Mutant gene OsNRAMP5 for reducing cadmium content in rice grains and application of mutant gene OsNRAMP5

Abstract

The invention discloses a mutant gene OsNRAMP5 capable of reducing cadmium content in rice grains and application of the mutant gene OsNRAMP5. The gene has a genome nucleotide sequence as shown in SEQ ID No.1, and the 8875149th nucleotide on the No.7 chromosome of the wild type of fragrant rice, namely the 10th exon splicing site of the LOCOs07g15370 (OsNRAMP5) gene, is mutated from A to T, so that the gene is subjected to variable splicing in the transcription process. The invention also discloses a protein coded by the nucleotide sequence and used for regulating and controlling low accumulation of cadmium in fragrant rice grains, and the protein has an amino acid sequence as shown in SEQ ID No.3. The OsNRAMP5 gene for regulating and controlling the accumulation of the harmful heavy metal element cadmium is cloned in the fragrant rice by utilizing a forward genetics technology, and the mutant gene not only can improve the content of fragrant substances in the fragrant rice, but also can greatly reduce the content of cadmium in the rice, so that the quality safety of the fragrant rice is ensured.

Description

Technical Field

[0001] The present invention belongs to the field of crop genetic breeding. Specifically, the present invention relates to a method for utilizing mutmap technology to clone and regulate a mutant gene for low accumulation of heavy metal element cadmium in aromatic rice. OsNRAMP5 and its encoded proteins and applications. Background Art

[0002] Cadmium (Cd) is listed as a primary pollutant affecting agricultural land safety in my country due to its strong migration capacity and high biotoxicity. Rice is a plant with a strong ability to absorb cadmium. Low concentrations of soil cadmium contamination can lead to reduced rice quality, while high concentrations can reduce rice yields. Selecting and breeding low-cadmium, high-quality varieties is a long-term goal for rice breeders. High-quality aromatic rice is widely favored by consumers and farmers for its rich aroma and high economic and nutritional value. Recent studies have shown that 2-acetyl-1-pyrroline (2-AP) is an effective flavor compound that plays a key role in producing the distinctive aromatic flavor and taste, thereby improving the quality characteristics of aromatic rice varieties (Mo et al., 2019). Cadmium contamination in rice fields not only reduces aromatic rice yields but also affects enzyme activity and precursor content in the 2-AP biosynthesis pathway (Imran et al., 2023), reducing the aroma of rice grains. Therefore, studying the molecular mechanism of fragrant rice's response to heavy metal cadmium stress is not only beneficial to alleviating the toxic effects of cadmium and increasing the yield of fragrant rice, but is also of great significance for ensuring the quality traits and quality safety of fragrant rice grains.

[0003] Studies have shown that the absorption, transport and distribution characteristics of rice for cadmium involve transmembrane transport of ions, chelation and transfer of cadmium. absorption, accumulation and distribution of cadmium in rice. So far, several genes related to cadmium absorption and accumulation in rice have been cloned, and the molecular genetic mechanism of cadmium accumulation in rice has been preliminarily elucidated. The absorption of cadmium by rice roots is mainly mediated by the symplasmic pathway of manganese transporter OsNRAMP5 into the root cells (Sasaki et al., 2012). Mutation of this gene can greatly reduce the absorption of cadmium by rice roots, and in turn reduce the cadmium content in rice grains. In addition, OsNRAMP1, heavy metal transporting ATPase (HMA) family proteins and zinc-iron transporting proteins (Zinc-regulated transporters and iron-regulated transporter like protein, ZIP) family play an important role in the absorption, compartmentalization and xylem loading of cadmium in roots (Chang et al., 2020; Tan et al., 2020; Takahashi et al., 2012). Cadmium mainly enters the grain through the phloem, and the stem node is the key site for the transfer of cadmium from xylem to phloem (Fujimaki et al., 2010). The plant cation / calcium super family (cation / calcium super family, CaCA) OsCCX2 protein expressed in the xylem region of the stem node vascular tissue of the stem node participates in the accumulation of cadmium in rice grains through the calcium transport pathway in the stem node (Hao et al., 2018). Uraguchi et al. identified a gene encoding a low-affinity cation transporter (low-affinity cation transporter, LCT) that mediates the transport of cadmium from the phloem of the stem node to the grain, regulating the cadmium content in the grain. The rice defensin protein OsCAL1 promotes the secretion of cadmium to the extracellular space by chelating cadmium in the cytoplasm, thereby reducing the cytoplasmic cadmium concentration, and at the same time promoting the long-distance transport of cadmium through the xylem vessels (Luo et al., 2018; Zhao et al., 2018). However, the current research on the physiological and molecular biological mechanisms of cadmium absorption and accumulation characteristics mainly focuses on indica rice and japonica rice, and the molecular mechanism of response to heavy metal cadmium stress and accumulation in high-quality rice such as fragrant rice with special quality traits is less studied. OsLCT1 encoding a low-affinity cation transporter (low-affinity cation transporter, LCT) that mediates the transport of cadmium from the phloem of the stem node to the grain, regulating the cadmium content in the grain. The rice defensin protein OsCAL1 promotes the secretion of cadmium to the extracellular space by chelating cadmium in the cytoplasm, thereby reducing the cytoplasmic cadmium concentration, and at the same time promoting the long-distance transport of cadmium through the xylem vessels (Luo et al., 2018; Zhao et al., 2018). However, the current research on the physiological and molecular biological mechanisms of cadmium absorption and accumulation characteristics mainly focuses on indica rice and japonica rice, and the molecular mechanism of response to heavy metal cadmium stress and accumulation in high-quality rice such as fragrant rice with special quality traits is less studied. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a mutant gene for reducing the cadmium content in rice grains OsNRAMP5and application. The application studies the low-cadmium accumulation characteristics of rice, and screens rice germplasm resources with high quality and low risk of cadmium at the same time on the basis of ensuring the quality of flavor.

[0005] To achieve the object of the present application, the present application provides OsNRAMP5 In the application of regulating the accumulation of cadmium in the grains of aromatic rice, the mutant lcf1 The mutant gene OsNRAMP5 A base at the 10th exon splicing site (8875149th nucleotide on chromosome 7) is mutated from A to T, causing variable splicing during transcription of the gene. The mutant lcf1 Two transcripts are generated, one of which contains a 283bp intron, causing premature termination of translation, resulting in a deletion of the amino acid sequence of the OsNRAMP5 protein and damage to the function. The mutant lcf1 The content of cadmium in the grains of aromatic rice is reduced by 90%, and the content of the key flavor substance 2-AP is higher than that of wild-type rice.

[0006] The coding amino acid sequence of the mutant gene lcf1 is shown in SEQ ID No. 3.

[0007] The application of the mutant gene OsNRAMP5 makes the rice contain the mutant gene OsNRAMP5 ; or the amino acid sequence encoded by the mutant gene OsNRAMP5 .

[0008] The application includes one or more of transgenic, hybridization, backcrossing or vegetative reproduction.

[0009] An expression cassette or a recombinant vector containing the mutant gene OsNRAMP5 . Advantages of the present application:

[0010] The grains of the mutant material OsNRAMP5 obtained by the present application are aromatic rice varieties, and the content of heavy metal cadmium in the rough rice of the mutant material is reduced by about 90% compared with the wild type, and the content of the key flavor substance is higher than that of the wild type. Therefore, the mutant lcf1 The gene mutation mode of the mutant lcf1 is beneficial to efficient cadmium reduction, ensuring rice quality safety and excellent quality. BRIEF DESCRIPTION OF DRAWINGS

[0011] OsNRAMP5 is the 2-AP quantitative chromatogram in the mutant ( Figure 1 ) and wild-type (WT) aromatic rice.

[0012] lcf1 is the 2-AP quantitative chromatogram in the mutantFigure 2 Comparison of the contents of five heavy metal elements in roots, stems and leaves of aromatic rice ( ) and wild type ( WT ).

[0013] lcf1 yes Figure 3 Gene location analysis. DETAILED DESCRIPTION

[0014] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0015] The process of obtaining a low-cadmium accumulation mutant in aromatic rice Approximately 450 g of wild-type seeds (M0) of long-grain aromatic rice R8 (a breeding material derived from Xiangyi Xiangzhan) were soaked in 1.0% ethyl methanesulfonate (EMS) at room temperature for 8 hours with stirring. The EMS solution was discarded and the seeds were rinsed with water for 4 hours. The mutagenized seeds were soaked, germinated, and sown, and routinely fertilized and watered. At maturity, 2–3 rice seeds were harvested from each plant and mixed to form M1 seeds.

[0016] In 2021, M1 seeds were soaked, germinated, and sown in paddy fields with a total cadmium content of 1.7 mg / kg and a pH of 5.6. Conventional fertilization and watering were maintained throughout the growing season. Upon maturity, M1 seeds were harvested individually, yielding a total of 2,008 plants (M2). The harvested rice grains were partially dehulled, and the contents of five heavy metals, including cadmium, in the brown rice were determined using inductively coupled plasma-mass spectrometry (ICP-MS). By measuring the heavy metal contents of the 2,008 individual plant grains, a rice line, R8-460, with low cadmium accumulation, was identified. The cadmium content in its brown rice was 0.035 mg / kg, which is lower than the cadmium limit of 0.2 mg / kg specified in the GB2762-2022 "Limits of Contaminants in Foods" standard. The cadmium content in the brown rice of the corresponding wild-type long-grain aromatic rice is 0.483 mg / kg.

[0017] In order to obtain a stably inherited fragrant rice material with low cadmium accumulation, the seeds of the obtained cadmium-accumulating materials were soaked and germinated in 2022, and then sown in different plots, and managed with conventional fertilizers and water. After maturity, the individual plants were harvested, and after the harvested rice grains were shelled, the content of heavy metal elements in the brown rice was determined by ICP-MS. The results showed that the cadmium content in the brown rice of the R8-460 mutant fragrant rice selected and planted in Fuyang and Chang'an, Hangzhou, ranged from 0.008 to 0.009 mg / kg and 0.088 to 0.109 mg / kg, respectively, while the cadmium content in the brown rice of the wild-type fragrant rice was 0.077 to 0.129 mg / kg and 4.215 to 6.81 mg / kg, respectively. The cadmium content in the mutant brown rice was significantly lower than that in the wild long-grain fragrant rice. The low cadmium accumulation mutant strain was further self-selected and planted in two cadmium-contaminated rice fields in Fuyang and Chang'an, Zhejiang, for verification. A multi-generationally stably inherited fragrant rice mutant with low cadmium accumulation was obtained, named Oslcf1 ( l lcf1 c ow f admium ).

[0018] Long-grain fragrant rice ingredients ragrant rice 1 Phenotypic analysis Harvested fragrant rice mutants grown in ordinary rice fields lcf1 The protein content and amylose content of the rice were higher than those of the wild type, and there were no significant differences in other rice qualities such as milling quality, appearance quality, cooking quality, and nutritional quality between the rice and the wild type (see Table 1).

[0019]

[0020] Wild type and mutant varieties of fragrant rice harvested from the field lcf1 The seeds of the rice were ground into brown rice and cooked into rice to extract the aromatic substance 2-AP. The aromatic substance content in the two materials was determined by gas chromatography-time of flight mass spectrometry. The results are as follows lcf1 As shown, the mutant Figure 1 The peak area of ​​the quantitative ion chromatogram of the aroma substances in the mutant was significantly larger than that of the wild type, indicating that lcf1 The content of 2-AP aroma compounds in aromatic rice is significantly higher than that in wild type rice. Cadmium pollution may lead to poor root development in rice, affecting nutrient absorption and transport, and thus affecting the supply of precursors for the synthesis of aroma compounds, indirectly affecting the formation of aroma compounds.

[0021] The mutant and wild type were planted in the rice fields of Fuyang, Hangzhou for two consecutive years, and the heavy metal content in the brown rice was measured after harvest. lcf1The cadmium content in brown rice was significantly lower than that in the wild type, approximately 90% lower, while the contents of other heavy metals were not significantly different from those in the wild type. Detailed results are shown in Table 2.

[0022]

[0023] Collecting fragrant rice mutants grown in the field lcf1 and wild-type seedlings, and the heavy metal contents in roots, stems and leaves were determined. lcf1 The contents of chromium, nickel, arsenic and lead in the roots, stems and leaves of the cultivar were not significantly different from those of the wild type, and the cadmium content was 85-98% lower than that of the wild type (see lcf1 ).

[0024] Aromatic rice mutant Figure 2 Identification of key genes for low cadmium accumulation Long-grain aromatic rice mutant lcf1 The material was hybridized with the wild type, and the cadmium content in the brown rice of all parents and F1 populations was determined to be 0.094-0.0114 mg / kg, which was close to the content of Nipponbare, and all showed normal cadmium accumulation characteristics. In the winter of that year, the F1 generation was self-pollinated in Hainan to obtain the F2 generation population. About 700 F2 generation seeds were randomly selected, soaked and germinated, and then sown in a hydroponic nutrient pool sieve floating on 0.1 μΜ CdCl2. After one month, the seedlings were harvested individually, and the cadmium content in the roots and leaves was measured. The results showed that the normal accumulation of cadmium in the roots of the 654 surviving F2 segregation population seedlings and the low accumulation of cadmium in the individual plants were a typical 3:1 (c 2 =2.24 <c 2 0.05=3.84) separation, indicating that rice lcf1 The specific traits of the mutant are controlled by a recessive single gene.

[0025] Further, 33 plants with low cadmium accumulation and 22 plants with high cadmium accumulation were selected from the F2 segregating population, leaf DNA was extracted, and a mixed pool of extremely low cadmium accumulation and extremely high cadmium accumulation DNA was constructed. The mixed pool and the two parents were subjected to whole genome sequencing.

[0026] DNA extraction was performed by CTAB method as follows: select rice seedling leaves, cut into 2 mL centrifuge tubes with steel balls. Put the centrifuge tube into liquid nitrogen for 30 s, then put it into the plant tissue crusher, shake for 60 s, and crush into powder. Add 750 μL CTAB extraction solution to the centrifuge tube. Put the sample into a constant temperature water bath at 65 ℃ for 30 min, and invert the centrifuge tube every 10 min to mix the sample thoroughly. Add 500 μL chloroform to the centrifuge tube, shake vigorously, and stand at room temperature for 5 min. Put the centrifuge tube into the centrifuge at room temperature at 12000 rpm for 10 min. Gently pipette 500 μL supernatant into a new 1.5 mL centrifuge tube, add 500 μL pre-cooled anhydrous ethanol, invert gently 3-5 times, and place in a -20 ℃ refrigerator for more than 30 min. Put the centrifuge tube in the centrifuge at 12000 rpm for 10 min. Gently pour off the supernatant, and then invert the centrifuge tube on the water absorption paper to dry. Add 200 μL ultrapure water to the centrifuge tube, and place it at room temperature until the precipitate is completely dissolved. Store it in a -20 ℃ refrigerator. Use a pipette to take 2 μL of the sample, and use NanoDrop 2000c (Thermo, USA) to detect the mass and concentration of the DNA.

[0027] After the DNA sample was detected to be qualified, the Covaris ultrasonic crusher was used to randomly break the DNA of the two parents and the two offspring extreme phenotype mixed pools, and then end repair, A tailing, sequencing adapter addition, fragment screening, PCR amplification, and purification steps were performed to complete the whole library preparation work. The final DNA library was obtained by PE150 high-throughput sequencing.

[0028] The sequencing produced a total of Raw data 36.301G, and after filtering, Clean data 36.046G was obtained. The Raw data of each sample was between 5,151.864 M and 15,449.023 M, with high sequencing quality (Q20 >= 96.11%, Q30 >= 90.26%), and GC content between 43.16% and 44.81%. In summary, the data volume of all samples was sufficient, the sequencing quality was qualified, the GC distribution was normal, and the library sequencing was successful.

[0029] Valid sequencing data were aligned to the reference genome using BWA software (parameters: mem -t 4 -k 32 -M). Alignment results were deduplicated using SAMTOOLS (parameters: rmdup). The reference genome is 374,471,240 bp in size. Alignment rates for all samples ranged from 95.75% to 97.2%. Average coverage depth to the reference genome (excluding the N region) ranged from 12.02X to 32.74X, with 1X coverage (coverage of at least one base) exceeding 89.87%. Alignment results were normal and suitable for subsequent variant detection and related analyses.

[0030] GATK was used to detect individual SNPs, and then a population analysis was performed based on the gvcf file. 2,712,086 candidate polymorphic marker sites were obtained between the two parents. According to the distribution of SNP-index on chromosomes in the offspring, it can be clearly seen that chromosome 7 is the chromosome where the candidate gene is located (see lcf1 (a) of the .

[0031] For candidate polymorphic marker sites, extract the annotation results of ANNOVAR and screen out candidate genes Figure 3 The candidate gene encodes the natural resistance-related macrophage protein OsNRAMP5, which is located on the plasma membrane of rice roots and is involved in Mn 2+ 、Cd 2+ and Fe 2+ The main transporter of Os07g0257200 and wild type lcf1 The DNA sequence of the candidate gene was verified and analyzed, and it was found that the nucleotide 8875149 on chromosome 7 mutated from A to T ( OsNRAMP5 (b)). This nucleotide is located at Figure 3 The splicing site of the tenth exon. Mutations at this site may cause variable splicing and affect the transcript of the gene. LOC_Os07g15370 and wild-type CDS, and separated by SDS-PAGE electrophoresis and sequenced, and the mutant was found lcf1 Contains two transcripts ( lcf1 (c) of the transcript 2, where transcript 2 contains a 283 bp intron ( Figure 3 Snapgene software was used to analyze the mutants. Figure 3 lcf1 Coding sequence analysis revealed a termination codon, TAA, at 982 nucleotides downstream of ATG, leading to premature translation termination, a deletion of the OsNRAMP5 amino acid sequence, and disruption of protein structure and function (as shown in SEQ ID No. 3).

[0032] The embodiments in the above description can be further combined or replaced, and the embodiments are merely used to describe the preferred embodiments of the present application, and are not intended to limit the concept and scope of the present application. Without departing from the design idea of the present application, various changes and improvements made by those skilled in the art to the technical solutions of the present application shall fall within the protection scope of the present application. The protection scope of the present application is given by the appended claims and any equivalent technical solutions thereof.

Claims

1. A mutant gene that reduces cadmium content in rice grains OsNRAMP5 , characterized in that, mutant lcf1 Mutated genes OsNRAMP5 The nucleotide sequence is shown in SEQ ID No.1, and the encoding nucleotide sequence is shown in SEQ ID No.

2. lcf1 The cadmium content in the grains of rice was significantly lower than that in the wild-type rice grains, while the key aroma substance 2-AP was higher than that in the wild-type rice.

2. The mutant gene involved in regulating the accumulation of heavy metal element cadmium in rice according to claim 1 OsNRAMP5 , characterized in that, The mutant gene OsNRAMP5 The amino acid sequence is shown in SEQ ID No.

3.

3. A mutant gene according to claim 1 OsNRAMP51 The application is characterized in that Making rice contain the mutant gene as claimed in claim 1 OsNRAMP5 ; Or make rice express the mutant gene as claimed in claim 2 OsNRAMP5 The amino acid sequence of the rice was modified, resulting in a significantly lower cadmium content than that in wild-type rice grains, while the key aroma substances were higher than those in wild-type rice.

4. The use according to claim 3, characterized in that The application methods include one or more of transgenic, hybridization, backcrossing or asexual reproduction.

5. An expression cassette or recombinant vector, characterized in that: Containing the mutant gene as claimed in claim 1 OsNRAMP5 .

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