Banana maPRX5 gene and application thereof in improving resistance of banana to fusarium wilt

Abstract

The application discloses a banana MaPRX5 gene and application thereof in improving resistance of banana to fusarium wilt, and belongs to the field of plant genetic engineering.The nucleotide sequence of the MaPRX5 gene is shown as SEQ ID NO:1. Foc Experimental results prove that overexpression of the MaPRX5 gene in banana can significantly inhibit Foc colonization and expansion of TR4 in corms, and effectively improve the resistance of banana to fusarium wilt.Molecular mechanism research shows that a transcription factor MaWRKY41 can specifically bind to the MaPRX5 promoter and activate the transcription activity thereof, constituting a regulation pathway responding to TR4 infection, thereby improving the resistance of banana to fusarium wilt.The MaPRX5 gene provided by the application can be used as an important gene resource for molecular breeding of banana resistance, and provides a new technical approach for solving the fusarium wilt hazard faced by the banana industry.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, and specifically relates to the banana MaPRX5 gene and its application in improving banana resistance to Fusarium wilt. Background Technology

[0002] Bananas are an important tropical and subtropical fruit crop globally, playing a crucial role in food security, fresh fruit trade, and agricultural economies in tropical regions. They are produced by *Fusarium oxysporum* strain 4, a specialized tropical race of *Fusarium oxysporum*. Fusarium oxysporum f. sp. cubense tropical race 4, abbreviated Foc Fusarium wilt caused by TR4 is currently the most serious soil-borne vascular disease affecting the banana industry. This pathogen can invade from the banana roots, spread through the corm to the vascular tissue of the pseudostem, causing browning of the vascular bundles, dysfunction of the vascular system, yellowing and wilting of leaves, and eventually death of the entire plant.

[0003] Currently, the control of banana wilt disease faces multiple technical bottlenecks. Regarding chemical control, due to… Foc TR4 is a systemic vascular disease, and conventional fungicides are difficult to achieve effective concentrations within the vascular tissue. Furthermore, long-term use of chemical agents poses risks of environmental pollution, food residues, and pathogen resistance. In terms of agricultural control measures, production mainly relies on comprehensive management methods such as quarantine isolation, timely removal of diseased plants, soil improvement, and crop rotation. Foc TR4 can survive in the soil as chlamydospores for decades, making it difficult to eradicate once an infected banana plantation is established. Regarding disease resistance breeding, since most banana cultivars are triploid, highly sterile, and primarily rely on asexual reproduction, traditional hybridization breeding is time-consuming and inefficient, making it difficult to develop new varieties that combine excellent agronomic traits with stable resistance to Fusarium wilt in a short period.

[0004] Plant cell walls are the first physical barrier against pathogen invasion. Lignin deposition in the cell wall enhances its mechanical strength and resistance to enzymatic degradation, thus limiting the penetration, spread, and colonization of pathogens within plant tissues. Class III peroxidases (PRXs) are the largest subfamily members of the plant peroxidase superfamily. These enzymes typically use hydrogen peroxide (H₂O₂) as an oxidant to catalyze the oxidative polymerization of monolignins such as coniferyl alcohol, producing G-lignin (guaiacyl lignin), which participates in lignin biosynthesis. Furthermore, PRXs also participate in the regulation of reactive oxygen species (ROS) homeostasis and the transduction of biotic stress signals in plants. Previous studies have shown that some members of the plant PRX family can influence plant resistance to pathogens by regulating the cell wall lignification process. However, different PRX members exhibit significant tissue expression specificity and functional differentiation characteristics. Currently, research on the molecular mechanisms by which banana-derived PRX genes participate in FocTR4 resistance is still relatively limited, especially lacking in-depth analysis of the functional verification and molecular regulatory mechanisms of the banana PRX5 (MaPRX5) gene in Fusarium wilt resistance.

[0005] In conclusion, screening and identifying banana gene resources with clear disease resistance functions, elucidating their molecular mechanisms of disease resistance, and applying them to the molecular breeding of Fusarium wilt-resistant banana varieties are of significant scientific importance and practical value. Summary of the Invention

[0006] The purpose of this invention is to provide the banana MaPRX5 gene and its application in improving banana resistance to Fusarium wilt, so as to solve the technical problems of lacking effective banana Fusarium wilt resistance-related gene resources and difficulty in rapidly creating new Fusarium wilt resistant banana germplasm through molecular breeding.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for improving the resistance of bananas to Fusarium wilt, the method comprising overexpressing the MaPRX5 gene in bananas to improve the resistance of bananas to Fusarium wilt, the nucleotide sequence of the MaPRX5 gene being shown in SEQ ID NO:1.

[0008] Furthermore, the wilt disease is caused by Fusarium oxysporum, specifically race 4 of the Cuban variant.

[0009] Furthermore, the amino acid sequence encoded by the MaPRX5 gene is shown in SEQ ID NO:2.

[0010] Furthermore, the overexpression of the MaPRX5 gene in bananas is achieved by introducing an expression vector integrating the nucleic acid molecule shown in SEQ ID NO:1 into the banana.

[0011] Secondly, this invention provides applications of overexpressing the MaPRX5 gene, wherein the application is any of the following: A1) Application in improving banana resistance to Fusarium wilt; A2) Application in the preparation of bananas resistant to Fusarium wilt; The nucleotide sequence of the MaPRX5 gene is shown in SEQ ID NO:1.

[0012] Furthermore, the application involves introducing an expression vector integrating the nucleic acid molecule shown in SEQ ID NO:1 into bananas, thereby improving the bananas' resistance to Fusarium wilt.

[0013] Thirdly, the present invention provides applications of biological materials related to the overexpression of the MaPRX5 gene, wherein the application is any of the following: B1) Application in improving banana resistance to Fusarium wilt; B2) Application in the preparation of bananas resistant to Fusarium wilt; The biomaterial is any one of the following C1) to C3): C1) An expression cassette containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO:1; C2) Recombinant vectors containing nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:1; C3) A recombinant microorganism containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO:1, or a recombinant microorganism containing the expression cassette described in C1), or a recombinant microorganism containing the recombinant vector described in C2), wherein the microorganism is Agrobacterium.

[0014] Furthermore, the application involves introducing an expression vector integrating the nucleic acid molecule shown in SEQ ID NO:1 into bananas, thereby improving the bananas' resistance to Fusarium wilt.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention cloned and verified the wilt resistance function of the banana MaPRX5 gene, providing a new candidate gene resource for molecular breeding of bananas wilt resistance, which helps to accelerate the breeding process of disease-resistant varieties.

[0016] (2) Through transient overexpression system and FocThe TR4-GFP fluorescent labeling inoculation experiment demonstrated that MaPRX5 overexpression can significantly inhibit pathogen colonization and alleviate disease symptoms, indicating that MaPRX5 has a clear functional value in improving banana resistance to Fusarium wilt.

[0017] (3) Using a yeast one-hybrid and dual-luciferase reporter system, this invention identified the transcriptional activation effect of the upstream transcription factor MaWRKY41 on MaPRX5, thereby revealing the molecular mechanism by which MaPRX5 regulates disease resistance. This regulatory pathway provides a new target for molecular breeding of disease resistance, and more efficient genetic improvement of disease resistance can be achieved by regulating key nodes in this pathway (such as overexpression of MaWRKY41, synergistic overexpression of MaWRKY41 and MaPRX5, etc.).

[0018] (4) The MaPRX5 gene provided by this invention can be used as an important gene resource for molecular breeding of banana disease resistance. New germplasm of banana resistant to Fusarium wilt can be created through overexpression technology, providing a new technical approach to solve the Fusarium wilt damage faced by the banana industry. Attached Figure Description

[0019] Figure 1 for Foc Figure 1 shows the relative expression level of MaPRX5 after TR4 infection and transient overexpression treatment; different lowercase letters indicate significant differences between treatments (P < 0.05).

[0020] Figure 2 Transient overexpression of MaPRX5 enhances the resistance of banana bulbs to... Foc The graph shows the resistance results to TR4 infection. White arrows indicate... Foc TR4 colonization signal.

[0021] Figure 3 For MaWRKY41 and MaPRX5 Promoter binding and its pair MaPRX5 Regulation of promoter transcriptional activity. (A) Yeast one-hybrid results showed that MaWRKY41 can interact with... MaPRX5 (B) Promoter binding; dual-luciferase results showed that MaWRKY41 significantly improved MaPRX5 Transcriptional activity of the promoter. "****" indicates highly significant differences. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0023] Example 1: Cloning of the banana MaPRX5 gene By analyzing the banana root tips FocTranscriptome data at different time points after TR4 infection were analyzed and selected. MaPRX5 Based on the MaPRX5 open reading frame sequence, specific primers P1 (5′-ATGGCTTTGGGAAGAGGAGAGATGT-3′, SEQ ID NO:3) and P2 (5′-TCAGTTGACTACTCTGCAGTTGGTA-3′, SEQ ID NO:4) were designed to amplify the first-strand cDNA from banana root tissue as a template. MaPRX5The coding region is 975 bp in length (SEQ ID NO:1,ATGGCTTTGGGAAGAGGAGAGATGTCGCTAGCCATCATTGTGGTCTTGTGTCTCAGCGCAATGGGGACAGAAGCCTATCTCAAAGTTGGCTTCTACTCCTACAGCTGCCCTAAAGCGGAGGAGATCGTTAAGGAGGAGCTTGATAAGGCTCTGCAGGAAGACAAGGGCATTGGCTCTGACCTTCTCAGGATGCACTTCCATGACTGTTTCGTGAGGGGTTGCGATGGCTCACTTCTCATTGACTCGACCAAGGACAATGCCGCTGAGAAGGATGGGAAACCCAACGAAACCGTTGAAGATGAAGGGTTTGAAGTCATCGACAAGGTCAAGGAGAGGTTGGAGGCTGAATGCAAAGGAACGGTCTCATGCGCAGACATTCTTGCATTCCTCGCCCGAGACAGTGTTGCACATTACGGAGGAGTTCACTACCCAGTCCCTGCCGGCAGAAGAGATGGAAGGATCTCCAGATCAAATGACACGATCGATCTCCCTCCTCCAACATTTAAACTTGGTAATCTCACGAAGTTGTTCGTCTCGAAAGGGTTGAGCCGTGACGACATGGTCGCTCTCTCAGGAGCACACACCATCGGCATCGCGCACTGTTCTGCCTTCTCCGACAGGCTCTACAACTTCAGCCAGACGGTGAAGGCCGACCCGAGCTTGGACCCGAACTATGCTTCCCAACTGAGGGGCGAGTGCCCAACTGGGAGCGATAACGAGGTGGACATGGATCCGCCAAGCCCCCTCACGTTCGACAGCAGCTACTACAAGAACCTCCTAGCGCACCGCGGACTCTTCACATCGGACCAGACCCTCATGTCCAAGCACGGTACCGCGACGTTGGTGAAGCGGTTTGCCAAAAAGCCTGCGCTCTTCAAGAAGAAGTTTGCAGTTGCCATGGTGAAGATGGGGAGCATCGGCGTCCTCACCGGTGAACAAGGCGAGATACGTACCAACTGCAGAGTAGTCAACTGA), which encodes 324 amino acid residues (SEQ IDNO:2, MALGRGEMSLAIIVVLCLSAMGTEAYLKVGFYSYSCPKAEEIVKEELDKALQEDKGIGSDLLRMHFHDCFVRGCDGSLLIDSTKDNAAEKDGKPNETVEDEGFEVIDKVKERLEAECKGTVSCADILAFLARDSVAHYGGVHYPVPAGRRDGRISRSNDT IDLPPPTFKLGNLTKLFVSKGLSRDDMVALSGAHTIGIAHCSAFSDRLYNFSQTVKADPSLDPNYASQLRGECPTGSDNEVDMDPPSPLTFDSSYYKNLLAHRGLFTSDQTLMSKHGTATLVKRFAKKPALFKKKFAVAMVKMGSIGVLTGEQGEIRTNCRVVN). The specific steps are as follows: 1. Take the tuber tissue of Cavendish banana at the five-leaf stage, grind it into powder with liquid nitrogen, and extract total RNA using a plant total RNA extraction kit.

[0024] 2. The extracted total RNA was reverse transcribed into first-strand cDNA using a reverse transcription kit.

[0025] 3. Using primers P1 and P2 as described above, the coding region sequence of the MaPRX5 gene was amplified using the synthesized cDNA as a template. The amplification conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ extension for 5 min.

[0026] 4. The PCR products obtained by amplification were detected by agarose gel electrophoresis, the target band was recovered, ligated into the cloning vector and transformed into competent E. coli cells, and positive clones were screened and sequenced for verification.

[0027] 5. The correctly sequenced MaPRX5 coding region fragment was ligated into the plant expression vector pCAMBIA1300-GFP to construct the recombinant expression vector pCAMBIA1300-MaPRX5-GFP for subsequent transient transformation function verification.

[0028] Example 2: Obtaining and validating MaPRX5 transient overexpression materials To verify MaPRX5 The function of the gene in banana resistance to Fusarium wilt was investigated by transient overexpression in banana bulb tissue using Agrobacterium-mediated transformation. MaPRX5 And detected by RT-qPCR MaPRX5The relative expression levels were determined to confirm the effectiveness of transient overexpression treatment, laying the foundation for subsequent validation of disease resistance. The specific steps are as follows: 1. Transform the pCAMBIA1300-MaPRX5-GFP recombinant plasmid constructed in Example 1 into Agrobacterium GV3101 competent cells, pick a single colony and inoculate it into liquid LB medium containing the corresponding antibiotic, and culture at 28°C with shaking for 18-24 h.

[0029] 2. Inoculate 1 mL of bacterial culture into 50 mL of liquid LB medium and continue culturing until the OD600 value of the bacterial culture reaches 0.6-0.8. Centrifuge at room temperature to collect the bacterial cells, discard the supernatant, and resuspend the bacterial cells in resuspension buffer. The preferred resuspension buffer is 10 mmol / L MES + 10 mmol / L MgCl2 + 200 μmol / L AS, pH 5.8, and incubate at room temperature for 2 h before use.

[0030] 3. Select Cavendish banana seedlings with uniform growth at the five-leaf stage. Inject the resuspended bacterial solution into the corm using a syringe, followed by a 30-minute vacuum permeation treatment; use an empty carrier bacterial solution as a control. After transformation and cultivation for 48 hours, use a green fluorescent label... Foc TR4-GFP strain was inoculated at a concentration of 1.0 × 10⁻⁶. 6 cfu / g soil.

[0031] 4. Twenty-one days after inoculation, bulb tissue was collected, total RNA was extracted, and reverse transcribed into cDNA. [The following text appears to be a separate, unrelated sentence:] ...using... MaPRX5 RT-qPCR was performed using specific primers (Table 1), and the expression levels were normalized using the internal reference gene Actin. The relative expression levels were calculated using... The method was calculated. One-way ANOVA combined with Tukey's test was used for significance analysis, with P < 0.05 as the criterion for statistical significance.

[0032] Table 1. Primer information for quantitative fluorescence detection

[0033] RT-qPCR test results are as follows Figure 1 As shown, the basal expression level of MaPRX5 was low in the control group without Foc TR4 infection. Foc Following TR4 infection, the expression level of MaPRX5 significantly increased, indicating that MaPRX5 can respond to... Foc TR4 infection. FocUnder TR4 infection conditions, the relative expression level of MaPRX5 in the transient overexpression group was significantly increased, approximately 2.0 times that of the uninfected control group, and the differences between treatments were significant (P < 0.05, indicated by different lowercase letters). These results demonstrate that transient overexpression of MaPRX5 is effective, and that the expression level of MaPRX5 is related to... Foc A positive correlation was found between TR4 infection and MaPRX5, suggesting that MaPRX5 plays a positive regulatory role in the banana response to Fusarium wilt infection.

[0034] Example 3: Transient overexpression of MaPRX5 enhances the resistance of banana bulbs to... Foc TR4 resistance To verify MaPRX5 The impact on resistance to banana wilt disease, after confirmation MaPRX5 After transient overexpression proved effective, each treatment group was vaccinated. Foc TR4 was used, and its disease resistance was evaluated through phenotypic observation of bulb sections and colonization detection of fluorescently labeled pathogens. The specific steps are as follows: 1. The pCAMBIA1300-MaPRX5 recombinant plasmid constructed in Example 1 was transformed into Agrobacterium GV3101 competent cells. Single colonies were picked and inoculated into liquid LB medium containing the corresponding antibiotics and cultured at 28°C with shaking for 18-24 h.

[0035] 2. Expand the bacterial culture until the OD600 value reaches 0.6-0.8, and collect the bacterial cells by centrifugation. Resuspend the bacterial cells in a resuspension solution containing 10 mmol / L MES + 10 mmol / L MgCl2 + 200 μmol / L AS, adjust the pH to 5.8, and let stand at room temperature for 2 h for later use.

[0036] 3. Select Cavendish banana seedlings with uniform growth at the five-leaf stage. Inject or vacuum infiltrate the Agrobacterium suspension described above into the bulb tissue, using plants transformed with the empty vector as a control. After transformation, culture for 48 h, and detect the results using GFP fluorescence. MaPRX5 Disease status in the overexpression group.

[0037] 4. Continue culturing after inoculation. Observe the disease phenotype of the plants 21 days after inoculation, and take bulbs for laser confocal microscopy to observe the colonization and spread of pathogens in the bulbs.

[0038] Phenotypic observation results showed that the bulb tissue of the uninoculated control group had normal color and no obvious browning was observed; Foc The vascular tissue of the bulbs in the TR4-infected group showed obvious browning and necrosis. Figure 2 );exist Foc Under TR4 infection conditions, the browning degree in the MaPRX5 transient overexpression group was significantly reduced. Fluorescence observation results showed that... FocStrong and widespread pathogen fluorescence signals were observed in the TR4 infection group, while MaPRX5 The fluorescence signal in the overexpression group was significantly weakened ( Figure 2 The above results indicate that overexpression MaPRX5 Able to inhibit Foc TR4 colonizes and spreads in banana bulbs, reduces bulb tissue lesions, and thus improves banana resistance to Fusarium wilt.

[0039] Example 4: Elucidation of the molecular regulatory mechanism of MaPRX5 To clarify MaPRX5 In the banana response Foc The transcriptional regulatory mechanism during TR4 infection was investigated using yeast one-hybrid and dual-luciferase reporter systems to verify the relationship between candidate transcription factor MaWRKY41 and... MaPRX5 Promoter interactions. The specific steps are as follows: 1. Amplification based on banana genome sequence MaPRX5 A 1500bp promoter fragment upstream of the start codon was ligated into the yeast one-hybrid bait vector pAbAi, resulting in the vector pAbAi- MaPRX5 -1pro; Connect the MaWRKY41 encoding region to the pGADT7 carrier, resulting in the pGADT7-MaWRKY41 carrier. Complete. MaPRX5 After promoter self-activation detection, pGADT7-MaWRKY41 is converted to a form containing... MaPRX5 In yeast strains with promoters, empty vectors were used as negative controls.

[0040] 2. The effects of MaWRKY41 on the response of dual-luciferase reporter assays were verified using a dual-luciferase reporter assay system. MaPRX5 The promoter's transcriptional activation function. Using pGreenII 62-SK-MaWRKY41 as the effector vector, pGreenII 0800-LUC- MaPRX5 pro was used as a reporter vector to co-transform Agrobacterium and then injected into tobacco leaves. The group co-transformed with the empty effect vector and the reporter vector served as a control. Luciferase activity was measured 48 h after infection, and the promoter activation level was characterized by the LUC / REN ratio.

[0041] Yeast one-hybrid results showed that pGADT7- MaWRKY41 and pAbAi- MaPRX5 The co-transformed yeast with -1pro grew normally on selective medium, while the negative control did not grow, indicating that the MaWRKY41 protein can interact with... MaPRX5 Specific binding to the promoter ( Figure 3 (A in the middle).

[0042] Dual-luciferase assay results showed that, compared with the empty vector control, MaWRKY41 and MaPRX5 The LUC / REN ratio of the promoter co-transformation group was significantly increased, indicating that MaWRKY41 can not only bind to the MaPRX5 promoter, but also significantly activate the transcriptional activity of the MaPRX5 promoter. Figure 3 (B in the middle).

Claims

1. A method for improving the resistance of bananas to Fusarium wilt, characterized in that, The method includes overexpressing the MaPRX5 gene in bananas to improve their resistance to Fusarium wilt, the nucleotide sequence of which is shown in SEQ ID NO:1; the Fusarium wilt is caused by Fusarium oxysporum var. columbia tropical race 4.

2. The method according to claim 1, characterized in that, The amino acid sequence encoded by the MaPRX5 gene is shown in SEQ ID NO:

2.

3. The method according to claim 1, characterized in that, The overexpression of the MaPRX5 gene in bananas was achieved by introducing an expression vector into bananas that integrates the nucleic acid molecule shown in SEQ ID NO:

1.

4. The application of overexpressing the MaPRX5 gene, characterized in that, The application is any one of the following: A1) Application in improving banana resistance to Fusarium wilt; A2) Application in the preparation of bananas resistant to Fusarium wilt; The nucleotide sequence of the MaPRX5 gene is shown in SEQ ID NO:1; The wilt disease is caused by Fusarium oxysporum, specifically Tropical Race 4 of the Cuban Specialist Formation.

5. The application according to claim 4, characterized in that, The application involves introducing an expression vector that integrates the nucleic acid molecule shown in SEQ ID NO:1 into bananas, thereby improving the bananas' resistance to Fusarium wilt.

6. Application of biomaterials related to overexpression of the MaPRX5 gene, characterized in that, The application is any one of the following: B1) Application in improving banana resistance to Fusarium wilt; B2) Application in the preparation of bananas resistant to Fusarium wilt; The biomaterial is any one of the following C1) to C3): C1) An expression cassette containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO:1; C2) Recombinant vectors containing nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:1; C3) A recombinant microorganism containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO:1, or a recombinant microorganism containing the expression cassette described in C1), or a recombinant microorganism containing the recombinant vector described in C2), wherein the microorganism is Agrobacterium; The wilt disease is caused by Fusarium oxysporum, specifically Tropical Race 4 of the Cuban Specialist Formation.

7. The application according to claim 6, characterized in that, The application involves introducing an expression vector that integrates the nucleic acid molecule shown in SEQ ID NO:1 into bananas, thereby improving the bananas' resistance to Fusarium wilt.

8. A banana resistant to Fusarium wilt, characterized in that, The wilt-resistant banana was obtained by overexpressing the MaPRX5 gene in bananas, the nucleotide sequence of which is shown in SEQ ID NO:1; the wilt disease is caused by Fusarium oxysporum var. coli Tropical Race 4.

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