Application of MaLSF1 gene in regulation and control of banana starch degradation and improvement of fruit quality

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a MaLSF1 gene in regulation and control of banana starch degradation and improvement of fruit quality. According to the banana fruit MaLSF1 gene provided by the invention, the content of total starch and amylopectin in banana fruits overexpressed with the MaLSF1 gene is remarkably reduced, and the change of the amylose content is not influenced. Expression of the MaLSF1 gene is inhibited in banana fruits and fruit slices, the content of total starch and amylopectin is remarkably increased, banana peel presents a stay green phenotype, and the storage period is obviously prolonged by about 2.5 days. The invention provides an important gene resource for improving the flavor quality and appearance quality of bananas, provides a theoretical basis for research and development of a banana fresh-keeping technology and cultivation of new storage-resistant varieties, and also provides a target gene for research and development of a fresh-keeping technology of fruits such as bananas and cultivation of new storage-resistant varieties of bananas.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the MaLSF1 gene in regulating banana starch degradation and improving fruit quality. Background Technology

[0002] The starch degradation metabolism of banana fruits directly affects their softness, sweetness, glutinous texture, nutritional quality, and storage life. Research on the molecular mechanisms of starch degradation metabolism is the theoretical basis for improving quality, developing preservation technologies, and cultivating new varieties with good storage resistance. Phosphoglucan phosphatases (PPs) are important enzymes in starch degradation. They can dephosphorylate glucans in starch, promoting the release of glucose and maltose by amylase (Dylan et al., 2014). PPs are a multi-enzyme complex, mainly consisting of three single enzymes: Starch Excess 4 (SEX4), Like SEX Four 1 (LSF1), and Like SEX Four 2 (LSF2) (Delatte et al., 2005). et al., 2005; Meekins et al., 2016; Gallois et al., 2025).

[0003] SEX4, LSF1, and LSF2 proteins have similar molecular structures, but they have different functions. SEX4 contains a dual-specific phosphatase (DSP) catalytic domain and a carbohydrate binding module (CBM). LSF1, in addition to the CBM and DSP domains, also contains a unique PDZ domain, while LSF2 contains only the DSP domain (Comparot-Moss et al., 2010; Santelia et al., 2011; Meekins et al., 2014; Ma et al., 2016; Carrillo et al., 2020). SEX4 is primarily responsible for the dephosphorylation of starch during starch degradation in Arabidopsis leaves, promoting its breakdown into glucose for energy production or further metabolic processes (Weise et al., 2012; Mak et al., 2021). Unlike SEX4, LSF2 lacks a carbohydrate-binding module; however, it can bind to starch and specifically hydrolyze the C3 phosphate group (Santelia et al., 2011; Wilkens et al., 2016; Salmodien et al., 2018; Huang et al., 2020). LSF1 may play a role in the nocturnal starch degradation process in Arabidopsis leaves (Liuet et al., 2023), but the function of LSF1 itself remains unclear. In particular, no reports have been found regarding LSF1 in fresh starch-converting fruits such as bananas, kiwifruit, and mangoes. Furthermore, it is unclear whether LSF1 possesses other novel functions. Therefore, research on the banana MaLSF1 gene has significant theoretical and practical value for improving fruit starch quality, breeding high-quality, long-storage banana varieties, and discovering its new functions. Summary of the Invention

[0004] This invention provides the application of the MaLSF1 gene in regulating banana starch degradation and improving fruit quality.

[0005] The technical solution of this invention is implemented as follows:

[0006] A banana fruit MaLSF1 gene that regulates the degradation of banana starch has the nucleotide sequence shown in SEQ ID NO:1.

[0007] The amino acid sequence of the protein encoded by the MaLSF1 gene in banana fruit described above is shown in SEQ ID NO:2.

[0008] Recombinant vectors, host bacteria, or expression cassettes containing the banana fruit MaLSF1 gene described above.

[0009] Furthermore, the recombinant vector comprises the original vector and the banana fruit MaLSF1 gene described above.

[0010] The original vector can be a vector commonly used in the field of gene recombination, such as a virus or plasmid. This invention does not limit its use. In one specific embodiment of this invention, the original vector is the pCAMBIA3300 vector plasmid or the pTRV2 vector plasmid; however, it should be understood that other plasmids or viruses can also be used.

[0011] Preferably, the original vector is the pCAMBIA3300 vector plasmid, and the nucleotide sequence shown in SEQ ID NO:1 is located between the Xba I and Kpn I restriction endonuclease sites of the pCAMBIA3300 vector plasmid.

[0012] Preferably, the original vector is a pTRV2 vector plasmid, and the nucleotide sequence shown in SEQ ID NO:1 is located between the two restriction endonuclease sites Xba I and Kpn I in the pTRV2 vector plasmid.

[0013] The above-mentioned application of the banana fruit MaLSF1 gene, or the protein, or the recombinant vector, host bacteria, or expression cassette in reducing the total starch content and / or amylose content of banana pulp.

[0014] The application of the banana fruit MaLSF1 gene, or the protein, or the recombinant vector, host bacteria, or expression cassette described above in any of the following:

[0015] (1) Application in reducing the total starch content of banana pulp;

[0016] (2) Reduce the application of amylose in banana fruit;

[0017] (3) Reduce the total starch content and / or amylose content of banana pulp without affecting the application of amylose content in banana fruit;

[0018] (4) Application in reducing the firmness of banana flesh.

[0019] Furthermore, the application overexpresses the banana fruit MaLSF1 gene.

[0020] 8. The use of the banana fruit MaLSF1 gene, or the protein, or the recombinant vector, host bacterium, or expression cassette described above, in any of the following:

[0021] (1) Application in increasing the total starch content and / or amylose content of banana fruits;

[0022] (2) Increase the total starch content and / or amylose content of banana fruit without affecting the application of amylose content in banana fruit;

[0023] (3) Applications that reduce the soluble sugar content, vitamin C content and / or soluble solids content of banana fruits;

[0024] (4) Application in increasing the organic acid content and / or sugar / acid ratio of banana fruit;

[0025] (5) Application in increasing the chlorophyll content of banana peel;

[0026] (6) Application in extending the storage period of banana fruits;

[0027] (7) Application in improving banana quality and / or in developing banana varieties that are resistant to storage.

[0028] Furthermore, in the aforementioned application, the expression of the MaLSF1 gene in banana fruit is suppressed.

[0029] A primer pair for amplifying the MaLSF1 gene in banana fruit, the nucleotide sequences of which are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0030] The beneficial effects of this invention are:

[0031] This invention provides a MaLSF1 gene for banana fruit. When introduced into banana fruit, compared to the control, bananas overexpressing the MaLSF1 gene exhibit accelerated starch degradation and significantly softer flesh; iodine-potassium iodide staining (an indicator of starch content) on fruit slices becomes lighter; MaLSF1 expression level increases; total starch and amylopectin content significantly decrease, and flesh firmness decreases, while having no effect on amylose content. Inhibiting MaLSF1 gene expression in banana fruit and its slices using a transient silencing vector (VIGS) results in decreased MaLSF1 expression, darker iodine-potassium iodide staining on banana fruit slices; a highly significant increase in total starch and amylopectin content, with no effect on amylose content; decreased soluble sugar, vitamin C, and soluble solids content, increased organic acid content and sugar / acid ratio, and increased total chlorophyll content in the peel, leading to a sluggish green phenotype in the banana peel and significantly extending the storage period by approximately 2.5 days. These results show that the MaLSF1 gene is a key gene that accelerates the degradation of total starch and amylopectin in banana fruit and affects pigment metabolism in the peel. This invention provides important gene resources for improving the flavor and appearance qualities of bananas or other plants, including their softness, sweetness, glutinous texture, and nutritional value. It also provides a theoretical basis for the development of banana preservation technologies and the breeding of new, storage-resistant varieties. Furthermore, it offers new target genes for the development of preservation technologies for starch-storing fruits like bananas and for molecular design breeding to cultivate new, storage-resistant banana varieties. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The nucleotide sequence diagram of the MaLSF1 gene (1,782 bp).

[0034] Figure 2 The amino acid sequence diagram of MaLSF1 (593 amino acids).

[0035] Figure 3 The analysis included the phenotype of banana fruit with transient overexpression of the MaLSF1 gene (A), iodine-potassium iodide staining of banana fruit slices (B), MaLSF1 gene expression level (C), total starch content (D), amylose content (E), and amylopectin content (F).

[0036] Figure 4 Analysis of banana fruit phenotype (A), banana fruit slice iodine-potassium iodide staining (B), MaLSF1 gene expression level (C), total starch content (D), amylose (E), and amylopectin content (F) for RNAi MaLSF1 gene expression. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0038] Example 1

[0039] The MaLSF1 gene from banana fruit was obtained using cDNA from Brazilian banana fruit as a template.

[0040] Primer-F: 5'-ATGGCCCTTATTCTCCAACTC-3'

[0041] Primer-R: 5'-TTACTTGGGAGCAAGCCTGATA-3'

[0042] Using primers, the MaLSF1 gene of banana fruit was amplified by PCR. Figure 1 and Figure 2 As shown, the nucleotide sequence of the MaLSF1 gene is 1,782 bp in length (with TAA at the end as a stop codon), and the amino acid sequence of the protein encoded by the MaLSF1 gene is shown in SEQ ID NO:2.

[0043] The PCR reaction system is as follows:

[0044]

[0045] The PCR amplification procedure is as follows:

[0046]

[0047] Example 2

[0048] Functional study of the MaLSF1 gene

[0049] I. Construction of overexpression vector for the MalSF1 gene

[0050] The method for constructing an overexpression vector for the MaLSF1 gene in banana fruit includes the following construction steps:

[0051] The above MaLSF1 nucleotide sequence was double-digested with two restriction endonucleases, Xba I and Kpn I, to digest the target fragment and the pCAMBIA3300 vector plasmid, respectively. The digested target fragment and the plant expression vector pCAMBIA3300 fragment were recovered, ligated, transformed, and sequenced to verify their correctness, thus obtaining the MaLSF1 overexpression vector.

[0052] II. Construction of the RNAi vector for the MaLSF1 gene

[0053] The method for constructing the MaLSF1 RNAi vector includes the following steps:

[0054] The nucleotide sequence of the MaLSF1 gene was double-digested with the same two restriction endonucleases, Xba I and Kpn I, on both the target fragment and the pTRV2 vector plasmid. The digested target fragment and the RNAi vector pTRV2 fragment were recovered, ligated, transformed, and sequenced to verify their correctness, thus obtaining the RNAi vector of the banana fruit starch degradation-related enzyme gene MaLSF1.

[0055] III. Transformation of banana fruits and banana fruit slices using overexpression vectors

[0056] The specific experimental steps for the conversion of banana fruit and banana fruit slices are as follows:

[0057] (1) Agrobacterium transformation of pCAMBIA3300-MaLSF1 overexpression vector

[0058] Take 200 μL of Agrobacterium tumefaciens GV3101 competent cells thawed on ice, add 2 μg of pCAMBIA3300-MaLSF1 recombinant plasmid, mix gently, and place in an ice bath for 30 min; transfer to liquid nitrogen and freeze for 3 min, then quickly incubate in a 37°C water bath for 5 min; add 800 μL of LYEP liquid medium and pre-culture at 28°C and 250 rpm for 4–5 h; transfer 300 μL of bacterial culture to YEP solid selective medium containing 50 mg / L Rif and spread evenly across the entire plate; place the plate at 28°C until the liquid is absorbed, invert the plate, and incubate at 28°C for 2–3 days; select single colonies for verification and detection; use the correctly transformed Agrobacterium culture for the next experiment.

[0059] (2) Agrobacterium tumefaciens-mediated genetic transformation of banana fruits

[0060] Immerse banana fruits in a beaker of 75% ethanol for 1 minute on a clean bench, agitating thoroughly during immersion, then rinse three times with sterile water; immerse in 20% sodium hypochlorite solution for 15 minutes, agitating thoroughly during immersion, then rinse three times with sterile water. Transfer 20 μL of Agrobacterium tumefaciens GV3101 culture transformed with the pCAMBIA3300-MaLSF1 recombinant vector to 10 mL of YEP liquid medium containing 50 mg / L kan and 50 mg / L Rif for overnight activation culture; transfer 1 mL of the activated culture to a fresh 50 mL of YEP liquid medium containing 50 mg / L kan and 50 mg / L Rif. The bacteria were cultured in Rif YEP liquid medium until OD600 = 0.6; the required concentration of bacterial suspension was transferred to a 50 mL sterile centrifuge tube on a clean bench, centrifuged at 6000 rpm for 5 min at 4 °C, the supernatant was discarded, and an equal volume (the volume of bacterial suspension before centrifugation) of MS liquid medium was added to resuspend the bacteria; the bacterial suspension was transferred to a 100 mL sterile Erlenmeyer flask, and 0.1% (the volume of the resuspended bacterial suspension) of acetylsuccinone (AS) was added and mixed thoroughly; the bacterial suspension was then injected into banana fruits using a syringe, and vacuum was applied for 15 min; the fruits were removed and placed on sterile filter paper to absorb excess bacterial suspension from the surface, and then transferred to an incubator for incubation at 25 °C in the dark for 3 days; the transformed banana fruits were used for phenotypic observation, iodine-potassium iodide staining, detection of total starch, amylose, amylopectin content, and MaLSF1 expression analysis.

[0061] (3) Agrobacterium tumefaciens-mediated genetic transformation of banana fruit slices

[0062] On a clean bench, banana fruit slices (1 mm thick) were immersed in a sterile centrifuge tube containing 5 mL of 75% ethanol for 1 min, agitating thoroughly during immersion, and then rinsed three times with sterile water. They were then immersed in a 20% sodium hypochlorite solution for 15 min, agitating thoroughly during immersion, and rinsed three times with sterile water. 20 μL of Agrobacterium tumefaciens GV3101 culture transformed with the pCAMBIA3300-MaLSF1 recombinant vector was transferred to 10 mL of YEP liquid medium containing 50 mg / L kan and 50 mg / L Rif and incubated overnight for activation. 1 mL of the activated culture was then transferred to a fresh 50 mL container containing 50 mg / L kan and 50 mg / L Rif. The bacteria were cultured in Rif YEP liquid medium until OD600 = 0.6. The bacterial suspension of the required concentration was transferred to a 50 mL sterile centrifuge tube on a clean bench and centrifuged at 6000 rpm for 5 min at 4 °C. The supernatant was discarded, and an equal volume (the volume of the bacterial suspension before centrifugation) of MS liquid medium was added to resuspend the bacteria. The bacterial suspension was transferred to a 100 mL sterile Erlenmeyer flask, and 0.1% (the volume of the resuspended bacterial suspension) of acetylsylgenin (AS) was added and mixed thoroughly. Then, banana fruit slices were transferred to Agrobacterium suspension and soaked for 15 min, shaking the suspension to ensure full contact between the fruit slices and the bacterial suspension. The slices were removed and placed on sterile filter paper to blot off excess bacterial suspension. They were then transferred to MS medium containing acetylsylgenin and incubated in the dark at 25 °C for 3 days. The co-cultured banana fruit slices were used for iodine-potassium iodide staining, detection of total starch, amylose, amylopectin content, and MaLSF1 expression analysis.

[0063] See Figure 3 As shown, compared with the control empty vector pCAMBIA3300, transient overexpression of pCAMBIA3300+MaLSF1 resulted in accelerated starch degradation in the fruit, and the flesh firmness of the fruit overexpressing the MaLSF1 gene was 0.15 kg / cm². 2 The control group had a pulp firmness of 0.43 kg / cm². 2 The pulp softened significantly at and near the injection hole of MalSF1. Figure 3 A), the iodine-potassium iodide staining of the fruit pulp became significantly lighter ( Figure 3 B); and the relative expression level of MaLSF1 increased by approximately 1.5 times, reaching a significant difference level (B). Figure 3 C).

[0064] Compared with the control empty vector pCAMBIA3300, transient overexpression of MaLSF1 reduced the total starch content, amylose content, and amylopectin content of the pulp by 1.87%, 0.30%, and 0.57%, respectively. Figure 3D-3F); and compared with the control empty vector pCAMBIA3300, the transient overexpression of MaLSF1 resulted in significant differences in the total starch and amylopectin content of the pulp. Figure 3 The differences in amylose content between D and 3F were not significant, indicating that the MaLSF1 gene mainly acts on the degradation of total starch and amylopectin, and does not affect the degradation and metabolism of amylose.

[0065] IV. Transformation of banana fruit slices using RNAi vector

[0066] The specific experimental steps for the conversion of banana fruit and its slices are as follows:

[0067] (1) Agrobacterium-mediated transformation of RNAi vector pTRV2-MaLSF1

[0068] Take 200 μL of Agrobacterium tumefaciens GV3101 competent cells thawed on ice, add 2 μg of pTRV2-MaLSF1 recombinant plasmid, mix gently, and place in an ice bath for 30 min; transfer to liquid nitrogen and freeze for 3 min, then quickly incubate in a 37°C water bath for 5 min; add 800 μL of YEP liquid medium and pre-culture at 28°C and 250 rpm for 4–5 h; transfer 300 μL of bacterial culture to YEP solid selective medium containing 50 mg / L rifampicin and 50 mg / L kanamycin, and spread evenly across the entire plate; place the plate at 28°C until the liquid is absorbed, invert the plate, and incubate at 28°C for 2–3 days, select single colonies for verification, and use the correctly transformed Agrobacterium culture for the next experiment.

[0069] (1) Agrobacterium tumefaciens-mediated genetic transformation of banana fruits

[0070] Immerse banana fruits in a beaker of 75% ethanol for 1 minute on a clean bench, shaking thoroughly during immersion, then rinse three times with sterile water; immerse in 20% sodium hypochlorite solution for 15 minutes, shaking thoroughly during immersion, then rinse three times with sterile water. Activate the Agrobacterium tumefaciens GV3101 bacterial culture transformed with the pTRV2-MaLSF1 recombinant vector overnight in 10 mL of YEP liquid medium containing 50 mg / L kan and 50 mg / L Rif at a pTRV1:pTRV2-MaLSF1 ratio of 1:5. Transfer 20 μL of the activated bacterial culture to 10 mL of YEP liquid medium containing 50 mg / L kan and 50 mg / L Rif. The bacteria were cultured in Rif YEP liquid medium until OD600 = 0.6; the required concentration of bacterial suspension was transferred to a 50 mL sterile centrifuge tube on a clean bench, centrifuged at 6000 rpm for 5 min at 4 °C, the supernatant was discarded, and an equal volume (the volume of bacterial suspension before centrifugation) of MS liquid medium was added to resuspend the bacteria; the bacterial suspension was transferred to a 100 mL sterile Erlenmeyer flask, and 0.1% (the volume of the resuspended bacterial suspension) of acetylsuccinone (AS) was added and mixed thoroughly; the bacterial suspension was then injected into banana fruits using a syringe, and vacuum was applied for 15 min; the fruits were removed and placed on sterile filter paper to absorb excess bacterial suspension from the surface, and then transferred to an incubator for incubation at 25 °C in the dark for 3 days; the transformed banana fruits were used for phenotypic observation, iodine-potassium iodide staining, detection of total starch, amylose, amylopectin content, and MaLSF1 expression analysis.

[0071] (3) Transformation of banana fruit slices with RNAi vector pTRV2-MaLSF1

[0072] Take 15 mL of the bacterial suspension that has turned golden yellow, centrifuge at 4600 rpm for 5 min and discard the supernatant, repeat once. Resuspend the bacterial cells in 1.0 mM MgCl2, centrifuge at 4600 rpm for 5 min and discard the supernatant. Resuspend the bacterial cells in 10 mM MgCl2, take 2 μL of the bacterial suspension and dilute it 100 times with 198 μl of MgCl2 to determine the OD value; dilute the bacterial suspension to OD600 = 0.6 according to the measured OD600 value. Add 10 mM Mes and 200 μM acetylsyleugenone at a ratio of pTRV1:pTRV2-MaLSF1 = 1:5, and incubate in the dark for 4–6 h. After standing for 2-3 hours, banana fruit slices (1 mm thick) were soaked for 15 minutes, during which the bacterial solution was shaken to ensure full contact between the fruit slices and the bacterial solution. The banana fruit slices were then removed and placed on sterile filter paper, and then transferred to MS medium containing acetylsuccinone and cultured in the dark at 25°C for 3 days. The co-cultured banana fruit slices were used for iodine-potassium iodide staining, detection of total starch, amylose, amylopectin content and MaLSF1 expression analysis.

[0073] V. Testing Methods

[0074] The detection methods for banana fruit slices overexpressing MaLSF1 or MaLSF1RNAi fruit slices are as follows.

[0075] (1) Iodine-potassium iodide staining analysis

[0076] Take thin slices of the stained banana fruit and place them in a petri dish. Pour in 0.2N iodine-potassium iodide solution and stain for 5 minutes. Wash off 3 times with sterile water, and air dry on absorbent paper for photographing.

[0077] (2) MaLSF1 gene qPCR analysis

[0078] Using MaLSF1 overexpression or RNAi banana fruit slice cDNA as templates, the expression of the MaLSF1 gene in banana fruit slices was analyzed. The reaction system and specific experimental methods are as follows:

[0079] The following components were added to a 200 μL PCR tube, and the reaction system is as follows:

[0080]

[0081] Mix thoroughly by pipetting, centrifuge briefly for a few seconds, and then amplify and detect the sample using a real-time quantitative PCR instrument (Mx3000P, Stratagene) with MaActin as an internal control gene. Each sample was repeated three times. The amplification reaction procedure is as follows:

[0082]

[0083] (3) Determination of total starch content

[0084] Using fruit slices overexpressing the MaLSF1 gene or treated with RNAi as material, each sample was replicated three times and thoroughly ground to determine the total starch content. The specific experimental procedures are as follows:

[0085] Weigh 0.1 g of the ground sample into a 15 mL centrifuge tube. Add 5 mL of 80% ethanol, vortex thoroughly to mix, centrifuge at 4000 rpm for 5 min, discard the supernatant, and then add 5 mL of deionized water to the precipitate for resuspending and washing. Centrifuge at 4000 rpm for 5 min, discard the supernatant, and then add 5 mL of 80% Ca(NO3)2 solution to the precipitate for resuspending. Extract in a boiling water bath for 10 min, cool, centrifuge at 4000 rpm for 10 min, and transfer the supernatant to a 25 mL volumetric flask. Resuspend the precipitate twice more with 80% Ca(NO3)2 solution, centrifuge, and combine the supernatants in the 25 mL volumetric flask. Make up to 25 mL with 80% Ca(NO3)2 solution and mix thoroughly. Take 1 mL of the extracted starch solution, add 80% Ca(NO3)2 solution to make up to 2 mL, add 100 μL of 0.01 N I2-KI solution, mix well, and measure the absorbance at a wavelength of 620 nm. Substitute OD620 into the total starch standard curve to determine the total starch content in the sample.

[0086] (4) Determination of amylose and amylopectin content

[0087] Weigh 0.1 g of the ground sample into a 50 mL centrifuge tube, add 1 mL of 95% ethanol and 9 mL of 1 mol / L NaOH, and incubate at 40 °C for 24 h. Repeat the process three times for each sample. Transfer the solution to a 100 mL volumetric flask, and dilute to 100 mL with distilled water, mixing thoroughly by inverting. Pipette 5 mL of the diluted solution into a new 100 mL volumetric flask, add 1 mL of 1 mol / L acetic acid and 2 mL of I₂-KI solution, and then dilute to 100 mL with distilled water, mixing thoroughly by inverting. Incubate at 30 °C for 30 min, and measure the absorbance at 620 nm. Substitute OD₆₂O into the amylose standard curve to determine the amylose content in the sample. The amylopectin content is equal to the total starch content minus the amylose content.

[0088] See Figure 4 As shown, compared with the control empty vector pTRV1+pTRV2, transient silencing of pTRV1+pTRV2-MaLSF1 expression resulted in a significantly greener peel color, slower starch degradation in the fruit, and a storage period extended by approximately 2.5 days. Figure 4 A); the iodine-potassium iodide staining of the fruit pulp became significantly darker ( Figure 4 B); and the relative expression level of MaLSF1 decreased by approximately 2.02-fold, reaching a highly significant level. Figure 4 C).

[0089] Compared with the control empty vector pTRV1+pTRV2, transient silencing of MaLSF1 expression increased the total starch content, amylose content, and amylopectin content of banana pulp by 4.76%, 0.31%, and 4.38%, respectively. Figure 4 D-3F); and compared with the control empty vector pTRV1+pTRV2, the transient silencing of MaLSF1 expression resulted in highly significant differences in the changes of total starch and amylopectin content in the pulp. Figure 4 D, 4F) had no effect on the change in amylose content.

[0090] Meanwhile, the effects of RNAi inhibition of MaLSF1 gene expression on soluble sugar content, vitamin C content, soluble solids content, organic acid content, sugar / acid ratio, and chlorophyll content of banana fruit were examined. The specific results are shown in Table 1.

[0091] Compared with the control empty vector, inhibiting MaLSF1 expression led to a decrease in soluble sugar content, vitamin C content, and soluble solids content, an increase in organic acid content and sugar / acid ratio, and an increase in total chlorophyll content in the pericarp.

[0092] Table 1. Effects of RNAi inhibition of MaLSF1 gene expression on banana fruit.

[0093] detection indicators pTRV1+pTRV2 pTRV1+pTRV2-MaLSF1 Soluble sugar content (Glug / g) 0.41 0.37 Vitamin C content (mg / 100g FM) 15.83 14.92 Soluble solids content (%) 14.80 14.07 Organic acid content (%) 0.12 0.18 Sugar / acid ratio 2.74:1 3.08:1 <![CDATA[Chlorophyll content (μg / g -1 )]]> 4.49 25.69 Storage period (days) 14.5 17.0

[0094] During the post-harvest ripening process of bananas, a significant positive correlation exists between the decrease in total starch content and the reduction in pulp firmness and the shortening of the storage period. The first step in banana starch degradation involves the formation of a binary complex between starch phosphorylase (PHO1) and glucan hydrated dikinase 1 (GWD1), which acts on the surface of starch granules, initiating the phosphorylation process and creating cracks on the starch granule surface to open channels for hydrolytic enzymes to degrade starch. Subsequently, multiple enzymes, including α-amylase, β-amylase, and LSF1, work synergistically to degrade the fruit starch, resulting in increased sugar content, softer texture, and a more glutinous mouthfeel. Furthermore, the total starch content decreases in ripe bananas, while the fructose and glucose contents increase significantly; therefore, the total starch content directly affects the sweetness of bananas. The decrease in total starch, amylose, and amylopectin content primarily leads to decreased banana firmness, a softer texture, improved glutinousness, increased sweetness, and a significantly shorter storage period.

[0095] In this invention, overexpression of the MaLSF1 gene reduces the total starch and amylopectin content of banana fruits, mainly leading to decreased fruit firmness, improved soft and chewy texture, and accelerated ripening. Inhibition of MaLSF1 expression increases total starch and amylopectin content, decreases soluble sugar, vitamin C, and soluble solids content, increases organic acid content and sugar / acid ratio, increases chlorophyll content in the peel, and results in a sluggish green peel phenotype. This delays post-harvest yellowing and softening, prolongs post-harvest ripening, and significantly extends the storage period by approximately 2.5 days.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A banana fruit method for regulating starch degradation in bananas. MaLSF1 Genes, characterized by, Its nucleotide sequence is shown in SEQ ID NO:

1.

2. The banana fruit as described in claim 1 MaLSF1 Gene-encoded proteins are characterized by, Its amino acid sequence is shown in SEQ ID NO:

2.

3. A banana fruit containing the banana fruit as described in claim 1 MaLSF1 Gene recombinant vectors, host bacteria, or expression cassettes.

4. The recombinant vector as described in claim 3, characterized in that, Includes the original carrier and the banana fruit as described in claim 1 MaLSF1 Gene; the original vector is either pCAMBIA3300 vector plasmid or pTRV2 vector plasmid.

5. The banana fruit as described in claim 1 MaLSF1 The use of the gene, or the protein of claim 2, or the recombinant vector, host bacterium, or expression cassette of claim 3, in any of the following: Application in reducing the total starch content of banana pulp; Reduce the application of amylose in banana fruit; It reduces the total starch content and / or amylose content of banana pulp without affecting the amylose content of banana fruit. Application in reducing the firmness of banana flesh.

6. The application as described in claim 5, characterized in that, In the aforementioned application, overexpression of banana fruit MaLSF1 Gene.

7. The banana fruit as described in claim 1 MaLSF1 The use of the gene, or the protein of claim 2, or the recombinant vector, host bacterium, or expression cassette of claim 3, in any of the following: Application in increasing the total starch content and / or amylose content of banana fruits; To increase the total starch content and / or amylose content of banana fruit without affecting the application of amylose content in banana fruit; Application in reducing the soluble sugar content, vitamin C content and / or soluble solids content of banana fruit; Applications in increasing the organic acid content and / or sugar / acid ratio of banana fruits; Application in increasing the chlorophyll content of banana peels; Application in extending the storage period of banana fruits; Applications in improving banana quality and / or developing banana varieties with good storage life.

8. The application as described in claim 7, characterized in that, In the aforementioned application, banana fruit is inhibited MaLSF1 Gene expression.

9. A method for increasing banana fruit yield MaLSF1 A primer pair for a gene, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:3 and SEQ ID NO:4.

Images