Maarf8 gene and application thereof in regulating banana male fertility

Abstract

The application discloses MaARF8 genes and application thereof in regulating banana male fertility. Through overexpression of the MaARF8 gene, the tomato appears the traits of late flowering, abnormal anther dehiscence, significantly reduced pollen amount, pollen grain deformity, extremely low pollen germination rate and significantly decreased male fertility; the scanning electron microscope results show that the pollen exine of the tomato with overexpression of the MaARF8 gene appears obvious defects compared with a control. Therefore, the application finds a MaARF8 gene related to male fertility, and provides a way for coping with complex and changeable cultivation environment, developing hybrid breeding and breaking through industrial difficulties.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry and molecular biology, specifically relating to the MaARF8 gene and its application in regulating male fertility in bananas. Background Technology

[0002] Bananas are a typical parthenocarpic horticultural crop, with high-quality male parents exhibiting low fertility. Therefore, evaluating male fertility in banana resources and identifying its regulatory genes is crucial. During the long-term domestication and breeding process of bananas, seedlessness (parthenocarpic) has been the primary selective trait, resulting in low fertility in most high-quality improved diploid male parents. This ultimately leads to low hybrid seed production, becoming a bottleneck restricting banana germplasm innovation and seed industry development. Therefore, identifying key genes regulating male fertility in bananas has become another key focus. Summary of the Invention

[0003] The purpose of this invention is to provide the MaARF8 gene and its application in regulating male fertility in bananas.

[0004] Previous studies have reported that pollen exine and inflorescence are closely related to pollen viability, and auxin-responsive factor ARF also participates in the regulation of male fertility in plants. However, research on whether and how ARF transcription factors regulate male fertility in bananas has not yet been reported. The main banana cultivar in my country is the AAA genotype. This study selected 126 diploid (AA) banana resources from both domestic and international sources as materials. On the one hand, pollen quantity and pollen viability were evaluated; on the other hand, genome-wide association analysis (GWAS) was used to identify the candidate gene MaARF8 (Macma4_05_g16680.1) regulating male fertility in bananas. Genetic transformation was used to verify that MaARF8 participates in the regulation of male fertility in plants, and the mechanism by which the MaARF8 gene regulates male fertility in bananas was preliminarily explored.

[0005] The main research results obtained in this paper are as follows:

[0006] 1. Evaluation of male fertility in diploid (AA) bananas: Among 126 banana resources, 83 had fewer than 10 pollen cells per anther, 14 had between 10 and 200 pollen cells per anther, and 29 had more than 200 pollen cells per anther. TTC staining results showed that 25 samples (86.21% of the varieties with high pollen counts) had pollen viability exceeding 50%, indicating a significant positive correlation between pollen count and pollen viability in bananas.

[0007] 2. MaARF8's regulation of pollen exine integrity affects male fertility in bananas: Genome-wide association analysis (GWAS) was performed using resequencing data from 126 accessions and pollen quantity traits. The results showed that the candidate region was located on chromosome 5 (Chr5: 13.57-13.77 Mb), containing 10 genes. Homologous gene alignment in Arabidopsis identified the auxin signaling transcription factor MaARF8 as a key candidate gene. Further validation revealed that tomatoes overexpressing MaARF8 exhibited significantly reduced male fertility, including delayed flowering, abnormal anther dehiscence, significantly reduced pollen quantity, pollen grain deformities, and extremely low pollen germination rates. Scanning electron microscopy showed that the pollen exine of tomatoes overexpressing MaARF8 showed significant defects compared to the control.

[0008] Therefore, the first objective of this invention is to provide the MaARF8 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] A second objective of this invention is to provide the protein encoded by the aforementioned MaARF8 gene.

[0010] A third objective of this invention is to provide the application of the aforementioned MaARF8 gene in regulating male fertility in plants.

[0011] The preferred approach is the application of overexpression of the MaARF8 gene in reducing male fertility in plants.

[0012] Preferably, the plant is a banana or a tomato.

[0013] The preferred application is in the overexpression of the MaARF8 gene in tomatoes, which results in delayed flowering, pollen malformation, abnormal pollen wall structure, significantly reduced pollen quantity, decreased seed setting rate, significantly reduced pollen viability and germination rate, and significantly reduced pollen quantity.

[0014] The fourth objective of this invention is to provide a method for reducing male fertility in plants by overexpressing the MaARF8 gene in plants.

[0015] This invention reveals that tomatoes overexpressing the MaARF8 gene exhibit significantly reduced male fertility, including delayed flowering, abnormal anther dehiscence, significantly decreased pollen quantity, pollen grain deformities, and extremely low pollen germination rates. Scanning electron microscopy results show that the pollen exine of tomatoes overexpressing the MaARF8 gene shows obvious defects compared to the control. Therefore, this invention discovers a MaARF8 gene related to male fertility, providing a pathway to address complex and variable cultivation environments, vigorously develop hybrid breeding, and overcome industrial challenges. Attached Figure Description

[0016] Figure 1 This is a GWAS association analysis diagram of male fertility in 126 diploid (AA) banana resources;

[0017] Figure 2 This is a positive identification image of MaARF8 overexpressing tomatoes;

[0018] Figure 3 These are phenotypic images of MaARF8-overexpressing tomato plants; Note: a is a phenotypic image of wild-type plants, with a scale bar of 10 cm; b is a phenotypic image of MaARF8-overexpressing tomato plants, with red boxes indicating inflorescences with reduced fruit set, and a scale bar of 10 cm; c is a magnified phenotypic image of a portion of an inflorescence of a MaARF8-overexpressing tomato plant.

[0019] Figure 4 These are floral organ diagrams of MaARF8-overexpressing tomatoes. Note: a and b are floral organ diagrams of WT and MaARF8-overexpressing tomatoes, respectively; c and d are anther and style diagrams of WT and MaARF8-overexpressing tomatoes, respectively.

[0020] Figure 5 These are phenotypic diagrams of MaARF8-overexpressing tomato fruits. Note: a is a statistical analysis diagram of the fresh weight and number of seeds per fruit of wild-type and MaARF8-overexpressing tomatoes; b is a phenotypic diagram of fruits and seeds of wild-type and MaARF8-overexpressing tomatoes; ns indicates P>0.1, no statistically significant difference, **** indicates P<0.0001, significant difference;

[0021] Figure 6 This is a graph showing the germination rate of MaARF8-overexpressing tomato seeds;

[0022] Figure 7 This is a graph showing the decrease in pollen viability in MaARF8-overexpressing tomatoes using TTC staining. Note: a and b are pollen viability staining graphs of wild-type tomatoes; c and d are pollen viability staining graphs of MaARF8-overexpressing tomatoes, with scale bars of 50 μm and 20 μm, respectively; e is the statistical analysis of tomato pollen viability. **** indicates P < 0.0001, a significant difference.

[0023] Figure 8 The graph shows the decrease in in vitro germination rate of MaARF8-overexpressing tomato pollen. Note: a is the in vitro germination graph of wild-type pollen; b is the in vitro germination graph of MaARF8-overexpressing tomato pollen; c is the statistical graph of germination rate of wild-type and MaARF8-overexpressing tomato pollen. **** indicates P<0.0001, which is a significant difference.

[0024] Figure 9 This is a diagram showing the pollen germination of tomatoes overexpressing MaARF8;

[0025] Figure 10These are scanning electron microscope (SEM) images of mature pollen grains from MaARF8-overexpressing tomatoes. Note: a and b are magnified at 3000x and 15000x magnification, respectively, for wild-type pollen grains; c and d are magnified at 3000x and 15000x magnification, respectively, compared to wild-type pollen grains. Scale bars are 10 μm and 1 μm, respectively. Detailed Implementation

[0026] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0027] Example 1:

[0028] 1. Materials and Methods

[0029] 1.1 Experimental Materials

[0030] 126 banana germplasm resources were planted at the Zhongluotan Experimental Base in Baiyun District, Guangzhou City, Guangdong Province, and the Banana Experimental Base in Dongguan City, Guangdong Province. The tomato variety used for genetic transformation and control was AC++ (Solanum lycopersicum Mill. var. Ailsa Craig), and the tobacco variety was Nicotiana benthamiana.

[0031] 1.2 Main Instruments and Equipment

[0032] PCR amplification instrument (BIO-RAD);

[0033] Gel Imaging System (BIO-RAD);

[0034] Zeiss AxioScope A1 fluorescence upright microscope;

[0035] ABI StepOnePlus™ Real-Time PCR System with Tower;

[0036] Biological Microscope ML31 (Mingmei);

[0037] High-pressure steam sterilizer;

[0038] Clean benches, shakers, incubators, etc.

[0039] 1.3 Main reagents and medicines

[0040] Genetic engineering reagents: Conventional restriction endonucleases were purchased from TaKaRa; Taq DNA polymerase, high-fidelity DNA polymerase, cDNA synthesis kit, and high-specificity dye-based quantitative PCR detection kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.; DNA gel recovery kit and DNA extraction kit were purchased from Magen; plasmid extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; TS-GelRed nucleic acid gel dye, DNA marker, etc. were purchased from Beijing Qingke Biotechnology Co., Ltd.; plant RNA extraction kit was purchased from Guangzhou Ruizhen Biotechnology Co., Ltd.; primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0041] Antibiotics: Ampicillin (Amp) and kanamycin (Kan) were purchased from Guangzhou Dingguo Biotechnology Co., Ltd.; Rifampicin (Rif) was purchased from Sigma-Aldrich.

[0042] Other experimental reagents: 2,3,5-triiodobenzoic acid (TIBA) and aniline blue (water-soluble) were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0043] 1.4 Preparation of main reagents and culture media

[0044] Pollen germination solid / liquid culture medium: 0.02% boric acid + 0.01% potassium nitrate + 0.02% magnesium sulfate + 0.03% calcium nitrate + 15% sucrose + 0.8% agar powder (solid), pH 5.8.

[0045] LB solid / liquid medium: 0.5% (w / v) yeast extract + 1% (w / v) tryptone + 1% (w / v) NaCl + agar powder (solid) 1.5 g / L.

[0046] Preparation of 5% Suc solution (containing 0.02% Silwet L-77): 50 g / L sucrose + 0.02% Silwet L-77.

[0047] 1% 2,3,5-Triphenyltetrazol chloride (TTC): Dilute with Tris buffer (HCl 0.15 M, pH 7.8) and store at 4°C protected from light.

[0048] 0.1% Aniline Blue Solution: 1.0 g / L aniline blue powder + 0.1M K3PO4·3H2O solution, adjust pH to 7.5, store at 4℃ protected from light, preferably prepared and used immediately.

[0049] Carno fixative: anhydrous ethanol: glacial acetic acid = 3:1.

[0050] 1.5 Strains and vectors required for the experiment

[0051] Strains: Escherichia coli strain DH5α (E. coli) was purchased from Beijing Qingke Biotechnology Co., Ltd., and Agrobacterium tumefaciens strain GV3101.

[0052] Carriers: pGBKT7, pGADT7.

[0053] 1.6 Evaluation of pollen fertility of banana germplasm resources

[0054] 1.6.1 Survey of Banana Pollen Quantity

[0055] 126 diploid (AA) banana germplasms were planted in Machong Town, Dongguan City and Zhongluotan Experimental Base, Baiyun District, Guangzhou City, with 15-45 plants per germplasm resource. Male pollen fertility was investigated regularly. During the male flowering period, male buds were cut from the stamens between 7:30 and 10:30 AM and immediately transported to the laboratory. Pollen grains from individual anthers were extracted from the stamens using a dissecting needle, placed on glass slides, and their pollen count was statistically analyzed under a microscope. The specific classification criteria were based on and modified from those of Dupe and Ortiz (1996), classifying pollen count per anther into three levels: 0 (low pollen yield, <10 grains per anther); 1 (medium pollen yield, 10-200 grains per anther); and 2 (high pollen yield, >200 grains per anther).

[0056] 1.6.2 Determination of banana pollen viability

[0057] Pollen viability is crucial for ensuring pollen-pistil interactions necessary for fertilization and seed formation in flowering plants. Pollen viability can be assessed using various methods, such as staining with reactive and nuclear dyes, in vitro and in vivo germination assays, and direct assessment of final seed set. Banana pollen viability assessment primarily involves acetyl carmine staining (Goigoux et al., 2013; Oselebe et al., 2014; Ssebuliba et al., 2008) and 2,3,5-triphenyltetrazolium chloride (TTC) staining (Soares et al., 2008; Soares et al., 2015). This study used TTC staining to detect banana pollen viability, staining pollen grains with TTC diluted to 1% using Tris buffer (HCl 0.15 M, pH 7.8). Pollen grains that stained light or dark red after TTC staining were considered viable, while unstained pollen grains were classified as inactive.

[0058] Because banana pollen is fresh and difficult to preserve, samples are typically collected at the farm between 8 and 10 AM and immediately returned to the laboratory for pollen viability testing. Pollen grains are collected from three anthers of the same stamen comb, evenly distributed on a glass slide, stained with TTC staining solution, covered with a coverslip, and observed under an optical microscope after 2 hours. To obtain good representativeness of pollen grain viability, the staining of 100 pollen grains on each slide is counted under an optical microscope, repeated three times. The pollen staining viability results are expressed as a percentage.

[0059] 1.6.3 In vitro germination of banana pollen

[0060] During the male flowering period, male inflorescences below the outermost bracts of the male buds were collected before 10:00 AM, placed in sterilized plastic bags, and quickly returned to the laboratory. Five flowers from the same stamen comb were randomly selected, and pollen grains were manually extracted using a dissecting needle or forceps and evenly distributed in a pollen germination medium. The medium consisted of 15% sucrose, 0.01% boric acid, 0.01% potassium nitrate, 0.03% calcium nitrate, and 0.02% magnesium sulfate by mass fraction, solidified with 0.8% agar, with the remainder being water. The pH was adjusted to 5.8, and the medium was autoclaved at 121°C for 20 minutes (Soares et al., 2008). The pollen grains were cultured in the dark at 28°C, and observed and photographed under an optical microscope after 24 hours. The pollen tube length being equal to or greater than the pollen grain diameter was used as the germination standard, and the pollen germination rate in each field of view was statistically analyzed. Three fields of view were randomly selected for each analysis, repeated three times, and the average value was calculated.

[0061] 1.7 Functional analysis of the banana MaARF8 gene

[0062] 1.7.1 GWAS Association Analysis

[0063] Genome-wide association analysis was performed using resequencing data and pollen quantity traits from 126 banana germplasm resources, and key candidate regions were predicted using multiple models. Based on the research progress on male fertility in model plants, 1-2 candidate genes were selected for functional verification.

[0064] 1.7.2 Banana DNA Extraction

[0065] Freshly sprouted banana leaves were immediately flash-frozen in liquid nitrogen and stored at -80°C. The extraction method was performed according to the instructions of the Novizan Fast Pure Plant DNA Isolation Mini Kit Box, and the obtained DNA was stored at -20°C.

[0066] 1.7.3 Amplification of the Target Fragment

[0067] Using banana cDNA as a template and MaARF8-QC-F and MaARF8-QC-R (Table 10) as primers, PCR amplification was performed using a high-fidelity enzyme. The PCR reaction system was prepared according to Table 1. The PCR reaction program was: 95℃ for 3 min, 95℃ for 30 s, 49℃ for 30 s, 72℃ for 90 s, 72℃ for 5 min, for 35 cycles.

[0068] Table 1. MaARF8 target fragment amplification reaction system

[0069]

[0070] 1.7.4 Purification and Recovery of Target Fragment

[0071] The PCR product of 1.73 was subjected to 1.2% agarose gel electrophoresis. The target fragment was recovered according to the instructions of the DP214 Universal DNA Purification and Recovery Kit of Tiangen Biotech (Beijing) Co., Ltd., and stored at -20℃ for later use.

[0072] 1.7.5 Enzyme digestion and ligation of the target fragment

[0073] The PCR products purified and recovered in 1.7.4 and the pCAMBIA1300 transformation vector were digested with enzymes according to the reaction system prepared in Table 2. After digestion at 37℃ for 60 min, the digested products were combined and purified with a PCR purification kit. The purified products were ligated according to the reaction system prepared in Table 3. After reaction at 20℃ for 60 min, they were used for subsequent transformation.

[0074] Table 2 Enzyme digestion reaction system

[0075]

[0076] Table 3 Connection Reaction System

[0077]

[0078] 1.7.6 Recombinant Transformation of Escherichia coli

[0079] 1) Thaw E. coli competent cells on ice at -80°C for half an hour in advance. Add 10 μL of the ligation product from 1.7.5 above to 100 μL of competent cells, gently tap the bottom of the centrifuge tube to mix well, and incubate on ice for 30 min.

[0080] 2) Heat shock at 42℃ for 70 seconds, then immediately place on ice and let stand for 2 minutes.

[0081] 3) Add 700 μL of antibiotic-free LB liquid medium to the centrifuge tube and incubate in the dark at 37℃ and 200 rpm for 1 h. Centrifuge at 5000 rpm for 1 min, remove 700-750 μL of supernatant, and spread the remaining 50-100 μL onto a plate.

[0082] 4) Gently blow on the bacteria before plating to suspend them. On a clean bench, use a pipette tip to plating 50-100 μL onto a plate containing Kan resistance.

[0083] 5) Invert the plate and incubate in the dark at 37℃ for 12-15 h.

[0084] 1.7.7 Screening and sequencing of positive clones

[0085] For preliminary screening of E. coli resistance, 1.0 mL of LB liquid medium containing 50 mg / L kan resistance was added to a 1.5 mL sterile centrifuge tube. Single colonies of recombinant transformed E. coli were picked with a sterile pipette tip and cultured on a shaker at 37℃ and 200 rpm for 6-8 h.

[0086] Bacterial culture PCR identification: Using specific primers MaARF8-jd-F and MaARF8-jd-R (Table 10), the PCR reaction system was prepared according to Table 4. PCR reaction program: 95℃ for 3 min, 95℃ for 30 s, 53℃ for 30 s, 72℃ for 90 s, 72℃ for 5 min; 35 cycles. After gel electrophoresis, bacterial culture samples containing the correct bands were sent to the company for sequencing.

[0087] Table 4. Bacterial PCR Reaction System

[0088]

[0089] 1.7.8 Plasmid Extraction

[0090] Add the correctly sequenced bacterial culture to LB medium containing 50 mg / L kan resistance, and incubate in the dark at 37°C and 200 rpm for 6-8 h. Then extract the plasmid according to the instructions of the plasmid extraction kit.

[0091] 1.7.9 Introduction of the banana MaARF8 gene expression vector into Agrobacterium

[0092] The expression vector (plasmid) constructed in section 1.7.8 above was transformed into Agrobacterium GV3101. The specific steps are as follows:

[0093] 1) Take Agrobacterium competent cells GV3101 stored at -80℃, thaw on ice for 30 min, add 5 μL of recombinant plasmid to the competent cells, gently tap the tube wall to mix, and then incubate on ice for 5 min. Then, perform liquid nitrogen rapid freezing for 5 min and water bath at 37℃ for 5 min in sequence.

[0094] 2) Add 700 μL of non-resistant liquid LB medium to a centrifuge tube on a clean bench and incubate at 28°C and 200 rpm for 2-3 h on a shaker.

[0095] 3) Centrifuge at 6000 rpm for 1 min, discard 700 μL of supernatant, and spread the remaining 100 μL onto a plate.

[0096] 4) Before plating, gently blow the bacteria to suspend them. On a clean bench, use a sterile pipette tip to evenly spread 100 μL of the resuspension onto a plate containing double antibiotics (Spec 50 mg / L + Rif 25 mg / L). Incubate in the dark at 28°C for 48-72 h.

[0097] 1.7.10 Screening and sequencing of positive clones

[0098] 1) Preliminary screening for Agrobacterium resistance: 1.0 mL of LB liquid medium containing Spec (50 mg / L) and Rif (25 mg / L) resistance was added to a sterilized 1.5 mL centrifuge tube. Single Agrobacterium colonies were picked with a sterilized pipette tip and cultured at 28℃ and 220 rpm for 12 h.

[0099] 2) PCR identification of bacterial culture: Using specific primers MaARF8-jd-F and MaARF8-jd-R as primers (Table 10), the PCR reaction system was prepared according to Table 5. PCR reaction program: 95℃ for 3 min, 95℃ for 30 s, 53℃ for 30 s, 72℃ for 90 s, 72℃ for 5 min, 35 cycles.

[0100] 3) Sample delivery for sequencing and bacterial culture preservation: Bacterial culture samples that pass gel electrophoresis are sent to the company for sequencing. Once sequencing is successful, the bacterial culture is preserved in 60% glycerol at -80℃ for later use.

[0101] Table 5. Bacterial PCR Reaction System

[0102]

[0103] 1.7.11 Stable genetic transformation of tomatoes

[0104] Tomatoes overexpressing the MaARF8 gene were obtained using Agrobacterium-mediated tomato genetic transformation. Specific tomato genetic transformation methods are described by Brian et al. (Brian et al., 2002).

[0105] 1.7.12 Positive Identification of Genetically Modified Tomatoes

[0106] DNA extraction from transgenic tomatoes: Young leaves from wild-type and T0 generation MaARF8 overexpressing tomatoes were immediately flash-frozen in liquid nitrogen and stored at -80℃ for later use. The specific extraction method is as described in 1.7.2 above, and the obtained DNA was stored at -20℃.

[0107] PCR amplification: The target gene was amplified using a high-fidelity enzyme with specific primers MaARF8-jd-F and MaARF8-jd-R (Table 10). The reaction system was prepared according to Table 6. PCR program: 95℃ for 3 min, 95℃ for 30 s, 58℃ for 30 s, 72℃ for 90 s, 72℃ for 5 min; 35 cycles. After amplification, gel electrophoresis was performed. Positive plants (overexpressing transgenic tomato lines) were planted in a greenhouse and managed with normal fertilizer and water.

[0108] Table 6 PCR Amplification System

[0109]

[0110] 1.7.13 Detection of MaARF8 gene expression level in overexpressed tomatoes

[0111] Total RNA extraction and reverse transcription: Total RNA was extracted from young leaves of wild-type and overexpression transgenic tomato lines, and cDNA was synthesized by reverse transcription. The obtained cDNA was used as a template to detect the expression level of MaARF8 in young tomato leaves. Specific RNA extraction methods were performed according to the instructions for the SteadyPure Plant RNA Extraction Kit from Guangzhou Ruizhen Biotechnology Co., Ltd. Reverse transcription of cDNA was performed according to Takara's Prime Script. TM Follow the instructions in the 1stStrand cDNA Synthesis Kit.

[0112] Real-time quantitative PCR: Based on the known CDS sequences, the required qRT-PCR primers were designed online using NCBI. The primers used in the experiment were ARF8-qPCR-F / ARF8-qPCR-R (Table 10). This experiment used Novizan's Taq Pro Universal SYBR qPCR Master Mix for real-time quantitative PCR. The specific method is as follows:

[0113] First, prepare the mixture in RNase-free centrifuge tubes according to Table 7, then set up the reaction system in the real-time quantitative PCR instrument according to Table 8, and finally calculate the relative expression level of the MaARF8 gene.

[0114] Table 7 Real-time quantitative PCR reaction solution

[0115]

[0116] Table 8 Real-time fluorescence PCR reaction procedure

[0117]

[0118] 1.8 Determination of MaARF8-overexpressing tomato pollen fertility-related indicators

[0119] 1.8.1 Tomato pollen viability detection

[0120] Positive MaARF8 overexpressing tomato plants (overexpressing transgenic tomato lines) were planted in a greenhouse and managed with normal fertilizer and water. Pollen from wild-type and MaARF8 overexpressing tomatoes was collected during the flowering period, stained with 0.1% TTC solution, observed under a microscope, and photographed. The experiment was repeated three times, following the steps in 1.6.2 above.

[0121] 1.8.2 In vitro germination of tomato pollen

[0122] Pollen from wild-type and MaARF8-overexpressing tomatoes was collected during the flowering period and transferred to germination medium. The mixture was then incubated in the dark at 25°C for 3 hours. Germination was defined as when the pollen tube was at least as long as the pollen grain diameter. Pollen germination was then observed.

[0123] 1.8.3 Intra-plantar germination of tomato pollen

[0124] Pollen tube germination was analyzed using aniline blue staining, with a 0.1% aniline blue solution as the working solution. The specific steps for observing pollen tube development are as follows:

[0125] Fixation: Before flowering, the stamens of tomatoes were removed, and pollen was collected from MaARF8 overexpressing tomatoes and wild-type tomatoes for pollination and isolation by bagging. The styles were collected 24 h after pollination, and three styles were placed in Carno fixative (25% ethanol: 75% glacial acetic acid) for fixation at room temperature for 24 h and stored at 4℃ for later use.

[0126] Softening: Discard the Carno fixative, rinse twice with ddH2O, add an appropriate amount of 2 mol / L NaOH solution, and place in a 45℃ constant temperature water bath for 1 h to soften.

[0127] Cleaning: After recovering the NaOH solution, add glacial acetic acid to neutralize the residual alkali solution (30 s), and finally rinse 5 times with ddH2O.

[0128] Aniline blue fluorescent staining: Immerse the transparent styles in 0.1% (w / v) aniline blue staining solution and stain overnight at room temperature in the dark.

[0129] Slide observation: Take the style of the flower onto a glass slide, drop a small amount of 50% glycerol on it to prepare the slide, and observe and photograph it under 360 nm ultraviolet excitation light.

[0130] 1.8.4 Scanning electron microscopy observation of tomato pollen morphology

[0131] Pollen from wild-type and MaARF8-overexpressing tomatoes during their flowering period was collected between 7 and 9 AM for experiments. The pollen was rapidly placed in pre-cooled 2.5% glutaraldehyde fixative and fixed for 2 h. After fixation, the samples were rinsed three times (15 min each time) with 0.1 M phosphate-buffered saline (PBS) (pH 7.4), followed by treatment with 30%, 50%, 70%, 80%, 90%, 100%, and 100% ethanol for 15 min each time. Subsequently, the samples were transferred to isoamyl acetate and immersed for 15 min to replace residual ethanol. The dehydrated samples were then subjected to supercritical CO2 drying in a critical point desiccator to prevent structural collapse. The dried samples were fixed onto conductive carbon-based double-sided tape and then sputtered with platinum (10 nm thickness, 30 s sputtering time). The ultrastructure was observed and photographed under a scanning electron microscope.

[0132] 1.9. Data Analysis and Processing

[0133] Data were processed using Excel and plotted using GraphPad Prism 9.5. Each experiment was performed in three biological replicates. The data in the plot are the mean ± SD (standard deviation).

[0134] 2 Results and Analysis

[0135] 2.1 Association between MaARF8 gene and banana pollen quantity trait

[0136] Genome-wide association analysis (GWAS) was performed on resequencing data and pollen quantity traits from 126 diploid (AA) banana natural populations. The results showed that the candidate region was located on chromosome 5, Chr5: 13.57-13.77 Mb, containing 10 genes (Table 9). Previous studies have indicated that auxin is involved in banana pollen maturation, influencing pollen germination rate and pollen tube elongation. Auxin signaling response factor (ARF) genes are key transcription factors regulating auxin signal transduction. Therefore, the auxin signaling transcription factor MaARF8 gene (Macma4_05_g16680.1) was identified as a key candidate gene. Figure 1 Analysis of the MaARF8 gene sequence showed that the full-length MaARF8 gene sequence is 1536 bp (nucleotide sequence as shown in SEQ ID NO.1), containing 17 exons and 16 introns, with a protein size of 58.12 kb, and exhibiting typical DNA binding and ARF structures.

[0137] Table 9. GWAS association analysis screened candidate genes associated with male fertility in bananas.

[0138]

[0139] 2.2 Evaluation of MaARF8 overexpression in tomatoes

[0140] To investigate the function of the MaARF8 gene, this study used overexpression to verify its effectiveness. The MaARF8 gene was overexpressed in tomato. Figure 2 As shown in Figure a, the target bands were amplified in the three antibiotic-selected tomato lines, named MaARF8-3, MaARF8-5, and MaARF8-8 (corresponding to lanes 1, 2, and 3; lane 4 is the positive control, and lane 5 is the negative control). Furthermore, to determine the expression of the MaARF8 gene in transgenic tomatoes, we performed real-time quantitative PCR analysis on the flowers of wild-type and transgenic tomatoes. The results showed that no MaARF8 gene expression was detected in wild-type tomatoes, while the relative expression levels of the three MaARF8 overexpressing tomatoes—MaARF 8-3, MaARF 8-5, and MaARF 8-8—were 0.26, 0.29, and 0.74 times higher than that of the tomato Actin gene, respectively. Figure 2 (b) in the middle.

[0141] The growth and development phenotypes of the three positive lines were then evaluated. The results showed that, compared with the wild type, MaARF8-overexpressing tomatoes exhibited leaf curling and shortened internode distance during the vegetative growth stage; and during the reproductive development stage, they showed a delayed inflorescence initiation node. The first inflorescence of the wild type started at the 7th-8th true leaf node, while the inflorescence of the MaARF8-overexpressing tomato started at the 15th-20th node, and the flowering time was delayed by about one month. Figure 3 The three positive lines showed similar phenotypes during both vegetative and reproductive growth stages, so the MaARF 8-8 line was selected as the subject of subsequent experiments.

[0142] Previous studies found that the MaARF8 gene was highly expressed in floral organs. Further investigation was conducted to determine whether overexpression of the MaARF8 gene affected the development of tomato floral organs. Experimental results showed that the external morphology of the floral organs in transgenic tomatoes (such as the size and shape of petals, sepals, and stamens) was similar to that of the wild type. Figure 4 (a and b in the text) There is no significant difference in appearance between the two, but there are significant differences in anther dehiscence, pollen quantity, and style length. The wild-type anthers can dehisce normally, and the style is level with the anther dehiscence. Under natural conditions, a large amount of pollen is scattered on the style. Figure 4 (c) In contrast, MaARF8 overexpression significantly reduces pollen in tomato anthers, and the style is shorter than the anther dehiscence. Under natural conditions, the amount of pollen in the style is extremely low. Figure 4 (d in the text)

[0143] Further statistical analysis revealed that the reduced pollen count led to a significantly lower fruit set rate in MaARF8-overexpressing tomatoes compared to the wild type: the wild type averaged 47 fruits per plant, while the MaARF8-overexpressing tomatoes produced only 5, a decrease of 89.36%. The fresh weight of individual fruits in MaARF8-overexpressing tomatoes was not significantly different from that of the wild type, but their seed set rate was significantly lower. Figure 5 MaARF8-overexpressing tomatoes had an average of 8.71 seeds per fruit, while wild-type fruits had an average of 104 seeds, representing a 91.63% decrease in seed set. Furthermore, MaARF8-overexpressing tomato seeds were shriveled compared to wild-type seeds, with a germination rate of 53.3%, significantly lower than the wild-type (90%). Figure 6 ).

[0144] The above results indicate that tomatoes overexpressing MaARF8 exhibit phenotypes such as delayed flowering, abnormal anther dehiscence, significantly reduced pollen quantity, and decreased seed setting rate.

[0145] 2.3 MaARF8 overexpression significantly reduced tomato pollen viability

[0146] Besides pollen quantity, pollen viability also affects tomato fruit set and seed set rate. To further understand the mechanism of reduced seed set rate in MaARF8-overexpressing tomatoes, this study used 1% TTC staining to detect pollen viability. Figure 7 The results showed that most of the pollen grains of wild-type tomatoes were plump and easily stained, with a pollen viability of 88.51%, while most of the pollen grains of MaARF8-overexpressing tomatoes were shriveled and dry, with a pollen viability of only 9.45%, indicating a significant decrease in pollen viability.

[0147] 2.4 MaARF8 overexpression significantly reduced the pollen germination rate of tomatoes.

[0148] Pollen viability of MaARF8-overexpressing tomatoes was assessed using pollen germination experiments. The results showed that the germination rate of MaARF8-overexpressing tomatoes was 3.80%, while the germination rate of wild-type pollen was 46.93%, indicating that the pollen viability of MaARF8-overexpressing tomatoes was lower than that of wild-type tomatoes. Figure 8 Furthermore, the TTC staining results also demonstrated that the pollen viability of MaARF8-overexpressing tomatoes was lower than that of wild-type tomatoes. Figure 8 The decrease in fruit setting and seed setting rate in tomatoes overexpressing MaARF8 also indicates a decrease in pollen viability.

[0149] To investigate the germination of MaARF8-overexpressing tomato pollen grains on the stigma, an in vivo pollen germination experiment was conducted using aniline blue staining. Since tomatoes are self-pollinating plants, stamens were removed before flowering, and pollen from both MaARF8-overexpressing and wild-type tomatoes was collected for pollination and bagging. 24 hours after pollination, the styles were collected for fixation and staining observation. Under ultraviolet light irradiation under a fluorescence microscope, the callose produced in the pollen tubes showed a blue color after staining. Figure 9 Tomato pollen overexpressing MaARF8 germinated in vivo but not on the style of the plant body or the wild-type WT (whole-rooted) style. Experimental results showed that in the wild-type self-pollination system, a large number of pollen tubes germinated, but no pollen germination was observed on the style of the cross between the wild-type female parent and the MaARF8-overexpressing male parent. Notably, pollen germination was also not detected on the style in the MaARF8-overexpressing tomato self-pollination system. However, when the MaARF8-overexpressing tomato was used as the female parent and crossed with the wild-type male parent, most of the pollen on the stigma of the female parent germinated normally, and the pollen tubes were long enough to extend into the style, a result quite similar to that of wild-type self-pollination. These results indicate that compared to the wild type, the pollen germination rate of MaARF8-overexpressing tomatoes is extremely low and lacks viability, suggesting that overexpression of the MaARF8 gene affects the viability of tomato pollen, but the underlying mechanism remains unclear.

[0150] 2.5 Ma ARF8 overexpression resulted in incomplete pollen walls in tomato flowers.

[0151] Significant pollen deformities were observed in MaARF8-overexpressing tomatoes under an optical microscope. To further analyze the differences between pollen from MaARF8-overexpressing tomatoes and wild-type plants, the morphology of pollen grains that flowered on the same day was observed using scanning electron microscopy (SEM). The results showed that wild-type pollen grains were oval, plump, with clear and evenly distributed germination furrows; the exine surface of wild-type pollen grains had evenly distributed spiny protrusions, and its exine reticulate structure had no obvious defects. Figure 10 (a, b) in the text; while MaARF8 overexpression in tomatoes resulted in significantly malformed pollen grains, exhibiting varying degrees of shriveling and collapse, underdeveloped, with a smooth outer wall structure, reticulate ornamentation covered by random cuticle and other substances, and an uneven, incomplete pollen wall. Figure 10 (c and d in the original text). It is speculated that the low pollen viability in tomatoes with MaARF8 overexpression may be due to abnormal pollen wall development.

[0152] 3. Discussion

[0153] In this study, firstly, genome-wide association analysis was used to identify the banana male fertility regulatory gene MaARF8. Secondly, the MaARF8 gene was overexpressed in tomato. Tomatoes overexpressing MaARF8 exhibited delayed flowering, pollen malformation, abnormal pollen wall structure, significantly reduced pollen viability and germination rate, and significantly reduced pollen quantity, among other phenotypes. This indicates that the MaARF8 gene is involved in regulating banana male fertility.

[0154] Table 10 Primers used in this invention

[0155] .

Claims

1. Overexpression MaARF8 The application of genes in reducing male fertility in plants, as described MaARF8 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the plant is tomato.

2. The application according to claim 1, characterized in that, Overexpression in tomatoes MaARF8 This gene is used in applications that cause delayed flowering, pollen malformation, abnormal pollen wall structure, reduced seed setting rate, significantly decreased pollen viability and germination rate, and significantly reduced pollen quantity in tomatoes.

3. A method for reducing male fertility in plants, characterized in that: Overexpression in plants MaARF8 Genes, as described MaARF8 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the plant is tomato.

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