Rice OsNRAMP5 gene mutant and application thereof

Abstract

The invention relates to the technical field of crop genetic breeding, and particularly discloses a rice OsNRAMP5 gene mutant and application thereof. According to the rice OsNRAMP5 gene mutant, a wild rice OsNRAMP5 gene coding sequence is used as a reference sequence, and the mutant comprises mutations that the 804th G base is mutated into A, the 806th to 952nd bases are deleted and / or the 984th T base is mutated into C. The invention provides a novel rice two-line sterile line cadmium low-accumulation mutant, which enriches low-cadmium accumulation related genes and related mutants, and provides a novel method for creating low-cadmium accumulation rice two-line sterile line germplasm.

Description

Technical Field

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

[0002] Cadmium (Cd) is a toxic heavy metal that 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. After entering the human body, it can accumulate for a long time, leading to bone damage, kidney disease, hematuria and other diseases, and further induce cancer and teratogenesis, which seriously threatens human health. With human activities such as mining, smelting and chemical industry and other industrial production, sewage irrigation, fertilizer application, etc., the Cd content in some cultivated land has increased significantly. Rice easily absorbs cadmium from the soil during its growth, resulting in 80% of rice cadmium content exceeding the standard in areas with high cadmium pollution, which brings huge hidden dangers.

[0003] In recent years, breakthroughs have been made in the study of the main control genes for the transport and accumulation of heavy metals such as cadmium in rice. OsNRAMP5 is the most important gene found so far that is involved in cadmium absorption in rice roots. Mutations in this gene block cadmium absorption in the roots, so that even in highly cadmium-polluted rice fields, the Cd accumulation in the OsNRAMP5 mutant is significantly reduced. It is a key target gene for low-cadmium rice breeding. OsNRAMP5 is mainly expressed in rice roots, including the parenchyma cells of the root epidermis, cortex, and vascular bundle near the xylem. It is also lowly expressed in tissues such as glume, leaves, and leaf sheaths, suggesting that OsNRAMP5 is not only involved in cadmium absorption, but also in the migration of cadmium in rice tissues. In addition, OsNRAMP5 is also involved in the absorption of manganese ions, which is a key element for rice growth and disease resistance. Therefore, OsNRAMP5 mutants often lead to a decrease in yield while reducing cadmium ions. Therefore, it is urgent to screen and apply mutant materials that reduce cadmium ions without affecting yield.

[0004] Although there are currently a variety of OsNRAMP5 mutants that can reduce cadmium accumulation in rice, there are relatively few types of mutations that can be applied to two-line sterile rice lines without causing a decrease in rice yield. It is still necessary to develop new mutants that can reduce cadmium accumulation without affecting yield, especially mutants based on two-line sterile rice lines. Summary of the invention

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

[0006] The present invention provides a rice OsNRAMP5 gene mutant, which uses the wild-type rice OsNRAMP5 gene coding sequence (NCBI-ID: 4342859, SEQ ID NO. 3) as a reference sequence, and the mutant comprises a mutation in which the 804th G base is mutated to A, the 806th to 952nd bases are deleted and / or the 984th T base is mutated to C.

[0007] The creation of low cadmium accumulation germplasm of two-line sterile rice must not only take into account the characteristics of low cadmium accumulation, but also consider not affecting the sterility threshold temperature, flowering habits and yield of the two-line sterile line during the mutagenesis and screening process, which further increases the difficulty of creation. In view of the shortcomings of the existing two-line sterile rice cadmium low accumulation germplasm, the present invention provides a new two-line sterile rice cadmium low accumulation mutant, which, after being introduced into wild-type rice by means known in the art, reduces the cadmium accumulation in brown rice and thus improves the safety of brown rice without affecting the yield.

[0008] The gene mutant of the present invention may only include a mutation in which the G base at position 804 is mutated to A and the bases at positions 806-952 are deleted; it may also only include a mutation in which the T base at position 984 is mutated to C; it may also include a mutation in which the G base at position 804 is mutated to A, the bases at positions 806-952 are deleted, and the T base at position 984 is mutated to C.

[0009] Preferably, the gene mutant of the present invention has a nucleotide sequence as shown in SEQ ID NO.1 (comprising three mutations at the same time).

[0010] The present invention also provides the protein encoded by the gene mutant.

[0011] The protein of the present invention uses the wild-type rice OsNRAMP5 amino acid sequence (SEQ ID NO. 4) as a reference sequence, and the protein comprises a mutation in which amino acids at positions 269-317 are deleted.

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

[0013] When the gene mutant of the present invention comprises a mutation in which the 804th G base mutates to A and the 806-952th bases are deleted, or comprises a mutation in which the 804th G base mutates to A, the 806-952th bases are deleted and the 984th T base mutates to C, its encoded protein sequence is shown in SEQ ID NO.2.

[0014] When the gene mutant of the present invention only comprises a mutation in which the 984th T base is mutated to a C, the encoded protein sequence is the same as the wild-type sequence (SEQ ID NO. 4).

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

[0016] The above expression cassette may be a DNA fragment obtained by connecting the gene mutant with an element that regulates its transcription or expression.

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

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

[0019] The present invention also provides the use of the gene mutant or protein or biological material in regulating rice cadmium accumulation or improving rice germplasm resources.

[0020] The present invention also provides the use of the above gene mutant or protein or biological material in preparing transgenic rice.

[0021] The invention also provides a method for reducing cadmium accumulation in rice, so as to make the rice produce the gene mutant.

[0022] In the method of the present invention, the gene mutants are produced in rice by gene editing, hybridization, backcrossing, selfing or asexual reproduction.

[0023] The beneficial effects of the present invention are at least: The present invention provides a novel rice two-line sterile line cadmium low accumulation mutant, which enriches low cadmium accumulation related genes and related mutants, and provides a new method for creating low cadmium accumulation rice two-line sterile line germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a comparison of the plants and rice panicles of the two-line sterile rice line with low cadmium accumulation mutant (L184S) and the wild type 18S-184.

[0025] Figure 2 The results are a comparison of the effects of OsNRAMP5 (wild type), OsNRAMP5-M1 (mutation at site 804 and deletion of sites 806-952), OsNRAMP5-M2 (mutation at site 984), and OsNRAMP5-M3 (mutation at site 804, deletion of sites 806-952, and mutation at site 984) on the growth of yeast strain Δycf1. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0027] 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.

[0028] The present invention firstly performs EMS mutagenesis treatment (M0 generation) on the intermediate material 18S-184 seeds of the dual-purpose nuclear sterile line of rice (derivative materials of Long S, which can be purchased from China Seed Group Co., Ltd.), and plants the treated seeds to obtain M1 generation plants. The M1 generation plants are self-pollinated to produce seeds (M2 generation), and the M2 generation plants are planted on cadmium-contaminated fields. After the harvested rice grains are shelled, the cadmium content in the brown rice is measured by an inductively coupled plasma spectrometer to screen low cadmium accumulation plants. The obtained rice low cadmium accumulation strain is further self-pollinated and screened, and planted and verified in 5 cadmium-contaminated rice fields in Hunan and Jiangxi, and finally a mutant homozygous single plant with extremely low cadmium accumulation and stable inheritance is obtained, which is named L184S and used for hybrid breeding and biotechnology research.

[0029] Example 1 This embodiment provides a gene mutant that controls low cadmium accumulation in rice grains, the mutation site contained in the mutant is located in the coding region of the rice OsNRAMP5 gene, including a mutation of two bases and a deletion of 147 bases. The cadmium content of rice grains containing this mutation type is significantly lower than that of rice grains without mutation, and does not affect the yield. The specific research process is as follows: 1. Process of obtaining rice mutants with low cadmium accumulation The seeds of the intermediate material 18S-184 of the dual-purpose nuclear sterile line of rice were soaked in clean water for 12 hours, and then soaked in 1.0% ethyl methanesulfonate (EMS) at room temperature for 12 hours, stirred during the period, separated from the EMS treatment solution and soaked in clean water for 4 hours. The rice seeds (M0 generation) treated with the mutation treatment were soaked, germinated, sown, and transplanted individually after 25 days of seedling age. According to conventional water and fertilizer management, M1 generation plants were obtained. The M1 generation plants were self-pollinated to produce seeds. When mature, 1 to 3 ears were harvested from each plant according to the fruit setting rate, and a total of 5120 individual plants were harvested. In addition to preserving individual plant seeds, 20 seeds were randomly taken from each plant and mixed into M2 seeds.

[0030] The following year, M2 seeds were soaked, germinated, and sown. Single seedlings at 25 days old were transplanted into cadmium-contaminated fields (total soil cadmium 10.0 mg / kg, pH 7.5), and water and fertilizer management was carried out according to routine methods. After maturity, individual plants were harvested, and a total of 33,576 plants were harvested. After the harvested rice grains were shelled, the cadmium content in brown rice was determined using an inductively coupled plasma spectrometer (ICP). A rice cadmium low accumulation material 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.

[0031] The seeds of the rice with low cadmium accumulation materials were soaked and germinated in the next year before sowing. 50 plants were transplanted to cadmium-contaminated fields (total cadmium in the soil 1.7 mg / kg, pH 5.7) at 25 days of age, and water and fertilizer management was carried out according to the conventional method. After maturity, the plants were harvested, and a total of 20 plants were harvested. After the harvested rice grains were shelled, the cadmium content in brown rice was determined by ICP. The cadmium content in brown rice of 3 rice plants was significantly lower than that of the wild type, which were 0.021 mg / kg, 0.025 mg / kg and 0.027 mg / kg respectively. Other agronomic traits such as flowering habits, single plant yield and rice quality did not change significantly with the wild type.

[0032] The three low-cadmium accumulation lines were further self-pollinated and screened, and the line with the best overall performance was selected for planting and verification in five cadmium-contaminated rice fields in Hunan and Jiangxi. Each test site was repeated three times, and 10 plants were taken from each repeat for yield and brown rice cadmium content determination. Finally, a rice mutant with extremely low cadmium accumulation and stable inheritance was obtained, named L184S (available from China National Seed Group Co., Ltd.). Compared with the wild type, this mutant has the low-cadmium characteristics without affecting the yield (Table 1). The comparison of mature plants and rice ears of the two-line sterile rice line low-cadmium accumulation mutant (L184S) and the wild type (18S-184) is shown in Figure 1. Figure 1 .

[0033] Table 1 Phenotypic characteristics of mutants and wild-type plants 2. Determination of the mutant gene of the low-cadmium accumulation mutant L184S The low-cadmium accumulation rice mutant L184S was hybridized with the wild type 18S-184, and all F1 plants did not show the low-cadmium accumulation characteristics. In the winter of the same year, the F1 generation was self-pollinated in Sanya, Hainan, to obtain the F2 generation segregation population. About 1,000 F2 generation seeds were randomly selected, soaked and germinated, and sown in a water culture pool screen floating on a cadmium concentration of 0.1M. After one month, the individual plants were 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 separation, indicating that the low-cadmium trait of the rice L184S mutant is controlled by a recessive single gene.

[0034] The F2 population of mutant L184S hybridized with wild type 18S-184 was further selected as the positioning population, and the isolated low cadmium accumulation materials were used for fine positioning. A set of SSR markers covering 12 rice chromosomes (a total of 630 pairs of primers, https: / / archive.gramene.org / markers / ) was used to perform a genome-wide scan of mutant L184S and wild type 18S-184, and a total of 98 pairs of differential primers were obtained. Next, these differential primers were used to perform linkage analysis verification on 60 low-cadmium materials in the F2 population. It was found that two pairs of molecular markers RM21300 and RM21332 on chromosome 7 had an obvious linkage relationship with the low cadmium trait, and then the SSR markers in this interval were encrypted, and finally this interval was narrowed to the physical distance of 33.7kb between RM21323 (physical position 8870028 of Nipponbare) and RM21328 (physical position 8903771 of Nipponbare). The localization interval contains OsNRAMP5, a major gene that controls cadmium absorption. It is speculated that the mutation of this gene causes the low-cadmium phenotype of L184S. Subsequently, primers were designed for PCR amplification and sequencing analysis of the promoter and gene regions of the OsNRAMP5 gene of the mutant L184S and its wild type 18S-184, and it was found that the nucleotide at position 804 downstream of the ATG of the OsNRAMP5 gene of the mutant L184S was mutated from G to A, the nucleotides at positions 806 to 952 (147 bases) were deleted, and the nucleotide at position 984 was mutated from T to C (the nucleotide sequence is shown in SEQ ID NO.1).

[0035] 3. Verification of the low cadmium accumulation mutant L184S Then, 240 materials were randomly selected from the F2 population, and sequencing analysis was performed on the 804 site, the 806-952 site and the 984 site, and genotyping was performed according to the sequencing results. The results showed that the base mutation at the 804 site and the large fragment deletion at the 806-952 site had a complete co-segregation phenomenon. Subsequently, 10 plants were randomly selected from 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 simultaneous mutations at the 804 and 984 sites, and planted in heavily cadmium-contaminated fields (soil cadmium concentration was 1.8 mg / kg and pH was 6.5). Cultivation management was carried out in the form of alternating dry and wet. After the rice was fully mature, the single plants were harvested, threshed, tested, and the brown rice was ground into powder. Referring to the national standard determination method (GB / T5009.15-2003), the cadmium content in brown rice was determined by ICP. The results showed that the cadmium content of the grains of the homozygous genotype with only the 804 / 806-952 mutation in L184S was 0.028 mg / kg; the cadmium content of the grains of the homozygous genotype with only the 984 mutation in L184S was 0.095 mg / kg; the cadmium content of the grains of the homozygous genotype with both the 804 / 806-952 and 984 mutations in L184S was 0.026 mg / kg; all the variant types had no significant effect on the yield (Table 2). This indicates that these homozygous variants can significantly reduce the cadmium content of the grains without affecting the rice yield.

[0036] Table 2 Phenotypic characteristics of cadmium content in grains and yield per plant of each genotype mutant 4. Analysis of genetic effects of loci 804, 806-952, and 984 in L184S To verify the genetic effect of the L184S mutation site, L184S was used as a donor, and hybridization and backcrossing were performed with Zhongzhonghui 2130 (available from China Seed Group Co., Ltd.), Huanghuazhan (commercially available), and R1627 (available from China Seed Group Co., Ltd.), respectively. 240 materials were randomly selected from their respective BC1F2 populations, and sequencing analysis was performed on the 804 site, 806-952 site, and 984 site. Genotyping was performed based on the sequencing results. The sequencing results showed that in all BC1F2 populations, the base mutation at the 804 site and the large fragment deletion at the 806-952 site were completely co-segregated. Subsequently, in each BC1F2 population, 10 plants were randomly selected from three genotypes of 804 loci (A / A, A / G, G / G), three genotypes of 984 loci (C / C, C / T, T / T) and single plants with mutations at both loci 804 and 984, and planted in heavily cadmium-contaminated fields (soil cadmium concentration was 1.8 mg / kg, pH was 6.5). Alternating dry and wet cultivation management was adopted. After the rice was fully mature, the single plants were harvested, threshed, tested, and brown rice was ground into powder. The cadmium content in brown rice was determined by ICP with reference to the national standard determination method (GB / T5009.15-2003). The results showed that the cadmium content of the grains of homozygous mutations at loci 804, 806-952 and 984 of L184S under different genetic backgrounds was significantly lower than that of other genotypes, and was lower than the national limit of 0.2 mg / kg. At the same time, these variants had no effect on rice yield under different genetic backgrounds (Table 3, Table 4).

[0037] Table 3 Phenotypic characteristics of cadmium content in grains of mutant genotypes under different genetic backgrounds Table 4 Yield characteristics of mutants of different genotypes under different genetic backgrounds To further evaluate the genetic effects of these mutation sites, a yeast cadmium tolerance growth experiment was conducted. The pYES2::OsNRAMP5 (wild type), pYES2::OsNRAMP5-M1 (containing the 804th site mutation G→A and the 806-952th site deletion), pYES2::OsNRAMP5-M2 (containing the 984th site mutation T→C), and pYES2::OsNRAMP5-M3 (the 804th site mutation, the 806-952th site deletion, and the 984th site mutation) vectors were constructed; then the pYES2, pYES2::OsNRAMP5, pYES2::OsNRAMP5-M1, pYES2::OsNRAMP5-M2, and pYES2::OsNRAMP5-M3 vectors were respectively transformed into the cadmium-sensitive mutant yeast strain Δycf1 (available from Shanghai Weidi Biotechnology Co., Ltd.), and the effects of different genotypes on yeast growth were analyzed. The results showed that in the medium without cadmium, there was no significant difference in the growth rate of yeast transformed into each mutant ( Figure 2 ); In the medium supplemented with 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 ( Figure 2 This indicates that the mutation of the OsNRAMP5 gene inhibits the ability to absorb and transport cadmium, thereby maintaining the normal growth of yeast.

[0038] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A rice OsNRAMP5 gene mutant, characterized in that: Taking the wild-type rice OsNRAMP5 gene coding sequence as a reference sequence, the mutant comprises a mutation in which the 804th G base is mutated to A, the 806th to 952nd bases are deleted and / or the 984th T base is mutated to C.

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

1.

3. The encoded protein of the gene mutant according to claim 1 or 2.

4. The protein according to claim 3, characterized in that The amino acid sequence of wild-type rice OsNRAMP5 protein is used as a reference sequence, and the protein comprises a mutation in which amino acids at positions 269-317 are deleted.

5. The protein according to claim 4, characterized in that It has the amino acid sequence shown in SEQ ID NO.

2.

6. A biological material comprising the gene mutant according to claim 1 or 2, characterized in that: The biological material is an expression cassette, a vector or a host cell.

7. Use of the gene mutant according to claim 1 or 2, the protein according to any one of claims 3 to 5, or the biological material according to claim 6 in regulating cadmium accumulation in rice or improving rice germplasm resources.

8. Use of the gene mutant according to claim 1 or 2, the protein according to any one of claims 3 to 5, or the biological material according to claim 6 in preparing transgenic rice.

9. A method for reducing cadmium accumulation in rice, characterized in that: The gene mutant according to claim 1 or 2 is produced in rice.

10. The method according to claim 9, characterized in that The gene mutant according to claim 1 or 2 is produced in rice by gene editing, hybridization, backcrossing, selfing or asexual reproduction.

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