A soybean root development-related gene GmERF3 and its application

Abstract

The present invention belongs to the fields of molecular biology and genetic engineering, and specifically relates to a soybean root development-related gene, GmERF3, and its applications. The full-length coding region sequence of GmERF3, which regulates soybean root development, is shown in the sequence listing as SEQ ID NO.1; the amino acid sequence is shown in the sequence listing as SEQ ID NO.2. The present invention provides a soybean root development-related gene and is used to construct an expression vector for the gene. The constructed plant expression vector is transformed into young soybean cotyledons via Agrobacterium rhizogenes-mediated transformation. The resulting gene-overexpressing transgenic hairy roots develop well, with root development indicators superior to those of the control group, indicating that the gene positively regulates soybean root development. The present invention provides a theoretical basis and technical means for improving soybean root development and has great application value.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and genetic engineering, and particularly relates to a soybean root development-related gene GmERF3 and an application thereof. Background Art

[0002] The root system is a vital tissue for plant growth and development. During plant growth, it not only provides nutrients such as water and inorganic salts but also synthesizes plant hormones and maintains rhizosphere microorganisms. When plants are subjected to abiotic stresses such as drought, low temperature, and salt stress, they adapt by changing the shape and number of their lateral roots to maintain normal growth. Soybean (Glycine max) is an important oilseed crop that plays a crucial role in agricultural production in my country. Soybean is a typical taproot crop, with a root system composed of a main root and branching lateral roots. The degree of root development is one of the key agronomic traits affecting soybean yield.

[0003] Ethylene-responsive factors (ERFs) are plant-specific transcription factors involved in various developmental stages and stress responses. AP2 / ERFs are widely distributed in plants and play key roles in plant growth and development, as well as resistance to biotic and abiotic stresses. The Arabidopsis AP2 / ERF transcription factor genes ERFII-1 and ERFII-2 are involved in lateral root growth and development. Overexpression of ERFII-1 and ERFII-2 significantly reduces lateral root sprouting density. Studies have shown that ERFs can activate ASA1 expression, increasing auxin accumulation in primary roots and inhibiting taproot elongation. In addition to promoting auxin biosynthesis, ERFs also promote auxin transport by directly upregulating PIN1 and AUX1 expression, leading to auxin accumulation in lateral root primordia. Furthermore, ERFs can inhibit ARF7 transcription, thereby downregulating the expression of cell wall remodeling genes and ultimately inhibiting lateral root emergence.

[0004] ERFs respond to various stresses by regulating stress-responsive genes. For example, in Arabidopsis, ERF96 upregulates pathology-related genes such as PDF and PR to defend against fungal pathogens. Group IXb ERFs upregulate CYP81F2 to promote the synthesis of fungicidal indole glucosinolates. In Solanum lycopersicum, ERF68 upregulates genes involved in programmed cell death, promoting cell death. In petunia, PhERF2 enhances plant resistance to waterlogging by upregulating the expression of the ADH1-2 alcohol dehydrogenase genes. In loquat, EjERF39 is induced by low temperature and upregulates the lignin synthesis gene Ej4CL1, promoting fruit lignification and alleviating chilling damage. In Vitis amurensis, VaERF092 is induced by low temperature and enhances the expression of VaWRKY33, thereby indirectly enhancing the plant's chilling resistance. VaERF3 in adzuki beans (Vigna angularis) and OsERF71 in rice (OsERF71) in drought stress can both induce proline accumulation in response to salinity and alkalinity stress, respectively, enhancing stress tolerance. In kidney beans (Phaseolus vulgaris), PvERF15 interacts with the promoter of the heavy metal-responsive transcription factor PvMTF-1 and regulates its expression, promoting cadmium tolerance in plants. Overexpression of the ERF transcription factor TdSHN1 in tobacco can enhance the activity of ROS-scavenging enzymes in wheat. Apple MdMYC2 can bind to the promoter region of MdERF3, activate MdERF3 transcription, influence ethylene production, and promote aluminum-induced growth inhibition. ERFs play an important regulatory role in plant responses to various environmental stresses.

[0005] Breakthrough progress has been made in the study of the structure, function, and mechanism of action of ERF transcription factors. However, the ERF family is numerous in plants, and the relationship between ERF sequence diversity and its functional diversity remains unclear. The regulatory mechanisms of ERFs on plant growth and development, stress response, and signal transduction require further exploration. A growing number of studies indicate that ERFs play a key role in plant responses to stress. However, in crops such as soybeans, the mechanisms by which ERFs regulate soybean root development and their involvement in stress responses are not fully understood. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a soybean root development-related gene GmERF3 and its application. The GmERF3 gene is isolated from soybean, a plant expression vector of the gene is constructed, and the gene is transformed into soybean cotyledons through the Agrobacterium rhizogenes-mediated method to obtain positive hairy roots, thereby realizing functional analysis of the GmERF3 gene.

[0007] The present invention is achieved by providing a soybean root development-related gene GmERF3, the coding region sequence of which is shown in SEQ ID NO.1.

[0008] Provided is a protein encoded by a soybean root development-related gene GmERF3, whose amino acid sequence is shown as SEQ ID NO.2.

[0009] Provided is a primer for amplifying soybean root development-related gene GmERF3, comprising:

[0010] ERF3-3301-F:5'-GA AGATCT ATGCGCCGAGGGA-3′;

[0011] ERF3-3301-R:5'-G GGTAAC CTCAGAGGCACAG-3';

[0012] Among them, the first 8 bases at the 5' end of ERF3-3301-F and ERF3-3301-R are required for constructing the overexpression vector and do not belong to the gene sequence of GmERF3. The underlined bases are enzyme cleavage sites, and the bases in italics are protected bases.

[0013] Provided is an overexpression vector containing soybean root development-related gene GmERF3, which is pCAMBIA3301-CaMV35S-GFP-GmERF3.

[0014] Provided is an application of an overexpression vector containing soybean root development-related gene GmERF3 for promoting plant root development.

[0015] Provided is an application of a soybean root development-related gene GmERF3 in regulating soybean root development.

[0016] Compared with the prior art, the advantages of the present invention are:

[0017] The present invention utilizes existing plant genetic engineering technology to clone the soybean root development-related gene GmERF3, and transfers the gene into soybean explants through an Agrobacterium-mediated method. Comparative analysis shows that GmERF3 overexpression leads to good root development, increased main root length, lateral root density, and root number, and improved root resistance indicators, indicating that the gene positively regulates the development of the soybean root system, providing a theoretical basis and technical means for improving soybean root resistance, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the expression cassette for constructing the pCAMBIA3301-EGFP-GmERF3 vector;

[0019] Figure 2 This is the amplification result of the full-length 636 bp coding region sequence of the GmERF3 gene, where M is DL 2000;

[0020] Figure 3 The results of double enzyme digestion of pMD19T-GmERF3 vector;

[0021] Figure 4 This is the sequencing result of the pCAMBIA3301-CaMV35S-GFP-GmERF3 recombinant vector;

[0022] Figure 5 A genetic transformation system for soybean hairy roots mediated by Agrobacterium rhizogenes with GFP selection marker;

[0023] Figure 6 (a) shows the phenotypic identification of hairy roots overexpressing GmERF3;

[0024] Figure 6(b) shows the statistics of root development indicators of GmERF3 overexpressing hairy roots;

[0025] Figure 6(c) shows the determination of antioxidant enzyme activity indicators in hairy roots with overexpression of GmERF3. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0027] Example 1: Cloning of the soybean GmERF3 gene

[0028] (1) Soybean cultivar 'Dongnong 50' was used as the test material.

[0029] (2) Total RNA was extracted using the RNAprep pure plant total RNA extraction kit and reverse transcribed to synthesize the first chain of cDNA.

[0030] (3) Gene cloning: To construct the instructions attached Figure 1 The GmERF3 overexpression vector shown in FIG was used to amplify the reverse transcribed cDNA first strand using primers ERF3-3301-F / ERF3-3301-R. The PCR product was recovered to obtain a 636 bp target fragment. The PCR results are shown in FIG. Figure 2 .

[0031] Example 2: Construction of plant expression vector

[0032] The purified gel-recovered product, the full-length GmERF3 sequence, was ligated into the pMD19T cloning vector. The ligation system was as follows: 5 μL Solution I, 4 μL target gene PCR product, 1 μL pMD19T. The reaction was carried out at 16°C overnight to obtain the pMD19T-GmERF3 recombinant cloning expression vector. The recombinant cloning expression vector was double-digested with Bgl II and BstE II. At the same time, the pCAMBIA3301-CaMV35S-GFP expression vector was double-digested to linearize it. The recombinant cloning vector / expression vector double enzyme digestion system was as follows: 7 μL pMD19T-GmERF3 / pCAMBIA3301-35S-GFP, 1 μL Bgl II, 1 μL BstE II, 1 μL 10×H buffer. The reaction was carried out at 37°C for 4 hours. The results of the double enzyme digestion of the recombinant cloning vector pMD19T-GmERF3 are shown in the table. Figure 3 Use T4 DNA ligase to connect the target gene and the final expression vector. The ligation system is: 0.5μL T4 DNA ligase, 1μL T4 DNA ligase buffer, 2μL linearized pCAMBIA3301-35S-GFP, 6.5μL target gene fragment, and react at 50℃ for 40min. The ligation product was transformed into Escherichia coli competent DH5α, and the plant expression vector pCAMBIA3301-CaMV35S-GFP-GmERF3 with the target gene was obtained by colony PCR, shaking, and plasmid extraction. The sequencing results are shown in Figure 4 Then, Agrobacterium rhizogenes R599 competent cells were transformed, and the obtained Agrobacterium rhizogenes containing the recombinant plasmid was used to transform soybean cotyledons.

[0033] Example 3: Acquisition and verification of transgenic soybean materials

[0034] (1) Transformation of soybean cotyledons with recombinant plasmids

[0035] The positive clone of Agrobacterium rhizogenes R599 containing pCAMBIA3301-CaMV35S-GFP-GmERF3 was selected and inoculated into MS liquid medium (containing 100 mg / L AS, 1 mg / L BA and 0.1 mg / L NAA) without any resistance. The concentration of the infection solution was controlled at OD 600 = 0.02-0.3, create wounds on the surface of young soybean cotyledons to destroy their vascular tissue, co-incubate with the infection solution for about 10 minutes, transplant the explants with the wound facing downwards into MS solid medium (containing 100 mg / L AS), seal with sealing film, and incubate in the dark at 26°C for 3 days. After washing the explants, move them with the wound facing upwards into rooting medium and incubate them in the light at 26°C for 2-4 weeks to induce hairy roots. The operating system is shown in the table. Figure 5 .

[0036] (2) Identification of GmERF3-overexpressing hairy roots

[0037] Fluorescence detection and phenotypic detection: The pCAMBIA3301-CaMV35S-GFP-GmERF3 overexpression vector contains a GFP tag, which is used to detect the transformation effect of specific genes in hairy roots. Soybean hairy roots infected only with pCAMBIA3301-CaMV35S-GFP served as a control. GFP fluorescence in soybean hairy roots was detected using a live imaging device to determine the expression of GmERF3. Fluorescence signals were detected in soybean hairy roots transformed with pCAMBIA3301-CaMV35S-GFP-GmERF3, indicating that the GmERF3 protein was successfully expressed in the hairy roots. The results are shown in Figure 2. Figure 5 (right).

[0038] Example 4: Effect of GmERF3 gene on soybean root development

[0039] GmERF3-overexpressing hairy roots were cultured and transferred to MS culture jars. Root morphology and root development indicators were observed, and changes in root antioxidant enzyme activity were measured. Figures 6(a), 6(b), and 6(c) show that hairy roots overexpressing GmERF3 developed well compared to the control. The taproot length and number of lateral roots in GmERF3-overexpressing hairy roots were significantly longer than those in the control, and the CAT and POD enzyme activities were higher, while the MDA level was lower. These results demonstrate that GmERF3 overexpression promotes soybean root development and improves root resistance.

Claims

1. A gene containing soybean root development-related genes GmERF3 The use of an overexpression vector is characterized in that The gene GmERF3 The coding region sequence is shown in SEQ ID NO. 1, and the application is for promoting soybean root development.

2. The soybean root development-related gene according to claim 1 GmERF3 The use of an overexpression vector is characterized in that The overexpression vector is pCAMBIA3301-CaMV35S-GFP- GmERF3 .

3. Overexpression of soybean root development-related genes GmERF3 The application in promoting soybean root development is characterized in that: The gene GmERF3 The coding region sequence is shown in SEQ ID NO.1.

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