CN112501180B - Gene OsABCG42 for regulating and controlling rice cadmium accumulation and encoding protein and application thereof - Google Patents

Abstract

The invention discloses a gene OsABCG42 for regulating and controlling rice cadmium accumulation, and a coding protein and application thereof, wherein the nucleotide sequence of the gene OsABCG42 is shown as SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO: 2, respectively. The invention improves the cadmium accumulation characteristic of the existing rice by mutating the gene OsABCG42 through mutagenesis technologies such as gene editing, EMS mutagenesis, radiation mutagenesis, spaceflight carrying and the like. The method combines molecular marker assisted selection, can also realize polygene polymerization to create a rice variety with low cadmium content, has simple operation and short period for improving and cultivating new varieties, and does not influence main agronomic characters.

Description

Gene OsABCG42 for regulating and controlling rice cadmium accumulation and encoding protein and application thereof

Technical Field

The invention relates to the technical field of rice genetic engineering, in particular to a gene OsABCG42 for regulating and controlling rice cadmium accumulation, and a coding protein and application thereof.

Background

Rice is one of the most main food crops in China, and more than half of the population takes rice as staple food. In recent years, the problem of cadmium exceeding of rice caused by cadmium pollution of cultivated land has attracted extensive attention of society. The investigation bulletin of the national soil pollution situation in 2014 shows that the exceeding standard rate of cadmium (Cd) pollution at cultivated land points in China reaches 7 percent. In southern areas of China, the cadmium exceeding rate of rice reaches 23%, and the safe production of rice is seriously threatened. Cadmium enriched in rice can enter human body through food chain, thus being harmful to human health. The problem of regulating and controlling and reducing the cadmium content of rice is urgently needed to be solved.

Aiming at the current situation that the cadmium pollution of the rice field is increasingly serious at present, researchers develop the research of screening a large amount of rice cadmium low-accumulation varieties. The cadmium accumulation of rice relates to a plurality of physiological processes such as cadmium absorption, transport, distribution and the like and a plurality of transport proteins. Most of the identified cadmium transporters are also main effective transporters of other metals such as manganese, zinc, iron, calcium and the like, so that the gene mutation reduces the cadmium content, but the growth and development are hindered because the balance of mineral elements in rice bodies is destroyed. The method improves the high cadmium accumulation characteristic of rice, and is expected to provide more gene resources and molecular breeding approaches which do not influence the agronomic characters for cultivating the low cadmium accumulation rice variety.

The ABC transporter (ATP-binding cassette transporters) family is one of the largest and oldest protein families known at the present stage, and comprises 8 subfamilies such as ABCB, ABCC and ABCG, and can transport various substrates such as metals, sugars, nucleotides, proteins and hormones. The heterologous expression of the OsABCG43 in the yeast can increase the cadmium tolerance of the yeast; the OsABCG36 is subjected to cadmium induced expression in rice roots, and the cadmium tolerance of plants is enhanced by discharging the cadmium of root cells to apoplast. However, relatively few reports of other ABC transporters participating in rice cadmium regulation are reported.

Disclosure of Invention

The technical problem to be solved by the invention is to find the functional gene capable of reducing the cadmium content of the rice and provide a molecular breeding way for the cadmium rice, which is in urgent need of solving the cadmium rice problem, and the cadmium rice has important theoretical value and practical significance. Therefore, the invention provides a gene OsABCG42 for regulating and controlling rice cadmium accumulation, and a coding protein and application thereof. The OsABCG42 gene can be applied to genetic improvement of rice cadmium accumulation traits and cultivation of rice varieties with low cadmium accumulation.

In order to achieve the aim, the invention provides a gene OsABCG42 for regulating and controlling rice cadmium accumulation, wherein the nucleotide sequence of the gene OsABCG42 is shown as SEQ ID NO: 1 is shown.

Further, the nucleotide sequence of the gene OsABCG42 is similar to that of SEQ ID NO: 1 has more than 80% homology and the amino acid sequence of the coded protein is shown as SEQ ID NO: 2, respectively.

Based on a general technical concept, the invention provides a coding protein of the gene OsABCG42, and the amino acid sequence of the coding protein is shown as SEQ ID NO: 2, respectively.

Based on a general technical concept, the invention provides application of the gene OsABCG42 or the coding protein in improving the cadmium accumulation characteristic of rice or cultivating a rice variety with low cadmium accumulation.

In the application, the activity of the coded protein is reduced or inactivated by mutating the gene OsABCG42, or the abundance of the coded protein is reduced by inhibiting the expression of the gene OsABCG42, so that the cadmium accumulation characteristic of rice is improved or the rice variety with low cadmium accumulation and reduced cadmium content of rice is cultivated.

More preferably, the modes for mutating the gene OsABCG42 or inhibiting the expression of the gene OsABCG42 comprise gene editing, EMS mutagenesis, radiation mutagenesis and aerospace carrying.

In the above application, more preferably, the gene editing is: the gene OsABCG42 is knocked out by using CRISPR/Cas9 and TALEN, or the promoter thereof is edited by using CRISPR/Cas9 and TALEN to inhibit the expression thereof.

The application is more preferably that the specific method for knocking out the gene OsABCG42 by using the CRISPR/Cas9 method is as follows:

s1, selecting a target sequence according to an exon sequence of a gene OsABCG42, and constructing a CRISPR/Cas9 recombinant vector containing the target sequence;

s2, introducing the CRISPR/Cas9 recombinant vector into rice callus to obtain a transgenic positive plant;

s3, breeding the positive plants, and separating out OsABCG42 function deletion mutants without transgenic ingredients from progeny plants to obtain the rice line with reduced cadmium content.

For the above use, more preferably, the target sequence comprises: as shown in SEQ ID NO: 3 and the DNA sequence shown in SEQ ID NO: 4.

Compared with the prior art, the invention has the advantages that:

1. the invention provides a gene OsABCG42 for regulating and controlling rice cadmium accumulation. The yeast heterologous expression identification shows that the yeast expressing the gene OsABCG42 is more sensitive to Cd stress compared with the yeast control of the empty vector. The OsABCG42 knockout in rice shows that the cadmium content of the overground part and the root system of the plant is obviously reduced under the water culture condition in the seedling stage; cadmium pollution field experiments show that the removal of OsABCG42 obviously reduces the cadmium content of the brown rice and has no obvious influence on main agronomic characters.

2. The invention provides an application of a gene OsABCG42 for regulating and controlling cadmium accumulation of rice in improving the cadmium accumulation characteristic of rice or cultivating a low-cadmium-accumulation rice variety, and the mutation of OsABCG42 can be realized through mutagenesis technologies such as gene editing, EMS mutagenesis, radiation mutagenesis, spaceflight carrying and the like, so that the cadmium accumulation characteristic of the existing rice is improved. The method combines molecular marker auxiliary selection, can also realize polygene polymerization to create rice varieties with low cadmium content, has simple operation and short period for improving and cultivating new varieties, does not influence main agronomic characters, and provides gene resources and technical support for breeding of rice with low cadmium accumulation.

Drawings

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.

FIG. 1 shows the structure of the gene OsABCG42 and the results of sequencing the mutation sites of the knockout strains OsABCG42-1 and OsABCG42-2 in example 2 of the present invention.

FIG. 2 shows the results of cadmium content measurements of knock-out strains osabcg42-1, osabcg42-2 and wild type aerial parts (a) and root systems (b) under seedling hydroponic conditions in example 3 of the present invention (where: indicates a significant difference at 0.01 level compared to wild type; and: indicates a significant difference at 0.05 level compared to wild type).

FIG. 3 shows the results of measuring the cadmium content in brown rice of the knockout strains osacg 42-1, osacg 42-2 and wild type wazhuan in example 3 of the present invention (where an x indicates a significant difference at 0.01 level compared with the wild type; and an x indicates a significant difference at 0.05 level compared with the wild type).

FIG. 4 is a result of identifying cadmium stress sensitivity of OsABCG42 yeast transformant in example 4 of the present invention.

FIG. 5 is a time-course growth curve of pYES2-OsABCG42 yeast transformants and wild-type control pYES2 yeast transformants in a liquid medium containing 0. mu.M and 5. mu.M cadmium in example 4 of the present invention.

Detailed Description

The invention is further described below with reference to specific preferred examples, without thereby limiting the scope of protection of the invention.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods. The methods in the following examples are conventional in the art unless otherwise specified.

Examples

The materials and equipment used in the following examples are commercially available.

Example 1:

the nucleotide sequence of a coding region of the gene OsABCG42 for regulating and controlling rice cadmium accumulation is shown as SEQ ID NO: 1, specifically:

ATGATACTGGGATTGGATATATGCGCGGACACGATCGTCGGCGACCAGATGCAGAGGGGGATCTCCGGTGGTCAGAAGAAACGCGTCACCACCGGTGAGATGATTGTCGGTCCAACAAAGGTTCTATTCATGGATGAGATATCAACTGGATTGGACAGCTCCACCACATTCCAGATTGTCAAATGCCTTCAGCAAATCGTGCACTTGGGCGAGGCAACCATCCTCATGTCACTCCTACAACCAGCCCCTGAGACTTTTGAGCTATTCGATGACATTATCCTACTGTCAGAAGGCCAGATTGTTTATCAGGGACCCCGCGAATACGTCCTTGAGTTCTTTGAGTCATGCGGATTCCGCTGCCCAGAGCGTAAGGGTACTGCAGACTTTCTTCAGGAGGTGACATCAAAGAAGGATCAGGAGCAGTATTGGGCCGACAAGCATAGGCCATACAGATACATTTCAGTCTCAGAATTTGCACAGAGATTTAAAAGGTTCCATGTTGGGCTCCAACTTGAGAATCATCTCTCAGTCCCATTTGATAAAACTCGTAGCCATCAGGCTGCTCTTGTCTTCTCGAAGCAATCGGTGTCAACAACAGAGCTCCTCAAGGCATCCTTTGCCAAGGAGTGGCTCCTCATTAAGCGCAACTCATTTGTGTACATCTTCAAGACCATACAGCTCATCATTGTAGCCCTTGTCGCGTCGACAGTGTTTCTTAGGACCCAGATGCACACAAGGAATTTAGATGATGGCTTTGTGTACATTGGAGCACTACTTTTCAGTCTGATTGTGAACATGTTCAATGGTTTCGCTGAGCTCTCTTTGACCATCACAAGGTTGCCAGTGTTCTTCAAGCACCGGGACCTCCTCTTCTACCCTGCTTGGATCTTCACTCTACCGAATGTTATTCTGAGAATCCCATTTTCCATCATCGAATCTATAGTCTGGGTGATTGTTACATACTACACTATAGGATTTGCCCCAGAGGCTGACAGATTTTTCAAGCAGTTGCTGCTAGTGTTCTTGATCCAGCAGATGGCAGGTGGCCTTTTCAGAGCAACTGCTGGTCTGTGCAGATCCATGATCATTGCTCAAACTGGAGGAGCCCTGGCCCTTCTCATATTTTTCGTTCTTGGAGGATTTCTTCTGCCAAAAGCATTCATCCCAAAATGGTGGATCTGGGGTTACTGGGTTTCACCACTGATGTACGGGTATAATGCTCTAGCAGTCAATGAATTCTACTCTCCTCGGTGGATGAACAAGTTTGTACTGGATAACAATGGCGTTCCTAAAAGACTAGGAATAGCTCTGATGGAAGGTGCCAACATCTTCACTGACAAAAATTGGTTCTGGATTGGAGCAGCAGGGCTCCTGGGTTTCACCATGTTTTTCAATGTGCTTTTCACTCTGTCACTCGTTTATCTGAATCCTCTGGGCAAACCACAAGCTGTCATATCTGAAGAAACTGCGAAGGAAGCGGAAGGCAATGGGGATGCAAGACATACAGTAAGAAATGGCAGCACAAAATCAAATGGTGGAAATCACAAAGAAATGAGGGAGATGAGATTGAGTGCTCGTCTGAGCAATAGTTCATCGAATGGAGTTTCACGACTGATGTCCATTGGTAGCAATGAAGCTGGTCCAAGAAGAGGAATGGTTCTTCCATTTACTCCTCTATCCATGTCTTTTGATGATGTGAACTACTATGTCGACATGCCTGCAGAAATGAAGCAGCAAGGAGTGGTGGATGATAGGCTCCAATTGTTACGTGACGTTACTGGATCATTTAGGCCTGCAGTGCTGACAGCACTCATGGGAGTCAGTGGAGCCGGAAAGACAACTCTTATGGATGTTTTGGCAGGAAGAAAGACTGGTGGTTACATTGAAGGAGATATGAGAATTTCTGGTTATCCTAAGAACCAAGAAACATTTGCAAGAATTTCTGGCTACTGTGAGCAAAACGATATCCATTCACCTCAGGTCACAGTTAGGGAGTCTTTGATATACTCTGCTTTCCTGCGCCTTCCAGAAAAAATAGGAGATCAAGAAATCACTGATGATATCAAGATTCAATTTGTTGATGAAGTTATGGAACTAGTGGAGCTCGACAATCTGAAGGATGCGTTAGTTGGCCTGCCTGGAATCACAGGGCTTTCAACAGAGCAAAGGAAGAGATTGACAATAGCAGTGGAGCTTGTTGCAAATCCCTCGATCATCTTCATGGATGAACCGACTTCAGGTCTTGATGCAAGAGCAGCAGCCATTGTTATGAGAACAGTGAGGAACACAGTTGACACTGGACGGACAGTGGTTTGCACAATTCACCAGCCAAGCATTGACATATTTGAGGCTTTTGATGAGCTGCTACTACTGAAAAGAGGAGGGCAGGTGATATACTCTGGGCAATTGGGTCGTAATTCCCAGAAAATGATTGAATATTTCGAGGCAATTCCTGGTGTGCCTAAAATCAAAGATAAGTACAATCCAGCTACATGGATGCTTGAGGTCAGTTCAGTTGCTGCGGAAGTACGCTTAAATATGGACTTTGCTGAGTACTATAAGACATCAGACCTGTACAAGCAAAACAAGGTATTGGTGAATCAGCTAAGTCAACCAGAGCCAGGAACATCAGATCTGCATTTTCCTACAAAATACTCTCAATCCACCATAGGGCAATTTAGGGCCTGCCTCTGGAAGCAATGGCTGACCTATTGGCGCAGCCCAGATTACAATCTTGTTAGATTTTCCTTCACTCTGTTCACAGCCTTGCTACTCGGCACCATCTTTTGGAAGATCGGCACCAAGATGGGAAATGCCAATTCTCTTAGAATGGTCATTGGAGCAATGTATACAGCAGTGATGTTTATCGGTATCAACAATTGTGCGACTGTGCAGCCAATCGTGTCGATTGAGAGAACAGTTTTCTACCGAGAGAGGGCTGCTGGGATGTACTCTGCTATGCCCTATGCCATTGCTCAGGTTGTCATGGAGATACCCTATGTGTTCGTCCAAACTGCATATTATACCCTCATTGTTTATGCCATGATGAGCTTCCAGTGGACAGCAGCCAAGTTCTTCTGGTTCTTCTTCGTCTCCTACTTCTCATTTCTCTACTTCACCTACTATGGTATGATGACGGTGGCAATCTCACCAAACCATGAGGTTGCAGCCATCTTTGCCGCGGCGTTCTATTCCTTGTTCAACCTATTCTCAGGATTCTTCATTCCGAGACCAAGGATTCCCAAATGGTGGATCTGGTACTACTGGCTTTGCCCATTGGCATGGACAGTGTATGGGCTCATAGTGACACAGTATGGAGACCTAGAACAAATCATCTCAGTCCCTGGCCAATCCAACCAGACAATCAGCTACTATGTTACTCATCATTTTGGATATCACAGGAAATTTATGCCAGTTGTTGCGCCGGTGCTCGTGCTCTTCGCTGTGTTTTTCGCGTTCATGTATGCCATTTGCATCAAGAAGTTGAACTTCCAACATCGATAG。

the rice transport protein OsABCG42 coded by the protein has an amino acid sequence shown in SEQ ID NO: 2, specifically:

MILGLDICADTIVGDQMQRGISGGQKKRVTTGEMIVGPTKVLFMDEISTGLDSSTTFQIVKCLQQIVHLGEATILMSLLQPAPETFELFDDIILLSEGQIVYQGPREYVLEFFESCGFRCPERKGTADFLQEVTSKKDQEQYWADKHRPYRYISVSEFAQRFKRFHVGLQLENHLSVPFDKTRSHQAALVFSKQSVSTTELLKASFAKEWLLIKRNSFVYIFKTIQLIIVALVASTVFLRTQMHTRNLDDGFVYIGALLFSLIVNMFNGFAELSLTITRLPVFFKHRDLLFYPAWIFTLPNVILRIPFSIIESIVWVIVTYYTIGFAPEADRFFKQLLLVFLIQQMAGGLFRATAGLCRSMIIAQTGGALALLIFFVLGGFLLPKAFIPKWWIWGYWVSPLMYGYNALAVNEFYSPRWMNKFVLDNNGVPKRLGIALMEGANIFTDKNWFWIGAAGLLGFTMFFNVLFTLSLVYLNPLGKPQAVISEETAKEAEGNGDARHTVRNGSTKSNGGNHKEMREMRLSARLSNSSSNGVSRLMSIGSNEAGPRRGMVLPFTPLSMSFDDVNYYVDMPAEMKQQGVVDDRLQLLRDVTGSFRPAVLTALMGVSGAGKTTLMDVLAGRKTGGYIEGDMRISGYPKNQETFARISGYCEQNDIHSPQVTVRESLIYSAFLRLPEKIGDQEITDDIKIQFVDEVMELVELDNLKDALVGLPGITGLSTEQRKRLTIAVELVANPSIIFMDEPTSGLDARAAAIVMRTVRNTVDTGRTVVCTIHQPSIDIFEAFDELLLLKRGGQVIYSGQLGRNSQKMIEYFEAIPGVPKIKDKYNPATWMLEVSSVAAEVRLNMDFAEYYKTSDLYKQNKVLVNQLSQPEPGTSDLHFPTKYSQSTIGQFRACLWKQWLTYWRSPDYNLVRFSFTLFTALLLGTIFWKIGTKMGNANSLRMVIGAMYTAVMFIGINNCATVQPIVSIERTVFYRERAAGMYSAMPYAIAQVVMEIPYVFVQTAYYTLIVYAMMSFQWTAAKFFWFFFVSYFSFLYFTYYGMMTVAISPNHEVAAIFAAAFYSLFNLFSGFFIPRPRIPKWWIWYYWLCPLAWTVYGLIVTQYGDLEQIISVPGQSNQTISYYVTHHFGYHRKFMPVVAPVLVLFAVFFAFMYAICIKKLNFQHR。

in this example, the sequence shown in SEQ ID NO: 1 has more than 80% homology, and the amino acid sequence of the coded protein is shown as SEQ ID NO: 2, the same results as in example 1 were obtained.

Example 2: obtaining and molecular identification of OsABCG42 knockout strain

The invention relates to a method for inhibiting expression of a gene OsABCG42, which comprises the following steps: the CDS of the OsABCG42 gene is edited by CRISPR/Cas9, so that the OsABCG42 generates nonsense mutation. The specific implementation method comprises the following steps:

(1) according to the selection principle of a target site of a CRISPR/Cas9 vector system, two 20bp sequences are selected from the third exon of OsABCG42 as target sequences for generating sgRNA.

The first target sequence is located at the antisense strand 189 bp along the downstream of the initiation codon ATG of the coding region of the OsABCG42 gene, and the sequence is CCAAGTGCACGATTTGCTGA (shown as SEQ ID NO: 3);

the second target sequence is located in 317-336 bp antisense chain at the downstream of the initiation codon ATG of the coding region of the OsABCG42 gene, and the sequence is GAACTCAAGGACGTATTCGC (shown as SEQ ID NO: 4).

Based on the above two target sequences, the linker primers were designed as follows:

42P1U3-F：ggcaccaagtgcacgatttgctga (shown in SEQ ID NO: 5);

42P1U3-R：aaactcagcaaatcgtgcacttgg (shown in SEQ ID NO: 6);

42P2U6-F：gccgaactcaaggacgtattcgc (shown in SEQ ID NO: 7);

42P2U6-R：aaacgcgaatacgtccttgagtt (shown in SEQ ID NO: 8).

Synthesizing the above linker primers, dissolving into 100 μ M mother liquor, respectively, taking 1 μ L of each, wherein 42P1U3-F and 42P1U3-R are mixed to obtain 42P1U3 mixed liquor, 42P2U6-F and 42P2U6-R are mixed to obtain 42P2U6 mixed liquor, diluting the 42P1U3 mixed liquor and 42P2U6 mixed liquor to 1 μ M, placing on PCR at 98 ℃ for 30S, moving to room temperature for cooling, and completing annealing to form the linker of two target sites.

References Ma XL, Zhang QY, Zhu QL, Liu W, Chen Y, Qiu R, Wang B, Yang ZF, Li HY, Lin YR, Xie YY, Shen RX, Chen SF, Wang Z, Chen YL, Guo JX, Chen LT, ZHao XC, Dong ZC, Liu YG. A robust CRISPR/Cas9 system for containment, high-efficiency multiplex gene editing in monochip and dicot plants, mol plants 2015,8(8): 1274-.

(2) The pCRISPR/Cas9-OsABCG42 recombinant vector is introduced into competent Agrobacterium strain EHA105, and Hubei Wuhanbo remote biotechnology limited company is entrusted to complete the rice genetic transformation work.

Briefly described as follows: selecting mature and full indica rice variety Huazhan seeds, shelling, sterilizing by sodium hypochlorite, filtering, and inoculating to an induction culture medium to induce callus; impregnating agrobacterium tumefaciens EHA105 bacterial liquid containing a pCRISPR/Cas9-OsABCG42 recombinant vector with Huazhan callus, transferring to a co-culture medium for dark culture at 24 ℃ for 3 days, cleaning the callus, transferring to a selection culture medium containing hygromycin for screening and culturing for 30 days; transferring the selected resistant callus to a pre-differentiation culture medium for 7-10 days, transferring to a differentiation culture medium for illumination culture, and transferring to a rooting culture medium for about 3 weeks when the plantlet grows to 2-4 cm; 7 strains of T were obtained₀Hardening off the seedlings for 3 days, transplanting the seedlings into soil, receiving the seeds, planting and breeding the first generation to obtain T₁And (5) plant generation.

(3) For T₁And carrying out target site genotype detection on the generation plant to identify the mutant plant, which is briefly described as follows:

CTAB method for extracting T₁And (3) designing a primer aiming at the target sequence of OsABCG42 in the DNA of the generation plant for amplifying a DNA fragment containing the target sequence, wherein the amplification primer is as follows:

ABCG42-CAS 9-F: GCACGTTTCCACTCCCGATA (shown as SEQ ID NO: 9);

ABCG42-CAS 9-R: TGCCTTGAGGAGCTCTGTTG (shown in SEQ ID NO: 10).

Amplifying the DNA fragment containing the target site by high-fidelity enzymatic PCR: the reaction system is as follows: 0.5. mu.L of DNA template, 1. mu.L of ABCG42-CAS9-F, 1. mu.L of ABCG42-CAS9-R, 4. mu.L of dNTP, 15. mu.L of 2xbuffer, 0.5. mu.L of KOD enzyme, plus ddH₂O to a total volume of 30. mu.L.

Reaction procedure: pre-denaturation at 95 ℃ for 3min, 30 cycles: denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 30s, and extension at 6 ℃ for 60 s.

And (4) carrying out electrophoresis on the PCR product and sequencing. Screening and sequencing single plants with single peak at the target site, directly comparing with wild type sequences, and analyzing the target site genotype. At T₁The generation plants are screened for homozygous knockout strains with mutation at target sites to cause nonsense mutation, transgenic elements such as Hpt and Cas9 are detected by a PCR method, knockout strains without transgenic elements such as osabcg42-1 and osabcg42-2 are further screened, and the mutation sequences are shown in figure 1.

Example 3: element content and main agronomic characters of OsABCG42 gene knockout strain

Detecting the Cd content of the overground part and the root system of the OsABCG42 knockout strain: soaking OsABCG42 knockout strains OsABCG42-1, OsABCG42-2 and wild type control Huazhan in seeds, accelerating germination, sowing in 96-well PCR plate with cut bottom, culturing in nutrient solution for 14 days, and respectively using 0.2 and 0.5 mu M CdCl₂Treating for 10 days, washing with tap water and deionized water for 3 times, separating aerial parts and root system, drying at 80 deg.C to constant weight, pulverizing, weighing, and adding HNO₃-HClO₄Digesting the mixed solution, and measuring the Cd content of the overground part and the root system by using an inductively coupled plasma MASs spectrometer (ICP-MAS).

The results are shown in FIG. 2, from which it is clear that: under the conditions of 0.2 mu M and 0.5 mu M cadmium stress, the cadmium content of the overground part and the root system of the knockout strains OsABCG42-1 and OsABCG42-2 are obviously reduced, which indicates that the OsABCG42 positively regulates the cadmium accumulation of rice.

The major agronomic traits of the OsABCG42 knockout strain are examined: when osabcg42-1, osabcg42-2 and wild type control Huazhan were planted in the cadmium contaminated field, it was found that the plant heights of osabcg42-1 and osabcg42-2, the effective ears of each plant, the number of grains per ear, the setting rate and the weight average of thousand grains were not significantly changed compared with the wild type control Huazhan, as detailed in table 1.

TABLE 1 major agronomic traits for OsABCG42 knock-out lines

Line of plants	Plant height (cm)	Effective ear of single plant	Total number of grains per ear	Percentage of fruit set (%)	Thousand Kernel weight (g)
						WT	102.8±0.8	24.8±1.5	162.6±13.3	88.3±1.3	19.5±0.7
osabcg42-1	102.1±1.7	23.8±2.8	158.2±3.9	85.5±3.8	19.2±0.7
						osabcg42-2	104.2±1.4	25.8±4.2	167.7±5.8	87.3±1.7	19.0±0.8

Detecting the cadmium content of the OsABCG42 knockout strain brown rice: ICP-MAS is adopted to detect the cadmium content of brown rice, the result is shown in figure 3, the cadmium content of brown rice of OsABCG42-1 and OsABCG42-2 is obviously reduced compared with that of wild type control, the fact that the OsABCG42 gene is knocked out is shown to reduce the cadmium content of rice, the main agronomic characters are not obviously affected, and the cadmium content of rice is positively regulated and controlled by OsABCG 42.

Example 4: yeast heterologous expression identification of cadmium transport activity of OsABCG42 gene

Constructing a yeast expression vector pYES2-OsABCG42 of the OsABCG42 gene, and transforming the vector into a cadmium sensitive Saccharomyces cerevisiae strain delta ycf. Briefly described as follows:

(1) the CDS of OsABCG42 is amplified by high-fidelity Taq enzyme PCR by taking the rice cDNA as a template, and the amplification primers are YES-ABCG42-F and YES-ABCG 42-R.

YES-ABCG 42-F: ggatccagtgtggtggaattcATGATACTGGGATTGGATATATGCG (shown in SEQ ID NO: 11);

YES-ABCG 42-R: accttcgaagggccctctagaCTATCGATGTTGGAAGTTCAACTTCT (shown in SEQ ID NO: 12).

(2) The CDS fragment of OsABCG42 was recovered by electrophoresis, the yeast expression vector pYES2 was digested with EcoR I and Xba I, the vector was linearized, and the linearized pYES2 and the CDS fragment of OsABCG42 were ligated by recombinant cloning (Vazyme Co., Ltd.)

II, a recombinant cloning kit, catalog number C112), transforming Escherichia coli, and obtaining a recombinant vector pYES2-OsABCG42 through colony PCR amplification and sequencing verification.

(3) The recombinant vector pYES2-OsABCG42 and the empty vector pYES2 are transformed into a cadmium sensitive yeast strain delta ycf by a lithium acetate transformation method, the delta ycf yeast strain transformed into the empty vector pYES2 is used as a control, yeast transformants with concentration gradient dilution are cultured on a yeast induction solid culture medium added with galactose and 0, 20 and 30 mu M cadmium at 30 ℃, and after 3 days, the growth vigor of bacterial plaques is observed and photographed.

The results are shown in FIG. 4, and the growth vigor of the yeast expressing OsABCG42 is consistent with that of the yeast transforming the empty vector under the condition of no cadmium stress; and under the cadmium stress of 20 and 30 mu M, compared with the yeast transformed with an empty vector, the growth of the yeast expressing the OsABCG42 is severely inhibited, which shows that the expression of the OsABCG42 gene in the yeast makes the yeast more sensitive to the cadmium stress.

(4) Culturing yeast transformant of recombinant vector pYES2-OsABCG42 and empty vector pYES2 on yeast liquid culture medium with glucose as carbon source to logarithmic growth phase, washing with sterile water for 3 times, and adjusting OD₆₀₀To 0.6, adding 5 μ L of the extract into 50mL yeast liquid culture medium containing galactose as carbon source and 0 μ M cadmium or 5 μ M cadmium, shaking at 30 deg.C and 200rpm, culturing for 0h, 3h, 6h, 9h, 12h, 24h, and 36h, and measuring OD₆₀₀The value is obtained.

As a result, as shown in FIG. 5, OD of yeast transformant expressing OsABCG42 was observed in the absence of cadmium stress₆₀₀Yeast transformants with values slightly lower than those for empty vector pYES2, whereas OD was slightly lower under 5uM Cd stress conditions₆₀₀The values were significantly lower than for the yeast transformant with the empty vector pYES 2.

In general, the rice OsABCG42 knockout strain is obtained by utilizing gene editing, and the rice OsABCG42 knockout strain is found through seedling-stage water culture experiments and cadmium-polluted field experiments, so that the cadmium content of the root system, the straw and the brown rice of the rice plant is remarkably reduced, the main agronomic characters are not remarkably influenced, and the fact that the OsABCG42 positively regulates and controls the cadmium accumulation of the rice is shown. The identification of yeast heterologous expression OsABCG42 shows that the yeast expressing OsABCG42 is more sensitive to Cd stress compared with the yeast control of an empty vector.

Based on the OsABCG42 gene provided by the invention, the cadmium accumulation characteristic of the existing rice can be improved through gene editing, EMS mutagenesis, radiation mutagenesis, aerospace carrying, molecular marker assisted breeding and other technologies, intermediate materials and rice varieties with low cadmium content of rice are cultivated and created, the operation is simple, the period for improving and cultivating new varieties is short, and the main agronomic characters are not influenced.

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many possible variations and modifications to the disclosed embodiments, or equivalent modifications, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.

Sequence listing

<110> research center for hybrid rice in Hunan province

<120> gene OsABCG42 for regulating and controlling rice cadmium accumulation, and encoding protein and application thereof

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3504

<212> DNA

<213> Rice (rice)

<400> 1

atgatactgg gattggatat atgcgcggac acgatcgtcg gcgaccagat gcagaggggg 60

atctccggtg gtcagaagaa acgcgtcacc accggtgaga tgattgtcgg tccaacaaag 120

gttctattca tggatgagat atcaactgga ttggacagct ccaccacatt ccagattgtc 180

aaatgccttc agcaaatcgt gcacttgggc gaggcaacca tcctcatgtc actcctacaa 240

ccagcccctg agacttttga gctattcgat gacattatcc tactgtcaga aggccagatt 300

gtttatcagg gaccccgcga atacgtcctt gagttctttg agtcatgcgg attccgctgc 360

ccagagcgta agggtactgc agactttctt caggaggtga catcaaagaa ggatcaggag 420

cagtattggg ccgacaagca taggccatac agatacattt cagtctcaga atttgcacag 480

agatttaaaa ggttccatgt tgggctccaa cttgagaatc atctctcagt cccatttgat 540

aaaactcgta gccatcaggc tgctcttgtc ttctcgaagc aatcggtgtc aacaacagag 600

ctcctcaagg catcctttgc caaggagtgg ctcctcatta agcgcaactc atttgtgtac 660

atcttcaaga ccatacagct catcattgta gcccttgtcg cgtcgacagt gtttcttagg 720

acccagatgc acacaaggaa tttagatgat ggctttgtgt acattggagc actacttttc 780

agtctgattg tgaacatgtt caatggtttc gctgagctct ctttgaccat cacaaggttg 840

ccagtgttct tcaagcaccg ggacctcctc ttctaccctg cttggatctt cactctaccg 900

aatgttattc tgagaatccc attttccatc atcgaatcta tagtctgggt gattgttaca 960

tactacacta taggatttgc cccagaggct gacagatttt tcaagcagtt gctgctagtg 1020

ttcttgatcc agcagatggc aggtggcctt ttcagagcaa ctgctggtct gtgcagatcc 1080

atgatcattg ctcaaactgg aggagccctg gcccttctca tatttttcgt tcttggagga 1140

tttcttctgc caaaagcatt catcccaaaa tggtggatct ggggttactg ggtttcacca 1200

ctgatgtacg ggtataatgc tctagcagtc aatgaattct actctcctcg gtggatgaac 1260

aagtttgtac tggataacaa tggcgttcct aaaagactag gaatagctct gatggaaggt 1320

gccaacatct tcactgacaa aaattggttc tggattggag cagcagggct cctgggtttc 1380

accatgtttt tcaatgtgct tttcactctg tcactcgttt atctgaatcc tctgggcaaa 1440

ccacaagctg tcatatctga agaaactgcg aaggaagcgg aaggcaatgg ggatgcaaga 1500

catacagtaa gaaatggcag cacaaaatca aatggtggaa atcacaaaga aatgagggag 1560

atgagattga gtgctcgtct gagcaatagt tcatcgaatg gagtttcacg actgatgtcc 1620

attggtagca atgaagctgg tccaagaaga ggaatggttc ttccatttac tcctctatcc 1680

atgtcttttg atgatgtgaa ctactatgtc gacatgcctg cagaaatgaa gcagcaagga 1740

gtggtggatg ataggctcca attgttacgt gacgttactg gatcatttag gcctgcagtg 1800

ctgacagcac tcatgggagt cagtggagcc ggaaagacaa ctcttatgga tgttttggca 1860

ggaagaaaga ctggtggtta cattgaagga gatatgagaa tttctggtta tcctaagaac 1920

caagaaacat ttgcaagaat ttctggctac tgtgagcaaa acgatatcca ttcacctcag 1980

gtcacagtta gggagtcttt gatatactct gctttcctgc gccttccaga aaaaatagga 2040

gatcaagaaa tcactgatga tatcaagatt caatttgttg atgaagttat ggaactagtg 2100

gagctcgaca atctgaagga tgcgttagtt ggcctgcctg gaatcacagg gctttcaaca 2160

gagcaaagga agagattgac aatagcagtg gagcttgttg caaatccctc gatcatcttc 2220

atggatgaac cgacttcagg tcttgatgca agagcagcag ccattgttat gagaacagtg 2280

aggaacacag ttgacactgg acggacagtg gtttgcacaa ttcaccagcc aagcattgac 2340

atatttgagg cttttgatga gctgctacta ctgaaaagag gagggcaggt gatatactct 2400

gggcaattgg gtcgtaattc ccagaaaatg attgaatatt tcgaggcaat tcctggtgtg 2460

cctaaaatca aagataagta caatccagct acatggatgc ttgaggtcag ttcagttgct 2520

gcggaagtac gcttaaatat ggactttgct gagtactata agacatcaga cctgtacaag 2580

caaaacaagg tattggtgaa tcagctaagt caaccagagc caggaacatc agatctgcat 2640

tttcctacaa aatactctca atccaccata gggcaattta gggcctgcct ctggaagcaa 2700

tggctgacct attggcgcag cccagattac aatcttgtta gattttcctt cactctgttc 2760

acagccttgc tactcggcac catcttttgg aagatcggca ccaagatggg aaatgccaat 2820

tctcttagaa tggtcattgg agcaatgtat acagcagtga tgtttatcgg tatcaacaat 2880

tgtgcgactg tgcagccaat cgtgtcgatt gagagaacag ttttctaccg agagagggct 2940

gctgggatgt actctgctat gccctatgcc attgctcagg ttgtcatgga gataccctat 3000

gtgttcgtcc aaactgcata ttataccctc attgtttatg ccatgatgag cttccagtgg 3060

acagcagcca agttcttctg gttcttcttc gtctcctact tctcatttct ctacttcacc 3120

tactatggta tgatgacggt ggcaatctca ccaaaccatg aggttgcagc catctttgcc 3180

gcggcgttct attccttgtt caacctattc tcaggattct tcattccgag accaaggatt 3240

cccaaatggt ggatctggta ctactggctt tgcccattgg catggacagt gtatgggctc 3300

atagtgacac agtatggaga cctagaacaa atcatctcag tccctggcca atccaaccag 3360

acaatcagct actatgttac tcatcatttt ggatatcaca ggaaatttat gccagttgtt 3420

gcgccggtgc tcgtgctctt cgctgtgttt ttcgcgttca tgtatgccat ttgcatcaag 3480

aagttgaact tccaacatcg atag 3504

<210> 2

<211> 1167

<212> PRT

<213> Rice (rice)

<400> 2

Met Ile Leu Gly Leu Asp Ile Cys Ala Asp Thr Ile Val Gly Asp Gln

1 5 10 15

Met Gln Arg Gly Ile Ser Gly Gly Gln Lys Lys Arg Val Thr Thr Gly

20 25 30

Glu Met Ile Val Gly Pro Thr Lys Val Leu Phe Met Asp Glu Ile Ser

35 40 45

Thr Gly Leu Asp Ser Ser Thr Thr Phe Gln Ile Val Lys Cys Leu Gln

50 55 60

Gln Ile Val His Leu Gly Glu Ala Thr Ile Leu Met Ser Leu Leu Gln

65 70 75 80

Pro Ala Pro Glu Thr Phe Glu Leu Phe Asp Asp Ile Ile Leu Leu Ser

85 90 95

Glu Gly Gln Ile Val Tyr Gln Gly Pro Arg Glu Tyr Val Leu Glu Phe

100 105 110

Phe Glu Ser Cys Gly Phe Arg Cys Pro Glu Arg Lys Gly Thr Ala Asp

115 120 125

Phe Leu Gln Glu Val Thr Ser Lys Lys Asp Gln Glu Gln Tyr Trp Ala

130 135 140

Asp Lys His Arg Pro Tyr Arg Tyr Ile Ser Val Ser Glu Phe Ala Gln

145 150 155 160

Arg Phe Lys Arg Phe His Val Gly Leu Gln Leu Glu Asn His Leu Ser

165 170 175

Val Pro Phe Asp Lys Thr Arg Ser His Gln Ala Ala Leu Val Phe Ser

180 185 190

Lys Gln Ser Val Ser Thr Thr Glu Leu Leu Lys Ala Ser Phe Ala Lys

195 200 205

Glu Trp Leu Leu Ile Lys Arg Asn Ser Phe Val Tyr Ile Phe Lys Thr

210 215 220

Ile Gln Leu Ile Ile Val Ala Leu Val Ala Ser Thr Val Phe Leu Arg

225 230 235 240

Thr Gln Met His Thr Arg Asn Leu Asp Asp Gly Phe Val Tyr Ile Gly

245 250 255

Ala Leu Leu Phe Ser Leu Ile Val Asn Met Phe Asn Gly Phe Ala Glu

260 265 270

Leu Ser Leu Thr Ile Thr Arg Leu Pro Val Phe Phe Lys His Arg Asp

275 280 285

Leu Leu Phe Tyr Pro Ala Trp Ile Phe Thr Leu Pro Asn Val Ile Leu

290 295 300

Arg Ile Pro Phe Ser Ile Ile Glu Ser Ile Val Trp Val Ile Val Thr

305 310 315 320

Tyr Tyr Thr Ile Gly Phe Ala Pro Glu Ala Asp Arg Phe Phe Lys Gln

325 330 335

Leu Leu Leu Val Phe Leu Ile Gln Gln Met Ala Gly Gly Leu Phe Arg

340 345 350

Ala Thr Ala Gly Leu Cys Arg Ser Met Ile Ile Ala Gln Thr Gly Gly

355 360 365

Ala Leu Ala Leu Leu Ile Phe Phe Val Leu Gly Gly Phe Leu Leu Pro

370 375 380

Lys Ala Phe Ile Pro Lys Trp Trp Ile Trp Gly Tyr Trp Val Ser Pro

385 390 395 400

Leu Met Tyr Gly Tyr Asn Ala Leu Ala Val Asn Glu Phe Tyr Ser Pro

405 410 415

Arg Trp Met Asn Lys Phe Val Leu Asp Asn Asn Gly Val Pro Lys Arg

420 425 430

Leu Gly Ile Ala Leu Met Glu Gly Ala Asn Ile Phe Thr Asp Lys Asn

435 440 445

Trp Phe Trp Ile Gly Ala Ala Gly Leu Leu Gly Phe Thr Met Phe Phe

450 455 460

Asn Val Leu Phe Thr Leu Ser Leu Val Tyr Leu Asn Pro Leu Gly Lys

465 470 475 480

Pro Gln Ala Val Ile Ser Glu Glu Thr Ala Lys Glu Ala Glu Gly Asn

485 490 495

Gly Asp Ala Arg His Thr Val Arg Asn Gly Ser Thr Lys Ser Asn Gly

500 505 510

Gly Asn His Lys Glu Met Arg Glu Met Arg Leu Ser Ala Arg Leu Ser

515 520 525

Asn Ser Ser Ser Asn Gly Val Ser Arg Leu Met Ser Ile Gly Ser Asn

530 535 540

Glu Ala Gly Pro Arg Arg Gly Met Val Leu Pro Phe Thr Pro Leu Ser

545 550 555 560

Met Ser Phe Asp Asp Val Asn Tyr Tyr Val Asp Met Pro Ala Glu Met

565 570 575

Lys Gln Gln Gly Val Val Asp Asp Arg Leu Gln Leu Leu Arg Asp Val

580 585 590

Thr Gly Ser Phe Arg Pro Ala Val Leu Thr Ala Leu Met Gly Val Ser

595 600 605

Gly Ala Gly Lys Thr Thr Leu Met Asp Val Leu Ala Gly Arg Lys Thr

610 615 620

Gly Gly Tyr Ile Glu Gly Asp Met Arg Ile Ser Gly Tyr Pro Lys Asn

625 630 635 640

Gln Glu Thr Phe Ala Arg Ile Ser Gly Tyr Cys Glu Gln Asn Asp Ile

645 650 655

His Ser Pro Gln Val Thr Val Arg Glu Ser Leu Ile Tyr Ser Ala Phe

660 665 670

Leu Arg Leu Pro Glu Lys Ile Gly Asp Gln Glu Ile Thr Asp Asp Ile

675 680 685

Lys Ile Gln Phe Val Asp Glu Val Met Glu Leu Val Glu Leu Asp Asn

690 695 700

Leu Lys Asp Ala Leu Val Gly Leu Pro Gly Ile Thr Gly Leu Ser Thr

705 710 715 720

Glu Gln Arg Lys Arg Leu Thr Ile Ala Val Glu Leu Val Ala Asn Pro

725 730 735

Ser Ile Ile Phe Met Asp Glu Pro Thr Ser Gly Leu Asp Ala Arg Ala

740 745 750

Ala Ala Ile Val Met Arg Thr Val Arg Asn Thr Val Asp Thr Gly Arg

755 760 765

Thr Val Val Cys Thr Ile His Gln Pro Ser Ile Asp Ile Phe Glu Ala

770 775 780

Phe Asp Glu Leu Leu Leu Leu Lys Arg Gly Gly Gln Val Ile Tyr Ser

785 790 795 800

Gly Gln Leu Gly Arg Asn Ser Gln Lys Met Ile Glu Tyr Phe Glu Ala

805 810 815

Ile Pro Gly Val Pro Lys Ile Lys Asp Lys Tyr Asn Pro Ala Thr Trp

820 825 830

Met Leu Glu Val Ser Ser Val Ala Ala Glu Val Arg Leu Asn Met Asp

835 840 845

Phe Ala Glu Tyr Tyr Lys Thr Ser Asp Leu Tyr Lys Gln Asn Lys Val

850 855 860

Leu Val Asn Gln Leu Ser Gln Pro Glu Pro Gly Thr Ser Asp Leu His

865 870 875 880

Phe Pro Thr Lys Tyr Ser Gln Ser Thr Ile Gly Gln Phe Arg Ala Cys

885 890 895

Leu Trp Lys Gln Trp Leu Thr Tyr Trp Arg Ser Pro Asp Tyr Asn Leu

900 905 910

Val Arg Phe Ser Phe Thr Leu Phe Thr Ala Leu Leu Leu Gly Thr Ile

915 920 925

Phe Trp Lys Ile Gly Thr Lys Met Gly Asn Ala Asn Ser Leu Arg Met

930 935 940

Val Ile Gly Ala Met Tyr Thr Ala Val Met Phe Ile Gly Ile Asn Asn

945 950 955 960

Cys Ala Thr Val Gln Pro Ile Val Ser Ile Glu Arg Thr Val Phe Tyr

965 970 975

Arg Glu Arg Ala Ala Gly Met Tyr Ser Ala Met Pro Tyr Ala Ile Ala

980 985 990

Gln Val Val Met Glu Ile Pro Tyr Val Phe Val Gln Thr Ala Tyr Tyr

995 1000 1005

Thr Leu Ile Val Tyr Ala Met Met Ser Phe Gln Trp Thr Ala Ala Lys

1010 1015 1020

Phe Phe Trp Phe Phe Phe Val Ser Tyr Phe Ser Phe Leu Tyr Phe Thr

1025 1030 1035 1040

Tyr Tyr Gly Met Met Thr Val Ala Ile Ser Pro Asn His Glu Val Ala

1045 1050 1055

Ala Ile Phe Ala Ala Ala Phe Tyr Ser Leu Phe Asn Leu Phe Ser Gly

1060 1065 1070

Phe Phe Ile Pro Arg Pro Arg Ile Pro Lys Trp Trp Ile Trp Tyr Tyr

1075 1080 1085

Trp Leu Cys Pro Leu Ala Trp Thr Val Tyr Gly Leu Ile Val Thr Gln

1090 1095 1100

Tyr Gly Asp Leu Glu Gln Ile Ile Ser Val Pro Gly Gln Ser Asn Gln

1105 1110 1115 1120

Thr Ile Ser Tyr Tyr Val Thr His His Phe Gly Tyr His Arg Lys Phe

1125 1130 1135

Met Pro Val Val Ala Pro Val Leu Val Leu Phe Ala Val Phe Phe Ala

1140 1145 1150

Phe Met Tyr Ala Ile Cys Ile Lys Lys Leu Asn Phe Gln His Arg

1155 1160 1165

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ccaagtgcac gatttgctga 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gaactcaagg acgtattcgc 20

<210> 5

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggcaccaagt gcacgatttg ctga 24

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aaactcagca aatcgtgcac ttgg 24

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gccgaactca aggacgtatt cgc 23

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aaacgcgaat acgtccttga gtt 23

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gcacgtttcc actcccgata 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgccttgagg agctctgttg 20

<210> 11

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggatccagtg tggtggaatt catgatactg ggattggata tatgcg 46

<210> 12

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

accttcgaag ggccctctag actatcgatg ttggaagttc aacttct 47

Claims (7)

1. The application of a gene OsABCG42 for regulating and controlling rice cadmium accumulation in improving rice cadmium accumulation characteristics or cultivating a rice variety with low cadmium accumulation is characterized in that the nucleotide sequence of the gene OsABCG42 is shown as SEQ ID NO: 1 is shown.

2. The use according to claim 1, wherein the amino acid sequence of the protein encoded by the gene OsABCG42 is as shown in SEQ ID NO: 2, respectively.

3. The use of claim 1, wherein the activity of the encoded protein is reduced or inactivated by mutating the gene OsABCG42, or the abundance of the encoded protein is reduced by inhibiting the expression of the gene OsABCG42, so that the cadmium accumulation property of rice is improved or a rice variety with low cadmium accumulation in rice is cultivated.

4. The use of claim 3, wherein the means for mutating the gene OsABCG42 or inhibiting the expression of the gene OsABCG42 comprises gene editing, EMS mutagenesis, radiation mutagenesis, aerospace carry-on.

5. The use of claim 4, wherein the gene editing is: the gene OsABCG42 is knocked out by using CRISPR/Cas9 and TALEN, or the promoter thereof is edited by using CRISPR/Cas9 and TALEN to inhibit the expression thereof.

6. The use of claim 5, wherein the specific method for knocking out the gene OsABCG42 by using CRISPR/Cas9 is as follows:

s1, selecting a target sequence according to an exon sequence of a gene OsABCG42, and constructing a CRISPR/Cas9 recombinant vector containing the target sequence;

s2, introducing the CRISPR/Cas9 recombinant vector into rice callus to obtain a transgenic positive plant;

s3, breeding the positive plants, and separating out OsABCG42 function deletion mutants without transgenic ingredients from progeny plants to obtain the rice line with reduced cadmium content.

7. The use of claim 6, wherein the target sequence comprises: as shown in SEQ ID NO: 3, or the nucleotide sequence shown as SEQ ID NO: 4.

Images