Mutant OsNRAMP5 gene and application thereof in regulation and control of rice cadmium accumulation

Abstract

The invention relates to the technical field of crop genetic breeding, and particularly discloses a mutant OsNRAMP5 gene and application thereof in regulation and control of rice cadmium accumulation. A mutant OsNRAMP5 gene is found through research, rice containing the mutant gene has the characteristic of low cadmium accumulation, and then the scheme of the invention is provided. The nucleotide sequence of the mutant OsNRAMP5 gene is shown as SEQ ID No.1. The mutant OsNRAMP5 gene can reduce cadmium without influencing agronomic traits, provides a new germplasm for cultivating a new variety of cadmium-low-accumulation rice, and has a relatively strong application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of crop genetic breeding, and in particular to a mutant OsNRAMP5 Genes and their applications in regulating cadmium accumulation in rice. Background Art

[0002] Cadmium (Cd) is a heavy metal element. The background Cd content in the soil is very low under natural conditions. However, human activities, such as mining, smelting and chemical industry, sewage irrigation, fertilizer application, etc., have led to a significant increase in the Cd content in some cultivated land. Rice absorbs Cd from rice fields and accumulates in rice. After people eat rice containing Cd, most of the Cd will be absorbed and accumulated in the body to produce toxicity, endangering human health. Cd accumulates in human organs, of which the accumulation in the kidneys and liver accounts for about 60% of the total Cd in the body. Long-term Cd toxicity can easily lead to impaired organ function, and severe cases can even induce cancer.

[0003] Environmental factors have an important impact on the form of Cd in paddy fields. For example, soil pH, redox state, the content of other ions, soil microorganisms, etc. will affect the content of free Cd, thereby affecting the Cd absorption of rice. Measures such as applying lime, selenium, silicon, etc., flooding throughout the process, and applying bacterial fertilizers containing beneficial microorganisms can passivate Cd in paddy fields, thereby effectively reducing the absorption of Cd by rice roots. They are important emergency agronomic measures, but there are some shortcomings in terms of sustainability, ease of operation, or environmental friendliness. To produce rice with a Cd content that meets the standard in Cd-contaminated paddy fields, it still mainly depends on the screening and cultivation of low-Cd rice varieties.

[0004] A large number of studies have shown that genetic factors play an important regulatory role in Cd absorption and transport. Under the same environmental conditions, there is genetic diversity in the ability of rice to absorb, transport and accumulate Cd. Using various forward and reverse genetics methods, dozens of Cd accumulation-related genes have been cloned and identified from rice. Among them, Cd transporter genes play a key role in Cd absorption and Cd distribution, such as the natural resistance-associated macrophage protein (NRAMP) family genes, among which OsNRAMP5 It is the most important gene involved in Cd absorption in rice roots discovered so far. Mutations in this gene block Cd absorption in the roots, making it possible to OsNRAMP5 The Cd accumulation in the mutant was also significantly reduced, which is a key target gene for low-Cd rice breeding; there are also P-type heavy metal ATPase (HMA) family genes, among which OsHMA3 and OsHMA2 They were successively identified as key genes for Cd transport. OsHMA3(qCdT7)It is the first Cd transporter gene cloned by QTL mapping, and plays a key role in regulating the transport of Cd from roots to the ground. There are also other transporter genes involved in Cd transport, such as the rice low-affinity cation transporter 1 gene OsLCT1 , iron-regulated transporters OsIRT1 etc. are all related to cadmium absorption and accumulation in rice.

[0005] Radiation-induced mutagenesis refers to the process of irradiating the seeds, plants, or certain organs and tissues of crops with different radiation sources to induce various mutations, and then selecting the heritable and excellent mutations with utilization value to cultivate new excellent varieties. Compared with traditional breeding methods, radiation-induced mutagenesis breeding has the advantages of high mutation rate, wide mutation spectrum, and short breeding period.

[0006] In order to enrich variety diversity, it is still necessary to develop new rice mutants and mutant genes that can reduce cadmium accumulation. Summary of the invention

[0007] One of the purposes of the present invention is to provide a new mutant and mutant gene which can make rice have the characteristics of low cadmium accumulation.

[0008] The present invention provides a mutant OsNRAMP5 The gene has a nucleotide sequence as shown in SEQ ID No.1.

[0009] The present invention is to treat the dry seeds of the conventional early rice variety Jiazao 17 with 250 Gy 60 Co-γ irradiation and 0.1% EMS (ethyl methanesulfonate) were combined for mutagenesis, and the induced population was planted to obtain M1 generation plants. The M1 generation plants were self-pollinated to produce seeds (M2 generation), and the M2 generation plants were planted in cadmium-contaminated fields. The cadmium content of the harvested rice was measured using the NX-300FA food metal detector. 345 plants with low cadmium accumulation were initially screened out from 12,000 individual plants. These plants were further self-pollinated and screened, and planted in cadmium-contaminated fields (M3) for verification. A single plant with a rice cadmium content significantly lower than that of the wild type Zhongjiazao 17 was screened out and named Zhongjiazao 17- cd1 , and used in hybrid breeding and biotechnology research.

[0010] Further, the present invention is from Zhongjia Zao 17- cd1 A gene mutation site that reduces the cadmium content in rice grains was found. OsNRAMP5 The AT bases at positions 1051 and 1052 downstream of the start codon ATG in the gene coding region are missing, resulting in a frameshift mutation and premature termination of translation. OsNRAMP5 Gene's Zhongjia Zao 17- cd1 The cadmium contents in rice plants and grains were lower than those in wild-type Zhongjiazao 17 rice.

[0011] The present invention also provides the mutant OsNRAMP5 The amino acid encoded by the gene has an amino acid sequence as shown in SEQ ID NO.2.

[0012] Compared with the wild-type amino acid, the amino acid of the present invention terminates translation prematurely at position 416.

[0013] The present invention also provides an expression cassette, a recombinant vector or a cell containing the mutant OsNRAMP5 Gene.

[0014] The present invention also provides the mutant OsNRAMP5 Application of genes or amino acids or expression cassettes, recombinant vectors or cells in regulating rice cadmium accumulation or improving rice germplasm resources.

[0015] The present invention also provides a method for reducing cadmium accumulation in rice, which allows the rice to contain the above mutant OsNRAMP5 gene or amino acid.

[0016] In the method of the present invention, the rice is made to contain the above-mentioned mutant by one or more methods of gene editing, hybridization, backcrossing, selfing or asexual reproduction. OsNRAMP5 gene or amino acid.

[0017] The beneficial effects of the present invention are at least: The invention provides a new mutant type capable of causing low cadmium accumulation in rice OsNRAMP5 Genes provide new opportunities for breeding rice varieties with low cadmium accumulation OsNRAMP5 The alleles and new cadmium-low accumulation rice germplasm can reduce cadmium without affecting agronomic traits, providing new germplasm resources for creating new low cadmium accumulation rice varieties and have strong application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The cadmium low accumulation mutant Zhongjiazao 17- cd1 of OsNRAMP5 Type of gene mutation.

[0019] Figure 2 For Zhongjia Early 17 and Zhongjia Early 17- cd1 Amino acid sequence alignment of OsNRAMP5.

[0020] Figure 3 For Zhongjia Zao 17 and Zhongjia Zao 17- cd1 phenotype of plants. DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] Example 1 1. Screening of rice mutants with low cadmium accumulation and identification of mutant genes In February 2021, the conventional early rice variety Zhongjiazao 17 was used as the mutagenic material and a dose of 250 Gy was used at the Nuclear Radiation Center of Liuyang Industrial Park in Hunan Province. 60 1 kg of dry seeds of Zhongjiazao 17 were irradiated with Co-γ rays, and the irradiated seeds were mutated with 0.1% EMS solution for 16 h. The mutated seeds were germinated and sown at the Longping High-Tech Guanshan Research Base, and the field was fertilized and watered normally. After maturity, one ear was harvested from each plant (M1), and a total of 4909 ears were harvested. One-third of the seeds from each ear were taken and sown at the Longping High-Tech Guanshan Research Base after germination in May 2022. They were transplanted to cadmium-contaminated fields (total cadmium content in the soil was 0.317 mg / kg, pH 5.42) at the 5-leaf 1-heart stage, and the field was fertilized and watered normally. After the seeds matured, they were harvested as single ears per plant (M2), and a total of 12,000 ears were harvested. After threshing, the rice grains of each ear were measured with the NX-300FA food metal detector to determine the cadmium content in the rice grains, and 345 materials with cadmium content of less than 0.1 mg / kg were preliminarily screened out. In April 2023, these 345 low-cadmium accumulation materials were planted in cadmium-contaminated fields (total soil cadmium content 0.317 mg / kg, pH 5.42) and harvested per plant (M3) per ear. The cadmium content of the rice was measured using an NX-300FA food metal detector, and one material with a cadmium content of less than 0.1 mg / kg was selected and named Zhongjia Zao 17- cd1 .

[0024] Since Zhongjia was 17- cd1 The cadmium content of rice is extremely low, so we conducted a mutation analysis on the genes that have been reported to affect cadmium absorption in rice. cd1 Next generation sequencing was performed to compare the whole genome of Zhongjia Zao 17 and Zhongjia Zao 17- cd1 Cadmium absorption-related genes OsNramp1, OsNramp2, OsNramp5, OsIRT1, OsLCT1, OsHMA2 and OsHMA3The mutation of cadmium absorption related genes OsNramp5 The 10th exon of the coding region was missing 2 bp, while the coding regions of other cadmium absorption-related genes were missing in Zhongjiazao 17 and Zhongjiazao 17- cd1 There was no variation between the two groups. cd1 of OsNRAMP5 The gene was verified by first-generation sequencing, and the results were consistent with the results of whole genome comparison analysis, that is, OsNRAMP5 The 10th exon of the gene is missing 2 bp ( Figure 1 ),correspond OsNRAMP5 The AT bases at positions 1051 and 1052 downstream of the start codon ATG in the coding region are missing, resulting in OsNRAMP5 The amino acid after position 350 changes, and the peptide chain terminates prematurely at position 416 ( Figure 2 ). Zhongjia Morning 17- cd1 Mutant gene OsNRAMP5 The nucleotide sequence is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2.

[0025] 2. Cadmium accumulation characteristics of the low-cadmium accumulation mutant Zhongjiazao17-cd1 In order to identify Zhongjia Zao 17- cd1 The cadmium accumulation characteristics of the Zhongjia Early 17- cd1 The seeds were sown at the Longping High-Tech Guanshan Research Base and transplanted to the cadmium accumulation characteristic identification pools (unpolluted pool, lightly polluted pool and heavily polluted pool) when they reached the 5-leaf 1-heart stage. The soil pH value, organic matter, and total cadmium, total arsenic, available manganese, available iron, available copper, and available zinc content of the cadmium accumulation characteristic identification pools are shown in Table 1. cd1 and wild type Zhongjiazao 17 were transplanted into three cadmium pools with different concentration gradients, with three replicates in each cadmium pool, and six plants were planted in each replicate, with a single plant size of 17 cm × 20 cm. After maturity, the roots, stems, leaves, leaf sheaths, and seeds were sampled, the roots were rinsed with water, and dried together with the stems, leaf sheaths, leaves, and seeds, and then sent to Hunan Huake Environmental Testing Company for testing of cadmium content.

[0026] Table 1 Soil parameters of cadmium accumulation characteristics identification pool As shown in Table 2, in the unpolluted, lightly polluted and heavily polluted cadmium accumulation characteristic identification pools, Zhongjia Zao 17- cd1The cadmium content of the grains was 0.012 mg / kg, 0.019 mg / kg and 0.028 mg / kg, respectively, all far below the limit standard value of 0.20 mg / kg. In the unpolluted, lightly polluted and heavily polluted cadmium accumulation characteristic identification pools, the cadmium content of rice in Zhongjia Zao 17 was 0.028 mg / kg, 0.240 mg / kg and 0.457 mg / kg, respectively. In the lightly polluted and heavily polluted cadmium accumulation characteristic identification pools, the cadmium content of the grains in Jiazao 17 exceeded the limit standard value. These results show that Zhongjia Zao 17- cd1 When planted in lightly polluted and heavily polluted cadmium accumulation characteristic identification pools, its grains have the characteristics of low cadmium accumulation.

[0027] Table 2 Zhongjia Zao 17 and Zhongjia Zao 17- cd1 Cadmium content in grains of different cadmium-contaminated pools Note: and It indicates that the differences between Zhongjiazao 17-cd1 and Zhongjiazao 17 reached significant levels (P<0.05) and extremely significant levels (P<0.01), respectively.

[0028] Further investigation was conducted on Zhongjiazao 17 and Zhongjiazao 17- cd1 The cadmium content in the roots, stems, leaf sheaths and leaves of Zhongjiazao 17 was tested to study the cadmium accumulation characteristics of different tissues of the plant. As shown in Table 3, the cadmium contents in the roots, stems, leaf sheaths and leaves of Zhongjiazao 17 were 9.305 mg / kg, 2.295 mg / kg, 0.589 mg / kg and 0.097 mg / kg, respectively. cd1 The cadmium contents in the roots, stems, leaf sheaths and leaves of the wild-type strain were 2.720 mg / kg, 0.033 mg / kg, 0.027 mg / kg and 0.022 mg / kg, respectively, which were significantly lower than those in the corresponding tissues of the wild-type strain Zhongjiazao17. cd1 The roots, stems, sheaths and leaves of this plant have the characteristics of low cadmium accumulation.

[0029] Table 3 Zhongjia Zao 17 and Zhongjia Zao 17- cd1 Cadmium content in different tissue parts (mg / kg) Note: These indicate that the differences between Zhongjiazao 17-cd1 and Zhongjiazao 17 reached extremely significant levels (P<0.01).

[0030] For the purpose of research, Jia Zao 17- cd1 of OsNRAMP5 Whether the mutation affects its agronomic traits, the present invention is in Zhongjia Zao 17- cd1 The plant height, tiller number, ear length, number of grains per ear, fruit setting rate, 1000-grain weight and yield per plant were investigated at maturity. cd1 There were no significant differences in plant height, tiller number, ear length, number of grains per ear, fruit setting rate, 1000-grain weight and yield per plant between Zhongjiazao 17 and Zhongjiazao 17 (Table 4). OsNRAMP5 The mutation had no significant effect on the agronomic and yield traits of Zhongjiazao 17-cd1. Zhongjiazao 17-cd1 could reduce the cadmium content in grains without affecting agronomic traits, and had high application value. cd1 The plant phenotype is shown in Figure 3 .

[0031] Table 4 Comparison of agronomic traits between Zhongjiazao 17-cd1 and Zhongjiazao 17 Note: The same letters after the data in the same column in the table indicate that there is no significant difference between the two data.

[0032] 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 mutant OsNRAMP5 A gene characterized by The nucleotide sequence is shown in SEQ ID No.

1.

2. The mutant according to claim 1 OsNRAMP5 The amino acid encoded by the gene is characterized in that The amino acid sequence is shown in SEQ ID NO.

2.

3. An expression cassette, recombinant vector or cell, characterized in that: Containing the mutant according to claim 1 OsNRAMP5 Gene.

4. The mutant according to claim 1 OsNRAMP5 Use of the gene or the amino acid described in claim 2 or the expression cassette, recombinant vector or cell described in claim 3 in regulating cadmium accumulation in rice.

5. The mutant according to claim 1 OsNRAMP5 Use of the gene or the amino acid described in claim 2 or the expression cassette, recombinant vector or cell described in claim 3 in improving rice germplasm resources.

6. A method for reducing cadmium accumulation in rice, characterized in that: Making rice contain the mutant according to claim 1 OsNRAMP5 The gene or the amino acid according to claim 2.

7. The method according to claim 6, characterized in that The rice contains the mutant type according to claim 1 by one or more methods of gene editing, transgenic, hybridization, backcrossing, selfing or asexual reproduction. OsNRAMP5 The gene or the amino acid according to claim 2.

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