Rice grain cadmium accumulation related gene OsTF11 and application thereof

Abstract

The invention discloses a rice grain cadmium accumulation related gene OsTF11 and application thereof. The invention belongs to the field of plant breeding, and particularly relates to a rice grain cadmium accumulation related gene OsTF11 and application thereof. The protein OsTF11 or a substance for regulating and controlling the activity or content of the protein or regulating and controlling the expression of the coding gene of the protein can be applied to any one of the following applications: 1) regulating and controlling the cadmium content in plant seeds; 2) application in preparation of a product for regulating and controlling the cadmium content in plant seeds; the invention relates to application of the CRISPR / Cas9 gene mutant in the field of rice breeding, application of the CRISPR / Cas9 gene mutant in the field of rice breeding, application of the CRISPR / Cas9 gene mutant in the field of rice breeding, application of the CRISPR / Cas9 gene mutant in the field of rice breeding, application of the CRISPR / Cas9 gene mutant in the field of rice breeding, and application of the CRISPR / Cas9 gene mutant in the field of rice breeding.

Description

Technical Field

[0001] The invention belongs to the field of plant breeding, and particularly relates to a rice grain cadmium accumulation-related gene OsTF11 and an application thereof. Background Art

[0002] Cadmium accumulation is a major hazard to crop and human health. When the cadmium content in plant tissues reaches 1.0 mg kg⁻¹, it will cause a series of physiological and metabolic disorders by hindering plant root growth and inhibiting the absorption of water and nutrients, such as the synthesis of proteins, sugars and chlorophyll, decreased photosynthetic intensity and changes in enzyme activity, causing plants to show symptoms such as faded leaf color, dwarfing, and delayed phenology, ultimately leading to reduced crop quality and yield, or even death. When the soil contains 0.43 mg kg⁻¹ of soluble cadmium, rice yields decrease by 10%; when the content is 8.1 mg kg⁻¹, rice yields decrease by 25%.

[0003] Rice is the first staple food crop in my country, but it is also the hardest hit area for dietary cadmium intake, contributing 58.6% of the intake. Cadmium has strong carcinogenic, teratogenic and mutagenic effects, which can cause great harm to the human body. The toxicity of cadmium to the human body is divided into acute toxicity and chronic toxicity. Acute toxicity is mainly manifested as lung damage, gastrointestinal irritation, general fatigue, muscle aches and collapse, etc.; chronic toxicity is mainly manifested as a series of damage to bones, liver, kidneys, immune system, genetics, etc., and induces a variety of cancers. Breeding low-cadmium rice can effectively reduce the cadmium content in rice, increase the total grain output, provide effective support for ensuring food security, and is of great significance to food security.

[0004] The accumulation of cadmium in rice is a process of root absorption and compartmentalization, xylem loading and transportation, cadmium distribution in rice stem nodes, and cadmium redistribution in leaves, forming an overall distribution pattern of cadmium with a trend of root > stem > leaf sheath > leaf > grain. Cadmium transporters are usually located in the plasma membrane and vacuole membrane and participate in the transmembrane transport of ions, which can realize the absorption, efflux and vacuole compartmentalization of cadmium in plant cells. In rice roots, multiple transporter family members have been identified to participate in cadmium transport, such as the NRAMP (natural resistance-associated macrophage protein) family, the HMA (heavy metal transporting ATPase) family, and the ZIP (zinc-iron transporter) family. Stem node cadmium transporters include ZIP and plant calcium / cation transporter superfamily. Cadmium does not have a specific transporter in rice. It accumulates in leaves along with other metal elements and is redistributed to grains during the reproductive growth period. At present, two leaf cadmium transporter genes, OsLCD and OsMTP1, have been identified. Transcription factors participate in rice's response to cadmium by regulating the spatiotemporal expression of cadmium-related genes in rice, but reports on related genes are limited. Summary of the invention

[0005] The main problem to be solved by the present invention is how to regulate the cadmium content in plant seeds and reduce cadmium pollution.

[0006] In order to solve the problems existing in the prior art, the present invention provides the use of a protein or a substance regulating the activity or content of the protein or regulating the expression of a gene encoding the protein in regulating the cadmium content in plant seeds.

[0007] The use of the protein or gene expression regulating substance provided by the present invention or the substance regulating the activity or content of the protein in any of the following: 1) Application of regulating cadmium content in plant seeds; 2) Application in the preparation of products for regulating the cadmium content in plant seeds; 3) Application in the cultivation of plants with altered cadmium content in their seeds; 4) Use in the preparation of products for cultivating plants with altered cadmium content in seeds; 5) Application in plant breeding; The protein is any of the following: a1) a protein having an amino acid sequence of SEQ ID No: 1; a2) a protein having the same function as the amino acid sequence shown in SEQ ID No: 1 after one or more amino acid residues are substituted and / or deleted and / or added; a3) a protein with an amino acid sequence of at least 75% identity with any of the amino acids specified in a1) or (a2) and having the same function; a4) A fusion protein obtained by connecting a tag to the end of any protein defined in a1) to (a3).

[0008] The protein described in a1) above is named OsTF11.

[0009] In order to facilitate purification or detection of the protein in a1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No: 1 in the sequence listing.

[0010] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0011] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0012] A person skilled in the art can easily mutate the nucleotide sequence encoding the protein OsTF11 of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the protein OsTF11 isolated by the present invention are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein OsTF11 and have the function of the protein OsTF11.

[0013] The above-mentioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0014] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence or a nucleotide sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching for a pair of amino acid sequences or nucleotide sequences to calculate the identity, then the value (%) of the identity can be obtained.

[0015] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0016] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0017] In the above application, the protein is derived from rice ( Oryza sativa L.).

[0018] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene encoding the protein OsTF11.

[0019] In the above, the substance regulating gene expression may be a substance that performs at least one of the following six types of regulation: 1) Regulation at the transcriptional level of the gene; 2) regulation after the transcription of the gene (i.e., regulation of the splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of the transport of mRNA of the gene from the nucleus to the cytoplasm); 4) Regulation of the translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0020] In the present invention, the regulation may be up-regulation, enhancement or increase. The regulation may also be inhibition, reduction or down-regulation.

[0021] Herein, the upregulation or enhancement or increase of the expression level of the gene encoding the protein mentioned above in the recipient plant, or / and the enhancement, improvement or upregulation of the activity and / or content of the gene encoding the above protein is achieved by introducing the gene encoding the above protein into the recipient plant.

[0022] Herein, regulating the expression of the coding gene of the protein may also be inhibiting, reducing or down-regulating the expression of the coding gene. Inhibiting, reducing or down-regulating the expression of the coding gene may be achieved by gene knockout or gene silencing.

[0023] The gene knock-out refers to the phenomenon of inactivating a specific target gene through gene editing technology. Gene knock-out inactivates a specific target gene by changing the DNA sequence, including but not limited to zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and CRISPR / Cas system. CRISPR (clustered regulatory interspaced short palindromic repeat) is a clustered regularly spaced short palindromic repeat sequence, which is a site in the genome containing multiple short repeat sequences. Cas9 protein can cut the target sequence recognized by crRNA-tracrRNA under the mediation of RNA.

[0024] The gene silencing refers to the phenomenon that the gene is not expressed or is underexpressed without damaging the original DNA. Gene silencing is based on the premise that the DNA sequence is not changed, so that the gene is not expressed or is underexpressed. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization and position effect, and the other is post-transcriptional gene silencing, that is, at the level after gene transcription, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, gene repression, RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition, etc.

[0025] In the above application, the substance regulating gene expression or the substance regulating the activity or content of the protein may be a biological material related to the protein mentioned above, and the biological material may be any of the following: c1) a nucleic acid molecule encoding the protein described above; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); c5) a transgenic plant cell line containing the nucleic acid molecule described in c1) or a transgenic plant cell line containing the expression cassette described in c2); c6) transgenic plant tissue containing the nucleic acid molecule described in c1) or transgenic plant tissue containing the expression cassette described in c2); c7) a transgenic plant organ containing the nucleic acid molecule described in c1) or a transgenic plant organ containing the expression cassette described in c2); e1) Nucleic acid molecules that inhibit, reduce or silence the expression of the protein encoding gene mentioned above; e2) an expression cassette containing the nucleic acid molecule described in e1); e3) a recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3); e5) a transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) transgenic plant tissue containing the nucleic acid molecule described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

[0026] In the above application, the nucleic acid molecule in c1) can be any of the following DNA molecules, d1) The nucleotide sequence is a DNA molecule shown in SEQ ID No: 3; d2) the coding region sequence is the DNA molecule shown in SEQ ID No: 2; d3) a DNA molecule that has 90% or more identity with the nucleotide sequence specified in d1) or d2) and encodes the protein described above; d4) A DNA molecule that hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the protein described above.

[0027] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0028] The vectors described herein are well known to those skilled in the art, including but not limited to: plasmids, phages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), Ti plasmids or viral vectors. Specifically, the vector may be BGK03.

[0029] Optionally, e3) the recombinant vector contains an expression cassette sequence of sgRNA SEQ ID No: 4, and the target site of the sgRNA is located at OsTF11 The second exon of the gene, the nucleotide sequence of the target of the sgRNA is 515-537 of SEQ ID No: 3 or 2951-2973 of SEQ ID No: 2. The nucleotides 1-245 of SEQ ID No: 4 are the promoter for initiating the transcription of the sgRNA gene, the sgRNA gene is located at 246-265 of SEQ ID No: 4 of the sequence list, and the nucleotides 342-346 are the terminators for terminating the transcription of the sgRNA gene.

[0030] The present invention also provides a method for reducing the cadmium content in plant seeds, the method comprising step P, wherein step P is to inhibit, reduce or silence the activity and / or content of the protein mentioned above in the target plant, or / and, inhibit, reduce or silence the expression level of the gene encoding the protein mentioned above, so as to reduce the cadmium content in the plant seeds.

[0031] In a specific embodiment, the method for inhibiting, reducing or silencing the cadmium content in plant seeds may include the following steps: A) Using CRISPR / Cas9 system to clone the gene encoding the OsTF11 protein in the recipient plant OsTF11 Gene editing is performed, and the OsTF11 The gene mutation causes the premature termination of protein translation, which reduces the cadmium content in the seeds of the target plant; B) The target plant is self-pollinated to obtain a homozygous plant, namely the target plant, and the cadmium content in the seeds of the target plant is higher than that of the recipient plant.

[0032] In the above method, the gene encoding the OsTF11 protein in the recipient plant is OsTF11 Gene editing may be performed by subjecting the coding gene of the protein in the plant genome (nucleotide sequence is shown in SEQ ID No: 2) to at least one of the following mutations: 1) replacing 5'-GCAGCTTGGACTATTTGACTGG -3' in the gene encoding the protein in the plant genomic DNA with 5'-GCAGCTTGGACTGACTGG -3', thereby knocking out the gene encoding the OsTF11 protein; 2) Replacing 5'-GCAGCCTTGGACTATTTTTGACTGG-3' in the gene encoding the protein in the plant genomic DNA, thereby knocking out the gene encoding the OsTF11 protein.

[0033] The present invention also provides a method for increasing the cadmium content in plant seeds, the method comprising step M, wherein step M is to enhance, increase or up-regulate the activity and / or content of the protein mentioned above in the target plant, or / and enhance, increase or up-regulate the expression level of the gene encoding the protein mentioned above, so as to increase the cadmium content in the plant seeds.

[0034] In the above method, reducing the expression level and / or activity of the gene encoding the protein OsTF11 in the target plant can be: using gene mutation, gene knockout, gene editing or gene knockdown technology to reduce or inactivate the activity of the gene encoding the protein OsTF11 in the genome of the target plant.

[0035] The present invention provides a method for cultivating plants with reduced cadmium content in seeds, comprising inhibiting or reducing or silencing the expression of the coding gene of the above-mentioned protein and / or the content and / or activity of the above-mentioned protein in the target plant, or / and inhibiting or reducing or silencing the activity and / or content of the coding gene of the above-mentioned protein, to obtain plants with reduced cadmium content in seeds.

[0036] In one embodiment of the present invention, the breeding method for cultivating plants with reduced cadmium content in seeds comprises the following steps: (1) constructing a recombinant expression vector for inhibiting, reducing or silencing the gene encoding the protein mentioned above; (2) The recombinant expression vector constructed in step (1) is introduced into a recipient plant to obtain a plant having a cadmium content in its grains lower than that of the recipient plant.

[0037] In the above method, the inhibition, reduction or silencing of the protein mentioned above may specifically be knocking out the protein.

[0038] In the above method, the knockout is achieved through the CRISPR / Cas9 system.

[0039] In the above method, the target of gene editing by the CRISPR / Cas9 system is positions 515-537 of SEQ ID No: 3 or positions 2951-2973 of corresponding SEQ ID No: 2.

[0040] In the present invention, the purpose of plant breeding may include cultivating plants with reduced cadmium content in seeds.

[0041] In the present invention, the plant may be as follows: N1) monocots or dicots; N2) Gramineae; N3) Gramineae; N4) Oryza plants; N5) Rice.

[0042] This study shows OsTF11 Genes have a significant impact on rice's cadmium accumulation ability, created by CRISPR / Cas9 ostf11 The gene mutant can effectively reduce the cadmium content in rice grains and improve the safety and quality of rice. This study provides important information for understanding the mechanism of cadmium accumulation in rice and also provides new elements for future rice breeding and food security. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 for OsTF11 CRISPR Strain construction and identification. a, Gene schematic diagram and target sequence of OsTF11; b, OsTF11 CRISPR mutation sequence.

[0044] Figure 2 for OsTF11 CRISPR The seedling phenotype of the strain. OsTF11 CRISPR Soil culture phenotype of the strain; b. OsTF11 CRISPR Plant height; c. OsTF11 CRISPR Cadmium content in seeds of different strains. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0046] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0047] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0048] BGK03 in the following examples has been recorded in: Lu Y, Ye X, Guo R, et al. Genome-wide Targeted Mutagenesis in Rice Using the CRISPR / Cas9 System. Mol Plant. 2017;10(9):1242-1245. The public can obtain the biological material from the applicant. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0049] The following examples were processed using SPSS11.5 statistical software, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.

[0050] Embodiment 1, OsTF11CRISPR Mutant construction OsTF11 The coding sequence (CDS) of the gene in the japonica rice variety Nipponbare is SEQ ID No: 2, and the encoded amino acid sequence is OsTF11 protein of SEQ ID No: 1. In the genomic DNA of Nipponbare, the genomic gene encoding the OsTF11 protein is shown in SEQ ID No: 3 of the sequence listing.

[0051] The CRISPR vector used was BGK03, and the specific construction process was as follows; 1) Design and generate gRNA target sequences. Use the Baige gRNA target design tool ( http: / / www.biogle.cn / index / excrispr ) Design and generate the gRNA target sequence: 5'-GCAGCCTTGGACTATTTGACTGG-3', the target sequence is located atOsTF11 The second exon region of the gene ( Figure 1 a), is the 515-537th position of SEQ ID No:3 or the 2951-2973th position of SEQ ID No:2.

[0052] 2) Preparation of oligo dimers.

[0053] UP Oligo: 5'-TGTGGTGCAGCCTTGGACTATTTGAC-3'; Low Oligo: 5'-AAACGTCAAATAGTCCAAGGCTGCA-3'.

[0054] Dissolve the synthesized oligo in water to 10 µM, mix according to the following reaction system, heat at 95°C for 3 minutes, and then slowly decrease the temperature to 20°C at a rate of about 0.2°C / second. The reaction system (20 µl) is: Buffer Anneal (concentration of 10 μM) 18 µl, UP Oligo (concentration of 10 μM) 1 µl, Low Oligo (concentration of 10μM) 1 µl, and add water to make up to 20 µl of H2O.

[0055] 3) Construct the oligo dimer into the BGK03 vector. Mix the components on ice according to the following reaction system, and react at room temperature (20°C) for 1 hour. The connection system is: BGK03 Vector 2 µl, Oligo dimer 1 µl, EnzymeMix 1 µl, add water to make up to 10 µl of H2O.

[0056] 4) E. coli transformation (A)After taking out the competent E. coli cells (Weidi Biotechnology, Catalog No.: DL1001) from the refrigerator, immediately place them on ice to thaw.

[0057] (B) Add the recombinant system to the competent cell suspension in a clean bench, mix the contents by gently pipetting, and place on ice for 30 min.

[0058] (C) Heat shock at 42°C for 90 s and incubate on ice for 3 min.

[0059] (D) Add 900 μl of liquid LB medium to a sterile centrifuge tube, mix well, and place in a shaker at 220 rpm at 37°C for 1-2 h.

[0060] (E) Centrifuge at 12,000 rpm for 1 min, remove the supernatant, add 200 μl LB liquid medium to resuspend, and use a spreading stick to spread the bacterial solution on LB solid medium containing 100 mg / L Kan+, and invert it in a 37°C incubator for overnight culture.

[0061] (F) Single clones were picked for sequencing, and strains with correct sequencing results were used for subsequent experiments.

[0062] The structure of the recombinant vector PBGK03-sgTF11 is described as follows: a DNA fragment with a sequence of SEQ ID No 5 is inserted between the 5'-gcgctgtcgcttgtgt-3' and 5'-ctagctctaaaaca-3' fragments of the starting vector BGK03, while keeping other sequences of the starting vector BGK03 unchanged to obtain a recombinant vector.

[0063] The recombinant vector PBGK03-sgTF11 contains the sgRNA expression cassette sequence SEQ ID No: 4, wherein nucleotides 1 to 245 of SEQ ID No: 4 are a promoter for initiating transcription of the sgRNA gene, the sgRNA gene is located at positions 246 to 265 of SEQ ID No: 4 in the sequence table, and nucleotides 342 to 346 are terminators for terminating transcription of the sgRNA gene.

[0064] Rice transformation used the Agrobacterium infection rice callus method, and the transformation background was Nipponbare. CRISPR vector construction and rice transformation were completed by Weimi Biotechnology Co., Ltd. The recombinant vector PBGK03-sgTF11 was inoculated with Agrobacterium EHA105 strain to obtain Agrobacterium EHA105 / PBGK03-sgTF11, and Agrobacterium EHA105 / PBGK03-sgTF11 was transformed into the rice variety Nipponbare by Agrobacterium infection rice callus method, successfully obtaining two OsTF11 CRISPR mutant strains: cr-osdof11- L3 (L3 for short) and cr-osdof11-L4 (Abbreviated as L4).

[0065] Sequence analysis revealed that ( Figure 1 Middle b): Compared with the genomic DNA of rice Nipponbare, the genes encoding OsTF11 protein in the two homologous chromosomes of L3 strains have undergone the following mutations: "5'-GCAGCCTTGGACTATTTGACTGG-3' (corresponding to positions 2951-2973 of SEQ ID No: 3, positions 515-537 of SEQ ID No: 2)" mutated to "5'-GCAGCCTTGGACTGACTGG-3'"; 4 bases "ATTT" were missing in the protein coding sequence, making OsTF11A frameshift mutation occurs, resulting in loss of function, thereby knocking out the gene encoding OsTF11 protein.

[0066] Compared with the genomic DNA of rice Nipponbare, the genes encoding OsTF11 protein in the two homologous chromosomes of L4 strains underwent the following mutations: "5'-GCAGCCTTGGACTATTTGACTGG-3' (corresponding to positions 2951-2973 of SEQ ID No: 3, positions 515-537 of SEQ ID No: 2)" mutated to "5'-GCAGCCTTGGACTATTTTTGACTGG-3'"; two bases "TT" were inserted into the protein coding sequence, making OsTF11 A frameshift mutation occurs, resulting in loss of function, thereby knocking out the gene encoding OsTF11 protein.

[0067] Embodiment 2, OsTF11CRISPR Field phenotype of strains The wild rice variety Nipponbare (Nip) and ostf11-L3, ostf11-L4 The materials were planted in cadmium-free and cadmium-contaminated soils for cadmium treatment experiments. The cadmium treatment experiments were as follows: the soil cadmium content in environment I was 0.8 mg / kg (abbreviated as EN1); the soil cadmium content in environment II was 1.2 mg / kg (abbreviated as EN2).

[0068] During the rice maturity period, the plant height was tested and found to be Figure 2 a and b): 1) In the field without cadmium pollution, the aboveground plant height of the mutant was 95.33±0.577 cm and 88.33±0.577 cm, respectively, which was significantly lower than the wild type 101±1 cm; 2) This phenomenon still existed in the two cadmium-contaminated fields. In environmental type I, the aboveground plant height of the mutant was 77.67±2.082 and 78.33±2.887 cm, respectively, which was significantly lower than the wild type 90.67±1.155 cm; in environmental type II, the aboveground plant height of the mutant was 86.7±1.044 and 81.67±2.517 cm, respectively, which was significantly lower than the wild type 96.33±3.51 cm. This result shows that OsTF11 The knockout of affected the normal growth of rice plants, and this phenomenon existed both in cadmium-free and normal cadmium-contaminated fields.

[0069] After the rice is mature, the individual plants are harvested, and after being thoroughly dried, the cadmium content of the grains is measured. The specific steps for testing the cadmium content of rice grains are as follows: 1) Sample treatment before digestion: Dry the rice grains at 80°C to constant weight and grind them using a vibrating ball mill; 2) Accurately weigh 0.2 g (accurate to 0.0001 g) of sample and place it in a glass tube, trying not to let the sample stick to the wall of the test tube; 3) Add 1 ml of nitric acid in a fume hood, seal and pre-digest overnight; 4) On the second day, place a small funnel at the mouth of the test tube and heat it in an electric digestion furnace for 9 hours. The temperature of the instrument is 200°C throughout the whole process. 5) After taking it out, let it cool down, then add distilled water to the sample in the digestion tube to make up to 15 ml; 6) Shake well and filter into a 10 ml plastic centrifuge tube, and use an inductively coupled plasma emission spectrometer iCAP6300 to determine the cadmium content.

[0070] For each batch, three blank controls and three standard substance positive controls (rice component analysis standard substances, Institute of Geophysical and Geochemical Exploration) were set up.

[0071] The results show that ( Figure 2 In the two environments, OsTF11 The cadmium content in the grains of CRISPR strains L3 and L4 was lower than that of the Nipponbare control. In environmental type I, the cadmium content in the grains of Nipponbare rice was about 0.072±0.007 mg / kg, while OsTF11 The cadmium content in the grains of CRISPR strains L3 and L4 was approximately 0.017±0.001 and 0.024±0.009 mg / kg. In environmental type II, the cadmium content in the grains of Nipponbare rice was approximately 0.139±0.006 mg / kg, while OsTF11 The cadmium contents in the grains of CRISPR strains L3 and L4 were approximately 0.078±0.007 and 0.068±0.002 mg / kg.

[0072] Statistical analysis showed that the difference was extremely significant (P = 0.01 and 0.024, environmental type Ⅰ; P = 0.0016 and 0.0036, environmental type Ⅱ). The above results show that: OsTF11 Gene knockout can significantly reduce cadmium accumulation in rice grains, providing important genetic resources for breeding low-cadmium rice varieties.

[0073] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of a protein or a substance that regulates the activity or content of the protein or regulates the expression of a gene encoding the protein in any of the following: 1) Application in regulating cadmium content in plant seeds; 2) Application in the preparation of products for regulating the cadmium content in plant seeds; 3) Application in the cultivation of plants with altered cadmium content in their seeds; 4) Use in the preparation of products for cultivating plants with altered cadmium content in seeds; 5) Application in plant breeding; The protein is any of the following: a1) a protein having an amino acid sequence of SEQ ID No: 1; a2) a protein having the same function as the amino acid sequence shown in SEQ ID No: 1 after one or more amino acid residues are substituted and / or deleted and / or added; a3) a protein with an amino acid sequence identity of more than 80% with that specified in a1) or a2) and having the same function; a4) A fusion protein obtained by connecting a tag to the end of any one of the proteins defined in a1) to a3).

2. The use according to claim 1, characterized in that: The protein is derived from rice.

3. The use according to claim 1 or 2, characterized in that: The substance that regulates the activity or content of the protein or regulates the expression of the gene encoding the protein is a biological material related to the protein in the application of claim 1 or 2, and the biological material is any one of the following: c1) a nucleic acid molecule encoding the protein; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); c5) a transgenic plant cell line containing the nucleic acid molecule described in c1) or a transgenic plant cell line containing the expression cassette described in c2); c6) transgenic plant tissue containing the nucleic acid molecule described in c1) or transgenic plant tissue containing the expression cassette described in c2); c7) a transgenic plant organ containing the nucleic acid molecule described in c1) or a transgenic plant organ containing the expression cassette described in c2); e1) a nucleic acid molecule that inhibits, reduces or silences the expression of the gene encoding the protein; e2) an expression cassette containing the nucleic acid molecule described in e1); e3) a recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3); e5) a transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) transgenic plant tissue containing the nucleic acid molecule described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

4. The use according to claim 3, characterized in that: c1) The nucleic acid molecule is a DNA molecule as shown below: d1) The nucleotide sequence is a DNA molecule shown in SEQ ID No: 3; d2) the coding region sequence is the DNA molecule shown in SEQ ID No: 2; d3) a DNA molecule that has 90% or more identity with the nucleotide sequence defined in d1) or d2) and encodes the protein described in claim 1; d4) A DNA molecule which hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the protein described in claim 1.

5. A method for reducing the cadmium content in plant seeds, characterized in that: The method comprises step P, wherein step P is to inhibit, reduce or silence the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and, to inhibit, reduce or silence the expression level of the gene encoding the protein described in claim 1 or 2, so as to reduce the cadmium content in the plant seeds.

6. A method for increasing the cadmium content in plant seeds, characterized in that: The method comprises step M, wherein step M is to enhance, increase or up-regulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhance, increase or up-regulate the expression level of the gene encoding the protein described in claim 1 or 2 to increase the cadmium content in the plant seeds.

7. A method for breeding plants with reduced cadmium content in seeds, characterized in that: The method comprises inhibiting, reducing or silencing the expression level of the gene encoding the protein described in claim 1 or 2 in a recipient plant, and / or the activity and / or content of the protein to obtain a plant with reduced cadmium content in the grains, wherein the cadmium content in the grains of the plant with reduced cadmium content is lower than that of the recipient plant.

8. The method according to claim 7, characterized in that: The steps include: (1) constructing a recombinant expression vector for inhibiting, reducing or silencing the gene encoding the protein described in claim 1 or 2; (2) The recombinant expression vector constructed in step (1) is introduced into a recipient plant to obtain a plant having a cadmium content in its seeds lower than that of the recipient plant.

9. The protein according to claim 1 or 2 and / or the biomaterial according to claim 3 or 4.

10. The use according to any one of claims 1 to 4, and / or the method according to any one of claims 5 to 8, characterized in that: The plant is any of the following: N1) monocots or dicots; N2) Gramineae; N3) Gramineae; N4) Oryza plants; N5) Rice.

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