Improved oryza plant or plant portion thereof, and method for producing improved oryza plant

Abstract

Disclosed are: an improved Oryza plant, or a plant portion thereof, having two copies of a gene encoding OsNramp5 protein, and a method for producing an improved Oryza plant. The method and method for producing an improved Oryza plant comprises: (I) a step for crossing Pokkali with an Oryza plant other than Pokkali to obtain an F1 individual; (II) a step for bringing about self-fertilization of the F1 individual to obtain an F2 individual; and (A) a step for confirming that the obtained F2 individual has two copies of a gene encoding OsNramp5 protein. The method also comprises: (I) a step for crossing Pokkali with an Oryza plant other than Pokkali to obtain an F1 individual; (II') as necessary a step for bringing about self-fertilization of the F1 individual to obtain an F2 individual; (III) a step for backcrossing the F1 individual or F2 individual with an Oryza plant other than Pokkali to obtain a BC1F1 individual; and (IV) as necessary a step for further repeating steps (II') to (III) n-1 times using the BC1F1 individual to obtain a BCnF1 individual (n represents an integer of 2 or more), wherein the method includes (A) a step for confirming that an individual obtained in at least one step selected from the group consisting of steps (I)-(IV) has two copies of a gene encoding OsNramp5 protein.

Description

Improved rice plant or plant part thereof, and method for producing improved rice plant

[0001] The present disclosure relates to an improved rice plant or a plant part thereof, a method for producing an improved rice plant, and the like.

[0002] Cadmium (Cd) is a heavy metal with the atomic symbol Cd, atomic number 48, and atomic weight 112.4. Cadmium is a zinc-group element with similar chemical behavior to zinc. It is always present in zinc ore, and is therefore recovered during zinc refining. Cadmium is an important metal used in various industrial products, such as electrodes for secondary batteries (nickel-cadmium batteries), pigments (cadmium yellow, red, orange, etc.), and Wood's alloy. However, it is known to be harmful to the human body, causing kidney dysfunction when absorbed. Cadmium-contaminated soil has been found in the vicinity of mines, refineries, and gold warehousing factories that handle cadmium. In Japan, cadmium-related environmental contamination has led to the outbreak of Itai-itai disease in the Jinzu River basin in Toyama Prefecture.

[0003] Cadmium soil contamination is believed to have progressed through mining development and associated wastewater. However, once cadmium accumulates in the soil, it is extremely difficult to remove. Methods for restoring contaminated soil include physical and civil engineering techniques such as soil addition, soil removal, and inversion, as well as methods for absorbing cadmium in the soil through plants. However, the former methods essentially only dilute the cadmium to reduce its concentration, while the latter methods do not adequately absorb cadmium, requiring a long time for their effectiveness to be seen, and thus fail to remove cadmium from the contaminated soil (see Non-Patent Document 1). As a result, cadmium in the contaminated soil migrates to plants in the environment without being removed. In particular, when soil in agricultural production areas is contaminated with cadmium, the cadmium in the agricultural products can be absorbed by humans and animals, potentially causing serious symptoms. Rice, in particular, is known to be one of the sources of foodborne cadmium contamination.

[0004] Rice (rice grains) is the staple food for half of the world's population and is a major source of cadmium intake. Cadmium intake from rice is reported to account for 40-65% of total cadmium intake, depending on the amount of rice consumed. Furthermore, rice accumulates more cadmium than other grains, such as barley and wheat. Furthermore, since rice is used as animal feed, animal feed, and pet food, reducing the transfer of cadmium from soil to rice grains is important not only for human health but also for animal health.

[0005] Japanese Patent Application Publication No. 2022-029767

[0006] Fertilizer Science, No. 42, 35-79 (2020) Plant Cell. 2012 May;24(5):2155-67. doi: 10.1105 / tpc.112.096925. Epub 2012 May 15.J Exp Bot. 2020 Sep 19;71(18):5705-5715. doi: 10.1093 / jxb / eraa287.

[0007] To date, several approaches have been tested to reduce cadmium migration, including soil dressing, application of amendments such as biochar, liming, and water management. However, while these approaches reduce cadmium solubility in soil, they have drawbacks such as low cost-effectiveness and time-consuming implementation. Furthermore, several genes involved in cadmium accumulation in rice have been identified, and so-called genetic engineering techniques have been developed to reduce cadmium accumulation by disrupting or overexpressing these genes. However, such genetic engineering techniques have not yet fundamentally solved the problem of cadmium accumulation due to factors such as reduced growth or yield and concerns about genetically modified foods. The present disclosure aims to provide a means for producing rice plants with low cadmium content (accumulation) in the aboveground parts of the plants.

[0008] Pokkali (World Rice Core Collection number WRC96; JP95066, JP247553) is a salt-tolerant plant of the Poaceae family, Oryza, cultivated in southern India. The present inventors discovered that Pokkali has a lower cadmium content in the aboveground parts compared to other Oryza plants. Furthermore, the present inventors discovered that Pokkali has two copies of the gene encoding the OsNramp5 protein. They then crossed Pokkali with a plant of the Oryza genus other than Pokkali that has only one copy of the gene encoding the OsNramp5 protein to produce an Oryza plant that has two copies of the gene encoding the OsNramp5 protein. They then discovered that a plant of the Oryza genus with low cadmium accumulation could be obtained, and further improvements were made.

[0009] The present disclosure encompasses, for example, the subject matter described in the following paragraphs. Item 1. An improved rice plant or plant part thereof having two copies of a gene encoding OsNramp5 protein. Item 2. An improved rice plant or plant part thereof according to Item 1, wherein the rice plant is Oryza sativa L. Item 3. An improved rice plant or plant part thereof according to Item 1, wherein the rice plant is a Japonica species. Item 4. An improved rice plant or plant part thereof according to any one of Items 1 to 3, wherein the plant part is a plant part other than a root or a processed product thereof. Item 5. A method for producing an improved rice plant, comprising: (I) crossing Pokkali with a rice plant other than Pokkali, and producing F １ (II) obtaining an individual; １ Self-fertilize the individual and F ２ (A) obtaining the individual; and ２ Item 6. A method for producing an improved Oryza plant, comprising: (I) crossing Pokkali with a plant of the genus Oryza other than Pokkali, and producing an F １ (II') optionally obtaining the F １ Self-fertilize the individual and F ２ (III) obtaining an individual; １ Individual or F ２The individual was backcrossed with the rice plant other than Pokkali, and BC １ F １ (IV) optionally, obtaining the BC １ F １ Repeat steps (II') to (III) n-1 times using the individual to obtain BC ｎ F １ Item 7. A method comprising the steps of: obtaining an individual (n is an integer of 2 or more); and (A) confirming that the individual obtained in at least one step selected from the group consisting of steps (I) to (IV) has two copies of the gene encoding the OsNramp5 protein. １ F １ Individual or BC ｎ F １ The individual was self-fertilized m-1 times and BC １ F ｍ Individual or BC ｎ F ｍ Item 6. The method according to Item 6, comprising a step of obtaining an individual (m represents an integer of 2 or more). Item 8. The method according to any one of Items 5 to 7, wherein the plant of the genus Oryza other than Pokkali is Oryza sativa. Item 9. The method according to any one of Items 5 to 7, wherein the plant of the genus Oryza other than Pokkali is a Japonica species. Item 10. A method for producing an improved Oryza plant, comprising: (I) crossing Pokkali with a plant of the genus Oryza other than Pokkali, and producing an F １ (II) obtaining an individual; １ Self-fertilize the individual and F ２ (A) obtaining the individual F obtained in step (II); ２ (III) confirming that the individual has two copies of the gene encoding the OsNramp5 protein; ２ The individual was backcrossed with the rice plant other than Pokkali, and BC １ F １ (IV) obtaining the individual; １ F １ Repeat steps (II), (A), and (III) n-1 times using the individual to obtain BC ｎ F １(V-1) a step of obtaining an individual (n represents an integer of 2 or more), ｎ F １ Self-fertilize the individual and BC ｎ F ２ (A) a step of obtaining an individual BC obtained in step (V-1) ｎ F ２ (V-2) confirming that the individual has two copies of the gene encoding the OsNramp5 protein; and ｎ F ２ The individual was self-fertilized x-2 times and BC ｎ F ｘ A method comprising the step of obtaining an individual (wherein x is an integer of 3 or greater).

[0010] According to the present disclosure, there are provided plants of the genus Oryza that have a significantly low cadmium content (accumulation amount) in above-ground parts even when cultivated in fields with high cadmium content, and means for producing the same.

[0011] Schematic diagram of population construction for QTL detection and mapping of shoot cadmium concentration in rice. a) Detection and contribution of two major QTLs for low shoot cadmium accumulation are shown. b) F obtained from the cross between Pokkali and Khau Tan Chiem to map the QTL gene detected in Figure 1a. ２ From F ６ A schematic diagram of the population is shown. c. BC obtained from the backcross of Pokkali and Koshihikari for fine mapping of the QTL genes detected in Fig. 1a. ３ F ２ and B.C. ４ F ２ A schematic diagram of the QTL gene involved in low cadmium accumulation in rice is shown. a) F obtained from the cross between Khau Tan Chiem (high cadmium) and Pokkali (low cadmium). ２ The results of mapping QTL genes using a population are shown. b. F obtained from the cross between Koshihikari (relatively high cadmium) and Pokkali (low cadmium). ２Figure 3 shows the results of mapping QTL genes using a population. Also shown are the cadmium concentrations in the aboveground parts of seedlings (21 days old) exposed to a solution containing 0.5 μM cadmium for 7 days. Data are mean ± standard deviation (SD) (n≧3). Significant differences were determined by ANOVA and are indicated with an asterisk (P<0.05). c. Differences in the QTL region between Koshihikari and Pokkali are shown. Figure 3 shows the results of analyzing the overlapping region of OsNramp5 in Pokkali. a. Schematic diagrams of the gene encoding the OsNramp5 protein in the genomes of Koshihikari and Pokkali are shown. b and c. Fragments amplified from Koshihikari and Pokkali using specific primers indicated by arrows in Figure 3a are shown. d. Schematic diagram of the markers used to determine the genome copy number of Pokkali. Gray indicates the overlapping region of OsNramp5. e. Figure 3 shows the genome copy numbers of various markers in Pokkali. The genome copy numbers were determined by RT-PCR using different primers shown in Figure 3d, and OsLsi1 was used as an internal standard. Data are means ± SD (n = 3). Figure 3 shows the results of sequence comparison of the coding region of OsNramp5 protein in Koshihikari and Pokkali. Figure 3 shows the results of amino acid alignment of OsNramp5 protein in Koshihikari and Pokkali. Figure 3 shows the results of comparison of genome copy number and expression pattern of OsNramp5 in Koshihikari and Pokkali. a. The genome copy number of OsNramp5 in Koshihikari and Pokkali. The genome copy number was determined by quantitative PCR using OsLsi1 as an internal standard. Data are means ± SD (n = 3). An asterisk indicates a significant difference from Koshihikari (*P < 0.05 by ANOVA test). b. 1 shows the results of analyzing OsNramp5 expression in different parts of the roots of Pokkali and Koshihikari. ２Seedlings (5 days old) grown in a greenhouse were exposed to 0.5 μM Cd for 12 hours, and then different regions, including the 0-1 cm and 1-2 cm regions from the root tip, were sampled and subjected to OsNramp5 expression analysis. Asterisks indicate significant differences from Koshihikari (*P<0.05, ANOVA test). Data represent mean ± SD (n=3). c and d. OsNramp5 expression in Koshihikari and Pokkali roots in response to Cd supply (c) and Mn deficiency (d). Roots from seedlings grown with or without 0.5 μM Cd for 1 day or 0.5 μM Mn for 3 days were harvested and RNA extracted. Expression levels were determined by quantitative RT-PCR. Histone H3 was used as an internal standard. Expression relative to Koshihikari is shown. Data represent mean ± SD (n=4). Asterisks indicate significant differences compared to Koshihikari (*P<0.05 ANOVA test). Circles indicate the original data for Koshihikari, and squares indicate the original data for Pokkali. Figure 7 shows the tissue and cellular localization and transport activity of OsNramp5 in Pokkali. a. Localization of OsNramp5 in Pokkali roots. b. The enlarged image within the dotted box in Figure 7a shows polar localization in the exodermis (ex) and endodermis (en). Primary roots of seedlings (4 days old) were sampled and immunostained with OsNramp5 antibody. Red indicates the signal from the OsNramp5 antibody, and cyan indicates cell wall autofluorescence. Scale bar = 100 μm. c. Transport activity of two OsNramp5 alleles. Empty vector pDR196, OsNramp5 derived from Koshihikari ５０５Ａ or OsNramp5 from Pokkali ５０５Ｔ Yeast cells (Cd-sensitive mutant Δycf1) into which the vector was introduced were spotted at different dilutions onto synthetic dropout (SD)-uracil medium with or without 4 μM Cd. ５０５Ａ or OsNramp5 ５０５Ｔ Yeast cells (Δsmf1) into which the vector was introduced were spotted at different dilutions onto SD-uracil medium with or without 5 mM EGTA. The plates were incubated at 30°C for 3 days. VC in c and d indicates the vector control. Koshihikari and BC ４ F ３The results of comparing the genome copy number, gene expression, and localization of OsNramp5 in the lines are shown. ４ F ３ The genome copy number of OsNramp5 in the lines is shown. The genome copy number was determined by quantitative PCR using OsLsi1 as an internal standard. Data are shown as mean ± SD (n = 3). Asterisks indicate significant differences from Koshihikari (*P < 0.05 ANOVA test). b. Koshihikari and BC ４ F ３ The figure shows the expression of OsNramp5 in the roots of the lines. Seedlings (12 days old) grown in half-concentration Kimura B aqueous solution were harvested and subjected to OsNramp5 expression analysis. Expression levels were determined by quantitative RT-PCR. Histone H3 was used as an internal standard. Expression relative to Koshihikari is shown. Data are mean ± SD (n = 4). Asterisks indicate significant differences from Koshihikari (*P < 0.05 ANOVA test). c and d. Koshihikari (c) and BC ４ F ３ Figure 8(d) shows the localization of OsNramp5 in the roots of line (d). Figure 8(e) shows the signal intensity of OsNramp5 in the exodermis and endodermis of lines (c) and (d). Primary roots from seedlings (4 days old) were collected and immunostained with OsNramp5 antibody. Red indicates the signal from the OsNramp5 antibody, and cyan indicates autofluorescence of the cell wall. Scale bar = 100 μm. OsNramp5 signal intensity was quantified using LAS X software, and the intensity relative to the exodermis of Koshihikari is shown. Data in (e) represent mean ± SD (n = 18). Asterisks indicate significant differences from Koshihikari (***P < 0.001 ANOVA test). Circles indicate Koshihikari, and squares indicate BC. ４ F ３ The original data for each strain are shown. ４ F ２ / BC ４ F ３ The results of physiological and agronomic evaluation of the lines are shown. a-c. Cadmium accumulation in roots and aboveground parts at different cadmium concentrations. Koshihikari and BC ４ F ３Seedlings (20 days old) of the lines were exposed to solutions with different concentrations ranging from 0.1 μM to 5 μM for one day. Cadmium concentrations in the shoots (a) and roots (b) were measured by ICP-MS, and the translocation rate of cadmium from the roots to the shoots (c) was calculated. In the figure, conc. indicates concentration, and DW indicates dry weight (wp). d-j. Koshihikari and BC ４ F ２ The results of a comparison of the agronomic traits of the lines are shown. d shows a photograph at harvest (scale bar is 15 cm). Grain yield per plant (e), dry weight of rice straw (f), cadmium concentration in brown rice and polished rice (g), cadmium concentration in rice straw (h), Mn concentration in brown rice (i), and Mn concentration in rice straw (j) were measured. Data in a-c and e-j show mean ± SD (n = 4). Asterisks indicate significant differences from Koshihikari (*P < 0.05 ANOVA test). Circles indicate Koshihikari, and squares indicate B and C. ４ F ３ / BC ４ F ２ The original data for each line is shown. ４ F ３ The deposition patterns of Mn and Cd in the roots of the lines are shown. a-d. Koshihikari (a, c) and BC ４ F ３ Mapping images of Mn (a, b) and Cd (c, d) in the roots of the lines (b, d) are shown (scale bar 200 μm). e. Koshihikari (c) and BC ４ F ３ The Cd signal intensity in line (d) is shown. Seedlings (5 days old) were exposed to a solution containing 0.5 μM Cd for 6 hours. Five independent roots from each line were sampled for Mn and Cd mapping by LA-ICP-MS. Signal intensity was quantified using iQuant2. Data in e show mean ± SD (n = 5). Asterisks indicate significant differences from Koshihikari (*P < 0.05 ANOVA test). Circles indicate Koshihikari, and squares indicate BC. ４ F ３ The original data for each line is shown. ４ F ３ The Cd and Mn concentrations in the root cell sap and xylem sap of the lines are shown. a and b. Koshihikari and BC ４ F３ The Cd (a) and Mn (b) concentrations in the root cell sap of the lines are shown. c and d. Koshihikari and BC ４ F ３ The concentrations of Cd (c) and Mn (d) in the xylem sap of the lines are shown. Seedlings (26 days old) were exposed to 0.5 μM Cd. After 24 hours, xylem sap and root cell sap were collected. The concentrations of Mn and Cd were measured by ICP-MS. Data are mean ± SD (n = 5). Asterisks indicate significant differences from Koshihikari (*P < 0.05 ANOVA test). Circles indicate Koshihikari, and squares indicate BC. ４ F ３ The original data for each strain is shown.

[0012] Each embodiment of the present disclosure will be described in more detail below. An improved Oryza plant or plant part thereof encompassed by the present disclosure has two copies of a gene encoding the OsNramp5 protein. In this specification, such a plant may be referred to as the "improved Oryza plant of the present disclosure," etc.

[0013] OsNramp5 (Oryza sativa natural resistance-associated macrophage protein 5) protein encodes a cadmium and manganese transporter and is responsible for the first step in the uptake of cadmium from the soil solution into root cells.

[0014] An example of the amino acid sequence of the OsNramp5 protein is the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence constituting the OsNramp5 protein derived from Pokkali, a plant of the genus Oryza.

[0015] Pokkali (World Rice Core Collection number WRC96; JP95066, JP247553) is a salt-tolerant plant of the genus Oryza in the family Poaceae cultivated in southern India. As shown in the Examples below, Pokkali has two copies of the gene encoding the OsNramp5 protein on chromosome 7 (in other words, two copies of the gene encoding the OsNramp5 protein on each of the paired chromosomes). Seeds of Pokkali can be obtained, for example, from the Gene Bank of the Ministry of Agriculture, Forestry and Fisheries of Japan.

[0016] The amino acid sequence of the OsNramp5 protein may be, for example, an amino acid sequence that has 85% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, so long as the protein function is not lost. The sequence identity may be, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. For example, the amino acid sequence constituting the OsNramp5 protein derived from Koshihikari, a plant of the Oryza genus, is an amino acid sequence in which the alanine at position 505 of the amino acid sequence set forth in SEQ ID NO: 1 is substituted with threonine, and has 99% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0017] Amino acid sequence identity can be calculated using the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: / / www.ncbi.nlm.nih.gov / BLAST / using default parameters.

[0018] The amino acid sequence of the OsNramp5 protein is preferably derived from Pokkali and shown in SEQ ID NO: 1, but may also be an amino acid sequence in which one or two to ten amino acids have been deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 1 during, for example, the process of breeding, as long as the protein's function is not lost. The upper or lower limit of this range may be 3, 4, 5, 6, 7, 8, or 9 amino acids. More specifically, it may be 2 to 9 amino acids.

[0019] An example of the nucleotide sequence of the gene encoding the OsNramp5 protein is the nucleotide sequence shown in SEQ ID NO: 2. The nucleotide sequence shown in SEQ ID NO: 2 represents the nucleotide sequence of the gene (cDNA) encoding the OsNramp5 protein derived from Pokkali.

[0020] The base sequence of the gene encoding the OsNramp5 protein is preferably a base sequence having a certain level of identity to the base sequence shown in SEQ ID NO: 2. The certain level of identity is, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. For example, the base sequence of the gene (cDNA) encoding the OsNramp5 protein derived from Koshihikari, a plant of the genus Oryza, is a sequence in which the cytosine at position 985 of the base sequence shown in SEQ ID NO: 2 is replaced with thymine, the thymine at position 1311 is replaced with cytosine, and the guanine at position 1513 is replaced with adenine.

[0021] The identity of nucleotide sequences can be calculated using commercially available analytical tools or those available via telecommunications lines (Internet), for example, software such as FASTA, BLAST, PSI-BLAST, and SSEARCH. Specifically, the main initial conditions generally used in BLAST searches are as follows: In other words, in Advanced BLAST 2.1, the nucleotide sequence identity value (%) can be calculated by performing a search using the blastn program with various parameters set to default values.

[0022] The improved Oryza plant of the present disclosure has two copies of the gene encoding the OsNramp5 protein, but may have three or more copies as long as the desired effects of the present disclosure are achieved. In this specification, the number of copies of a gene refers to the total of two genes located on each of a pair of chromosomes as one copy. In other words, the improved Oryza plant of the present disclosure has two copies of the OsNramp5 protein-encoding gene on each of the pair of chromosomes (a total of four copies) (homozygote), but may also have two copies of the OsNramp5 protein-encoding gene on one of the pair of chromosomes (heterozygote), as long as the desired effects of the present disclosure are achieved.

[0023] The improved Oryza plant of the present disclosure has two copies of the gene encoding the OsNramp5 protein, and the two copies may be a single type or a combination of two types. In other words, the improved Oryza plant of the present disclosure only needs to have two copies of the gene encoding at least one type of OsNramp5 protein. In particular, in the improved Oryza plant of the present disclosure, at least one copy of the gene encoding the OsNramp5 protein is preferably a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 2. Alternatively, for example, both copies may be polynucleotides consisting of the nucleotide sequence set forth in SEQ ID NO: 2.

[0024] The improved Oryza plant of the present disclosure can have two copies of the gene encoding the OsNramp5 protein on at least one chromosome selected from the group consisting of chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In particular, it is preferable that the plant have two copies of the gene encoding the OsNramp5 protein on chromosome 7.

[0025] The method for measuring the copy number of the gene encoding the OsNramp5 protein in the improved Oryza plant of the present disclosure is not particularly limited, and examples thereof include a sequence analysis method, and a method using PCR or RT-PCR to compare the copy number of the gene encoding the OsNramp5 protein with that in Pokkali.

[0026] Examples of Oryza plants include Asian rice (Oryza sativa), African rice, and Oryza glaberrima. Of these, Asian rice is preferred. Examples of Asian rice include japonica species (O. sativa subsp. japonica) and indica species (O. sativa subsp. indica). Of these, japonica species is preferred. Examples of Japonica varieties include Koshihikari, Koshihikari BL, Akitakomachi, Sasanishiki, Hitomebore, Kinuhikari, Milky Queen, Tsuyahime, Hinohikari, Aichi no Kaori, Ichihomare, Oborozuki, Kaze Sayaka, Kinuhikari, Kirara 397, Ginga no Shizuku, Koshibuki, Kin'iro no Kaze, Sakihokore, Satoyama no Tsubu, Shinnosuke, and Seiten. Some of the names include Noboliki, Date Masayume, Tsugaru Romance, Tsukiakari, Tsuyahime, Tentakaku, Ten no Tsubu, Donpishari, Nanatsuboshi, Nikomaru, Haenuki, Hatsushimo, Hyakumankoku, Fusakogane, Fukkuriinko, Futomi Tomi, Masushigura, Mineasahi, Mori no Kumasan, Yukiwakamaru, Yume Obako, Yumepirika, Yumemizuho, ​​Akebono, Omachi and Asahi.

[0027] As used herein, the term "improved rice plant" refers to a rice plant having a genomic region that is not derived from Pokkali. In other words, as used herein, "improved rice plant" does not include Pokkali.

[0028] The improved Oryza plant of the present disclosure may be a genetically modified plant or a non-genetically modified plant, with a non-genetically modified plant being preferred.

[0029] Examples of plant parts include plant parts other than roots and processed products thereof. Examples of plant parts other than roots include seeds (e.g., rice grains, seed rice, etc.) and leaves. For example, above-ground parts may also be used. Examples of processed plant parts other than roots include polished rice, brown rice, cooked rice, rice flour, processed rice products (including no-wash rice), straw, etc.

[0030] The improved Oryza plants of the present disclosure can be produced by the production methods of the present disclosure described below.

[0031] It has been reported that knockout of the gene encoding the OsNramp5 protein reduces cadmium accumulation in rice grains by more than 90%, but often leads to growth inhibition and yield loss, especially under manganese-limited conditions (Non-Patent Document 2). This is because OsNramp5 also functions as the primary transporter of the essential metal manganese, and knockout of this gene is thought to cause Mn deficiency in rice. Conversely, overexpression of OsNramp5 under the control of the rice Actin1 or maize Uniquitin promoter unexpectedly reduced cadmium accumulation in both the shoots and rice grains, but also reduced grain yield and manganese concentration in the grains. Although the cause is unclear, this effect of overexpression may be due to increased abundance, altered cellular localization, or loss of polarity of the overexpressed OsNramp5 protein (Non-Patent Document 3). As shown in the Examples below, the improved Oryza plants of the present disclosure have reduced cadmium content in straw, brown rice, polished rice, etc., even when cultivated in a cadmium-contaminated field, without substantially affecting yield. It is speculated that the duplication (two copies) of the gene encoding the OsNramp5 protein in the improved Oryza plants of the present disclosure increases the uptake of cadmium and manganese into root cells, resulting in manganese competing with and inhibiting the translocation of cadmium to the aboveground parts, thereby reducing the cadmium content in the aboveground parts.

[0032] The present disclosure provides: (I) crossing Pokkali with a plant of the genus Oryza other than Pokkali to produce F １ (II) obtaining an individual; １ Self-fertilize the individual and F ２ (A) obtaining the individual; and ２ The present disclosure also encompasses a method for producing an improved Oryza plant, the method comprising the steps of: (I) crossing Pokkali with an Oryza plant other than Pokkali to produce F １ (II') optionally obtaining the F １ Self-fertilize the individual and F ２(III) obtaining an individual; １ Individual or F ２ The individual was backcrossed with the rice plant other than Pokkali, and BC １ F １ (IV) optionally, obtaining the BC １ F １ Repeat steps (II') to (III) n-1 times using the individual to obtain BC ｎ F １ (A) confirming that the individuals obtained in at least one step selected from the group consisting of steps (I) to (IV) have two copies of the gene encoding the OsNramp5 protein. In this specification, these methods may be referred to as "the production methods of the present disclosure."

[0033] In the production method of the present disclosure, the Oryza plant used for crossbreeding with Pokkali (the Oryza plant may be referred to as the "parent Oryza plant") is not particularly limited as long as it is a Oryza plant other than Pokkali. For example, any generally available Oryza plant can be used. Examples of Oryza plants other than Pokkali include Asian rice (Oryza sativa), African rice, and Oryza glaberrima. Of these, Oryza is preferred. Examples of Asian rice include the japonica species (O. sativa subsp. japonica) and the indica species (O. sativa subsp. indica). Of these, the japonica species is most preferred. Examples of Japonica varieties include Koshihikari, Koshihikari BL, Akitakomachi, Sasanishiki, Hitomebore, Kinuhikari, Milky Queen, Tsuyahime, Hinohikari, Aichi no Kaori, Ichihomare, Oborozuki, Kaze Sayaka, Kinuhikari, Kirara 397, Ginga no Shizuku, Koshibuki, Kin'iro no Kaze, Sakihokore, Satoyama no Tsubu, Shinnosuke, and Seiten. Some of the names include Noboliki, Date Masayume, Tsugaru Romance, Tsukiakari, Tsuyahime, Tentakaku, Ten no Tsubu, Donpishari, Nanatsuboshi, Nikomaru, Haenuki, Hatsushimo, Hyakumankoku, Fusakogane, Fukkuriinko, Futomi Tomi, Masushigura, Mineasahi, Mori no Kumasan, Yukiwakamaru, Yume Obako, Yumepirika, Yumemizuho, ​​Akebono, Omachi and Asahi.

[0034] In step (I), the method for crossbreeding Pokkali with a plant of the genus Oryza other than Pokkali is not particularly limited. It can be performed according to or in accordance with a known method. Furthermore, the method for cultivating the plant from seeds can also be a method according to or in accordance with a known cultivation method.

[0035] The method of the present disclosure further comprises: (II) preparing the F １ Let the individual self-pollinate (also called self-pollination) to produce F ２ In this specification, when step (II) is an optional step, it may be referred to as step (II').

[0036] In step (II), F １The method for self-pollinating individuals is not particularly limited. It can be carried out according to or in accordance with known methods. Furthermore, the method for cultivating seeds obtained by cross-pollination can also be a method according to or in accordance with known cultivation methods.

[0037] The production method of the present disclosure includes (III) the F obtained in step (I). １ The individual or F obtained in step (II) ２ The individual was backcrossed (backcross; BC) with a plant of the genus Oryza other than Pokkali, and the BC １ F １ The method may include a step of obtaining the individual. １ Individual, or F ２ The method for backcrossing the individual with a plant of the genus Oryza other than Pokkali is not particularly limited. It can be carried out according to or in accordance with a known method. Furthermore, the method for cultivating the seeds obtained by crossing can also be a method according to or in accordance with a known cultivation method. In other words, the production method of the present disclosure includes: (I) crossing Pokkali with a plant of the genus Oryza other than Pokkali, and producing F １ (III) obtaining an individual; and １ The individual was backcrossed with the rice plant other than Pokkali, and BC １ F １ (A) obtaining the BC obtained; １ F １ The method of the present disclosure may further comprise the step of: (I) crossing Pokkali with a plant of the genus Oryza other than Pokkali, and producing an F １ (II) obtaining an individual; １ Self-fertilize the individual and F ２ (III) obtaining an individual; and ２ The individual was backcrossed with the rice plant other than Pokkali, and BC １ F １ (A) obtaining the individual F ２ Individuals and / or BCs １ F １The method of the present disclosure may include a step of confirming that the individual has two copies of the gene encoding the OsNramp5 protein. Among others, the method of the present disclosure preferably includes steps (I), (II), and (III).

[0038] The method of the present disclosure further comprises: (IV) optionally adding the BC １ F １ Repeat steps (II') to (III) n-1 times using the individual to obtain BC ｎ F １ The expression "n" may include a step of obtaining an individual. n represents an integer of 2 or more. For example, n may be 3, 4, 5, or 6. Of these, n is preferably 4 or more. In particular, the production method of the present disclosure includes steps (I), (II), (III), and (IV), and preferably repeats steps (II) to (III) in step (IV). In other words, the production method of the present disclosure includes the following steps: (I) crossing Pokkali with a plant of the genus Oryza other than Pokkali, and obtaining F １ (II) obtaining an individual; １ Self-fertilize the individual and F ２ (III) obtaining an individual; ２ The individual was backcrossed with the rice plant other than Pokkali, and BC １ F １ (IV) obtaining the individual; and １ F １ Repeat steps (II) to (III) n-1 times using the individual to obtain BC ｎ F １ (A) obtaining an individual F (where n is an integer of 2 or more); ２ Individual, BC １ F １ Individual, BC ｎ－１ F2 individuals and BC ｎ F １Preferably, the method further comprises a step of confirming that at least one individual selected from the group of individuals has two copies of the gene encoding the OsNramp5 protein. That is, the production method of the present disclosure preferably uses Pokkali as the novo parent, and preferably uses a plant of the genus Oryza other than Pokkali as the recurrent parent. By repeating step (IV), the genome of the improved Oryza plant obtained by the production method of the present disclosure can be made closer to the genome of the parent Oryza plant, except for having two copies of the gene encoding the OsNramp5 protein.

[0039] The method of the present disclosure also includes (V) the BC １ F １ Individual or BC ｎ F １ The individual was self-fertilized m-1 times and BC １ F ｍ Individual or BC ｎ F ｍ The expression "m" may include a step of obtaining an individual. m represents an integer of 2 or more. For example, m may be 3, 4, or 5. Of these, m is preferably 2 or 3. Of these, the production method of the present disclosure includes steps (I), (II), (III), (IV), and (V), and preferably repeats steps (II) to (III) in step (IV). In other words, the production method of the present disclosure includes the following steps: (I) crossing Pokkali with a plant of the genus Oryza other than Pokkali, and obtaining F １ (II) obtaining an individual; １ Self-fertilize the individual and F ２ (III) obtaining an individual; ２ The individual was backcrossed with the rice plant other than Pokkali, and BC １ F １ (IV) obtaining the BC １ F １ Repeat steps (II) to (III) n-1 times using the individual to obtain BC ｎ F １ (V) obtaining an individual (n is an integer of 2 or more); and ｎ F １ The individual was self-fertilized m-1 times and BC ｎ Fｍ (A) obtaining an individual F (where m is an integer of 2 or more); ２ Individual, BC １ F １ Individual, BC ｎ－１ F2 individuals, BC ｎ F １ Individuals and B.C. ｎ F ｍ Preferably, the method further comprises a step of confirming that at least one individual selected from the group consisting of has two copies of the gene encoding the OsNramp5 protein.

[0040] In step (V), BC １ F １ Individual or BC ｎ F １ The method for self-pollinating individuals is not particularly limited. It can be carried out according to or in accordance with known methods. Furthermore, the method for cultivating seeds obtained by cross-pollination can also be a method according to or in accordance with known cultivation methods.

[0041] Furthermore, the production method of the present disclosure preferably includes the step of (A) confirming that the obtained individual has two copies of the gene encoding the OsNramp5 protein.

[0042] The above description can be applied to the gene encoding the OsNramp5 protein. Of the two copies of the gene encoding the OsNramp5 protein, at least one copy is preferably a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2. For example, both copies may be polynucleotides consisting of the nucleotide sequence shown in SEQ ID NO: 2, or one copy may be a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 and the other copy may be a gene encoding the OsNramp5 protein derived from a parent Oryza plant.

[0043] The method for confirming that the plant has two copies of the gene encoding the OsNramp5 protein is not particularly limited, and examples thereof include a sequence analysis method, and a method of comparing the copy number of the gene encoding the OsNramp5 protein in Pokkali or a parent plant of the genus Oryza by PCR or RT-PCR.

[0044] In the production method of the present disclosure, step (A) can be performed on the individual obtained in step (II), (III), (IV), and / or (V). For example, when the production method of the present disclosure includes step (II), the F obtained in step (II) can be ２ The method may further include a step of confirming that the individual has two copies of the gene encoding the OsNramp5 protein. In other words, step (A) can be performed after step (II). For example, when the method of the present disclosure includes step (III), the BC obtained in step (III) can be used. １ F １ The method may further comprise a step of confirming that the individual has two copies of the gene encoding the OsNramp5 protein. In other words, step (A) can be performed after step (III). For example, when the method of the present disclosure includes step (IV), the BC obtained in step (IV) can be ｎ F １ For example, when the method of the present disclosure includes step (IV) and steps (II) to (III) are repeated n-1 times in step (IV), the BC obtained in step (II) in step (IV) may be used. ｎ－１ F ２ The method may further include a step of confirming that the individual has two copies of the gene encoding the OsNramp5 protein. In other words, step (A) can be performed after step (IV) and / or after step (II) in step (IV). For example, when the method of the present disclosure includes step (V), the BC obtained in step (V) can be used. １ F ｍ Individual or BC ｎ F ｍThe method may further include a step of confirming that the individual has two copies of the gene encoding the OsNramp5 protein. In other words, step (A) can be performed during and / or after step (V).

[0045] The production method of the present disclosure may include, for example, step (A) multiple times. For example, it may include step (A) 2 to 7 times. For example, it may include step (A) 3 times, 4 times, 5 times, or 6 times. Of these, it is preferable that step (A) be included 4 or more times.

[0046] The method of production of the present disclosure is ２ It is preferable to include a step (A) of confirming that the individual has two copies of the gene encoding the OsNramp5 protein. ２ Individual, BC obtained in step (II) in step (IV) ｎ－１ F ２ The individual and BC obtained in step (V) ｎ F ２ It is preferable to carry out step (A) on at least one individual selected from the group consisting of individuals.

[0047] In a preferred embodiment of the production method of the present disclosure, for example, the method comprises: (I) crossing Pokkali with a plant of the genus Oryza other than Pokkali, and producing F １ (II) obtaining an individual; １ Self-fertilize the individual and F ２ (A) obtaining the individual F obtained in step (II); ２ (III) confirming that the individual has two copies of the gene encoding the OsNramp5 protein; ２ The individual was backcrossed with the rice plant other than Pokkali, and BC １ F １ (IV) obtaining the BC １ F １ Repeat steps (II), (A), and (III) n-1 times using the individual to obtain BC ｎ F １ (V-1) a step of obtaining an individual (n represents an integer of 2 or more), ｎ F１ Self-fertilize the individual and BC ｎ F ２ (A) a step of obtaining an individual BC obtained in step (V-1) ｎ F ２ (V-2) confirming that the individual has two copies of the gene encoding the OsNramp5 protein; and ｎ F ２ The individual was self-fertilized x-2 times and BC ｎ F ｘ An example of such a method includes a step of obtaining an individual (where x is an integer of 3 or more). The above description can be applied to n. For example, x may be 4, 5, or 6. Of these, 3 is preferable.

[0048] The production method of the present disclosure can produce the improved rice plant of the present disclosure. The production method of the present disclosure can produce a rice plant with a low cadmium content. Furthermore, the production method of the present disclosure can produce a safe rice plant with a low cadmium content that produces rice seeds with a low cadmium content even in soil with a high cadmium content. Furthermore, such rice plants have the excellent characteristic of having a low cadmium content in plant parts other than the roots.

[0049] Furthermore, the agricultural products that are the subject of the present disclosure are not necessarily limited to plants of the genus Oryza, but can also be applied to agricultural products other than plants of the genus Oryza that accumulate cadmium, and examples of such agricultural products include, but are not limited to, wheat, barley, rye, buckwheat, corn, soybean, rhizome, spinach, garland chrysanthemum, leek, onion, garlic, lily of the valley, asparagus, radish, carrot, burdock, taro, mountain yam, potato, sweet potato, cucumber, tomato, eggplant, and okra.

[0050] The effect of cadmium reduction in the present disclosure is not limited to a specific mechanism, but may be exerted via metal ion transporter proteins. Examples of such metal ion transporters (transporting metals) include AtNramp1 (Fe, Cd, Mn), AtNramp3 (Mn, Fe, Cd), AtNramp4 (Mn, Fe, Cd), AtNramp6 (Cd), OsNramp1 (Fe, Cd), OsNramp5 (Mn, Cd), TcNramp3 (Zn, Cd), TcNramp4 (Zn, Cd), TgMIP1 (Cd, Co, Zn, Ni), ZIP IRT1 (Fe, Zn, Mn, Cd), TcZNT (Zn, Cd), cation / H ＋ Antiporters include, but are not limited to, CAX2 (Ca, Mn, Cd), ABC transporter ID17 (PC-Cd, GS-Cd), OsHMA2(Cd), OsHMA4(Cd), OsLCT1(Cd), or equivalents thereof.

[0051] In particular, the cadmium-reducing plants and methods for cadmium reduction disclosed herein are characterized by containing or using a gene encoding OsNramp5 or a protein equivalent thereto, including orthologous proteins of Nramp5 such as soybean GmNramp5, wheat Nramp5 (TaNramp5A, 5B, 5D, TdNramp5A, 5B), and barley HvNramp5.

[0052] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Furthermore, the present disclosure encompasses any and all combinations of the constituent elements described herein as long as they do not deviate from the intended purpose of the present disclosure.

[0053] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure may be combined in any way to specify the subject matter included in the present disclosure, as long as the combination does not deviate from the intended purpose of the present disclosure. In other words, the present disclosure includes all subject matter consisting of all combinations of the combinable characteristics described in this specification.

[0054] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, experiments were performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "% by mass."

[0055] <Plant Materials and Growth Conditions> Rice (Oryzo saliva L.) seeds were soaked in water for 2 days in a dark place at 30°C, and then soaked in 0.5M CaCl ２ The seedlings were then transferred to a net floating in the solution. Five-day-old seedlings were transferred to 1.2 L plastic pots containing half-concentrated Kimura B solution (pH 5.6) (Plant Physiol. 2002 Dec;130(4):2111-7. doi: 10.1104 / pp.010348..). The nutrient solution was changed every two days. The plants were grown in a greenhouse at 25-30°C under natural light. Each experiment had at least three biological replicates, as indicated in the figure legends.

[0056] <Physiological Characterization of Three Rice Cultivars> To clarify the physiological characteristics of three cultivars, including Khau Tan Chiem (Japonica), Koshihikari (Japonica), and Pokkali (Indica), used in QTL analysis and gene mapping, the seedlings (21 days old) prepared as described above were exposed to 0.2 μM, 0.5 μM, or 1 μM cadmium for 7 days, or to 0.5 μM cadmium for 1, 3, 5, and 7 days. Roots were treated with 5 mM cold CaCl. ２ After washing three times with aqueous solution, the roots and aerial parts were harvested.

[0057] QTL detection and fine mapping of candidate genes: The F obtained from a cross between Pokkali and Khau Tan Chiem was analyzed according to the method described in New Phytol. 2009;182(3):644-653. (doi: 10.1111 / j.1469-8137.2009.02784.x. Epub 2009 Feb 27.) ２ A total of 96 Fs were used for measuring cadmium concentrations and extracting DNA. ２ The lines were used. Seedlings were exposed to 0.5 μM cadmium for 7 days. The genotypes of the lines at 345 SNP marker loci were determined using 768-plex SNPs for the Illumina GoldenGate BeadArray technology platform (Illumina Inc.). QTL analysis was performed using the composite interval mapping method using the software QTL Cartographer version 2.5. The threshold corresponding to P = 5% was determined using 1,000 permutations. The location of the QTL was determined as the position where the peak logarithm of the odds score exceeded the threshold. To map the major QTL on chromosome 7, a total of 14 polymorphic markers were designed and recombinant individuals were selected (Table 1).

[0058]

[0059] To fine-map the QTL gene, Pokkali was crossed with Koshihikari and backcrossed with Koshihikari three or four times to obtain the BC. ３ F ２ or B.C. ４ F ２ F ２ ~F ６ (Pokkali x KhauTan Chiem), BC ３ F ２ and B.C. ４ F ２More than 10,000 plants from the Pokkali × Koshihikari cross were genotyped and the QTL genes were mapped (Figure 1). Genes within the QTL region were sequenced using the BigDye sequencing kit (Applied Biosystems) on a SeqStudio Genetic Analyser with the primers listed in Table 2.

[0060]

[0061] To confirm the 19.2-kb overlapping region in Pokkali, primer walking was performed using different primers listed in Table 3 (Fig. 3). The overlapping and original regions of OsNramp5 in Pokkali were sequenced independently. The PCR products were directly sequenced using the primers listed in Table 2.

[0062]

[0063] <Creation of Transgenic Lines> A complementation test was performed to confirm whether the gene in the QTL region of Pokkali reduces cadmium accumulation in aboveground parts. A 9.6-kb fragment containing the second OsNramp5 promoter (7,363 bp), the OsNramp5 coding sequence (1,617 bp), and the downstream sequence (614 bp after the translation termination site) amplified from Pokkali genomic DNA was inserted into the pPZP2H-lac vector. The sequences of the primers used to prepare the construct are shown in Table 3. This construct was introduced into Koshihikari callus by Agrobacterium tumefaciens (strain EHA101)-mediated transformation. Five transgenic lines were obtained, and three lines showing doubled expression of OsNramp5 were selected for further analysis. To compare the expression level of OsNramp5 and the amount of cadmium accumulated in the shoots between the transgenic lines and Koshihikari, 21-day-old seedlings of Koshihikari and three independent complementation lines (T2) were exposed to 0.5 μM cadmium for 1 day. Roots and shoots were harvested and analyzed for OsNramp5 expression and cadmium levels in the shoots were quantified.

[0064] <Determination of genome copy number of OsNramp5> Pokkali, Koshihikari or BC ４ F ２ Genomic DNA (50 ng) isolated from leaves of the lines according to the method of Murray and Thompson (Nucleic Acids Res. 1980 Oct 10; 8(19): 4321-4325. doi: 10.1093 / nar / 8.19.4321) was used as a template. The relative genome copy number of OsNramp5 or the above markers was determined by quantitative PCR using Thunderbird SYBR qPCR Mix (Toyobo Co., Ltd.). OsLsi1, a single-copy gene encoding a rice genomic Si transporter, was used as an internal standard. The genome copy number of OsNramp5 or the markers was normalized based on the cycle threshold of OsLsi1. The primer sequences for OsNramp5 and OsLsi1 used are shown in Table 3.

[0065] <Gene Expression Analysis> To compare the spatial expression of OsNramp5 in different root regions of Koshihikari and Pokkali, 0.5 mM CaCl ２ Seedlings (5 days old) grown in BC were exposed to 0.5 μM cadmium for 12 hours, and different root regions, including 0-1 cm and 1-2 cm from the root tip, were collected for OsNramp5 expression analysis. To examine the response of OsNramp5 expression to Mn deficiency in Koshihikari and Pokkali roots, seedlings (9 days old) were cultured in a nutrient solution with or without 0.5 μM manganese. After 3 days, roots were harvested for RNA extraction. The response of OsNramp5 expression to cadmium was examined by exposing seedlings (11 days old) to 0.5 μM cadmium or no cadmium for 1 day. ４ F ３To compare OsNramp5 expression with other lines, roots from 12-day-old seedlings grown in half-concentrated Kimura B solution were harvested. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) and converted to cDNA using ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo Co., Ltd.). OsNramp5 expression was determined by quantitative RT-PCR using Thunderbird SYBR qPCR mix (Toyobo Co., Ltd.) on a CFX96 (Bio-Rad). Histone H3 was used as an internal control. Relative expression was normalized using the ΔΔCt method using CFX Manager software (Bio-Rad). The primer sequences used for gene expression analysis are shown in Table 3.

[0066] <Immunostaining of OsNramp5 and OsLsi1> Immunostaining of OsNramp5 and OsLsi1 was performed using Pokkali, Koshihikari, and BC according to Yamaji and Ma (Plant Physiol. 2007 Mar;143(3):1306-13. doi: 10.1104 / pp.106.093005. Epub 2007 Jan 26.). ４ F ３ The study was performed on three lines. Germinated seedlings were exposed to a half-concentration Kimura B solution for two days, and then primary roots from these lines were harvested and immunostained using OsNramp5 or OsLsi1 antibodies. Fluorescence from the secondary antibody (Alexa Fluor 555 goat anti-rabbit IgG; Invitrogen) was observed using a confocal laser scanning microscope TCS SP8x (Leica Microsystems). Signal intensity was quantified using 18 independent images using LAS X software (Leica Microsystems).

[0067] <Transporter activity of OsNramp5 in yeast> A yeast assay was performed to compare the transport activity of OsNramp5 from Pokkali and Koshihikari. ５０５Ａ and OsNramp5 ５０５ＴThe open reading frames (ORFs) of these genes were amplified from the cDNAs of Koshihikari and Pokkali, respectively. The primers used are shown in Table 3. These two ORFs were inserted into the pDR196 vector (Addgene). After sequence confirmation, these two plasmids or an empty vector were introduced into yeast according to the manufacturer's protocol (Sc easy comp transformation kit, Invitrogen). The cadmium-sensitive mutant Δycf1 was used for the cadmium transport activity assay, and the Mn uptake-deficient yeast mutant Δsmf1 was used for the Mn transport activity assay (Nat. Commun. 2013;4:2442. doi: 10.1038 / ncomms3442.). To evaluate cadmium tolerance, the empty vector pDR196, OsNramp5, and the OsNramp5 vector were used. ５０５Ａ or OsNramp5 ５０５Ｔ Yeast expressing the plasmids were spotted at different dilutions onto SD-uracil medium with or without 4 μM cadmium. Complementation of the Δsmf1 phenotype was achieved by reducing Mn availability by chelating Mn at varying dilutions in SD-uracil medium with or without 5 mM ethylene glycol tetraacetic acid (EGTA). Plates were incubated at 30°C for 3 days and photographed. To quantify Mn and Cd transport activity, liquid experiments were performed using the yeast mutant Δycf1. Yeast cells transfected with different constructs were pre-cultured overnight in liquid medium, pelleted by centrifugation, and washed three times with Milli-Q water (Millipore). The resulting pellet was resuspended in the same liquid medium in the absence and presence of 1 μM Cd or 3 μM Mn. Uptake experiments were performed at 30°C and 200 rpm. The cells were shaken horizontally at 4°C for 1 hour, and at the end of the experiment, the cells were resuspended in cold 1 mM CaCl ２ The dry weight of the yeast cells was recorded after drying in an oven at 70°C for 24 hours, and the yeast was washed three times with 5% HNO for Cd and Mn determination. ３ The effect of different Mn concentrations on cadmium uptake by yeast cells was also investigated. ５０５Ａ or OsNramp5 from Pokkali ５０５ＴYeast cells transfected with Δycf1 (Cd-sensitive mutant) were spotted at various dilutions onto SD-uracil medium (basal 3 μM Mn) supplemented with or without 2 μM Cd. Mn was added at different concentrations ranging from 0 μM to 100 μM. The plates were incubated at 30°C for 3 days and then photographed.

[0068] <BC ４ F ３ Phenotypic analysis of strains > Koshihikari and BC ４ F ３ To compare the amount of cadmium accumulated between the two strains, we first conducted a concentration-dependent experiment in a hydroponic solution. Seedlings (20 days old) were exposed to aqueous solutions containing different cadmium concentrations, ranging from 0 μM to 5 μM, for 1 day. Before harvesting, the roots were treated with 5 mM cold CaCl. ２ The roots were washed three times with the aqueous solution and then separated from the above-ground parts with a razor. Samples of the roots and above-ground parts were used for cadmium measurement, as described below. ４ F ２ Seedlings (14 days old) of the line were placed in 120 kg of cadmium-contaminated soil (16.4 mg Cd kg －１ The plants were then transplanted into containers filled with water. The plants were grown under submerged conditions in a greenhouse under natural light until the heading stage. They were then cultivated under high-altitude conditions. After four months, all lines were harvested and photographed. Straw, brown rice, and polished rice grains were subjected to mineral element analysis as described below. Polished rice grains were prepared using a Kett grain polisher (Pearless; Kett Scientific Laboratory Co., Ltd.). Taste evaluation was performed using two instrumental methods that were significantly correlated with sensory taste scores. Taste values ​​were measured using a cooked rice taste analyzer STA1A (Satake Corporation) based on the amount of near-infrared light reflected and transmitted by cooked rice grains. The physical properties of cooked rice grains, such as the hardness of the grain surface and the whole grain, and the stickiness of the grain surface and the whole grain, were measured using a Tensipresser MyBoy texture analyzer (manufactured by Takemoto Electric Co., Ltd.) by high compression, low compression, and continuous step compression methods.

[0069] <Detection of Mn and Cd deposition patterns by LA-ICP-MS> Koshihikari and BC ４ F ３Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was performed to compare the distribution patterns of Mn and Cd in the roots of the lines. ２ Five-day-old seedlings grown in aqueous solution were treated with half-concentration Kimura B solution containing 0.5 μM Mn and 0.5 μM Cd for 6 hours, after which primary roots were sampled for Mn and Cd mapping by LA-ICP-MS according to the method described in Plant J. 2019 Sep;99(6):1254-1263. doi: 10.1111 / tpj.14410. Epub 2019 Jun 26. Five independent roots were used for each line. Cd and Mn signals were acquired using an Agilent Technologies 8900 ICP-MS instrument (Agilent Technologies) operated in helium mode. The time course of the raster-scanned ICP-MS output was converted to an elemental image, and the signal intensity of the exine was quantified using iQUANT2 software.

[0070] <Analysis of root cell sap and xylem sap> Koshihikari and BC ４ F ３ To compare the Mn and Cd concentrations in the cell sap and xylem sap of the lines, seedlings (26 days old) were exposed to 0.5 μM Cd and 0.5 μM Mn for 24 hours. The nutrient solution containing 0.5 μM Cd was replaced 4 hours before collecting xylem sap. For xylem sap collection, the above-ground part was excised with a razor blade 2 cm above the junction of the root and above-ground part, and xylem sap was collected from the cut surface using a micropipette for 30 minutes. After collecting xylem sap, the roots were soaked in 5 mM cold CaCl ２ The roots were washed three times with PBS, blotted, placed on a filter in a tube, and frozen in liquid nitrogen. After thawing at room temperature, the roots were centrifuged at 20,600 g for 10 minutes to extract the root cell sap.

[0071] <Measurement of mineral element concentrations> All plant samples were dried in an oven at 70°C for 3 days. The dried samples were digested with concentrated nitric acid (60%, w / w). The mineral element concentrations in the digestion fluid, xylem sap, and root cell sap were measured using ICP-MS (7700X; Agilent Technologies).

[0072] <Statistical Analysis> Using SPSS 22 (SPSS Inc.), ANOVA and Duncan's tests were applied to test for differences between lines and treatments at P<0.05.

[0073] <Pokkali exhibits extremely low levels of cadmium accumulation in aboveground parts> The present inventors previously investigated intervarietal differences in cadmium accumulation in aboveground parts using 146 accessions from a rice core collection representative of rice genetic diversity and found a 13-fold difference in cadmium concentrations in aboveground parts. Among the accessions with low cadmium accumulation, the indica variety Pokkali (WRC96; JP95066, JP247553) exhibited the lowest cadmium levels in aboveground parts. The present inventors first clarified the physiological characteristics of Pokkali by comparing it with Khau Tan Chiem (japonica) and Koshihikari (japonica), which were used to create the mapping population. When grown in soil for the same period as Koshihikari, Pokkali was significantly taller. Furthermore, the aboveground biomass of Pokkali was 4.5 times that of field-grown Koshihikari. Analysis of cadmium accumulation showed that at cadmium concentrations between 0.2 μM and 1.0 μM in the nutrient solution, Pokkali accumulated significantly less cadmium in its aboveground parts than Khau Tan Chiem and Koshihikari. Furthermore, time-dependent experiments showed that Pokkali consistently accumulated lower cadmium in its aboveground parts than the other two lines, regardless of the cadmium exposure time. These results confirm that Pokkali can suppress cadmium accumulation in its aboveground parts, regardless of the cadmium concentration or exposure time.

[0074] <QTL detection and mapping of low cadmium accumulation gene> To perform QTL analysis for low cadmium accumulation in Pokkali, F ２A population was created. Analysis of the genotype and cadmium accumulation in the aboveground parts revealed two QTLs on chromosomes 1 and 7. These two QTLs explained 23.5% and 52.4%, respectively, of the total cadmium accumulation in the aboveground parts (Fig. 1a). To map the gene for the major QTL on chromosome 7, we first performed F1 of the Pokkali × Khau Tan Chiem population. ２ From F ６ A total of 14 polymorphism makers (Table 1) were designed for large-scale screening of recombinant plants using F (Fig. 1b). The QTL was first mapped to a 287.8 kilobase (kb) region between SSR markers RM21321 and RM5436 (Fig. 2a). ５ ~F ６ Using advanced progeny of this strain, the QTL was further refined to a 24-kb region between markers CHR7-8878 and CHR7-8902 (Fig. 1a). To further refine this region, another mapping population was generated from a cross between Pokkali and Koshihikari.

[0075] Because Koshihikari is an elite variety with good eating quality in Japan, this population was also used in the first attempt to develop low-cadmium varieties in the Koshihikari background. ３ F ２ Population and BC ４ F ２Using the population (Fig. 1c), we further defined the candidate gene in a 4.8-kb region between two single-nucleotide polymorphism (SNP) markers, SNP-8881 and SNP-8886, located upstream of the original OsNramp5 gene (Fig. 2b, c). Next, we sequenced this region in Pokkali and Koshihikari using specific primers and found a 19.2-kb insertion in Pokkali (Fig. 2c and Fig. 3a). Furthermore, this insertion contained another OsNramp5 gene, indicating a tandem duplication of OsNramp5 in Pokkali (Fig. 1c, Fig. 3a, b). Using different primer pairs (Fig. 3a), we were able to detect one and two copies of the gene encoding the OsNramp5 protein in Koshihikari and Pokkali, respectively (Fig. 3b, c). Furthermore, primer walking confirmed that OsNramp5 in Pokkali is duplicated (Fig. 3d, e). Although there were no differences in the sequence of OsNramp5 between the two copies in Pokkali, when compared with OsNramp5 in Koshihikari, there were three SNPs in the coding region (Fig. 4). In Pokkali, only one SNP, G to A at position 1,513 (from ATG), was found to be an Ala. ５０５ From Thr ５０５ This resulted in an amino acid substitution to the OsNramp5 promoter (Figure 5). To confirm this QTL region, we created transgenic lines by introducing a 9.6-kb fragment containing the 7,363-base pair (bp) second OsNramp5 promoter from Pokkali, the 1,617-bp OsNramp5 coding sequence, and the 614-bp native terminator from Koshihikari into the Koshihikari background. The transgenic lines showed approximately two-fold higher OsNramp5 expression than Koshihikari, and the transgenic lines also showed lower cadmium concentrations in the shoots than Koshihikari. These results clearly demonstrate that low Cd accumulation in Pokkali is associated with the duplication of OsNramp5.

[0076] Detection of OsNramp5 duplication in the rice core collection. We investigated the genome copy number of OsNramp5 in 132 varieties obtained from the rice core collection, which covers most of the genetic and phenotypic diversity of the original population. As a result, we found that only Pokkali had a duplicated OsNramp5 gene.

[0077] The above mapping results revealed that the gene encoding the OsNramp5 protein is duplicated in Pokkali (Fig. 2). To further confirm this result, we first compared the genome copy number of OsNramp5 between Pokkali and Koshihikari. Consistent with the mapping results (Fig. 2), we found that the copy number of OsNramp5 in Pokkali was twice that of Koshihikari (Fig. 6a). Therefore, we investigated the effect of this duplication on OsNramp5 expression. Spatial expression analysis in roots revealed that OsNramp5 expression levels were higher in the mature root region (1-2 cm) than in the root tip region (0-1 cm) in both Pokkali and Koshihikari (Fig. 6b). In the mature root zone, OsNramp5 expression levels were approximately two-fold higher in Pokkali than in Koshihikari (Fig. 6b). The expression level of OsNramp5 was unaffected by exposure to cadmium and manganese deficiency in both Pokkali and Koshihikari (Fig. 6c, d), indicating that the response of OsNramp5 to cadmium or manganese deficiency was unchanged in Pokkali. However, the expression level of OsNramp5 in Pokkali roots was always approximately twice that of Koshihikari roots (Fig. 6c, d).

[0078] <Localization and Transport Activity of OsNramp5> To investigate whether the duplication of the gene encoding OsNramp5 protein alters the cell specificity of the localization of the encoded protein, we performed immunostaining in Pokkali using an antibody specific for OsNramp5 (Plant Cell. 2012 May;24(5):2155-67. doi: 10.1105 / tpc.112.096925. Epub 2012 May 15.). OsNramp5 was polarly localized to the distal side of both the exodermis and endodermis in the primary roots of Pokkali (Fig. 7a, b). We also investigated whether amino acid substitutions in OsNramp5 affect its transporter activity for cadmium. OsNramp5 from Koshihikari ５０５Ａ and OsNramp5 from Pokkali ５０５Ｔ was expressed in yeast. As a result of measuring the transport activity, OsNramp5 ５０５Ａ and OsNramp5 ５０５Ｔ Both OsNramp5 and OsNramp5 were found to transport cadmium and manganese (Fig. 7c, d). ５０５Ａ and OsNramp5 ５０５Ｔ There was no difference in the Cd and Mn transport activity between the two OsNramp5 proteins. Furthermore, we investigated the effect of different Mn concentrations on cadmium uptake by the two OsNramp5 proteins. ５０５Ａ or OsNramp5 ５０５Ｔ Growth of yeast expressing OsNramp5 was similarly inhibited in the presence of cadmium. As the Mn concentration increased, cadmium toxicity decreased in yeast expressing both proteins, further indicating competition between Cd and Mn for Nramp5. These results indicate that this single amino acid substitution in OsNramp5 does not affect its transport activity for both Cd and Mn.

[0079] <Introduction of OsNramp5 into Koshihikari> To develop a rice variety with low cadmium accumulation, the duplicated Nramp5 from Pokkali was introduced into Koshihikari by backcrossing, resulting in a BC with the Pokkali allele, i.e., two copies of the gene encoding the OsNramp5 protein (two copies of the gene encoding the OsNramp5 protein on each of the paired chromosomes). ４ F ２ Lineage and BC ４ F ３ Koshihikari is the most popular rice variety in Japan, but it accumulates relatively high amounts of cadmium. ４ F ３ The lines had double the genome copy number and expressed OsNramp5 in the roots (Fig. 8a, b). ４ F ３ OsNramp5 in the BC line was polarly localized in the distal parts of the root epidermis and endodermis, similar to Koshihikari (Fig. 8c, d). However, the abundance of OsNramp5 in both tissues was significantly higher than that in the BC line. ４ F ３ The protein abundance of the root Si transporter Lsi1, which was the same as that used to detect OsNramp5, was also compared. ４ F ３ In the lineage, Lsi1 abundance was comparable in both the exodermis and endodermis.

[0080] Koshihikari and BC ４ F ３ The amount of cadmium accumulated in the lines was compared in a dose-response experiment. ４ F ３ In the lines, the Cd concentration in the shoots increased linearly as the Cd concentration in the external solution increased from 0 μM to 5 μM (Fig. 9a). ４ F ３ Compared with Koshihikari, the BC line accumulated significantly less cadmium in the shoots under all cadmium conditions tested (Fig. 9a). On the other hand, the cadmium concentration in the roots was significantly lower in the BC line than in Koshihikari. ４ F ３The results showed that the BC ４ F ３ In the BC line, the translocation of cadmium from roots to shoots was significantly reduced (Fig. 9c). １ F １ A decrease in cadmium concentration was confirmed in individual plants compared to Koshihikari.

[0081] When grown in pots with cadmium-contaminated soil, BC ４ F ２ The lines showed growth similar to that of Koshihikari (Fig. 9d). ４ F ２ Agronomic traits such as plant height, number of burrs, straw dry weight, fertility, number of grains per plant, thousand-grain weight, and grain yield per plant were compared among the lines, but no significant differences were observed in these traits (Fig. 9e, f). ４ F ２ The Cd concentrations in brown rice and rice straw of the lines were reduced by 64% and 44%, respectively, compared to Koshihikari (Fig. 9g, h). ４ F ２ The Cd concentration in the lines was 67% lower than that in Koshihikari (Fig. 9g). ４ F ２ Although the concentrations of other mineral elements, including Mg, P, K, Ca, Fe, Cu, and Zn in brown rice and rice straw, were 27% and 74% higher in the BC line than in Koshihikari (Fig. 9i, j), respectively. ４ F ２ was comparable between strains.

[0082] B.C. ４ F ２ To investigate whether the taste changes between strains, Koshihikari and BC ４ F ２ The taste scores of the lines, stickiness and hardness of cooked rice grains were compared. ４ F ２ The results were comparable across strains.

[0083] <Elucidation of the mechanism of low cadmium accumulation> The above results revealed that duplication of the Cd / Mn transporter gene OsNramp5 doubled its expression in roots, resulting in reduced Cd accumulation in the shoots, brown rice, and polished rice (Fig. 2 and Fig. 9a, g, h). To explore this mechanism, we compared the distribution patterns of Mn and Cd in roots using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Mapping revealed that BC ４ F ３ The roots of the BC lines accumulated more Mn and Cd than those of Koshihikari (Fig. 10a, b). ４ F ３ In the lines, Cd accumulated in the root exodermis significantly more (1.7-fold) than in the lines (Fig. 10c-e). These results suggest that the uptake of cadmium was ４ F ３ This confirms that cadmium was retained in the roots, especially in the exodermis, of the lines, resulting in reduced translocation of cadmium to the xylem. Furthermore, the Mn and Cd concentrations in the root cell sap and xylem sap of Koshihikari and BC were ４ F ３ The Mn and Cd concentrations in the root cell sap were higher in BC than in Koshihikari (Fig. 11a-d). ４ F ３ The results were significantly higher in the BC lineage (Fig. 11a, b). ４ F ３ This coincided with the increased expression of OsNramp5 in the BC lines (Fig. 8b). ４ F ３ The Mn concentration in the BC line was 65% lower than that in Koshihikari (Fig. 11c). ４ F ３ The Cd concentration in the root cell sap was much higher than that in the xylem sap (Fig. 11a, b), whereas the Cd concentration in the xylem sap was much lower than that in the root sap (Fig. 11c, d). These results suggest that high levels of Mn in root cells hinder the translocation of Cd to the aboveground parts.

Claims

1. An improved rice plant or plant part thereof having two copies of the gene encoding the OsNramp5 protein.

2. The improved Oryza plant or plant part thereof according to claim 1, wherein the Oryza plant is Oryza sativa L.

3. The improved Oryza plant or plant part thereof according to claim 1, wherein said Oryza plant is a Japonica species.

4. An improved rice plant or a plant part thereof according to claim 1 or 2, wherein the plant part is a plant part other than a root or a processed product thereof.

5. A method for producing an improved rice plant, comprising: (I) crossing Pokkali with a rice plant other than Pokkali, and obtaining F １ (II) obtaining an individual; １ Self-fertilize the individual and ２ (A) obtaining the individual; and ２ confirming that the individual has two copies of the gene encoding the OsNramp5 protein.

6. A method for producing an improved rice plant, comprising: (I) crossing Pokkali with a rice plant other than Pokkali, and obtaining F １ (II') optionally obtaining an individual; １ Self-fertilize the individual and ２ (III) obtaining an individual; １ Individual or F ２ The individual was backcrossed with the rice plant other than Pokkali to obtain BC １ F １ (IV) optionally obtaining the individual; and １ F １ The steps (II') to (III) are repeated n-1 times using the individual, and BC ｎ F １ The method includes the steps of: obtaining an individual (n is an integer of 2 or more); and (A) confirming that the individual obtained in at least one step selected from the group consisting of steps (I) to (IV) has two copies of a gene encoding OsNramp5 protein.

7. (V) The above BC １ F １ Individual or BC ｎ F １ The individual was selfed m-1 times and BC １ F ｍ Individual or BC ｎ F ｍ The method according to claim 6, further comprising the step of obtaining an individual, wherein m is an integer of 2 or more.

8. The method according to claim 5 or 6, wherein the Oryza plant other than Pokkali is Oryza sativa.

9. The method according to claim 5 or 6, wherein the Oryza plant other than Pokkali is a Japonica species.

10. A method for producing an improved rice plant, comprising: (I) crossing Pokkali with a rice plant other than Pokkali, and obtaining F １ (II) obtaining an individual; １ Self-fertilize the individual and ２ (A) obtaining an individual F obtained in step (II); ２ (III) confirming that the individual has two copies of a gene encoding the OsNramp5 protein; ２ The individual was backcrossed with the rice plant other than Pokkali to obtain BC １ F １ (IV) obtaining the individual; １ F １ Repeat steps (II), (A), and (III) further n-1 times using the individual to obtain BC ｎ F １ (V-1) obtaining an individual (n is an integer of 2 or more); ｎ F １ Self-fertilize the individual and BC ｎ F ２ (A) obtaining an individual BC obtained in step (V-1); ｎ F ２ (V-2) confirming that the individual has two copies of a gene encoding the OsNramp5 protein; and ｎ F ２ The individual was selfed x-2 times to produce BC ｎ F ｘ Obtaining an individual, wherein x is an integer of 3 or greater.

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