Rice osnramp5 gene mutant and application thereof

Abstract

The application relates to the field of crop genetic breeding technology, and particularly discloses a rice OsNRAMP5 gene mutant and application thereof. The rice OsNRAMP5 gene mutant takes a wild-type rice OsNRAMP5 gene coding sequence as a reference sequence, and the mutant contains mutations of a G base at the 804th position being mutated into A, bases at the 806th-952nd positions being deleted, and / or a T base at the 984th position being mutated into C. The application provides a new rice two-line sterile line cadmium low-accumulation mutant, enriches low-cadmium-accumulation-related genes and related mutants, and provides a new method for creating a low-cadmium-accumulation rice two-line sterile line germplasm.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of crop genetic breeding, in particular to a rice OsNRAMP5 gene mutant and application thereof. BACKGROUND

[0002] Cadmium (Cd) is a toxic heavy metal, which can enter the human body through the food chain and have toxic effects on the human immune, urinary, nervous and other systems. The half-life of cadmium is as long as 10-30 years, which can accumulate in the human body for a long time, leading to bone damage, kidney disease, hematuria and other diseases, and further inducing cancer and teratogenicity, seriously threatening human health. With human activities such as mining, smelting, chemical industry, industrial production, sewage irrigation, fertilizer application, etc., the Cd content of some farmland has increased significantly. Rice is easy to absorb cadmium elements from the soil during growth, resulting in 80% of rice cadmium content exceeding the standard in high cadmium pollution areas, which brings great hidden dangers.

[0003] In recent years, the research on the main control genes of cadmium and other heavy metals in rice has made a breakthrough. OsNRAMP5 is the most important gene involved in the absorption of cadmium in rice roots so far. The mutation of this gene blocks the absorption of cadmium in roots, so that the accumulation of Cd in OsNRAMP5 mutants is significantly reduced even in high cadmium pollution paddy fields, which is a key target gene for low cadmium rice breeding. OsNRAMP5 is mainly expressed in rice roots, including root epidermis, cortex and parenchyma cells near xylem, as well as low expression in husks, leaves and leaf sheaths, etc., suggesting that OsNRAMP5 not only participates in the absorption of cadmium, but also participates in the migration of cadmium in rice tissues. In addition, OsNRAMP5 also participates in the absorption of manganese ions, which is a key element for rice growth and disease resistance, so the OsNRAMP5 mutant often leads to yield reduction while reducing cadmium ions. Therefore, it is urgent to screen and apply mutant materials that can reduce cadmium ions without affecting yield.

[0004] Although there are many OsNRAMP5 mutants that can reduce the accumulation of cadmium in rice, there are few mutants that can be applied to rice two-line sterile lines and do not lead to yield reduction, so it is still necessary to develop new mutants that can reduce cadmium accumulation and do not affect yield, especially mutants based on rice two-line sterile lines. SUMMARY

[0005] One of the purposes of the present application is to provide a new rice two-line sterile line cadmium low-accumulation mutant and its application.

[0006] The application provides a rice OsNRAMP5 gene mutant, which takes a wild type rice OsNRAMP5 gene coding sequence (NCBI-ID: 4342859, SEQ ID NO. 3) as a reference sequence, and the mutant contains a mutation of G base at position 804 to A, deletion of bases at positions 806-952, and / or a mutation of T base at position 984 to C.

[0007] The creation of a rice two-line sterile line cadmium low-accumulation germplasm not only needs to consider the characteristics of low cadmium accumulation, but also needs to consider that the sterile starting temperature, flowering habit and yield characteristics of the two-line sterile line are not affected during the mutagenesis and screening process, which further increases the difficulty of creation. In view of the deficiencies of the existing rice two-line sterile line cadmium low-accumulation germplasm, the application provides a new rice two-line sterile line cadmium low-accumulation mutant, which is introduced into wild type rice by means known in the art, and after the introduction, the yield is not affected while the cadmium accumulation in the rice brown rice is reduced to improve the safety of the brown rice.

[0008] The gene mutant of the application can only contain the mutation of G base at position 804 to A and the deletion of bases at positions 806-952; can only contain the mutation of T base at position 984 to C; or can contain the mutation of G base at position 804 to A, the deletion of bases at positions 806-952 and the mutation of T base at position 984 to C.

[0009] Preferably, the gene mutant of the application has a nucleotide sequence as shown in SEQ ID NO. 1 (containing three mutations).

[0010] The application also provides a protein encoded by the above-mentioned gene mutant.

[0011] The protein of the application takes a wild type rice OsNRAMP5 amino acid sequence (SEQ ID NO. 4) as a reference sequence, and the protein contains a mutation of deletion of amino acids at positions 269-317.

[0012] Preferably, the protein of the application has an amino acid sequence as shown in SEQ ID NO. 2.

[0013] When the gene mutant of the application contains the mutation of G base at position 804 to A and the deletion of bases at positions 806-952, or contains the mutation of G base at position 804 to A, the deletion of bases at positions 806-952 and the mutation of T base at position 984 to C, the encoded protein sequence is as shown in SEQ ID NO. 2.

[0014] When the gene mutant of the application contains only the mutation of T base at position 984 to C, the encoded protein sequence is the same as the wild type sequence (SEQ ID NO. 4).

[0015] The application also provides a biological material comprising the above-mentioned gene mutant, wherein the biological material is an expression cassette, a vector or a host cell.

[0016] The above-mentioned expression cassette can be a DNA fragment obtained by connecting the gene mutant with an element for regulating transcription or expression thereof.

[0017] The above-mentioned vector can be a cloning vector or an expression vector.

[0018] The above-mentioned host cell can be a microbial cell or a non-propagating plant cell.

[0019] The application also provides application of the above-mentioned gene mutant or protein or biological material in regulating cadmium accumulation in rice or improving rice germplasm resources.

[0020] The application also provides application of the above-mentioned gene mutant or protein or biological material in preparing transgenic rice.

[0021] The application also provides a method for reducing cadmium accumulation in rice, which produces the above-mentioned gene mutant.

[0022] In the method of the application, the above-mentioned gene mutant is produced in rice by gene editing, hybridization, backcrossing, selfing or vegetative propagation.

[0023] The application has at least the following beneficial effects:

[0024] The application provides a new rice two-line sterile line cadmium low-accumulation mutant, which enriches genes related to low cadmium accumulation and related mutants, and provides a new method for creating a low-cadmium-accumulation rice two-line sterile line germplasm. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a comparison diagram of plants and panicles of a rice two-line sterile line cadmium low-accumulation mutant (L184S) and wild type 18S-184.

[0026] Figure 2 is a comparison of effects of OsNRAMP5 (wild type), OsNRAMP5-M1 (mutation at position 804 and deletion at positions 806-952), OsNRAMP5-M2 (mutation at position 984) and OsNRAMP5-M3 (mutation at position 804, deletion at positions 806-952 and mutation at position 984) on growth of a yeast strain Δycf1. DETAILED DESCRIPTION

[0027] The preferred embodiments of the present application will be described in detail below with examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications and substitutions to the present application without departing from the spirit and principles of the present application.

[0028] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0029] The present application first mutagenized the intermediate material 18S-184 seeds (a derivative of Long S, available from China Seed Group Co., Ltd.) of a rice dual-purpose cytoplasmic male sterile line (M0 generation) with EMS, and planted the treated seeds to obtain M1 plants. The M1 plants were selfed to produce seeds (M2 generation), and the M2 plants were planted in cadmium-contaminated fields. The harvested rice grains were dehulled, and the cadmium content in the brown rice was determined by inductively coupled plasma spectrometry to screen for low-cadmium-accumulating plants. The obtained low-cadmium-accumulating rice lines were further selfed and screened, and were planted in five cadmium-contaminated rice fields in Hunan and Jiangxi for verification. Finally, a mutant homozygous single plant with extremely low cadmium accumulation in rice and stable heredity was obtained, named L184S, and was used for hybrid breeding and biotechnological research.

[0030] Example 1

[0031] The present embodiment provides a gene mutant for controlling low cadmium accumulation in rice grains, which contains a mutation site in the coding region of the rice OsNRAMP5 gene, comprising a two-base mutation and a 147-base deletion. The cadmium content in rice grains containing this mutation type is significantly lower than that in rice grains without the mutation, and does not affect the yield. The specific research process is as follows:

[0032] 1. Obtaining process of rice low-cadmium-accumulating mutant

[0033] The seeds of the intermediate material 18S-184 of a rice dual-purpose cytoplasmic male sterile line were soaked in water for 12 hours, and then treated with 1.0% ethyl methanesulfonate (EMS) at room temperature for 12 hours with stirring. The treated seeds (M0 generation) were soaked, germinated, and sown, and then transplanted at a 25-day seedling age according to conventional water and fertilizer management to obtain M1 plants. The M1 plants were selfed to produce seeds, and at maturity, 1-3 ears were harvested from each plant according to the seed setting rate, for a total of 5120 single plants. In addition to the preservation of single plant seeds, 20 seeds were randomly taken from each plant to mix into M2 seeds.

[0034] The next year, the M2 seeds were soaked, germinated, and sown, and 25 d seedlings were transplanted to a cadmium-polluted field plot (soil total cadmium 10.0 mg / kg, pH 7.5) at a single plant, and managed according to the conventional water and fertilizer. After maturation, a total of 33576 plants were harvested. After harvesting, the rice grains were hulled, and the cadmium content in the brown rice was determined by inductively coupled plasma spectrometry (ICP). A cadmium low-accumulation material of rice was obtained, with a brown rice cadmium content of 0.015 mg / kg, while the corresponding wild type 18S-184 variety had a brown rice cadmium content of 0.93 mg / kg.

[0035] The obtained cadmium low-accumulation material of rice seeds were soaked, germinated, and sown the next year, and 25 d seedlings were transplanted to a cadmium-polluted field plot (soil total cadmium 1.7 mg / kg, pH 5.7) at a single plant, and managed according to the conventional water and fertilizer. After maturation, a total of 20 plants were harvested. After harvesting, the rice grains were hulled, and the cadmium content in the brown rice was determined by ICP, of which the brown rice cadmium content of 3 plants was significantly lower than that of the wild type, being 0.021 mg / kg, 0.025 mg / kg and 0.027 mg / kg, respectively, and other agronomic traits such as flowering habit, yield per plant and rice quality had no significant change compared with the wild type.

[0036] The 3 cadmium low-accumulation lines were further self-crossed and screened, and the best one was selected for verification in 5 cadmium-polluted rice fields in Hunan and Jiangxi, with 3 repetitions at each test site, and 10 plants were taken for yield and brown rice cadmium content determination at each repetition, and finally a mutant with extremely low cadmium accumulation in rice and stable heredity was obtained, named L184S (available from China Seed Group Co., Ltd.). The mutant has low cadmium characteristics without affecting yield compared with the wild type (Table 1). The comparison of mature plants and rice panicles between the cadmium low-accumulation mutant (L184S) and the wild type (18S-184) is shown in Figure 1 .

[0037] Table 1 Phenotypic characteristics of mutant and wild type plants

[0038]

[0039] 2. Determination of the mutation gene of cadmium low-accumulation mutant L184S

[0040] The cadmium low-accumulation rice mutant L184S was crossed with wild type 18S-184, and all F1 plants did not show low cadmium accumulation characteristics. In the winter of the same year, the F1 generation was selfed in Sanya, Hainan, and the F2 segregation population was obtained. About 1000 seeds of the F2 generation were randomly selected, soaked, and germinated, and then sown in a floating screen in a hydroponic tank with a cadmium concentration of 0.1M. After one month, the single plant was harvested, and the cadmium content in the leaves and roots was determined by ICP. The normal plants and low cadmium accumulation plants in the F2 segregation population showed a typical 3:1 segregation, indicating that the low cadmium trait of the rice L184S mutant is controlled by a recessive single gene.

[0041] Further selection of the F2 population of the mutant L184S crossed with the wild type 18S-184 as a positioning population, and the separated cadmium low-accumulation material was used for fine mapping. A set of SSR markers covering 12 chromosomes of rice (a total of 630 pairs of primers, https: / / archive.gramene.org / markers / ) was used for whole genome scanning of the mutant L184S and the wild type 18S-184, and a total of 98 pairs of differential primers were obtained. Next, 60 low-cadmium materials in the F2 population were subjected to linkage analysis verification using these differential primers. It was found that two pairs of molecular markers RM21300 and RM21332 on chromosome 7 had a significant linkage relationship with the low cadmium trait, and then the SSR markers in this interval were encrypted, and finally the interval was narrowed down to a physical distance of 33.7kb between RM21323 (physical position of Nipponbare 8870028) and RM21328 (physical position of Nipponbare 8903771). The positioning interval contains OsNRAMP5, a major gene controlling cadmium uptake, and it is speculated that the mutation of this gene leads to the low cadmium phenotype of L184S. Subsequently, primers were designed for PCR amplification and sequencing analysis of the OsNRAMP5 gene promoter and gene region of the mutant L184S and its wild type 18S-184, and it was found that the mutant L184S had a G to A mutation at nucleotide 804 downstream of the ATG of the OsNRAMP5 gene, a deletion of nucleotides 806 to 952 (147 bases), and a T to C mutation at nucleotide 984 (nucleotide sequence shown as SEQ ID NO. 1).

[0042] 3. Verification of cadmium low-accumulation mutant L184S

[0043] Subsequently, 240 rice plants were randomly selected from the F2 population, and sequencing analysis was performed at loci 804, 806-952, and 984. Genotyping was then performed based on the sequencing results. The results showed that the base mutation at locus 804 and the large fragment deletion at locus 806-952 exhibited complete co-segregation. Subsequently, 10 plants were randomly selected from each of the three 804 locus genotypes (A / A, A / G, G / G), the three 984 locus genotypes (C / C, C / T, T / T), and the plant with simultaneous mutations at both loci 804 and 984. These plants were then planted in heavily cadmium-contaminated fields (soil cadmium concentration 1.8 mg / kg, pH 6.5). Alternating wet and dry cultivation management was adopted. After the rice was fully mature, individual plants were harvested, threshed, tested, and milled. The cadmium content in the brown rice was determined using ICP according to the national standard determination method (GB / T5009.15-2003). The results showed that the homozygous genotype with mutations only at sites 804 / 806-952 in L184S had a grain cadmium content of 0.028 mg / kg; the homozygous genotype with mutations only at site 984 in L184S had a grain cadmium content of 0.095 mg / kg; and the homozygous genotype with simultaneous mutations at sites 804 / 806-952 and 984 in L184S had a grain cadmium content of 0.026 mg / kg. All mutation types had no significant effect on yield (Table 2). This indicates that these homozygous variations can significantly reduce grain cadmium content without affecting rice yield.

[0044] Table 2 Phenotypic characteristics of grain cadmium content and yield per plant for each genotype mutant.

[0045]

[0046] 4. Genetic effect analysis of loci 804, 806-952, and 984 in L184S.

[0047] To verify the genetic effect of L184S mutation site, L184S was used as a donor to cross and backcross with Zhongzhuanghuazhan (available from China Seed Group Co., Ltd.), Huanghuazhan (commercially available) and R1627 (available from China Seed Group Co., Ltd.), respectively. Then, 240 plants were randomly selected from each of the BC1F2 populations, and the 804 site, 806-952 site and 984 site were sequenced and analyzed. According to the sequencing results, genotyping was performed. The sequencing results showed that in all BC1F2 populations, the base mutation of the 804 site and the large fragment deletion of the 806-952 site were completely segregated. Subsequently, in each BC1F2 population, 10 plants were randomly selected from each of the three 804 site genotypes (A / A, A / G, G / G), the three 984 site genotypes (C / C, C / T, T / T) and the single plants with mutations in both the 804 site and the 984 site, respectively, and were planted in a severe cadmium-contaminated field (soil cadmium concentration was 1.8 mg / kg, pH was 6.5). Dry-wet alternation was used for cultivation management. After the rice was completely mature, single plants were used for seed collection, threshing, seed testing, and brown rice milling. According to the national standard determination method (GB / T5009.15-2003), ICP was used to determine the cadmium content in brown rice. The results showed that the homozygous mutation of the 804 site, 806-952 site and 984 site of L184S in different genetic backgrounds was significantly lower than that of other genotypes, and was lower than the national limit standard of 0.2 mg / kg. At the same time, these variations had no effect on the yield of rice in different genetic backgrounds (Table 3, Table 4).

[0048] Table 3 Phenotypic characteristics of grain cadmium content of different genotypes of mutant in different genetic backgrounds

[0049]

[0050] Table 4 Yield characteristics of different genotypes of mutant in different genetic backgrounds

[0051]

[0052] To further evaluate the genetic effects of these mutation sites, yeast cadmium tolerance growth experiments were performed. The pYES2::OsNRAMP5 (wild type), pYES2::OsNRAMP5-M1 (containing the 804th site mutation G→A and the 806-952 site deletion), pYES2::OsNRAMP5-M2 (containing the 984th site mutation T→C) and pYES2::OsNRAMP5-M3 (the 804th site mutation, the 806-952 site deletion and the 984th site mutation) vectors were constructed, respectively; then the pYES2, pYES2::OsNRAMP5, pYES2::OsNRAMP5-M1, pYES2::OsNRAMP5-M2 and pYES2::OsNRAMP5-M3 vectors were transformed into the cadmium-sensitive mutant yeast strain Δycf1 (available from Shanghai Widi Biotechnology Co., Ltd.), respectively, and then the effects of different genotypes on yeast growth were analyzed. The results showed that in the medium without added cadmium, the growth rate of yeast transformed with each mutant was not significantly different (left panel in FIG. 8); in the medium with added 20 μM cadmium, the growth rate of yeast transformed with OsNRAMP5 wild type was much lower than that of yeast transformed with OsNRAMP5-M1, OsNRAMP5-M2 and OsNRAMP5-M3 (right panel in FIG. 8). This indicates that after mutation of the OsNRAMP5 gene, the ability of cadmium absorption and transport is inhibited, thereby maintaining the normal growth of yeast. Figure 2 Figure 2

[0053] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, are within the scope of the present application claimed.​​

Claims

1. A type of rice OsNRAMP 5-gene mutant, characterized by, Wild-type rice OsNRAMP5 The nucleotide sequence of the gene is the basic sequence. The mutant is a mutation in which the G base at position 804 is changed to A and the bases at positions 806-952 are deleted, or the mutant is a mutation in which the G base at position 804 is changed to A, the bases at positions 806-952 are deleted, and the T base at position 984 is changed to C; the wild-type rice OsNRAMP5 The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

2. The gene mutant according to claim 1, characterized in that, The nucleotide sequence of the gene mutant is shown in SEQ ID NO.

1.

3. The protein encoded by the gene mutant of claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID NO.

2.

4. A biomaterial comprising the gene mutant of claim 1 or 2, characterized in that, The biomaterial is an expression cassette, vector, or host cell.

5. The application of the gene mutant of claim 1 or 2, the protein of claim 3, or the biomaterial of claim 4 in reducing cadmium accumulation in rice.

6. The use of the gene mutant of claim 1 or 2, the protein of claim 3, or the biomaterial of claim 4 in the preparation of transgenic rice.

7. A method for reducing cadmium accumulation in rice, characterized in that, To induce the gene mutant of claim 1 or 2 in rice.

8. The method according to claim 7, characterized in that, Rice can be induced to produce the gene mutants described in claim 1 or 2 by means of gene editing, hybridization, backcrossing, self-pollination or asexual reproduction.

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