affect rice growth and development.
By editing the rice OsABCB8 gene, a CRISPR/Cas9 editing vector was designed, inserted into the target site, and nucleotide mutations were performed, resulting in the loss of function of the OsABCB8 gene, thereby reducing cadmium accumulation.
Effectively reduce the cadmium content in rice, cultivate rice varieties with reduced cadmium absorption, and safeguard food security and human health.
Smart Images
Figure CN118879726B_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 OsABCB8 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] Cadmium (Cd) is a toxic heavy metal. Although its background levels in the environment are low, its long half-life makes it difficult to decompose and it is easily absorbed by plants. This leads to symptoms such as yellowing leaves, stunted growth, disordered growth, and reduced yield in plants. Furthermore, Cd absorbed by plants can accumulate in the human body through the food chain, causing serious damage to internal organs and the immune system, leading to diseases such as osteoporosis, kidney stones, and pancreatic cancer.
[0003] Among major cereal crops, rice has a strong ability to adsorb the heavy metal cadmium (Cd). Severe cadmium stress not only leads to reduced grain yields but also accumulates in the human body through the food chain, posing a carcinogenic risk. Therefore, breeding low-Cd-accumulation rice varieties is an economical and effective way to safely utilize Cd-contaminated farmland and solve the "Cd rice" problem. Currently identified rice Cd transport proteins are often also major transport proteins for other metal ions. Genetic modification of these proteins to reduce Cd content in rice usually results in impaired mineral element homeostasis within the mutants, thus adversely affecting rice growth and development. Therefore, improving the accumulation characteristics of rice for the heavy metal cadmium and cultivating low-cadmium-accumulation rice varieties still requires more genetic resources and breeding methods.
[0004] In recent years, research has revealed that the ABC (ATP-binding cassette) transporter family in plants plays a crucial role in the absorption and transport of the heavy metal cadmium (Cd). Members of the ABC transporter family are widely distributed in organisms, participating in the transport of sugars, auxins, and metal ions, and playing a significant role in regulating plant responses to external environmental factors and resisting abiotic stresses. In Arabidopsis thaliana, AtABCC1, AtABCC2, and AtABCC3 chelate and isolate Cd into vacuoles through thiol complexes formed with ligands such as GSH or PC, thereby reducing Cd toxicity to plant cells. Researchers have also found that OsABCB36 in rice can influence Cd tolerance by regulating Cd excretion from roots. However, few ABC transporter family genes related to Cd transport have been identified in rice. Therefore, identifying target genes that can reduce cadmium content in rice can provide genetic resources for low-cadmium rice breeding and has 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 is a mutant sequence of the rice OsABCB8 gene, which is obtained by mutating the nucleotide sequence of the rice OsABCB8 gene; the mutation includes substitution, deletion and / or addition of one or more nucleotides in the rice OsABCB8 gene.
[0007] The rice OsABCB8 gene has the nucleotide sequence described in (I) or (II):
[0008] (I) The nucleotide sequence shown in SEQ ID NO: 1;
[0009] (II) A nucleotide sequence that has more than 80% homology with the nucleotide sequence shown in SEQ ID NO: 1 at a non-mutation site and encodes a protein with the same function.
[0010] Optionally, the mutant sequence is selected from at least one of the following sequences:
[0011] (1) The nucleotide sequence obtained by deleting the GTCTGG fragment from position 6 to position 11 of the target site I of the rice OsABCB8 gene, as shown in SEQ ID NO: 6.
[0012] (2) The nucleotide sequence shown in SEQ ID NO: 7 was obtained by inserting a base G between the 11th and 12th bases of the target site I of the rice OsABCB8 gene.
[0013] (3) The nucleotide sequence shown in SEQ ID NO: 8 was obtained by inserting a base T between the 11th and 12th bases of the target site I sequence of the rice OsABCB8 gene;
[0014] The nucleotide sequence of target site I is shown in SEQ ID NO: 3.
[0015] 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: 9, SEQ ID NO: 10 and / or SEQ ID NO: 11.
[0016] The third aspect of this disclosure is a vector, which is a CRISPR / Cas9 editing vector, wherein the vector is inserted with target site I and produces a mutant sequence as described in the first aspect;
[0017] The nucleotide sequence of target site I is shown in SEQ ID NO: 3.
[0018] 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;
[0019] The nucleotide sequence of target site I is shown in SEQ ID NO: 3.
[0020] The fifth aspect of this disclosure is a method for reducing the cadmium content in rice, the method comprising: gene editing of the rice OsABCB8 gene to mutate the nucleotide sequence of the rice OsABCB8 gene and to change the protein encoded by the OsABCB8 gene.
[0021] The mutations include substitutions, deletions, and / or additions of one or more nucleotides in the rice OsABCB8 gene.
[0022] Optionally, the gene editing method includes:
[0023] S1. Design a CRISPR / Cas9 editing vector based on target site I of the rice OsABCB8 gene;
[0024] 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.
[0025] 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.
[0026] The nucleotide sequence of target site I is shown in SEQ ID NO: 3;
[0027] The sequences of the primers are shown in SEQ ID NO:4 and SEQ ID NO:5.
[0028] Optionally, in step S1, the mutation includes one of the following:
[0029] (a) Deletion of the GTCTGG segment from position 6 to position 11 of the target site I of the rice OsABCB8 gene;
[0030] (b) Insert a base G between the 11th and 12th bases of the target site I sequence of the rice OsABCB8 gene;
[0031] (c) Insert a base T between the 11th and 12th bases of the target site I sequence of the rice OsABCB8 gene.
[0032] The sixth aspect of this disclosure provides the application of the rice OsABCB8 gene and / or the protein encoded by the rice OsABCB8 gene in regulating cadmium accumulation in rice and / or breeding rice varieties with reduced cadmium uptake.
[0033] The seventh aspect of this disclosure provides the application of mutant sequences of the rice OsABCB8 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.
[0034] Through the above-described technical solution, this disclosure achieves the loss of function of the OsABCB8 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 address the problem of excessive cadmium content in rice, which is of great significance for solving food security issues. It also provides technical support for further in-depth research on the molecular mechanisms of cadmium absorption, translocation, and accumulation in rice.
[0035] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description
[0036] 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:
[0037] Figure 1 This refers to the OsABCB8 gene structure and mutant type in the examples.
[0038] Figure 2 Gene-edited rice osabcb8 1 , osabcb8 2 and osabcb8-3 Cadmium content test results for mutants and wild-type (WT) cadmium. Detailed Implementation
[0039] 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.
[0040] The first aspect of this disclosure is a mutant sequence of the rice OsABCB8 gene, which is obtained by mutation of the nucleotide sequence of the rice OsABCB8 gene; the mutation includes substitution, deletion and / or addition of one or more nucleotides in the rice OsABCB8 gene.
[0041] The rice OsABCF1 gene has the nucleotide sequence described in (I) or (II):
[0042] (I) The nucleotide sequence shown in SEQ ID NO: 1;
[0043] (II) A nucleotide sequence that has more than 80% homology with the nucleotide sequence shown in SEQ ID NO: 1 at a non-mutation site and encodes a protein with the same function.
[0044] The amino acid sequence of the protein encoded by the rice OsABCB8 gene is shown in SEQ ID NO: 2.
[0045] In this disclosure, the inventors have made a surprising discovery that the OsABCB8 gene (accession number: LOC_Os01g74470) can regulate the accumulation of cadmium in rice. This disclosure discloses that by mutating the nucleotide sequence of the rice OsABCB8 gene to cause the gene to lose its function, the accumulation of cadmium in rice can be effectively reduced, and rice varieties with reduced cadmium content can be cultivated.
[0046] According to this disclosure, sequences having more than 80% homology with the nucleotide sequence shown in SEQ ID NO: 1 at a non-mutation site include sequences having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the nucleotide sequence shown in SEQ ID NO: 1 at a non-mutation site, wherein the aforementioned homologous sequences can encode proteins with the same function as the nucleotide sequence shown in SEQ ID NO: 1.
[0047] In one specific embodiment of this disclosure, the mutant sequence is selected from at least one of the following sequences:
[0048] (1) The nucleotide sequence obtained by deleting the GTCTGG fragment from position 6 to position 11 of the target site I of the rice OsABCB8 gene, as shown in SEQ ID NO: 6.
[0049] (2) The nucleotide sequence shown in SEQ ID NO: 7 was obtained by inserting a base G between the 11th and 12th bases of the target site I of the rice OsABCB8 gene.
[0050] (3) The nucleotide sequence shown in SEQ ID NO: 8 was obtained by inserting a base T between the 11th and 12th bases of the target site I sequence of the rice OsABCB8 gene;
[0051] The nucleotide sequence of target site I is shown in SEQ ID NO: 3.
[0052] 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, based on designed primers, the mutant sequences were detected, selecting mutant lines with deletions of the GTCTGG fragment or insertions of G or T bases. The deletion of the GTCTGG fragment or the insertion of G or T bases into the nucleotide sequence of the rice OsABCB8 gene caused corresponding changes in the codon sequence and composition after the insertion site, resulting in the premature appearance of the stop codon and premature termination of the encoded protein. The structure and function of the protein encoded by the mutant sequence were altered. Furthermore, the inventors of this disclosure also discovered that homozygous mutant plants with functional defect mutations in the OsABCB8 gene obtained through transgenics, and those with deletions of the GTCTGG fragment or insertions of G or T bases in the OsABCB8 gene nucleotide sequence, had significantly lower cadmium content in their rice grains than the wild-type cadmium content. This can be used to analyze the biological function of the OsABCB8 gene in rice and has potential application value in agricultural development.
[0053] 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: 9, SEQ ID NO: 10 and / or SEQ ID NO: 11.
[0054] According to this disclosure, the nucleotide sequence shown in SEQ ID NO: 6 encodes the protein shown in SEQ ID NO: 9; the nucleotide sequence shown in SEQ ID NO: 7 encodes the protein shown in SEQ ID NO: 10; and the nucleotide sequence shown in SEQ ID NO: 8 encodes the protein shown in SEQ ID NO: 11.
[0055] The third aspect of this disclosure is a vector, which is a CRISPR / Cas9 editing vector, wherein the vector is inserted with target site I and produces a mutant sequence as described in the first aspect;
[0056] The nucleotide sequence of target site I is shown in SEQ ID NO: 3.
[0057] 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;
[0058] The nucleotide sequence of target site I is shown in SEQ ID NO: 3.
[0059] In this disclosure, the genetically engineered bacterium can be Agrobacterium EHA105.
[0060] The fifth aspect of this disclosure is a method for reducing the cadmium content in rice, the method comprising: gene editing of the rice OsABCB8 gene to mutate the nucleotide sequence of the rice OsABCB8 gene and to change the protein encoded by the OsABCB8 gene.
[0061] The mutations include substitutions, deletions, and / or additions of one or more nucleotides in the rice OsABCB8 gene.
[0062] In this disclosure, gene editing of the rice OsABCB8 gene alters the protein encoded by the OsABCB8 gene, thereby reducing or eliminating the expression level of the OsABCB8 gene and decreasing the activity of the protein encoded by the rice OsABCB8 gene. This is beneficial for cultivating rice varieties with reduced cadmium absorption and effectively solves the problem of excessive cadmium content in rice.
[0063] In one specific embodiment of this disclosure, the gene editing method includes:
[0064] S1. Design a CRISPR / Cas9 editing vector based on target site I of the rice OsABCB8 gene;
[0065] 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.
[0066] 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.
[0067] The nucleotide sequence of target site I is shown in SEQ ID NO: 3;
[0068] The sequences of the primers are shown in SEQ ID NO:4 and SEQ ID NO:5.
[0069] In the above embodiments, CRISPR-Cas9 technology was used to construct mutant plants, and three different types of mutant plants were screened for subsequent experiments. After planting the mutant plants in soil contaminated with the heavy metal cadmium, the Cd content of individual plant grains was measured. The results showed that the cadmium content in the mutant plant grains was significantly lower than that in the wild type. This indicates that OsABCB8 participates in regulating the absorption and translocation of Cd in rice. The mutant strain disclosed in this paper can significantly reduce the heavy metal cadmium content in rice grains, contributing to food security and human health.
[0070] In a preferred embodiment of this disclosure, in step S1, the mutation includes one of the following:
[0071] (a) Deletion of the GTCTGG segment from position 6 to position 11 of the target site I of the rice OsABCB8 gene;
[0072] (b) Insert a base G between the 11th and 12th bases of the target site I sequence of the rice OsABCB8 gene;
[0073] (c) Insert a base T between the 11th and 12th bases of the target site I sequence of the rice OsABCB8 gene.
[0074] The sixth aspect of this disclosure provides the application of the rice OsABCB8 gene and / or the protein encoded by the rice OsABCB8 gene in regulating cadmium accumulation in rice and / or breeding rice varieties with reduced cadmium uptake.
[0075] The seventh aspect of this disclosure provides the application of mutant sequences of the rice OsABCB8 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.
[0076] In this disclosure, OsABCB8 participates in the transport of the heavy metal cadmium in rice, increasing the rice's sensitivity to cadmium. Improving OsABCB8 can optimize the cadmium accumulation characteristics of rice and reduce the amount of cadmium accumulated in rice grains. Breeding rice varieties with reduced cadmium absorption using mutants of the rice OsABCB8 gene has potential application value in agriculture.
[0077] The present disclosure is further described in detail below through examples.
[0078] 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.
[0079] Example 1
[0080] 1. Construction of OsABCB8 mutant plants.
[0081] The OsABCB8 gene has a coding region of 4194 bp. CRISPR-Cas9 genome knockout system was used to create mutant plants of the OsABCB8 gene. First, CRISPR target sites (sequence shown in SEQ ID NO: 3) were designed based on the coding region sequence, and the pCRISPR-OsABCB8 plasmid with OsABCB8-specific target was constructed. Then, mutant plants were constructed by infecting wild-type Nipponbare rice callus with Agrobacterium strain. Finally, homozygous mutant plants were screened by PCR, and specific primers were designed:
[0082] Forward primer F: 5'-ATTGGGAACCATGAGTGTGCT-3' (SEQ ID NO: 4); Reverse primer R: 5'-GGCCTCTAGTGATGTGCGG-3' (SEQ ID NO: 5).
[0083] After PCR amplification and sequencing of the designed target sites, three independent homozygous mutant plants were selected. osabcb8-1 SEQ ID NO: 6; osabcb8-2 SEQ ID NO: 7 and osabcb8-3 (SEQ ID NO: 8) The following experiments were performed. Target sites and mutation types for wild-type and mutant strains are as follows: Figure 1 As shown.
[0084] 2. Analysis of Cd content in OsABCB8 mutant plants.
[0085] Wild-type WT and osabcb8-1 , osabcb8-2 and OsABCB8-3 The mutants were transplanted to an experimental field in Beishan Town, Changsha County, Hunan Province. The soil there was slightly acidic (pH 5.4-6.2) with a Cd content of 1.8 mg / kg. Field planting was conducted using a randomized block design with 3-5 replicates. Except for intermittent irrigation, all other field management practices were standard. Rice grains were harvested from individual plants and then treated using ICP-MS. MS was used to determine the Cd element content. The results are as follows: Figure 2 As shown, "*" indicates a comparison with the wild type. p <0.05; "**" indicates compared to the wild type, p <0.01. osabcb8 1 , osabcb8 2 and osabcb8 3 The Cd content in the seeds of the mutant plants was significantly lower than that of the wild type, indicating that... osabcb8 1 , osabcb8 2 and osabcb8 3 The mutation reduced the accumulation of Cd in rice grains.
[0086] The above examples show that OsABCB8 participates in the transport of the heavy metal cadmium in rice, increasing the rice's sensitivity to cadmium. Improving OsABCB8 can optimize the cadmium accumulation characteristics of rice and reduce the amount of heavy metal cadmium accumulated in rice.
[0087] 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.
[0088] 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.
[0089] 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 content in rice, characterized in that, The method includes: modifying the nucleotide sequence of the rice OsABCB8 gene by gene editing, thereby altering the protein encoded by the OsABCB8 gene. The mutations include the substitution, deletion and / or addition of one or more nucleotides in the rice OsABCB8 gene; The gene editing method includes: S1. Design a CRISPR / Cas9 editing vector based on target site I of the rice OsABCB8 gene; 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. 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 target site I is shown in SEQ ID NO: 3; The sequences of the primers are shown in SEQ ID NO: 4 and SEQ ID NO: 5; The nucleotide sequence of the rice OsABCB8 gene is shown in SEQ ID NO:
1.
2. The method according to claim 1, wherein, The mutation includes one of the following: (a) Deletion of the GTCTGG segment from position 6 to position 11 of the target site I of the rice OsABCB8 gene; (b) Insert a base G between the 11th and 12th bases of the target site I sequence of the rice OsABCB8 gene; (c) Insert a base T between the 11th and 12th bases of the target site I sequence of the rice OsABCB8 gene.
3. The application of the method according to any one of claims 1-2 in reducing cadmium accumulation in rice and / or cultivating rice varieties with reduced cadmium uptake.