CN111996199B

CN111996199B - Arabidopsis thaliana seed iron accumulation regulatory gene INO and coding protein and application thereof

Info

Publication number: CN111996199B
Application number: CN202010973235.4A
Authority: CN
Inventors: 郑绍建; 孙鹂; 李桂新; 丁忠杰
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-09-16
Filing date: 2020-09-16
Publication date: 2022-04-15
Anticipated expiration: 2040-09-16
Also published as: CN111996199A

Abstract

The invention provides an arabidopsis seed iron accumulation regulatory gene INO as well as a coding protein and application thereof, belonging to the technical field of plant genetic engineering. The nucleotide sequence of the arabidopsis seed iron accumulation regulatory gene INO is shown as SEQ ID NO. 1 in the sequence table. The arabidopsis seed iron accumulation regulatory gene INO encodes a 231 amino acid protein. Experiments prove that the gene INO negatively regulates the iron loading amount in plant seeds, so that the seed iron accumulation regulation gene INO or the coding protein thereof is applied to the regulation of the plant seed iron accumulation; meanwhile, the accumulation of iron in seeds can be obviously promoted by down-regulating the expression of INO, and the resistance of the plants to the iron-deficiency environment in the seedling stage is improved. Therefore, the invention also provides the application of the seed iron accumulation regulatory gene INO or the coding protein thereof in the biological strengthening of iron or in the improvement of the iron deficiency resistance of plants.

Description

Arabidopsis thaliana seed iron accumulation regulatory gene INO and coding protein and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to an arabidopsis thaliana seed iron accumulation regulatory gene INO, and a coding protein and application thereof.

Background

Iron is a necessary microelement for the life and growth of organisms, but because the effectiveness of iron in soil and the iron content in seeds are very low, the organisms are easy to lack iron^[1]. Thus, iron deficiency is the most common nutritional deficiency disorder, and its resulting iron deficiency anemia remains a worldwide concern to dateProblem (2)^[2]. According to the recommended intake of dietary nutrients for Chinese residents^[3]It was shown that the recommended daily iron intake for adult males is 12mg, and for adult females 20mg/d more. Seeds, particularly seeds of cereal crops, are human staple food. However, seeds act as heterotrophic organs, and their storage of iron depends both on the input of the parent as a source and on the ability of the seeds themselves as a sink to take up iron. Although iron deficiency of plants can be relieved by applying iron fertilizer in agricultural production, the effect is limited because soluble iron is easily fixed by soil and is ineffective; in addition, iron is less mobile in plants, and seeds are generally the lowest iron content in all organs of plants, especially the kernels of cereal crops as staple food. It is also the more serious reason that causes iron deficiency anemia in the wide developing countries that use grains as staple food^[4]. Research shows that the content of the trace elements in the seeds depends on the absorption of root systems, the migration in vivo and the loading to the seeds^[5]Wherein the loading into the seed determines to a large extent the iron content in the seed. Therefore, the process of loading iron into seeds by a plant matrix and the regulatory elements thereof are the key points for solving the problems, and a possible improvement way is provided for improving the content of iron in the seeds by optimizing the loading process of the iron through a genetic engineering technology.

Transcription factors are eukaryotic organisms, and a group of transcription factors can be specifically combined with a specific sequence at the 5' end upstream of a gene, so that protein molecules for regulating and controlling the expression of the gene at a specific time and space play an important role in regulating and controlling the expression of downstream genes. In higher plants, a number of transcription factors are involved in the uptake, transport and storage of plant iron. Taking Arabidopsis as an example, iron deficiency inducing transcription factor FIT and other four basic helix-loop-helix (bHLH) family transcription factors can jointly act to regulate and control the downstream ferric reductase FRO2 and ferrous transporter IRT1 to absorb iron^[6]. In addition, another bHLH transcription factor, popeye (pye), helps maintain iron homeostasis by modulating the ferroportin ZIF1, FRO3, and NAS4^[7]. Recent studies have shown that there are still more bHLH genes, such as bHLH34/104/105/115/121, that present a complex transcriptional regulatory network,regulating downstream transporters involved in iron uptake and homeostasis in plants^[8-11]This also shows that the transcription factor plays an important regulatory role in different links of plant iron nutrition. However, in the process of plant seed formation, how the transcription factor regulates the transport protein to load iron element from the mother into the seed has not been reported.

The exointegument of plant seed is a co-plastic extension of phloem, and the nutrients are discharged to the developing seed through exointegument after reaching the end of phloem^[12]. The research result shows that NRT1.6 transfers nitrate to developing seeds through a co-plastic continuum formed by phloem and periderm in early embryonic development^[13]. SWEET15 expressed on the periintegument can also be used for introducing carbohydrate as C source into seeds^[14]. It is shown that the delivery of nutrients into developing seeds through the phloem and integumentary complex is a common route. Using Perls/DAB staining, which can characterize the iron distribution, it was observed that a small amount of iron was uniformly distributed in the embryo at the early stage of the embryo development, and as the embryo matured, iron was also heavily imported as other nutrient elements, and finally accumulated in endoderm cells around the original vascular system^[15]. It has been reported that the transcription factor INO is the exointegument in the early stage of seed development^[16]Medium expression and strong expression in early embryo development, and the gene expression is reduced along with the gradual development of the embryo. However, there is no report that the transcription factor INO participates in the iron transport process of plant embryos at present.

Reference to the literature

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

[2] The influence of the slow release of iron on the iron nutrition status and brain stem auditory evoked potential of premature infants [ D ]. Zhejiang university, 2018.

[3] Chinese residents have reference dietary nutrient intake [ M ] 2001.

[4]POTTIER M,DUMONT J,MASCLAUX-DAUBRESSE C,et al.Autophagy is essential 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 is essential 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 Factor POPEYE Regulates Response to Iron Deficiency in Arabidopsis Roots[J].The Plant Cell,2010,22(7):2219-2236.

[8]LIANG G,ZHANG H,LI X,et al.bHLH transcription factor bHLH115 regulates iron homeostasis in Arabidopsis thaliana[J].Journal of Experimental Botany,2017,68(7):1743-1755.

[9]ZHANG J,LIU B,LI M,et al.The bHLH Transcription Factor bHLH104 Interacts 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 and bHLH104,regulate iron homeostasis in Arabidopsis thaliana[J].Plant Physiology,2016,170(4):2478.

[11]GAO F,ROBE K,BETTEMBOURG M,et al.The Transcription Factor bHLH121 Interacts with bHLH105(ILR3)and its Closest Homologs to Regulate Iron Homeostasis in Arabidopsis[J].The Plant Cell,2019:541-2019.

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

[13]ALMAGRO A,LIN S H,TSAY Y F.Characterization of the Arabidopsis nitrate transporter NRT1.6 reveals a role of nitrate in early embryo development[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.

[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 OUTER regulates abaxial-adaxial patterning in Arabidopsis ovules[J].Genes&Development,1999,13(23):3160.

Disclosure of Invention

In view of the above, the present invention aims to provide an arabidopsis thaliana seed iron accumulation regulatory gene INO, and a coding protein and an application thereof, and it is clear that the gene INO plays an important regulatory role in the loading of iron in plant seeds.

The invention provides an arabidopsis seed iron accumulation regulatory gene INO, wherein the nucleotide sequence of the INO is shown as SEQ ID NO. 1 in a sequence table.

The invention provides a primer for amplifying an iron accumulation regulatory gene INO of an arabidopsis seed, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2 in a sequence table; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3 in the sequence table.

The invention provides a coding protein of an arabidopsis seed iron accumulation regulatory gene INO, and the amino acid sequence of the coding protein is shown as SEQ ID NO. 4 in a sequence table.

The invention provides an application of the arabidopsis seed iron accumulation regulatory gene INO, the primer or the coding protein in plant seed iron accumulation regulation.

Preferably, the plant seed comprises arabidopsis thaliana.

Preferably, the plant seed comprises a mature embryo.

Preferably, the mature embryo is collected from arabidopsis pod 4-6 days after flowering.

The invention provides an application of the arabidopsis seed iron accumulation regulatory gene INO, the primer or the coding protein in bioaugmentation of iron.

The invention provides an application of the arabidopsis seed iron accumulation regulatory gene INO, the primer or the coding protein in improving the iron deficiency resistance of plants.

The arabidopsis seed iron accumulation regulatory gene provided by the invention is a transcription factor INO. The transcription factor INO is cloned from a model plant Arabidopsis thaliana, a low-expression mutant line of the gene shows that the iron content in seeds is obviously increased, and an over-expression line of the gene shows that the iron content in the seeds is obviously reduced. The invention proves that INO can inhibit the loading of iron to the seeds in the early development stage of the embryo so as to avoid the influence of the accumulation of excessive iron on the normal development of the embryo by generating ROS; the down regulation of the expression of INO can obviously promote the accumulation of iron in seeds and simultaneously improve the resistance of plants to the iron-deficient environment in the seedling stage. The results show that the expression of INO in the early development stage of the embryo is controlled at a reasonable level through gene operation, so that the aims of not causing poison caused by excessive iron and increasing the accumulation level of iron in seeds are fulfilled, and the healthy growth of plants in the iron-deficient environment and the improvement of iron nutrition of human beings in the seedling stage of the plants are facilitated. The gene INO provided by the invention has important significance for improving the iron nutrition of crop seeds by adopting a biological strengthening means.

Drawings

FIG. 1 is a schematic diagram of binary vector 35s-pCAMBIA 1301;

FIG. 2 is a schematic representation of the transgenic vector pOEINO;

FIG. 3 is a graph comparing the expression levels of INO genes of wild-type and INO over-expression transgenic lines;

FIG. 4 is a comparison of seed iron staining of wild type, INO low expressing mutants and overexpressing transgenic lines; wherein the length of each scale is 500 mu m;

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

Detailed Description

The invention provides an arabidopsis seed iron accumulation regulatory gene INO, wherein the nucleotide sequence of the INO is shown as SEQ ID NO. 1 in a sequence table. In the invention, the INO is positioned in the 86-781 coding region of the INO full-length cDNA, and the length of the nucleotide sequence is 696 bp.

The invention provides a primer for amplifying an iron accumulation regulatory gene INO of an arabidopsis seed, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2(5'-TGGTACCTACACACACACTCTCTATGACAAAG-3') in a sequence table; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3(5'-CGGATCCTCCCAAATTGTTATTACTCAAATGG-3') in the sequence table.

The source of the gene INO is not particularly limited in the present invention, and a gene acquisition scheme known in the art, such as gene cloning or artificial synthesis, may be adopted. Preferably, the gene cloning method uses the cDNA of the mature embryo of arabidopsis thaliana as a template, and the primer is adopted to carry out PCR amplification to obtain a PCR amplification product, namely the gene. The reaction procedure for the PCR amplification is preferably as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 10 seconds; annealing at 57 ℃ for 30 seconds; extension at 68 ℃ for 1 min for 30 cycles; final extension at 68 ℃ for 7 min.

The invention provides a coding protein of an arabidopsis thaliana seed iron accumulation regulatory gene INO, wherein the coding protein is a sequence consisting of 231 amino acids, and the amino acid sequence of the coding protein is shown as SEQIDNO:4 in a sequence table.

Based on the negative regulation effect of the arabidopsis seed iron accumulation regulatory gene INO on the iron content in seeds, the invention provides the application of the arabidopsis seed iron accumulation regulatory gene INO, the primer or the coding protein in the plant seed iron accumulation regulation.

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

cloning the gene INO into a plant constitutive overexpression vector to obtain a recombinant expression vector;

transforming the recombinant expression vector into plant seeds by agrobacterium mediation, culturing, screening and harvesting the seeds of the first transgenic generation (T1 generation).

In the present invention, the plant constitutive overexpression vector is preferably binary vector pCAMBIA1301 containing promoter CaMV 35S. The method for cloning is not particularly limited in the present invention, and a method for cloning a gene into a vector, which is well known in the art, may be used. The method of transformation is not particularly limited in the present invention, and Agrobacterium-mediated transformation methods well known in the art may be used.

In the present invention, the application of the arabidopsis seed iron accumulation regulatory gene INO is applicable to all types of plant seeds. In order to illustrate the regulation mode of the INO, the invention takes model plant Arabidopsis thaliana as a material to carry out an experiment, and the amplified gene is marked as AtINO.

In the present invention, the plant seed preferably comprises mature embryos, since in the early embryonic development stage of the plant, only a small amount of iron is distributed in the embryo, and when the embryo develops to maturity, iron, as well as other nutrient elements, is also substantially imported and eventually accumulated in endodermal cells surrounding the original vasculature. The mature embryo is not particularly limited in the present invention, and a mature embryo well known in the art may be used. In the embodiment of the present invention, the mature embryo is preferably collected from arabidopsis pod 4-6 days after flowering is completed.

In the invention, because the gene INO has negative regulation and control effect on the iron content in plant seeds, the overexpression of the gene INO reduces the iron content in the seeds, and therefore, the expression level of the gene INO in the plant seeds is inhibited to improve the iron content in the seeds. By controlling the expression quantity of the appropriate gene INO, the method can improve the iron content of the plant seeds, ensure that the iron cannot be poisoned, use the obtained plant seeds as the biological strengthened iron, greatly simplify the industrial production operation, develop the channel of the biological strengthened iron, provide a new production idea for the biological strengthened iron and greatly reduce the production cost.

The arabidopsis seed iron accumulation regulatory gene INO provided by the present invention and its encoded protein and use are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

In particular, the invention is funded by a research and development project (2016YFD0100704) of the department of science and technology.

Example 1

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

And (2) sowing the arabidopsis thaliana seeds with the disinfected surfaces in an 1/2MS solid culture medium, performing vernalization for 2-3 days at 4 ℃ in the dark, transferring to a light condition for culture for 7 days, performing soil transfer culture, after the bolting arabidopsis thaliana flowers, shearing young green pod fruits which bloom for 4-6 days by using scissors for extracting RNA, and performing reverse transcription by using a kit to synthesize cDNA (complementary deoxyribonucleic acid) as a template for subsequent gene cloning.

Respectively designing an upstream primer and a downstream primer according to the sequencing result of the whole genome of the arabidopsis which is disclosed at present:

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

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

PCR amplification was performed using KOD FX enzyme (TOYOBO Co., Ltd.) according to the following reaction procedure: pre-denaturation: 94 ℃ for 2 minutes; denaturation: 10 seconds at 98 ℃; annealing at 57 ℃ for 30 seconds; extension at 68 ℃ for 1 min (30 cycles); final extension: 68 ℃ for 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

By utilizing a double-enzyme digestion and connection method of DNA fragments, a cauliflower mosaic virus constitutive promoter CaMV35S is positively inserted into a multiple cloning site by utilizing enzyme digestion sites SacI and KpnI, so that the promoter CaMV35S is successfully connected to the vector, and the vector 35s-pCAMBIA1301 (shown in figure 1) which can be used for constructing a constitutive overexpression transgenic material is obtained by transformation.

Using primers: 5'-TGGTACCTACACACACACTCTCTATGACAAAG-3' (SEQ ID No:2) and INO-R: 5'-CGGATCCTCCCAAATTGTTATTACTCAAATGG-3' (SEQ ID No:3), using the cDNA sequence obtained in the above example 1 as a template, and referring to the PCR amplification reaction program in the above example 1, the coding region sequence of the Arabidopsis transcription factor gene AtINO containing enzyme cleavage sites at both ends was amplified. The sequence coding the region of the Arabidopsis thaliana transcription factor gene AtINO was ligated to pMD19T according to the instructions for use of the vector pMD19T produced by Takara, and then ligated to the inserted promoter CaMV35S after the sequence coding the Arabidopsis thaliana transcription factor gene AtINO was excised from the pMD19T vector by the KpnI and BamHI double digestion and ligation, to obtain a transgenic vector pOEINO (see FIG. 2) for promoting the Arabidopsis thaliana gene AtINO by the promoter CaMV 35S.

Example 3

Transformation method of arabidopsis thaliana

Adding 0.5 mu g of the binary transgenic vector pOEINO plasmid prepared in the example 2 into competent cells of a strain GV3101 of Agrobacterium tumefaciens (Agrobacterium tumefaciens), sequentially carrying out ice bath for 5 minutes, liquid nitrogen for 5 minutes, water bath for 5 minutes at 37 ℃ and ice bath for 5 minutes, adding nonreactive LB into a shaker at 28 ℃ to activate for 1 hour, and obtaining the Agrobacterium tumefaciens strain containing the binary plasmid vector. The GV3101 strain containing binary plasmid vector is used to transform Arabidopsis thaliana, and the specific steps are as follows:

agrobacterium containing the binary plasmid vector was cultured in LB medium containing 50mg/L kanamycin (Kan) and 50mg/L rifampicin (Rif) with shaking at 28 ℃ overnight to an absorbance of 1.0 at OD 600, centrifuged at 4000rpm for 15min to collect the cells, and resuspended in 1/2MS medium containing 50g/L sucrose. Selecting wild type (Col-0) arabidopsis thaliana which is subjected to bolting and partial flowering as a transgenic material, subtracting mature pods, preserving flowers and buds, adopting a vacuumizing transformation method to dip-dye the overground part of the arabidopsis thaliana into the prepared bacterial liquid, performing vacuum extraction for 5min, culturing for 24h under the conditions of darkness and 23 ℃, screening resistant seedlings on 1/2MS culture medium containing 50mg/L hygromycin for 1 week, and harvesting a transgenic first generation (T1 generation) seed after transplanting soil culture. T1 generation seeds were screened for additional generations on 1/2MS medium containing 50mg/L hygromycin to obtain homozygous transgenic T2 generation material (INO ox 1 and INO ox 2).

Example 4

Molecular detection method for target gene expression

Sampling tender whole seedlings of wild type and over-expression transgenic plants, extracting RNA, carrying out reverse transcription, and carrying out fluorescent real-time quantitative PCR detection by adopting SYBR Green real-time PCR Master Mix of TOYOBO company, wherein the gene of Actin2 is used as an internal reference; the detection system and primers used were as follows:

the used fluorescent real-time quantitative PCR reaction primers are as follows:

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 for fluorescent real-time quantitative PCR was as follows:

pre-denaturation: 1 minute at 95 ℃; and (3) PCR circulation: at 95 ℃ for 15 seconds; 60 ℃ for 15 seconds; 72 ℃ for 45 seconds (40 cycles).

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

the results are shown in FIG. 3. After detection, the INO gene is 6000-7000 times highly expressed in the over-expression transgenic plant compared with the non-transgenic plant.

Example 5

The gene is indeed involved in seed iron loading, as in comparative experiments as follows:

detecting the iron content in the seeds:

arabidopsis thaliana, a representative plant of the brassicaceae family, has its seed ripened, endosperm disappeared, and all nutrients stored in the mature embryo, so that in order to determine whether there is a difference in the total amount of iron loaded into the seed, we performed histochemical examination of iron in the mature embryo using prussian blue staining. Respectively disinfecting the wild type, low-expression mutant materials (INO-1 and INO-2) of an INO gene of arabidopsis thaliana purchased from an arabidopsis thaliana biological resource center (ABRC) and seeds of an overexpression transgenic material of the gene (namely, the seeds obtained in the embodiment 3 of the invention) with 75% alcohol by mass concentration, cleaning the seeds for 3-5 times with sterilized water, soaking the seeds in the sterilized water for 2-3 hours, carefully peeling off the seeds with tweezers, soaking the taken embryos in a Prussian blue dye solution (4% K)₄Fe(Cn)₆And 4% HCl stock solution in a volume ratio of 1: 1) and evacuated for 15 minutes and then incubated for 15 minutes at room temperature, the embryos are transferred to sterilized water for washing and are photographed and observed under a microscope.

The results show that the iron content in transgenic over-expressed (INO-ox) Arabidopsis thaliana is obviously reduced compared with that in the seeds of wild type control, while the iron content in the seeds of low-expression mutant lines of INO genes is obviously higher than that in the wild type, and particularly, see FIG. 4, the gene INO is shown to be actually involved in the regulation and control of the iron loading process of the seeds.

Example 6

The content of iron element in the seeds is quantitatively analyzed by an inductively coupled plasma mass spectrometer (ICP-MS), 1000 seeds of wild type and low expression mutant materials of INO gene and over-expression transgenic materials of the gene are respectively put into 500 mu l of concentrated nitric acid for high-temperature digestion at 220 ℃, ultrapure water is added to the seeds for constant volume to 5ml after digestion is clear and transparent, and the seeds are uniformly mixed and then are put on a machine for determination.

The results show that the iron content in seeds of low-expression mutant lines of the INO gene is obviously higher than that of wild type seeds, compared with wild type control, the iron content in the seeds of transgenic over-expression (INO-ox) is obviously lower, and as shown in FIG. 5, the influence of the gene INO on the iron content of the seeds is verified again.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that modifications can be made by those skilled in the art without departing from the principle of the present invention, and these modifications should also be construed as the protection scope of the present invention.

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Claims

1. The application of an arabidopsis seed iron accumulation regulatory gene INO, a primer for amplifying the arabidopsis seed iron accumulation regulatory gene INO or a coding protein of the arabidopsis seed iron accumulation regulatory gene INO in plant seed iron accumulation regulation, wherein the nucleotide sequence of the INO is shown as SEQ ID NO 1 in a sequence table;

the amino acid sequence of the encoded protein is shown as SEQ ID NO. 4 in the sequence table;

the primers comprise an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2 in the sequence table; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3 in the sequence table.

2. Use according to claim 1, wherein the plant seed is an Arabidopsis seed.

3. Use according to claim 1 or 2, wherein the plant seed is a mature embryo.

4. The use of claim 3, wherein the mature embryos are harvested from Arabidopsis pods at 4-6 days after flowering.

5. The application of an arabidopsis seed iron accumulation regulatory gene INO, a primer for amplifying the arabidopsis seed iron accumulation regulatory gene INO or a coding protein of the arabidopsis seed iron accumulation regulatory gene INO in bioaugmentation iron, wherein the nucleotide sequence of the INO is shown as SEQ ID NO. 1 in a sequence table;

6. The application of an arabidopsis seed iron accumulation regulatory gene INO, a primer for amplifying the arabidopsis seed iron accumulation regulatory gene INO or a coding protein of the arabidopsis seed iron accumulation regulatory gene INO in improving the iron deficiency resistance of plants, wherein the nucleotide sequence of the INO is shown as SEQ ID NO 1 in a sequence table;