Banana maZAT12 gene and application thereof in improving resistance of banana to fusarium wilt

Abstract

The application discloses a banana MaZAT12 gene and application thereof in improving resistance of bananas to fusarium wilt, and belongs to the technical field of genetic engineering. Foc The application proves that overexpression of the MaZAT12 gene in bananas can significantly inhibit colonization and expansion of TR4 in corms, and effectively improve the resistance of bananas to fusarium wilt. Molecular mechanism research shows that MaZAT12, as an upstream transcriptional regulatory factor, can specifically bind to a MaPR1-1 promoter related to defense, and significantly activate the transcriptional activity of the promoter, thereby positively regulating the expression of MaPR1-1 and enhancing the immune response of bananas to the fusarium wilt pathogen. The MaZAT12 gene provided by the application can be used as an important gene resource for molecular breeding of bananas against diseases, 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 genetic engineering technology, specifically relating to the banana MaZAT12 gene and its application in improving banana resistance to Fusarium wilt. Background Technology

[0002] Bananas are an important tropical and subtropical economic crop worldwide, produced by Fusarium oxysporum, specifically Tropical Race 4 of the Cuban Specialist Formation (… Fusarium oxysporum f. sp. cubense tropical race 4, Foc Fusarium wilt caused by TR4 is one of the most serious soil-borne diseases currently threatening the banana industry. This pathogen can invade the plant through the roots and further spread in the vascular tissue of the corm and pseudostem, leading to damage to the plant's vascular system, yellowing and wilting of leaves, and even death of the entire plant. This is especially true for major banana varieties such as Cavendish. Foc TR4 is generally quite sensitive, which seriously restricts the stable development of the banana industry.

[0003] Although banana wilt disease has significant agricultural damage and economic impact, current treatments... Foc The control of TR4 still lacks safe, stable, and efficient chemical methods, and production mainly relies on quarantine, cultivation management, and the breeding of disease-resistant varieties. However, bananas are polyploid crops with complex genetic backgrounds, and they mostly use asexual reproduction. Traditional breeding methods are time-consuming and inefficient, making it difficult to quickly meet the demand for creating disease-resistant varieties. Therefore, identifying key genes with clear disease resistance functions at the molecular level and applying them to the genetic improvement of banana disease resistance has become an important technical approach to solving the damage caused by banana wilt disease.

[0004] Existing research indicates that plant resistance to pathogen infection involves multiple biological processes, including pathogen recognition, reactive oxygen species bursts, plant hormone signal transduction, and regulation of pathogenesis-related protein expression. Among these, ZAT12-type proteins, belonging to the C2H2-type zinc finger transcription factor family, typically contain zinc ion-binding domains and play crucial regulatory roles in plant responses to abiotic and biotic stresses. PR1-type pathogenesis-related protein genes are classic marker genes in plant immune responses and play a vital role in pathogen infection defense. However, current research on the origin of bananas... MaZAT12 Research on the function of genes in resistance to Fusarium wilt is still relatively limited, especially regarding their role as upstream transcriptional regulators in activating downstream defense genes and enhancing banana resistance through overexpression. Foc Systematic studies on TR4 resistance have not yet been found in the current technology. MaZAT12 Genes with clearly defined disease-resistant functions are used in technical solutions to improve plant resistance to Fusarium wilt. Summary of the Invention

[0005] The purpose of this invention is to provide the banana MaZAT12 gene and its application in improving banana resistance to Fusarium wilt. The MaZAT12 gene was cloned from banana, identified as encoding a C2H2 type zinc finger transcription factor, and its function and molecular mechanism in banana resistance to Fusarium wilt were revealed for the first time. Experiments showed that overexpression of MaZAT12 significantly activated the expression of the defense-related gene MaPR1-1 and effectively improved banana resistance to Fusarium wilt. Foc TR4 resistance. The technical solution of this invention fills the gap in the research on key regulatory genes for banana resistance to Fusarium wilt, and provides new genetic resources and technical support for molecular breeding of bananas for disease resistance.

[0006] 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 MaZAT12 gene in bananas to improve the resistance of bananas to Fusarium wilt, the nucleotide sequence of the MaZAT12 gene being shown in SEQ ID NO:2.

[0007] Furthermore, the wilt disease is caused by Fusarium oxysporum, specifically race 4 of the Cuban variant.

[0008] Furthermore, the amino acid sequence encoded by the MaZAT12 gene is shown in SEQ ID NO:1.

[0009] Furthermore, the overexpression of the MaZAT12 gene in bananas is achieved by introducing an expression vector integrating the nucleic acid molecule shown in SEQ ID NO:2 into the banana.

[0010] Secondly, this invention provides applications of overexpressing the MaZAT12 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 MaZAT12 gene is shown in SEQ ID NO:2.

[0011] Furthermore, the application involves introducing an expression vector integrating the nucleic acid molecule shown in SEQ ID NO:2 into bananas, thereby improving the bananas' resistance to Fusarium wilt.

[0012] Thirdly, the present invention provides applications of biomaterials related to the overexpression of the MaZAT12 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:2; C2) Recombinant vectors containing nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:2; C3) A recombinant microorganism containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO:2, 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.

[0013] Furthermore, the application involves introducing an expression vector integrating the nucleic acid molecule shown in SEQ ID NO:2 into bananas, thereby improving the bananas' resistance to Fusarium wilt.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention cloned the MaZAT12 gene from banana and revealed that it encodes a C2H2 type zinc finger transcription factor with a zinc ion binding domain, which can be used as a functional gene for banana resistance to Fusarium wilt.

[0015] (2) This invention demonstrates through Agrobacterium-mediated transient transformation experiments that overexpression of MaZAT12 can significantly inhibit Foc The colonization of TR4 in banana bulbs significantly delayed the onset of disease symptoms and reduced the rate of pathogen infection and spread, indicating that MaZAT12 has a clear functional value in improving the resistance of bananas to Fusarium wilt.

[0016] (3) Through multi-level experiments such as RT-qPCR, yeast one-hybrid and dual luciferase reporter system, this invention has confirmed that MaZAT12, as an upstream transcriptional regulator, can bind to the promoter of MaPR1-1 gene and significantly activate its transcriptional activity, thereby revealing the molecular mechanism by which MaZAT12 achieves disease resistance regulation by activating the expression of defense genes.

[0017] (4) The MaZAT12 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

[0018] Figure 1 for MaZAT12 Transient overexpression enhances the effect of banana bulbs on Foc Resistance to TR4 infection. CK was the blank control. Foc TR4 represents inoculation with Fusarium wilt pathogen in bananas, and OE-MaZAT12 represents transient overexpression. MaZAT12 Post-vaccinationFoc TR4, OE-Empty represents the empty vector control after inoculation. Foc TR4. The white arrow indicates the GFP marker. Foc TR4 colonization signal. Compared with the empty vector control group, the OE-MaZAT12 group showed reduced browning of bulbs and weakened green fluorescence signal, indicating overexpression. MaZAT12 Able to inhibit Foc TR4 colonizes banana bulbs and enhances banana resistance to Fusarium wilt.

[0019] Figure 2 for MaZAT12 and MaPR1-1 Relative expression level analysis. RT-qPCR results showed that, compared with the CK group, Foc TR4 processing improved MaZAT12 and MaPR1-1 level of expression Foc TR4 expression was further increased in the transient transformation group of MaZAT12. Different lowercase letters indicate significant differences (P < 0.05).

[0020] Figure 3 for MaZAT12 and MaPR1-1 Promoter interaction and transcriptional activation analysis. (A) shows the results of a yeast one-hybrid assay. MaZAT12 Can be with MaPR1-1 (B) shows the promoter binding; (B) is the result of dual-luciferase assay. MaZAT12 It can significantly improve MaPR1-1 Transcriptional activity of the promoter. * indicates significant difference. Detailed Implementation

[0021] 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.

[0022] Example 1: Banana MaZAT12 Cloning of genes Bioinformatics analysis of banana single-cell transcriptome data and a reference genome was used to screen for key responses to banana wilt disease. MaZAT12 Gene. Specific primers P1 (5′-ATGAAGAGTCCAAGAGCCGACGAAG-3′, SEQ ID NO:3) and P2 (5′-CTAACTACGGTTTAAATGAACGTTG-3′, SEQ ID NO:4) were designed based on the open reading frame sequence of this gene. Using banana bulb first-strand cDNA as a template, [the primers were applied to the gene]. MaZAT12 The coding region sequence of the gene was amplified by PCR to obtain... MaZAT12The full-length coding sequence of the gene is 498 bp (SEQ ID NO: 2, ATGAAGAGTCCAAGAGCCGACGAAGACGATGCAGAGCTGCTGCTGTCGCTCTCCTGCGGGGCGACGACGGCGAAGACCCACAGTGAGCCGAGGGCGGCGGTCGGTGTCTTCGAGTGCAAGACGTGC AGTCGCCAATTCCCGACGTTCCAAGCGTCGGCGGCCACCGGACGAGCCACAAGCGCCCGAGAACCGATCGTTCCGTCTCCAAACGTAGCGTGCATCGGTGCTCCATCTGTGGCATGAATTTCGCGATGGG GCAAGCTTTGGGCGGCCACATGCGCCGCCATAAGCCCACGGCCGAGGAGTACTCGAAACAGACTCATCAGAAGAAGGAATCGAGCATGGCTGGTCTACAGATTGACGTCTCCGATGATGATCGGATTGTAGCCCATACGAAGGGATTCGAGGGATCTTCTTCCGGAAACGGTTTCCAAGTAACTGCGGTTAAGATGACACACCTCGTCCCCGCGATCAACGTTCATTTAAACCGTAGTTAG), derived from the open reading frame sequence, MaZAT12. The amino acid sequence, consisting of 165 amino acid residues (SEQ ID NO:1, MKSPRADEDDAELLLSLSCGATTAKTHSEPRAAVGVFECKTCSRQFPTFQALGGHRTSHKRPRTDRSVSKRSVHRCSICGMNFAMGQALGGHMRRHKPTAEEYSKQTHQKKESSMAGLQIDVSDDDRIVAHTKGFEGSSSGNGFQVTAVKMTHLVPAINVHLNRS). 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.

[0023] 2. The extracted total RNA was reverse transcribed into first-strand cDNA using a reverse transcription kit.

[0024] 3. Using primers P1 and P2 as described above, the coding region sequence of the MaZAT12 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.

[0025] 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.

[0026] 5. Sequencing the correct sequence MaZAT12 The coding region fragment was ligated into the plant expression vector pCAMBIA1300-GFP to construct the recombinant expression vector pCAMBIA1300-MaZAT12-GFP, which was used for subsequent transient transformation function verification.

[0027] 6. Sequencing results show that the cloned... MaZAT12 The gene encodes a C2H2 type zinc finger transcription factor with a typical zinc ion binding domain, which can be used as a functional gene related to banana resistance to Fusarium wilt for subsequent analysis.

[0028] Example 2: MaZAT12 Transient overexpression and disease resistance verification in bananas To verify MaZAT12 The function of the gene in banana resistance to Fusarium wilt was investigated using Agrobacterium-mediated transformation. MaZAT12 Transiently converted to banana bulb tissue for overexpression, followed by inoculation Foc TR4 was used to verify the colonization of pathogens through phenotypic observation and laser confocal microscopy. MaZAT12 Its role in improving the disease resistance of bananas. The specific steps are as follows: 1. Transform the pCAMBIA1300-MaZAT12-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. 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.

[0032] The results show that MaZAT12 The overexpression vector showed a clear GFP fluorescence signal in both banana root and bulb tissues, indicating successful transient transformation. Figure 1 Further phenotypic observations and confocal analysis showed that, compared to the empty vector control, MaZAT12 Overexpressing plants after inoculation Foc After 21 days, the green fluorescence signal in the bulbs of TR4 was significantly weakened, the rate of pathogen infection and spread was reduced, and the onset of symptoms was significantly delayed, indicating that overexpression of TR4 was a significant indicator. MaZAT12 Effectively inhibit Foc TR4 colonization and spread in banana corms significantly improves banana resistance to Fusarium wilt. Figure 1 ).

[0033] Example 3: RT-qPCR Validation MaZAT12 Regulatory role of defense-related gene expression To further verify MaZAT12 To enhance the molecular mechanism of banana resistance to Fusarium wilt, extract... MaZAT12 Transient overexpression plants and empty vector control plants Foc Total RNA was collected from the bulb tissue after TR4 inoculation, and changes in the expression of defense-related genes were analyzed using RT-qPCR. The specific steps are as follows: 1. at Foc 21 days after TR4 inoculation, samples were collected. MaZAT12 Total RNA was extracted from banana bulb tissues in the overexpression group and the empty vector control group, and then reverse transcribed into cDNA.

[0034] 2. Design MaZAT12 And the quantitative fluorescent primers for candidate defense-related genes (Table 1), the candidate genes including MaPR1-1 Standardization was performed using the internal reference gene Actin.

[0035] Table 1. Primer information for quantitative fluorescence detection

[0036] 3. RT-qPCR was performed using the SYBR Green real-time fluorescence kit. Relative expression levels were measured using... The method was used to calculate the variance, and one-way ANOVA combined with Tukey's test was used for significance analysis. P < 0.05 was used as the criterion for significance.

[0037] RT-qPCR results showed that MaZAT12 In the overexpression group MaZAT12 The transcription level of the gene was significantly higher than that of the empty vector control group, indicating that the overexpression treatment was successful. Meanwhile, the transcription level of disease-related protein genes... MaPR1-1 The expression level in MaZAT12 Significantly increased in the overexpression group ( Figure 2 ).

[0038] Example 4: Yeast one-hybrid and dual-luciferase verification MaZAT12 right MaPR1-1 Regulatory role of promoters To further verify MaZAT12 To investigate the regulatory relationships of downstream defense-related genes, this embodiment employs a yeast one-hybrid and dual-luciferase reporter system. MaZAT12 and MaPR1-1 The binding of the promoter and its transcriptional activation function were verified. The specific steps are as follows: 1. Amplification based on banana genome sequence MaPR1-1 The promoter-1500bp fragment was ligated into the yeast one-hybrid vector pAbAi and the dual-luciferase reporter vector pGreenII 0800-LUC, respectively, to construct the recombinant vectors pAbAi-MaPR1-1pro and pGreenII 0800-LUC-MaPR1-1pro. Simultaneously, the... MaZAT12 The coding region sequence of the gene was ligated into the yeast expression vector pGADT7 and the effector vector pGreenII 62-SK to construct the recombinant vectors pGADT7-MaZAT12 and pGreenII 62-SK-MaZAT12, respectively.

[0039] 2. Verification using the yeast one-hybrid method MaZAT12 and MaPR1-1 Promoter binding relationship. First, pAbAi-MaPR1-1pro was transformed into Y1HGold yeast competent cells, and bait strains were screened. Then, pGADT7-MaZAT12 was transformed into the above bait strains, with empty vector pGADT7 as a negative control. Yeast growth was observed by culturing on a deficiency medium containing an appropriate concentration of AbA.

[0040] 3. Validation using a dual-luciferase reporter system. MaZAT12 right MaPR1-1 Transcriptional activation of the promoter was investigated. pGreenII 62-SK-MaZAT12 was used as the effector vector, and pGreenII 0800-LUC-MaPR1-1pro was used as the reporter vector. These vectors were co-transformed into Agrobacterium and then injected into Tobacco Benzoenta leaves. A control group was formed by co-transformation with both empty effector and reporter vectors. Luciferase activity was measured 48 h after infection, and the LUC / REN ratio was used to characterize the promoter activation level.

[0041] Analysis of cis-acting elements shows that, MaPR1-1 The promoter contains defense response-related components such as TGACG and TGACGGT.

[0042] Yeast one-hybrid results showed that co-transformed yeast containing pGADT7-MaZAT12 and pAbAi-MaPR1-1pro could grow normally on selective medium, while the negative control could not grow, indicating that... MaZAT12 Proteins can interact with MaPR1-1 Specific binding to the promoter ( Figure 3 (A in the middle).

[0043] Dual-luciferase assay results showed that, compared with the empty vector control, MaZAT12 and MaPR1-1 The significantly increased LUC / REN ratio in the promoter cotransformation group indicates that... MaZAT12 Able to activate MaPR1-1 promoter transcriptional activity ( Figure 3 (B in the middle).

[0044] The above results indicate that MaZAT12 Not only can it improve the banana's effect on Foc TR4 induces resistance to Fusarium wilt and can directly bind to and activate upstream transcriptional regulators. MaPR1-1 The promoter positively regulates the expression of defense-related genes, thereby enhancing the immune response of bananas to Fusarium wilt pathogens.

Claims

1. A method for improving the resistance of bananas to Fusarium wilt, characterized in that, The method includes overexpressing the MaZAT12 gene in bananas to improve the bananas' resistance to Fusarium wilt, the nucleotide sequence of which is shown in SEQ ID NO:2; the Fusarium wilt is caused by Fusarium oxysporum var. copus tropical race 4.

2. The method according to claim 1, characterized in that, The amino acid sequence encoded by the MaZAT12 gene is shown in SEQ ID NO:

1.

3. The method according to claim 1, characterized in that, The overexpression of the MaZAT12 gene in bananas was achieved by introducing an expression vector into bananas that integrates the nucleic acid molecule shown in SEQ ID NO:

2.

4. The application of overexpression of the MaZAT12 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 MaZAT12 gene is shown in SEQ ID NO:2; 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:2 into bananas, thereby improving the bananas' resistance to Fusarium wilt.

6. Application of biomaterials related to overexpression of the MaZAT12 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:2; C2) Recombinant vectors containing nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:2; C3) A recombinant microorganism containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO:2, 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:2 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 MaZAT12 gene in bananas; the nucleotide sequence of the MaZAT12 gene is shown in SEQ ID NO:2; the wilt disease is caused by Fusarium oxysporum, specifically Tropical Race 4.

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