An E3 ubiquitin ligase gene SlRZFP34 and its application

Abstract

The present invention discloses an E3 ubiquitin ligase gene SlRZFP34 , which is derived from tomato, and its nucleotide sequence is shown in SEQ ID NO:1. The expression level of this gene in tomato root tissue is higher than that in stems and leaves; its expression is induced by infection with Fusarium oxysporum; this gene is expressed in tobacco leaves, increasing tobacco's resistance to infection with Fusarium oxysporum; this gene has application potential in tomatoes to resist infection with Fusarium oxysporum.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a tomato E3 ubiquitin ligase gene. SlRZFP34 Genes and their application in improving plant disease resistance. Background Art

[0002] tomato( Solanum lycopersicum Tomatoes, also known as tomatoes or tomatoes, are an important vegetable crop cultivated worldwide. Their fruits are used in a wide variety of food preparations. Besides their delicious taste, tomatoes contain a variety of vitamins, lycopene, beta-carotene, and other nutrients beneficial to human health. However, with the rapid development of the tomato industry, it is also facing increasing threats from pathogenic microorganisms, resulting in significant economic losses to agricultural production. Therefore, in-depth research on tomato disease resistance genes and improving tomato disease resistance and stress tolerance through genetic engineering and other means are currently important issues.

[0003] Fusarium wilt is a plant disease that causes the plant to die and wilt due to infection by pathogenic bacteria. Fusarium wilt can cause severe yield losses in tomatoes and is a global soil-borne disease caused by infection with the Fusarium genus fungus. Fusarium oxysporum ) belongs to the genus Fusarium. Fusarium oxysporum includes more than 100 host-specific strains and is a soil-borne plant pathogen that causes serious wilt diseases on important economic crops worldwide. This fungus is extremely pathogenic and can infect a variety of plants, causing plant disease and even death. When Fusarium oxysporum infects a plant, it enters the plant body through the roots, gradually spreads to the stem, and forms a pink mold layer at the base of the stem. Conidia and conidia are produced in large quantities under humid conditions, blocking the vascular bundles, resulting in obstruction of water and nutrient transport in the plant, and causing extensive wilt, necrosis, and yellowing of the aboveground parts of the plant. Ultimately, the disease causes the plant to decline rapidly, accelerates fruit ripening, and causes plant death.

[0004] Plants are able to mount a defensive response against potential pathogens through their innate immune system. This system consists of two primary layers: the first, PTI (pathogen-triggered immunity), considered the first line of defense in plant immunity; and the second, ETI (effector-triggered immunity). At the PTI level, plants utilize specific receptors located on their cell surfaces, known as plant pattern recognition receptors (PRRs), to precisely recognize pathogen-associated molecular patterns (PAMPs), enabling them to detect the presence of pathogens. The ETI response is often associated with a hypersensitive response (HR), in which programmed cell death occurs at the site of infection, thereby limiting the spread of the pathogen. PTI and ETI complement each other, forming two lines of defense in the plant immune system, protecting plants from pathogens and, as such, are receiving increasing attention.

[0005] In addition to the innate immune response, plants also possess a more in-depth and complex immune mechanism, namely the plant systemic acquired immune response (SAR). SAR is an inducible defense mechanism that enables leaves to produce immune responses against a variety of pathogens, including fungi, bacteria, and viruses. Under the high pressure of pathogen infection, SAR confers an adaptive advantage to plants. Previous studies have shown that several small metabolites in Arabidopsis recognize long-distance SAR migration signals, including salicylic acid (SA), methyl salicylate, azelaic acid, jasmonic acid, abietane-diterpenoid-dehydroabietal, 3-phosphoglycerol, and the protein DIR1. These metabolites are transported from the primary infected tissue to distal tissues, which is crucial for the activation of SAR. The petiole secretions of the first inoculated leaves contain vascular bundle migration signals that can induce SAR in young plants.

[0006] Ubiquitination is a post-translational protein modification that not only controls the stability and localization of substrate proteins but also influences their function. Ubiquitination plays an essential role in plant defense against disease. Its highly dynamic ubiquitination reaction can rapidly adjust the plant's defense gene network. In this process, E3 ubiquitin ligases play a key role in substrate recognition. Based on their catalytic mechanisms and structural characteristics, E3 ubiquitin ligases mainly include the RING domain family, the HECT domain family, the RBR domain family, and the U-box protein family. RING-type E3 ubiquitin ligases play a crucial role in plant-pathogen interactions. Studies have shown that Arabidopsis thaliana ATL9, as a RING-type E3 ubiquitin ligase, is involved in regulating plant resistance to vegetative pathogens. When pathogens infect plants, plants induce the expression of ATL9 to counteract the pathogen's inhibitory proteins, thereby promoting an immune response. Overexpression of the CaRFP1 gene in Arabidopsis thaliana increased the susceptibility of transgenic plants to tomato bacterial leaf spot. This may be due to the protein acting as an E3 ubiquitin ligase, which reduces the expression levels of disease-related genes such as PR-2 and PR-5. EIRP degrades the transcription factor VpWRKY11 through the ubiquitin-proteasome system, participating in the disease resistance response of Vitis pseudoreticulate, and improving the resistance of Vitis rapae in East China to pathogens.

[0007] Higher plants harbor a large and diverse array of E3 ubiquitin ligases, particularly RING-type E3 ubiquitin ligases. However, relatively little research has been conducted on their specific functions, especially in important fruit and vegetable crops like tomatoes, where functional studies on E3 ubiquitin ligases and their interacting proteins are even more scarce. Summary of the Invention

[0008] The present invention provides an E3 ubiquitin ligase gene SlRZFP34 , whose nucleotide sequence is shown in SEQ ID NO: 1, encoding a protein with an amino acid sequence shown in SEQ ID NO: 2; which is obtained from tomato ( Ailsa Craig ) plant, the full-length gene sequence was 936 bp, and the encoded SlRZFP34 protein consisted of 311 amino acids.

[0009] Another object of the present invention is to transform the ubiquitin ligase gene SlRZFP34 Application in improving plant resistance to Fusarium oxysporum Fusariumoxysporum ) medium resistance.

[0010] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0011] 1. Roots, stems, and leaves were collected from tomato plants grown in a greenhouse at the College of Life Sciences and Technology, Kunming University of Science and Technology, Yunnan Province. The expression characteristics of E3 ubiquitin ligase genes were analyzed using qRT-PCR. The results showed that compared with stems and leaves, eight tomato E3 ubiquitin ligase genes showed stable expression levels in roots, including SlRZFP34 The expression level of the gene in roots was significantly higher than that in stems and leaves. SlRZFP34 The expression level of the gene was significantly upregulated by Fusarium oxysporum infection. SlRZFP34 The expression level of the gene was significantly decreased, indicating that SlRZFP34 Genes play important role in tomato resistance to blight;

[0012] 2. Extract total RNA from tomato plants, reverse transcribe it into cDNA and use it as a template. Design primers based on the gene sequence to amplify SlRZFP34 The amplified fragment was homologously recombined with the pCAMBIA1300-GFP vector after enzyme digestion and transformed into Escherichia coli DH5 α Then, a plate containing kanamycin was coated, and positive colonies were picked for sequencing verification, and the gene sequence encoding the SlRZFP34 protein was identified, the sequence of which is shown in SEQ ID NO: 1. The protein is encoded by 311 amino acids, and the amino acid sequence is shown in SEQ ID NO: 2;

[0013] 3. To confirm SlRZFP34 The subcellular localization of genes was determined by repeated freeze-thaw method. SlRZFP34 -GFP recombinant plasmid was transformed into Agrobacterium tumefaciens LBA4404 strain to obtain recombinant Agrobacterium strain containing recombinant expression plasmid. PCR detection of Agrobacterium transformants showed that a 936bp band could be amplified, indicating that the plasmid was successfully transformed into Agrobacterium. SlRZFP34 -GFP recombinant plasmid was injected into tobacco leaves; after culture, the leaves were observed using a laser confocal microscope; the results showed that the expression SlRZFP34 -GFP fusion protein cells, the green fluorescence signal is mainly distributed in the cytoplasm, cell membrane and cell nucleus; this indicates SlRZFP34 The gene expression product is widely distributed in the cell, indicating that the SlRZFP34 protein may perform multiple functions in different parts of the cell;

[0014] 4. To explore SlRZFP34 The role of genes in plant disease resistance was investigated by injecting pCAMBIA1300-GFP and pCAMBIA1300-SlRZFP34 -GFP Agrobacterium solution was injected, and 2 days later, Fusarium oxysporum spore solution was inoculated to observe the resistance response of tobacco leaves to pathogens. SlRZFP34 -GFP-containing Agrobacterium solution on tobacco leaves inoculated with Fusarium oxysporum spores showed mild rot, while the leaves of the control group injected with Agrobacterium containing pCAMBIA1300-GFP showed more severe rot. The lesion area of ​​the control group leaves was much larger than that of the experimental group, with an area of ​​177 mm. 2 , while the experimental group was only 19mm 2 This result shows that SlRZFP34 Overexpression of the gene significantly improved tobacco's resistance to Fusarium oxysporum.

[0015] The advantages and technical effects of the present invention are as follows:

[0016] Tomato E3 ubiquitin ligase gene of the present invention SlRZFP34 The expression product is located in the cell membrane, cytoplasm and nucleus. SlRZFP34 Gene overexpression can enhance tobacco's disease resistance. SlRZFP34 may enhance plant disease resistance by interacting with its target protein. This efficient and environmentally friendly approach to genetic engineering can enhance plant disease resistance, and has important implications for breeding disease-resistant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram showing the analysis results of the expression characteristics of 8 E3 ubiquitin ligase genes in tomato roots, stems, and leaves;

[0018] Figure 2 This is the expression result of eight E3 ubiquitin ligase genes in the roots of the disease-resistant tomato line Motelle after 0 h, 6 h, 12 h, and 24 h of infection with Fusarium oxysporum;

[0019] Figure 3 The expression results of eight E3 ubiquitin ligase genes at 0 h, 6 h, 12 h, and 24 h after Fusarium oxysporum infection of the roots of the susceptible tomato line Moneymaker;

[0020] Figure 4 For the present invention SlRZFP34 PCR identification diagram of gene amplification and transformation of Agrobacterium tumefaciens LBA4404; Figure A is SlRZFP34 Electrophoresis diagram of gene amplified fragments; Figure B is pCAMBIA1300- SlRZFP34 -PCR identification of Agrobacterium LBA4404 transformed with GFP recombinant plasmid;

[0021] Figure 5 This is a diagram showing the subcellular localization analysis of the SlRZFP34 protein in tobacco. GFP indicates green fluorescence; Bright indicates bright field; DAPI indicates nuclear localization dye; and Merged indicates the superposition of green fluorescence, bright field, and nuclear localization dye. Scale bar: 20 μm.

[0022] Figure 6 The phenotype and lesion area comparison of transgenic tobacco leaves infected with Fusarium oxysporum of the present invention for 5 days, wherein Figure A shows that the tobacco leaves were injected with pCAMBIA1300-GFP and pCAMBIA1300- SlRZFP第一行34 -GFP Agrobacterium liquid, and 2 days later inoculated with Fusarium oxysporum spore liquid, the tobacco leaves' resistance response to the pathogen; Figure B is a statistical chart of the size and area of ​​leaf lesions. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below by way of examples, but the content of the present invention is not limited thereto. Unless otherwise specified, the methods in the present examples are operated according to conventional methods, and the reagents used are conventional reagents or reagents prepared according to conventional methods unless otherwise specified.

[0024] Example 1: Analysis of expression characteristics of tomato E3 ubiquitin ligase genes in different tissues and after infection with Fusarium oxysporum

[0025] 1. Preparation of Fusarium oxysporum spore suspension

[0026] Spread 20-30 μL of the preserved Fusarium oxysporum spore solution on a PDA plate and activate it at 28°C for 4 days. Select a single colony from the plate and inoculate it into 100 mL of YPD liquid culture medium. Culture it at 28°C with shaking for 4 days. Filter the spore suspension into a 50 mL centrifuge tube using a sterilized 350-mesh nylon cloth. Remove the supernatant by centrifugation. Then add 1 mL of sterile water to the centrifuge tube and resuspend the spores. Count the spores in the spore suspension using a hemocytometer and adjust the concentration of the spore suspension to 10 8 / mL;

[0027] 2. Select the disease-resistant tomato strain Motelle and the disease-susceptible tomato strain Moneymaker. When the tomato seedlings grow 4 to 5 true leaves, remove them from the soil, rinse the roots of the tomatoes, and use scissors to wound the roots of the tomato plants. Divide the disease-resistant tomato plants Motelle and the disease-susceptible tomato plants into 4 groups (6 plants / group). 8The tomato seedlings were immersed in a spore suspension of 1000 ng / mL for 30 min, and then re-transplanted into the soil. 0.2 g of roots, stems, and leaves of the disease-resistant tomato line Motelle and the susceptible tomato line Moneymaker were collected 0 h, 6 h, 12 h, and 24 h after spore suspension treatment, respectively. The samples were quickly frozen in liquid nitrogen and then transferred to a -80°C freezer for storage.

[0028] 3. Extraction and reverse transcription of RNA from tomato roots, stems and leaves

[0029] Total RNA from tomato samples was extracted using the Trizol method and reverse transcribed according to the instructions of the TaKaRa reverse transcription kit to obtain tomato cDNA.

[0030] 4. Quantitative real-time PCR (qRT-PCR) was used to analyze gene transcription levels. qRT-PCR primers were designed based on the sequences of eight tomato E3 ubiquitin ligase genes: the forward primer for SlCSU1, qp-SlCSU1-F, was 5'-GTTGTGTTGCCAGAAAGGGC-3', and the reverse primer, qp-SlCSU1-R, was 5'-TGTATGAGCAGCTAACTTTCTTTGG-3'; the forward primer for SlRNF4, qp-SlRNF4-F, was 5'-CCTCTTTGACAGCTGGAGCA-3', and the reverse primer, qp-SlRNF4-R, was 5'-TAGACGCACGGTCCTCAGAA-3'; the forward primer for SlRZFP34, qp-SlRZFP 34-F is 5'-ATCGGAGTTGAAAGGCTTGT-3', and the reverse primer qp-SlRZFP34-R is 5'-ATCGGAGTTGAAAGGCTTGT-3'; the forward primer qp-SlUPL5-F of SlUPL5 is 5'-TTCTTTCCAAATCCAGCGTCG-3', and the reverse primer qp-SlUPL5-R is 5'-ACACGCGGTCAAAGACAATG-3'; the forward primer qp-SlUPL6-F of SlUPL6 is 5'-CAGAACTCTGCTGCCCTCAA -3', the reverse primer qp-SlUPL6-R is 5'-ACTGAAGCACTGCCTGTCAA-3'; the forward primer qp-SlUPL7-F of SlUPL7 is 5'-GGGTCAATAATCAAACCCGCT-3', the reverse primer qp-SlUPL7-R is 5'-GATCCTCGCAATGAAACCTGG-3'; the forward primer qp-SlRIN2-F of SlRIN2 is 5'-TTTGTCTGAGATCTTCACAGTTTTT-3', the reverse primer qp-SlRIN2-R is 5'-C CGCAACCAGTCCACCTAAT-3'; the forward primer qp-SlRGLG3-F of SlRGLG3 is 5'-TCAACGTGCAGCCATCAAAA-3', and the reverse primer qp-SlRGLG3-R is 5'-TGTTGTCAAAAGCTCTGAAGGA-3'; the forward primer qp-SlRGLG4-F of SlRGLG4 is 5'-TTTGGGTTTGGTGATGCGAC-3', and the reverse primer qp-SlRGLG4-R is 5'-ACTGGCCCGTAAGATGTTGG-3';qRT-PCR reactions were performed using the tomato actin gene SlActin as an internal reference. Each reaction was repeated three times. The expression level of the E3 ubiquitin ligase gene in tomato tissues not treated with Fusarium oxysporum was used as a control. The expression level of the tomato E3 ubiquitin ligase gene under each treatment was calculated using the 2-ΔΔCt method.

[0031] The results showed that tomato E3 ubiquitin ligase genes showed stable expression levels in roots compared with stems and leaves. SlRZFP34 The expression level of the gene in roots was significantly higher than that in stems and leaves ( Figure 1 ). Tomato plants of the disease-resistant strain Motelle and the susceptible strain Moneymaker were infected with Fusarium oxysporum. In the disease-resistant strain, SlRZFP34 The expression level of α-glucan was significantly upregulated, with the highest expression at 24h, which was 3.68 times that of the non-infected Fusarium oxysporum ( Figure 2 In susceptible strains, after infection with Fusarium oxysporum SlRZFP34 The expression level of the gene was significantly reduced, and the expression levels at 6 h, 12 h, and 24 h were reduced by 14.29 times, 25 times, and 20 times, respectively, compared with those without Fusarium oxysporum infection. Figure 3 ), which shows SlRZFP34 It plays an important role in tomato's resistance to Fusarium oxysporum infection.

[0032] Example 2: SlRZFP34 Gene amplification and construction of plant expression vectors

[0033] Design amplification SlRZFP34 Primers for gene fragments, forward primer GFP- SlRZFP34 -F is 5'-TACGAACGATACTCGACCCCATGGGAGACATAGTGATTGAACA-3', reverse primer GFP- SlRZFP34 -R is 5'-CTAGAGTCGACGGATCCCCCTCCACCTCTTGTTTGGCGGG-3'. Using tomato cDNA as a template, amplification of 936 bp SlRZFP34 Gene fragments ( Figure 4 A). The amplified SlRZFP34 The gene was recovered and connected to the enzyme-cut pCAMBIA1300-GFP vector for homologous recombination. After transformation into E. coli DH5α, it was sent to Sangon Biotech Co., Ltd. for sequencing verification. The plasmid with the correct sequencing was the successfully constructed plant expression vector pCAMBIA1300- SlRZFP34 -GFP. SlRZFP34-GFP plasmid was transformed into Agrobacterium LBA4404, and the bacterial solution was subjected to PCR amplification to obtain a band of the correct size, indicating that the plasmid was successfully transformed into Agrobacterium ( Figure 4 B).

[0034] Example 3: Subcellular localization analysis of SlRZFP34 protein

[0035] The pCAMBIA1300- SlRZFP34 -GFP recombinant plasmid, pCAMBIA1300-GFP Agrobacterium solution was injected into tobacco leaves, pCAMBIA1300-GFP and pCAMBIA1300- SlRZFP34 -GFP was transiently expressed in tobacco cells and observed using a laser confocal microscope after culturing for 2 days; the results are shown in Figure 2. Figure 5 As shown, pCAMBIA1300- SlRZFP34 In tobacco plants expressing the -GFP plasmid, the green fluorescence signal is mainly distributed in the cytoplasm, cell membrane, and nucleus. SlRZFP34 The gene expression product is widely distributed in the cell, and the SlRZFP34 protein may perform multiple functions in different regions of the cell.

[0036] Example 4: SlRZFP34 increases tobacco resistance to Fusarium oxysporum

[0037] The pCAMBIA1300- SlRZFP34 -GFP Agrobacterium liquid 300μL, after injection 2 days inoculated with Fusarium oxysporum spore suspension 300μL, after inoculation 5 days, observe the tobacco leaves resistance to pathogens. Figure 6 As shown, the cells containing pCAMBIA1300- SlRZFP34 -GFP-containing Agrobacterium solution on tobacco leaves, mild rot occurred near the area inoculated with Fusarium oxysporum, while the control group of tobacco leaves injected with Agrobacterium solution containing pCAMBIA1300-GFP showed more severe rot ( Figure 6 A), and injected with pCAMBIA1300- SlRZFP34 -GFP Agrobacterium slurry tobacco leaf lesion area is much smaller than the control group ( Figure 6 B). The experimental results show that SlRZFP34 说明:原文中“SlRZFP34 ”直接翻译为“SlRZFP34”感觉不太符合专利文本的准确表达习惯,推测可能是“SlRZFP第一行34”,你可根据实际情况调整。或者你能提供更多关于这个文本的背景信息,以便我更准确翻译。 The expression of the gene can significantly improve tobacco's resistance to Fusarium oxysporum.

Claims

1. An E3 ubiquitin ligase gene SlRZFP34 In improving tobacco resistance to Fusarium oxysporum ( Fusarium oxysporum ) resistance, characterized in that: The E3 ubiquitin ligase gene SlRZFP3 The nucleotide sequence is shown in SEQ ID NO: 1.

Images