Rice grain cadmium accumulation related gene OsCd38 and application thereof

Abstract

The invention discloses a rice grain cadmium accumulation related gene OsCd38 and application thereof. The invention belongs to the technical field of biology, and particularly relates to a rice grain cadmium accumulation related gene OsCd38 and application thereof. The protein or the 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: 1) regulating and controlling the cadmium content in plant seeds; the invention discloses an OsLCd38 gene mutant, a preparation method of the OsLCd38 gene mutant, a preparation method of the OsLCd38 gene mutant, a preparation method of the OsLCd38 gene mutant, a preparation method of the OsLCd38 gene mutant, a preparation method of the OsLCd38 gene mutant, a preparation method of the OsLCd38 gene mutant, a preparation method of the OsLCd38 gene mutant, a preparation method of the OsLCd38 gene mutant, a preparation method of the OsLCd38 gene mutant, a preparation method of the OsLCd38 gene mutant and a preparation method of the OsLCd38 gene mutant,

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and specifically relates to genes related to cadmium accumulation in rice grains. OsCd38 and its applications. Background Art

[0002] Cadmium is a toxic heavy metal element with a half-life of 8-30 years in the human body. Rice is one of the most important food crops in the world and the staple food of more than half of the population in my country. Rice grains and processed foods are widely consumed throughout the country. Rice has the characteristic of easily accumulating cadmium, and its edible parts are more likely to accumulate cadmium than other crop varieties such as soybeans, barley, corn, and wheat. Rice is the crop with the largest proportion of cadmium intake in the diet of Chinese residents, and its contribution accounts for an average of more than 55.8% of the total dietary cadmium intake. Systematically assessing the risk of cadmium pollution in rice and breeding rice germplasm with low cadmium accumulation are of practical significance for ensuring my country's food security and people's health. Summary of the invention

[0003] The main problem to be solved by the present invention is how to regulate the cadmium content in plant seeds and reduce cadmium pollution.

[0004] 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.

[0005] 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 their 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 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).

[0006] The name of the protein described in a1) above is OsLCd38.

[0007] 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: 2 in the sequence listing.

[0008] 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.

[0009] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0010] Those skilled in the art can easily mutate the nucleotide sequence encoding the protein OsLCd38 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 OsLCd38 isolated from the present invention are 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 OsLCd38 and have the function of the protein OsLCd38.

[0011] The above-mentioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0012] 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.

[0013] 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.

[0014] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0015] In the above application, the protein is derived from rice ( Oryza sativa L.).

[0016] 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 OsLCd38.

[0017] 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).

[0018] In the present invention, the regulation may be up-regulation, enhancement or increase. The regulation may also be inhibition, reduction or down-regulation.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] In the above application, the substance regulating the expression of the gene 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).

[0024] 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: 1; 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.

[0025] 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.

[0026] 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.

[0027] Optionally, e3) the recombinant vector contains a sgRNA expression cassette sequence SEQ ID No: 4, and the target site of the sgRNA is located at OsLCd38The third exon of the gene, the target sequence of the sgRNA is 1661-1680 of SEQ ID No: 3 or 527-546 of SEQ ID No: 2. The nucleotides 1-245 of SEQ ID No: 4 are the promoters 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 348-352 are the terminators for terminating the transcription of the sgRNA gene.

[0028] 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.

[0029] 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.

[0030] In the above method, the inhibition, reduction or silencing of the protein mentioned above may specifically be knocking out the protein.

[0031] In the above method, the knockout is achieved through the CRISPR / Cas9 system.

[0032] In the above method, the target of gene editing by the CRISPR / Cas9 system is positions 1661-1680 of SEQ ID No: 3 or positions 527-546 of corresponding SEQ ID No: 2.

[0033] In the present invention, the purpose of plant breeding may include cultivating plants with reduced cadmium content in seeds.

[0034] 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.

[0035] 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 OsLCd38 protein in the recipient plant OsLCd38 Gene editing is performed, and the OsLCd38 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.

[0036] In the above method, the gene encoding the OsLCd38 protein in the recipient plant is OsLCd38 Gene editing may be performed by subjecting the protein encoding gene in the plant genome (nucleotide sequence is shown in SEQ ID No: 3) to at least one of the following mutations: 1) replacing 5'- CATTTGATGCTGCTGCT-3' in the gene encoding the protein in the plant genomic DNA with 5'- CATTTGTGCTGCT-3', thereby knocking out the gene encoding the OsLCd38 protein; 2) Replacing 5'-AACTTGGGTTT-3' in the gene encoding the protein in the plant genomic DNA with 5'-ACTTGGGTTT-3', thereby knocking out the gene encoding the OsLCd38 protein.

[0037] The present invention also provides a method for increasing the cadmium content in cells, the method comprising step M, wherein step M is to enhance, increase or up-regulate the activity and / or content of the protein described above in the target cells, or / and enhance, increase or up-regulate the expression level of the gene encoding the protein described above, so as to increase the cadmium content in the cells.

[0038] The cell may be a yeast cell.

[0039] The yeast may be yeast strain BY4741.

[0040] In the present invention, the plant may be as follows: N1) monocots or dicots; N2) Gramineae; N3) Gramineae; N4) Oryza plants; N5) Rice.

[0041] OsLCd38 identified in this study is a cadmium transporter that has a significant impact on the cadmium accumulation capacity of rice. OsLCd38The 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

[0042] Picture 1 for OsLCd38 Transgenic yeast phenotype. OsLCd38 Transgenic yeast phenotype; b. OsLCd38 Cadmium content in transgenic yeast.

[0043] Picture 2 for OsLCd38 Soil culture phenotype of CRISPR strains. OsLCd38 Photos of CRISPR strains grown in soil; b. OsLCd38 CRISPR strain plant height; c, OsLCd38 Cadmium content in grains of CRISPR strains. DETAILED DESCRIPTION

[0044] 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.

[0045] The experimental methods in the following examples, unless otherwise specified, are conventional methods and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0046] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

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

[0048] In the following embodiments pAG413 The carrier has been described in: Characterization of a novel arsenite long-distance transporter from arsenic hyperaccumulator fern Pteris vittata. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0049] The BGK03 vector in the following example has been described 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 experiment of the present invention and cannot be used for other purposes.

[0050] The yeast strain BY4741 in the following examples has been described in: Yan H, Xu W, Zhang T, Feng L, Liu R, Wang L, Wu L, Zhang H, Zhang X, Li T, Peng Z, Jin C, Yu Y, Ping J, MaM, He Z. Characterization of a novel arsenite long-distance transporter from arsenic hyperaccumulator fern Pteris vittata. New Phytol. 2022 Mar;233(6):2488-2502. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0051] 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.

[0052] Embodiment 1, OsLCd38 Gene cloning The Tiangen Plant Genomic DNA Extraction Kit (TIANGEN, DP305) was used to extract genomic DNA from the leaves of rice variety Nipponbare seedlings according to the instructions. The specific primers F / R were designed using Primer Premier 5.0 software, F: 5'-ATGGAGGTGTTCTACTAC-3', R: 5'-CTATGGGTTTAACGGCTCAT-3', and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0053] The PCR amplification system (25 μL) contained: 2 μL template DNA, 1 μL upstream and downstream primers (10 μmol / L), 12.5 μL 2×Taq PCR Master Mix, and ddH2O to 25 μL. The PCR reaction program was: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 90 s, 35 cycles; 72°C final extension for 10 min. After the PCR product was detected by 1% agarose gel electrophoresis, the target band was cut and recovered, and sequencing was used to verify the target gene clone.

[0054] OsLCd38 The coding sequence (CDS) of the gene in the rice variety Nipponbare is SEQ ID No: 2, and the encoded amino acid sequence is OsLCd38 protein of SEQ ID No: 1. In the genomic DNA of Nipponbare, the genomic sequence encoding OsLCd38 protein is shown in SEQ ID No: 3.

[0055] Example 2: Yeast transformation and identification of yeast resistance to manganese and cadmium 1. Vector construction Using homologous recombination OsLCd38 Gene construction to pAG413 In the carrier.

[0056] First, Vazyme Phanta Max Super-Fidelity DNA Polymerase (Vazyme, P505) was used to amplify OsLCd38 Gene fragments, primers are designed to add pAG413 Vector homologous sequence.

[0057] F: 5'-gaaaaaaccccggat tctaga ATGGAGGTGTTCTACTAC-3', R: 5'-ga ctcgag CTATGGGTTTAACGGCTCAT-3'.

[0058] The PCR product was checked by 1% agarose gel electrophoresis, and the target fragment was purified using a gel recovery kit (TIANGEN, DP209).

[0059] The pAG413 plasmid was linearized by restriction endonuclease SmaI (NEB, R0141) and purified by gel recovery. The homologous recombination reaction was performed using ClonExpress II One Step Cloning Kit (Vazyme, C112). The reaction system (10 μL) contained: 50 ng of linearized vector, purified PCR product fragment (molar ratio 1:3), 2 μL of 5×CE II Buffer, 1 μL of Exnase II, and ddH2O was added. The recombinant product was transformed into Escherichia coli DH5α competent cells, positive clones were screened by ampicillin resistance, and the correct recombinant vector was obtained after plasmid extraction and sequencing verification. pAG413-OsLCd38 .

[0060] Recombinant vector pAG413-OsLCd38 The structure is described as follows: pAG413 Insert a DNA fragment with the sequence of SEQ ID No: 2 between the 5'-gaaaaaaccccggattctaga-3' and 5'-gactcgag-3' fragments of pAG413 The recombinant vector is obtained while other sequences remain unchanged. pAG413-OsLCd38 The vector can express OsLCd38 protein, whose amino acid sequence is SEQ ID No:1.

[0061] 2. Yeast transformation Yeast transformation was performed using the Coolaber Yeast Transformation Kit (Cat. No. PYK0051): 3. Analysis of cadmium resistance on yeast plates 1) Transform the recombinant yeast (containing the recombinant vector pAG413-OsLCd38 ) single colonies and containing pAG413 The control yeast with empty vector was inoculated into 5 mL SD-His liquid medium and cultured at 30°C and 200 rpm with shaking for 16 h until the logarithmic growth phase.

[0062] 2) Use a UV spectrophotometer (Thermo Scientific, NanoDrop 2000) to measure the OD of the culture medium. 600 According to the test results, the bacterial solution was diluted with sterile water to OD 600 = 1.0. Use gradient dilution method to prepare 10 0 , 10⁻¹, 10⁻², and 10⁻³ (each time, 50 μL of bacterial solution was added to 450 μL of sterile water).

[0063] 3) In the clean bench, take 3 μL of yeast solution with different concentration gradients and sequentially drip onto SD-His plate culture medium (Coolaber, KA0031) containing different concentrations of CdCl2. At the same time, use SD-His plate without CdCl2 as a control. Place the plate in a 30℃ constant temperature incubator for 3-5 days, and observe and record the growth of the colonies.

[0064] Will OsLCd38 Gene construction into yeast expression vector pAG413GAL The results showed that: on cadmium-free medium, pAG413GAL-OsLCd38 There was no significant difference in the growth of transformed yeast compared with wild-type yeast; however, when 20 μM CdCl2 was added to the medium, an increase in the growth of transformed yeast was observed. OsLCd38 The sensitivity of yeast to cadmium was significantly increased ( Picture 1 (a).

[0065] Inductively coupled plasma mass spectrometry was used to determine the cadmium content in the BY4741 control group and transgenic yeast cells. The results showed that ( Picture 1 b), expression OsLCd38 The accumulation of cadmium in yeast cells was significantly higher than that in wild-type controls. This finding suggests that OsLCd38 Expression in BY4741 yeast cells promoted the accumulation of intracellular cadmium, resulting in increased sensitivity of yeast to cadmium ions.

[0066] Example 3: Construction of OsLCd38 CRISPR strain 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'-GCCTAGTTGCGATCATCGCT-3', the target sequence is located at OsLCd38 The third exon region of the gene is from position 1661 to position 1680 of SEQ ID No:3 or corresponding to position 527 to position 546 of SEQ ID No:2.

[0067] 2) Preparation of Oligo Dimer Synthesized UP Oligo: 5'-TGTGGCCTAGTTGCGATCATCGCT-3'; Low Oligo: 5'-AAACAGCGATGATCGCAACTAGGC-3'. 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 reduce the temperature to 20°C at 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 10 μM) 1 μL, add water to make up to 20 μL of H2O.

[0068] 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: 2 μL BGK03 vector, 1 μL oligo dimer, 1 μL Enzyme Mix, and add water to make up to 10 μL H2O.

[0069] 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.

[0070] (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.

[0071] (C) Heat shock at 42°C for 90 s and incubate on ice for 3 min.

[0072] (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.

[0073] (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.

[0074] (F) Single clones were picked for sequencing, and strains with correct sequencing results were used for subsequent experiments.

[0075] The structure of the recombinant vector PBGK03-sgLCd38 is described as follows: a DNA fragment with a sequence of 5'-GCCTAGTTGCGATCATCGCT-3' 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 the recombinant vector.

[0076] The recombinant vector PBGK03-sgLCd38 contains the sgRNA expression cassette sequence SEQ ID No: 4, 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 positions 246-265 of SEQ ID No: 4 in the sequence table, and nucleotides 348-352 are the terminator for terminating the transcription of the sgRNA gene.

[0077] Rice transformation was carried out by the Agrobacterium-infected rice callus method, and the transformation background was Nipponbare. Agrobacterium was the EH105 strain (Zhuangmeng Biotechnology, ZC142-1), and CRISPR vector construction and rice transformation were completed by Biotech Co., Ltd.

[0078] Through the Agrobacterium infection rice callus method, two gene knockout lines, cr- oslcd38 -L1 (abbreviated as L1) and cr- oslcd38 -L2 (abbreviated as L2).

[0079] Sequence analysis found that compared with the genomic DNA of rice Nipponbare, oslcd38-L1 In the two homologous chromosomes of the strain, the gene encoding OsLCd38 protein has undergone the following mutation: "5'-CATTTGATGCTGCTGCT-3' (corresponding to positions 1733-1749 of SEQ ID No: 3, positions 599-615 of SEQ ID No: 2)" mutated to "5'-CATTTGTGCTGCT-3'"; the protein coding sequence lacked 4 bases "ATGC", making OsLCd38 A frameshift mutation occurs, resulting in loss of function, thereby knocking out the gene encoding OsLCd38 protein.

[0080] Compared with the genomic DNA of rice Nipponbare, oslcd38-L2 In the two homologous chromosomes of the strain, the gene encoding OsLCd38 protein has undergone the following mutation: "5'-AACTTGGGTTT-3' (corresponding to positions 1708-1718 of SEQ ID No: 3, positions 574-584 of SEQ ID No: 2)" mutated to "5'-ACTTGGGTTT-3'"; the protein coding sequence lacked one base "A", making OsLCd38 A frameshift mutation occurs, resulting in loss of function, thereby knocking out the gene encoding OsLCd38 protein.

[0081] Example 4: Field phenotype of OsLCd38 CRISPR strain Wild-type rice and oslcd38-L1, oslcd38-L2 The materials were planted in cadmium-contaminated soil for cadmium treatment experiments. The cadmium content in the soil was 0.8 mg / kg.

[0082] 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.

[0083] For each batch, three blank controls and three standard substance positive controls (standard substance for cadmium component analysis in rice flour, GBW08510, National Center for Standard Material Research) were set up.

[0084] During the rice maturity period, the plant height and cadmium content in brown rice were tested simultaneously. The results showed that ( Picture 2 (a and b): There was no significant difference in plant height between the mutant and the wild type in the cadmium-free and cadmium-contaminated fields.

[0085] The results of the analysis of cadmium content in rice grains showed that ( Picture 2 Middle c): The cadmium content in the grains of OsLCd38 CRISPR strains L1 and L2 was lower than that of the Nipponbare control. The cadmium content in the grains of Nipponbare rice was about 0.139 mg / kg, while the cadmium content in the grains of OsLCd38 CRISPR strains L1 and L2 was 0.089 mg / kg and 0.079 mg / kg respectively. The cadmium ion concentration in the grains of the CRISPR strains was reduced by about 36% and 43%. Statistical analysis showed that this difference was extremely significant. In summary: OsLCd38Gene knockout can significantly reduce cadmium accumulation in rice grains, providing important genetic resources for breeding low-cadmium rice varieties.

[0086] 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 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.

6. The method according to claim 5, 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.

7. 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.

8. A method for increasing the cadmium content in cells, 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 cell, 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 cell.

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 7, 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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