Method for inducing capsaicin synthesis by a light signal transcription factor caBBX2

Abstract

The application provides a method for inducing capsaicin synthesis by using a light signal transcription factor CaBBX2, and specifically comprises an experimental method for studying the influence of silencing CaBBX2 gene on capsaicin synthesis in a living plant. The CaBBX2 gene is silenced in a living pepper, and the contents of capsaicin, dihydrocapsaicin and total capsaicin and the expression changes of capsaicin synthesis related genes in the pepper fruits at 16d, 25d, 33d and 38d after pollination are observed and analyzed. The experimental results show that the content of capsaicin is significantly reduced compared with the empty control group after the CaBBX2 gene is silenced, which indicates that CaBBX2 is related to the biosynthesis of capsaicin, and the expression of capsaicin synthesis related genes is also reduced to different degrees with the silencing of CaBBX2, further verifying that CaBBX2 can affect the synthesis of capsaicin by regulating the expression of capsaicin synthesis related genes.

Description

Technical Field

[0001] This invention belongs to the field of plant research, specifically to the experimental field of capsaicin synthesis research, and more specifically, to an experimental method for verifying the influencing factors of capsaicin synthesis induced by the light signal transcription factor CaBBX2. Background Technology

[0002] Transient silencing and transient overexpression are two commonly used techniques in biological experiments, particularly in gene function research, disease model establishment, and drug screening. Transient transformation does not integrate the foreign gene into the chromosome of the target recipient; it is expression at the cellular level, and the transformed gene can be directly observed and identified. It has been widely used in plants. Transient silencing refers to infecting a host plant with a viral vector carrying the target gene cDNA. This activates the plant's own immune system, causing the viral RNA to degrade and simultaneously producing microRNAs containing the endogenous target gene. These microRNAs specifically and complementaryly bind to homologous RNAs in the cytoplasm, leading to the degradation of homologous mRNAs, thus resulting in post-transcriptional gene silencing. Transient overexpression refers to introducing a foreign gene into cells through some method, enabling it to be expressed efficiently within a short period of time, thereby studying the gene's function or impact. This technique typically uses plasmids, viral vectors, etc., to introduce the target gene into cells. It can be applied to the analysis of promoters and regulatory elements, such as studying the effects of different promoters or regulatory elements on gene expression, and to protein function research, using overexpression of specific proteins to study their intracellular function and mechanism of action.

[0003] Chili peppers are a common vegetable in our daily lives and also a traditional Chinese medicine, known for their warming and digestive properties. Modern research shows that chili peppers have anti-inflammatory, antiviral, and anti-cancer effects, as well as strong antioxidant and immunomodulatory effects. They can promote the production of cytokines, protect the liver, and enhance the body's immunity. Capsaicin is a unique secondary metabolite of Capsicum plants and is an important factor determining the quality of chili peppers. Pure capsaicin (or similar substances) can be worth tens of thousands of yuan per kilogram. Therefore, increasing the capsaicin content can significantly increase the economic value of chili peppers.

[0004] Capsaicin synthesis involves two pathways: phenylpropane metabolism and fatty acid metabolism. There are five main types of capsaicin in chili peppers: capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin. Capsaicin and dihydrocapsaicin together account for about 90% of the total capsaicin and provide about 90% of the spiciness and heat sensation. Their content directly affects the spiciness of chili peppers and chili products.

[0005] Light signal transcription factors (BBX) refer to members of the BBX family in plants that can respond to light signals and regulate the expression of related genes. CaBBX2 participates in a variety of biological processes in plants, including light signal transduction and growth and development regulation. At present, there is no relevant experimental evidence to support the conclusion that light signal transcription factor CaBBX2 can induce capsaicin synthesis. Therefore, a new technology is needed to improve this situation. Summary of the Invention

[0006] To address the problems in the prior art, the present invention provides a method for inducing capsaicin synthesis by the phototransmission factor CaBBX2, thereby solving the problems in the prior art.

[0007] This invention is implemented through the following technical solution: a method for inducing capsaicin synthesis by the light signal transcription factor CaBBX2, specifically an experimental method for the effect of silencing the CaBBX2 gene in living plants on capsaicin synthesis; the experimental steps are as follows:

[0008] Construction of S1.CaBBX2-TRV2 silencing vector: A specific fragment of CaBBX2 was obtained from the pepper CaBBX2 gene sequence. Using pepper cDNA as a template, the vector was constructed... Flash Master Mix high-fidelity enzymes were used to amplify a specific fragment of CaBBX2 via PCR, followed by purification and recovery to obtain CaBBX2-TRV2. The TRV2 vector was double-digested using restriction endonucleases Xba I and Sma I: the reaction reagents were added separately, the mixture was pipetted and centrifuged briefly, and the vector was confirmed to be cut by gel electrophoresis. The digested TRV2 vector was then purified and recovered. CaBBX2-TRV2 was ligated to the digested TRV2 vector using homologous recombination ligase. The recombinant vector was transformed into *E. coli* DH5α using a heat shock method. Positive single colonies were identified using TRV2 universal primers, sequenced, and analyzed. Successfully sequenced colonies were amplified, and plasmids were extracted using a plasmid kit to obtain the recombinant plasmid TRV:CaBBX2 silencing vector.

[0009] Preparation of inoculum materials A and B for the S2.TRV:CaBBX2 silencing vector group and the TRV:00 empty vector control group: The TRV:CaBBX2 silencing vector, TRV:00 empty vector, and TRV1 helper expression vector were plated on LB solid medium containing kanamycin and rifampin, respectively. After incubation in an inverted manner, single colony PCR detection was performed. Single colonies with positive results were picked and inoculated into liquid LB medium containing kanamycin and rifampin for overnight amplification culture. The bacterial culture was then transferred to a medium containing the corresponding antibiotics. The bacteria were propagated in liquid LB medium. The OD value of the bacterial solution was measured using a UV spectrophotometer. The cells were centrifuged and collected. The cells were washed with pre-cooled ddH2O and resuspended in infection solution A containing acetylsyleugenol to obtain infection solution B containing TRV:CaBBX2 silencing vector and TRV:00 empty vector infection solution B1, and TRV1 helper expression vector infection solution B2. The cells were incubated in the dark at room temperature. Then, infection solutions B and B1 were mixed with infection solution B2 to obtain infection solution raw material A and control group infection solution raw material B.

[0010] S3. Treatment of Zunla No. 1 chili pepper: When the cotyledons of Zunla No. 1 chili pepper grow out, use a needleless syringe to draw up the dyeing solution raw material A and dyeing solution raw material B and inject them into the back of the chili pepper cotyledons respectively.

[0011] S4. Post-infection treatment: Improve the silencing efficiency of peppers by first subjecting them to darkness and low light, and then to normal light.

[0012] S5. Pollination and sampling: When the four mother peppers are in bloom, artificial pollination is carried out and the pollination time is recorded. After pollination, the pollinated pepper fruits are taken, the fruit stalks and seeds are removed, the pericarp and placenta are cut into pieces and quick-frozen in liquid nitrogen to obtain samples for measuring capsaicin content and gene expression.

[0013] The experimental method for the interaction between S6.CaBBX2 and capsaicin synthesis genes (AT3, COMT, ACS, PAL3) is as follows: A vector was constructed, and the gene promoter fragment was ligated into the pAbai vector. The KpnI recognition sequence—GGTACC—and the adapter were added to the front primer of the gene promoter fragment, and the XhoI recognition sequence—TCTAGA—was added to the front primer. Using the genomic DNA of pepper CM334 as a template, the gene promoter fragment was amplified and ligated into the pAbai vector via heat shock transformation of *E. coli* as bait. pAbai was digested with BstBI and BbsI enzymes. The recombinant plasmid was added along with phosphorylase AP, and a one-step linearization and dephosphorylation reaction was performed. The reaction product was transferred into Y1HGold yeast cells and plated on SD-Ura medium to obtain single clones. Single clones were selected and transferred to YPDA liquid medium for shaking and propagation to obtain bacterial suspension. The bacterial suspension was collected by centrifugation. The pGADT7 empty vector plasmid and pGADT7:CaBBX2 recombinant vector were transferred into the above-mentioned single-clone yeast strain containing the promoter-PAbai vector and plated on SD / -Ura-Leu medium to obtain positive clones. Spotting was performed on SD / -Ura-Leu medium containing a gradient of Aureobasidin A (AbA) antibiotic concentrations (0-200 ng / mL) to screen for AbA concentrations that prevented pGADT7 from growing normally but allowed pGADT7:CaBBX2 to grow.

[0014] S7. Gene expression level detection: RNA was extracted from the sample molecules in step S5, and the RNA quality was checked by agarose gel electrophoresis. The RNA concentration and purity were detected by UV spectrophotometer. The obtained RNA was reverse transcribed into cDNA using reverse transcription reagent and stored at 4℃. Real-time quantitative qRT-PCR experiment: The synthesized cDNA first strand was diluted 3 times as a template, and the CaBBX2 gene was detected by primer pair consisting of qCaBBX2 upstream primer and qCaBBX2 downstream primer. Capsicum actin was used as the internal reference gene, and the primer for the internal reference gene was designed as qActin.

[0015] S8. Determination of capsaicin: Capsaicin was determined by high performance liquid chromatography;

[0016] S9. Evaluation of experimental data: First, spotting was performed on SD-Ura-Leu medium to screen for the AbA concentration at which pGADT7 could not grow normally, but pGADT7:CaBBX2 could grow, proving that CaBBX2 can interact with this gene;

[0017] Secondly, based on the capsaicin content data of the TRV:CaBBX2 silenced vector group and the TRV:00 empty vector control group at different times after pollination, the accumulation of capsaicin reached its peak 33 days after pollination of live peppers. After silencing the CaBBX2 gene, the capsaicin content was significantly reduced relative to TRV2, which can be evaluated as a correlation between CaBBX2 and capsaicin biosynthesis.

[0018] Finally, based on the data on the expression levels of capsaicin synthesis-related genes at different time points after pollination of the TRV:CaBBX2 silencing vector group and the TRV:00 empty vector control group, CaBBX2 was effectively silenced with a silencing efficiency of 40%-60%. Furthermore, with the silencing of CaBBX2, the expression levels of capsaicin synthesis-related genes also decreased to varying degrees. In the data at 33 days, the expression levels of CaCOMT and CaAT3 even decreased by 70% compared with the control group. It can be evaluated that CaBBX2 can affect capsaicin synthesis by regulating the expression of capsaicin synthesis-related genes.

[0019] Furthermore, the PCR reaction program in step S1 is as follows: 98℃ for 10s, 55℃ for 5s annealing, 72℃ for 5s·1kb, 34 cycles;

[0020] PCR reaction system: Mix 25μL, F 2μL, R 2μL, template 2μL, then add ddH2O to 50μL;

[0021] The reaction reagents consist of 20 μL plasmid DNA, 5 μL 10×NEBbuffer, 1.5 μL Xba I, 1.5 μL Sma I, and 22 μL ddH2O.

[0022] The reaction method after instantaneous centrifugation is to react at 37°C for 3 hours.

[0023] Furthermore, the specific steps of step S2 are as follows:

[0024] The TRV:CaBBX2 silencing vector, TRV:00 empty vector, and TRV1 helper expression vector were plated on LB solid medium containing 100 mg / mL kanamycin and rifampin, respectively, and incubated upside down at 28°C for 2-3 days before colony PCR detection. Single clones with positive results were picked and inoculated into liquid LB medium containing 100 mg / mL kanamycin and rifampin for overnight amplification culture, followed by shaking treatment at 28°C and 200 rpm.

[0025] The OD value of the bacterial suspension was measured using a UV spectrophotometer until the OD600 was between 0.8 and 1.0. The bacterial cells were centrifuged, collected, and washed twice with pre-cooled ddH2O. The bacterial cells were then resuspended in infection solution A containing 150 μM acetylsyleugenone to obtain infection solution B containing the TRV:CaBBX2 silencing vector, and TRV:00 empty vector infection solution B1 and TRV1 helper expression vector infection solution B2. The solutions were incubated in the dark at room temperature for 3 hours. Then, infection solutions B and B1 were mixed with infection solution B2 at a ratio of 1:1 to obtain infection solution raw material A and control group infection solution raw material B. Infection solution A specifically consisted of 10 mM MES, 10 mM MgCl2, and pH = 5.5.

[0026] Furthermore, the specific steps of step S4 are as follows:

[0027] The peppers were first cultured in the dark at 18°C ​​for 56 hours, then subjected to light at an intensity of 50 μmol·m⁻¹. -2 ·s -1 Adapted to low light conditions for 24 hours, then subjected to a 12-hour light cycle, day / night temperature of 22 / 20℃, and light intensity of 200 μmol·m⁻². -2 ·s -1 The plants are cultivated and grown in the seedling room under the specified conditions. When the peppers begin to grow flower buds, the four new leaves around the fruiting position of the pepper are silenced according to step S3 to ensure the silencing effect.

[0028] Furthermore, the specific steps of step S5 are as follows:

[0029] Pepper fruits were collected at 16, 25, 33, and 38 days after pollination, with 25-30 fruits at each time point. The fruit stalks and seeds were removed, and the pericarp and placenta were cut into small pieces and flash-frozen in liquid nitrogen to obtain samples for later use.

[0030] Further, the one-step linearization and dephosphorylation reaction system in step S6 is as follows: 10×Fast DigstBuffer, 2 μL; BstBI and BbsI, 1 μL each; AP, 1 μL; pAbai recombinant plasmid, 10 μL; ddH2O, 5 μL; the one-step linearization and dephosphorylation reaction program is: 37℃, 10 min; 65℃, 15 min; the culture conditions of the SD / -Ura medium and the SD / -Ura-Leu medium are: 30℃ constant temperature incubator, 2-3 days; the reaction conditions for the bacterial culture obtained by shaking are 30℃, 180 rpm, 16 h.

[0031] Furthermore, the specific reaction parameters for step S7 are as follows:

[0032] The qRT-PCR reaction system using 10 μL of internal reference gene was as follows: 5 μL SYBR, 3.4 μL ddH2O, 0.3 μL 10 μM PrimerF, 0.3 μL 10 μM PrimerR, and 1 μL cDNA. The qRT-PCR reaction conditions using 10 μL of internal reference gene were: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 10 s, 40 cycles, 58℃ annealing for 45 s, and 72℃ extension for 30 s. The relative gene expression level was analyzed using a relative quantification method. -ΔΔCt The method is used for calculation, and step S7 is repeated three times to obtain the final experimental data.

[0033] Furthermore, the specific steps of step S8 are as follows:

[0034] Accurately weigh 10g of the sample into a 50mL centrifuge tube, add 25mL of 95% ethanol solution, shake, extract with an ultrasonic extractor for 30min, filter, and collect the filtrate;

[0035] The filter residue, along with the filter paper, was extracted again with 25 mL of 95% ethanol solution using an ultrasonic extractor for 30 min. After filtration, the filtrate was collected and the process was repeated once more.

[0036] The filtrates collected from the three filtrations were combined, concentrated to 25 mL using a rotary evaporator, transferred to a new 50 mL centrifuge tube, and brought to a final volume of 30 mL with 95% ethanol solution. After filtration through a 0.45 μm organic phase filter membrane, the solution was subjected to chromatographic analysis.

[0037] The prepared test solution was determined according to chromatographic conditions, and the final content of capsaicin, dihydrocapsaicin, and total capsaicin was calculated by using the peak area integral value.

[0038] The chromatographic reference conditions were as follows: column: Ultimate ODS-3 (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile: water = 7:3, filtered through a 0.45 μm filter before use and degassed; flow rate: 1 mL / min; UV detector: wavelength 280 nm; injection volume: 10 μL.

[0039] The preparation process of the standard working solutions is as follows: weigh natural capsaicin and dihydrocapsaicin and prepare them into 1 mg / mL stock solutions with methanol, and then dilute them to prepare a series of standard working solutions with mass concentrations of 0.2 mg / L, 1 mg / L, 10 mg / L, 20 mg / L, 40 mg / L, 80 mg / L and 160 mg / L.

[0040] Beneficial effects

[0041] This invention silences the CaBBX2 gene in live Zunla No. 1 chili peppers, using a TRV:00 empty vector control group. Through measurements of capsaicin content and expression levels, and experiments on the interaction between CaBBX2 and capsaicin synthesis genes, it was demonstrated that CaBBX2 can interact with capsaicin synthesis. Furthermore, the capsaicin content at different time points after pollination with CaBBX2 gene silence was significantly lower than that under TRV:00, further verifying that the CaBBX2 gene can affect capsaicin synthesis. With CaBBX2 silencing, the expression levels of capsaicin synthesis-related genes also decreased to varying degrees, further verifying that CaBBX2 can influence capsaicin synthesis by regulating the expression of capsaicin synthesis-related genes. Attached Figure Description

[0042] Figure 1 This is a bar graph showing the changes in capsaicin content in live chili peppers after silencing the CaBBX2 gene for 16, 25, 33, and 38 days, according to an embodiment of the present invention.

[0043] Figure 2 This is a bar graph showing the changes in dihydrocapsaicin content in live chili peppers after silencing the CaBBX2 gene for 16, 25, 33, and 38 days, according to an embodiment of the present invention.

[0044] Figure 3 This is a bar graph showing the changes in total capsaicin content in live chili peppers after silencing the CaBBX2 gene for 16, 25, 33, and 38 days, according to an embodiment of the present invention.

[0045] Figure 4 This is a bar graph showing the changes in the expression levels of capsaicin synthesis-related genes after 16, 25, 33, and 38 days of in vivo gene silencing of CaBBX2 in an embodiment of the present invention.

[0046] Figure 5 This is a microbial community diagram of PAbai:AT3+PGADT7 and PAbai:AT3+PGADT7:CaBBX2 loaded into SD-Ura-Leu medium containing antibiotic concentration gradients of 0 and 200 ng / mL, according to an embodiment of the present invention.

[0047] Figure 6 This is a microbial community diagram of PAbai:PAL3+PGADT7 and PAbai:PAL3+PGADT7:CaBBX2 loaded into SD-Ura-Leu medium containing antibiotic concentration gradients of 0 and 120 ng / mL, according to an embodiment of the present invention.

[0048] Figure 7This is a microbial community diagram of PAbai:ACS+PGADT7 and PAbai:ACS+PGADT7:CaBBX2 loaded into SD-Ura-Leu medium containing antibiotic concentration gradients of 0 and 120 ng / mL, according to an embodiment of the present invention.

[0049] Figure 8 The bacterial community diagram of PAbai:COMT+PGADT7 and PAbai:COMT+PGADT7:CaBBX2 loaded into SD-Ura-Leu medium containing antibiotic concentration gradients of 0 and 200 ng / mL is shown in an embodiment of the present invention.

[0050] Figure 9 This is a bar graph showing the changes in capsaicin content in isolated pepper fruits after transient overexpression of the CaBBX2 gene for 12 hours, 1 day, 3 days, 5 days, and 7 days, according to an embodiment of the present invention.

[0051] Figure 10 This is a bar graph showing the changes in dihydrocapsaicin content in isolated pepper fruits after transient overexpression of the CaBBX2 gene for 12 h, 1 d, 3 d, 5 d, and 7 d, according to an embodiment of the present invention.

[0052] Figure 11 This is a bar graph showing the changes in total capsaicin content in isolated pepper fruits after transient overexpression of the CaBBX2 gene for 12 h, 1 d, 3 d, 5 d, and 7 d, according to an embodiment of the present invention.

[0053] Figure 12 This is a bar graph showing the changes in the expression of chili-related genes synthesized over 3 days after transient overexpression of the CaBBX2 gene in isolated chili fruits according to an embodiment of the present invention.

[0054] Figure 13 This is a bar graph showing the expression changes of related genes in isolated pepper fruits after transient overexpression of the CaBBX2 gene for 7 days, according to an embodiment of the present invention.

[0055] Figure 14 Bar graphs showing the changes in capsaicin content in isolated pepper fruits after transiently silencing the CaBBX2 gene for 12 hours, 1 day, 3 days, 5 days, and 7 days, according to an embodiment of the present invention.

[0056] Figure 15 Bar graph showing the changes in dihydrocapsaicin content in isolated pepper fruits after transiently silencing the CaBBX2 gene for 12 hours, 1 day, 3 days, 5 days, and 7 days, according to an embodiment of the present invention.

[0057] Figure 16 This is a bar graph showing the changes in total capsaicin content in isolated pepper fruits after transiently silencing the CaBBX2 gene for 12 hours, 1 day, 3 days, 5 days, and 7 days, according to an embodiment of the present invention.

[0058] Figure 17This is a bar graph showing the changes in the expression of chili-related genes synthesized after transiently silencing the CaBBX2 gene in isolated chili fruits for 3 days, according to an embodiment of the present invention. Detailed Implementation

[0059] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0060] Example 1

[0061] Effects of silencing the CaBBX2 gene in living plants on capsaicin synthesis

[0062] The experimental steps are as follows:

[0063] Construction of S1.TRV:CaBBX2 silencing vector: A specific fragment of CaBBX2 was obtained from the pepper CaBBX2 gene sequence. Using pepper cDNA as a template, the vector was constructed... Flash Master Mix high-fidelity enzymes were used to amplify a specific fragment of CaBBX2 via PCR, followed by purification and recovery to obtain CaBBX2-TRV2. The TRV2 vector was double-digested using restriction endonucleases Xba I and Sma I: the reaction reagents were added separately, the mixture was pipetted and centrifuged briefly, and the vector was confirmed to be cut by gel electrophoresis. The digested TRV2 vector was then purified and recovered. CaBBX2-TRV2 was ligated to the digested TRV2 vector using homologous recombination ligase. The recombinant vector was transformed into *E. coli* DH5α using a heat shock method. Positive single colonies were identified using TRV2 universal primers, sequenced, and analyzed. Successfully sequenced colonies were amplified, and plasmids were extracted using a plasmid kit to obtain the recombinant plasmid TRV:CaBBX2 silencing vector.

[0064] Preparation of inoculum materials A and B for the S2.TRV:CaBBX2 silencing vector group and the TRV:00 empty vector control group: The TRV:CaBBX2 silencing vector, TRV:00 empty vector, and TRV1 helper expression vector were plated on LB solid medium containing kanamycin and rifampin, respectively. After incubation in an inverted manner, single colony PCR detection was performed. Single colonies with positive results were picked and inoculated into liquid LB medium containing kanamycin and rifampin for overnight amplification culture. The bacterial culture was then transferred to a medium containing the corresponding antibiotics. The bacteria were propagated in liquid LB medium. The OD value of the bacterial solution was measured using a UV spectrophotometer. The cells were centrifuged and collected. The cells were washed with pre-cooled ddH2O and resuspended in infection solution A containing acetylsyleugenol to obtain infection solution B containing TRV:CaBBX2 silencing vector and TRV:00 empty vector infection solution B1, and TRV1 helper expression vector infection solution B2. The cells were incubated in the dark at room temperature. Then, infection solutions B and B1 were mixed with infection solution B2 to obtain infection solution raw material A and control group infection solution raw material B.

[0065] S3. Treatment of Zunla No. 1 chili pepper: When the cotyledons of Zunla No. 1 chili pepper grow out, use a needleless syringe to draw up the dyeing solution raw material A and dyeing solution raw material B and inject them into the back of the chili pepper cotyledons respectively.

[0066] S4. Post-infection treatment: Improve the silencing efficiency of peppers by first subjecting them to darkness and low light, and then to normal light.

[0067] S5. Pollination and sampling: When the four mother peppers are in bloom, artificial pollination is carried out and the pollination time is recorded. After pollination, the pollinated pepper fruits are taken, the fruit stalks and seeds are removed, the pericarp and placenta are cut into pieces and quick-frozen in liquid nitrogen to obtain samples for measuring capsaicin content and gene expression.

[0068] The experimental method for the interaction between S6.CaBBX2 and the capsaicin synthesis gene is as follows: Vector construction: The promoter fragment of the gene was ligated into the pAbai vector. The KpnI recognition sequence—GGTACC—and the adapter were added to the front primer of the promoter fragment, and the XhoI recognition sequence—TCTAGA—was added to the front primer. Using CM334 genomic DNA as a template, the promoter fragment of the gene was amplified and ligated into the pAbai vector via heat shock transformation of *E. coli* as bait. The pAbai recombinant plasmid was digested with BstBI and BbsI enzymes, and simultaneously added... Phosphorylase AP was linearized and dephosphorylated in a one-step process. The reaction product was transferred into Y1HGold yeast cells and plated on SD / -Ura medium to obtain single clones. Single clones were selected and cultured in YPDA liquid medium by shaking to obtain bacterial suspension. 2 mL of bacterial suspension was centrifuged to collect the cells. The pGADT7 empty vector and pGADT7:CaBBX2 recombinant vector were transferred into the above-mentioned single-clone yeast strain containing the promoter-PAbai vector and plated on SD / -Ura-Leu medium to obtain positive clones. Spotting was performed on SD / -Ura-Leu medium containing a gradient of Aureobasidin A (AbA) antibiotic concentrations (0-200 ng / mL) to screen for AbA concentrations that prevented pGADT7 from growing normally but allowed pGADT7:CaBBX2 to grow.

[0069] S7. Gene expression level detection: RNA was extracted from the sample molecules in step S5, and the quality of RNA was checked by agarose gel electrophoresis. The concentration and purity of RNA were detected by UV spectrophotometer. The obtained RNA was reverse transcribed into cDNA using reverse transcription reagent and stored at 4℃. Real-time quantitative PCR experiment was performed: the synthesized cDNA first strand was diluted 3 times as a template, and the CaBBX2 gene was detected using a primer pair composed of qCaBBX2 upstream primer and qCaBBX2 upstream primer. Capsicum actin was used as the internal reference gene, and the primer for the internal reference gene was designed as qActin.

[0070] S8. Determination of capsaicin: Capsaicin was determined by high performance liquid chromatography;

[0071] S9. Evaluation of experimental data: First, on the SD / -Ura-Leu medium after spotting, the AbA concentrations that pGADT7 could not grow normally, but pGADT7:CaBBX2 could grow were screened.

[0072] like Figures 5-8 The image shows the bacterial community of the recombinant plasmids PAbai:AT3, PAbai:PAL3, PAbai:ACS, PAbai:COMT and their empty vectors constructed by the method in step S6, loaded into culture media with different antibiotic concentration gradients.

[0073] in, Figure 5 The two images on the left prove that PGADT7 and PGADT7:CaBBX2 were successfully transferred into Y1Hgold bacteria containing PAbai:AT3, and the activity of the bacteria was not a problem. In the two images on the right, PGADT7 (negative control) did not grow, while PGADT7:CaBBX2 could grow normally, which suggests that CaBBX2 interacts with AT3.

[0074] Figure 6 The two images on the left prove that PGADT7 and PGADT7:CaBBX2 were successfully transferred into Y1Hgold bacteria containing PAbai:PAL3, and the activity of the bacteria was not a problem. In the two images on the right, PGADT7 (negative control) did not grow, while PGADT7:CaBBX2 could grow normally, which suggests that CaBBX2 interacts with PAL3.

[0075] Figure 7 The two images on the left prove that PGADT7 and PGADT7:CaBBX2 were successfully transferred into Y1Hgold bacteria containing PAbai:ACS, and the activity of the bacteria was not a problem. In the two images on the right, PGADT7 (negative control) did not grow, while PGADT7:CaBBX2 could grow normally, which suggests that CaBBX2 interacts with COMT.

[0076] Figure 8 The two images on the left demonstrate that PGADT7 and PGADT7:CaBBX2 were successfully transferred into Y1Hgold bacteria containing PAbai:COMT, and the bacteria's activity was not affected. In the two images on the right, PGADT7 (negative control) did not grow, while PGADT7:CaBBX2 could grow normally, which suggests that CaBBX2 interacts with ACS.

[0077] In summary, it can be demonstrated that CaBBX2 can interact with genes related to capsaicin synthesis.

[0078] Secondly, such as Figure 1-3As shown, based on the capsaicin content data of the TRV:CaBBX2 silencing vector group and the TRV:00 empty vector control group at different times after pollination, capsaicin accumulation reached its peak 33 days after pollination of live peppers. After silencing the CaBBX2 gene, the capsaicin content was significantly reduced relative to TRV2, which can be evaluated as a correlation between CaBBX2 and capsaicin biosynthesis.

[0079] Finally, as Figure 4 As shown, data on the expression levels of capsaicin synthesis-related genes at different time points after pollination of the TRV:CaBBX2 silencing vector group and the TRV:00 empty vector control group showed that CaBBX2 was effectively silenced, with a silencing efficiency of 40%-60%. Furthermore, with the silencing of CaBBX2, the expression levels of capsaicin synthesis-related genes also decreased to varying degrees. In the data at 33 days, the expression levels of CaCOMT and CaAT3 even decreased by 70% compared to the control group. It can be evaluated that CaBBX2 can affect capsaicin synthesis by regulating the expression of capsaicin synthesis-related genes.

[0080] In this embodiment, the PCR reaction program in step S1 is as follows: 98℃ for 10s, 55℃ for 5s annealing, 72℃ for 5s·1kb, 34 cycles; PCR reaction system: Mix 25μL, F 2μL, R 2μL, template 2μL, then add ddH2O to 50μL; reaction reagents are plasmid DNA 20μL, 10×NEBbuffer 5μL, Xba I 1.5μL, Sma I 1.5μL, ddH2O 22μL; the reaction method after instantaneous centrifugation is 37℃ for 3h.

[0081] In this embodiment, the specific steps of step S2 are as follows:

[0082] TRV:CaBBX2 silencing vector, TRV:00 empty vector, and TRV1 helper expression vector were plated on LB solid medium containing 100 mg / mL kanamycin and rifampin, respectively, and incubated upside down at 28°C for 2-3 days. Colony PCR was then performed. Positive single clones were picked and inoculated into liquid LB medium containing 100 mg / mL kanamycin and rifampin for overnight amplification. The culture was then shaken at 28°C and 200 rpm. The OD value of the bacterial culture was measured using a UV spectrophotometer until... The OD600 was between 0.8 and 1.0. After centrifugation and collection of bacterial cells, the cells were washed twice with pre-cooled ddH2O. The cells were then resuspended in infection solution A containing 150 μM acetylsylcholine to obtain infection solution B containing the TRV:CaBBX2 silencing vector and TRV:00 empty vector infection solution B1, and TRV1 helper expression vector infection solution B2. The cells were incubated in the dark at room temperature for 3 hours. Then, infection solutions B and B1 were mixed with infection solution B2 at a ratio of 1:1 to obtain infection solution raw material A and control group infection solution raw material B. Infection solution A specifically contained 10 mM MES, 10 mM MgCl2, and pH = 5.5.

[0083] In this embodiment, the specific steps of step S4 are as follows:

[0084] The peppers were first cultured in the dark at 18°C ​​for 56 hours, then subjected to light at an intensity of 50 μmol·m⁻¹. -2 ·s -1 Adapted to low light conditions for 24 hours, then subjected to a 12-hour light cycle, day / night temperature of 22 / 20℃, and light intensity of 200 μmol·m⁻². -2 ·s -1 The plants are cultivated and grown in the seedling room under the specified conditions. When the peppers begin to grow flower buds, the four new leaves around the fruiting position of the pepper are silenced according to step S3 to ensure the silencing effect.

[0085] In this embodiment, the specific steps of step S5 are as follows:

[0086] Pepper fruits were collected at 16, 25, 33, and 38 days after pollination, with 25-30 fruits at each time point. The fruit stalks and seeds were removed, and the pericarp and placenta were cut into small pieces and flash-frozen in liquid nitrogen to obtain samples for later use.

[0087] In this embodiment, the one-step linearization and dephosphorylation reaction system in step S6 is as follows: 10×Fast DigstBuffer, 2 μL; BstBI and BbsI, 1 μL each; AP, 1 μL; pAbai recombinant plasmid, 10 μL; ddH2O, 5 μL; the one-step linearization and dephosphorylation reaction program is: 37℃, 10 min; 65℃, 15 min; the culture conditions of SD / -Ura medium and SD / -Ura-Leu medium are: 30℃ incubator, 2-3 days; the reaction conditions for the bacterial culture obtained by shaking are 30℃, 180 rpm, 16 h.

[0088] In this embodiment, the specific reaction parameters of S7 are as follows:

[0089] The qRT-PCR reaction system using 10 μL of internal reference gene was as follows: 5 μL SYBR, 3.4 μL ddH2O, 0.3 μL 10 μM PrimerF, 0.3 μL 10 μM PrimerR, and 1 μL cDNA. The qRT-PCR reaction conditions using 10 μL of internal reference gene were: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 10 s, 40 cycles, 58℃ annealing for 45 s, and 72℃ extension for 30 s. The relative expression level of the gene was calculated using the relative quantification method 2-ΔΔCt. Step S7 was repeated three times to obtain the final experimental data.

[0090] In this embodiment, the specific steps of step S8 are as follows:

[0091] Accurately weigh 10g of sample into a 50mL centrifuge tube, add 25mL of 95% ethanol solution, shake, and extract using an ultrasonic extractor for 30min. Filter and collect the filtrate. Extract the residue along with filter paper again using an ultrasonic extractor with 25mL of 95% ethanol solution for 30min, filter, and collect the filtrate. Repeat this process once more. Combine the filtrates collected from the three filtrations, concentrate to 25mL using a rotary evaporator, transfer to a new 50mL centrifuge tube, and dilute to 30mL with 95% ethanol solution. Filter through a 0.45μm organic phase filter membrane and perform chromatographic analysis. Determine the content of capsaicin, dihydrocapsaicin, and total capsaicin according to the chromatographic conditions, using the peak area integral value for quantification. Calculate the final content of capsaicin, dihydrocapsaicin, and total capsaicin. The chromatographic reference conditions are: Column: Ultimate ODS-3 (4.6mm×250mm, 5μm); Mobile phase: acetonitrile:water = 7:3, filtered through a 0.45μm filter before use and degassed; Flow rate: 1mL / min; UV detector: wavelength 280nm; Injection volume: 10μL; The preparation process of the standard working solutions is as follows: weigh natural capsaicin and dihydrocapsaicin and prepare them into 1mg / mL stock solutions with methanol, and then dilute them to prepare a series of standard working solutions with mass concentrations of 0.2mg / L, 1mg / L, 10mg / L, 20mg / L, 40mg / L, 80mg / L and 160mg / L.

[0092] In step S1, the upstream primer sequence of CaBBX2-TRV2, the downstream primer sequence of CaBBX2-TRV2, the upstream primer sequence of TRV2, and the downstream primer sequence of TRV2 are respectively: TGAGTAAGGTTACCGAATTCTCTAGAAAGGCAGAGAATTGAGTTTCCAG;

[0093] TTTAATGTCTTCGGGACATGCCCGGGTCATGATTGTTGCCACTACGCA;GATAATGGTTTGGTGGTC;GTTTAATGTCTTCGGGAC.

[0094] The CDS sequence for CaBBX2 Capana08g002625 is:

[0095] ATGAGAACTCTCTGTGATGTTTGTGAAAGTGCTGCTGCTATACTTTTCTGTGCTGCTGATGAGGCTGCTCTCTGCCGTTCCTGTGACCAAAAGGTCCATATGTGCAACAAGCTTGCAAGTCGACATGTAAGAGTTGGTCTTGCTGCCCCCAGTAAAATCCAGCGTTGCGACATATGTGAAAATGCACCTGCTTTTTTTTATTGTGAGATCGATGGAAGTTCCCTTTGTTTGCAATGTGATATGATTGTCCATGTTGGAGGTAAAAGAACCCATGGAAGATATCTCCTTATAAGGCAGAGAATTGAGTTTCCAGGGGATAAATTGGGCCCTTCAAATGAGCTAGGATTGCCATCTACAGAACAAGGTGATGGAAGAAGGGAGCCTGCACAGCCCTTTAAGCTCCCTATGATAGACAATCACCAACCAAACAGGGAGATTGCTATGGCAGGGTTAGAAAATAACGTGAATAACAGCGTGAAAATGGAGAATGAGTTAATTGACCTTAATTCCAGGCCTCAAAGGATACATGGTCAAACGTCAAATAATCAGGAACAAGGAATGGACATGCGTAGTGGCAACAATCATGAGTCAGTCGGCGTGGTTCCTGATGGACCCTTCAAAAGAGAACCAGAGAAGTGA。

[0096] The amino acid sequence of CaBBX2 is as follows:

[0097] MRTLCDVCESAAAILFCAADEAALCRSCDQKVHMCNKLASRHVRVGLAAPSKIQRCDICENAPAFFYCEIDGSSLCLQCDMIVHVGGKRTH GRYLLIRQRIEFPGDKLGPSNELGLPSTEQGDGRREPAQPFKLPMIDNHQPNREIAMAGLENNVNNSVKMENELIDLNSRPQRIHGQTSNN QEQGMDMRSGNNHESVGVVPDGPFKREPEK*。

[0098] Sequencing of the gene promoter fragment of the reaction product of constructing the vector in step S6 is as follows:

[0099] The AT3-PAbai-F primer sequence is AGCTTGAATTCGAGCTCGGTACCGAAAAAAGACAAGTAAATTGAGAAATCAA;

[0100] The AT3-PAbai-R primer sequence is ACATACAGAGCACATGCCTCGAGGATATTATAAATGGGTGATATCTTTCCA;

[0101] The primer sequence for PAL3-PAbai-F is AGCTTGAATTCGAGCTCGGTACCGCTCTCCGTACCAAGATTTTTTTGT;

[0102] The PAL3-PAbai-R primer sequence is ACATACAGAGCACATGCCTCGAGGAAGATTAATCTTGAACGTTGGATTGCA;

[0103] The primer sequence for ACS-PAbai-F is AGCTTGAATTCGAGCTCGGTACCATACTCAAATGTCATATGTTCTCTTTT

[0104] ACS-PAbai-R primer sequence: ACATACAGAGCACATGCCTCGAGACCCTAAGATGTAATATAATTACATAACT;

[0105] The COMT-PAbai-F primer sequence is AGCTTGAATTCGAGCTCGGTACCTGTTTGAATTCATACACCGGGC;

[0106] The COMT-PAbai-R primer sequence is ACATACAGAGCACATGCCTCGAGATTTTCGACGTCAAAGATCGATACCA;

[0107] The primer sequence for CaBBX2-AD-F is GGACAGCCCACCACCCATATGATGAGAACTCTCTGTGATGTTTGT;

[0108] The CaBBX2-AD-R primer sequence is CTGGTGATTTCAGCGCCCGGGCTTCTCTGGTTCTCTTTTGAAGGG.

[0109] The upstream primer sequence of the internal reference primer qActin, the downstream primer sequence of qActin, the upstream primer sequence of qCaBBX2, and the downstream primer sequence of qCaBBX2 in S7 are: GACTGACCTAACTGATAACCTGAT; CTCTCAGCACCAATGGTAATAACTT;

[0110] CTCTCTGCCGTTCCTGTGA; GGCAGCAAGACCAACTCTTA.

[0111] This embodiment demonstrates, through comparative experiments and experiments on the interaction between CaBBX2 and capsaicin synthesis genes, that CaBBX2 can interact with capsaicin synthesis-related genes. By silencing the CaBBX2 gene in living plants, experimental data on capsaicin content and capsaicin synthesis-related gene expression levels in Zunla No. 1 chili pepper fruits at 16, 25, 33, and 38 days after CaBBX2 silencing were obtained. The results showed that after silencing the CaBBX2 gene, the capsaicin content was significantly downregulated relative to TRV:00. With the silencing of CaBBX2, the expression levels of capsaicin synthesis-related genes also decreased to varying degrees, confirming that CaBBX2 can affect capsaicin synthesis by regulating the expression of capsaicin synthesis-related genes.

[0112] Example 2

[0113] Experimental method for transient overexpression of chili peppers in vitro

[0114] Includes the following steps:

[0115] The construction of the S1.35S:CaBBX2-flag overexpression vector is as follows:

[0116] The full-length CaBBX2 (CDS) was amplified by PCR using a high-fidelity enzyme, and then purified and recovered to obtain CaBBX2-35-flag;

[0117] The 35S:flag vector was digested with the restriction endonuclease EcoRI. 20 μL of plasmid DNA, 5 μL of 10×NEBbuffer, 3 μL of EcoRI, and 22 μL of ddH2O were added, and the mixture was pipetted and briefly centrifuged. The reaction was carried out at 37°C for 3 h. After confirming that the vector was cut by gel electrophoresis, the digested 35S:flag vector was purified and recovered.

[0118] The CaBBX2-35-flag vector was ligated with the enzyme-digested 35S:flag vector using homologous recombination ligase to obtain the recombinant vector 35S:CaBBX2-flag.

[0119] The recombinant vector was transformed into Escherichia coli DH5α using the heat shock method. Positive single colonies were identified using the 35S:flag universal primer, and then sequenced and analyzed. Finally, the successfully sequenced colonies were amplified and the plasmid was extracted using a plasmid kit to obtain the recombinant plasmid overexpression vector 35S:CaBBX2-flag.

[0120] S2. Preparation of original inoculum solutions A and B for the overexpression vector 35S:CaBBX2-flag group and the 35S:flag empty vector control group: Construction, the successfully constructed overexpression vector 35S:CaBBX2-flag and 35S:flag empty vector were transformed into GV3101 Agrobacterium competent cells, plated on LB solid medium containing 100 mg / mL spectinomycin and incubated upside down at 28℃ for 2-3 days, followed by colony PCR detection;

[0121] Selected positive single clones were inoculated into liquid LB medium containing 100 mg / mL spectinomycin for overnight amplification culture. The culture was shaken at 28°C and 200 r / min. The OD value of the bacterial culture was measured using a UV spectrophotometer until the OD600 was between 0.8 and 1.0. The cells were then centrifuged, collected, and washed twice with pre-cooled ddH2O.

[0122] The bacterial cells were resuspended in infection solution A (10mM MES, 10mM MgCl2, pH=5.5) containing 150μM acetylsylsyringone to obtain stock solution A containing the overexpression vector 35S:CaBBX2-flag and stock solution B containing the empty vector control 35S:flag. The solutions were then incubated in the dark at 28℃ for 3h.

[0123] S3. Treatment of disease-free Ganjiao 19 pepper fruits: Disease-free Ganjiao 19 pepper fruits of uniform size and color were collected and laid flat on a clean, sterilized laboratory table to cool overnight at a room temperature of 23℃. The fruits and stems were wiped with paper towels soaked in disinfectant alcohol for disinfection. 1 mL syringes were used to draw undiluted infection solutions A and B and injected into the pepper stems. Infection solutions A and B were injected into the placenta of the peppers. Sampling times were 12 h, 1 d, 3 d, 5 d, and 7 d after injection. 25 pepper fruits were collected at each time point. The sampling sites were the upper half of the peel and placenta of the Ganjiao pepper fruit. The samples were flash-frozen in liquid nitrogen, mixed evenly, and ground for later use. Four 0.5 g samples from each treatment were weighed into 2 mL centrifuge tubes as molecular samples. Blue ink was used as a negative control test to confirm that injection from the stem can inject the infection solution into the placenta.

[0124] S4. Gene Expression Detection: RNA was extracted from the molecular sample obtained in step S3 using the Trizol method. RNA quality was assessed using agarose gel electrophoresis, and RNA concentration and purity were detected using a UV spectrophotometer. The obtained RNA was reverse transcribed into cDNA using reverse transcription reagent and stored at 4℃. Real-time quantitative PCR (qRT-PCR) of gene expression: The synthesized cDNA first strand was diluted 3-fold as a template, and a primer pair consisting of the qCaBBX2 upstream and downstream primers was used to detect the CaBBX2 gene. Using capsicum actin as an internal reference gene, primer qActin was designed. A 10 μL qRT-PCR reaction system for the internal reference gene was used: 5 μL SYBR, 3.4 μL ddH2O, 0.3 μL 10 μM PrimerF, 0.3 μL 10 μM PrimerR, and cDNA. 1 μL; the qRT-PCR reaction conditions using 10 μL of internal reference gene were: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 10 s, 40 cycles, 58℃ annealing for 45 s, and 72℃ extension for 30 s. The relative gene expression level was analyzed using the relative quantification method. -ΔΔCt The method is used for calculation, and step S7 is repeated three times to obtain the final experimental data.

[0125] S5. Capsaicin Determination Method: Capsaicin is determined by high-performance liquid chromatography (HPLC), specifically including the following steps:

[0126] Accurately weigh 10g of sample into a 50mL centrifuge tube, add 25mL of 95% ethanol solution, shake, extract with an ultrasonic extractor for 30min, filter, and collect the filtrate; extract the residue along with the filter paper again with 25mL of 95% ethanol solution using an ultrasonic extractor for 30min, filter, collect the filtrate, and repeat once more;

[0127] The filtrates collected from the three filtrations were combined, concentrated to 25 mL using a rotary evaporator, transferred to a new 50 mL centrifuge tube, and brought to a final volume of 30 mL with 95% ethanol solution. After filtration through a 0.45 μm organic phase filter membrane, the solution was subjected to chromatographic analysis.

[0128] The prepared test solution was determined according to chromatographic conditions, and the final content of capsaicin, dihydrocapsaicin, and total capsaicin was calculated by using the peak area integral value.

[0129] S6. For example Figure 9-13 As shown, the experimental data results are evaluated as follows:

[0130] Based on the capsaicin content data of the overexpression vector 35S:CaBBX2-flag group and the 35S:flag empty vector control group, after transient overexpression of the CaBBX2 gene in detached pepper fruits, except for 12 hours, the capsaicin, dihydrocapsaicin, and total capsaicin contents were significantly increased compared with the control group 35S:flag. This can be evaluated as transient overexpression of CaBBX2 increasing the spiciness of pepper fruits.

[0131] Since capsaicin synthesis involves two pathways: phenylpropane metabolism and fatty acid metabolism, gene expression detection data from chili fruits transiently overexpressing 3d and 7d showed that the genes with significant differences between 3d and 7d were different. This is because these genes exist at different locations in the same pathway, and the gene expression response should be faster than the secondary metabolite response.

[0132] Based on the capsaicin expression data of the overexpression vector 35S:CaBBX2-flag group and the 35S:flag empty vector control group, transient overexpression of the CaBBX2 gene in detached pepper fruits can increase the expression level of CaBBX2 by more than two times. It can be evaluated that the overexpression vector 35S:CaBBX2-flag can also increase the expression level of capsaicin synthesis-related genes to varying degrees.

[0133] In this embodiment, the chromatographic reference conditions for step S5 are: chromatographic column: Ultimate ODS-3 (4.6mm×250mm, 5μm);

[0134] Mobile phase: acetonitrile:water = 7:3, filtered through a 0.45μm filter membrane before use to degas; flow rate: 1mL / min; UV detector: wavelength 280nm; injection volume: 10uL; the preparation process of the standard working solutions is as follows: weigh natural capsaicin and dihydrocapsaicin and prepare them into 1mg / mL stock solutions with methanol, and then dilute them to prepare a series of standard working solutions with mass concentrations of 0.2mg / L, 1mg / L, 10mg / L, 20mg / L, 40mg / L, 80mg / L and 160mg / L.

[0135] In step S1, the upstream primer sequence of CaBBX2-35-flag, the downstream primer sequence of CaBBX2-35-flag, the upstream primer sequence of 35S:flag, and the downstream primer sequence of 35S:flag are as follows:

[0136] GATGACGATGACAAGGAATTCATGAGAACTCTCTGTGATGTTTTGT,

[0137] GTCCTTGTAATCCATGAATTCCTTCTCTGGTTCTCTTTTGAAGGG, ACGCACAATCCCACTATCCTTC, CATAAAAATACGATAGTAACGGGTG.

[0138] This embodiment uses a comparative experimental method, employing transient overexpression of CaBBX2 in isolated peppers, to obtain experimental data on capsaicin content and expression levels in disease-free Ganjiao No. 19 peppers at 12h, 1d, 3d, 5d, and 7d after CaBBX2 overexpression in isolated peppers. The results show that transient overexpression of CaBBX2 can increase the spiciness of pepper fruits and also increase the expression levels of capsaicin synthesis-related genes to varying degrees.

[0139] Example 3

[0140] Transient silencing experiment method for isolated peppers

[0141] In this implementation, steps S1, S2, S4, and S5 are the same as in Example 2.

[0142] The difference between this embodiment and Embodiment 2 lies in the treatment of disease-free Ganjiao 19 pepper fruits in S3. Specifically, the following steps are taken: Disease-free Ganjiao 19 pepper fruits of uniform size and color are collected and laid flat on a clean, sterilized laboratory table to cool overnight at a room temperature of 23 degrees Celsius. The fruit body and stem are wiped with paper towels soaked in disinfectant alcohol for disinfection. Injection solutions B and B1, respectively, into the pepper stem using a 1mL syringe. The infection solutions are then injected into the placenta of the pepper. Sampling times are 12 hours, 1 day, 3 days, 5 days, and 7 days after injection. At each time point, 25 pepper fruits are collected, sampling the upper half of the peel and placenta of the Ganjiao fruit. The samples are flash-frozen in liquid nitrogen, thoroughly mixed, and ground for later use. Four 0.5g samples from each treatment are weighed into 2mL centrifuge tubes as molecular samples.

[0143] The difference between this embodiment and Embodiment 2 lies in the evaluation of experimental data in S6, such as... Figure 14-17 As shown, the details are as follows:

[0144] According to the capsaicin content data of the silencing vector TRV:CaBBX2 group and the empty control group TRV:00, after transient silencing of detached peppers, the capsaicin, dihydrocapsaicin and total capsaicin contents were significantly reduced compared with the control group TRV:00. This can be evaluated as transient silencing of CaBBX2 can reduce the spiciness of pepper fruits.

[0145] Based on the capsaicin-related gene detection data of the TRV:CaBBX2 group and the TRV:00 empty vector control group, since the difference between the experimental group and the control group was greatest after transient silencing for 3 days, samples 3 days after transient silencing were selected for RNA extraction and capsaicin synthesis-related gene detection. It can be seen that after transient silencing CaBBX2, the expression levels of CaBBX2 and capsaicin synthesis-related genes decreased significantly. It can be evaluated that transient silencing CaBBX2 will reduce the expression level of capsaicin synthesis-related genes, thereby reducing the capsaicin content.

[0146] Based on the above embodiments, by combining the experimental methods of silencing and overexpressing the CaBBX2 gene in isolated peppers with the experimental method of silencing the CaBBX2 gene in live peppers, a comprehensive evaluation shows that the CaBBX2 gene affects capsaicin synthesis. CaBBX2 affects capsaicin synthesis by regulating the expression of genes related to capsaicin synthesis.

[0147] CaBBX2 binds to the promoter of the capsaicin synthesis gene, thereby promoting the expression of the capsaicin synthesis gene and ultimately increasing capsaicin synthesis.

Claims

1. A method for inhibiting capsaicin synthesis by the phototransmission factor CaBBX2, characterized in that: Silencing the CaBBX2 gene in chili peppers reduces capsaicin synthesis.

2. The method for inhibiting capsaicin synthesis by the optical signal transcription factor CaBBX2 according to claim 1, characterized in that: The experimental steps for silencing the CaBBX2 gene in chili peppers to reduce capsaicin synthesis are as follows: S1. Construction of CaBBX2-TRV2 silencing vector: Using pepper cDNA as a template, the CDS sequence of CaBBX2 was amplified by PCR and ligated with the TRV2 vector that had been double-digested with restriction endonucleases Xba I and Sma I for homologous recombination. The ligation product was transformed into Escherichia coli DH5α and positive clones were identified. After sequencing verification, the plasmid was extracted to obtain the TRV:CaBBX2 silencing vector. S2. Preparation of Infection Solutions: The TRV:CaBBX2 silencing vector, TRV:00 empty vector, and TRV1 helper expression vector were inoculated into LB solid medium containing kanamycin and rifampin, respectively. After inverted culture, positive single clones were screened by single colony PCR. The positive single clones were amplified and cultured. After amplification, the bacterial cells were collected by centrifugation. The bacterial cells were resuspended in an infection solution containing acetylsuccinone to obtain TRV:CaBBX2 silencing vector infection solution, TRV:00 empty vector infection solution, and TRV1 helper expression vector infection solution. Each infection solution was allowed to stand at room temperature in the dark. Then, the TRV:CaBBX2 silencing vector infection solution and TRV:00 empty vector infection solution were mixed with the TRV1 helper expression vector infection solution at a ratio of 1:1 to obtain the experimental group infection solution and the control group infection solution. S3. Treatment of pepper plants: Select Zunla No. 1 peppers at the cotyledon stage, and inject the infection solution of the experimental group and the infection solution of the control group into the back of the cotyledons of the peppers using a needleless syringe; S4. Silencing efficiency optimization: After the infected pepper plants have adapted to low temperature, darkness and weak light, they are then transferred to normal cultivation conditions. When the pepper plants show buds, four new leaves around the budding position of the pepper are selected for silencing treatment. S5. Pollination and sample preparation: Artificial pollination was carried out during the flowering period of the four mother flowers of the pepper plant. Pepper fruits at different time points after pollination were selected, the fruit stalks and pepper seeds were removed, the pericarp and placenta were cut into pieces and then quick-frozen with liquid nitrogen to obtain samples for determining capsaicin content and gene expression level. S6 Effect Verification Test: The effect of CaBBX2 on capsaicin synthesis was verified by the interaction experiment between CaBBX2 and capsaicin synthesis gene, gene expression level detection, and capsaicin content determination.

3. The method for inhibiting capsaicin synthesis by the optical signal transcription factor CaBBX2 according to claim 2, characterized in that: In step S4, the low-temperature darkness condition is 18°C ​​darkness; the light intensity of the weak light is 50 μmol·m⁻¹. m-2 · s-1 .

4. The method for inhibiting capsaicin synthesis by the optical signal transcription factor CaBBX2 according to claim 2, characterized in that: In step S6, the interaction experiment between CaBBX2 and the capsaicin synthesis gene specifically involves: the capsaicin synthesis gene being AT3, COMT, ACS, or PAL3; ligating the promoter fragment of the above gene to the pAbai vector; adding a KpnI recognition sequence and adapter to the front primer of the promoter fragment, and adding an XhoI recognition sequence to the front primer; using the genomic DNA of pepper CM334 as a template, amplifying the gene promoter fragment by PCR; constructing a bait vector by heat shock transformation of E. coli; digesting the pAbai recombinant plasmid with BstBI and BbsI enzymes, and simultaneously adding phosphorylase AP; and then... Enzyme digestion products were linearized and dephosphorylated in a one-step process. The treated reaction products were then transferred into Y1HGold yeast cells and plated on SD / -Ura medium to obtain single clones. Single clones were selected and propagated on YPDA liquid medium, and the cells were collected by centrifugation. The pGADT7 empty vector and the pGADT7:CaBBX2 recombinant vector were respectively transferred into single-clone yeast strains containing the promoter-PAbai vector and plated on SD / -Ura-Leu medium to obtain positive clones. Spotting experiments were performed on SD / -Ura-Leu medium containing gradient concentrations of Aureobasidin A (AbA) antibiotic to screen for AbA concentrations at which pGADT7 could not grow normally but pGADT7:CaBBX2 could grow.

5. The method for inhibiting capsaicin synthesis by the optical signal transcription factor CaBBX2 according to claim 2, characterized in that: In step S6, the gene expression level detection specifically involves: extracting RNA from the sample molecule from step S5, and detecting the RNA concentration and purity; after reverse transcribing the RNA into cDNA, detecting the CaBBX2 gene by real-time quantitative qRT-PCR; wherein capsicum actin is used as an internal reference gene.

6. The method for inhibiting capsaicin synthesis by the optical signal transcription factor CaBBX2 according to claim 2, characterized in that: In step S6, the capsaicin content is determined by: extracting capsaicin from the sample in step S5, and determining the capsaicin content using high performance liquid chromatography, with natural capsaicin and dihydrocapsaicin as the standards.

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