Application of a coding gene for improving plant iron deficiency traits and promoting iron intake

Abstract

The present invention relates to the application of a gene encoding a plant for improving iron deficiency traits and promoting iron uptake, and belongs to the field of bioengineering technology. A DNA sequence for improving plant iron deficiency traits and promoting iron uptake, shown in SEQ ID No: 1 in the sequence table, is transferred into a plant, wherein the plant is Arabidopsis thaliana. The specific operation of the present invention is to transfer the DNA sequence for improving plant iron deficiency traits and promoting iron uptake, shown in SEQ ID No: 1 in the sequence table, into a wild-type plant through a floral dip method, so that the DNA sequence is overexpressed in the wild-type plant, and the plant exhibits tolerance to iron deficiency. The gene for improving plant iron deficiency traits and promoting iron uptake of the present invention can provide genetic resources and technical support for breeding crops for tolerance to iron deficiency stress.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a gene for improving plant iron deficiency traits and promoting iron uptake. At the same time, it relates to using the gene to improve plant iron deficiency traits, thereby possibly positively regulating the plant's iron deficiency stress response. Background Art

[0002] Iron (Fe) is a metallic element with an average relative atomic mass of 55.845. It accounts for 4.75% of the Earth's crust, ranking fourth in content after oxygen, silicon, and aluminum. Iron is an essential trace element for the human body. Iron exists primarily in the body as a protein-bound form, forming transferrin in plasma, or as hemoglobin, which primarily supplies oxygen to the body. An adult's body contains 4-6g of iron. The body primarily absorbs iron from food. Iron-containing foods undergo a series of transformations in the stomach to form ferrous iron, which is then absorbed by the small intestine. Free iron ions are toxic to cells, and therefore do not exist in the human body. This is a characteristic that distinguishes iron from other metallic elements. Iron levels in the human body vary with age, weight, gender, and nutritional status. In my country, the anemia rate among people aged six and over is close to 10%, with iron deficiency being the primary cause.

[0003] Iron is primarily concentrated in chloroplasts in plants, where it participates in chloroplast formation and is essential for chlorophyll formation and photosynthesis, promoting plant photosynthesis. Iron is present in high concentrations in soil, primarily as trivalent iron. Its low solubility in alkaline soils limits its uptake by plant roots. Although plants have evolved effective mechanisms to absorb and utilize iron from the soil over the course of evolution to maintain iron homeostasis and regulate iron uptake, transport, and storage, iron is difficult to transfer from mature to young tissues within the plant and must rely on transport through the xylem. To meet the plant's iron needs, a continuous supply of available iron is essential throughout all stages of growth and development. Therefore, iron deficiency is a common nutritional stress in plants. Iron deficiency can severely hinder normal plant growth and development by impacting photosynthesis, mitochondrial respiration, nutrient transport, and immunity. Furthermore, plants are a major food source for humans, providing a variety of essential nutrients, including iron.

[0004] Currently, foliar fertilization is the common method for addressing plant iron deficiency. This involves spraying ferrous sulfate and ferric fulvate to improve plant iron status. However, traditional fertilization methods are costly and inefficient, and cannot effectively address plant iron deficiency. The biological approach analyzes the mechanisms of iron absorption and transport in plants and, through molecular biology, improves these capabilities. This is an efficient and environmentally friendly approach.

[0005] The WRKY family of plant zinc-finger transcriptional regulators is an important member of the plant transcription factor family. Genes in this family are involved in various plant life processes and play a vital role in plant resistance to biotic and abiotic stresses. WRKY proteins can activate or repress transcription and usually have abundant potential transcriptional activation and repression domains. Some WRKY factors have both functions. Generally, WRKY transcription factor family members exert transcriptional regulation by binding to the W-box of their target gene promoters. Although the functions of many WRKY transcription factor family members have been revealed, the role of the WRKY47 gene in plant iron deficiency response has not been revealed.

[0006] Faced with the global problem of plant iron deficiency, finding genes that improve plant iron deficiency traits and promote iron uptake and clarifying their functions are of great theoretical and practical significance. Summary of the Invention

[0007] The purpose of the present invention is to provide an application of a coding gene for improving plant iron deficiency traits and promoting iron intake.

[0008] The invention relates to an application of a gene encoding a plant for improving iron deficiency traits and promoting iron uptake, wherein a DNA sequence for improving plant iron deficiency traits and promoting iron uptake shown in SEQ ID No: 1 is transferred into a plant, wherein the plant is Arabidopsis thaliana.

[0009] The specific operations of the present invention are as follows:

[0010] The DNA sequence for improving plant iron deficiency traits and promoting iron uptake shown in SEQ ID No: 1 in the sequence table was transferred into wild-type plants by the floral dipping method, so that it was overexpressed in the wild-type plants, and the plants showed tolerance to iron deficiency.

[0011] The beneficial technical effects of the present invention are embodied in the following:

[0012] 1. The present invention constructs an overexpression vector 35S:WRKY47 Then, Agrobacterium was used as a carrier to infect the flowers. WRKY47 Overexpression of the gene in wild-type Arabidopsis thaliana resulted in a tolerance phenotype under iron deficiency stress. Figure 5 ; Its chlorophyll (see Figure 6 ) and fresh weight (see Figure 7 ) were significantly higher than those in the wild type; and the plant contained significantly higher iron than that in the wild type (see Figure 8 The coding gene for improving plant iron deficiency traits and promoting iron uptake can provide genetic resources and technical support for breeding improved plant iron deficiency traits and increasing iron accumulation in crops.

[0013] 2. The present invention is based on the genome sequence published by the Arabidopsis thaliana database, WRKY47 (AT4G01720) is a member of the WRKY transcription factor family. This family of genes is involved in plant growth and development processes such as lateral root development, flowering, and aging, and plays a vital role in plant resistance to biotic and abiotic stresses. WRKY47 Plants with gene deletion showed sensitivity to iron deficiency, which indicated that WRKY47 The gene is involved in the regulation of iron deficiency tolerance. To this end, we studied the function of this gene and found that WRKY47 When the gene is missing, iron deficiency treatment will reduce the fresh weight, chlorophyll content and iron content of the plant. WRKY47 When the gene is overexpressed, iron deficiency treatment increases the fresh weight, chlorophyll content and iron content of the plant. WRKY47 The gene is involved in regulating the iron content in Arabidopsis, thereby regulating the plant's tolerance to iron deficiency and accumulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 for WRKY47 Phenotypic analysis of mutant plants under iron supplementation and iron deficiency.

[0015] Figure 2 for WRKY47 Chlorophyll content detection diagram of mutant plants under iron supplementation and iron deficiency.

[0016] Figure 3 for WRKY47 Fresh weight analysis of mutant plants under iron supplementation and iron deficiency.

[0017] Figure 4 for WRKY47 Iron content analysis of mutant plants under iron supplementation and iron deficiency.

[0018] Figure 5 for WRKY47 Phenotypic analysis of overexpression plants under iron supplementation and iron deficiency.

[0019] Figure 6 for WRKY47 Chlorophyll content detection diagram of overexpression plants under iron supplementation and iron deficiency.

[0020] Figure 7 for WRKY47 Fresh weight analysis of overexpressing plants under iron supplementation and iron deficiency.

[0021] Figure 8 for WRKY47 Iron content analysis of overexpression plants under iron supplementation and iron deficiency. DETAILED DESCRIPTION

[0022] The present invention will be further described below by way of embodiments with reference to the accompanying drawings.

[0023] The gene encoding the improved plant iron deficiency trait and promoted iron uptake shown in SEQ ID No: 1 in the sequence list is obtained from Arabidopsis thaliana mutant seeds obtained from the American Arabidopsis germplasm resource bank to screen mutants that respond to plant iron deficiency stress.

[0024] Example 1. Screening and functional analysis of iron deficiency response mutants

[0025] Mutant seeds obtained from the Arabidopsis Germplasm Resource Center in the United States were cultured on iron-deficient medium. Plants sensitive to iron deficiency were obtained from them and identified as WRKY47 Loss-of-function mutants, named wrky47-1 (Seed number: SALK_046806). To more reliably analyze the function of this gene mutant, we obtained another WRKY47 The mutant plants were named wrky47-2 (Seed number: SALK_123305). WRKY47 The iron deficiency stress phenotype of the gene loss-of-function mutant was analyzed. WRKY47 The gene function loss mutant was sown on iron-supplemented and iron-deficient solid culture medium at the same time and placed in a 22℃ constant temperature greenhouse (photoperiod of 16 hours light and 8 hours dark) for vertical cultivation. After seven days of observation, it was found that the mutant grown on the iron-supplemented solid culture medium WRKY47 There was no significant difference between the mutant and the wild-type plant (WT), but the mutant grown on iron-deficient solid medium WRKY47 The root length of the mutant was significantly shorter than that of the wild-type plant (WT), and WRKY47 The leaves of the mutant were significantly yellower than those of the wild-type plant (WT).

[0026] See also Figure 1 , wild type (WT) and wrky47-1 , wrky47-2 The seeds were sown simultaneously in 90 mm diameter culture dishes on solid culture medium with or without iron, and placed in a 22°C constant temperature and light incubator (with a photoperiod of 16 hours light and 8 hours dark) for vertical culture. After one week, the following observations were made: WRKY47 The mutant and wild-type plants (WT) were cultured vertically on culture dishes and directly seeded on MS and -Fe culture media respectively. The comparison photos of the vertical culture for one week under normal light conditions are shown in Fig. Figure 1 ; Among them, MS medium was used as control, WT and WRKY47 Mutants in chlorophyll (see Figure 2 ), fresh weight (see Figure 3 ), iron content (see Figure 4There was no significant difference in the above aspects. When the plants were directly planted on the medium without iron, WRKY47 The mutant showed obvious iron deficiency sensitivity. WRKY47 The chlorophyll content and fresh weight of the mutant were significantly higher than those of the WT. WRKY47 The mutant was significantly more sensitive to iron deficiency stress than the WT.

[0027] Example 2: Cultivation of iron-deficiency-tolerant Arabidopsis

[0028] 1. WRKY47 Gene overexpression transgenic lines WRKY47-OE3, OE7 Acquisition

[0029] To further verify the function of this gene in the regulation of plant iron deficiency stress, we constructed WRKY47 Gene overexpression vector ( 35S:WRKY47 First, amplify the target fragment. Wild-type Arabidopsis thaliana plants were cultured normally on MS medium for two weeks. Total RNA was extracted and reverse-transcribed to synthesize cDNA. PCR was performed using the synthesized cDNA as a template to amplify a sufficient amount of the target product. A second amplification was performed using the PCR product as a template to introduce restriction sites. The PCR product was then digested and recovered with the vector pCAMBIA1301. The purified target DNA fragment and the vector were then ligated overnight using T4 DNA ligase. The ligation solution was transferred into DH5α medium, and positive clones were screened and sequenced. Once sequencing results were confirmed, the clones were transformed into Agrobacterium tumefaciens GV3101 by electroporation. The transformed Agrobacterium GV3101 was activated and plated on LB medium containing two antibiotics (Kan and Gen). Single colonies were randomly selected and expanded in LB medium containing two antibiotics (Kan and Gen), and the plasmid was extracted. The recombinant vector was identified by double enzyme digestion with Kpn I and Hind III, and wild-type Arabidopsis plants were transformed using the floral dipping method to obtain WRKY47 Gene overexpression transgenic lines.

[0030] 2. WRKY47 Comparison of iron deficiency tolerance between overexpressing transgenic plants and wild-type plants

[0031] See also Figure 5 , wild type (WT) and WRKY47-OE3 and OE7 At the same time, they were sown in a 90 mm diameter culture dish, with either iron-added or iron-free solid culture medium, and placed in a 22°C constant temperature light incubator (with a photoperiod of 16 hours of light and 8 hours of darkness) for vertical cultivation. After one week, the following transgenic plants were observed: WRKY47-OE3 and OE7Comparison photos of wild-type plants cultured vertically, directly seeded on medium containing or not containing iron and cultured vertically under normal light conditions for one week (see Figure 5 ), in which MS medium was used as control. WT and WRKY47-OE3 、 OE7 There was no significant difference; when the plants were directly planted on a medium without iron, WRKY47-OE3 and OE7 Compared with WT, it showed obvious tolerance to iron deficiency stress.

[0032] 3. WRKY47 Detection of chlorophyll content in overexpressing transgenic plants

[0033] See also Figure 6 , WRKY47 Overexpressing transgenic plants and wild-type plants (WT) were cultured vertically on culture dishes, directly seeded on iron-supplemented and iron-deficient culture media, and cultured vertically under normal light conditions for one week. WRKY47 The chlorophyll content of overexpressing plants and wild-type plants (WT) was significantly higher than that of plants grown on iron-supplemented medium. WRKY47 There was no significant difference in chlorophyll content between the overexpression plants and the wild-type plants (WT); however, under iron-deficient culture conditions, WRKY47 Chlorophyll content in overexpressing plants (see Figure 6 ) were significantly higher than those in the wild type.

[0034] 4. WRKY47 Fresh weight analysis of overexpression plants under iron supplementation and iron deficiency

[0035] See also Figure 7 , WRKY47 Overexpressing transgenic plants and wild-type plants (WT) were cultured vertically on culture dishes and directly seeded on iron-supplemented and iron-deficient culture media and cultured vertically under normal light conditions for one week. WRKY47 The fresh weight of overexpressing transgenic plants and wild-type plants (WT). The results showed that under normal conditions, WRKY47 There was no significant difference in fresh weight between the overexpressing transgenic plants and the wild type; however, under iron deficiency conditions, WRKY47 The fresh weight of the overexpressing transgenic plants was significantly higher than that of the wild type.

[0036] 5. WRKY47 Analysis of iron content in overexpression plants under iron supplementation and iron deficiency

[0037] See also Figure 8 , WRKY47Overexpressing transgenic plants and wild-type plants (WT) were cultured vertically on culture dishes and directly seeded on iron-supplemented and iron-deficient culture media and cultured vertically under normal light conditions for one week. WRKY47 The iron content of overexpressing transgenic plants and wild-type plants (WT). The results showed that under normal conditions, WRKY47 The iron content of the overexpressing transgenic plants was not significantly different from that of the wild type; however, under iron deficiency conditions, WRKY47 The iron content of the overexpressing transgenic plants was significantly higher than that of the wild type.

Claims

1. Use of the WRKY47 gene in improving plant iron deficiency traits and promoting plant iron uptake, characterized by: The DNA sequence of the WRKY47 gene is shown in SEQ ID No: 1; the application includes transferring the gene shown in SEQ ID No: 1 into a plant, so that the plant exhibits tolerance to iron deficiency; the plant is Arabidopsis thaliana.

2. The use according to claim 1, wherein: The application comprises transferring the gene shown in SEQ ID No: 1 into wild-type plants by floral dipping method, so that the gene is over-expressed in the wild-type plants, and the plants show tolerance to iron deficiency.

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