Banana disease-resistant new gene and use thereof

Abstract

Banana disease-resistant new gene and use thereof, the present application provides a new gene affecting the resistance of banana fruit to pathogenic fungi and use thereof, and belongs to the technical field of genetic engineering. The coding region of the gene encodes an amino acid sequence as shown in SEQ ID NO. 2. The expression of the banana disease-resistant gene increases in the process of banana fruit responding to fusarium infection, indicating that the disease-resistant gene plays an important role in the process of banana responding to pathogenic bacteria infection. The present application causes the resistance of banana to fusarium proliferatum to be significantly enhanced through overexpression of the disease-resistant gene. Therefore, the expression of the disease-resistant gene and the encoded protein can be used as an important candidate target site for designing and screening new antifungal preservatives, and applied in the field of prevention and control of postharvest fungal diseases of fruits.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a novel disease-resistant gene for bananas and its application in defending against Fusarium moniliforme infection. Background Technology

[0002] Bananas (Musa acuminata) are a major global fruit, ranking first in fresh fruit trade. However, bananas are highly susceptible to diseases during storage and transportation, causing significant economic losses. Crown rot is one of the most important post-harvest diseases of bananas, leading to a significant decline in post-harvest nutrition and quality, posing considerable difficulties for post-harvest storage and transportation of bananas in my country, and gradually becoming one of the major post-harvest diseases. Fusarium proliferatum is one of the main pathogens causing post-harvest crown rot in bananas and can produce fumonisin. Therefore, Fusarium proliferatum infection severely reduces the post-harvest storage quality of bananas and affects human dietary health. Therefore, identifying disease-resistant genes in banana fruits and controlling pathogen infection through the regulation of these genes is of great significance for mitigating the further spread of post-harvest crown rot, maintaining banana quality, and extending storage life. Summary of the Invention

[0003] Based on the problems existing in the background art, the purpose of this invention is to provide a banana disease resistance-related gene and its uses.

[0004] On one hand, the present invention provides a novel gene that enhances the disease resistance of banana fruits. The protein-coding region of the disease resistance gene encodes the amino acid sequence shown in SEQ ID NO.2.

[0005] In a preferred embodiment, the protein coding region of the disease-resistant gene of the present invention is a nucleotide sequence as shown in SEQ ID NO.1, or a sequence that is completely complementary to the nucleotide sequence shown in SEQ ID NO.1.

[0006] On the other hand, the present invention provides the application of the aforementioned disease-resistant gene or its encoded protein or biological material containing the aforementioned disease-resistant gene in improving the disease resistance of bananas.

[0007] On the other hand, the present invention provides the application of the aforementioned disease-resistant gene or its encoded protein or biological material containing the aforementioned disease-resistant gene in the breeding of disease-resistant banana varieties.

[0008] In a preferred embodiment, the biological material containing the aforementioned disease-resistant gene includes an expression cassette containing the disease-resistant gene, a recombinant vector, or a recombinant microorganism.

[0009] In the preferred embodiment, the disease resistance refers to resistance to crown rot.

[0010] In a further preferred embodiment, the disease resistance refers to resistance to crown rot caused by Fusarium moniliforme.

[0011] In a preferred embodiment, the improvement of banana disease resistance is achieved by increasing the expression level of the aforementioned disease resistance gene or its encoded protein, and the cultivation of disease-resistant banana varieties is achieved by increasing the expression level of the aforementioned disease resistance gene or its encoded protein to cultivate banana varieties with improved disease resistance.

[0012] In a preferred embodiment, the increase in the expression level of the disease resistance gene or its encoded protein is achieved by constructing an overexpression vector of the aforementioned disease resistance gene and transforming bananas using Agrobacterium-mediated genetic transformation to obtain bananas with overexpression of the disease resistance gene.

[0013] In the aforementioned scheme, banana can refer to a growing banana plant, or it can refer to the detached or non-detached tissues or organs of a banana.

[0014] In the aforementioned scheme, the aforementioned disease-resistant gene plays a role as a regulated / induced target in inhibiting Fusarium effusum infection of bananas.

[0015] On the other hand, the present invention provides a method for improving the disease resistance of banana fruit, characterized by including the step of increasing the expression level of disease resistance genes and / or the protein expression level of disease resistance genes in banana peel to achieve the improvement of disease resistance, wherein the protein coding region of the disease resistance gene encodes an amino acid sequence as shown in SEQ ID NO.2.

[0016] In a preferred embodiment, the improvement of the expression level of the disease resistance gene and / or the protein expression level of the disease resistance gene in the banana peel is achieved by constructing an overexpression vector of the disease resistance gene and transforming bananas using Agrobacterium-mediated genetic transformation to obtain banana fruits with overexpression of the disease resistance gene.

[0017] In the preferred embodiment, the disease resistance refers to resistance to crown rot.

[0018] In a further preferred embodiment, the disease resistance refers to resistance to crown rot caused by Fusarium moniliforme.

[0019] On the other hand, the present invention provides a method for reducing the pathogenicity of Fusarium moniliforme to banana fruit, characterized in that the infection of banana fruit by Fusarium moniliforme is reduced by increasing the mRNA expression level or protein expression level of disease resistance genes in banana peel.

[0020] On the other hand, the present invention provides the application of disease-resistant genes in screening antifungal preservatives for bananas. The application includes the following steps: preserving bananas with a test preservative, and analyzing the expression of disease-resistant genes in the treated bananas. The invention is characterized by using the expression level of the disease-resistant gene in bananas as a screening index for antifungal preservatives, and judging the preservation effect of the test preservative based on its promotion of the expression of the disease-resistant gene. The disease-resistant gene encodes an amino acid sequence as shown in SEQ ID NO.2.

[0021] In a preferred embodiment, the antifungal preservative can promote the expression of the disease-resistant genes in the banana peel.

[0022] In another preferred embodiment, the antifungal preservative can enhance the activity of the disease-resistant protein in bananas, as shown in SEQ ID NO.2.

[0023] Compared with the prior art, the present invention has the following advantages: 1) This invention is the first to discover the new disease-resistant gene in bananas and its use in controlling banana diseases.

[0024] 2) This invention provides a novel method for reducing the pathogenicity of Fusarium moniliforme to banana fruit, namely, by increasing the mRNA expression level or protein expression level of disease resistance genes in banana peel to enhance the disease resistance of bananas. Attached Figure Description

[0025] The following detailed description, in conjunction with the accompanying drawings and specific embodiments, illustrates the banana disease resistance-related genes of the present invention, their uses, and their beneficial effects.

[0026] Figure 1 The graph shows the expression levels of the disease resistance gene in the peel of bananas in the control group and those with overexpression (OE). Different letters on the bars represent highly significant differences (p<0.01).

[0027] Figure 2 The results show the performance of banana peels overexpressing disease resistance genes and wild-type bananas after inoculation with pathogens. Figure A shows the growth phenotype, and Figure B shows the statistics of plaque diameter. Different letters on the bars in the figures represent highly significant differences (p<0.01). Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0030] The nucleotide sequence of the banana disease resistance gene in the example is shown in SEQ ID NO.1: .

[0031] The amino acid sequence of the banana disease-resistant protein is shown in SEQ ID NO.2: MDAHRVAEAPLPTAERRCSDEPRNKCETDAGAQASASACFDCNICLDFAVEPVVTLCGHLYCWPCIYRWLQQADGAAPQQCPVCKAALHHDALVPLYGGGRHGAKKPRPDLELPRRPP PAHPPSPGANEEQRYPETRPHSRRHHQHPNHGWEYTSGATRVIHSTAGGLGLVMAVLPWVFRNQEWPGIYYSSPYHMGGDVGSGRLRRQEMELKRSLHQIWLFLVCCAVVCLLLF.

[0032] Example 1: Transient overexpression of disease resistance gene in banana peel 1. Construction of overexpression vectors The banana disease resistance gene shown in SEQ ID NO.1 was cloned into the pUBQ10 vector using the forward primer: 5'-TATGGCGCGCCCATGGACGCCCACCGGGTAGC-3' and the reverse primer: 5'-GCGACTAGTGAATAAGAGCAGGCACACAAC-3'.

[0033] 2. Transformation of Agrobacterium 1) Select positive clones of the disease resistance gene and transform them into Agrobacterium. Incubate on LB solid medium containing spectinomycin (working concentration 100 mg / L) and rifampin (working concentration 25 mg / L) at 28°C for 2-3 days. Select single clones and transfer them to 1 ml of liquid LB medium (containing 100 mg / L spectinomycin + 25 mg / L rifampin), and shake for one day. Transfer 500 μl of the above bacterial culture to 100 ml of LB medium (containing 100 mg / L spectinomycin and 25 mg / L rifampin), and shake at 28°C and 200 rpm for approximately 20 hours until the bacterial culture turns orange-yellow.

[0034] 2) Pour the above bacterial culture into a 50 ml centrifuge tube, centrifuge at 5000g at room temperature for 5 min to collect the bacterial cells, discard the supernatant, add 20 ml of resuspension buffer to resuspend the precipitate, then transfer it to a tissue culture flask and add resuspension buffer to 100 ml. Adjust the OD600 of the bacterial culture to 0.8~1.0 and incubate in the dark for 3 h.

[0035] 3) Select healthy bananas (Musa acuminata AAA group, cv. Cavendish) without mechanical damage, wash them thoroughly with clean water, then soak them in 0.5% sodium hypochlorite solution for 3 minutes for disinfection, and air dry them. Cut the bananas horizontally into several sections, remove the peels and cut them longitudinally into small squares. Place them in the infiltration solution (i.e., the bacterial solution in step 2) until the infiltration solution just covers the peels. Place them in a vacuum dryer and permeate under -80 kPa negative pressure for 15 minutes. Remove the peels, discard the bacterial solution, rinse with sterile water, and take peel samples to verify whether transient overexpression is successful.

[0036] 3. Analysis of disease resistance gene expression Banana peels from both the control group and the bananas transfected with the disease-resistant gene were extracted. RNA was extracted and cDNA was reverse transcribed. Forward primers: 5'-GAGTGACCGTTTCAGCTCCA-3' and reverse primers: 5'-AACGAGGAACAACGGTACCC-3' were designed, and gene expression was analyzed using quantitative real-time PCR. Gene expression analysis revealed that the expression level of the disease-resistant gene was upregulated in the transfected banana peels. Figure 1 This indicates that the gene was successfully overexpressed in banana peel.

[0037] Example 2: Effects of disease resistance gene overexpression on disease resistance in banana fruit Fusarium proliferatum was cultured on a PDA plate at 28°C for 7 days. The spores were washed with sterile water and filtered through gauze. The filtrate was collected, and the spore concentration was adjusted to 1×10⁻⁶ using a spore counter. 6 1 spore / ml. Wild-type and whole bananas that successfully overexpressed the disease resistance gene using the same method as in Example 1 (the whole banana peel fragment was replaced in Example 1 for infection, and successful overexpression was confirmed by quantitative fluorescence) were taken, washed thoroughly with sterile water, and then air-dried. Fusarium moniliformes was inoculated using the needle-punching method: 10 μl of the above spore solution was dropped onto the needle-punched area of ​​the banana fruit. All banana fruits were stored in an incubator at 25°C and 85% relative humidity, and the disease development of the bananas was observed.

[0038] Three days after inoculation, it was first observed by the naked eye that Fusarium moniliforme grew slowly on the banana peel overexpressing the disease resistance gene. Figure 2 A), and the diameter of fungal plaques on the peel of bananas overexpressing the disease resistance gene is significantly smaller than that of wild-type bananas. Figure 2 B). This indicates that the disease-resistant gene is an important disease-resistant factor in banana peel. Increasing the expression level of this disease-resistant gene in bananas can improve the banana's ability to resist diseases caused by Fusarium moniliforme. This gene can be used in disease-resistant breeding of bananas.

[0039] Example 3: Screening method for antifungal preservatives for bananas 1. Banana fruit Select healthy bananas that are harvested on a sunny day, are 80% ripe, and are free from mechanical damage and pests.

[0040] 2. Treatment with antifungal preservatives The bananas from step 1 were randomly divided into two groups. The first group was the treatment group, in which the bananas were treated with the tested preservative. The second group served as the control group.

[0041] 3. Analysis of disease resistance gene expression Banana peels were collected after processing, and RNA and cDNA were extracted and synthesized. Forward primer: 5'-GAGTGACCGTTTCAGCTCCA-3' and reverse primer: 5'-AACGAGGAACAACGGTACCC-3' were designed. The expression of the aforementioned disease resistance gene (nucleotide sequence as shown in SEQ ID NO.1) was analyzed by real-time PCR.

[0042] By analyzing gene expression, the tested preservatives whose expression levels of the disease-resistant genes in the treatment group were higher than those in the control group were selected as candidate antifungal preservatives. This method can quickly screen antifungal preservatives and narrow down the candidate range of antifungal preservatives.

[0043] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of a disease-resistant gene or its encoded protein, or biological material containing a disease-resistant gene, in improving the disease resistance of bananas, characterized in that, The disease-resistant gene encodes the amino acid sequence shown in SEQ ID NO.2; the disease resistance is resistance to crown rot caused by Fusarium moniliforme; the improvement of banana disease resistance is achieved by increasing the expression level of the disease-resistant gene or its encoded protein in the banana peel.

2. The application of disease-resistant genes or their encoded proteins or biological materials containing disease-resistant genes in the breeding of disease-resistant banana varieties, characterized in that, The disease-resistant gene encodes the amino acid sequence shown in SEQ ID NO.2; the disease resistance is resistance to crown rot caused by Fusarium moniliforme; the cultivation of disease-resistant banana varieties is achieved by increasing the expression level of the disease-resistant gene or its encoded protein in the banana peel to cultivate banana varieties with improved disease resistance.

3. The application according to claim 1 or 2, characterized in that, The biological materials containing disease-resistant genes include expression cassettes, recombinant vectors, or recombinant microorganisms containing disease-resistant genes.

4. In the application according to claim 1 or 2, the increase in the expression level of the disease resistance gene or its encoded protein in banana peel is achieved by constructing an overexpression vector of the disease resistance gene and transforming the banana peel using Agrobacterium-mediated genetic transformation.

5. A method for improving the disease resistance of banana fruits, characterized in that, The method includes steps to enhance disease resistance by increasing the expression level of disease resistance genes and / or the protein expression level of disease resistance genes in banana peels, wherein the disease resistance gene encodes an amino acid sequence as shown in SEQ ID NO. 2, and the disease resistance is resistance to crown rot caused by Fusarium moniliforme.

6. The method according to claim 5, wherein the increase in the expression level of the disease resistance gene and / or the protein expression level of the disease resistance gene in the banana peel is achieved by constructing an overexpression vector of the disease resistance gene and transforming the banana peel using Agrobacterium-mediated genetic transformation.

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