Mutant gene osnramp5 of mutant yb58 inhibiting cadmium absorption of rice, recombinant vector, expression cassette, cell containing the gene and application thereof

Abstract

The application belongs to the field of crop genetic breeding and biotechnology, and provides a mutant gene of a mutant YB58 for inhibiting cadmium absorption of rice OsNRAMP5 and a recombinant vector, an expression cassette, a cell containing the gene and application of the mutant YB58 mutant gene OsNRAMP5 has a nucleotide sequence as shown in SEQ ID NO. 2, wherein the mutant belongs to OsNRAMP5 a loss-of-function mutant of OsYB58, which has weaker cadmium ion transport capacity and has the function of significantly inhibiting cadmium absorption.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of crop genetic breeding and biotechnology, and relates to a mutant gene of a mutant YB58 for inhibiting cadmium absorption of rice OsNRAMP5 and a recombinant vector, an expression cassette, a cell containing the gene and application thereof. BACKGROUND

[0002] Cadmium (Cd) is a heavy metal element with high toxicity, which widely exists in industrial wastewater, mine tailings and cadmium-containing fertilizers. With the acceleration of global industrialization, the problem of cadmium pollution in farmland is becoming increasingly serious. According to the statistics of the Food and Agriculture Organization (FAO) of the United Nations, about 10% of the global arable land is contaminated by heavy metals to varying degrees, and cadmium pollution is particularly prominent. Rice, as the main food crop for more than half of the world's population, is prone to absorb and accumulate cadmium in the soil due to its special physiological characteristics (such as developed root system and flooding environment promoting cadmium activation). Studies have shown that the enrichment coefficient of cadmium in rice grains is 2-10 times that of wheat and corn, resulting in rice becoming the main way for humans to intake cadmium. Long-term consumption of cadmium-exceeding rice can cause serious health problems such as "itching disease", kidney damage, osteoporosis and cancer. Therefore, cultivating cadmium-low-accumulation rice varieties is the most economical and effective way to solve the problem of "cadmium rice".

[0003] At present, the technologies for treating cadmium pollution in rice mainly include soil remediation, agronomic regulation and genetic improvement: soil remediation technology is to adjust the soil pH by adding lime, organic matter or passivation agents (such as zeolite, biochar) to reduce the biological availability of cadmium. However, this method is costly, its effect is restricted by soil type and climate conditions, and it is difficult to achieve economic and large-scale application in lightly contaminated areas. The agronomic regulation technology is to reduce cadmium absorption by using water management (such as dry-wet alternate irrigation) or intercropping with remediation plants (such as pteris multifida). However, agronomic measures are highly dependent on planting patterns, and may affect rice yield and quality. Genetic improvement technology is to screen or create cadmium-low-accumulation rice varieties, which is considered as the most economical and sustainable solution. Studies have shown that the absorption, transport and accumulation of cadmium in rice are regulated by multiple genes, and there are significant differences among varieties. By improving key genes through molecular marker-assisted breeding or gene editing technology, the cadmium content in grains can be reduced. In recent years, through quantitative trait locus (QTL) positioning and functional genomics research, scholars at home and abroad have identified a number of key genes that regulate cadmium accumulation in rice. For example, OsNRAMP5Natural Resistance-Associated Macrophage Protein 5 (Genbank, Accession No. AP004176.2, 20080216, see 29957-37219) encodes natural resistance-associated macrophage protein, which is the main transporter of cadmium and manganese absorption in rice roots. Japanese scholar Ishikawa et al. found that the presence of natural resistance-associated macrophage protein can enable rice to normally perform cadmium absorption function, and the average value of cadmium absorption is 1.73 mg / kg. The cadmium content of brown rice of three low-cadmium mutants obtained by ion beam mutagenesis is significantly reduced. In addition, the function-enhanced allele of OsHMA3 Heavy Metal ATPase 3 can isolate cadmium in the root cell vacuole and inhibit its loading into the xylem. Chinese scholars found that the function-enhanced allele of OsHMA3 can significantly reduce the accumulation of cadmium in grains. Other genes such as OsLCD Low Cadmium, OsIRT1 Iron-Regulated Transporter 1, etc. also affect the cadmium content in grains by regulating the absorption or distribution of cadmium. Although the above research has made some progress, there are still some bottlenecks in the existing technology. Traditional hybrid breeding combined with phenotype screening is time-consuming and laborious, and the low-cadmium trait is easily disturbed by environmental factors and lacks stability. In view of the urgent need for cadmium pollution control in rice, combined with functional genomics and molecular breeding technology, the development of new germplasm with low-cadmium characteristics and agronomic advantages is the key direction of current research.

[0004] In recent years, domestic and foreign scholars have made significant progress in the innovation of low-cadmium germplasm in rice through mutagenesis breeding technology. For example, CN115961075A discloses a OsNRAMP5 gene mutant with 12 base deletions at position 8878413-8878424 on chromosome 7; CN108794608A finds a point mutant Lcd1 that causes a mutation in codon 236. These mutants all show significant reduction in cadmium accumulation, providing valuable genetic resources for low-cadmium rice breeding.

[0005] However, the existing disclosed technology still has the following limitations: (1) Most mutants are based on conventional varieties, and lack of targeted improvement of sterile lines, which is the core material of hybrid rice breeding; (2) The combining ability of mutants and restorer lines and their performance in hybrid combinations lack systematic research; (3) The specific markers suitable for molecular marker assisted selection are insufficient. In view of these problems, the present application creates a new low-cadmium sterile line mutant YB58 by radiation mutagenesis technology, and systematically studies its application value. For the first time, a stable low-cadmium mutant is created in the sterile line, a functional molecular marker closely linked to the mutation site is developed, and the application value of the mutant in hybrid breeding is systematically evaluated. These achievements provide a new idea for improving the cadmium accumulation characteristics of sterile lines by mutagenesis breeding technology, and have important significance for realizing the safe production of hybrid rice in cadmium-polluted rice fields. SUMMARY

[0006] The technical problem to be solved by the present application is a mutant gene of a mutant YB58 for inhibiting cadmium absorption of rice OsNRAMP5 has a nucleotide sequence as shown in SEQ ID NO. 2, wherein the base at 1039 nucleotides downstream of ATG is mutated from G of the wild type to C, resulting in that the 347th codon GGA of the coding region is mutated to CGA, and the cadmium absorption capacity of the mutant YB58 is significantly lower than that of the wild type rice Y58S.

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

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

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

[0010] The application of the expression cassette, the recombinant vector or the cell containing the mutant gene OsNRAMP5 in the function of inhibiting cadmium absorption of rice.

[0011] 1. The present application first proves that the unit point mutation of the amino acid can significantly inhibit the cadmium absorption capacity of rice compared with the existing low-cadmium rice varieties.

[0012] 2. The present application provides a mutant gene of a rice mutant YB58 OsNRAMP5 , and a method for breeding low-cadmium rice varieties using the gene, which also provides a new gene resource for breeding low-cadmium rice varieties, and is beneficial to the breeding of low-cadmium rice varieties, so as to ensure 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] Put the sealed 96-well plate into a water bath which is adjusted to 65℃ for 1-1.5 hours, during which the plate is taken out several times and mixed well on a vortex shaker.

[0027] After the end of the warm bath, take out the sealed 96-well plate and put it into a refrigerated centrifuge, adjust the speed to 4000 rpm and the temperature to 4℃, and centrifuge for 10 minutes.

[0028] Take out the centrifuged 96-well plate and transfer 400ul of the supernatant to a 2ml purified 96-well plate using a semi-automatic 96-well pipettor.

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

[0030] Place the washing solution plate at the 2-3 plate position of the extraction, and place the dissolved DNA plate containing 150ul of eluent at the 4 plate position.

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

[0032] Example 2

[0033] (1) Quality control: use Qubit fluorometric quantifier to detect the concentration of DNA sample; use 1% agarose gel electrophoresis to detect the integrity of DNA sample (see Figure 1 ), and use the qualified sample for library preparation. The DNA obtained in step (1) is subjected to WGS whole genome sequencing library construction, and the library construction is carried out according to the library construction process (see Figure 2 ). Including the following.

[0034] (2) DNA fragmentation, end repair and quality control: use fragmentation enzyme to cut and fragment the DNA sample, repair the cut ends, and add A base to the 3' end. Use 2% agarose gel electrophoresis to detect the effect of DNA fragmentation, and the sample with obvious bright band at 300-500bp is used for subsequent reaction.

[0035] (3) Adapter ligation and quality control: ligate sequencing adapters to fragmented DNA, and purify the ligation product using magnetic beads. The purified product is detected by Qubit fluorometric quantifier to detect the concentration of ligation product, and the qualified sample is used for subsequent reaction.

[0036] (4) Fragment amplification, selection and quality control: use PCR to amplify the ligation product, and use magnetic beads to select the PCR product. The product after fragment selection is detected by Qubit fluorometric quantifier to detect the library concentration, 2% agarose gel electrophoresis is used to detect the fragment size, and Qsep400 biological analyzer is used to verify the library fragment size.

[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] The M4 generation seeds are sowed according to the strains, and the cadmium contaminated farmland is selected as the screening environment, and the M4 generation plants are planted by using the random block design, and 30 plants are planted for each strain. After the rice matures, the single plant is harvested, and the cadmium content in the brown rice is analyzed by using the inductively coupled plasma mass spectrometer (ICP-MS). The cadmium content in the brown rice of the wild type Y58S in the B test point is 1.183 mg / kg, which is 6 times of the national limited standard 0.2 mg / kg; and the cadmium content in the brown rice of the mutant YB58 is 0.0419 mg / kg (Table 1), which indicates that the mutant YB58 has the characteristics of low cadmium absorption.

[0049] Table 1. Cadmium content analysis of the mutant YB58 and the original parent Y58S brown rice

[0050] Traits YB58 Y58S Lotianliangyou 1 Region A (1.20 mg / kg, pH 5.60) 0.0583 1.7310 0.0586 Region B (0.86 mg / kg, pH 5.48) 0.0419 1.1830 0.0437

[0051] The above is only the preferred embodiment of the present application, and does not limit the present application in any form. Although the present application has been disclosed as above, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical content without departing from the scope of the present application, and any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application are still within the scope of the present application.

[0052] Mutant gene of the mutant YB58 OsNRAMP5 Full-length genome sequence (SEQ ID NO: 1, including intron and exon):

[0053]

[0054] Mutant gene of mutant YB58 OsNRAMP5 The cDNA sequence (SEQ ID NO: 2) of the mutant YB58 is:

[0055]

[0056] Mutant gene of mutant YB58 OsNRAMP5 Encoded amino acid sequence (SEQ ID NO: 3):

[0057] MEIERESSERGSISWRASAAHDQDAKKLDADDQLLMKEPAWKRFLAHVGPGFMVSLAYLDPGNLETDLQAGANHRYELLWVILIGLIFALIIQSLAANLGVVTGRHLAEICKSEYPKFVKIFLWLLAELAVIAADIPEVIGTAFAFNILFHIPVWVGVLITGTSTLLLLGLQKYGVRKLEFLISMLVFVMAACFFGELSIVKPPAKEVMKGLFIPRLNGDGATADAIALLGALVMPHNLFLHSALVLSRKTPASVRGIKDGCRFFLYESGFALFVALLINIAVVSVSGTACSSANLSQEDADKCANLSLDTSSFLLKNVLGKSSAIVYGVALLASGQSSTITGTYARQYIMQGFLDIRMRKWLRNLMTRTIAIAPSLIVSIIGGSRGAGRLIIIASMILSFELPFALIPLLKFSSSKSKMGPHKNSIYIIVFSWFLGLLIIGINMYFLSTSFVGWLIHNDLPKYANVLVGAAVFPFMLVYIVAVVYLTIRKDSVVTFVADSSLAAVVDAEKADAGDLAVDDDEPLPYRDDLADIPLPR.

Claims

1. A mutant gene of a rice mutant YB58, characterized in that, OsNRAMP5 The nucleotide sequence of the gene is shown as SEQ ID NO.

2. ​ 2. The mutant gene of the rice mutant YB58 according to claim 1 OsNRAMP5 The encoded protein is characterized in that, The amino acid sequence is shown as SEQ ID NO.

3.

3. The mutant gene of the rice mutant YB58 according to any one of claims 1-2 OsNRAMP5 or the application of the encoded protein thereof in inhibiting the cadmium absorption function of rice.

4. An expression cassette, recombinant vector or host cell, characterized in that, Mutant gene containing the mutant YB58 of claim 1 OsNRAMP5 .

5. Use of the expression cassette, the recombinant vector or the host cell of claim 4 in inhibiting cadmium absorption function of rice.

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