Application of a coding gene for enhancing iron accumulation in plants and tolerating iron deficiency stress

Abstract

The present invention relates to the application of a coding gene for enhancing iron accumulation in plants and tolerating iron deficiency stress, belonging to the technical field of bioengineering. Knocking out the coding gene for enhancing iron accumulation in plants and tolerating iron deficiency stress shown in SEQ ID No: 1 in the sequence listing, that is, not expressing it in plants, the plants show tolerance to iron deficiency, and the plant is Arabidopsis thaliana. The specific operation is as follows: The coding gene for enhancing iron accumulation and tolerating iron deficiency stress shown in SEQ ID No: 1 in the sequence listing is knocked out in Arabidopsis thaliana by the method of T-DNA insertion. RCD1 ). The present invention enables the above-mentioned gene for enhancing iron accumulation in plants and tolerating iron deficiency stress not to be expressed in plants by gene knockout, so that the plant body shows tolerance to iron deficiency. The gene for enhancing iron accumulation in plants and tolerating iron deficiency stress of the present invention can provide gene resources and technical support for breeding crops tolerant to iron deficiency stress.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a coding gene of a protein related to enhanced iron accumulation in plants. At the same time, it relates to using this gene to enhance the tolerance of plants to iron-deficient environments, thereby positively regulating the plant's response to iron-deficiency stress. Background Art

[0002] Metal elements are required during the growth processes of animals and plants, and iron is one of the essential metal elements. At the same time, iron is also one of the most common metals in daily life. Iron is an essential trace element for the survival of humans, animals, and plants. In plants, iron is the trace element with the largest demand, and it plays an important role in the life activities of plants, such as participating in a series of life activity processes such as the synthesis of plant chlorophyll, photosynthesis, and respiration. Iron is also a relatively abundant element on Earth, but due to its special chemical properties, its solubility in soil is extremely low, resulting in plants being extremely difficult to obtain sufficient iron for growth and development from the soil, leading to a large number of plants facing iron-deficiency stress globally. Plant iron deficiency usually occurs in alkaline or calcareous soils. Mild iron deficiency in plants will affect growth, resulting in slow synthesis and reduced content of chlorophyll, thus affecting photosynthesis; in the case of severe iron deficiency, chlorophyll cannot be synthesized, the leaf color turns yellow, the photosynthesis process is blocked, the plant biomass decreases, seriously hindering growth; and plants are the main food source for humans and animals, and the lack of iron in the edible parts of plants will indirectly lead to a reduction in the iron intake of humans and animals, resulting in a decline in the immunity of humans and animals and the frequent occurrence of iron-deficiency anemia. Iron deficiency is one of the most common hidden hunger in the world, because iron deficiency is not easily detected, so the harm caused by iron deficiency to humans and animals is even more serious.

[0003] Plant iron deficiency will lead to serious consequences, so it is very important to analyze the molecular mechanism of plant iron absorption and transport. Through the analyzed molecular mechanism, using molecular breeding techniques, genes that play an important role in plant iron-deficiency response are modified by corresponding methods, so as to improve the tolerance of crops to iron-deficiency stress, thereby improving the iron-deficiency tolerance ability of crops, increasing the iron content of crops, and fundamentally solving the problem of plant iron deficiency.

[0004] Using the model organism Arabidopsis thaliana as an experimental material to study the functions of genes related to plant iron-deficiency response. Most genes of Arabidopsis thaliana can be found in other plants, and any findings related to Arabidopsis thaliana can be applied to other plant studies. Therefore, using Arabidopsis thaliana as the research object can achieve the experimental expected goals faster and better, and can greatly shorten the experimental time and simplify the experimental conditions.

[0005] Radical-induced cell death 1 ( RCD1, AT1G32230) is one of the members of the Radical-induced cell death 1-related protein family (SRO protein family), which is widely involved in the growth and development process of plants and plays a crucial role in plant resistance to biotic and abiotic stresses. The Radical-induced cell death 1 (RCD1) protein consists of a conserved globular domain called WWE and a putative PARP domain, and WWE is predicted to mediate specific protein-protein interactions. However, the exact functions of these domains in the Radical-induced cell death 1 ( RCD1 ) gene are still unclear. Previous studies have found that Radical-induced cell death 1 ( RCD1 ) interacts with transcription factors such as the STO protein and can confer salt tolerance when ectopically expressed in yeast. Microarray analysis of rcdl-1 mutants determined that the expression levels of several abiotic stress-responsive genes were lower in the mutants. These studies suggest that Radical-induced cell death 1 ( RCD1 ) is an important regulator of plant stress responses, but so far, it is unclear whether Radical-induced cell death 1 ( RCD1 ) is involved in the response to iron deficiency stress in plants. SUMMARY OF THE INVENTION

[0006] The object of the present invention is to provide an application of a coding gene for enhancing iron accumulation in plants and tolerating iron deficiency stress.

[0007] An application of a coding gene for enhancing iron accumulation in plants and tolerating iron deficiency stress, the coding gene shown in SEQ ID No: 1 in the sequence listing is mutated in plants by T-DNA insertion, that is, it is not expressed in plants, and the plants show tolerance to iron deficiency. The plant is Arabidopsis thaliana.

[0008] The further technical solution is as follows:

[0009] Agrobacterium can integrate its own genome into plants. Agrobacterium carrying the T-DNA sequence is transfected into the flowers of Arabidopsis thaliana by the floral dip method, so that the T-DNA sequence is inserted into the coding gene shown in SEQ ID No: 1 in the sequence listing for enhancing iron accumulation in plants and tolerating iron deficiency stress, damaging the structure of the Radical-induced cell death 1 ( RCD1 ) gene and making it unable to be normally expressed, thus achieving knockout of the Radical-induced cell death 1 ( RCD1 ) gene in Arabidopsis thaliana.

[0010] The beneficial technical effects of the present invention are reflected in the following aspects:

[0011] 1. In the present invention, by RCD1), Gene knockout can significantly improve the ability of plants to tolerate iron deficiency stress, as shown in Figure 2 ; under iron deficiency stress, during the growth process of plants, more chlorophyll is synthesized compared to ordinary plants, as shown in Figure 3 ; having a higher iron content than ordinary plants, as shown in Figure 7 . The coding gene for enhancing iron accumulation in plants and tolerating iron deficiency stress according to the present invention can provide gene resources and technical support for breeding crops tolerant to iron deficiency environments and increasing iron accumulation in crops.

[0012] 2. According to the genomic sequence published in the Arabidopsis database, Radical-induced cell death 1 ( RCD1 , AT1G32230 ) is one of the members of the SRO protein family, widely involved in the growth and development process of plants, and plays a crucial role in the plant's resistance to biotic and abiotic stresses. The applicant found that the mutant plants with the deletion of the Radical-induced cell death 1 ( RCD1 ) gene showed iron deficiency tolerance under iron deficiency treatment, indicating that the Radical-induced cell death 1 ( RCD1 ) gene is involved in the regulation of iron deficiency tolerance. Therefore, we studied the function of this gene, and the research results showed that when the Radical-induced cell death 1 ( RCD1 ) gene was deleted, iron deficiency treatment increased the fresh weight, chlorophyll content, and iron content of the plants. When the Radical-induced cell death 1 ( RCD1 ) gene was overexpressed, iron deficiency treatment decreased the fresh weight, chlorophyll content, and iron content of the plants. This shows that the Radical-induced cell death 1 ( RCD1 ) gene is involved in regulating the iron content of Arabidopsis thaliana, and thus regulates plant iron accumulation and tolerance to iron deficiency stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is RCD1 Schematic diagram of the T-DNA insertion site of the mutant.

[0014] Figure 2 is RCD1 Phenotype analysis diagram of the mutant plants under iron addition and iron deficiency.

[0015] Figure 3 is RCD1 Chlorophyll content detection diagram of the mutant plants under iron addition and iron deficiency.

[0016] Figure 4 is RCD1 qRT-PCR analysis diagram of the overexpressed transgenic lines.

[0017] Figure 5 is RCD1 Phenotype analysis diagram of the overexpressed plants under iron addition and iron deficiency.

[0018] Figure 6For RCD1 Detection chart of chlorophyll content in overexpressing plants

[0019] Figure 7 For RCD1 Analysis chart of iron content in related plants under iron addition and iron deficiency Specific implementation manners

[0020] The present invention will be further described below in conjunction with the accompanying drawings through embodiments

[0021] The coding gene shown in SEQ ID No: 1 in the sequence listing for enhancing plant iron accumulation and tolerating iron deficiency stress is to screen mutants responsive to plant iron deficiency stress from Arabidopsis thaliana mutant seeds obtained from the Arabidopsis Biological Resource Center in the United States

[0022] Example 1: Screening and functional analysis of iron deficiency-responsive mutants

[0023] A large number of mutant seeds obtained from the Arabidopsis Biological Resource Center in the United States were cultured on an iron-deficient medium. Plants tolerant to iron deficiency were obtained therefrom, and it was identified that this mutant was RCD1 a gene function loss mutant, named rcd1- 1 (seed number: SALK_043480). To more reliably analyze the function of this gene mutant, we obtained another RCD1 mutant plant named rcd1-2 (seed number: SALK_046241). Referring to Figure 1 , the T-DNA insertion sites in rcd1-1 and rcd1-2 were determined by polymerase chain reaction amplification and sequencing alignment. Referring to Figure 2 , phenotypic analysis of iron deficiency stress was performed on wild type (WT) and RCD1 gene function loss mutants, that is, wild type (WT) and RCD1 gene function loss mutants were sown simultaneously on solid media with iron addition and iron deficiency, and vertically cultured in a constant temperature greenhouse at 22°C (photoperiod: 16 hours of light, 8 hours of darkness). After seven days, the results showed that there was no obvious difference between the RCD1 mutant growing on the solid medium with iron addition and the wild type plant (WT), while the root length of the RCD1 mutant growing on the iron-deficient solid medium was significantly longer than that of the wild type plant (WT), and the leaves of the RCD1 mutant were significantly greener than those of the wild type plant (WT). Referring to Figure 3 , by detecting the chlorophyll of each group of plants in Figure 2 , it was found that the wild type (WT) and RCD1 cultured on the normal mediumThere was no significant difference in chlorophyll of the gene function loss mutants; while the chlorophyll of the gene function loss mutants cultured on iron-deficient medium was significantly higher than that of the wild-type plants (WT). RCD1 The chlorophyll of the gene function loss mutants was significantly higher than that of the wild-type plants (WT).

[0024] Example 2 RCD1 Analysis of the response of overexpressing plants to iron deficiency stress

[0025] 1. RCD1 Obtaining of the gene overexpression transgenic lines RCD1-OE1, OE2 To further verify the function of this gene in the regulation of plant iron deficiency stress, we constructed a gene overexpression vector. First, the target fragment was amplified. The PART-27 plasmid was extracted by a plasmid extraction kit and digested and recovered. Then the recovered and purified target DNA fragment and the digested vector were ligated with a homologous recombination enzyme. The above ligation solution was transferred into DH5α, and positive clones were detected and screened for sequencing. After the sequencing results were confirmed to be correct, they were transferred into Agrobacterium tumefaciens GV3101 by electroporation. The electroporated Agrobacterium tumefaciens GV3101 was activated and spread on an LB medium plate containing double antibiotics. Single colonies were randomly selected, expanded in an LB culture solution containing double antibiotics and the plasmids were extracted. The recombinant vector was identified to be correct by double digestion with Kpn I and Xho I, and the wild-type Arabidopsis plants were transformed by the floral dip method to obtain the gene overexpression transgenic lines.

[0026] To further verify the function of this gene in the regulation of plant iron deficiency stress, we constructed a RCD1 gene overexpression vector. First, the target fragment was amplified. The PART-27 plasmid was extracted by a plasmid extraction kit and digested and recovered. Then the recovered and purified target DNA fragment and the digested vector were ligated with a homologous recombination enzyme. The above ligation solution was transferred into DH5α, and positive clones were detected and screened for sequencing. After the sequencing results were confirmed to be correct, they were transferred into Agrobacterium tumefaciens GV3101 by electroporation. The electroporated Agrobacterium tumefaciens GV3101 was activated and spread on an LB medium plate containing double antibiotics. Single colonies were randomly selected, expanded in an LB culture solution containing double antibiotics and the plasmids were extracted. The recombinant vector was identified to be correct by double digestion with Kpn I and Xho I, and the wild-type Arabidopsis plants were transformed by the floral dip method to obtain RCD1 gene overexpression transgenic lines.

[0027] 2. RCD1 Identification of the transcriptional level of overexpressing transgenic plants and comparison of iron deficiency tolerance with wild-type plants

[0028] See Figure 4 , the transcriptional level of the overexpressing transgenic plants was identified, and finally RCD1 and OE1 were selected for the next experiment. See OE2 , the wild type (WT) and Figure 5 and RCD1-OE1 and OE2 were sown simultaneously in a petri dish with a diameter of 90 mm. The medium was solid medium with and without iron, and they were vertically cultured in a constant temperature light incubator at 22 °C (photoperiod: 16 hours of light, 8 hours of darkness). After one week, it could be observed that: the transgenic plants RCD1-OE1 and OE2 were vertically cultured with the wild-type plants, and the comparison photos of directly sowing on the medium containing or not containing iron and vertically culturing for one week under normal light conditions; among them, the MS medium was used as a control. There was no significant difference between the WT and RCD1-OE1, OE2 plants growing on the MS medium; directly sowing on the medium without iron and culturing,RCD1-OE1, OE2 all showed obvious iron deficiency sensitive traits. See Figure 6 , by detecting Figure 5 the chlorophyll of each group of plants in RCD1- OE1 and OE2 , it was found that there was no obvious difference in the chlorophyll of the wild type (WT) cultured on the normal medium, RCD1-OE1 and OE2 the chlorophyll was significantly lower than that of the wild type plants (WT).

[0029] 3. Analysis of iron content in wild type and RCD1 related materials

[0030] See Figure 7 , RCD1 The mutants, overexpressing plants and wild type plants (WT) were vertically cultured on the culture dish, directly seeded on the iron-supplemented and iron-deficient media respectively, and vertically cultured for one week under normal light conditions. The iron content of each material under different conditions was detected respectively. The results showed that under normal conditions, RCD1 there was no obvious difference in the iron content between the mutants and the wild type (WT), RCD1 nor was there an obvious difference in the iron content between the overexpressing plants and the wild type (WT); while under iron-deficient conditions, RCD1 the iron content in the mutants was significantly higher than that of the wild type (WT) plants, while RCD1 the iron content in the overexpressing plants was significantly lower than that of the wild type (WT) plants.

Claims

1. Use of a coding gene for enhancing iron accumulation in plants and tolerating iron deficiency stress, characterized in that: By knocking out the gene shown in Sequence Listing SEQ ID No: 1, the inhibitory effect of the gene shown in Sequence Listing SEQ ID No: 1 on plant iron deficiency tolerance is relieved, and the plant shows tolerance to iron deficiency. The plant is Arabidopsis thaliana.

2. The use according to claim 1, characterized in that: The coding gene shown in Sequence Listing SEQ ID No: 1 for enhancing plant iron accumulation and tolerating iron deficiency stress is knocked out in Arabidopsis thaliana by the method of T-DNA insertion.

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