Mutant gene OsNRAMP5 of mutant YB58 for inhibiting cadmium absorption of rice, recombinant vector containing gene, expression cassette, cell and application of mutant gene OsNRAMP5

Abstract

The invention belongs to the technical field of crop genetic breeding and biology, and provides a mutant gene OsNRAMP5 of a mutant YB58 for inhibiting cadmium absorption of rice, a recombinant vector containing the gene, an expression cassette, a cell and application of the gene OsNRAMP5, the mutant gene OsNRAMP5 of the mutant YB58 has a nucleotide sequence shown in SEQ ID NO.2, the mutant belongs to a function deletion mutant of OsNRAMP5, and the gene OsNRAMP5 has a nucleotide sequence shown in SEQ ID NO.1. The compound has weaker cadmium ion transport capacity and has a function of remarkably inhibiting cadmium absorption.

Description

Technical Field

[0001] This invention belongs to the field of crop genetics and breeding and biotechnology, and relates to a mutant gene of YB58 that inhibits cadmium absorption in rice. OsNRAMP5 And recombinant vectors, expression cassettes, cells containing this gene and their applications. Background Technology

[0002] Cadmium (Cd) is a highly toxic heavy metal element widely found in industrial wastewater, mine tailings, and cadmium-containing fertilizers. With the acceleration of global industrialization, cadmium pollution in farmland is becoming increasingly serious. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), approximately 10% of the world's arable land is contaminated with heavy metals to varying degrees, with cadmium pollution being particularly prominent. Rice, as the staple crop for more than half of the world's population, readily absorbs and accumulates cadmium in the soil due to its unique physiological characteristics (such as a well-developed root system and the fact that flooding promotes cadmium activation). Studies have shown that the cadmium accumulation coefficient of rice grains is 2-10 times that of wheat and corn, making rice a major route of cadmium intake for humans. Long-term consumption of rice with excessive cadmium levels can lead to serious health problems such as Itai-itai disease, kidney damage, osteoporosis, and cancer. Therefore, cultivating low-cadmium-accumulation rice varieties is the most economical and effective way to solve the "cadmium rice" problem.

[0003] Currently, technologies for controlling cadmium pollution in rice mainly fall into three categories: soil remediation, agronomic regulation, and genetic improvement. Soil remediation involves adjusting soil pH by adding lime, organic matter, or passivating agents (such as zeolite and biochar) to reduce the bioavailability of cadmium. However, these methods are costly, their effectiveness is limited by soil type and climate conditions, and they are difficult to implement economically feasible large-scale applications in lightly polluted areas. Agronomic regulation techniques use water management (such as alternating wet and dry irrigation) or intercropping with remediation plants (such as centipede grass) to reduce cadmium absorption. However, agronomic measures are highly dependent on planting patterns and may affect rice yield and quality. Genetic improvement techniques involve screening or creating rice varieties with low cadmium absorption; this method is considered the most economical and sustainable solution. Studies have shown that the absorption, translocation, and accumulation of cadmium in rice are regulated by multiple genes, with significant differences between varieties. Improving key genes through molecular marker-assisted breeding or gene editing technologies can target and reduce grain cadmium content. In recent years, domestic and international scholars have identified several key genes regulating cadmium accumulation in rice through quantitative trait locus (QTL) mapping and functional genomics studies. For example, OsNRAMP5The gene encoding Natural Resistance-Associated Macrophage Protein 5 (Genbank, accession number: AP004176.2, 20080216, see positions 29957-37219) encodes a natural resistance-associated macrophage protein that is the main transporter for cadmium and manganese uptake in rice roots. Japanese researchers Ishikawa et al. found that its presence enables rice to perform normal cadmium uptake, with an average uptake of 1.73 mg·kg⁻¹. However, the brown rice cadmium content of three low-cadmium mutants obtained through ion beam mutagenesis was significantly reduced. Furthermore, vacuolar membrane localization... OsHMA3 HeavyMetal ATPase 3 can isolate cadmium in root cell vacuoles, inhibiting its loading into the xylem. Chinese scholars have discovered... OsHMA3 The functionally enhanced alleles can significantly reduce cadmium accumulation in grains. Other genes such as OsLCD (Low Cadmium) OsIRT1 Iron-Regulated Transporter 1 and others have also influenced grain cadmium content by regulating cadmium absorption or distribution. Despite these advancements, existing technologies still face limitations. Traditional hybridization breeding combined with phenotypic screening is time-consuming and labor-intensive, and the low-cadmium trait is easily affected by environmental factors, resulting in insufficient stability. Addressing the urgent need for cadmium pollution control in rice, developing new germplasm that combines low-cadmium characteristics with agronomic advantages, using functional genomics and molecular breeding techniques, is a key research focus.

[0004] In recent years, scholars both domestically and internationally have made significant progress in the innovation of rice germplasm with low cadmium accumulation through mutation breeding technology. For example, CN115961075A discloses a 12-base deletion at positions 8878413-8878424 on chromosome 7. OsNRAMP5 Gene mutants; CN108794608A discovered a point mutant, Lcd1, which causes a mutation at codon 236. These mutants all exhibited significant reductions in cadmium accumulation, providing valuable genetic resources for low-cadmium rice breeding.

[0005] However, existing publicly available technologies still have the following limitations: (1) Most mutants are created based on conventional varieties, lacking targeted improvement of the male-sterile line, a core material for hybrid rice breeding; (2) The combining ability of mutants and restorer lines and their performance in hybrid combinations lack systematic research; (3) There is insufficient development of specific markers suitable for marker-assisted selection. To address these issues, this invention created a novel low-cadmium male-sterile mutant, YB58, using radiation mutagenesis technology and systematically studied its application value. For the first time, a stably inherited low-cadmium mutant was created in a male-sterile line. Functional molecular markers closely linked to the mutation site were developed, and the application value of the mutant in hybrid breeding was systematically evaluated. These achievements provide new ideas for improving the cadmium accumulation characteristics of male-sterile lines using mutagenesis breeding technology, and are of great significance for achieving safe production of hybrid rice in cadmium-contaminated paddy fields. Summary of the Invention

[0006] The technical problem this application aims to solve is: a mutant gene of the YB58 mutant that inhibits cadmium absorption in rice. OsNRAMP5 Having the nucleotide sequence shown in SEQ ID NO.2, in which the base 1039 downstream of ATG is mutated from G to C in the wild type, resulting in the GGA to CGA mutation in the 347th codon of the coding region, the mutant YB58 has a significantly lower cadmium uptake capacity than the wild type rice Y58S.

[0007] The mutant gene of the rice mutant YB58 OsNRAMP5 The gene is characterized by having a nucleotide sequence as shown in SEQ ID NO.2.

[0008] The mutant gene of the rice mutant YB58 OsNRAMP5 The gene is characterized in that the amino acid sequence encoded by the gene is shown in SEQ ID NO.3.

[0009] An expression cassette, recombinant vector, or cell, characterized in that it contains the mutant gene of the YB58 mutant. OsNRAMP5 .

[0010] The one containing the mutated gene OsNRAMP5 The application of expression cassettes, recombinant vectors, or cells in inhibiting cadmium absorption in rice.

[0011] 1. This invention is the first to demonstrate that a single point mutation of this amino acid can significantly inhibit the cadmium absorption capacity of rice compared with existing low-cadmium rice varieties.

[0012] 2. This invention provides a mutant gene for rice mutant YB58. OsNRAMP5 The gene and the method of using it to breed low-cadmium rice varieties also provide new genetic resources for breeding low-cadmium rice germplasm, which is conducive to the breeding of low-cadmium rice varieties and thus ensures the safety of rice production. Attached Figure Description

[0013] Figure 1 : Mixed-pool DNA quality control electrophoresis image of the M2 generation of mutants.

[0014] Figure 2 Flowchart for constructing resequencing libraries.

[0015] Figure 3 : In the cadmium-low accumulation mutant YB58 OsNRAMP5 Types of gene mutations.

[0016] Figure 4 Plant phenotype of the cadmium-low accumulation mutant YB58. Detailed Implementation

[0017] The present invention will now be further described with reference to embodiments, but it is not limited to any one of these embodiments or similar examples.

[0018] Example 1

[0019] In this implementation case, wild-type rice was found in the NCBI database based on the agronomic traits of the breeding target. OsNRAMP5 Gene.

[0020] Healthy seeds of the main rice variety Y58S were selected as mutagenesis materials, and physical mutagenesis was performed using 60Co-γ rays. The seeds were evenly spread in an irradiation disk, and the irradiation dose was set to 300 Gy (absorbed dose rate 1.0-2.0 Gy / min). M1 generation seeds were obtained after irradiation.

[0021] M1 generation seeds are soaked in water for 12 hours, then germinated at a constant temperature of 25℃ until they show white sprouts. They are then sown in the field and harvested as single ears at maturity, with each single plant yielding one ear of seeds, thus obtaining M2 generation seeds.

[0022] M2 generation seeds were soaked, germinated, and sown in 200-well seedling trays. When the seedlings reached the 3-leaf stage, mixed pools were constructed by sampling at 96 seedlings / pool, and genomic DNA was extracted from the mixed pools, including the following: 20-30 mg was placed in a 96-well plate and freeze-dried.

[0023] Two 4mm steel balls were added to the 96-hole plate of the vacuum-evacuated blade using a bead separator, and then covered with a matching silicone film.

[0024] Place the 96-well plate in a high-throughput tissue grinder, adjust the speed to 1400 rpm and grind for 3 minutes. The grinding time can be increased until the leaflets are crushed.

[0025] Take out the 96-well plate and add 600 μL of CTAB extraction solution to each well using a pipette. After sealing with heat-sealing film, place it on a vortex mixer and shake appropriately to mix.

[0026] Place the sealed 96-well plate in a water bath at 65°C for 1-1.5 hours, removing it several times during this period and placing it on a vortex mixer to mix it properly.

[0027] After the warm bath, remove the sealed 96-well plate and place it in a refrigerated centrifuge. Set the speed to 4000 rpm and the temperature to 4°C, and centrifuge for 10 minutes.

[0028] Remove the centrifuged 96-well plate and use a semi-automatic 96-well pipette to transfer 400 μL of the supernatant to 2 mL of purified 96-well plate.

[0029] Add an equal volume of magnetic bead mixing solution to a 2 ml purification 96-well plate containing 400 μL, and place it into the ME480 extraction instrument.

[0030] Place the washing buffer plate in positions 2 and 3 after extraction, and place the DNA-dissolving plate containing 150 μL of elution buffer in position 4.

[0031] Run the ME-480 extraction program, which takes about 30 minutes to complete. Cover the DNA plate with a membrane for storage, and the extraction process is complete.

[0032] Example 2

[0033] (1) Quality control: The concentration of DNA samples was detected using a Qubit fluorescence quantitative quantitation instrument; the integrity of DNA samples was detected using 1% agarose gel electrophoresis. Figure 1 Samples that pass quality control are used for library preparation. The DNA obtained in step (1) is subjected to WGS whole genome sequencing for library construction, following the library construction process ( Figure 2 The library is constructed, including the following.

[0034] (2) DNA fragmentation, end repair and quality control: DNA samples were fragmented using a fragmentation enzyme, the enzyme ends were repaired, and an A base was added to the 3' end. The effect of DNA fragmentation was detected by 2% agarose gel electrophoresis. Samples with a clear bright band at 300-500bp were used for subsequent reactions.

[0035] (3) Adapter ligation and quality control: Sequencing adapters were ligated to fragmented DNA, and the ligation products were purified using magnetic beads. The concentration of the purified products was detected using a Qubit fluorescence quantitative PCR instrument, and samples with qualified concentrations were used for subsequent reactions.

[0036] (4) Fragment amplification, selection and quality control: The ligation products were amplified by PCR, and the PCR products were screened for fragments using magnetic beads. The library concentration of the selected products was detected by Qubit quantitative PCR instrument, the fragment size was detected by 2% agarose gel electrophoresis, and the library fragment size was verified by Qsep400 bioanalyzer.

[0037] (5) Circularization and quality control: After denaturing the linear library into single strands, circularization was performed. Uncircularized linear DNA molecules were digested to obtain single-stranded circular libraries. The concentration of the single-stranded circular libraries was detected using a Qubit fluorescence quantitative quantitation system. If the concentration was within acceptable limits, subsequent reactions were carried out.

[0038] (6) DNB preparation and quality control: Single-stranded circular DNA molecules are replicated through rolling circle replication to form a DNA nanosphere (DNB) containing more than 300 copies. The concentration of DNB is detected using a Qubit fluorescence quantitative quantification instrument. If the concentration is qualified, subsequent reactions can proceed.

[0039] (7) Sequencing: DNB is loaded into the sequencing chip using the MGIDL-T7 loading device and sequenced using the combined probe anchoring polymerization technology.

[0040] Bioinformatics and data analysis were performed on the obtained DNA library, including the following.

[0041] a. Data preprocessing and quality control: This includes filtering the raw sequencing data, detecting sequencing data contamination, and assessing the quality of sequencing data.

[0042] b. Use FASTP (Chen et al. 2018) to perform quality control on the offline data.

[0043] c. Use Sentieon (Kendig et al. 2019) to compare and detect mutations in the offline data to obtain the mutation results for each individual in the population.

[0044] Analysis revealed a mixed pool in Os NRAMP Consistent mutations were present in the coding regions of all 5 genes. Figure 3 The ATG start codon at position +1039 undergoes a mutation from G to C (G→C), resulting in a mutation from glycine (Gly) to arginine (Arg) at codon position 347 in the coding region. Further Sanger sequencing of each individual strain in the pool was used to verify the homozygosity of this site in individual strains.

[0045] Example 3

[0046] The M2 homozygous mutant single plant ( Figure 4 Transplanted to the experimental field, cultivated under conventional fertilizer and water management, and self-pollinated with cold water to obtain M3 seeds.

[0047] All M3 generation seeds were sown and cultivated using conventional fertilizer and water management. M3 plants were then self-pollinated and seeded, and M4 seeds were harvested per plant.

[0048] M4 generation seeds were sown in cadmium-contaminated farmland as screening environments, and M4 generation plants were planted using a randomized block design, with 30 plants per line. After the rice matured, individual plants were harvested, and the cadmium content of brown rice was analyzed using inductively coupled plasma mass spectrometry (ICP-MS). The cadmium content in the grains of wild-type Y58S at test site B was 1.183 mg / kg, exceeding the national limit of 0.2 mg / kg by 6 times; while the cadmium content in the grains of mutant YB58 was 0.0419 mg / kg (Table 1), indicating that mutant YB58 has the characteristic of low cadmium absorption.

[0049] Table 1. Analysis of cadmium content in brown rice of mutant YB58 and original parent Y58S Properties YB58 Y58S Lianliangyou No.1 Zone A (1.20 mg / kg, pH 5.60) 0.0583 1.7310 0.0586 Zone B (0.86 mg / kg, pH 5.48) 0.0419 1.1830 0.0437

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

[0051] The mutant gene of YB58 OsNRAMP5 Full-length genome sequence (SEQ ID NO:1, containing introns and exons):

[0052] The mutant gene of YB58 OsNRAMP5 cDNA sequence (SEQ ID NO:2):

[0053] Mutant gene of mutant YB58 OsNRAMP5 The encoded amino acid sequence (SEQ ID NO:3): MEIERESSERGSISWRASAAHDQDAKKLDADDQLLMKEPAWKRFLAHVGPGFMVSLAYLDPGNLETDLQAGANHRYELLWVILIGLIFALIIQSLAANLGVVTGRHLAEICKSEYPKFVKIFLWLLAELAVIAADIPEVIGTAFAFNILFHIPVWVGVLITGTSTLLLLGLQKYGVRKLEFLISMLVFVMAACFFGELSIVKPPAKEVMKGLFIPRLNGDGATADAIALLGALVMPHNLFLHSALVLSRKTPASVRGIKDGCRFFLYESGFALFVALLINIAVVSVSGTACSSANLSQEDADKCANLSLDTSSFLLKNVLGKSSAIVYGVALLASGQSSTITGTYARQYIMQGFLDIRMRKWLRNLMTRTIAIAPSLIVSIIGGSRGAGRLIIIASMILSFELPFALIPLLKFSSSKSKMGPHKNSIYIIVFSWFLGLLIIGINMYFLSTSFVGWLIHNDLPKYANVLVGAAVFPFMLVYIVAVVYLTIRKDSVVTFVADSSLAAVVDAEKADAGDLAVDDDEPLPYRDDLADIPLPR。

Claims

1. A mutant gene of rice mutant YB58 OsNRAMP5 Its characteristics are, The nucleotide sequence of this gene is shown in SEQ ID NO.

2.

2. The mutant gene of rice mutant YB58 as described in claim 1 OsNRAMP5 Its characteristics are, The amino acid sequence encoded by this gene is shown in SEQ ID NO.

3.

3. The mutant gene of rice mutant YB58 as described in any one of claims 1-2 OsNRAMP5 Application in inhibiting cadmium absorption in rice.

4. An expression cassette, recombinant vector, or cell, characterized in that, The mutant gene containing the mutant YB58 as described in claim 1 or 2 OsNRAMP5 .

5. The application of the expression cassette, recombinant vector, or cell described in claim 4 in inhibiting cadmium absorption in rice.

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