Application of OsNRAMP3 gene in improving iron content in rice seeds

Abstract

The present invention discloses OsNRAMP3 The invention relates to the application of genes in increasing the iron content of rice seeds, belonging to the field of biotechnology. The invention obtains rice seeds by cloning plant genomes. OsNRAMP3 gene, constructed the pCAMBIA1301 overexpression vector, and obtained OsNRAMP3 Gene overexpression strains OX1 and OX2 , analyzed OsNRAMP3 The iron content in the seeds of the gene overexpression strains was OsNRAMP3 The iron content in the seeds of the overexpression strain was higher than that of the wild type, revealing OsNRAMP3 The role of genes in iron transport provides new genetic resources for breeding iron-rich crop varieties.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to OsNRAMP3 Application of genes in improving iron content of rice seeds. Background Art

[0002] Metal transporters play a role in maintaining metal homeostasis. With the progress of molecular genetic analysis, many membrane proteins, including channels, pumps and transporters, have been found to be involved in the transport and homeostasis of metals, among which the NRAMP (natural resistance-associated macrophage protein) family genes play an important role in metal transport.

[0003] NRAMP proteins constitute a highly conserved family of integral membrane proteins involved in the transport of divalent metals. NRAMP genes have been found in a wide range of organisms, including bacteria, fungi, plants, and animals. The first member of this family, NRAMP1 (SLC11A1), has been characterized in mice as a resistance gene against endogenous pathogens such as Leishmania, Mycobacterium, and Salmonella. Many studies have demonstrated that NRAMP1 is a key component of mammalian host resistance to endogenous bacterial infection. In infected macrophages, NRAMP1 is rapidly recruited to the maturing phagosomal membrane, and mutations in this transporter lead to increased susceptibility to bacterial infection. NRAMP1 regulates the transport of essential divalent metals such as Fe 2+ and Mn 2+ ), but the exact function of NRAMP1 in host resistance remains controversial. DCT1 / DMT1 / NRAMP2 (SLC11A2), as the second NRAMP gene discovered in mammals, is a H + / Divalent metal co-transporter involved in intestinal iron absorption and iron release from endosomes during the transferrin cycle.

[0004] In recent years, phytohormones have been identified in various plant species. NRAMP So far, two genes have been identified and partially characterized in Arabidopsis NRAMP There are six members of the gene family. AtNramp1 Expression is induced by iron deficiency and controls plant iron homeostasis in roots. AtNramp3 and AtNramp4 It is also induced by iron deficiency and promotes the transport of iron ions in the vacuole, while overexpression of this gene does not significantly change the iron content. AtNramp3 and AtNramp4 It plays an important role in manganese homeostasis in adult Arabidopsis plants. However, no studies have shown that overexpression OsNRAMP3 Gene can increase iron content in rice seeds. Summary of the invention

[0005] The object of the present invention is to provide a OsNRAMP3 Application of genes in improving iron content of rice seeds.

[0006] In order to achieve the above object, the technical solution of the present invention is summarized as follows:

[0007] Based on the rice whole genome sequencing published on the official website of the National Rice Data Center (https: / / www.ricedata.cn / gene / ), rice OsNRAMP3 Nucleotide sequence information of the gene (GenBank: AK070574.1, LOC_AK070574). OsNRAMP3 The nucleotide sequence of the gene coding frame is 1653 bp long and consists of 550 amino acids. Its sequence can be obtained through the official website of the National Rice Data Center.

[0008] The present invention also constructs a series of expression vectors, and the expression vectors or transgenic plant lines containing the above-mentioned genes and host cells containing the vectors also fall within the protection scope of the present invention in terms of the function of increasing the iron content of plants.

[0009] The functions of the genes protected by the present invention include not only the above OsNRAMP3 Genes, including OsNRAMP3 Homologous genes with higher homology (eg, homology higher than 85%; more preferably higher than 90%; more preferably higher than 95%; more preferably higher than 98%) have functions in iron enrichment.

[0010] The present invention identifies the method of transient transformation of rice protoplasts OsNRAMP3 According to the subcellular localization of OsNRAMP3 The CDS gene sequence of GFP:: OsNramp3 -pBI221 transient expression vector was used to transiently transform rice protoplasts and laser confocal microscopy revealed that the OsNRAMP3 protein was localized on the vacuole membrane.

[0011] The present invention discloses OsNRAMP3 The gene is expressed in the roots, stems, leaves, and seeds of rice, with higher expression levels in the stems, leaves, and embryos; OsNRAMP3 The gene is induced to express by iron deficiency, which is manifested in the following aspects: OsNRAMP3 The gene expression levels were higher than those under iron culture.

[0012] The present invention discloses OsNRAMP3 The gene has the biological function of transporting iron, which is manifested in: OsNRAMP3-pDR196, using the growth rate of the defective yeast DEY1453 on medium containing different iron concentrations, found that OsNRAMP3 The ability to restore the growth of defective yeast indicates that OsNRAMP3 Has the ability to transport Fe.

[0013] The present invention discloses OsNRAMP3 The biological function of the gene in transporting iron in plants is specifically manifested in: constructing a successful plant expression vector and using the Agrobacterium-mediated rice transformation system to introduce the target gene into wild-type rice ( Oryzasativa cv. Kitaake), OsNRAMP3 Overexpression in rice caused changes in the growth state of rice, which occurred both in the absence of iron and in the presence of iron. Moreover, the iron content in the seeds of the overexpressing rice strains was significantly higher than that of wild-type rice, up to 3.2 times higher.

[0014] Specifically, the gene editing vector can be introduced into the target plant. In the method, the gene editing vector can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant tissue can be cultivated into a plant.

[0015] In order to improve the excellent traits of plants, the present invention also protects a new plant breeding method, the method is: by increasing the activity of OsNRAMP3 protein in the target plant, obtaining a plant whose seed iron content is higher than that of the target plant; or by overexpressing OsNRAMP3 Gene, to obtain plants whose seeds have higher iron content than the target plant;

[0016] Wherein, the target plant of the present invention is rice.

[0017] In addition, the present invention also discloses a method for increasing the iron content in plant seeds, comprising: OsNRAMP3 -pCAMBIA1301 overexpression vector, transform the constructed overexpression vector into plant tissues or plant cells, OsNRAMP3 Genes are overexpressed in plant tissues or plant cells. OsNRAMP3 The iron content of the gene overexpression strain was increased.

[0018] In the present invention, there is no particular limitation on the plants applicable to the present invention, as long as they are suitable for gene transformation operations, such as various crops, flower plants, or forestry plants, etc. The plants can be, for example (but not limited to): dicotyledons, monocotyledons or gymnosperms.

[0019] As a preferred embodiment, the "plant" includes but is not limited to rice, and any plant having the gene or a gene homologous thereto is applicable.

[0020] The "plant" mentioned in the present invention includes the whole plant, its parent and progeny plants and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues and organs, and our target gene or nucleic acid is present in these different parts. The "plant" mentioned here also includes plant cells, suspension cultures, callus, embryos, meristem regions, gametophytes, sporophytes, pollen and microspores, and similarly, each of the aforementioned objects contains the target gene / nucleic acid.

[0021] The present invention includes any plant cell, or any plant obtained or obtainable by the method therein, and all plant parts and propagules thereof. This patent also includes transfected cells, tissues, organs or whole plants obtained by any of the aforementioned methods. The only requirement is that the progeny exhibit the same genotypic or phenotypic characteristics, and the progeny obtained using the method of this patent have the same characteristics.

[0022] The invention also extends to the harvestable parts of the plants as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. It also further relates to other derivatives of the plants after harvest, such as dry granules or powders, oils, fats and fatty acids, starch or proteins. The invention also relates to foods or food additives obtained from the relevant plants.

[0023] Advantages of the present invention:

[0024] The present invention obtains rice by cloning plant genome OsNRAMP3 Gene, constructed OsNRAMP3 -pCAMBIA1301 overexpression vector, obtained by Agrobacterium infection OsNRAMP3 Gene overexpression strains OX1 and OX2 , analyzed OsNRAMP3 The iron content in the seeds of the gene overexpression strains was OsNRAMP3 The iron content in the seeds of the overexpression strain was higher than that of the wild type, revealing OsNRAMP3 The role of genes in iron transport provides new genetic resources for breeding iron-rich crop varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 yes OsNRAMP3 Gene cloning status, Figure 1 In the figure, A is the electrophoresis diagram of gene amplification products, and B is OsNramp3 -pMD18-T restriction enzyme cutting verification diagram.

[0026] Figure 2 yes OsNRAMP3 Schematic diagram of gene structure.

[0027] Figure 3 yes OsNRAMP3 Fluorescence detection results of genes. Figure 3 In the figure, A and B are schematic diagrams of the constructed transient expression vectors, C1 is a cell image under the green fluorescence signal detection field, C2 is a cell image under the red fluorescence signal detection field, and C3 is a cell image under the green and red signal combined field.

[0028] Figure 4 yes OsNRAMP3 tissue expression.

[0029] Figure 5 yes OsNRAMP3 Genetic yeast experiments, Figure 5 In the figure, A is the yeast growth experiment and B is the yeast growth rate comparison chart.

[0030] Figure 6 yes OsNRAMP3 Acquisition of overexpression materials, Figure 6 In the figure, A: rice embryonic callus; B: transformation and screening; C: differentiation culture; D: rooting culture; E: greenhouse growth.

[0031] Figure 7 for OsNRAMP3 Identification of overexpressed material, Figure 7 In, A: OsNRAMP3 -Schematic diagram of pCAMBIA1301 overexpression vector; B: OsNRAMP3 Northern analysis of overexpression lines OX1 and OX2 and wild-type rice as control materials; C: OsNRAMP3 SDS-PAGE and Western analysis of overexpression lines OX1 and OX2 and wild-type rice kitaake (abbreviated as k); D: OsNRAMP3 Detection of iron content in seeds of overexpressing lines OX1 and OX2 and wild-type rice. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, the specific experimental methods involved in the following examples are all conventional methods or implemented under the conditions recommended by the manufacturer's instructions unless otherwise specified.

[0033] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can all be purchased from the market.

[0034] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0035] Unless otherwise specified, the implementation of the present invention will use conventional botanical techniques, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination and bioinformatics techniques that are obvious to those skilled in the art. These techniques are fully explained in the published literature. In addition, the methods of DNA extraction, construction of phylogenetic trees, gene editing methods, construction of gene editing vectors, and obtaining gene-edited plants used in the present invention can be achieved by using methods already disclosed in existing literature, except for the methods used in the following examples.

[0036] The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-stranded or double-stranded structures. These nucleic acids or polynucleotides include gene coding sequences, antisense sequences and regulatory sequences of non-coding regions, but are not limited to these. These terms include a gene. "Gene" or "gene sequence" is widely used to refer to a functional DNA nucleic acid sequence. Therefore, a gene may include introns and exons in a genomic sequence, and / or include a coding sequence in a cDNA, and / or include a cDNA and its regulatory sequences. In specific embodiments, such as with respect to an isolated nucleic acid sequence, it is preferably assumed to be cDNA.

[0037] Before introducing the specific embodiments, in order to facilitate those skilled in the art to understand the relevant research and development of the present application in detail, the principles, some experimental materials, detection methods, etc. of the present invention are described below in conjunction with the accompanying drawings:

[0038] 1. Extraction and reverse transcription of total RNA from plant materials

[0039] Take 0.2 g of plant material and grind it into off-white powder in liquid nitrogen for experiment. Add 600 µL Buffer EL to the powder, shake for 1 min, centrifuge for 5 min (12000 rpm) immediately after the powder is completely mixed, transfer the supernatant to adsorption column III and centrifuge for 1 min (12000 rpm), transfer the supernatant to a new tube and add 300 µL of anhydrous ethanol to adjust the column environment, add the mixture to adsorption column V in batches after thorough mixing, centrifuge for 30 s (12000 rpm), and discard the filtrate. Then add RWA and RWB to adsorption column V step by step and centrifuge to remove impurities (RWB needs to be washed twice). After washing, replace the new collection tube and centrifuge the empty column for 2 min (12000 rpm), dry at room temperature, and then add water to elute (low-temperature high-speed centrifugation for 2 min). After elution, the total RNA is temporarily stored on ice or frozen in liquid nitrogen and stored at -80℃.

[0040] After measuring the concentration of the extracted total RNA with Nanodrop, the RNA template was denatured according to the amount of 5 µg total RNA, and the PCR program was set (heating at 65℃ for 5 min, cooling at 12℃ for 5 min). After the program was completed and cooled, the denatured product was taken out and ice bathed for 2 min. After ice bathing, gDNA wiper Mix was added and mixed, and incubated at 42℃ for 2 min. RT Mix, HiScript IIIEnzyme Mix, Oligo (dT) 20VN and RNase-free ddH2O were added to the mixed solution in sequence. After mixing the above liquids with a pipette, the PCR program was set (heating at 25℃ for 5 min, heating at 37℃ for 45 min, and treating at 85℃ for 5s). The cDNA product after removal can be used for subsequent experiments or stored at -80℃.

[0041] 2. Vector Construction

[0042] Overexpression vector construction process:

[0043] RNA was extracted from wild-type rice materials, and the total RNA was measured with Nanodrop for concentration and then reverse transcribed into cDNA. At the same time, the cDNA sequence of LOC_AK070574 was downloaded from the official website of the National Rice Data Center (https: / / www.ricedata.cn / gene / ), and specific primers were designed using DNAMAN and Primer express3.0. The cDNA of the wild-type Japanese early-maturing japonica rice variety Kitaake was used as a template and cloned using PCR reaction (PCR reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, 37 cycles; 72℃ extension for 5 min, 12℃ cooling for 5 s)OsNRAMP3 The coding sequence of the gene was determined by electrophoresis of the PCR reaction product using 1% agarose gel. The agarose gel electrophoresis diagram is shown in Figure 1 As shown in A. Figure 1 Figure B is the restriction enzyme verification image after construction on the pMD18-T plasmid. The subsequent gene sequencing showed that the sequencing results were correct. The target band was recovered using Tiangen's DNA recovery and purification kit and the recovered target gene was connected to pCAMBIA1301 (connection at 55°C for 30 min). Figure 2 yes OsNRAMP3 Schematic diagram of the gene structure, which contains 13 introns and 13 exons; Figure 7 A is constructed OsNRAMP3- Schematic diagram of the structure of the pCAMBIA1301 overexpression vector.

[0044] 3. Subcellular localization of OsNRAMP3 protein

[0045] First, rice protoplasts were prepared: yellow rice seedlings that had been cultured in the dark for 14±2 days and were in good growth condition were selected. The young stems were cut into 1±0.5 mm thin segments with a blade and transferred to a plate containing enzymatic solution (Cellulase RS, 1.5% (w / v); Macerozyme R10, 0.7% (w / v); D-Mannitol, 0.4 M; CaCl2, 20 mM; MES-KOH (pH 5.7), 20 mM; BSA, 0.1% (w / v); KCl, 20 mM.), and the plate was placed in a 30℃ constant temperature shaker (60±20 rpm) away from light for 6±2 h. Slowly add W5 buffer equal to the volume of enzymatic solution to the completely digested cell suspension, and gently shake the plate to mix the liquid to terminate the digestion. The mixed cell suspension was filtered through a 300-mesh cell sieve into a pre-cooled round-bottom centrifuge tube (slow grinding), centrifuged at 4°C for 2 min (speed 200 g), and 5 mL of W5 buffer was added along the tube wall after discarding the supernatant. The centrifuge tube was gently spun to resuspend the protoplast precipitate at the bottom of the tube, and placed on ice to settle naturally for 30 min. After removing the supernatant, the rice protoplast preparation was completed.

[0046] Secondly, rice protoplast transformation (protected from light throughout the process): Add the MMG buffer required for transformation (MgCl2, 15 mM; D-Mannitol, 0.4 M; MES-KOH (pH 5.7), 4 mM) along the wall of the prepared rice protoplasts, and gently rotate the centrifuge tube until the protoplasts are resuspended in the buffer. At the same time, the pCambia1300-35S-CTO1-GFP plasmid and pCambia1300-35S-CTO1-RFP to be transformed are divided into 2 mL round-bottom centrifuge tubes (total plasmid volume 10-20 µg, volume not exceeding 20 µL), add the corresponding volume of protoplast cell suspension during transformation and stir well, then add the PEG transformation solution (D-Mannitol, 0.2 M; CaCl2, 100 mM; PEG4000, 40% (w / v)) prepared in advance, stir well and leave at room temperature for 15 min. When the transformation reaction is completed, add 4 volumes of W5 buffer (CaCl2, 125 mM; NaCl, 154 mM; KCl, 5 mM; MES-KOH (pH 5.7), 2 mM) to terminate the reaction, centrifuge at room temperature for 2 min (speed 100g), remove the supernatant and add 1 mL of W5 buffer to resuspend the protoplasts in the centrifuge tube. Then lay the centrifuge tube flat on a plate and culture it at 28°C for 12±4 h. The next day, take out the overnight cultured sample, let it settle naturally, remove 700 µL of supernatant, and resuspend the protoplasts with the remaining buffer. Take a small amount of protoplast suspension and place it under a fast super-resolution laser confocal microscope for imaging observation and take pictures. In this experiment, the red fluorescence signal of AtNRAMP3 protein fused with red fluorescent protein reported in Arabidopsis was used as a control for vacuole membrane localization to observe the localization of OsNRAMP3 protein. The results are shown in Figure 3 As shown: A is a schematic diagram of the structure of the mCherry-AtNRAMP3 transient expression vector, and B is a schematic diagram of the structure of the OsNRAMP3-GFP transient expression vector. The green fluorescence signal of the OsNRAMP3-GFP fusion protein in rice leaf cells ( Figure 3 C1) and the red fluorescence signal of mCherry-AtNRAMP3 localized to the vacuole membrane ( Figure 3 C2) completely overlaps, such as Figure 3 As shown in C3, this signal distribution indicates that the OsNRAMP3-GFP fusion protein is localized in the vacuole membrane.

[0047] 4. OsNRAMP3 Tissue expression analysis

[0048] For research OsNRAMP3The expression pattern of genes was detected by RT-PCR. OsNRAMP3 The expression of the gene in these tissues was found to be expressed in roots, stems, leaves and seeds, with slightly higher expression levels in stems and leaves, and lower expression levels in roots and seeds. Further real-time PCR was used to detect the expression of the gene in seedlings, roots, stems, leaves, flowers, embryos and endosperms ( Figure 4 ),Discover OsNRAMP3 It is expressed in all these tissues, with higher expression levels in stems, leaves and embryos and lower expression levels in other parts.

[0049] 5. Yeast Growth Experiment

[0050] Construction of yeast transformation vector: Amplification of 1.1 kb from rice mRNA by RT-PCR OsIRT1 and OsNRAMP3 During amplification, the primers were designed with EcoR Ⅰ. Sal I site, after sequencing is correct, use EcoR Ⅰ and Sal Ⅰ enzyme digestion;

[0051] Take 1 μg of pDR196 plasmid and use Hind III and Sal I digestion, after 0.8% agarose gel electrophoresis, the linear vector fragment was recovered and purified; the vector and the target fragment were ligated at a molar ratio of 1:3, and the reaction condition was 16°C overnight. The ligation product was transformed into E. coli DH5α competent cells, and the positive clones were identified; the recombinant plasmid was used for yeast transformation experiments after double restriction digestion, PCR and sequencing.

[0052] Yeast transformation: Streak the yeast strain DEY1453 on a YPD plate and culture it in a 28°C incubator for 2-3 days; pick a single clone of bacteria and place it in 5mL YPD liquid medium, shake it on a shaker at 28°C, then transfer it to 50mL YPD and shake it until the OD value is about 1; centrifuge at 3000rpm for 5min to collect the bacteria; rinse with 25mL sterile water, vortex to mix, and centrifuge to collect the bacteria; vortex with 25mL sterile water again, and dispense 1mL into 1.5mL centrifuge tubes; centrifuge at 6000rpm for 5min, remove the supernatant, add 100μL one-step buffer and vortex to mix; add 6μL carrier DNA and mix (first boil the carrier DNA for 20min and cool it on ice); add 1μL carrier DNA and mix; incubate at 45°C for 30min, shaking once every 10min. The incubated yeast was evenly spread on the corresponding Drop-out medium and cultured in a 28°C incubator for 3 days; the grown colonies were screened and re-streaked to obtain single clones; the colonies transformed with only the vector plasmid (as a negative control) and the transformedOsIRT1 (as a positive control) and OsNRAMP3 The colonies were transferred to the Drop-out culture medium with 20 μM Fe ions. When the bacteria reached an OD value of 1, they were spotted on the Drop-out plates with 0, 5, 20 and 100 μM Fe ions, respectively, to observe the transformation. OsNRAMP3 Whether DEY1453-deficient yeast can restore the ability to transport iron. Figure 5 As shown, Figure 5 A in the middle is a yeast growth experiment. Figure 5 B is a comparison of yeast growth rates. OsNRAMP3 The DEY1453-deficient yeast was able to restore the growth ability of the defective yeast in 0μM culture medium, and its growth rate in drop-out medium with 0, 5, 20 and 100μM Fe ions was much faster than that of the defective yeast.

[0053] 6. Genetic material creation

[0054] The wild type Japanese early-maturing japonica rice variety Kitaake was used as the starting plant. OsNRAMP3 The pCAMBIA1301 overexpression vector containing the 35S promoter was constructed by using the CDS sequence of the gene. The constructed expression vector was introduced into Agrobacterium, and the rice callus was infected by Agrobacterium. Figure 6 Middle A), the callus tissue obtained was used to further induce regenerated shoots ( Figure 6 Middle B: transformation and screening; Figure 6 Middle C: differentiation culture; Figure 6 Middle D: rooting culture; Figure 6 Middle E: greenhouse growth), obtained through resistance screening OsNRAMP3 Gene overexpression lines (OX1, OX2).

[0055] Take leaves of wild-type strain (WT) and overexpression strain (OX1, OX2) for RNA extraction, add 10μg RNA to 1 / 2 volume of loading buffer (if one more drop of EB is added, the loading buffer is reduced to 1 / 3 volume of loading), mix well and heat at 70℃ for 10min, put on ice immediately, and centrifuge after it is cool enough. Electrophoresis at 80v for about 3hr, during which time prepare membrane, absorbent paper, salt bridge, and filter paper. After running the electrophoresis, wash the gel with 2×SSC for 10min. Cut HybondN with a length 1mm larger than the gel. +Membrane nylon membrane and two filter papers. After the nylon membrane is moistened from top to bottom with ddH2O, the membrane is then immersed in an appropriate 10xSSC (10xSSC: 1.5MNaCl, 0.16MNa3C6H5O7.2H2O, pH 7.0) transfer solution for at least 5 minutes. Add an appropriate volume of transfer solution to the tray and build a transfer bridge, from bottom to top: absorbent paper, gel (with the sample well facing down), nylon membrane, two filter papers, glass plate, 400 g weight. Use a glass rod to drive away the bubbles between the layers, and cut off a corner of the nylon membrane as a mark. Transfer the membrane for 8-12 hours, changing the filter paper several times during this period. Place the wet, unstained nylon membrane on a dry filter paper and irradiate at a wavelength of 254nm at 1.5J / cm 2 The Northern detection results are as follows: Figure 7 As shown in B.

[0056] Pick OsNRAMP3 About 0.1 g of the leaves of the gene overexpression material were placed in a mortar, and 400 μL of whole protein extract (E buffer: 125 mM Tris-HCl, pH 8.8; 50 mM Na2S2O5; 1% (w / v) SDS; 10% (w / v) Glycerol) was added to the mortar. After being fully ground, the mixture was transferred to a 1.5 mL centrifuge tube, shaken for 15 min, and centrifuged at room temperature (12,000 g, 10 min). The supernatant was aspirated into a new 1.5 mL centrifuge tube to obtain OsNRAMP3 Total protein solution of overexpressed materials; if the solution is still turbid, repeat the above centrifugation process and collect the supernatant for Western blot. Clean and prepare the relevant tools for preparing polyacrylamide gel in advance, and prepare 15% polyacrylamide gel: the upper layer is the concentration gel, the concentration (acrylamide) is 4.5%, and the lower layer is the separation gel, the concentration (acrylamide) is 15%. Prepare the gel in proportion and wait for it to solidify. Prepare 1× SDS-PAGE electrophoresis buffer and pre-cool it in a 4℃ refrigerator. Add the extracted OsNRAMP3 Add loading buffer (5 mM Tris-HCl, pH 8.8; 1.2% (w / v) SDS; 1% (w / v) Glycerol; 1% (w / v) β-Mercaptoethanol; 0.0001% (w / v) Bromophenol blue) to the total protein solution of the overexpressed material, heat it at high temperature for 10 min to completely denature it, then add the sample to the gel loading well using a loader and start electrophoresis (10 mA per gel, 4-6h). Determine the position of the target protein based on the position of the bromophenol blue indicator and protein marker, and stop electrophoresis when the target protein runs to 2 / 3 of the gel ( Figure 7C). At the same time, prepare the transfer buffer and soak the filter paper and nitrocellulose membrane in the transfer buffer in advance. Carefully remove the gel from the glass plate and soak it in the transfer buffer. Place the filter paper, gel, nitrocellulose membrane, and filter paper in order, buckle the transfer clip, and start the transfer (current 180 mA, time 1.5 h). After the transfer is completed, take out the nitrocellulose membrane with protein, put it in 8% skim milk at room temperature for 1 h, and then replace the skim milk with TTBS (50 mMTris-HCl, pH 7.4; 150 mM NaCl; 0.05% (v / v) Tween-20) to wash the blocked nitrocellulose membrane (wash 3 times in total, 5 min each time). Transfer the washed nitrocellulose membrane to 1% skim milk (containing the primary antibody) and incubate it overnight at 4°C. After recovering the primary antibody, wash the nitrocellulose membrane with TTBS (wash 5 times in total, 5 min each time). Transfer the washed nitrocellulose membrane to 1% skim milk (containing secondary antibody) and incubate at room temperature for 1 h. After incubation, wash the nitrocellulose membrane with TTBS (4 times, 5 min each time). Mix equal volumes of chemiluminescent solution A and B on plastic wrap; after 1 min, place the membrane with the protein side facing down to fully contact the mixture; after 1 min, move the membrane to another plastic wrap, remove all the residual liquid, wrap it, and put it in the X-ray film folder. In a darkroom, pour 1× developer and fixer into plastic trays respectively; take out the X-ray film under red light and cut it to the appropriate size with a paper cutter (1 cm larger than the length and width of the film); open the X-ray film folder, put the X-ray film on the film, do not move it, cover the X-ray film folder, and start timing; adjust the exposure time appropriately according to the strength of the signal, generally 1 to 5 minutes, or choose to press the film multiple times at different times to achieve the best effect; after the exposure is completed, open the X-ray film folder, take out the X-ray film, and quickly immerse it in the developer for development. When obvious strips appear, stop the development immediately. The development time is generally 1-2 minutes (20-25℃). When the temperature is too low (below 16℃), the development time needs to be appropriately extended; after the development is completed, immediately immerse the X-ray film in the fixer, and the fixing time is generally 5 to 10 minutes until the film is transparent; rinse off the residual fixer with tap water and dry it at room temperature. It should be noted that when moving the film for development and fixing, try to hold the film by a corner and do not scratch the film with your fingernails. OsNRAMP3 The NRAMP3-FLAG protein in the gene overexpression material was stably expressed under the drive of the CaMV 35S promoter.

[0057] 7. Determination of iron content in mature rice seeds

[0058] The mature dry seeds of wild-type rice and overexpression lines OX1 and OX2 were ground into powder after removing the seed coat. 0.01-0.3g of dried and ground material was weighed on an analytical balance and the mass was recorded. The weighed material was transferred to a microwave digestion tube, and 5mL of nitric acid (which can be left overnight) and 2mL of hydrogen peroxide were added to the tubes. After standing for 20 minutes, the tubes were placed on a microwave digester. After digestion, the solution was cooled to room temperature and the volume of the digestion solution was adjusted to a 100mL volumetric flask with distilled water. The adjusted solution was filtered to remove the residue. The Fe, Mn, Zn, Cu and other ion contents in the filtered solution were determined using inductively coupled plasma emission spectroscopy (iCAP).

[0059] Result calculation: ion content (μg / g) = test result (μg / mL) × 100mL (extract volume) × 0.001 / sample mass (g). The test results are as follows: Figure 7 As shown in D, the iron content in the seeds of the overexpression rice line was 3.2 times higher than that of the wild-type rice.

[0060] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. OsNRAMP3 The application of the gene in increasing the iron content of rice seeds is characterized in that: Said OsNRAMP3 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: By building OsNRAMP3 -pCAMBIA1301 overexpression vector, obtained OsNRAMP3 A gene overexpression strain, wherein the iron content in the seeds of the overexpression strain is higher than that of the wild type.

3. The use according to claim 2, characterized in that: Using wild-type rice as the starting plant, the constructed overexpression vector was introduced into Agrobacterium, and the rice callus was infected by Agrobacterium. The obtained callus was used to induce regenerated seedlings, and resistance screening was performed to obtain OsNRAMP3 Gene overexpression lines.

Images