Application of ca-btb36 gene or protein coded thereby in regulating plant type of pepper, recombinant vector and method for regulating plant type of pepper

Abstract

This application relates to the field of plant biology technology and provides a CaBTB36 Application of genes or their encoded proteins in regulating pepper plant architecture, recombinant vectors, and methods for regulating pepper plant architecture. CaBTB36 The gene nucleotide sequence is shown in SEQ ID NO.1, and its encoded protein sequence is shown in SEQ ID NO.2. The recombinant vector contains... CaBTB36 Methods for regulating pepper plant architecture by silencing specific gene segments: CaBTB36 This gene significantly reduces plant height, hypocotyl length, and first internode length in chili peppers. This invention, through virus-induced gene silencing (VIGS) technology, successfully inhibits this gene in chili peppers for the first time. CaBTB36 Gene expression. Experimental results show that silencing... CaBTB36 After gene modification, the plant height, hypocotyl length, and first internode length of the pepper plants were significantly reduced, and the overall plant type was noticeably dwarfed and compact.

Description

Technical Field

[0001] This application relates to the field of plant biology technology, specifically to a... CaBTB36 Application of genes or their encoded proteins in regulating chili plant architecture, recombinant vectors, and methods for regulating chili plant architecture. Background Technology

[0002] Plant architecture is a comprehensive reflection of a plant's phenotypic characteristics, including plant height, branching pattern, and the morphological characteristics and spatial arrangement of leaves and floral organs. The structure of these agronomic traits determines a plant's growth and development pattern, its adaptability to the environment, and its yield potential. Dwarfing is an important agronomic trait in higher plants. In annual crops, dwarfing not only significantly enhances the plant's resistance to lodging but also promotes intensive cultivation and effectively increases the harvest index. Currently, with the continuous expansion of chili pepper cultivation and the increasing market demands for yield and quality, how to achieve more efficient planting management through plant growth regulation has become a research hotspot. Among these, the dwarfing characteristics of chili peppers have attracted considerable attention. Dwarfed chili peppers often exhibit stronger lodging resistance, improve land use efficiency, utilize light energy more rationally, facilitate mechanized management and harvesting, and reduce labor costs while increasing chili pepper productivity. Therefore, breeding dwarf chili pepper varieties has become an important direction for improving planting efficiency, and identifying the key genes that regulate chili pepper dwarfing is the core approach to achieving this goal.

[0003] In recent years, several key genes involved in the regulation of plant height and their mechanisms of action have been reported. For example, in Arabidopsis thaliana... AtCPS Alleles AtCPS-168 Mutation can lead to ga1-168 The mutant exhibits dwarfing, shortened hypocotyl, late-flowering phenotype, and reduced GA synthesis; overexpression GA2ox Arabidopsis thaliana, switchgrass and transgenic JcGA2ox Arabidopsis thaliana all exhibited dwarfism; mutations in the YUCCA gene (a key enzyme gene for auxin synthesis) led to plant dwarfism; the dominant dwarfing gene DS-4 (encoding BnaC05.IAA7) in rapeseed, due to a substitution of conserved motif amino acids, blocked its interaction with TIR1, inducing dwarfism, and was highly expressed in multiple organs of the mutant; overexpression in rice OsARF19 This leads to stunted plant growth. OsARF12 Edited mutants showed significantly reduced plant height and shortened internodes near the base; overexpression of PcPIN-L increased plant height in tobacco; and the rice BR synthase gene... CYP90A3 / CYP90A4 The double mutant is a BR-deficient type, exhibiting dwarfism that can be restored by exogenous application of 24-epibrassin sterol; cotton GhCYP85A2-1 Plant height was significantly reduced after silencing, and overexpression was observed. GhBIN2 Both cotton and Arabidopsis thaliana are dwarfed; overexpression SlRR6The increased height of tomato plants can be reversed by exogenous application of paclobutrazol or IAA, and SlRR6 and SlSAUR58 Interactions can lead to overexpression of a dwarfed phenotype.

[0004] BTB (Broad-Complex, Tramtrack, and Bric-a-brac) domain proteins are a class of multifunctional proteins widely found in eukaryotes. Through their conserved protein-protein interaction interfaces, they play a crucial "bridge" or "hub" role in core biological processes such as the ubiquitin-proteasome pathway, transcriptional regulation, and cytoskeleton assembly. In plants, the BTB gene family has numerous members, and their functions often depend on chimerism with other domains such as ANK and MATH. This allows them to participate in complex networks involving photomorphogenesis, hormone signal transduction (such as auxin and abscisic acid), and responses to abiotic stresses, profoundly influencing plant growth, development, and adaptability.

[0005] Plant architecture is a key agronomic trait determining crop yield potential and planting density, and its development is precisely regulated by the synergistic interaction of genetic programs and environmental signals. Recent studies have shown that BTB proteins are important components in the plant architecture regulatory network. Research, exemplified by the TRU1 gene in maize and the OsBTB series in rice, reveals that BTB proteins can precisely influence processes such as apical dominance, axillary bud initiation and elongation by mediating the ubiquitination and degradation of specific regulatory factors, thereby directly determining the branching pattern and overall structure of plants. These findings not only elucidate the molecular basis of crop domestication and improvement but also provide key targets for molecular design breeding.

[0006] However, current research on the function of plant BTB genes is highly concentrated on model plants or major crops such as maize, rice, and Arabidopsis thaliana. For chili peppers, a globally important vegetable and economic crop, although the BTB gene family has been preliminarily identified at the genomic level, the functions of the vast majority of members remain unknown. Of particular interest is the fact that chili peppers have specific requirements for plant architecture (such as the number of branches, plant spread, and stem strength) to adapt to different cultivation methods and mechanization needs, but the key genes controlling these traits have not been fully explored. Whether and how chili pepper BTB genes participate in plant architecture is an important scientific question that has not yet been explored. Summary of the Invention

[0007] The present invention aims to solve the above-mentioned problems in the prior art and provide a solution. CaBTB36 Application of genes or their encoded proteins in regulating chili plant architecture, recombinant vectors, and methods for regulating chili plant architecture.

[0008] To achieve the above objectives, the first aspect of this application provides a CaBTB36 The application of genes or their encoded proteins in regulating pepper plant architecture, the CaBTB36 The gene nucleotide sequence is shown in SEQ ID NO.1; its encoded protein sequence is shown in SEQ ID NO.2.

[0009] In the above-mentioned application, preferably, the plant type includes the plant height, hypocotyl length, and first internode length.

[0010] Of the above applications, the preferred method is to reduce [the risk of infection] through virus-induced gene silencing technology. CaBTB36 Gene expression levels and / or activity in chili peppers.

[0011] In the above-described application, preferably, the virus-induced gene silencing technology includes the following steps:

[0012] (1) Constructing a structure containing CaBTB36 Viral silencing vectors containing gene-specific fragments, wherein the gene-specific fragments are selected from... CaBTB36 The 554-854 bp region of the gene coding region; (2) The virus silencing vector from step (1) is introduced into Agrobacterium to obtain recombinant Agrobacterium; (3) Infect pepper tissues or organs with the recombinant Agrobacterium.

[0013] The above-mentioned applications, preferably, are as follows: CaBTB36 Primer pairs for amplifying gene-specific fragments include: Forward primer: TGCGGAAAAAGAGGACAAGG; Reverse primer: AATCATTTGGTTGGTCACAA.

[0014] In the above application, preferably, the virus silencing vector is the pTRV2 vector, the Agrobacterium is the GV3101 strain, the OD600 of the Agrobacterium bacterial suspension at infection is 0.6-0.8, and the Agrobacterium carrying the pTRV1 helper vector and the Agrobacterium carrying the pTRV2- CaBTB36 The Agrobacterium vectors were mixed at a volume ratio of 1:1.

[0015] A second aspect of this application provides a method for silencing CaBTB36 A recombinant vector containing genes. CaBTB36 Specific segments of genes, said specific segments being selected from CaBTB36 The 554-854 bp region of the gene coding region.

[0016] Preferably, the recombinant vector described above is pTRV2- CaBTB36 The above primers were used to amplify the resulting product. CaBTB36 pTRV2- was obtained by inserting a gene-specific fragment into the pTRV2 vector. CaBTB36 .

[0017] A third aspect of this application provides a method for regulating the plant architecture of chili pepper plants by silencing [the plant's] plant form. CaBTB36 The gene can significantly reduce the plant height, hypocotyl length, and first internode length of chili peppers.

[0018] The preferred method described above involves silencing genes using virus-induced gene silencing technology. CaBTB36 The virus-induced gene silencing technology includes the following steps: (1) Constructing a structure containing CaBTB36 Viral silencing vectors containing gene-specific fragments, wherein the gene-specific fragments are selected from... CaBTB36 The 554-854 bp region of the gene coding region; (2) The virus silencing vector from step (1) is introduced into Agrobacterium to obtain recombinant Agrobacterium; (3) Infect pepper tissues or organs with the recombinant Agrobacterium.

[0019] Compared with the prior art, this application has the following beneficial effects: (1) This invention successfully inhibits the virus-induced gene silencing (VIGS) technique in chili peppers for the first time. CaBTB36 Gene expression. Experimental results show that silencing... CaBTB36 Following gene modification, the plant height, hypocotyl length, and first internode length of chili peppers were significantly reduced, resulting in a noticeably dwarfed and compact plant type. This discovery fills a gap in research on the role of the BTB family genes in regulating plant type in chili peppers, providing important functional genes and theoretical basis for subsequent precise improvement of chili pepper plant type using molecular breeding methods.

[0020] (2) This invention achieves its effect through silence. CaBTB36 Genes have enabled chili pepper plants to simultaneously shorten plant height, hypocotyl length, and first internode length while maintaining normal growth and development. This dwarf and compact plant type offers the following agricultural production advantages: Lower plant height lowers the plant's center of gravity, relatively increasing stem bending resistance and effectively reducing lodging losses caused by wind, rain, or excessive fruit load; the smaller plant size reduces the plant's footprint and shading effect, promoting intensive cultivation and improving land utilization and light energy efficiency; and it facilitates mechanized operations: the uniform height and compact structure of the dwarf plants facilitate mechanized sowing, fertilization, spraying, and harvesting, significantly reducing labor costs. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application 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 some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 For "Zhangshugang" chili peppers and CaBTB36 Comparison of gene-silenced pepper plants: a) pTRV2 control and pTRV2- 45 days after Agrobacterium injection. CaBTB36 Plant photos; b is the pTRV2 control and pTRV2. -CaBTB36 Relative expression levels of target genes under treatments of pTRV2-PDS and pTRV2-PDS; c represents plant height; d represents hypocotyl length; e represents first internode length; scale bar = 5 cm; data are mean ± SD (n = 10), ns (p>0.05). Note: ns indicates no statistical significance (P>0.05). P<0.05, P<0.01, P<0.001, P < 0.0001 (t-test). Detailed Implementation

[0023] To facilitate understanding of this application, the following description will be more comprehensive and detailed in conjunction with the accompanying drawings and preferred embodiments, but the scope of protection of this application is not limited to the following specific embodiments.

[0024] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of this application.

[0025] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0026] Example 1: CaBTB36 Gene cloning and sequence analysis 1. Extraction of total RNA from pepper leaves The leaves of Zhangshugang pepper were thoroughly ground into powder using liquid nitrogen grinding. All pipette tips, centrifuge tubes, and other equipment used in the experiment were RNase-free. Gloves and masks were worn throughout the procedure, and the equipment was disinfected with 75% alcohol. RNA extraction was performed according to the instructions for the EZNA™ Plant RNA Kit.

[0027] 2. Cloning of the target gene Using cDNA from Zhangshugang pepper leaves as a template, specific primers were designed based on known gene sequences using PrimerPremier 5.0 software: CaBTB36-F (SEQ ID NO.7): ATGTCATCCGATAACTGTTC; CaBTB36-R (SEQ ID NO. 8): CTAGCCCTTGCTCACCATGGT.

[0028] The target gene CDS sequence was amplified using PrimeSTAR® high-fidelity enzyme. The RT-PCR amplification program was as follows: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 30 s, 57 °C annealing for 30 s, 72 °C extension for 90 s, for 30 cycles; 72 °C final extension for 10 min, and storage at 12 °C.

[0029] 3. Gel recovery and purification After the PCR reaction, electrophoresis loading buffer was added to the amplification products for agarose gel electrophoresis. The electrophoresis results were observed using a gel imaging system. A gel block with clear bands and a length consistent with the target gene size was excised and placed in a 2 mL sterile centrifuge tube. The gel was then purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (9762). The specific steps are as follows: (1) Add Buffer GM, which is 3 times the volume of the glue block, to the centrifuge tube containing the glue block; (2) Solve the gel at 37 ℃ for 10 min. After the gel block is completely melted, transfer the sol to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the lower layer of filtrate. (3) Add 700 μL of Buffer WB (pre-added with 56 mL of anhydrous ethanol) to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate; (4) Repeat the above washing steps once; (5) Centrifuge the empty adsorption column at 12,000 rpm for 1 min to completely remove residual washing liquid; (6) Transfer the adsorption column to a brand new 1.5 mL centrifuge tube, add 30 μL of sterile ddH2O to the center of the adsorption column membrane, and let it stand at room temperature for 1 min to elute the DNA. (7) The concentration and purity of the recovered DNA were determined using a micro spectrophotometer, and the sample was stored at -20 ℃ for later use.

[0030] 4. T-carrier connection To stably preserve the gene fragment, the cloned target fragment needs to be ligated into the pMD19-T vector (6013, TaKaRa). The obtained DNA product was first tailed using a DNA A-Tailing Kit (6109, TaKaRa), and the PCR amplification program was set to: 72 °C for 20 min, followed by 4 °C for 2 min. Long-term storage was maintained at -20 °C. The system is shown in Table 1.

[0031] Table 1: Reaction system with A-tailing

[0032] Subsequently, the A-tailed product was ligated to the pMD19-T support. The ligation reaction system was as follows, and the reaction was carried out at 16 °C for 30 min.

[0033] Table 2: Connection Reaction System

[0034] 5. Transformation of competent cells and identification of positive strains The prepared ligation product was added to 100 μL of *E. coli* DH5α competent cells, gently mixed, and incubated on ice for 35 min. Then, the cells were heat-shocked in a 42 ℃ water bath for 90 s, and quickly removed and cooled on ice for 5 min. In a clean bench, 500 μL of antibiotic-free LB broth (formulation: 5 g tryptone, 2.5 g yeast extract, 5 g NaCl, diluted to 500 mL with distilled water, autoclaved) was added, and the cells were incubated at 37 ℃ and 200 rpm for 1 h with shaking to complete cell recovery.

[0035] Take an appropriate amount of the revived bacterial culture and spread it evenly on LB solid medium containing ampicillin resistance (formula: 5 g tryptone, 2.5 g yeast extract, 5 g NaCl, 7.5 g agar powder, diluted to 500 mL with distilled water, autoclaved, cooled, and then ampicillin antibiotic added). Seal the plate, invert it, and incubate overnight at 37 ℃. The next day, pick a plump single colony in a clean bench and inoculate it into LB liquid medium containing ampicillin resistance. Incubate at 37 ℃ with shaking at 200 rpm until the bacterial culture becomes noticeably turbid.

[0036] Using turbid bacterial culture as a template, PCR identification of the bacterial culture was performed using common Taq enzyme and universal primers. The PCR amplification instrument program was set as follows: 95 °C for 5 min; 95 °C for 30 s–55 °C for 30 s–72 °C for 2 min (30 cycles); 72 °C for 5 min. The reaction system is shown in Table 3 below.

[0037] Table 3: Reaction System

[0038] Primer sequences: M13-F (T-loaded universal primers, SEQ ID NO.16): CGCCAGGGTTTTCCCAGTCACGAC; CaBTB36-R (SEQ ID NO. 8): CTAGCCCTTGCTCACCATGGT.

[0039] 6. Gene sequence bioinformatics analysis Sequencing results analysis showed that the full-length CDS sequence of the CaBTB36 gene is 1050 bp, and the relative molecular mass of the encoded amino acid sequence is 39.93 kDa. Conserved domain prediction results indicated that the protein encoded by this gene contains a typical BTB / POZ conserved domain, suggesting that this gene belongs to the BTB family.

[0040] CaBTB36 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0041] SEQ ID NO.1

[0042] SEQ ID NO.2 MSSDNCSGDIDLFSGEMSETDIQVVTSGGLRIPAHSAVLASSSTVLENILVRPEKLRSSERTIRILGVPCDAVSVFIRFLYSSKCTEEQMERYGIHLLALSHVYLVPQLKQTCTKGLAKRLTIENAVDVLQLARLCDAPDLYLRSMKFLSSNFKKVEETEGWKFLQDHDPLLELE ILQFMDEAELRKKRTRRHKRELSLYLQLSEAMDCLEHICTEGCTSVGPCDKEPSYETQPCSKFDTCQGLQLLIRHFATCKRRTNGSCLRCKRMWQILRLHSSICDQPNDCRVPLCRQFKLKVQQRGDDDLWKSLVRKVVSARAMSSLSLPKRKRVDEPKMDLGHHEVMNFRLAA.

[0043] Example 2: CaBTB36 Virus-induced gene silencing (VIGS) in genetically modified peppers 1. Target fragment selection and primer design Using the VIGS online tool on the Solanaceae Genome website (http: / / vigs.solgenomics.net), select... CaBTB36 A specific fragment within the coding sequence (CDS) was identified, with the target region being 554-854 bp of the CDS. Specific amplification primers were designed, and adapter sequences were added to the 5' ends of both the forward and reverse primers. The primer sequences are as follows: Forward primer (SEQ ID NO.3): TGCGGAAAAAGAGGACAAGG; Reverse primer (SEQ ID NO.4): AATCATTTGGTTGGTCACAA; Connector sequence: Forward connector (SEQ ID NO.5): CTGTGAGTAAGGTTACCGAATTC; Reverse connector (SEQ ID NO.6): CGCGTGAGCTCGGTACCGGATCC.

[0044] Simultaneously, a pTRV2-PDS positive control vector was set up. A 300 bp fragment from the pepper PDS gene (phytopenic oleoresin dehydrogenase gene), confirmed by specificity analysis, was used as the positive control silencing fragment, the nucleotide sequence of which is shown in SEQ ID NO.11. PDS gene-specific upstream and downstream primers were designed, and adapter sequences matching the ends of the pTRV2 linearized vector were added to the 5' ends of the upstream and downstream primers, respectively, to enable homologous recombination ligation of the amplified fragment with the pTRV2 vector digested with EcoRI and BamHI. The amplification primers for the PDS gene fragment are as follows: CaPDS-F (SEQ ID NO.9): AGGTCTTCTTTGGGAACTGATAG; CaPDS-R (SEQ ID NO. 10): AGACAAACCACCCAAACCTGC.

[0045] PDS positive control silenced fragment (SEQ ID NO.11): AGGTCTTCTTTGGGAACTGATAGTCAAGATGGTTGCTCGCAAAGGAATTCGTTATGTTTTGGTGGTAGTGACTCAATGAGTCATAGGTTAAAGATTCGTAATCCCCATTCCATAACGAGAAGATTGGCTAAGGATTTCCGGCCTTTAAAG GTTGTTTGCATTGATTATCCAAGGCCAGAGCTAGACAATACAGTTAACTATTTGGAGGCTGCATTCTTATCATCATTCCGATCTTCTCCGCGCCCAACCAAACCACTGGAGATTGTTATTGCTGGTGCAGGTTTGGGTGGTTTGTCT.

[0046] 2. Vector linearization and target fragment amplification The pTRV2 vector was double-digested with EcoRI and BamHI (37 ℃, 3 h), and the linearized vector was recovered after electrophoresis. Using chili cDNA as a template, PCR amplification was performed using the aforementioned primers. The PCR reaction mixture (50 μL) consisted of 2 μL template cDNA, 2 μL each of forward and reverse primers (10 μM), 25 μL 2×Taq Master Mix, and ddH2O to a final volume of 50 μL. Reaction conditions were: 95 ℃ pre-denaturation for 5 min; 95 ℃ denaturation for 15 s, 58 ℃ annealing for 15 s, and 72 ℃ extension for 30 s, for a total of 35 cycles; and a final extension at 72 ℃ for 5 min. The PCR products were identified by 1.5% agarose gel electrophoresis, and the target fragment was recovered using a Novizan product purification kit.

[0047] 3. Construction and identification of recombinant plasmids The linearized pTRV2 vector and the recovered PCR product were ligated homologously using the In-Fusion HD Cloning Kit (Clontech) according to the manufacturer's instructions. The ligation product was transformed into *E. coli* DH5α and plated on LB agar plates containing kanamycin (50 μg / mL), incubated overnight at 37 °C. Single colonies were picked for colony PCR identification, and plasmids were extracted from positive clones for sequencing verification. The correctly sequenced recombinant plasmid was named pTRV2- CaBTB36 .

[0048] 4. Construction of pTRV2-PDS positive control recombinant plasmid To verify the effectiveness of the VIGS system, a positive control recombinant plasmid for PDS gene silencing was constructed simultaneously. The PDS gene fragment was amplified using cDNA from Zhangshugang pepper leaves as a template. The PCR reaction system and amplification program were the same as those used in the VIGS system. CaBTB36 The target fragments were amplified identically; the PCR products were identified and purified by 1.5% agarose gel electrophoresis, and then homologously ligated with the linearized pTRV2 vector according to the In-FusionHD Cloning Kit instructions; the ligation products were transformed into Escherichia coli DH5α, plated on LB plates containing kanamycin (50 μg / mL), and incubated overnight at 37 ℃; single colonies were picked for colony PCR identification, plasmids were extracted from positive clones and sequenced for verification, and the recombinant plasmid with correct sequencing was named pTRV2-PDS.

[0049] 5. Preparation of Agrobacterium-mediated transformation and infection solution pTRV1 helper vector, pTRV2 empty vector (negative control), and pTRV2- were added. CaBTB36 The pTRV2-PDS recombinant plasmid was transformed into Agrobacterium GV3101 competent cells. The cells were plated on LB agar plates containing kanamycin (100 μg / mL) and rifampin (50 μg / mL) and incubated at 28 °C for 48 h. Single colonies were picked for PCR identification; positive colonies were used for subsequent experiments.

[0050] Select a single positive Agrobacterium colony and inoculate it into 1 mL of LB liquid medium (containing 100 μg / mL Kan + 50 μg / mL Rif), and incubate at 28 ℃ and 200 rpm with shaking for 12–16 h. Transfer the culture to induction LB medium (100 μg / mL Kan + 50 μg / mL Rif + 10 mmol / L MES + 20 μmol / L AS) at a ratio of 1:20, and continue incubation at 28 ℃ and 200 rpm for 12–16 h.

[0051] Centrifuge the cultured bacterial suspension at 5000 rpm for 10 min, discard the supernatant, and collect the bacterial cells. Resuspend the cells in infection buffer (using sterile water as stock solution, 10 mmol / L MES, 10 mmol / L MgCl2, 200 μmol / L As, pH=5.6) until the OD600 is 0.6-0.8. Incubate at room temperature for 3 h. Before infection, Agrobacterium containing pTRV1 is separately inoculated with empty vector containing pTRV2, pTRV2-PDS, and pTRV2- CaBTB36 Agrobacterium was mixed at a volume ratio of 1:1 to form the following three groups of infection solutions: pTRV2 empty vector control group, pTRV2- CaBTB36 Experimental group and pTRV2-PDS positive control group.

[0052] 6. Infection and cultivation of chili pepper plants The tested chili pepper variety was 'Zhangshugang'. After disinfection, seeds were sown in seedling substrate and cultured in a light-controlled incubator (temperature 25 ℃ / 20 ℃, photoperiod 16 h light / 8 h dark). When the seedlings were about two weeks old (cotyledons fully expanded but before the first true leaf emerged), Agrobacterium infection solution was injected into the abaxial surface of the cotyledons using a 1 mL needleless syringe. The injected plants were then placed in a 18 ℃ incubator for one day in the dark, and then transferred to normal light conditions (25 ℃, 16 h light / 8 h dark). Ten plants were injected per treatment, repeated three times. The pTRV2-PDS positive control group was compared with the pTRV2 empty vector control group, and the pTRV2- CaBTB36 The experimental groups were treated synchronously with the same seedling age, the same cotyledon back injection method, and the same culture conditions. Phenotypic changes in the new leaves of the pTRV2-PDS positive control group were observed 14-21 days after Agrobacterium infection injection. The positive control group showed stable and obvious photobleaching, white spots, or white leaves, while the pTRV2 empty vector control group did not show the same whitening phenotype and remained normal green. These results indicate that the TRV-VIGS system was effectively activated in this batch of pepper plants.

[0053] 7. CaBTB36 Detection of gene silencing efficiency Forty-five days after Agrobacterium injection, pTRV2 empty vector control group and pTRV2- CaBTB36 Total RNA was extracted from young leaves of the experimental group and the pTRV2-PDS positive control group after flash freezing in liquid nitrogen. Genomic DNA was removed by treatment with DNase I. cDNA was then obtained by reverse transcription.

[0054] Using the capsicum actin gene as an internal control, a design was developed. CaBTB36 Gene-specific quantitative primers: CaBTB36-QF (SEQ ID NO.12):AGGAACCTCCCTACGAGACG; C aBTB36 -QR (SEQ ID NO.13):TGCATCGCAAACAACTTCCA.

[0055] The positive control primers for PDS gene quantification are as follows: PDS-QF (SEQ ID NO. 14): CTGGAGGCAAGGGATGTTCT; PDS-QR (SEQ ID NO.15):TTCTCCTGGCTTGTTTGGCA; qRT-PCR reactions were performed on a real-time quantitative PCR instrument, using 2... -ΔΔCt Method calculation CaBTB36 Relative gene expression levels. Three technical replicates were set for each sample, and three biological replicates were set for each treatment.

[0056] qRT-PCR results ( Figure 1 As shown in b), the results show that pTRV2- CaBTB36 Gene expression levels in the treatment group were significantly lower than those in the control group, and PDS gene expression levels in the pTRV2-PDS treatment group were lower than those in the pTRV2 empty vector control group. P<0.05, P < 0.0001 (t-test).

[0057] 8. Plant type phenotypic determination of silent plants (1) 45 days after Agrobacterium injection, the growth status of plants in each group was observed. Compared with the control (pTRV2 empty vector), pTRV2- CaBTB36 The plants as a whole exhibit a noticeable dwarfing phenomenon, with a more compact plant shape. Figure 1 (a)

[0058] (2) Select 10 control plants and 10 silent plants with uniform growth. Each plant was measured three times, and the average value was taken. The measurement indicators and methods are as follows: Plant height: Measure the vertical distance (cm) from the base of the plant to the apical growing point using a ruler.

[0059] Hypocotyl length: The straight-line distance (cm) from the point where the cotyledons attach to the base of the plant root, measured with a ruler.

[0060] First internode length: The straight-line distance (cm) from the point where the cotyledon attaches to the point where the first lateral branch sprouts above it, measured with a ruler.

[0061] Measurement results as follows Figure 1 As shown in ce.

[0062] Using TRV2 plants as a blank control, TRV2- CaBTB36 The plants were used as the experimental group, and statistical analysis was performed on the growth phenotypic indicators of the two groups of pepper plants. The results showed that compared with the control group TRV2, TRV2- CaBTB36 The experimental group plants were 6.04 cm shorter in height on average, and the overall plants were stunted. The hypocotyl length was significantly shorter than that of the control group, with an average decrease of 1.25 cm, which inhibited the elongation of the seedlings. At the same time, the length of the first internode in the experimental group was also significantly shorter than that in the TRV2 control group, with an average decrease of 0.52 cm, which hindered the elongation of the plant internodes.

[0063] The final results show that CaBTB36 Silent plants are noticeably shorter, with a significant reduction in plant height, hypocotyl length, and first internode length. This phenomenon results in a more compact overall plant shape for peppers, enhancing the plant's resistance to lodging.

[0064] The above are merely preferred embodiments of this application. It should be noted that this application is not limited to the above embodiments. For those skilled in the art, several improvements and modifications can be made without departing from the principles of this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should also be considered within the scope of protection of this application.

Claims

1. CaBTB36 The application of genes or their encoded proteins in regulating pepper plant architecture, characterized by, The CaBTB36 The gene nucleotide sequence is shown in SEQ ID NO.1; its encoded protein sequence is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The plant type includes the plant height, hypocotyl length, and first internode length.

3. The application according to claim 1, characterized in that, Reduced by viral-induced gene silencing technology CaBTB36 Gene expression levels and / or activity in chili peppers.

4. The application according to claim 3, characterized in that, The virus-induced gene silencing technology includes the following steps: (1) Constructing a structure containing CaBTB36 Viral silencing vectors containing gene-specific fragments, wherein the gene-specific fragments are selected from... CaBTB36 gene The 554-854 bp region of the coding area; (2) The virus silencing vector from step (1) is introduced into Agrobacterium to obtain recombinant Agrobacterium; (3) Infect pepper tissues or organs with the recombinant Agrobacterium.

5. The application according to claim 4, characterized in that, The CaBTB36 Primer pairs for amplifying gene-specific fragments include: Forward primer: TGCGGAAAAAGAGGACAAGG; Reverse primer: AATCATTTGGTTGGTCACAA.

6. The application according to claim 4, characterized in that, The virus silencing vector is pTRV2, and the Agrobacterium is strain GV3101. At infection, the OD600 of the Agrobacterium culture is 0.6-0.

8. Furthermore, Agrobacterium carrying the pTRV1 helper vector and Agrobacterium carrying the pTRV2-... CaBTB36 The Agrobacterium vectors were mixed at a volume ratio of 1:

1.

7. A tool for silence CaBTB36 A gene recombination vector, characterized in that, The recombinant vector contains CaBTB36 A specific segment of a gene, wherein the specific segment is selected from... CaBTB36 The 554-854 bp region of the gene coding region.

8. The recombinant vector as described in claim 7, characterized in that, The recombinant vector is pTRV2- CaBTB36 ,Will CaBTB36 Amplification primer pairs for gene-specific fragments CaBTB36 pTRV2- was obtained by inserting a gene-specific fragment into the pTRV2 vector. CaBTB36 The CaBTB36 Primer pairs for amplifying gene-specific fragments include: Forward primer: TGCGGAAAAAGAGGACAAGG; Reverse primer: AATCATTTGGTTGGTCACAA.

9. A method for regulating the plant architecture of chili peppers, characterized in that, By silencing in chili plants CaBTB36 Genes that reduce the plant height, hypocotyl length, and first internode length of chili peppers.

10. The method as described in claim 9, characterized in that, Silencing through viral-induced gene silencing technology CaBTB36 The virus-induced gene silencing technology includes the following steps: (1) Constructing a structure containing CaBTB36 Viral silencing vectors containing gene-specific fragments, wherein the gene-specific fragments are selected from... CaBTB36 The 554-854 bp region of the gene coding region; (2) The virus silencing vector from step (1) is introduced into Agrobacterium to obtain recombinant Agrobacterium; (3) Infect pepper tissues or organs with the recombinant Agrobacterium.

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