A method for rapidly screening low-cadmium mutants in a rice mutant library using the OsNramp5 gene

Abstract

The invention belongs to the technical field of rice breeding and specifically relates to a method of using OsNramp5 A method for rapid screening of low-cadmium mutants in a rice mutant library. OsNramp5 Inactivation of gene function can generate mutants with low cadmium trait, providing a method for utilizing OsNramp5 A method for quickly screening low-cadmium mutants in a rice mutant library. The method is to establish a mutant library population seed, then plant and culture it, perform phenotypic detection on the seedlings after a certain period of culture, screen candidate target mutants, perform mutation detection on the OsNramp5 gene sequence of the target mutant by sequencing to quickly lock the target mutant, and finally identify the cadmium accumulation characteristics of the target mutant to screen and obtain low-cadmium mutants. The method of the invention has low cost and simple process, only needs to perform germination screening on the seeds, and the mutants obtained by screening are all homozygous, which can save the time of obtaining homozygous individuals through additional generation reproduction and the variety breeding process, thereby improving efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of rice breeding and specifically relates to a method of using OsNramp5 A method for rapid screening of low-cadmium mutants in a rice mutant library. Background Art

[0002] There is a prominent cadmium pollution problem in the rice-growing areas in southern my country. Cultivating and planting low-cadmium rice varieties is currently the most economical and quickest way to solve the cadmium pollution problem in rice. The main gene loci used in the cultivation of low-cadmium rice in China are OsNramp5 Functional inactivation mutant alleles of genes. Since Japanese scholars have shown OsNramp5 Gene function inactivation mutation can significantly reduce the cadmium content in grains without affecting other agronomic traits {Ishikawa S, Ishimaru Y, Igura M, et al. Ion-beam irradiation, gene identification, and marker-assisted breeding in the development of low-cadmium rice[J]. Proc Natl AcadSci USA, 2012, 109(47):19166-19171}, OsNramp5 The breeding value of genes has begun to be valued, and different domestic researchers have successively created new OsNramp5Gene mutants to quickly breed low-cadmium rice varieties {References: Hu Liming, Peng Yan, Zheng Wenjie, et al. Using CRISPR / Cas9 technology to create low-cadmium accumulation fragrant rice[J]. Hybrid Rice, 2021, 36(06):77-83; Long Qizhang, Huang Yonglan, Tang Xiuying, et al. Using CRISPR / Cas9 to knock out OsNramp5 gene to create low-cadmium indica rice[J]. Chinese Rice Science, 2019,33(05):407-420; Yang Ch, Zhang Y, Huang Cf. Reduction in cadmium accumulationin japonica rice grains by CRISPR / Cas9-mediated editing of OsNRAMP5[J].Journal of Integrative Agriculture, 2019, 18(3):688-697; Liu S, Jiang J, LiuY, et al. Characterization and Evaluation of OsLCT1 and OsNramp5 Mutants Generated Through CRISPR / Cas9-Mediated Mutagenesis for Breeding Low Cd Rice[J]. Rice Science, 2019, 26(2):88-97;Tang L, Mao B, Li Y, et al. Knockout ofOsNramp5 using the CRISPR / Cas9 system produces low Cd-accumulating indicarice without compromising yield[J]. Sci Rep, 2017, 7(1):14438}。Gene editing is currently the fastest and most efficient method for targeted mutation induction. However, according to the Regulations on the Safety Administration of Agricultural Genetically Modified Organisms, gene-edited crops are still regarded as genetically modified crops and are subject to regulation. The time when they can be used in production is still unclear, forcing breeders to abandon this method and turn to traditional mutagenesis to meet the urgent demand for low-cadmium varieties. Since mutagenesis is random and non-directional, specific types of mutants need to be screened from a larger mutant library. If low-cadmium rice varieties are screened by measuring the cadmium content of rice, the workload and cost will be enormous, which is unaffordable for general research teams and is rarely used.TILLING technology (Targeting Induced Local Lesions IN Genomes) is a traditional method for rapid and efficient screening of target gene mutants. OsNramp5 Gene mutants can also be screened out from the mutant library through the TILLING method. Researchers from the Hunan Academy of Agricultural Sciences have developed a method (also a type of TILLING technology) to screen mutants of specific genes through high-efficiency mutagenesis (C heavy ion beam) plus high-throughput screening (mixed pool second-generation sequencing) and have successfully screened out OsNramp5 Mutants can be selected and cultivated into low-cadmium rice varieties with application value. However, the workload and cost of screening mutants of specific genes from a large-scale population of more than 10,000 plants using this method are still high. In order to speed up the cultivation of low-cadmium rice varieties, it is still necessary to find simpler and cheaper methods. Summary of the invention

[0003] The purpose of the present invention is to solve the deficiencies of the prior art and provide a OsNramp5 The method for rapidly screening low-cadmium mutants in a rice mutant library specifically adopts the following technical scheme:

[0004] A use of OsNramp5 A method for rapidly screening low-cadmium mutants in a rice mutant library. OsNramp5 The internationally recognized gene identifier of the gene is at least one of Os07g0257200, LOC_Os07g15370 or LOC4342859, and the low-cadmium mutant is OsNramp5 Mutants with inactivated gene function and low-cadmium traits;

[0005] The above method specifically comprises the following steps:

[0006] S1: Screening mutant seeds from the mutant library and placing them in a 96-well PCR (Polymerase Chain Reaction) reaction plate;

[0007] S2: Soak the seeds of the mutant library population in a culture solution lacking manganese, then germinate the seeds and culture the seedlings at 20 ℃-35 ℃;

[0008] S3: After the seedlings are cultured until the sixth leaf is fully extended, phenotypic testing is performed to screen candidate target mutants;

[0009] S4: First-generation sequencing of candidate target mutants OsNramp5The gene sequence is tested for mutations to quickly identify the target mutant strain, and then the cadmium accumulation characteristics of the target mutant strain are identified to confirm its low-cadmium trait, thereby obtaining a low-cadmium mutant;

[0010] In each batch of S1 above, positive and negative controls were added, and other varieties with known backgrounds were selected. OsNramp5 The non-functional mutant seeds were used as positive controls, and the original wild-type varieties derived from the mutant library were used as negative controls.

[0011] Among them, the above OsNramp5 The internationally recognized gene identification symbol is Os07g0257200 (Rice Annotation Project database, abbreviated as RAP), LOC_Os07g15370 (Rice Genome Annotation Project database, abbreviated as RGAP) or LOC4342859 (NCBI-Gene database).

[0012] The indica rice variety Wushan Si Miao obtained by gene editing in the present invention has a genetic background OsNramp5 The mutant and wild-type control were subjected to a manganese deficiency hydroponic experiment at the seedling stage. OsNramp5 The gene mutant exhibits yellow leaf phenotype from the moment the fifth complete leaf emerges, and all leaves thereafter show this phenotype. Leaves earlier than this are normal, and the yellow leaf symptom is striped, i.e. the veins remain green, but the leaf surface between the veins turns yellow, and even completely white in the later stage. The reason is OsNramp5 The protein encoded by the gene is the main transporter of manganese. Its mutation will cause a significant reduction in manganese absorption. Under manganese-deficient hydroponic conditions, japonica rice OsNramp5 The mutant seedlings showed a dwarf and striped yellow leaf phenotype (i.e., a manganese-deficient phenotype). Therefore, in the above method, when the two leaves newly grown by the positive control showed a yellow-green striped leaf phenotype with green veins and yellow mesophyll between the veins, the mutant seedlings screened by hydroponic culture were observed one by one, and those with phenotypes similar to the positive control were candidate target mutants. Generally, under manganese-free hydroponic conditions, OsNramp5 The mutant seedlings began to show yellow leaf phenotype when the fifth complete leaf was outstretched.

[0013] When the mutant seeds are placed in the leaky PCR reaction wells in the above step S1, the embryo end faces downward, one seed is placed in one well, and 12 seeds are screened for one strain, occupying one row of the PCR plate. When the seeds are small (narrower), the embryo end of the seeds placed in the PCR reaction wells will leak out of the bottom wells or even the entire seeds will leak out of the bottom wells, it is necessary to pad a layer of fine-pore gauze at the bottom of the PCR plate, and use a layer of plastic mesh with a larger pore size and a harder texture to hold the gauze against the gauze, and then use three rubber bands to tighten the two layers of pad mesh and the PCR plate in the middle and at both ends of the PCR plate, so that the gauze is tightly attached to the bottom of the PCR without leaving a gap to prevent the seed embryo from leaking out, while not affecting the extension of the seed roots. The gauze aperture is preferably just enough to prevent the seed embryo end from leaking out, such as a household polyester fiber screen with an aperture of 1 mm-1.5 mm, and the outer cover mesh has a mesh diameter of 0.5 cm-2 cm, and the texture is harder and not easy to deform.

[0014] In the above step S2, the PCR reaction plate is a leaky bottom PCR plate, and the hole diameter is 2 mm-3 mm. The PCR reaction plate is placed in a specific container, which is designed to be a rectangular parallelepiped, 45 cm long, 13 cm wide, and 12 cm high. It is made of black opaque polyvinyl chloride (PVC) material, and the plate thickness is 0.5 cm. The inner sides of the two side walls of the long side are provided with PCR plate brackets, and the upper edge of the bracket is 2 cm away from the upper edge of the container side wall. Each bracket is composed of a horizontal and two vertical polyvinyl chloride material square strips pasted and fixed on the inner side of the side wall. The horizontal PVC square strip of the bracket is the same length as the inner side of the side wall, 1 cm wide, and 0.5 cm thick. The vertical PVC square strip of the bracket is located below the two ends of the horizontal square strip. The bracket can make the protruding edge parts on both sides of the PCR plate rest in the container without slipping, and the PCR plate can be translated on it.

[0015] In the above step S2, natural light is used to germinate the seeds and culture the seedlings at 20°C-35°C. Fresh culture medium is replaced on the 15th day after soaking the seeds, and the culture medium is replaced once a week thereafter to maintain sufficient nutrients and avoid excessive reproduction of green algae, thereby ensuring the healthy growth of the seedlings. During this period, attention should be paid to replenishing the water lost due to evaporation.

[0016] As a further preferred embodiment, the mutant library is a high-frequency mutant library, which obtains mutant seeds through at least one physical and chemical mutagenesis of cobalt 60 radiation mutagenesis, EMS chemical mutagenesis, and high-energy heavy ion beam mutagenesis.

[0017] As a further preferred embodiment, the mutagenesis method is a combined mutagenesis by cobalt 60 radiation and EMS immersion, the radiation dose of cobalt 60 radiation is 300 Gy, and the EMS treatment uses a 1% (V / V) EMS aqueous solution.

[0018] As a further preferred embodiment, the mutant seeds are M1 generation seeds or M2 generation seeds; the M1 generation seeds are seeds harvested from M0 generation plants, and the M2 generation seeds are seeds harvested from M1 generation plants.

[0019] As a further preferred embodiment, the culture medium lacking manganese is Yoshida culture medium from which manganese sulfate is removed. The culture medium is prepared with ultrapure water having a resistance value of 18.2 MΩ.

[0020] As a further preferred embodiment, the cadmium accumulation characteristic identification in S4 is carried out on cadmium-contaminated soil with a cadmium content in the range of 0.5 mg / kg-6 mg / kg; within this concentration range OsNramp5 The cadmium content of the mutant brown rice did not exceed 0.2 mg / kg, while that of the control was above 0.4 mg / kg. The difference between the control and the mutant was quite significant, and the difference was not easily masked by sampling or measurement errors.

[0021] As a further preferred embodiment, the candidate target mutant OsNramp5 Gene sequencing for mutation detection includes the following steps:

[0022] Step 1: Use segmented PCR to quantify mutants and wild-type OsNramp5 The gene genome and its promoter fragments are amplified to obtain PCR products;

[0023] Step 2: Detect the PCR product in step 1 by agarose gel electrophoresis and recover and purify the target fragment;

[0024] Step 3: Send the purified target fragment to sequencing, and the sequencing primer is the same as the primer used for fragment amplification;

[0025] Step 4: Compare the sequencing results with the wild-type variety sequence, and infer its genetic effect based on the sequence differences between the mutant and the wild type, the sequence mutation and molecular biology knowledge.

[0026] In the above step 1, the PCR primers, amplified fragment information and key amplification conditions are described in Table 1. The PCR was performed using Beijing Polymer 2×M5 Taq HiFi PCR Mix reagent. The amounts of different components of the PCR amplification system and the amplification temperature program were performed using the conditions recommended in the reagent manual. The annealing temperatures used in the PCR temperature programs for different fragments are shown in Table 1.

[0027] Table 1 OsNramp5 Gene and promoter mutation detection amplification fragment information, PCR primers used and annealing temperature

[0028]

[0029] 1The first base of the start codon in the gene coding region is defined as the +1 position, and -1 is the upstream adjacent base.

[0030] The beneficial effects of the present invention are as follows: the experimental process of the method provided by the present invention is very simple, and only the germination screening of seeds is required, and the technical requirements for the experimenters are not high. All the work can be completed by ordinary workers under the guidance of technicians. The equipment and reagents used in the method are very cheap and easy to purchase or customize from the market, and the equipment can be used repeatedly, so the cost of the method provided by the present invention is very low, and the mutants obtained by the method provided by the present invention are all homozygous, which can save the time of obtaining homozygous individuals by multiplication, accelerate the variety breeding process, and improve efficiency. In addition, the present invention avoids the need to screen mutants in rice by TILLING in the traditional method, and generally requires the size of the mutant population to have tens of thousands of individual plants. In the process, the genomic DNA of each individual is extracted, the target gene is amplified, and the PCR product mutation is detected. The process is complicated and the workload is huge. At the same time, these methods have high technical requirements for the experimenters, often rely on special equipment, and are costly. In addition, the mutants obtained by screening are generally heterozygous mutants, and the probability of obtaining homozygous mutants is very low. A series of problems. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Shown is a through-bottom, non-skirted PCR plate;

[0032] Figure 2 Shown is a schematic diagram of a hydroponic tank;

[0033] Figure 3 Shown are rice seedlings grown in a manganese-free culture medium for 5 weeks;

[0034] Figure 4 The results obtained by screening are shown OsNramp5 Mutation of mutant genes; Figure A shows the sequence mutation, the numbers above the bases indicate the positions of the bases on the gene ORF, and Figure B shows the sequencing peak diagram;

[0035] Figure 5 The bottom view of a PCR plate without skirts is shown, with a layer of household polyester fiber mosquito screen with a pore size of 1 mm and a layer of polyethylene plastic mesh with a pore size of 0.6 cm on the bottom. The mesh and the PCR plate are fixed by three rubber bands. DETAILED DESCRIPTION

[0036] The concept, specific structure and technical effects of the present invention will be clearly and completely described below in combination with the embodiments and drawings to fully understand the purpose, scheme and effect of the present invention.

[0037] Example 1

[0038] A sort of OsNramp5 The method for rapidly screening low-cadmium mutants in a rice mutant library comprises the following steps:

[0039] (1) Construction of mutant library

[0040] a. Selecting the stable indica rice strain Z183 bred by the inventor's research group as the mutagenesis background material for mutagenesis treatment;

[0041] b. Mutagenesis method: In order to increase the frequency of mutagenesis, the combined mutagenesis method of "Co 60 radiation + EMS immersion" was used; the Co 60 radiation was entrusted to the Institute of Crop and Nuclear Technology Utilization of Zhejiang Academy of Agricultural Sciences, with a radiation dose of 300 Gy. The irradiated seeds were soaked in pure water at 25 °C for 24 hours, the water was filtered out, and the seeds were soaked in 1% (V / V) EMS aqueous solution in a fume hood for another 4 hours. The EMS aqueous solution was poured out (collected in a waste liquid tank), and then stirred and washed with pure water for 5 times, each time for 5 minutes. The waste liquid was collected in a waste liquid tank, and then rinsed with tap water in a sink for 2 hours before sowing in the field; the waste liquid containing EMS was mixed with equal volumes of 0.1M NaOH and 20% Na2S2O3 solution and allowed to stand for 24 hours for harmless treatment and then poured out;

[0042] c. Rice propagation: Plant rice in the field, harvest each plant after it matures, and harvest one main ear from each plant. A total of 10,358 plants are harvested, and the seeds are directly used for OsNramp5 For mutant screening, each single strain was defined as one material.

[0043] (2) Screening of mutant library

[0044] a. Preparation of leaky bottom PCR plate: Use a dedicated PCR plate cutter (Ningbo Xinzhi Instrument Co., Ltd.) to flush cut and punch holes on the bottom of a conventional non-skirted PCR plate, with a hole diameter of about 2 mm. Figure 1 shown.

[0045] b. Customization of hydroponic tank: The hydroponic tank is designed to be rectangular, 45cm long, 13cm wide, and 12cm high. It is made of black opaque polyvinyl chloride (PVC) material with a thickness of 0.5cm. PCR plate holders are placed on the inner sides of the two side walls of the long side. The upper edge of the holder is 2.2cm away from the upper edge of the container side wall. Each holder is composed of a horizontal and two vertical polyvinyl chloride square strips pasted and fixed on the inner side of the side wall. The horizontal PVC square strip of the holder is the same length as the inner side of the side wall, 0.9cm wide and 0.5cm thick. The vertical PVC square strip of the holder is located directly below the two ends of the horizontal square strip. The holder can make the protruding edges on both sides of the PCR plate rest in the container without slipping, and the PCR plate can be translated on it. Figure 2, the arrow indicates the PCR plate bracket on the inner side of the long side wall. During hydroponic culture, both ends of the PCR plate are placed on the bracket to prevent it from sliding. One hydroponic tank can hold 5 PCR plates;

[0046] c. Arrangement of seeds on the PCR plate: Use tweezers to pick up seeds and place them into the PCR wells with the embryo end facing downwards. One seed is placed into each well. A total of 12 seeds are screened for one strain, occupying one row of the PCR plate. Each plate is numbered, and the sample number placed in each row is recorded;

[0047] d. Preparation of culture medium: Yoshida culture medium lacking manganese was used for hydroponics. The manganese-deficient culture medium powder (article number NSP1040-Mn) was purchased from Beijing Coolbo Technology Co., Ltd. The culture medium was prepared with ultrapure water with a resistance value of more than 16MΩ (purchased from Shanghai Jingchun Water Treatment Technology Co., Ltd.), and 463.4mg of reagent powder was added per liter of water. The pH value of the culture medium was adjusted to 5.0 with solid sodium hydroxide; in order to prevent yellowing of seedlings in the early stage of germination, 0.2g of urea (analytical grade) was added per liter of culture medium. The culture medium was ready for use;

[0048] e. Germination and hydroponic culture: Place the protruding edges of the PCR plates with the seeds to be screened on the internal brackets of the hydroponic tank. A maximum of 5 PCR plates can be placed in one hydroponic tank, with one side placed close together and the other side left empty for adding culture medium;

[0049] Pour the culture solution into the water tank. The water level should just cover the seeds. After soaking for a week, most seeds will germinate and young shoots will emerge from the tube mouth.

[0050] The minimum temperature during germination and hydroponic culture must be in the range of 20-35 degrees Celsius. If it is too low, germination will be slow, and the buds will rot if the water level is not well controlled. In Nanchang, Jiangxi, the suitable screening period is generally from mid-May to early July, and from early September to the end of October. In July and August, the temperature is high, the culture medium is overheated, and the roots are prone to lack of oxygen and death.

[0051] Hydroponics must be carried out in an environment that is sheltered from rain and sun. Rainwater may supplement manganese to the seedlings and cause screening failure. Direct sunlight may cause the culture solution temperature to be too high, resulting in root hypoxia and burning of the seedlings.

[0052] During the hydroponic period, birds and mice must be prevented from harming the plants. Fresh culture medium should be replaced on the 15th day after soaking the seeds, and then once a week. During this period, attention should be paid to replenishing the water lost due to evaporation. When screening at high temperatures, an aerator should be used to oxygenate the culture medium.

[0053] Each batch of processing requires the setting of positive and negative controls, and the selection of other varieties with known background OsNramp5 The non-functional mutant seeds were used as positive controls, and the original wild-type varieties derived from the mutant library were used as negative controls; Figure 3 Five-week-old hydroponic seedlings are shown.

[0054] f. Screening target mutants: After 30 days of germination, observe whether the new leaves of the positive control turn yellow, and compare them with the negative control. OsNramp5 The mutant began to show the manganese deficiency yellow leaf phenotype from the 5th complete leaf, while the wild type did not show the typical manganese deficiency yellow leaf phenotype even when it grew to 8 leaves. When 2-3 leaves turned yellow, the mutant library materials to be screened could be observed one by one. In general, the manganese deficiency yellow leaf phenotype is manifested as yellow-green stripes, the veins are green, and the mesophyll tissue between the veins is yellow or even dead and faded to white. Those with a leaf phenotype similar to the positive control yellow leaf phenotype are determined as candidate target mutants.

[0055] In this example, after the screening of 6846 mutant materials, it was found that two individual plants in one material (12 seeds germinated and 10 grew into seedlings) had typical manganese deficiency yellow leaf phenotype, which was similar to the positive control.

[0056] h. Detection of candidate target mutants OsNramp5 Gene sequence variation: The candidate target mutant strains and wild-type control seedlings were transferred to normal soil for planting. Generally, new leaves turned green as soon as they grew. Leaf samples were collected after the seedlings grew strong and DNA was extracted. OsNramp5 Gene fragments are identified and sequenced, and candidate mutant sequences are compared with controls to obtain sequence mutation information.

[0057] In this embodiment, the PCR primers, amplified fragment information and key amplification conditions used in the experiment are described in Table 1. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. (Changsha Branch), and the PCR was performed using Beijing Polymer 2×M5 Taq HiFiPCR Mix reagent (manufacturer: Beijing Polymer Biotechnology Co., Ltd.; item number: MF002). The amounts of different components of the PCR amplification system are shown in Table 2, the amplification temperature program is shown in Table 3, and the annealing temperatures used in the PCR temperature program for different fragments are shown in Table 1. The PCR product was detected by agarose gel electrophoresis, and the target fragment was recovered and purified by gel and sent to Beijing Qingke Biotechnology Co., Ltd. (Changsha Branch) for sequencing. The sequencing primers were the same as the primers used for fragment amplification.

[0058] Table 2 Volume of PCR components

[0059]

[0060] Table 3 PCR temperature program

[0061]

[0062] In this example, after sequencing and sequence comparison, it was found that the candidate target mutant sequence had a 64 bp sequence deletion compared with the wild type, which occurred at 3688-3751 bp of the gene open reading frame (ORF), located at the junction of the exon and intron. The mutant and wild type sequence comparison and sequencing peak diagram are shown in Figure 4 .

[0063] j. Identification of cadmium accumulation performance of target mutants: The mutants and wild-type plants were planted in soil with a cadmium content of 4.5 mg / kg. The seeds were harvested and shelled to test the cadmium content of brown rice. It was found that the cadmium content of the wild-type control was 2.67 mg / kg, and the cadmium content of the low-cadmium mutant was 0.159 mg / kg. The cadmium-reducing effect of the mutant was very obvious compared with the wild-type, indicating that the low-cadmium mutant was successfully found.

[0064] Example 2

[0065] PCR Plate Germination Method for Small or Narrow Grain Rice Seeds

[0066] When the seeds are small or narrow, the embryo end of the seeds will leak out of the bottom hole or even the whole seed will leak out of the bottom hole when placed in the PCR reaction well. The solution in this case is to pad a layer of 1 mm household polyester fiber mosquito screen at the bottom of the PCR plate, and use a layer of 0.6 cm polyethylene plastic mesh to hold the screen against the screen, and then use three rubber bands to tighten the two layers of screen and the PCR plate in the middle and both ends of the PCR plate (see Figure 5 ), so that the gauze is tightly attached to the bottom of the PCR without leaving a gap to prevent the embryo from leaking out, while not affecting the extension of the seed roots. The rest of the operations are the same as in Example 1.

[0067] Although the description of the present invention has been quite detailed and specifically describes several described embodiments, it is not intended to be limited to any of these details or embodiments or any particular embodiment, but should be regarded as providing a broad possible interpretation of these claims in view of the prior art by reference to the appended claims, thereby effectively covering the intended scope of the present invention. In addition, the above description of the present invention is based on the embodiments foreseeable by the inventor, and its purpose is to provide a useful description, and those non-substantial changes to the present invention that have not yet been foreseen may still represent equivalent changes to the present invention.

Claims

1. A kind of utilization OsNramp5 A method for rapidly screening low-cadmium mutants in a rice mutant library, characterized in that: Said OsNramp5 The internationally recognized gene identifier of the gene is at least one of Os07g0257200, LOC_Os07g15370 or LOC4342859, and the low-cadmium mutant is OsNramp5 The gene function is inactivated to obtain a mutant with low cadmium trait; the method specifically comprises the following steps: S1: Screening mutant seeds from the mutant library and placing them in a 96-well PCR (Polymerase Chain Reaction) reaction plate; S2: Soak the seeds of the mutant library population in a culture solution lacking manganese, then germinate the seeds and culture the seedlings at 20 ℃-35 ℃; S3: After the seedlings are cultured until the sixth leaf is fully extended, phenotypic testing is performed to screen candidate target mutants; S4: First-generation sequencing of candidate target mutants OsNramp5 The gene sequence is tested for mutations to quickly identify the target mutant strain, and then the cadmium accumulation characteristics of the target mutant strain are identified to confirm its low-cadmium trait, thereby obtaining a low-cadmium mutant; In each batch of S1, positive and negative controls were added to select other varieties with known backgrounds. OsNramp5 The non-functional mutant seeds were used as positive controls, and the original wild-type varieties derived from the mutant library were used as negative controls; The culture medium deficient in manganese is Yoshida culture medium from which manganese sulfate has been removed; The identification of cadmium accumulation characteristics in S4 was carried out on cadmium-contaminated soils with cadmium contents ranging from 0.5 mg / kg to 6 mg / kg.

2. The method according to claim 1, characterized in that: The mutant library is a high-frequency mutant library, which obtains mutant seeds through at least one physical and chemical mutagenesis of cobalt 60 radiation mutagenesis, EMS chemical mutagenesis, and high-energy heavy ion beam mutagenesis.

3. The method according to claim 2, characterized in that The mutagenesis method was a combination of cobalt 60 radiation and EMS immersion.

4. The method according to claim 3, characterized in that The radiation dose of cobalt-60 irradiation was 300 Gy.

5. The method according to claim 3, characterized in that: The EMS immersion solution is a 1% by volume EMS aqueous solution.

6. The method according to claim 1, characterized in that The PCR reaction plate is a leaky bottom PCR plate with a hole diameter of 2 mm-3 mm.

7. The method according to claim 1, characterized in that Candidate target mutants OsNramp5 Gene sequencing for mutation detection includes the following steps: Step 1: Use segmented PCR to quantify mutants and wild-type OsNramp5 The gene genome and its promoter fragments are amplified to obtain PCR products; Step 2: Detect the PCR product in step 1 by agarose gel electrophoresis and recover and purify the target fragment; Step 3: Send the purified target fragment to sequencing, and the sequencing primer is the same as the primer used for fragment amplification; Step 4: Compare the sequencing results with the wild-type variety sequence, and infer its genetic effect based on the sequence differences between the mutant and the wild type, the sequence mutation and molecular biology knowledge.

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