CN116891861A - Application of plant AtRTH gene in iron absorption and transport - Google Patents

Abstract

The present invention relates to the field of biotechnology, and in particular to the application of AtRTH gene in plant iron absorption and transport. The nucleotide sequence of the AtRTH gene is shown in SEQ ID NO.1; the AtRTH gene is highly expressed in roots, stems and leaves of Arabidopsis vegetative organs; GUS staining results show that it is expressed in roots, stems and leaves. Expressed and mainly expressed in vascular tissues; the AtRTH gene is used to regulate the absorption and transport of iron by plants. By knocking out the AtRTH gene or down-regulating its expression, the sensitivity of Arabidopsis to high concentrations of iron is reduced, thereby improving the root length, leaf chlorophyll content and plant viability of Arabidopsis under high concentrations of iron salts. The technical solution of the present invention is of great significance for using EMS mutagenesis and/or gene editing to breed plants that are resistant to high concentrations of iron salts and to obtain plants with high iron content in plants.

Description

Application of plant AtRTH gene in iron absorption and transport

Technical Field

The invention relates to the field of biotechnology, in particular to application of an AtRTH gene in iron absorption and transportation of plants.

Background

Iron (Fe) is a very important trace element in organisms, is the highest trace element required by human bodies, exists in a plurality of tissues of the human bodies, is a main constituent element of various proteins, and participates in various biochemical reactions in the human bodies. Iron deficiency may lead to a decrease in human hemoglobin, which may cause other diseases. About 20 million people worldwide have iron deficiency, and women and children are more prevalent, as counted by the world's defense organization. Incomplete statistics show that nearly 3 hundred million people in China lack iron.

Iron is one of 16 nutritional elements necessary for plant growth and development, is a cofactor for more than 300 enzymes in plants, and plays an important role in many physiological processes, such as: respiration, photosynthesis, nitrogen fixation, and the like. Iron deficiency in plants causes reduced chlorophyll synthesis, inhibition of photosynthesis and respiration, and ultimately leads to stunted plant growth and development, reduced biomass accumulation, and reduced yield. Excessive iron can cause Fenton reaction, and cause hydroxyl poisoning. The content of iron element in the soil is rich and is much higher than that of other trace elements, the average content is up to 3.2%, but the multi-alkaline calcareous soil in northwest China has higher pH value and the concentration of soluble ferrous iron is very low (about 10) ^-15 mol/L), the iron content is much less than that required by normal growth and development of general cropsTherefore, crop growth shows iron deficiency phenomena with different degrees, while the south soil is acid, extremely strong in reducibility and high in iron solubility, so that plants are often poisoned by absorbing excessive iron.

Plants are one of the main channels for human body to ingest iron nutrition, and iron deficiency affects plant growth and indirectly leads to iron deficiency of human beings. Although we can increase the content of soluble iron in soil by applying fertilizer, the long-term large-scale application of fertilizer has relatively large harm to soil, so the most efficient, safe and direct method for increasing effective iron in plants is to research the molecular mechanism of iron absorption and transportation of plants, genetically improve crops, and increase the iron content of edible parts of crops so as to reduce the incidence of iron deficiency of human beings.

Ethylene is an important plant hormone and the receptor ETR1, upon receiving ethylene, causes a series of ethylene reactions, such as triplex reactions, via the ethylene signal transduction pathway. Arabidopsis RTE1 is a newly discovered upregulator of the receptor ETR1, and its family has only two members, RTE1 and RTH (RTE 1-HOMOLOG). Further studies have found that RTH interacts with RTE1 and modulates ethylene signaling through RTE 1.

Therefore, the influence of RTH on plant iron transport and the application of RTH in crop molecular breeding are urgently required to be explored.

Disclosure of Invention

The invention provides an application of a plant AtRTH gene in iron absorption and transportation. The invention obtains the coding sequence of the arabidopsis gene AtRTH, obtains a mutant rth-1 by using an EMS mutagenesis method, and simultaneously proves that the AtRTH gene plays a role in iron absorption and transportation, and reduces the sensitivity of the arabidopsis to high-concentration iron and the iron absorption and transportation capacity by knocking out or down-regulating the expression of the AtRTH gene, thereby improving the root length, the leaf chlorophyll content and the plant viability of the arabidopsis under high-concentration iron salt, and being beneficial to the growth of plants in the environment with extremely strong reducibility and large iron solubility of the iron salt in the acid soil in the south.

The nucleotide sequence of the AtRTH gene is shown as SEQ ID NO. 1.

The AtRTH gene is expressed higher in roots, stems and leaves of the arabidopsis thaliana vegetative organ; GUS staining results showed expression in roots, stems and leaves and mainly in vascular tissues; the AtRTH gene is used for regulating and controlling iron absorption and transportation of plants; atRTH mediates up-regulated expression of FIT, AHA and YSL genes by ethylene and affects the uptake and transport of iron by plants.

Furthermore, the sensitivity of the arabidopsis to high-concentration iron is reduced, the iron absorption and transport capacity and the iron content in plants are reduced by knocking out the AtRTH gene or down regulating the expression of the AtRTH gene, so that the root system length, the leaf chlorophyll content and the plant viability of the arabidopsis under high-concentration ferric salt are improved.

Furthermore, the AtRTH gene is knocked out or the expression of the AtRTH gene is regulated down, so that the AtRTH gene is suitable for application under the conditions of high iron solubility and easiness in poisoning of plants.

Furthermore, the sensitivity of arabidopsis to high-concentration iron and the iron absorption and transport capacity are improved by establishing an AtRTH over-expression line and/or establishing a complementary line, so that the iron content in plants is improved.

Furthermore, an AtRTH over-expression line is established and/or a complementary line is established, so that the method is suitable for application under the conditions of low iron solubility and iron deficiency of plants.

Furthermore, the nucleotide sequence of the AtRTH mutant plant rth-1 obtained by knocking out the AtRTH gene is shown as SEQ ID NO. 2.

Further, the 322 th base in SEQ ID No.2 is changed from C to T, resulting in the conversion of the original glutamine (CAA) to the termination code (TAA).

Further, the amplification primer sequences for identifying sequence 2 are:

rth1-1-Seq-F:CATCTCGTTCTTCCAGTTC(SEQ ID NO.3)

rth1-1-Seq-R:ATAGACGGTTCAGGTTGTT(SEQ ID NO.4)。

a cultivation method of arabidopsis thaliana resistant to high-concentration ferric salt is characterized by comprising the following steps: the cultivation method can be EMS mutagenesis, screening plants meeting the conditions after the mutagenesis, and carrying out offspring breeding to a homozygous line; and/or utilizing gene editing to obtain a vector containing the expression of the knocked-out sequence 1, transferring into agrobacterium to obtain a recombinant strain, infecting a target plant, and culturing to obtain the high-concentration ferric salt-resistant arabidopsis.

Preferably, the high concentration ferric salt is 200-600 mu M.

Compared with the prior art, the invention has the advantages that:

through the tissue expression specificity analysis of RTH, the AtRTH mainly plays a role in absorption and transportation parts; through Fe treatment response analysis, atRTH is expressed as being inhibited by iron; analysis of iron sensitivity of wild Col-0 and mutant rth-1 Arabidopsis thaliana shows that the root length of the mutant rth-1 Arabidopsis thaliana is obviously increased compared with that of the wild Arabidopsis thaliana under high concentration (600 mu M) iron, which indicates that AtRTH mutation reduces the sensitivity of plants to iron, has good growth vigor under high concentration ferric salt environment and can increase the chlorophyll content of leaves; obtaining a mutant rth-1 knocked out of the AtRTH gene by an EMS mutagenesis mode; the influence on the overexpression of AtRTH and the root length of a complementary line is explored through ferric salt treatment; the impact of the attth knockout line and over-expression development was explored by iron salt treatment.

To sum up, the application of the plant AtRTH gene in iron absorption and transportation and an arabidopsis thaliana resistant to high-concentration iron salt and a cultivation method thereof are obtained. The technical scheme of the invention has important significance for breeding high-concentration ferric salt-resistant plants and obtaining plants with high iron content in plants by using an EMS mutagenesis and/or gene editing mode.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is evident that the drawing figures in the following description are only one embodiment of the invention, and that other implementation drawings may be derived from the drawing figures provided without inventive effort for a person skilled in the art.

Fig. 1: the embodiment of the invention shows the AtRTH in roots, stems, rosette leaves, stem leaves, seeds and flowers;

fig. 2: the embodiment of the invention discloses an AtRTH Promoter-GUS vector construction and agrobacterium transformation, A: amplifying an electrophoresis chart by using an AtRTH promoter; b: pCambia1391 (z) double cleavage; c: the PCR detection result of the escherichia coli bacterial liquid; d: sequencing results of pCambia1391 (z) -proth; e: PCR detection results of agrobacterium tumefaciens bacteria liquid;

fig. 3: the GUS staining chart of the embodiment of the invention comprises the following steps: seedling; b: the overground part of the seedling; c: lateral roots; d: root tip; e: root hairs; f: a root-dimension pipe column; g: a supporting leaf; h: air holes; i: veins of the leaves;

fig. 4: the embodiment of the invention is relative expression of AtRTH under the treatment of ferric salt;

fig. 5: root length of the secondary Col-0 and rth-1 in the embodiment of the invention under different iron concentrations;

fig. 6: root length of Ler and rth-2 in the embodiment of the invention under different iron concentrations;

fig. 7: the two AtRTH genes are over-expressed and complementation system is constructed; a: screening over-expression T0 generation; b: the expression level of AtRTH in Col-0 and over-expression lines; c: screening the T0 generation of the complementation line; d: the expression level of AtRTH in Col-0 and complementary lines;

fig. 8: the ferric salt treatment of the embodiment of the invention affects the root system of the over-expression system;

fig. 9: the influence of the ferric treatment on the development of different strains of arabidopsis thaliana is achieved; effect of different concentrations of iron treatment on Col-0, rth-1, OE3 and OE24 seedling growth; b: effect of iron treatment on Col-0, rth-1 and OE24 development, 400. Mu.M EDTA-Fe ³⁺ Irrigation; c: effect of iron treatment on Col-0, rth-1 and OE24 development, clear water control; d: effect of iron treatment on chlorophyll content of Col-0, rth-1 and OE24 leaves; e: effect of iron treatment on Col-0, rth-1 and OE24 leaf plasma membrane permeability;

fig. 10: quantitative expression analysis of genes related to ferroferric absorption and transport is carried out;

fig. 11: the three AtRTH mutant provided by the embodiment of the invention detects the iron content under different concentrations of iron;

fig. 12: quantitative expression analysis of iron absorption and transport related genes under three ACC treatment in the embodiment of the invention.

Detailed Description

The invention is further illustrated by the following examples in conjunction with the accompanying drawings:

experimental materials:

1. strains and vectors:

the carriers used for the experiments were as follows: the overexpression vector pCambia1300-221-HA, the cloning vector pMD18-T, the GUS expression vector pCambia1391 (Z).

2. Plant material:

arabidopsis thaliana (Arabidopsis thaliana): wild Col-0 and Ler, an arabidopsis mutant rth-1 taking Col-0 as a background, wherein the arabidopsis mutant rth-1 is an AtRTH knockout plant of the wild Col-0, and the mutants rth-1 and the AtRTH knockout refer to the same type of arabidopsis;

an arabidopsis mutant rth-2 with Ler as a background and over-expression lines OE3 and OE24 with wild Col-0 as a background; complementary Re20 and Re36.

3. Reagent(s)

1) T4 DNA Ligase, restriction enzymes EcoR I and Sal I, etc. were purchased from Takara Shuzo China (Takara).

2) Plasmid extraction kits, total plant RNA extraction kits and common agarose gel recovery kits were purchased from Nanjinouzan biotechnology Co., ltd; reverse transcription kit (PrimeScript RT reagent Kit with gDNA Eraser) and real-time quantitative PCR (qRT-PCR) enzyme (TAKARA TB Green Premix Ex Taq II, RA 820) were purchased from Takara Shuzo China (Takara).

3) MS powder, EMS powder and agar powder used for plant culture are purchased from Shanghai Biotechnology engineering service Co.

4) The construction of the vector and the detection of the primer for gene expression and the sequencing of gene clone were completed by Qingdao qinghao biotechnology Co.

4. Configuration of culture Medium

1/2MS medium: 2.2g of MS powder is weighed, 0.5g of MES and 10g of sucrose are added, the pH is adjusted to 5.7-5.8, the volume is fixed to 1L, and if a solid culture medium is prepared, 7.5g of agar powder is added.

Example one AtRTH Gene expression Pattern analysis

1.1 Tissue-specific expression analysis of the attth gene

Taking root, stem, rosette leaf, stem leaf and flower of Col-0 growing for 42 days under long sunshine condition, extracting RNA for reverse transcription, analyzing the relative expression quantity of AtRTH gene in the material by qRT-PCR,

the RNA extraction method (Trizol method) is specifically as follows:

1) Placing plant materials of RNA to be extracted into a mortar for removing RNase by burning with alcohol, and grinding into powder under liquid nitrogen (directly used in the following experiment or stored in an ultralow temperature refrigerator of-80 ℃ for standby);

2) After the liquid nitrogen volatilizes, immediately transferring 100-200 mg of plant powder into a 1.5mL EP tube, then rapidly adding 1mL of TRIZOL extracting solution, and carrying out vortex vibration to enable a sample to be fully dissolved into the extracting solution, and standing for 5min at room temperature;

3) Centrifuging at 12,000rpm at 4 ℃ for 10min, transferring 0.8mL of supernatant to a new 1.5mL EP tube, adding 0.2mL of chloroform, shaking vigorously and mixing for 15sec, and standing at room temperature for 2-5 min;

4) Centrifuging at 12,000rpm at 4deg.C for 10min, transferring 0.4mL supernatant into new 1.5mL EP tube, adding 0.4mL isopropanol, gently turning over for 15 times, mixing, and standing at-20deg.C for 30min;

5) Centrifuging at 12,000rpm at 4℃for 10min, discarding the supernatant, washing the pellet twice with 0.9mL of 75% ethanol;

6) Removing the supernatant, uncovering the supernatant, drying RNA in an ultra-clean workbench for about 2-5 min, adding 40 mu LRNase-Free water, and fully dissolving in water bath at 60 ℃ for 10min;

7) The NanoDrop One is used for measuring the OD value and concentration of the RNA sample, the A260/A230 reaches more than 2.0, and the A260/A280 reaches preferably 1.7-2.0; in addition, agarose gel electrophoresis can be performed to detect RNA quality.

The reverse transcription method comprises the following steps:

the obtained RNA was reverse transcribed into cDNA in two steps using a reverse transcription kit (PrimeScript RT reagent Kit with gDNA Eraser), and a first system reagent was added according to Table 1.

Total RNA dosage is related to the concentration of the Total RNA, and the added amount is generally 1.5 mug, so that the added volume is confirmed to be 1-7 mug, and the first system is subjected to a constant-temperature water bath at 42 ℃ for 3min;

TABLE 1 reverse transcription first reaction System

After the completion of the first system reaction, a second system reagent was added (Table 2). After being gently mixed, the mixture is placed in a PCR instrument, and the procedures are as follows: the reverse transcription cDNA was obtained by storing at 42℃for 15min,85℃for 1min and 10℃for the subsequent experiments.

TABLE 2 reverse transcription second reaction System

The experimental results are shown in FIG. 1, in which roots (Rt), stems (St), rosettes leave (Rl), stems and leaves make leave (Cl), seeds (Se) and flowers (Fl);

the AtRTH gene is expressed in all parts of Col-0, wherein the expression is higher in the root, stem and leaf of the vegetative organ, thus the AtRTH possibly plays a role in the process of nutrient absorption and transport of plants.

1.2 AtRTH Promoter-GUS

1.2.1 Construction of AtRTH Promoter-GUS expression vector

A promoter fragment of 2060bp of the AtRTH gene was amplified using Arabidopsis genome DNA as a template and Sal I and EcoR I as primers (FIG. 2A).

Cloning, namely, carrying out gel recovery on an AtRTH amplification product and a carrier enzyme-digested fragment by using a Sal I and EcoR I double-enzyme carrier pCambia1391 (z) (fig. 2B), connecting the products in a water bath at 50 ℃ for 50min, converting the products into escherichia coli, selecting single colonies, shaking the single colonies in a 1.5mL EP tube, carrying out bacterial liquid PCR (polymerase chain reaction) detection after about 4h, and carrying out test on positive expression strains containing recombinant plasmids to obtain recombinant plasmids with normal sequencing results (fig. 2C and D). After the AtRTH Promoter-GUS expression vector is successfully established, plasmid is extracted from recombinant plasmid with correct detection result, GV3101 agrobacterium is transformed, single colony is selected for bacterial liquid PCR detection (figure 2E), 14 drops of glycerol are added into 1mL of the agrobacterium which is detected to be normal, and after uniform mixing, liquid nitrogen is rapidly frozen for bacterial preservation at minus 80 ℃.

1.2.2 Screening and GUS staining of AtRTH Promoter-GUS Arabidopsis plants

The Col-0 is infected by agrobacterium carrying pCambia1391 (z) -proRTH vector, and the harvested seeds are screened to obtain transgenic pure line plants pRTH-GUS for subsequent experiments. pRTH-GUS seed surface was sterilized and cultured on 1/2MS medium for 10 days, and GUS staining was performed, as a result, it was found that it was expressed in roots, stems and leaves and mainly in vascular tissues (FIG. 3), indicating that it might be involved in the transport of substances. In addition, it is expressed in root tips, lateral roots, root hairs, supporting leaves and air holes (fig. 3), suggesting that it may be involved in the absorption of substances.

1.3 response analysis of ferric salt treatment

1/2MS culture medium is used to grow Col-0 seedlings for two weeks in the presence of EDTA-Fe of 0, 50, 100, 200, 400 and 600 mu M ³⁺ In liquid 1/2MS medium for 0, 1, 3, 6, 12 and 24 hours, extracting RNA of the material at each time point, and determining the expression pattern of the AtRTH by using qRT-PCR.

As a result, it was found that under the non-iron salt (0. Mu.M) treatment conditions, atRTH was not changed with the increase of the treatment time, and that 50 to 600. Mu.M EDTA-Fe was used ³⁺ Under the treatment, the expression of the AtRTH gene is obviously reduced in the treatment for 1-12 h, and the phenomenon of restoring the expression in the treatment for 24h (figure 4) shows that the expression of the AtRTH gene is inhibited by the treatment of ferric salt.

Example two study of the effects of AtRTH on iron absorption transport

2.1 Effect of ferric salt treatment on Arabidopsis root length

The above experiments indicate that the AtRTH gene may be involved in iron uptake and transport, whereas roots are the major organ for plant nutrient uptake, thus Arabidopsis wild-type Col-0 and mutant rth-1 were grown for 10d on medium containing iron at concentrations of 0, 50, 100, 200, 400 and 600. Mu.M.

As a result, it was found that the rth-1 root length was greater than Col-0 at 0. Mu.M, the rth-1 root length was comparable to Col-0 at 50. Mu.M, and the rth-1 root length was greater than Col-0 at 200 to 600. Mu.M (FIG. 5), indicating that the rth-1 mutant root length was insensitive to high concentration iron relative to Col-0.

Verification experiments the same analysis was performed with wild type Ler and mutant rth-2 (Ler background), and as a result, like Col-0 and rth-1, the rth-2 mutant root length was insensitive to iron at high concentrations relative to Ler (fig. 6).

The above results indicate that the knockout of the AtRTH gene reduces the sensitivity of plant root length to high concentration iron.

2.2 AtRTH overexpression and complementation line construction

AtRTH vector agrobacteria are used for invasion of Col-0 and rth-1, harvested T0 generation seeds are placed on a 1/2MS culture medium containing 15mg/L Hyg (hygromycin) to obtain positive transgenic plants (figures 7A and C), the positive transgenic plants are moved into soil for cultivation to obtain harvested T1 generation seeds, the selection is continued on a 1/2MS plate containing Hyg, seedlings with the positive seedling and negative seedling ratio of 3:1 are selected for transplanting, the T2 generation seeds are harvested, and the selection is continued on a 1/2MS plate containing Hyg to obtain pure line plants.

RNA in the pure transgenic strain is extracted, after reverse transcription, qRT-PCR is used for detecting the expression quantity of the AtRTH genes, and the result shows that the expression quantity of the AtRTH genes of No.3 and No. 24 in the Col-0 transgenic strain is higher (FIG. 7B), and the two strains are named as OE3 and OE24; among the rth-1 transgenic mutants, the AtRTH gene expression levels of No. 20 and No. 36 were high (FIG. 7D), and these two lines were designated as Re20 and Re36.

Thus, the plants OE3 and OE24 of the AtRTH over-expression system are obtained, and the plants Re20 and Re36 of the AtRTH complementary system are obtained.

2.3 Effect of ferric salt treatment on AtRTH overexpression and complementing root length

Col-0, rth-1, OE3, OE24, re20 and Re36 were inoculated on a 1/2MS medium containing no iron, grown for 3 days, and transferred to a 1/2MS medium with iron concentrations of 0, 50, 100, 200, 400 and 600. Mu.M, respectively, and root length changes were observed for 7 days.

The result shows that: at 0 mu M, rth-1 root is longest; at 50 and 100 mu M, the root lengths of the 6 materials are equivalent; at 200-600. Mu.M, rth-1 root length was greater than Col-0 and the overexpressing and complementing lines were shorter than Col-0 (FIG. 8).

The complementary line restored the sensitivity of rth-1 to high-concentration iron, and the overexpressing line and the complementary line were more sensitive to high-concentration iron than Col-0, so that the change in expression of the AtRTH gene had an effect on the sensitivity of Arabidopsis to high-concentration iron.

2.4 Effect of ferric salt treatment on AtRTH knockout line and over-expression development

After the wild Col-0, atRTH knockout line rth-1, over-expression line OE3 and OE24 seeds were surface sterilized, they were inoculated on 1/2MS medium with iron concentrations of 0, 50, 400 and 600. Mu.M and observed by photographing after 14 d.

As a result, it was found that the difference in the growth conditions of the four materials under the low-concentration iron treatment (0 and 50. Mu.M) was not obvious; at an iron concentration of 400 mu M, the growth and development of the four materials are obviously inhibited, but the rth-1 growth is obviously superior to Col-0, and the OE3 and OE24 are obviously inferior to Col-0; at 600. Mu.M iron, the four materials were further inhibited from growing and developing, with Col-0 and rth-1 growing at comparable vigor, with little OE3 and OE24 developing (FIG. 9A). Next, we sprinkled Col-0, rth-1 and OE24 seeds on wet filter paper, vernalized at 4deg.C for 48h and spotted in a small flowerpot, and watered Col-0, rth-1 and OE24 respectively with 400 μM EDTA-Fe after three weeks of culture ³⁺ Watering the four weeks, and continuing to water respectively and 400 mu M EDTA-Fe in the fifth week ³⁺ Photograph 42d and chlorophyll and plasma membrane permeabilities were measured.

The results were as follows: among Col-0, rth-1 and OE24 leaves without ferric salt treatment, OE24 is slightly green, and Col-0 and rth-1 are basically consistent; the chlorophyll content and plasma membrane permeability of the three are not different. 400 mu M EDTA-Fe ³⁺ Among the treated Col-0, rth-1 and OE24 leaves, OE24 leaves were most severely green-deprived, had the lowest chlorophyll content, had the highest plasma membrane permeability, whereas rth-1 leaves were relatively most green, had the highest chlorophyll content, had the lowest plasma membrane permeability (FIG. 9B-E), indicating that rth-1 was insensitive to high concentrations of iron relative to Col-0, while OE24 was too sensitive to Col-0, consistent with the root growth phenotype.

The AtRTH knockout system rth-1 can keep good growth vigor of plants under the treatment of high-concentration (400 mu M) ferric salt, and has the advantages of greenest leaves, highest chlorophyll content and lowest plasma membrane permeability.

Example III molecular mechanism study of AtRTH affecting iron absorption transport

3.1 analysis of expression of genes involved in iron uptake and transport

In order to explore the molecular mechanism of the AtRTH for influencing iron absorption and transportation, we analyze the expression changes of iron absorption and transportation related genes in different Arabidopsis plants, and as a result, find that the expression of iron absorption related genes AHA, FIT, FRO and IRT in rth-1 is reduced, and the expression of AHA, FIT, FRO and IRT in an over-expression line OE24 is increased; the iron transport-related genes FPN1, MIT, NRAMP3, NRAMP4 and YSL were expressed down in rth-1, and the expression of FPN1, MIT, NRAMP3, NRAMP4 and YSL was increased in the over-expression line OE24 (FIG. 10), indicating that iron absorption transport capacity was decreased in iron rth-1, and the over-expression line was enhanced.

3.2 analysis of iron content

Firstly, the surfaces of wild Col-0, atRTH knockout line rth-1, over-expression line OE3 and OE24 seeds are disinfected and then are sown on a 1/2MS culture medium without iron, after vernalization for more than 48 hours in a refrigerator with the temperature of 4 ℃, the seeds are placed on an illumination incubator for 3 days, then are transferred to a 1/2MS flat plate containing 0, 50 and 200 mu M iron salt, are vertically cultivated until the materials are obtained for 14 days, and are dried at the temperature of 60 ℃, and the iron content in the strain is measured. The over-expression lines OE3 and OE24 have iron content higher than Col-0 and rth-1 has the lowest iron content under different concentrations; the Col-0 iron content increased by 27.6%, the rth-1 iron content increased by 21.4% and the OE3 and OE24 iron contents increased by 77.0% and 30.0%, respectively, at iron concentrations from 0. Mu.M to 50. Mu.M; the iron concentration increased from 50. Mu.M to 200. Mu.M with 33.0% increase in Col-0 iron content, only 24.3% increase in rth-1 iron content, 75.0% and 47.0% increase in OE3 and OE24 iron content, respectively, and all showed an upward trend with increasing iron concentrations Col-0, rth-1, OE3 and OE24 iron content, demonstrating that AtRTH affects iron absorption by Arabidopsis (FIG. 11).

3.3 Analysis of expression of genes related to iron uptake transport after ACC treatment (Col-0, rth-1 and OE)

Col-0, rth-1, OE3 and OE24 seedlings grown for 15d with 0 and 20. Mu. MACC treatments, respectively, were analyzed for changes in expression of genes involved in iron uptake transport, and as a result, it was found that in Col-0, the expression of YSL, FIT and AHA genes was significantly up-regulated as compared with the time points of the respective control groups (without ACC treatment) in ACC treatments, indicating that these three genes were induced to be expressed by ACC treatments; in rth-1, the YSL, FIT and AHA gene expression were not greatly different in ACC treatment 1, 3, 6, 12 and 24h compared to the respective control (non-ACC treated) time points, indicating that in rth-1, these three genes were not ACC-induced; in OE3 and OE24, the expression levels of YSL, FIT and AHA are already high under untreated conditions, and after ACC treatment, the expression is also induced by up-regulation; from this, it can be speculated that the deletion of RTH blocks the induction of YSL, FIT and AHA by ACC, and that overexpression of RTH upregulates expression of YSL, FIT and AHA (fig. 12). The MIT, FPN1, NRAMP3 and NRAMP4 genes were not greatly different in the Col-0, rth-1, OE3 and OE24 gene expression at the same time points of ACC treatment and non-treatment, indicating that MIT, FPN1, NRAMP3 and NRAMP4 were not induced to be expressed by ACC treatment.

FIT and AHA are plant iron uptake-related genes and YSL is a plant iron transport-related gene, thus indicating that RTH can mediate ethylene to promote up-regulated expression of FIT, AHA and YSL genes and influence the plant iron uptake and transport.

The present invention has been described above by way of example, but the present invention is not limited to the above-described embodiments, and any modifications or variations based on the present invention fall within the scope of the present invention.

Claims (10)

1. The application of the AtRTH gene in regulating and controlling the iron absorption and transport of arabidopsis thaliana is characterized in that the nucleotide sequence of the AtRTH gene is shown as SEQ ID NO. 1;

   the AtRTH gene is expressed higher in roots, stems and leaves of the arabidopsis thaliana vegetative organ; GUS staining results showed expression in roots, stems and leaves and mainly in vascular tissues; the AtRTH gene is used for regulating and controlling iron absorption and transportation of plants.
2. 2. The use of the attth gene according to claim 1 for regulating iron absorption and transport in arabidopsis thaliana, wherein the sensitivity of arabidopsis thaliana to high concentration iron is reduced by knocking out the attth gene or down regulating its expression, thereby improving root length, leaf chlorophyll content and plant viability of arabidopsis thaliana under high concentration iron salt.
3. 3. The use of the attth gene according to claim 2 for controlling iron absorption and transport in arabidopsis thaliana, wherein the attth gene is knocked out or down-regulated, and is suitable for use in cases where iron solubility is high and plants are susceptible to poisoning.
4. 4. The use of the AtRTH gene according to claim 1 for regulating iron absorption and transport in arabidopsis, wherein the sensitivity of arabidopsis to high concentration iron is improved and the iron absorption and transport capacity is improved by establishing an AtRTH overexpression line and/or establishing a complementation line, thereby improving the iron content in plants.
5. 5. The use of the AtRTH gene according to claim 4 for controlling iron absorption and transport in Arabidopsis thaliana, wherein an AtRTH overexpression line is established and/or a complementation line is established, which is suitable for use in the case of low iron solubility and iron deficiency in plants.
6. 6. The application of the AtRTH gene in regulating and controlling iron absorption and transport of arabidopsis thaliana according to claim 2, wherein the nucleotide sequence of an AtRTH mutant plant rth-1 obtained by knocking out the AtRTH gene is shown as SEQ ID No. 2.
7. 7. The use of the AtRTH gene according to claim 6, wherein the 322 th base in SEQ ID No.2 is changed from C to T, resulting in the conversion of original glutamine (CAA) to termination code (TAA).
8. 8. The use of the AtRTH gene according to claim 6 for regulating iron absorption and transport in Arabidopsis thaliana, wherein the amplification primer sequences for identifying sequence 2 are shown in SEQ ID NO.3 and SEQ ID NO. 4.
9. 9. A cultivation method of arabidopsis thaliana resistant to high-concentration ferric salt is characterized by comprising the following steps: the cultivation method can be EMS mutagenesis, screening plants meeting the conditions after the mutagenesis, and carrying out offspring breeding to a homozygous line; and/or

   And (3) carrying out gene editing to obtain a vector containing the expression of the knocked-out SEQ ID NO.1, transferring into agrobacterium to obtain a recombinant strain, infecting a target plant, and culturing to obtain the high-concentration ferric salt-resistant arabidopsis.
10. 10. The method for cultivating high-concentration ferric salt-resistant Arabidopsis thaliana according to claim 9, wherein the high-concentration ferric salt is 200-600. Mu.M.