Application of BbRbohF gene in regulation and control of shedding of trigonotis triangularis inflorescence

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to application of a BbRbohF gene in regulation and control of shedding of a trigonotis triangularis inflorescence. The nucleotide sequence of a CDS (Coding Sequence) region of the BbRbohF gene disclosed by the invention is as shown in SEQ ID No. 1. The BbRbohF gene provided by the invention can induce ROS accumulation by adjusting the ROS level under dark stress treatment so as to promote the shedding of the trigonometric plum blossom sequence, and after the gene is silenced, the cumulative shedding rate of the trigonometric plum blossom sequence can be effectively reduced.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically involving BbRbohF Application of genes in regulating bougainvillea inflorescence abscission. Background Technology

[0002] Organ abscission is a common phenomenon in the plant kingdom, a precise and highly programmed process of cell separation. Plants actively shed their reproductive and vegetative organs to adapt to different developmental stages or in response to external stimuli. This is a result of long-term evolution and is of great significance for species reproduction and dispersal. The rational use of organ abscission can also increase yield and improve crop quality. However, in an agricultural environment, premature or abnormal abscission means reduced productivity and a significant decrease in economic value. For ornamental plants, premature abscission of floral organs severely impacts their commercial and ornamental value.

[0003] Bougainvillea ( Bougainvillea Bougainvillea (spp.), also known as paper flower, is native to Brazil and Peru in South America. It is commonly referred to as a member of the genus Bougainvillea in the family Nyctaginacea. Bougainvillea Bougainvillea is a type of ornamental plant that thrives in bright light. During long-distance transport, it is susceptible to organ drop due to the stress of dark environments. In my country, bougainvillea is a typical example of a plant transported from the south to the north; potted bougainvillea often suffers severe inflorescence drop after long-distance transport, hindering its sale. Furthermore, prolonged exposure to low light levels indoors also easily leads to inflorescence drop. Therefore, minimizing the impact of dark environments during transport on the number of inflorescences is a pressing technical problem that needs to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide a method that can effectively control the shedding of bougainvillea inflorescences. BbRbohF Gene.

[0005] This invention provides a method for regulating the abscission of bougainvillea inflorescences. BbRbohF Genes, the ones mentioned BbRbohF

[0006] The present invention also provides the technical solutions described above. BbRbohF Application of genes in regulating bougainvillea inflorescence abscission.

[0007] Preferably, the regulation includes: overexpressing the above-described technical solution. BbRbohF Genes promote the abscission of bougainvillea inflorescences.

[0008] The present invention also provides a method for inhibiting the abscission of bougainvillea inflorescences, the method comprising: inhibiting the abscission described in the above technical solution. BbRbohF Gene expression.

[0009] Preferably, the inhibition includes silencing the expression of the BbRbohF gene described in the above technical solution.

[0010] Preferably, the primers for silencing the expression of the BbRbohF gene described in the above technical solution include BbRbohF-VIGS-F and BbRbohF-VIGS-R; the nucleotide sequence of BbRbohF-VIGS-F is shown in SEQ ID No. 6, specifically: 5'-GAGGTACAACAAAATTCGAATTCCACAA-3'; the nucleotide sequence of BbRbohF-VIGS-R is shown in SEQ ID No. 7, specifically: 5'-AGCCATTAGTTTAGCGCACATTTGATTGA-3'.

[0011] The beneficial effects of this invention are: Under stress conditions, the disruption of redox homeostasis in plants often leads to excessive accumulation of ROS, which in turn triggers plant organ abscission. The invention provides... BbRbohF Under dark stress, the gene can regulate ROS levels and induce ROS accumulation, thereby promoting the abscission of bougainvillea inflorescences. Silencing the gene can effectively reduce the cumulative abscission rate of bougainvillea inflorescences. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0013] Figure 1 A schematic diagram of the opening levels of bougainvillea inflorescences and the peduncle abscission zone (PAZ); Figure 2 Bougainvillea under the coercion of darkness Rboh Expression trends of gene family members in PAZ; Figure 3 For the present invention BbRbohF Gene( BbRboh22232 ) and Arabidopsis Rboh Family member phylogenetic tree analysis; Figure 4 for BbRbohF Gene sequence analysis; Figure 5 for BbRbohF Protein sequences and other species RbohF Protein sequence alignment results; Figure 6 PAZs under MV spraying treatment BbRbohF The expression; Figure 7 Bougainvillea BbDODA Silent bract phenotype; Figure 8 Under dark conditions BbRbohF Expression and cumulative shedding rate of silent plants; Figure 9 Bougainvillea BbRbohF NBT and DAB staining of silent plant tissues; Figure 10 This is a schematic diagram of the overexpression vector; Figure 11 The bracts of the overexpressing plant under ultraviolet light; Figure 12 for BbRbohF Expression and ROS level detection in bracts of overexpressing plants. Detailed Implementation

[0014] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0015] Example 1 Use one-year-old 'Water Red' bougainvillea ( Bougainvillea × buttiana 'MissManila' cuttings were purchased from the Tropical Crop Germplasm Resources Institute, Chinese Academy of Tropical Agricultural Sciences, as experimental material. The plants (with pots) were approximately 40cm tall and 20cm wide.

[0016] Based on the number of open bracts and the size of the bracts, 'Water Red' is divided into four grades, such as... Figure 1 As shown in (a~d), Figure 1The scale bar is 1cm. (a), (b), (c), and (d) represent S1, S2, S3, and S4 levels, respectively: S1 level: bracts approximately 1cm long, perianth tube not open; S2 level: bracts 2-3cm long, perianth tube not open; S3 level: bracts 3-4cm long, perianth tube fully open; S4 level: bracts 3-4cm long, perianth tube completely withered and curled. Under natural conditions, inflorescences usually abscission at S4, with S3 being the turning point. The peduncle (PAZ) of bougainvillea is morphologically distinct from the surrounding tissues, such as... Figure 1 As shown in (e).

[0017] Ten entries were downloaded from the Arabidopsis thaliana database. Rboh Family protein sequences were used to screen 10 candidate bougainvillea sequences using protein sequence BLAST. BbRboh The protein sequences are shown in Table 1.

[0018] Table 1 BbRboh Gene family member information

[0019] As can be seen from Table 1, most BbRbohs The mRNA sequence length is over 3000 bp, with the longest being 3849 bp. BbRboh22232 Below 3000bp are available. BbRboh10847 / 03619 / 07796 The shortest of them is BbRboh10847 It is only 1039bp. It can be seen that... BbRboh The significant differences in mRNA sequence length among gene families suggest functional diversity, potentially adapting to different cellular environments or stress conditions.

[0020] The 'Water Red' S2 grade inflorescences were marked, and then the bougainvillea plants were subjected to dark stress treatment. Starting on the day of treatment, samples were taken from the PAZ of the marked inflorescences at 18:00 on days 0, 2, 4, and 6: holding the inflorescence, the near end of the PAZ was cut off 2-3 mm with clean short-nose scissors, and then the far end was quickly cut off 2-3 mm. 15-20 PAZ tissues were taken as a replicate, and 7 replicates were taken. The tissues were wrapped in aluminum foil and placed in liquid nitrogen for storage at ultra-low temperature (-80℃) for later use.

[0021] RNA was extracted from all samples using the TRIzol method and then stored in an ultra-low temperature freezer (-80℃) for later use.

[0022] All RNAs were reverse transcribed using the HiScript IV All-in-one Ultra RT SuperMix for qPCR kit (Vazyme) to obtain their cDNA.

[0023] The cDNA was diluted 3-fold and subjected to qRT-PCR. The expression levels of each gene in Table 1 were analyzed to screen for key genes. The primer sequences are as follows: BbRbohF-OE-F: 5'-TCTCTCACTCTCCCAAGTCCC-3' (SEQ ID No. 2); BbRbohF-OE-R: 5'-GCATGATTTGTGCTCCGC-3' (SEQ ID No. 3).

[0024] The system for qRT-PCR reaction is shown in Table 2, and the reaction procedure is shown in Table 3.

[0025] Table 2 qRT-PCR reaction system

[0026] Table 3 qRT-PCR reaction procedure

[0027] Table 1 BbRboh03619 and BbRboh18404 The candidate genes were excluded from the screening process due to excessively low expression levels. The expression levels of the remaining candidate genes are as follows: Figure 2 As shown, * indicates a significant difference (two-tailed Student's t-test, ** P <0.01); the results show that only BbRboh22232 With increasing days of dark stress treatment, expression levels on days 2 and 6 were significantly higher than in the control under natural conditions. In summary, [the following was observed]: BbRboh22232 As the target gene, it will be used for subsequent functional verification experiments.

[0028] Phylogenetic analysis of Rboh family proteins from Arabidopsis thaliana and Bougainvillea was performed, such as... Figure 3 As shown, BbRboh22232 with Arabidopsis AtRbohF Their homology is most recent, therefore, BbRboh22232 Named BbRbohF .

[0029] right BbRbohF Full-length sequence analysis was performed, and the visualization results are as follows: Figure 4 As shown, BbRbohF The gene is 3802 bp in length, with a CDS region of 2775 bp that encodes 924 amino acids.

[0030] Comparison with protein sequences from other species showed that... BbRbohF The protein sequence contains four highly conserved domains ( Figure 5The N-terminus, from N to C, are NADPH_Ox (PF08414), Ferric_reduct (PF01794), FAD_binding_8 (PF08022), and NAD_binding_6 (PF0802), representing plants. Rbohs Four typical conserved domains, consistent with previous reports (Huetal., 2020). They are responsible for electron transport and ROS generation.

[0031] Example 2 In this embodiment, the virus-induced gene silencing (VIGS) and overexpression experiments were performed on potted annual bougainvillea 'Water Red' or 'Big Red' varieties. 'Big Red' bougainvillea ( Bougainvillea × buttiana 'San Diego Red'); Bougainvillea 'Scarlet' and 'Water Red' are both derived from Bougainvillea 'Bater' ( Bougainvillea × buttiana It is a cultivated variety of which is closely related to the plant.

[0032] Before the infection experiment, the plants were first induced to flower and then cared for. The specific steps are as follows: a. Pruning: After the inflorescences that came with the plant have completely fallen off, cut off the inflorescence stalks and large leaves, avoiding damage to the buds; cut off excessively long branches, leaving 2-3 buds at the bottom of each branch; cut off dead and messy branches to ensure full sunlight exposure; b. Controlled watering and fertilization: Follow the principle of small amounts and multiple times. Before the new inflorescence emerges, wait until the leaves soften and droop (about 5 days) and then apply a light water-fertilizer (1 / 1000 potassium dihydrogen phosphate). There is no need to water thoroughly. c. Once the plant has flower buds, stop applying any liquid fertilizer, apply solid slow-release fertilizer, resume watering, follow the principle of watering only when the soil is dry, and ensure the plant receives plenty of sunlight.

[0033] It takes about 30 days for the plant to produce inflorescences. Use the plants for experiments in a timely manner according to their growth stages to avoid missing the infection stage.

[0034] S2-level inflorescences were labeled and then treated with methylviologen (MV), a ROS inducer. Starting from the day of treatment, samples were taken from the PAZ of the labeled inflorescences at 18:00 on days 0, 1, 2, and 3. 15–20 PAZ tissues were considered a replicate, and five replicates were used for the target gene. BbRbohF The expression detection and sampling method are the same as in Example 1.

[0035] The presence of 0.2 mM MV in PAZ tissue was detected by qRT-PCR. BbRbohF The expression. For example... Figure 6As shown in the figure, * indicates a significant difference (two-tailed Student's t-test, *P<0.05); the results show that on the 2nd and 3rd day after treatment, the expression level of the treated cells was significantly upregulated compared with the control, and the expression level peaked on the 2nd day.

[0036] Selection BbDODA As a positive control gene for VIGS. The gene for Bougainvillea peruviana 'Thimma' was obtained via NCBI. BpDODA2-1 The mRNA sequence of the gene (LC542875.1) was used to select a conserved 405 bp fragment within the ORF box as a silencing fragment. Primers were designed using SnapGene software, and the primer sequences are as follows: BbDODA-VIGS-F: 5'-TCACACTTGGATGGTGAACACCACTATA-3' (SEQ ID No. 4); BbDODA-VIGS-R: 5'-ATCCATTGTTAACACGAGAAATCAGCTC-3' (SEQ ID No. 5).

[0037] The mRNA sequence of the target gene was cloned using cDNA from PAZs subjected to 'Water Red' dark stress on day 6 as a template, referring to the sequence identified in Example 1. BbRbohF Design primers based on mRNA sequences. BbRbohF The silenced fragment is 468 bp long and contains a partially 3' UTR-specific fragment; the overexpression fragment is 3237 bp long and contains the complete ORF frame. Primer sequences are as follows: BbRbohF-VIGS-F: 5'-GAGGTACAACAAAATTCGAATTCCACAA-3' (SEQ ID No. 6); BbRbohF-VIGS-R: 5'-AGCCATTAGTTTAGCGCACATTTGATTGA-3' (SEQ ID No. 7).

[0038] Reagents used: Vazyme Phanta Max Super-Fidelity DNApolymerase; Instrument used: Analytik Jena Biometra Tadvanced 96SG; Reaction system and procedure are as follows: Table 4. Target gene mRNA sequence cloning system

[0039] Table 5. Procedure for Cloning Target Gene mRNA Sequences

[0040] The amplified product was added to 10× loading buffer and then subjected to 1% agarose gel electrophoresis. After separation and purification to obtain the band of the target length, the gel was cut and recovered using the Cwbio Gel Extralation Kit. Buffer EB was used as a blank control. The concentration was detected, the information was recorded, and the product was stored at -20°C for later use.

[0041] TA Cloning The fragment cloned from the cDNA template was ligated to pTOPO using the TOPO-TA / Blunt Lightning Cloning Kit (GeneBetter) for convenient sequencing correction. The reaction system is as follows: Table 6 TA Cloning reaction system

[0042] The reaction system was gently mixed and allowed to bind at room temperature for 5 minutes.

[0043] E. coli transformation Remove 100 μL of Tolobio's DH5α chemocompetent cells from the -80℃ freezer, thaw them slowly on ice, and perform the conversion of the ligation product. The specific steps are as follows: a. Once the DH5α chemocompetents have melted to an ice-water mixture state, add the ligation product (≤10μL) to a clean bench, gently tap the bottom of the tube to mix, quickly insert it into ice, and let it stand and incubate for 30min. b. Heat shock: Quickly transfer the centrifuge tubes to a 42°C metal bath and let them stand for 90 seconds. Then quickly place them in ice and let them stand for 3 minutes. c. Recovery: In a clean bench, add 900 μL of blank LB medium, invert to mix, and incubate at 37°C and 220 rpm for 45 min in a shaker. d. Plate coating: Centrifuge at 5000×g for 1 min to collect bacteria, discard 900μL of supernatant, mix the bottom bacterial cells by pipetting, and evenly spread them on LB plates corresponding to the plasmid resistance. e. Culture: Invert the plate in a 37°C biochemical incubator and culture overnight.

[0044] bacterial PCR After single colonies grow on plates, pick them up with a 10 μL sterile pipette tip and pipette them into 200 μL of the corresponding antibiotic LB medium. Once the bacteria fall into the medium, discard the pipette tip. Incubate at 37℃ and 220 rpm for 3–5 h with shaking. Perform bacterial PCR. For bidirectional clones (TA clones), use upstream and downstream primers for the vector; for unidirectional clones (homologous recombination), use a combination of upstream (downstream) primers for the vector and downstream (upstream) primers for the gene clone to improve screening accuracy. Bacterial PCR was performed using 2×Magic Green Taq SuperMix, and the reaction system is as follows: Table 7 Bacterial PCR Reaction System

[0045] Table 8. PCR reaction procedure for bacterial culture

[0046] After the PCR products were detected by 1% agarose gel electrophoresis, the positive clone bacterial solutions were sent to Qingke for sequencing.

[0047] The target fragment was ligated to the silencing vector pTRV2 and the overexpression vector pCAMBIA1300-35S+35S-eGFP using homologous recombination.

[0048] After correction, the target fragment was used as a template by PCR to clone a recombinant fragment containing homologous sequences corresponding to the restriction enzyme sites. The concentrations of the recombinant fragment and the linearized vector were determined using a Thermo Micro UV-Vis spectrophotometer, and the recombination reaction was carried out at a molar ratio of 4:1 using the Vazyme ClonExpress II One-Step Cloning Kit.

[0049] After overnight shaking culture of the correctly sequenced bacterial culture, some was stored at -80℃ with a bacterial culture: 50% glycerol ratio of 1:1, while the rest was used for plasmid extraction using the PurePlasmid Mini Kit (Cwbio) to obtain the silencing vector pTRV2- BbRbohF and pTRV2- BbDODA .

[0050] VIGS In this embodiment, VIGS is mediated by GV3101 (Tolobio), using pTRV1, pTRV2 empty vectors, and pTRV2- BbRbohF pTRV2- BbDODA Transformation GV3101 infected bougainvillea 'Water Red' or 'Big Red' potted plants.

[0051] Agrobacterium infection of bougainvillea When the inflorescence of 'Water Red' or 'Big Red' is prior to or at the S1 level, VIGS shaking and infection can be performed. The specific operation steps are as follows: a. Prepare YEB plates and liquid culture medium, adding 1 / 1000 Kan and 1 / 2500 Rif; b. Stirring: Take out the corresponding frozen bacteria at -80℃ and thaw them slowly on ice. Stir the bacteria in three zones on a YEB plate and incubate at 28℃ for 36 hours. c. Small shake: In a 1.5 mL centrifuge tube, pick up a single colony with a 10 μL pipette tip and add it to 100 μL of YEB medium. Incubate at 28 °C and 200 rpm for 14 h. d. In-process shaking: Take 100 μL of the positive small bacterial culture obtained by PCR verification into a 50 mL centrifuge tube, add it to 10 mL of LYEB medium, and incubate at 28 °C and 200 rpm for 14 h. e. Large shaking: Add 2 mL of medium-shaking bacterial culture to 200 mL of YEB medium in a 500 mL Erlenmeyer flask, and incubate at 28 °C and 200 rpm for 14 h until OD600 = 0.6~0.8, indicating that the strain is in the logarithmic growth phase; f. Dispense the large-volume bacterial culture into 50mL centrifuge tubes, centrifuge at 4200rpm for 15min to collect bacteria, and discard the supernatant. g. Resuspend the bacterial cells in the infection solution (each 100 mL system contains 1 mL 1M MgCl2 + 1 mL 1M MES + 0.4 mL 50 μM AS, pH 5.6), OD600 = 0.8; the resuspended pTRV2, pTRV2-BbRbohF, and pTRV2-BbDODA bacterial cells were mixed with the resuspended pTRV1 bacterial cells at a 1:1 ratio, with a volume of approximately 4 L, and then poured into a 5 L beaker and incubated at 28 °C for 3 h; h. Remove the inflorescences above S1 in advance, and wrap the mouth of the flower pot with plastic wrap to prevent the substrate from falling into the dye solution; add 1 / 2000 Tween20 to each group of dye solutions and stir well. i. In a cylindrical container with a height of 50cm and a diameter of 38cm, place the bougainvillea plant upside down, use a support of appropriate height to hold the pot in place, and try to immerse the upper part of the plant in the infusion solution. j. Turn on the vacuum pump and wait for the negative pressure to rise to 0.8 MPa. Maintain this pressure for 10 minutes and then slowly introduce air for 15 minutes. You will see that the inflorescence and leaves are obviously waterlogged, which indicates that the penetration is sufficient. k. Rinse the infected parts of the plant with clean water, gently shake off excess water, transfer the plant to a dark incubator at 18℃, set the humidity to 100%, and incubate for 48 hours. After that, transfer it to an open-air environment for normal maintenance and management.

[0052] After the inflorescences have grown to S2 (approximately 12 days) in an open-air environment, the positive control gene will be recorded. BbDODA The inflorescence silencing phenotype; the target gene BbRbohF Silent plants and controls were transferred to a climate chamber (temperature 26℃, humidity 60%, light duration 0h) for dark stress treatment. Absence rate and ROS level were then measured (S2). For sampling, short-nose scissors were used to cut the inflorescence close to the base, which was then wrapped in aluminum foil and placed in liquid nitrogen. Five replicates were taken, with one inflorescence per replicate, for the target gene. BbRbohF Expression detection.

[0053] While silencing the target gene, use BbDODA Silent 'Water Red' and 'Big Red' plants were used as positive controls. DODA is a key enzyme in the betaine synthesis pathway, and its silencing causes bleaching of bougainvillea bracts. Figure 7 As shown, Figure 7 (a) is the silent bract phenotype of Bougainvillea 'Water Red' BbDODA, where white spots appear on the silent 'Water Red' Bougainvillea bracts; Figure 7 The bracts in the middle (b~e) are silent 'big red' and also show a spotted white phenotype, indicating the successful establishment of the VIGS system.

[0054] Using qRT-PCR to analyze bract samples from silent and control plants BbRbohF The expression level was detected, and the results were as follows: Figure 8 , where 'a' represents the condition under darkness BbRbohF Silent bracts express; b indicates dark conditions. BbRbohF Cumulative abscission rate of silent plants; * indicates significant difference (two-tailed Student's t-test, * P <0.05,** P <0.01); c represents the condition under darkness. BbRbohF Silent plant phenotype, scale bar = 5cm.

[0055] like Figure 8 As shown in (a), the expression levels in silenced plants significantly decreased on days 2 and 4 of the dark stress treatment, indicating that TRV silencing was effective. Subsequently, statistical analysis was performed... BbRbohF The cumulative abscission rate of silent plants, such as Figure 8 As shown in (b-c), on day 6 of the dark stress treatment, the cumulative shedding rate of the silent plants was significantly lower than that of the control. Therefore, it can be inferred that: BbRbohF It accelerated inflorescence drop during the dark stress treatment of bougainvillea.

[0056] This experiment simultaneously measured the ROS levels in both silent plants and control plants. Figure 9 As shown, 'a' represents the O2 accumulated in the NBT (nitro blue tetrazolium) colored PAZ and bracts.- b represents the H2O2 accumulated in the PAZ and bracts colored by DAB (3,3'-diaminobenzidine).

[0057] Depend on Figure 9 It can be seen that in the PAZ (peduncle abscission zone), the O2 of the control plants was higher. - H2O2 began to accumulate significantly on day 4, while silent plants showed no obvious coloration; ROS accumulation in bracts was similar to that in PAZ, with the most significant coloration occurring on day 6. From this, we can further infer that: BbRbohF Darkness stress treatment promoted bougainvillea inflorescence abscission by regulating ROS levels.

[0058] Overexpression experiment In this embodiment, the overexpression assay was a transient transfection mediated by the GV3101 strain containing pCAMBIA1300::35S-BbRbohF+35S-eGFP. Figure 10 pCAMBIA1300::35S-Empty+35S-eGFP was used as a control, and the method is as follows: a. Preparation of infection materials: The infection target is the 'Shuihong' plant whose inflorescence has grown to S2 after flowering. One pot of control and one pot of treatment are infected. Before infection, the inflorescences other than S2 are removed. b. The specific shaking procedure and infection solution preparation method are the same as for VIGS. Prepare 4L of resuspension for both the control and treatment. c. After infection, the cells were transferred to an incubator and cultured in the dark at 18°C ​​and 100% humidity for 12 hours. Then the parameters were changed to 26°C, 60% humidity, and 12 hours of darkness / 12 hours of light. After 48 hours of culture, the phenotype was observed and samples were taken.

[0059] eGFP fluorescence was observed under UV light to confirm transformation efficiency, and photographs were taken for recording. ROS levels in bracts were sampled and analyzed. Inflorescences were cut off close to the base using short-nose scissors, with 8 inflorescences from each of the control and treatment groups used as replicates for the target gene. BbRbohF Expression detection.

[0060] This embodiment uses the strong promoter 35S to overexpress [the gene] in the bougainvillea inflorescence. BbRbohF The gene and vector were appended with 35::eGFP. Fluorescence was observed under ultraviolet light, as shown... Figure 11 As shown, a represents the blank control; b represents the negative control; and c represents the overexpression control. BbRbohF Processing; Scale = 1cm. From Figure 11 As can be seen, the blank control showed no obvious fluorescence, while the negative control and overexpression treatment both showed fluorescence, indicating that GV3101 infection was complete and the vector could be expressed normally in the bracts.

[0061] qRT-PCR was performed on inflorescence samples from overexpressing and control plants, and the results are as follows: Figure 12 As shown, where a represents BbRbohF Results of expression level detection in bracts of overexpressing plants; b indicates BbRbohF Results of ROS level detection in bracts of overexpressing plants.

[0062] like Figure 12 As shown in Figure a, in overexpressing plants BbRbohF The significant upregulation indicates that the overexpression system is effective. Similarly, DAB and NBT were used to detect the ROS levels in the bracts, such as... Figure 12 As shown in Figure b, the overexpressing bracts accumulated significantly more ROS than the control, fully demonstrating that... BbRbohF It can synthesize ROS within the plant.

[0063] As can be seen from the above embodiments, BbRbohF It is a key gene that regulates the accumulation of ROS in bougainvillea induced by dark stress. It can increase the ROS level in bougainvillea PAZ and inflorescence, thereby promoting inflorescence abscission. Silencing this gene can effectively reduce the cumulative abscission rate of bougainvillea inflorescences.

[0064] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for regulating the abscission of bougainvillea inflorescences BbRbohF Genes, characterized by, The BbRbohF The nucleotide sequence of the CDS region of the gene is shown in SEQ ID No.

1.

2. The claim 1 BbRbohF Application of genes in regulating bougainvillea inflorescence abscission.

3. The application according to claim 2, characterized in that, The regulation includes: overexpression of the substance described in claim 1. BbRbohF Genes promote the abscission of bougainvillea inflorescences.

4. A method for inhibiting the abscission of bougainvillea inflorescences, characterized in that, The method includes: suppressing the method described in claim 1. BbRbohF Gene expression.

5. The method according to claim 4, characterized in that, The suppression includes silencing as described in claim 1. BbRbohF Gene expression.

6. The method according to claim 5, characterized in that, Silence as described in claim 1 BbRbohF Primers for gene expression include BbRbohF -VIGS-F and BbRbohF -VIGS-R; the BbRbohF The nucleotide sequence of -VIGS-F is shown in SEQ ID No.

6. BbRbohF The nucleotide sequence of -VIGS-R is shown in SEQ ID No. 7.

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