CN111996199A - Arabidopsis thaliana seed iron accumulation regulatory gene INO and coding protein and application thereof - Google Patents

Abstract

The invention provides an Arabidopsis thaliana seed iron accumulation regulating gene INO and its encoded protein and application, belonging to the technical field of plant genetic engineering. The nucleotide sequence of the Arabidopsis thaliana seed iron accumulation regulatory gene INO is shown in SEQ ID NO: 1 in the sequence listing. A protein encoding 231 amino acids of the Arabidopsis thaliana seed iron accumulation regulatory gene INO. The experiment of the present invention proves that the gene INO negatively regulates the iron loading in plant seeds, so the application of the seed iron accumulation regulation gene INO or its encoded protein in the regulation of iron accumulation in plant seeds; at the same time, by down-regulating the expression of INO, iron can be significantly promoted Accumulation in seeds also improves the resistance of plants to iron-deficiency environments at the seedling stage. Therefore, the present invention also provides the application of the seed iron accumulation regulating gene INO or its encoded protein in bio-enhancing iron or improving the resistance of plants to iron deficiency.

Description

An Arabidopsis thaliana seed iron accumulation regulatory gene INO and its encoded protein and applications

technical field

The invention belongs to the technical field of plant genetic engineering, and in particular relates to an Arabidopsis thaliana seed iron accumulation regulation gene INO and its encoded protein and application.

Background technique

Iron is an indispensable trace element for organisms to maintain life and growth, but due to the availability of iron in the soil and the low iron content in grains, it is easy to cause iron deficiency in living organisms ^[1] . Therefore, iron deficiency is the most common nutritional deficiency disease, and iron-deficiency anemia caused by it is still a worldwide concern ^[2] . According to the "Recommended Intake of Dietary Nutrients for Chinese Residents" ^[3] , the recommended daily intake of iron for adult men is 12 mg, while that for adult women is more than 20 mg/d. Seeds, especially those of cereal crops, are staples of human food. However, as a heterotrophic organ, the iron storage of seeds depends not only on the input of the parent as a source, but also on the ability of the seed as a sink to absorb iron itself. Although iron deficiency in plants can be alleviated by applying iron fertilizers in agricultural production, the effect is limited because soluble iron is easily fixed by soil and ineffective; in addition, iron has poor mobility in plants, and seeds are usually all plant organs The lowest iron content in the grain, especially the grain of the staple food. This is also an important reason why iron deficiency anemia is more serious in the vast developing countries where cereals are the main food ^[4] . Studies have shown that the content of trace elements in grains depends on root absorption, body migration and loading to seeds ^[5] , and the loading of seeds to a large extent determines the content of iron in seeds. Therefore, clarifying the process of iron loading from plant parent to seeds and its regulatory elements is the key to solving the problem, and it also provides a possible improvement approach to improve the iron content in seeds by optimizing the iron loading process through genetic engineering technology.

Transcription factors are a group of protein molecules that can specifically bind to specific sequences upstream of the 5' end of genes in eukaryotes, thereby regulating the expression of genes in specific time and space, and play an important role in regulating the expression of downstream genes. There are multiple transcription factors involved in plant iron uptake, transport and storage in higher plants. Taking Arabidopsis as an example, the iron deficiency-inducible transcription factor FIT and four other transcription factors of the basic helix-loop-helix (bHLH) family can work together to regulate the downstream ferric reductase FRO2 and ferrous transporter IRT1. The absorption of iron ^[6] . In addition, another bHLH transcription factor, POPEYE (PYE), helps maintain iron homeostasis by regulating the iron transporters ZIF1, FRO3 and NAS4 ^[7] . Recent studies have shown that there are many more bHLH genes, such as bHLH34/104/105/115/121, with a complex transcriptional regulatory network that regulates downstream transporters involved in iron uptake and homeostasis in plants ^[8- 11], which also indicates that transcription factors play important regulatory roles in different aspects of plant iron nutrition. However, it has not yet been reported how transcription factors regulate the transport of iron from the parent into seeds during plant seed formation.

The outer integument of plant seeds is a co-plastic extension of the phloem, through which nutrients are unloaded into the developing seed after reaching the phloem terminal ^[12] . The results showed that NRT1.6 transmits nitrate to developing seeds through the co-plastic continuum formed by the phloem and the outer integument during early embryonic development ^[13] . At the same time, SWEET15 expressed in the outer integument can also import carbohydrates as C sources into seeds ^[14] . This indicates that it is a common way to transport nutrients into developing seeds through the phloem and the outer integument complex. Using Perls/DAB staining, which can characterize iron distribution, it was observed that in the early stage of plant embryo development, a small amount of iron was evenly distributed in the embryo. Accumulates in the endoderm cells around the primitive vascular system ^[15] . It has been reported that the transcription factor INO is expressed in the outer integument ^[16] in the early stage of seed development, and is strongly expressed in the early stage of embryo development, and the gene expression decreases as the embryo gradually develops. However, there is no report on the involvement of the transcription factor INO in the iron transport process in plant embryos.

references

[1]JEONG J,GUERINOT M L.Homing in on iron homeostasis in plants[J].Trends in Plant Science,2009,14(5):280-285.

[2] Xu Lin. Effects of preventive iron supplementation on iron nutritional status and brainstem auditory evoked potentials in premature infants [D]. Zhejiang University, 2018.

[3] Reference intake of dietary nutrients for Chinese residents [M]. 2001.

[4] POTTIER M, DUMONT J, MASCLAUX-DAUBRESSE C, et al. Autophagy isessential for optimal translocation of iron to seeds in Arabidopsis [J]. Journal of Experimental Botany, 2019, 70(3): 859-869.

[5] POTTIER M, DUMONT J, MASCLAUX-DAUBRESSE C, et al. Autophagy isessential for optimal translocation of iron to seeds in Arabidopsis [J]. Journal of Experimental Botany, 2018, 70(3): 859-869.

[6]BRUMBAROVA T,BAUER P,IVANOV R.Molecular mechanisms governing Arabidopsis iron uptake[J].Trends in Plant Science,2015,20(2):124-133.

[7]LONG T A,TSUKAGOSHI H,BUSCH W,et al.The bHLH Transcription FactorPOPEYE Regulates Response to Iron Deficiency in Arabidopsis Roots[J].ThePlant Cell,2010,22(7):2219-2236.

[8] LIANG G, ZHANG H, LI X, et al.bHLH transcription factor bHLH115regulates iron homeostasis in Arabidopsis thaliana[J].Journal of ExperimentalBotany,2017,68(7):1743-1755.

[9]ZHANG J,LIU B,LI M,et al.The bHLH Transcription Factor bHLH104Interacts with IAA-LEUCINE RESISTANT3 and Modulates Iron Homeostasis in Arabidopsis[J].The Plant Cell,2015,27(3):787.

[10]LI X,ZHANG H,AI Q,et al.Two bHLH transcription factors,bHLH34 andbHLH104,regulate iron homeostasis in Arabidopsis thaliana[J].PlantPhysiology,2016,170(4):2478.

[11] GAO F, ROBE K, BETTEMBOURG M, et al. The Transcription Factor bHLH121Interacts with bHLH105(ILR3) and its Closest Homologs to Regulate IronHomeostasis in Arabidopsis [J]. The Plant Cell, 2019:541-2019.

[12] STADLER R, LAUTERBACH C, SAUER N. Cell-to-Cell Movement of GreenFluorescent Protein Reveals Post-Phloem Transport in the Outer Integument and Identifies Symplastic Domains in Arabidopsis Seeds and Embryos[J].PlantPhysiology,2005,139(2) :701.

[13]ALMAGRO A,LIN S H,TSAY Y F.Characterization of the Arabidopsisnitrate transporter NRT1.6 reveals a role of nitrate in early embryodevelopment[J].The Plant Cell,2008,20(12):3289.

[14] CHEN L, LIN I W, QU X, et al. A Cascade of Sequentially Expressed Sucrose Transporters in the Seed Coat and Endosperm Provides Nutrition for the Arabidopsis Embryo [J]. The Plant Cell, 2015, 27(3):607.

G,CURIEC,et al.Identification of theEndodermal Vacuole as the Iron Storage Compartment in the Arabidopsis Embryo[J].Plant Physiology,2009,151(3):1329.[15] ROSCHZTTARDTZ H,

G,CURIEC,et al.Identification of the Endodermal Vacuole as the Iron Storage Compartment in the Arabidopsis Embryo[J].Plant Physiology,2009,151(3):1329.

[16]VILLANUEVA J M,BROADHVEST J,HAUSER B A,et al.INNER NO OUTERregulates abaxial-adaxial patterning in Arabidopsis ovules[J].Genes&Development,1999,13(23):3160.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide an Arabidopsis thaliana seed iron accumulation regulation gene INO and its encoded protein and application, and it is clear that the gene INO plays an important regulatory role in the loading of iron and iron in plant seeds.

The present invention provides an Arabidopsis thaliana seed iron accumulation regulating gene INO, the nucleotide sequence of the INO is shown in SEQ ID NO: 1 in the sequence listing.

The present invention provides a primer for amplifying the Arabidopsis thaliana seed iron accumulation regulation gene INO, including an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is as shown in SEQ ID NO: 2 in the sequence table. The nucleotide sequence of the downstream primer is shown in SEQ ID NO: 3 in the sequence listing.

The present invention provides the encoded protein of the Arabidopsis thaliana seed iron accumulation regulation gene INO, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 4 in the sequence table.

The present invention provides the application of the Arabidopsis thaliana seed iron accumulation regulation gene INO, the primer or the encoded protein in the regulation of plant seed iron accumulation.

Preferably, the plant seeds include Arabidopsis thaliana.

Preferably, the plant seeds comprise mature embryos.

Preferably, the mature embryos are collected from Arabidopsis pods 4-6 days after flowering.

The present invention provides the application of the Arabidopsis thaliana seed iron accumulation regulating gene INO, the primer or the encoded protein in bio-enhancing iron.

The present invention provides the application of the Arabidopsis thaliana seed iron accumulation regulating gene INO, the primer or the encoded protein in improving the resistance of plants to iron deficiency.

The Arabidopsis thaliana seed iron accumulation regulation gene provided by the invention is a transcription factor INO. The transcription factor INO was cloned from the model plant Arabidopsis thaliana. The low-expression mutant line of the gene showed a significant increase in iron content in seeds, while the overexpression line of this gene showed a significant decrease in the iron content in seeds. The present invention proves that INO can inhibit the loading of iron into seeds in the early stage of embryonic development, so as to avoid the accumulation of excessive iron and affect the normal development of embryos by generating ROS; while down-regulating the expression of INO can significantly promote the accumulation of iron in seeds, and at the same time improve the Resistance of plants to iron-deficiency environments at the seedling stage. The above results show that the expression of INO in the early embryonic development can be controlled at a reasonable level through genetic manipulation, so as to achieve the purpose of not causing the toxicity caused by excessive iron, but also increasing the accumulation level of iron in the seeds, which is beneficial to the seedling stage of the plant itself. Healthy growth in iron-deficient environments and improved iron nutrition in humans. The gene INO provided by the present invention has great significance for improving the iron nutrition of crop seeds by means of biofortification.

Description of drawings

Fig. 1 is the schematic diagram of binary vector 35s-pCAMBIA1301;

Fig. 2 is the schematic diagram of transgenic vector pOEINO;

Fig. 3 is a graph showing the comparison of INO gene expression between wild-type and INO overexpression transgenic lines;

Figure 4 is a comparison chart of seed iron staining of wild-type, INO low-expression mutant and over-expression transgenic lines; the length of the scale is 500 μm;

Figure 5 is a graph comparing seed iron content of wild-type, INO low-expressing mutants, and over-expressing transgenic lines.

Detailed ways

The present invention provides an Arabidopsis thaliana seed iron accumulation regulating gene INO, the nucleotide sequence of the INO is shown in SEQ ID NO: 1 in the sequence listing. In the present invention, the INO is located in the coding region 86-781 of the full-length cDNA of INO, and the length of the nucleotide sequence is 696 bp.

The present invention provides a primer for amplifying the Arabidopsis thaliana seed iron accumulation regulation gene INO, including an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is such as SEQ ID NO: 2 ( 5'-TGGTACCTACACACACACTCTCTATGACAAAG-3'); the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 3 (5'-CGGATCCTCCCAAATTGTTATTACTCAAATGG-3') in the sequence listing.

The present invention has no special limitation on the source of the gene INO, and a gene acquisition scheme known in the art can be used, such as gene cloning or artificial synthesis. The method for gene cloning preferably takes the mature embryo cDNA of Arabidopsis as a template, and uses the primers for PCR amplification, and the PCR amplification product obtained is the gene. The reaction procedure of the PCR amplification is preferably as follows: pre-denaturation at 94°C for 2 minutes; denaturation at 98°C for 10 seconds; annealing at 57°C for 30 seconds; extension at 68°C for 1 minute, 30 cycles; final extension at 68°C for 7 minutes.

The present invention provides the encoded protein of the Arabidopsis thaliana seed iron accumulation regulation gene INO, the encoded protein is a sequence consisting of 231 amino acids, and the amino acid sequence thereof is shown in SEQ ID NO: 4 in the sequence table.

Based on the negative regulation effect of the Arabidopsis seed iron accumulation regulating gene INO on the iron content in seeds, the present invention provides the Arabidopsis thaliana seed iron accumulation regulating gene INO, the primer or the encoded protein in the regulation of iron accumulation in plant seeds Applications.

In the present invention, the method for reducing iron content in the plant seeds preferably comprises the following steps:

The gene INO is cloned into a plant constitutive overexpression vector to obtain a recombinant expression vector;

The recombinant expression vector is transformed into plant seeds with the mediation of Agrobacterium, cultured, screened, and the transgenic first generation (T1 generation) seeds are harvested.

In the present invention, the plant constitutive overexpression vector is preferably a binary vector pCAMBIA1301 containing the promoter CaMV35S. The method of cloning is not particularly limited in the present invention, and a method known in the art for cloning a gene into a vector can be used. The method of the transformation is not particularly limited in the present invention, and the transformation method mediated by Agrobacterium well known in the art can be used.

In the present invention, the application of the Arabidopsis seed iron accumulation regulating gene INO is applicable to all types of plant seeds. In order to illustrate the regulation mode of the INO, the present invention uses the model plant Arabidopsis thaliana as the material to conduct the test, and the amplified gene is denoted as AtINO.

In the present invention, due to the early stage of plant embryo development, only a small amount of iron is distributed in the embryo. When the embryo develops and matures, iron, like other nutrients, is also input in large quantities, and finally accumulates in the interior surrounding the primitive vascular system. In germ layer cells, the plant seed therefore preferably comprises mature embryos. In the present invention, the mature embryos are not particularly limited, and mature embryos known in the art may be used. In the embodiment of the present invention, the mature embryos are preferably collected from Arabidopsis pods 4-6 days after flowering.

The present invention provides the application of the Arabidopsis thaliana seed iron accumulation regulating gene INO, the primer or the encoded protein in bio-enhancing iron.

In the present invention, since the gene INO has a negative regulatory effect on the iron content in plant seeds, and overexpressing the gene INO reduces the iron content in the seeds, the expression of the gene INO in the plant seeds is inhibited in order to increase the iron content in the seeds. By controlling the expression level of the appropriate gene INO, it can not only increase the iron content of plant seeds, but also ensure that it will not cause poisoning caused by iron. The obtained plant seeds are used as bio-fortified iron, which greatly simplifies industrial production operations and opens up channels for bio-fortified iron. , to provide a new production idea for biofortified iron and greatly reduce the production cost.

The present invention provides the application of the Arabidopsis thaliana seed iron accumulation regulating gene INO, the primer or the encoded protein in improving the resistance of plants to iron deficiency.

The Arabidopsis thaliana seed iron accumulation regulating gene INO and its encoded protein and application will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

In particular, the present invention is funded by the key research and development plan project of the Ministry of Science and Technology (2016YFD0100704).

Example 1

Cloning method of transcription factor INO gene involved in regulation of seed iron

The surface-sterilized Arabidopsis thaliana seeds were planted in 1/2MS solid medium, vernalized for 2-3 days in the dark at 4°C, then moved to light conditions for 7 days, then transferred to the soil for cultivation, and waited for the bolted Arabidopsis to bloom. , using scissors to cut the young green pods 4 to 6 days after flowering for RNA extraction, and using a kit to perform reverse transcription to synthesize cDNA as a template for subsequent gene cloning.

According to the published whole genome sequencing results of Arabidopsis thaliana, the upstream and downstream primers were designed respectively:

Upstream primer: 5'-ATGACAAAGCTCCCCAACATGAC-3' (SEQ ID No: 5);

Downstream primer: 5'-TCCCAAATTGTTATTACTCAAATGGAG-3' (SEQ ID No: 6).

PCR amplification was carried out using KOD FX enzyme of TOYOBO company. The reaction program of PCR amplification was as follows: pre-denaturation: 94°C, 2 minutes; denaturation: 98°C, 10 seconds; annealing at 57°C, 30 seconds; extension at 68°C, 1 minute ( 30 cycles); final extension: 68°C, 7 minutes. The reaction system of PCR amplification is as follows:

The PCR amplification product was sent to sequencing to obtain the CDS sequence of AtINO (SEQ ID NO: 1).

Example 2

Construction method of constitutive overexpression transgenic vector

A cauliflower mosaic virus constitutive promoter CaMV35S was inserted into the multi-cloning site by using the method of double-enzyme cleavage and ligation of the DNA fragment using the enzyme cleavage sites SacI and KpnI, so that the promoter CaMV35S was successfully connected to the vector. The vector 35s-pCAMBIA1301 which can be used to construct constitutive overexpression transgenic material was obtained by transformation (see Figure 1).

Using primers: INO-F: 5'-TGGTACCTACACACACACTCTCTATGACAAAG-3' (SEQ ID No: 2) and INO-R: 5'-CGGATCCTCCCAAATTGTTATTACTCAAATGG-3' (SEQ ID No: 3), the cDNA obtained in Example 1 above was used Using the sequence as a template, referring to the PCR amplification reaction procedure in Example 1 above, amplify the Arabidopsis thaliana transcription factor gene AtINO coding region sequence containing restriction sites at both ends. According to the instructions of the pMD19T vector produced by Takara Company, the coding region sequence of the Arabidopsis transcription factor gene AtINO was connected to pMD19T, and then the coding region sequence of the Arabidopsis transcription factor gene AtINO was ligated with KpnI and BamHI double digestion and ligation. After being excised from the pMD19T vector and connected to the inserted promoter CaMV35S, the transgenic vector pOEINO (see Fig. 2 ), which promotes the Arabidopsis gene AtINO from the promoter CaMV35S, is obtained.

Example 3

Transformation Methods of Arabidopsis

0.5 μg of the binary transgenic vector pOEINO plasmid prepared in Example 2 was added to the competent cells of Agrobacterium tumefaciens strain GV3101, followed by ice bath for 5 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes and ice bath for 5 minutes. , add anti-anti-LB and activate in a shaker at 28°C for 1 h to obtain Agrobacterium strains containing binary plasmid vectors. Use the prepared GV3101 strain containing binary plasmid vector to transform Arabidopsis thaliana, and the specific steps are as follows:

The Agrobacterium containing the binary plasmid vector was cultured in LB medium containing 50 mg/L kanamycin (Kan) and 50 mg/L rifampicin (Rif) at 28°C with shaking overnight to the absorbance value of OD 600 The value was 1.0, and the cells were collected by centrifugation at 4000 rpm for 15 min, and resuspended in 1/2 MS medium containing 50 g/L sucrose. And select the wild-type (Col-0) Arabidopsis thaliana that has been bolted and partially completed flowering as the transgenic material, subtract the mature pods, retain the flowers and buds, and use the vacuum transformation method to infuse the above-mentioned Arabidopsis thaliana aerial part. In the prepared bacterial solution, after vacuum extraction for 5 minutes, cultured for 24 hours in the dark at 23 °C, and screened on 1/2MS medium containing 50 mg/L hygromycin for 1 week to obtain resistant seedlings, transplanted into soil for culture Then the transgenic generation (T1 generation) seeds were harvested. Homozygous transgenic T2 generation material (INO ox 1 and INO ox 2) were obtained after T1 generation seeds were screened for another generation on 1/2MS medium containing 50 mg/L hygromycin.

Example 4

Molecular assays for target gene expression

The wild-type and overexpressed transgenic plants were sampled to extract RNA from young whole plantlets. After reverse transcription, fluorescence real-time quantitative PCR was performed with TOYOBO's SYBR Green Realtime PCR Master Mix, and the Actin2 gene was used as an internal reference. The detection system and primers used are as follows:

The primers used in the real-time quantitative PCR reaction were:

qINO-F: 5'-TTGGGCCCATTTTCCTCCAG-3' (SEQ ID NO:7)

qINO-R: 5'-GCCTTTCTCTCTCGGAACCC-3' (SEQ ID NO: 8)

qActin2-F: 5'-GGTAACATTGTGCTCAGTGGTGG-3' (SEQ ID NO: 9)

qActin2-R: 5'-AACGACCTTAATCTTCATGCTGC-3' (SEQ ID NO: 10).

The reaction procedure of real-time quantitative PCR is as follows:

Pre-denaturation: 95°C, 1 minute; PCR cycles: 95°C, 15 seconds; 60°C, 15 seconds; 72°C, 45 seconds (40 cycles).

The reaction system of real-time quantitative PCR is as follows:

The results are shown in Figure 3. After testing, it was found that compared with the non-transgenic lines, the INO gene was 6000-7000 times higher expressed in the overexpressed transgenic plants.

Example 5

This gene is indeed involved in seed iron loading, as shown in the following comparative experiments:

Detection of iron content in seeds:

Arabidopsis is a representative plant of the cruciferous family. After the seeds mature, the endosperm disappears, and all nutrients are stored in the mature embryo. Therefore, in order to determine whether the total amount of iron loaded into the seeds is different, we used Prussian blue. The staining method was used for histochemical detection of iron in mature embryos. The wild-type, the low-expression mutant materials (ino-1 and ino-2) of the Arabidopsis INO gene and the over-expressing transgenic materials (namely this gene) were purchased from the Arabidopsis Biological Resource Center (ABRC). After the seeds obtained in Invention Example 3) were surface sterilized with a mass concentration of 75% alcohol, rinsed with sterile water for 3-5 times, soaked in sterile water for 2 to 3 hours, and then carefully peeled off the seed coat with tweezers, The removed embryos were soaked in Prussian blue staining solution (4% K ₄ Fe(Cn) ₆ and 4% HCl stock solution were mixed in a volume ratio of 1:1), vacuumed for 15 minutes, and then incubated at room temperature for 15 minutes. The isolated embryos were transferred to sterile water for washing and photographed under a microscope.

The results showed that the iron content in the seeds of the transgenic overexpressed (INO-ox) Arabidopsis thaliana was significantly lower than that of the wild-type control, while the iron content in the seeds of the mutant lines with low expression of the INO gene was significantly higher than that of the wild type, as shown in Figure 4. The gene INO is indeed involved in the regulation of the seed iron loading process.

Example 6

The iron content in seeds was quantitatively analyzed by inductively coupled plasma mass spectrometry (ICP-MS), and 1000 seeds of wild-type, low-expression mutant material of INO gene and transgenic material with over-expression of this gene were collected respectively. , put it in 500 μl of concentrated nitric acid and cook at 220°C at high temperature. After the digestion is clear and transparent, add ultrapure water to make up the volume to 5ml, and then measure on the machine after mixing.

The results showed that the iron content in the seeds of the mutant lines with low expression of the INO gene was significantly higher than that of the wild type, while the iron content in the seeds of the transgenic overexpression (INO-ox) was significantly lower than that of the wild type control, as shown in Figure 5, and the gene was verified again. INO affects seed iron content.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, some improvements can be made without departing from the principles of the present invention, and these improvements should also be regarded as the present invention. the scope of protection of the invention.

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Claims (9)

1. an Arabidopsis thaliana seed iron accumulation regulation gene INO, is characterized in that, the nucleotide sequence of described INO is as shown in SEQ ID NO:1 in the sequence listing. 2. a primer for amplifying the described Arabidopsis thaliana seed iron accumulation regulation gene INO of claim 1, comprising upstream primer and downstream primer, it is characterized in that, the nucleotide sequence of described upstream primer is such as SEQ in sequence listing ID NO: 2; the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 3 in the sequence listing. 3. The encoded protein of the Arabidopsis thaliana seed iron accumulation regulation gene INO according to claim 1, wherein the amino acid sequence of the encoded protein is as shown in SEQ ID NO: 4 in the sequence listing. 4. The application of the Arabidopsis thaliana seed iron accumulation regulation gene INO according to claim 1, the primer according to claim 2 or the encoded protein according to claim 3 in the regulation of plant seed iron accumulation. 5. The application according to claim 4, wherein the plant seeds comprise Arabidopsis thaliana. 6. The application according to claim 4 or 5, wherein the plant seeds comprise mature embryos. 7 . The application according to claim 6 , wherein the mature embryos are collected from Arabidopsis pods 4 to 6 days after flowering. 8 . 8. The application of the Arabidopsis thaliana seed iron accumulation regulating gene INO according to claim 1, the primer according to claim 2 or the encoded protein according to claim 3 in biofortified iron. 9. The application of the Arabidopsis thaliana seed iron accumulation regulating gene INO according to claim 1, the primer according to claim 2 or the encoded protein according to claim 3 in improving the resistance of plants to iron deficiency.

Images