A gene encoding for enhancing the tolerance of plants to iron deficiency stress and application thereof

Abstract

The application belongs to the technical field of bioengineering, and provides an encoding gene for enhancing the tolerance of plants to iron deficiency stress and application thereof. The application solves the problem that soybeans are restricted in growth in an iron deficiency environment, especially in calcareous soil and soda saline-alkali soil, by mining and applying the encoding gene (nucleotide sequence as shown in SEQ ID No. 1) of a soybean GmMYB14 transcription factor. Experiments prove that the gene can significantly enhance the tolerance of plants to iron deficiency stress after overexpression in Arabidopsis thaliana and soybeans. The application provides a key gene resource for molecular breeding: GmMYB14 effectively promotes the absorption and utilization of iron in plants by regulating the phenylpropanoid metabolic pathway, which not only can improve the planting adaptability of soybeans in low-iron soil, but also can improve the yield and quality of soybeans, thereby providing strong technical support for expanding the planting area of soybeans in iron deficiency soil areas, and having important significance for relieving the insufficient production capacity of soybeans in China and improving the self-sufficiency rate.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to an encoding gene for enhancing the tolerance of plants to iron deficiency stress and application. BACKGROUND

[0002] Iron (Fe) is the most demanding trace element in the growth and development of soybeans, which not only participates in multiple biological processes such as respiration and photosynthesis, but also is an important component of soybean nodule nitrogenase and soybean hemoglobin, and is an important influencing factor determining nodule nitrogen fixation activity. Although the total iron content in the earth's crust is high, the bioavailability of iron is generally low in well-aerated calcareous soil or soda saline soil, which leads to plants often being in a state of iron deficiency. Iron deficiency can cause crops to exhibit yellowing of new leaves and a decrease in photosynthetic rate at the seedling stage, and can also lead to a decrease in nodule formation and activity in the underground part, and even death in severe cases, which greatly affects the yield and quality of soybeans and brings huge economic losses to agricultural production. Therefore, in agricultural production, iron deficiency stress is one of the key factors limiting crop yield, and it is reported that more than one-third of the world's arable land is at risk of iron deficiency. The traditional solution to soybean iron deficiency stress is to apply iron fertilizer, which can temporarily alleviate the symptoms of soybean iron deficiency, but has the disadvantages of low utilization rate and high environmental risk. With the development of molecular biology, molecular breeding technology provides a revolutionary approach to breaking through the bottleneck of iron deficiency stress. This technology can fundamentally improve the adaptability of plants to iron deficiency environment by precisely targeting the regulation of the plant's own iron absorption and utilization mechanism.

[0003] Soybean is an important "food and oil" dual-purpose crop and is an indispensable strategic resource in global agricultural economy. In recent years, with the improvement of people's living standards, the demand for soybeans has increased. However, China's soybean production capacity is insufficient, and it mainly relies on imports, so it is urgent to expand the soybean planting area and improve its yield and quality. With the in-depth research of soybean and other crop genomes, transcription factor engineering has become a core strategy for improving crop stress tolerance. Among them, MYB transcription factors have been proven to be able to coordinately regulate multiple abiotic stress response pathways, and have become an important resource for molecular breeding. The application of molecular breeding can not only significantly reduce the dependence of agricultural production on chemical fertilizers, but also quickly and directionally improve soybean traits, so the molecular design and breeding targeting MYB network will become the only way to solve the problem of land alkalization and iron nutrition disorder.

[0004] MYB transcription factors (especially R2R3-MYB subfamily) are the core regulators of phenylpropanoid metabolism, which precisely control the temporal and spatial expression of lignin, flavonoids and other branch pathways by binding to cis-elements in the target gene promoter. In soybean, at least multiple abiotic stress-induced R2R3-MYB transcription factor genes (such as GmMYB111, GmMYB76, GmMYB117) have been identified, which show specific expression patterns in salt stress, cold tolerance and other adversity. As the core hub of plant secondary metabolism, the phenylpropanoid metabolic pathway plays a multiple physiological role in iron deficiency stress response. On the one hand, the pathway can promote the reduction and absorption of Fe 3+ in the root system by synthesizing coumarin substances as iron-reducing chelators; on the other hand, it can affect root architecture by regulating lignin deposition and change the nutrient foraging ability. Studies have shown that under iron deficiency stress, the phenylpropanoid metabolic pathway in soybean roots will be rapidly activated, and differential expression of key enzyme genes (such as PAL, 4CL) of phenylpropanoid synthesis can be detected in a short time of iron deficiency. At the metabolite level, the phenylpropanoid metabolic pathway of iron-efficient soybean is continuously up-regulated, and its products can chelate iron ions in the soil and maintain their mobility in the body.

[0005] In view of the current low soybean production capacity, low self-sufficiency rate, and high dependence on foreign countries in China, increasing soybean yield and quality, expanding planting area and other issues are of great concern. Traditional iron fertilizer application methods have low utilization rate and high environmental risk, and although they can temporarily alleviate iron deficiency symptoms, they are not a long-term solution. Although molecular breeding technology provides a direction for solving iron deficiency stress and other problems, there is still a lack of genes that can simultaneously improve soybean's alkali tolerance, iron deficiency tolerance, and iron content. Therefore, the present application provides a coding gene for enhancing plant tolerance to iron deficiency stress and its application. SUMMARY

[0006] The present application aims to provide a coding gene for enhancing plant tolerance to iron deficiency stress and its application, which aims to solve the problems raised in the background art.

[0007] The object of the present application is achieved by the following technical solutions:

[0008] A coding gene of a soybean GmMYB14 transcription factor GmMYB14 application in enhancing plant tolerance to iron deficiency stress, the nucleotide sequence of the coding gene GmMYB14 is shown as SEQ ID No. 1.

[0009] Further, the plant is soybean or Arabidopsis.

[0010] Further, the application is to enhance the planting ability of soybean in low-iron soil, including calcareous soil and soda saline soil.

[0011] Further, the DNA sequence of the coding gene GmMYB14 includes a DNA sequence having more than 90% homology with the sequence shown in SEQ ID NO. 1 and encoding the same functional protein.

[0012] A pro35S::GmMYB14 overexpression vector comprising a coding gene GmMYB14 with the nucleotide sequence shown in SEQ ID NO. 1, and the overexpression vector is selected from pCAMBIA3301-35S-GmMYB14 or PTF101-35S-GmMYB14. pro35S::GmMYB14

[0013] A recombinant bacterium for enhancing the tolerance of plants to iron deficiency stress, comprising the above-mentioned pro35S::GmMYB14 overexpression vector, selected from Escherichia coli DH5α or Agrobacterium GV3101; wherein the Escherichia coli DH5α is used for cloning and preservation of the overexpression vector, and the Agrobacterium GV3101 is used for mediating transformation of the overexpression vector into plants.

[0014] A primer pair for constructing the above-mentioned pro35S::GmMYB14 overexpression vector, comprising:

[0015] 3301-MYB14-F and 3301-MYB14-R, respectively shown in SEQ ID No. 3 and SEQ ID No. 4;

[0016] or 101-MYB14-F and 101-MYB14-R, respectively shown in SEQ ID No. 5 and SEQ ID No. 6.

[0017] A method for enhancing the tolerance of plants to iron deficiency stress, comprising:

[0018] transforming a coding gene GmMYB14 with the nucleotide sequence shown in SEQ ID NO. 1 into plant cells by the above-mentioned pro35S::GmMYB14 overexpression vector, so that the coding gene GmMYB14 is overexpressed in plants;

[0019] cultivating the transformed cells to obtain whole plants, and screening single copy homozygous line plants according to Mendelian inheritance law.

[0020] Further, the plants are soybeans or Arabidopsis thaliana; the Arabidopsis thaliana is transformed by a flower dipping method; and the soybean is transformed by a cotyledon node transformation method mediated by Agrobacterium GV3101.

[0021] Compared with the prior art, the beneficial effects of the present application are:

[0022] ​The application mines and applies a coding gene of a soybean GmMYB14 transcription factor GmMYB14 The application solves the core problem that soybean growth is limited in iron deficiency environment, especially in calcareous soil and soda saline soil. Experiments prove that the overexpression of the gene in Arabidopsis and soybean can significantly enhance the tolerance of plants to iron deficiency stress. The application breaks through the drawbacks of low utilization and high environmental risk of traditional iron fertilizer application, provides key gene resources for molecular breeding: GmMYB14 promotes the absorption and utilization of iron in plants by regulating the phenylpropanoid metabolic pathway, which not only improves the planting adaptability of soybean in low-iron soil, but also improves the yield and quality, and provides strong technical support for expanding the planting area of soybean in iron deficiency soil. This achievement lays a foundation for cultivating iron deficiency tolerant crops, reducing the dependence of agriculture on chemical fertilizers, and promoting the development of precision breeding, which has important significance for alleviating the insufficient production capacity of soybean in China and improving the self-sufficiency rate. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 For GmMYB14 The agarose gel electrophoresis map of gene PCR amplification and the structure map of gene; wherein A is GmMYB14 The agarose gel electrophoresis map of gene PCR amplification; B is GmMYB14 The structure map of gene.

[0024] Figure 2 For GmMYB14 The schematic diagram of gene overexpression vector construction; wherein A is the schematic diagram of Arabidopsis overexpression vector pCAMBIA3301-35S-GmMYB14 construction; B is the schematic diagram of soybean overexpression vector PTF101-35S-GmMYB14 construction.

[0025] Figure 3 The wild type Col-0 and pro35s::GmMYB14 The growth conditions of each overexpression strain of Arabidopsis wild type Col-0 and GmMYB14 (AOE-GmMYB14) under different iron treatment conditions for 7 days; wherein A is the growth phenotype; B is the expression level in the heterologous overexpression strain of Arabidopsis; C is the root length; D is the total chlorophyll content; E is the relative fluorescence value of root exudates; F is the aboveground biomass; G is the root biomass; H is the root iron content; I is the aboveground iron content.

[0026] Figure 4 The wild type Williams 82 and pro35s::GmMYB14 The treatment conditions of two overexpression strains of soybean wild type Williams 82 and GmMYB14Expression level; C is SPAD value; D is total chlorophyll content; E is root exudate relative fluorescence value; F is aboveground biomass; G is root biomass; H is aboveground iron content. DETAILED DESCRIPTION

[0027] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below, but it cannot be understood as limiting the scope of the present application.

[0028] The specific implementation of the present application is described in detail below in combination with specific examples. The experimental methods in the examples are all conventional methods unless otherwise specified.

[0029] Example 1: (1) By analyzing the transcriptome data of soybeans under iron deficiency stress treatment, a key transcription factor GmMYB14 (its coding gene GmMYB14 is named Glyma.15g259400 ) regulating phenylpropanoid metabolic pathway was mined, and relevant sequence information was obtained from the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov). Among them, the amino acid sequence of the transcription factor GmMYB14 is shown in SEQ ID No. 2, the nucleotide sequence of its coding gene GmMYB14 is shown in SEQ ID No. 1, and the structure diagram of the gene is shown in B of Figure 1 . In the experiment, the total RNA of the roots of soybean wild type Williams 82 after iron deficiency treatment was extracted, and the cDNA of the gene GmMYB14 was obtained by PCR amplification. The PCR reaction system refers to Table 1, the reaction program refers to Table 2, and the primers used refer to Table 3. The PCR amplification product was identified by agarose gel electrophoresis, and the results are shown in A of Figure 1 . It can be seen that there is a clear band at about 1146 bp, and the sequencing result of the amplification product is consistent with SEQ ID No. 1.

[0030] SEQ ID No. 1:

[0031]

[0032] SEQ ID No. 2:

[0033] MGKGRAPCCDKTQVKRGPWSPAEDLKLIAFIQKYGHENWRALPKQAGLLRCGKSCRLRWINYLRPDVKRGNFTPEEEETIIRLHKALGNKWSKIASGLPGRTDNEIKNVWNTHLKKRLAPKKVSESSADESKPESSITSSSSSSSESFFSNERPNSPKTTTPSNNEFNDQAEKLSVEDKMDKDSEKQLSNELVNITEDPKESSVSLSSVESNIVNTSQIVEHKPEEEQLASPLPYLGHYDVGSILEEVDKPNHLIEIPWEPDYDFWKLLDDDNSLGSFQSNEVQLGEFPAIQNIILGEEGVHDAEAKKWSHDFENEFGVVGGIKESSKDHFLPKNYAVEPEMDHTQAFDFDDIDMARPESELDFGYIQLWPSCPQNNNTSL*.

[0034] Table 1 PCR reaction system for vector construction

[0035]

[0036] Table 2 PCR reaction program for amplifying target fragment

[0037]

[0038] Table 3 Primers for expression vector construction

[0039]

[0040] (2) Construction of Arabidopsis thaliana GmMYB14 gene overexpression vector pCAMBIA3301-35S-GmMYB14, the vector construction schematic diagram is shown in pro35S::GmMYB14 Figure 2A; construction of soybean Figure 2 gene overexpression vector PTF101-35S-GmMYB14, the vector construction schematic diagram is shown in GmMYB14 Figure 2B. The specific method is as follows: the pro35S::GmMYB14 gene overexpression vector PTF101-35S-GmMYB14, the vector construction schematic diagram is shown in Figure 2 Figure 2B. The specific method is as follows: the GmMYB14When the gene is constructed into a plant expression vector, a 35S enhanced promoter (used in this embodiment) or other enhanced promoters, inducible promoters can be added before the transcription initiation nucleotide. The cDNA fragment of the soybean wild type Williams 82 root after iron deficiency treatment was used as a template, and the cDNA fragment was amplified by Takara PrimeSTAR HS enzyme GmMYB14 ; after the amplification product was identified by agarose gel electrophoresis (see Figure 1 A), the gel recovery was performed according to the steps of TIANGEN general gel recovery kit instructions. The recovered cDNA fragment was constructed into the linearized pCAMBIA3301-35S-NOS vector treated with SpeI restriction endonuclease and the linearized PTF101-35S-NOS vector treated with XbaI and SacI restriction endonuclease downstream of the 35S promoter, and the connection process was performed according to the instructions of 2x Seamless Cloning Mix. The ligation product was transformed into E. coli DH5α competent cells, and incubated at 37°C for 16h. Single colonies were selected and cultured at 37°C, 200rpm for 12h. After positive clones were identified by bacterial liquid PCR, sequencing alignment was performed. The bacterial liquid of successful sequencing was added with an equal volume of 50% glycerol and stored at -80°C for long-term preservation. The corresponding pro35S::GmMYB14 overexpression vectors were named as pCAMBIA3301-35S-GmMYB14 and PTF101-35S-GmMYB14, respectively.

[0041] Plasmid acquisition: 100µL of the above stored bacterial liquid was added into 100ml LB liquid medium containing the corresponding antibiotics. The corresponding antibiotic for pCAMBIA3301-35S-GmMYB14 was kanamycin (Kana), and the corresponding antibiotic for PTF101-35S-GmMYB14 was spectinomycin (Spec). The culture was incubated at 37°C, 200rpm for 12h. The plasmid was extracted according to the steps of TIANGEN plasmid extraction kit instructions, and the GmMYB14 gene pro35S::GmMYB14 overexpression vectors were obtained.

[0042] (3) The above constructed pro35S::GmMYB14The overexpression vector is transformed into Agrobacterium GV3101 (in this embodiment, Agrobacterium-mediated transformation is used, and in addition, Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, and other conventional biological methods can also be used), and the specific method is as follows: 40 μL of Agrobacterium competent cells (sterile operation) is slowly thawed on ice, 2 μL of extracted plasmid (about 200 ng) is added, ice bath for 5 min; liquid nitrogen freezing for 5 min; 37°C heat shock for 5 min; 1 mL of LB liquid medium is added, and incubated at 200 rpm and 28°C for 2 h; the incubated bacterial solution is spread on LB solid plate containing the corresponding antibiotics (Rifampicin Rif and Kanamycin Kana), and incubated at 28°C for 2 days. Single colonies are selected for colony PCR identification, and the bacterial solution is added with an equal volume of 50% glycerol after bacterial solution PCR identification is positive, and is stored at -80°C for long-term preservation.

[0043] (4) Arabidopsis and soybean are transformed by using pCAMBIA3301-35S-GmMYB14 and PTF101-35S-GmMYB14 expression vectors respectively.

[0044] For Arabidopsis transformation, the flower dipping method is used, and the detailed method is as follows: Agrobacterium containing pCAMBIA3301-35S-GmMYB14 expression vector is first inoculated into 5 mL of LB (kanamycin Kana and rifampicin Rif) liquid medium at a ratio of 1:10, and incubated at 28°C and 200 rpm overnight (12-16 h); then 3 mL of Agrobacterium solution is transferred into 300 mL of LB (containing plasmid corresponding antibiotics and Rif) liquid medium at a ratio of 1:10, and incubated at 28°C and 200 rpm overnight (12-16 h), so that the OD 600 reaches 0.8-1.1; then the bacterial solution is poured into a 400 mL centrifuge bottle, centrifuged at 4000 rpm and room temperature for 15 min (Beckman high-speed centrifuge Avanti JXN-26), the supernatant is removed, and the bacterial body is collected; resuspended in 250 mL of infiltration solution, and place the Arabidopsis to be transformed therein, and maintain for 1 min; the soaked plant is taken out after dark treatment in a turnover box (or tray) for 24 h, and is further cultured in a greenhouse. The first generation seeds are screened and identified for positive plants on ½MS solid medium containing glufosinate resistance (to facilitate identification and screening of transgenic plants), the positive plants are collected, and the positive plants meeting the Mendelian segregation genetic law 3:1 are further propagated and collected as single plants, and homozygous identification is performed, and the overexpression plant is obtained at the gene expression level.

[0045] For soybean transformation, the PTF101-35S-GmMYB14 expression vector was transformed into soybeans using an Agrobacterium-mediated cotyledonary node genetic transformation system. After screening for positive plants, they were propagated in a laboratory greenhouse and then at a transgenic seed propagation base the following year. Foliar spraying with a 25 μg / mL glufosinate solution was used to confirm positive plants. Before conducting iron deficiency experiments, each seedling underwent glufosinate spraying and expression level verification. Subsequent experiments were conducted only after confirming the plants as positive.

[0046] (5) Perform molecular testing on transgenic lines to verify GmMYB14 Gene expression status. For the Arabidopsis pCAMBIA3301-35S-GmMYB14 transgenic lines, gene expression was detected in each line using qRT-PCR. GmMYB14 The relative expression levels of genes, the results are as follows: Figure 3 As shown in Figure B, wild-type Arabidopsis thaliana (Col-0) GmMYB14 Gene expression levels were extremely low, while in the overexpressing Arabidopsis homozygous lines #10, #13, #14, and #19, GmMYB14 Gene expression levels were high, with line #10 showing the highest expression level. qRT-PCR was used to detect gene expression in the soybean PTF101-35S-GmMYB14 transgenic lines. GmMYB14 The relative expression levels of genes, the results are as follows: Figure 4 As shown in Figure B, wild-type soybean (WT) GmMYB14 The gene expression level was extremely low, while in the overexpressing homozygous soybean lines #13 and #17, GmMYB14 Gene expression levels were high, with the highest expression level observed in line #13. These results indicate... GmMYB14 The gene can be successfully and efficiently transcribed and expressed in Arabidopsis thaliana overexpression lines and soybean overexpression lines.

[0047] (6) Phenotypic observation, physiological indicators and iron content detection of transgenic lines.

[0048] overexpression GmMYB14 Arabidopsis thaliana lines exhibited greater iron deficiency tolerance and iron utilization than Col-0 under iron deficiency treatment (½ MS, 0 μM Fe) (see [reference needed]). Figure 3 Compared to Col-0, overexpressing Arabidopsis thaliana exhibits more developed root systems, milder iron deficiency chlorosis symptoms, and less inhibited aboveground growth. It also shows phenotypic characteristics such as higher root length, higher total chlorophyll content, more fluorescent phenolic root exudates, higher aboveground biomass, higher root biomass, higher root iron content, and higher aboveground iron content. Specifically, overexpression... GmMYB14 It alleviated the aboveground chlorosis symptoms and root growth inhibition caused by iron deficiency in Arabidopsis thaliana (see...). Figure 3(A) Average root length of wild-type Arabidopsis thaliana after iron deficiency treatment (see [reference]). Figure 3 The root length of the transgenic Arabidopsis thaliana overexpressing the C group was 3.31 cm. The overexpressing homozygous lines #10, #13, #14, and #19 had root lengths of 4.03 cm, 3.69 cm, 3.55 cm, and 3.65 cm, respectively. Iron-deficient transgenic plants showed a root length increase of 7.1%–21.6% compared to wild-type plants. The total chlorophyll content of wild-type Arabidopsis thaliana after iron deficiency treatment (see...) Figure 3 The iron deficiency chlorophyll content (CBD) was 0.64 mg / g in the control group, while the CBD levels in the overexpressing Arabidopsis homozygous lines #10, #13, #14, and #19 were 1.01 mg / g, 0.72 mg / g, 0.78 mg / g, and 0.85 mg / g, respectively. The total chlorophyll content in the iron-deficient transgenic plants increased by 12.6%–57.5% compared to the wild-type plants. The relative fluorescence values ​​of root exudates from iron-deficient wild-type Arabidopsis (compared to the exudate amount under normal treatment conditions) are shown in the figure. Figure 3 The mean E (emission rate) was 1.76, while the mean values ​​for the overexpressing Arabidopsis homozygous lines #10, #13, #14, and #19 were 3.91, 2.50, 2.40, and 3.05, respectively. The fluorescence values ​​of root exudates from iron-deficient transgenic plants increased by 35.9%–121.7% compared to wild-type plants. The average aboveground biomass of wild-type Arabidopsis after iron deficiency treatment (see...) Figure 3 The iron deficiency concentration (F) was 1.03 mg / plant, while the overexpressing homozygous Arabidopsis lines #10, #13, #14, and #19 had concentrations of 1.36 mg / plant, 1.21 mg / plant, 1.33 mg / plant, and 1.32 mg / plant, respectively. The aboveground biomass (fresh weight) of the iron-deficient transgenic plants increased by 18.2%–32.2% compared to the wild-type plants. The average root biomass of the wild-type Arabidopsis after iron deficiency treatment (see...) Figure 3 The iron content (G) in the transgenic plants was 0.42 mg / plant. The overexpressing homozygous Arabidopsis lines #10, #13, #14, and #19 had iron content of 0.63 mg / plant, 0.45 mg / plant, 0.51 mg / plant, and 0.49 mg / plant, respectively. Iron-deficient transgenic plants showed a 7.2%–50.0% increase in root biomass (underground fresh weight) compared to wild-type plants. Iron content in wild-type Arabidopsis roots after iron deficiency treatment (see...) Figure 3 The iron content in the roots of the transgenic Arabidopsis thaliana (H) was 85.63 μg / g. The overexpressing homozygous lines #10, #13, #14, and #19 had iron content of 141.83 μg / g, 93.35 μg / g, 252.30 μg / g, and 120.22 μg / g, respectively. The iron content in the roots of the iron-deficient transgenic plants was 9.0%–194.6% higher than that of the wild-type plants. (See the aboveground iron content of the wild-type Arabidopsis thaliana after iron deficiency treatment). Figure 3The iron content in transgenic plants treated with iron deficiency was 60.00 μg / g. The iron content in the aboveground parts of the transgenic plants was 43.5%-101.1% higher than that of the wild-type plants. The homozygous overexpressing Arabidopsis thaliana lines #10, #13, #14 and #19 were 97.85 μg / g, 120.66 μg / g, 86.39 μg / g and 86.08 μg / g, respectively.

[0049] overexpression GmMYB14 Soybean lines grown under low-iron hydroponic conditions (modified ¼ Hoagland's hydroponic nutrient solution containing 1 μM Fe-EDTA) showed greater tolerance to iron deficiency than wild-type (WT) soybeans (see [link to article]). Figure 4 This manifests in milder chlorosis in the terminal leaves, higher SPAD values, higher total chlorophyll content, more fluorescent phenolic root exudates, and higher aboveground iron content. Specifically, under iron-deficient conditions, the aboveground growth of overexpressing soybean lines is less inhibited than that of wild-type soybeans, while wild-type soybeans show more pronounced yellowing of the terminal leaves (see...). Figure 4 (A), SPAD value of wild-type soybeans after iron deficiency treatment (see...) Figure 4 The SPAD value of iron-deficient transgenic soybean was 11.24, while that of overexpression homozygous lines #13 and #17 was 24.77 and 16.3, respectively. The SPAD value of iron-deficient transgenic plants was 45.0%-120.0% higher than that of wild-type soybean. The total chlorophyll content of wild-type soybean after iron deficiency treatment (see...) Figure 4 The iron deficiency concentration (D) was 0.29 mg / g, while the overexpression homozygous lines #13 and #17 had concentrations of 1.27 mg / g and 0.80 mg / g, respectively. The total chlorophyll content of the iron-deficient transgenic plants was 170.8%–332.6% higher than that of wild-type soybean. The relative fluorescence values ​​of root exudates from iron-deficient wild-type soybeans (compared to the exudate amount under normal treatment conditions) are shown in [reference needed]. Figure 4 The iron content (E) of the transgenic soybean root exudate was 4.51, while that of the overexpression homozygous lines #13 and #17 was 175.81 and 41.88, respectively. The fluorescence value of the root exudate from iron-deficient transgenic plants increased by 830%-3800% compared to wild-type soybean. (See iron-deficient treatment of wild-type soybean aerial parts iron content...) Figure 4 The iron content (H) in the transgenic soybean was 47.04 μg / g, while the overexpression homozygous lines #13 and #17 had 67.96 μg / g and 61.57 μg / g, respectively. The iron-deficient transgenic plants showed a 30.9%-44.5% increase in aboveground iron content compared to wild-type soybean. Under normal growth conditions, the aboveground biomass of wild-type soybean (see...) Figure 4 The root biomass of wild-type soybean (F13) was 12.60 g, while that of overexpression homozygous lines #13 and #17 was 4.21 g and 7.63 g, respectively. After iron deficiency treatment, the aboveground biomass of wild-type soybean was 6.83 g, while that of overexpression homozygous lines #13 and #17 was 3.67 g and 5.33 g, respectively. Under normal growth conditions, the root biomass of wild-type soybean (see...)Figure 4 The iron deficiency stress (G) content in transgenic soybean was 3.92 g, while that in homozygous overexpression lines #13 and #17 was 1.28 g and 2.46 g, respectively. After iron deficiency treatment, the underground biomass of wild-type soybean was 2.11 g, while that in homozygous overexpression lines #13 and #17 was 0.72 g and 1.82 g, respectively. These data show that compared to wild-type soybean, transgenic soybean plants have lower biomass. Under normal growth conditions, the aboveground biomass of transgenic plants is 33.4%-60.6% of that of wild-type soybean, and the root biomass is 32.7%-62.6%. However, after iron deficiency treatment, the aboveground biomass of transgenic plants is 53.7%-78.1% of that of wild-type soybean, and the root biomass is 34.3%-86.1%. This indicates that iron deficiency stress has a smaller impact on the biomass of transgenic plants than on wild-type soybean.

[0050] This invention constructs using genetic engineering technology. pro35S::GmMYB14 Overexpression vectors (pCAMBIA3301-35S-GmMYB14 and PTF101-35S-GmMYB14) were transformed into wild-type Arabidopsis Col-0 plants using the flower-dip method and into wild-type soybean Williams 82 plants using Agrobacterium-mediated cotyledon node transformation, respectively. GmMYB14 The gene is overexpressed in wild-type plants, thereby enabling the plants to exhibit iron deficiency tolerance. This invention is the first to discover gene overexpression in Arabidopsis thaliana and soybean. GmMYB14 The transgenic lines showed significantly enhanced tolerance to iron deficiency. Compared to the wild type, overexpression of this gene not only increased plant biomass, root length, and leaf chlorophyll content under iron-deficient conditions but also significantly increased plant iron content. Based on functional validation of this gene in Arabidopsis and soybean, it is speculated that it may have a similar effect in enhancing iron deficiency tolerance in other monocotyledonous plants (such as rice, wheat, and maize) and dicotyledonous plants (such as rapeseed, tobacco, cucumber, tomato, poplar, turfgrass, and alfalfa). This invention is of great significance for elucidating the mechanism of iron deficiency stress tolerance in soybean and provides genetic resources and technical support for expanding soybean cultivation in low-iron soils (especially calcareous and soda-saline soils).

[0051] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. The gene encoding the soybean GmMYB14 transcription factor overexpression GmMYB14 Its application in enhancing plant tolerance to iron deficiency stress is characterized by, The encoding gene GmMYB14 The nucleotide sequence is shown in SEQ ID No. 1; the plant is soybean or Arabidopsis thaliana.

2. The application according to claim 1, characterized in that, The application aims to enhance the adaptability of soybeans to planting in low-iron soils, including calcareous soils and soda saline-alkali soils.

3. The application of a recombinant bacterium in enhancing plant tolerance to iron deficiency stress, characterized in that, The recombinant bacteria contain pro35S::GmMYB14 Overexpression vector, the pro35S::GmMYB14 The overexpression vector contains the coding gene with the nucleotide sequence shown in SEQ ID NO.

1. GmMYB14 And the pro35S::GmMYB14 The overexpression vector was selected from pCAMBIA3301-35S- GmMYB14 or PTF101-35S-GmMYB14 ; The recombinant bacteria are selected from Agrobacterium GV3101; wherein Agrobacterium GV3101 is used to mediate the transformation of the overexpression vector into plants; the plants are soybean or Arabidopsis thaliana.

4. The application according to claim 3, characterized in that, Used to construct the pro35S::GmMYB14 Primer pairs for overexpression vectors include: The encoded sequences of 3301-MYB14-F and 3301-MYB14-R are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively. Alternatively, 101-MYB14-F and 101-MYB14-R, with encoded sequences as shown in SEQ ID No. 5 and SEQ ID No. 6, respectively.

5. A method for enhancing the tolerance of plants to iron deficiency stress, characterized in that, include: The gene encoding the nucleotide sequence shown in SEQ ID NO.1 GmMYB14 pass pro35S::GmMYB14 The overexpression vector was transformed into plant cells, enabling the encoding gene to be expressed. GmMYB14 Overexpression in plants; After culturing the transformed cells, complete plants were obtained, and single-copy homozygous lines were screened according to Mendel's laws of inheritance. The plants were soybean or Arabidopsis thaliana; Arabidopsis thaliana was transformed using the flower-dip method; and soybean was transformed using the cotyledonary node method mediated by Agrobacterium GV3101.

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