Method for inducing synthesis of capsaicin by optical signal transcription factor CaBBX2

Abstract

The invention provides a method for inducing synthesis of capsaicin by using an optical signal transcription factor CaBBX2, and particularly relates to an experimental method for the influence of a living plant silencing CaBBX2 gene on synthesis of capsaicin. Capsicum fruits after pollination for 16 days, 25 days, 33 days and 38 days are taken to observe and analyze the content of capsaicin, dihydrocapsaicin and total capsaicin and the change condition of capsaicin synthesis related gene expression, and experimental results show that after the CaBBX2 gene is silenced, the content of capsaicin is remarkably reduced relative to that of a no-load control group, which indicates that the CaBBX2 and the biosynthesis of capsaicin have a certain correlation, and along with the silencing of the CaBBX2, the content of capsaicin is remarkably reduced relative to that of a no-load control group. The expression quantity of the capsaicin synthesis related gene is also reduced to different degrees, and further verification shows that the CaBBX2 can influence the synthesis of capsaicin by regulating the expression of the capsaicin synthesis related gene.

Description

Technical Field

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

[0002] Transient silencing and transient overexpression are two commonly used techniques in biological experiments, especially in the fields of gene function research, disease model establishment and drug screening. Transient transformation does not integrate exogenous genes into the chromosomes of the target receptor. It is a cellular level expression. After transformation, it can be directly observed and identified. It has been widely used in plants. Transient silencing refers to the infecting host plants with viral vectors carrying the target gene cDNA, activating the plant's own immune system to promote the degradation of viral RNA, and producing microRNA containing endogenous target genes. These microRNAs specifically complement and bind to homologous RNA in the cytoplasm, resulting in the degradation of homologous mRNA, thereby causing post-transcriptional gene silencing; transient overexpression refers to the introduction of exogenous genes into cells by some method, so that they are efficiently expressed in a short period of time, so as to study the function or effect of the gene. This technology usually uses plasmids, viral vectors, etc. to introduce target genes 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 protein function research, using overexpression of specific proteins to study their functions and mechanisms in cells.

[0003] Chili pepper is a common vegetable in our daily life. It is also a traditional Chinese medicine with the effects of warming the middle and dispelling cold, stimulating appetite and digestion. Modern research shows that chili pepper has anti-inflammatory, antiviral, anti-cancer and other effects. It also has strong antioxidant and immunomodulatory effects, which can promote the production of cytokines, protect the liver, and improve the body's immunity. Capsaicinoids are secondary metabolites unique to plants of the genus Capsicum and are an important factor in determining the quality of chili peppers. The value of pure capsaicinoids (substances) is as high as tens of thousands of yuan per kilogram. Therefore, increasing the content of capsaicinoids can significantly increase the economic value of chili peppers.

[0004] There are two pathways for the synthesis of capsaicin: phenylpropanoid metabolism and fatty acid metabolism. There are five main types of capsaicin in peppers, namely: Capsaicin, Dihydrocapsaicin, Nordihydrocapsaicin, Homocapsaicin and Homodihydrocapsaicin. Capsaicin and dihydrocapsaicin account for about 90% of the total capsaicin content and also provide about 90% of the spiciness and heat. Their content directly affects the spiciness of peppers and pepper products.

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

[0006] In view of the above problems in the prior art, the present invention provides a method for inducing capsaicin synthesis by a light signal transcription factor CaBBX2, thereby solving the above problems in the prior art.

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

[0008] S1. Construction of CaBBX2-TRV2 silencing vector: The specific fragment of CaBBX2 was obtained from the pepper CaBBX2 gene sequence, and pepper cDNA was used as a template. Flash Master Mix high-fidelity enzyme was used to amplify the specific fragment of CaBBX2 by PCR, and then purified and recovered to obtain CaBBX2-TRV2; restriction endonucleases Xba I and Sma I were used to double-digest the TRV2 vector: reaction reagents were added separately, pipetted and mixed, and the mixture was reacted by instant centrifugation. After the vector was confirmed to be cut by gel running, it was purified and recovered to obtain the cut TRV2 vector; CaBBX2-TRV2 was connected to the cut TRV2 vector by homologous recombination ligase, and the recombinant vector was transformed into Escherichia coli DH5α by heat shock method. The positive single colonies were identified by TRV2 universal primers, and sequencing and analysis were performed. The colonies with successful sequencing were expanded and the plasmids were extracted with a plasmid kit to obtain the recombinant plasmid TRV:CaBBX2 silencing vector;

[0009] S2. Preparation of impregnation liquid raw materials A and B for TRV:CaBBX2 silencing vector group and TRV:00 empty control group: The TRV:CaBBX2 silencing vector, TRV:00 empty vector and TRV1 auxiliary expression vector were respectively plated on LB solid culture medium containing kanamycin and rifampicin, inverted for culture, and single colony PCR detection was performed. The single clone with positive test results was picked and inoculated into liquid LB culture medium containing kanamycin and rifampicin for overnight amplification culture, and then the bacterial liquid was drawn from it to the liquid LB culture medium containing the corresponding antibiotics. The cells were propagated in liquid LB medium, the OD value of the bacterial solution was determined by ultraviolet spectrophotometer, the cells were centrifuged and collected, the cells were washed with pre-cooled ddH2O, and the cells were resuspended with infection solution A containing acetosyringone to obtain infection solution B containing TRV:CaBBX2 silencing vector and TRV:00 empty infection solution B1, and TRV1 auxiliary expression vector infection solution B2, and the cells were allowed to stand at room temperature in the dark, and then the infection solution B and the infection solution B1 were mixed with the infection solution B2 to obtain the infection solution raw material A and the control group infection solution raw material B;

[0010] S3. Treatment of spicy pepper No. 1: When spicy pepper No. 1 grows cotyledons, the infection liquid is drawn with a syringe without a needle into the raw material A and the raw material B of the pepper cotyledons;

[0011] S4. Treatment after infection: Improve the silencing efficiency by applying darkness, weak light and then normal light to peppers;

[0012] S5. Pollination and sampling: artificial pollination was performed when the four-mother bucket was flowering, and the pollination time was recorded on a sign. After pollination, the pepper fruits were taken after pollination, the fruit stalks and pepper seeds were removed, the peel and placenta were cut into pieces and quickly frozen in liquid nitrogen to obtain samples for the determination of capsaicin content and gene expression;

[0013] The experimental method for the interaction between S6.CaBBX2 and capsaicin biosynthesis genes (AT3, COMT, ACS, PAL3) is as follows: construct a vector, connect the promoter fragment of the gene to the pAbai vector, add the recognition sequence of KpnI - GGTACC and the linker in front of the front primer of the promoter fragment of the gene, add the recognition sequence of XhoI - TCTAGA in front of the back primer, use the genomic DNA of pepper CM334 as a template, amplify the promoter fragment of the gene, transform Escherichia coli by heat shock, connect it to the pAbai vector as bait, and cut pAbai with BstBI and BbsI. bai recombinant plasmid, and add phosphorylase AP at the same time, linearize and dephosphorylate in one step, transfer the reaction product into Y1HGold yeast cells, and spread it on SD-Ura medium to obtain a single clone; select a single clone to YPDA liquid medium for shaking and propagation to obtain a bacterial liquid, and aspirate the bacterial liquid to collect the bacteria by centrifugation; transfer the pGADT7 empty plasmid and the pGADT7:CaBBX2 recombinant vector into the above-mentioned monoclonal yeast strain containing the promoter-PAbai vector, and spread it on SD / -Ura-Leu medium to obtain a positive clone; spot on SD / -Ura-Leu medium with Aureobasidin A (AbA) antibiotic concentration gradient (0-200ng / mL) to screen the AbA concentration at which pGADT7 cannot grow normally but pGADT7:CaBBX2 can grow;

[0014] S7. Gene expression detection: extract RNA from the sample molecule sample in step S5, check the RNA quality with agarose gel electrophoresis, and detect RNA concentration and purity with UV spectrophotometer; use reverse transcription reagent to reverse transcribe the obtained RNA into cDNA and store it at 4°C; gene expression real-time quantitative qRT-PCR experiment: use the first chain of synthesized cDNA diluted 3 times as a template, and use the primer pair consisting of qCaBBX2 upstream primer and qCaBBX2 downstream primer to detect CaBBX2 gene; use pepper actin as the internal reference gene, and the primer designed for the internal reference gene is qActin;

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

[0016] S9. Experimental data evaluation: First, by spotting on SD-Ura-Leu medium, we screened the AbA concentration at which pGADT7 could not grow normally but pGADT7:CaBBX2 could grow, proving that CaBBX2 could interact with the gene;

[0017] Secondly, the capsaicin content data of the TRV:CaBBX2 silencing vector group and the TRV:00 empty control group at different times after pollination showed that the accumulation of capsaicin reached its peak 33 days after pollination of live peppers. After silencing the CaBBX2 gene, the content of capsaicin was significantly downregulated compared with TRV2, which can be evaluated as a relationship between CaBBX2 and the biosynthesis of capsaicin.

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

[0019] Furthermore, the PCR reaction program in step S1 was: 98°C for 10 s, 55°C for annealing for 5 s, 72°C for 5 s·1 kb, 34 cycles;

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

[0021] The reaction reagents are plasmid DNA 20 μL, 10×NEB buffer 5 μL, Xba I 1.5 μL, Sma I 1.5 μL, ddH2O 22 μL;

[0022] The reaction after instantaneous centrifugation is carried out 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 auxiliary expression vector were plated on LB solid medium containing 100 mg / mL kanamycin and rifampicin, respectively, and inverted cultured at 28°C for 2-3 days before colony PCR detection; single clones with positive test results were picked and inoculated into liquid LB medium containing 100 mg / mL kanamycin and rifampicin for overnight amplification culture, and shaken at 28°C and 200 r / min;

[0025] Use an ultraviolet spectrophotometer to measure the OD value of the bacterial solution until OD600 is between 0.8-1.0, centrifuge and collect the bacteria, wash the bacteria twice with pre-cooled ddH2O, and resuspend the bacteria in infection solution A containing 150 μM acetosyringone to obtain infection solution B containing TRV:CaBBX2 silencing vector and TRV:00 empty infection solution B1, TRV1 auxiliary expression vector infection solution B2, and stand in the dark at room temperature for 3 hours, then mix the infection solution B and the infection solution B1 with the infection solution B2 in a 1:1 ratio to obtain the infection solution raw material A and the control group infection solution raw material B, wherein the infection solution A is specifically 10mM MES, 10mM MgCl2, 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 h and then exposed to light at 50 μmol·m -2 ·s -1 The mice were adapted to low light conditions for 24 h, and then to a 12-h photoperiod, a day / night temperature of 22 / 20°C, and a light intensity of 200 μmol·m -2 ·s -1 Cultivate and grow the peppers in a nursery room under the conditions of; when the peppers begin to grow flower buds, silence the four new leaves around the pepper node according to step S3 to ensure the silencing effect.

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

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

[0030] Furthermore, in step S6, the one-step linearization and dephosphorylation reaction system is: 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 procedure is: 37°C, 10 min; 65°C, 15 min; the culture conditions of the SD / -Ura medium and the SD / -Ura-Leu medium are: 30°C constant temperature box, 2-3 d; the reaction conditions of the bacterial solution obtained by shaking the culture are 30°C, 180 rpm, 16 h.

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

[0032] The qRT-PCR reaction system using 10 μL of internal reference gene was as follows: SYBR 5 μL, ddH2O 3.4 μL, 10 μM PrimerF 0.3 μL, 10 μM PrimerR 0.3 μL, and cDNA 1 μL. The qRT-PCR reaction conditions using 10 μL of internal reference gene were as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 10 s, 40 cycles, 58°C annealing for 45 s, and 72°C extension for 30 s. The relative expression of genes was analyzed by relative quantitative analysis method 2 -ΔΔCt The method is used to calculate, and step S7 is repeated three times to obtain the final test data.

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

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

[0035] The filter residue and filter paper were extracted again with 25 mL of 95% ethanol solution by ultrasonic extraction for 30 min, filtered, and 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, fixed to 30 mL with 95% ethanol solution, filtered through a 0.45 μm organic phase filter membrane, and then subjected to chromatographic analysis;

[0037] The prepared test solution was measured according to the chromatographic conditions, and the peak area integral value was used for quantification to calculate the final contents of capsaicin, dihydrocapsaicin and total capsaicin.

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

[0039] The preparation process of the standard working solution is as follows: weigh natural capsaicin and dihydrocapsaicin, respectively, and use methanol to prepare a 1 mg / mL stock solution, and dilute it into 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] The present invention performs silencing treatment on CaBBX2 of living Zunla No. 1 pepper, adopts a TRV:00 empty control group, and through the determination of capsaicin content and expression amount and the interaction experiment between CaBBX2 and capsaicin synthesis gene, proves that CaBBX2 can interact with capsaicin synthesis, and evaluates the capsaicin content at different times after pollination of the gene of CaBBX2 silenced in living plants. After the CaBBX2 gene is silenced, the capsaicin content is significantly downregulated relative to TRV:00, further verifying that the CaBBX2 gene can affect capsaicin synthesis, and along with the silencing of CaBBX2, the expression amount of capsaicin synthesis-related genes is also reduced to varying degrees, and also verifies that CaBBX2 can affect capsaicin synthesis by regulating the expression of capsaicin synthesis-related genes. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0044] Figure 3 This is a bar graph showing the change in total capsaicin content of a living pepper after silencing the CaBBX2 gene for 16 days, 25 days, 33 days, and 38 days according to an embodiment of the present invention;

[0045] Figure 4 This is a bar graph showing the expression changes of genes related to capsaicin synthesis after 16d, 25d, 33d, and 38d of pollination of the gene-silenced CaBBX2 in vivo according to an embodiment of the present invention;

[0046] Figure 5 This is a bacterial flora diagram of PAbai:AT3+PGADT7 and PAbai:AT3+PGADT7:CaBBX2 loaded into SD-Ura-Leu medium containing 0 and 200 ng / mL antibiotic concentration gradient according to one embodiment of the present invention;

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

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

[0049] Figure 8 This is a bacterial flora diagram of PAbai:COMT+PGADT7 and PAbai:COMT+PGADT7:CaBBX2 loaded with SD-Ura-Leu medium containing 0 and 200 ng / mL antibiotic concentration gradient according to one embodiment of the present invention;

[0050] Fig. 9 This is a bar graph showing the changes in capsaicin content in isolated pepper fruit after transient overexpression of the CaBBX2 gene for 12h, 1d, 3d, 5d, and 7d according to an embodiment of the present invention;

[0051] Fig.10 This is a bar graph showing the changes in dihydrocapsaicin content in isolated pepper fruit 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] Fig.11 This is a bar graph showing the changes in total capsaicin content in isolated pepper fruit 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;

[0053] Fig.12 This is a bar graph showing the expression changes of pepper-related genes synthesized by transient overexpression of CaBBX2 gene in isolated pepper fruit for 3 days according to one embodiment of the present invention;

[0054] Fig.13 This is a bar graph showing changes in gene expression related to transient overexpression of the CaBBX2 gene in in vitro pepper fruit for 7 days according to an embodiment of the present invention;

[0055] Fig.14 This is a bar graph showing the changes in capsaicin content in isolated pepper fruit after transient silencing of the CaBBX2 gene for 12 hours, 1 day, 3 days, 5 days, and 7 days according to an embodiment of the present invention;

[0056] Fig.15 This is a bar graph showing the changes in dihydrocapsaicin content in isolated pepper fruit after transient silencing of the CaBBX2 gene for 12 hours, 1 day, 3 days, 5 days, and 7 days according to an embodiment of the present invention;

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

[0058] Fig.17This is a bar graph showing the expression changes of pepper-related genes synthesized 3 days after the CaBBX2 gene was transiently silenced in in vitro pepper fruit according to an embodiment of the present invention. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below with reference to the accompanying drawings and embodiments of the specification.

[0060] Embodiment 1

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

[0062] The experimental steps are as follows:

[0063] S1.TRV: CaBBX2 silencing vector construction: The specific fragment of CaBBX2 was obtained from the pepper CaBBX2 gene sequence, and pepper cDNA was used as a template. Flash Master Mix high-fidelity enzyme was used to amplify the specific fragment of CaBBX2 by PCR, and then purified and recovered to obtain CaBBX2-TRV2; restriction endonucleases Xba I and Sma I were used to double-digest the TRV2 vector: reaction reagents were added separately, pipetted and mixed, and the mixture was reacted by instant centrifugation. After the vector was confirmed to be cut by gel running, it was purified and recovered to obtain the cut TRV2 vector; CaBBX2-TRV2 was connected to the cut TRV2 vector by homologous recombination ligase, and the recombinant vector was transformed into Escherichia coli DH5α by heat shock method. The positive single colonies were identified by TRV2 universal primers, and sequencing and analysis were performed. The colonies with successful sequencing were expanded and the plasmids were extracted with a plasmid kit to obtain the recombinant plasmid TRV:CaBBX2 silencing vector;

[0064] S2. Preparation of impregnation liquid raw materials A and B for TRV:CaBBX2 silencing vector group and TRV:00 empty control group: The TRV:CaBBX2 silencing vector, TRV:00 empty vector and TRV1 auxiliary expression vector were respectively plated on LB solid culture medium containing kanamycin and rifampicin, inverted for culture, and single colony PCR detection was performed. The single clone with positive test results was picked and inoculated into liquid LB culture medium containing kanamycin and rifampicin for overnight amplification culture, and then the bacterial liquid was drawn from it to the liquid LB culture medium containing the corresponding antibiotics. The cells were propagated in liquid LB medium, the OD value of the bacterial solution was determined by ultraviolet spectrophotometer, the cells were centrifuged and collected, the cells were washed with pre-cooled ddH2O, and the cells were resuspended with infection solution A containing acetosyringone to obtain infection solution B containing TRV:CaBBX2 silencing vector and TRV:00 empty infection solution B1, and TRV1 auxiliary expression vector infection solution B2, and the cells were allowed to stand at room temperature in the dark, and then the infection solution B and the infection solution B1 were mixed with the infection solution B2 to obtain the infection solution raw material A and the control group infection solution raw material B;

[0065] S3. Treatment of spicy pepper No. 1: When spicy pepper No. 1 grows cotyledons, the infection liquid is drawn with a syringe without a needle into the raw material A and the raw material B of the pepper cotyledons;

[0066] S4. Treatment after infection: Improve the silencing efficiency by applying darkness, weak light and then normal light to peppers;

[0067] S5. Pollination and sampling: artificial pollination was performed when the four-mother bucket was flowering, and the pollination time was recorded on a sign. After pollination, the pepper fruits were taken after pollination, the fruit stalks and pepper seeds were removed, the peel and placenta were cut into pieces and quickly frozen in liquid nitrogen to obtain samples for the determination of capsaicin content and gene expression;

[0068] S6. The experimental method for the interaction between CaBBX2 and capsaicin biosynthesis gene is as follows: construct the vector: connect the promoter fragment of the gene to the pAbai vector, add the recognition sequence of KpnI - GGTACC and the linker in front of the front primer of the promoter fragment of the gene, add the recognition sequence of XhoI - TCTAGA in front of the back primer, use the genomic DNA of CM334 as a template, amplify the promoter fragment of the gene, heat-shock transform Escherichia coli and connect it to the pAbai vector as bait, cut the pAbai recombinant plasmid with BstBI and BbsI, and add Phosphorylase AP, one-step linearization and dephosphorylation reaction, the reaction product is transferred into Y1HGold yeast cells, spread on SD / -Ura medium to obtain a single clone; the single clone is selected to YPDA liquid medium for shaking and propagation to obtain a bacterial liquid, and 2 mL of the bacterial liquid is centrifuged to collect the bacteria; the pGADT7 empty plasmid and the pGADT7:CaBBX2 recombinant vector are transferred into the above-mentioned monoclonal yeast strain containing the promoter-PAbai vector, spread on SD / -Ura-Leu medium, and a positive clone is obtained; the AbA concentration at which pGADT7 cannot grow normally but pGADT7:CaBBX2 can grow is screened by spotting on SD / -Ura-Leu medium in the presence of Aureobasidin A (AbA) antibiotic concentration gradient (0-200 ng / mL).

[0069] S7. Detection of gene expression: extract RNA from the sample molecule in step S5, test the RNA quality with agarose gel electrophoresis, and detect RNA concentration and purity with UV spectrophotometer; reverse transcribe the obtained RNA into cDNA using reverse transcription reagent and store at 4°C; use real-time quantitative qRT-PCR experiment of gene expression: use the first chain of synthesized cDNA diluted 3 times as template, and use the primer pair consisting of qCaBBX2 upstream primer and qCaBBX2 upstream primer to detect CaBBX2 gene; use pepper actin as internal reference gene, and design the primer of internal reference gene as qActin;

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

[0071] S9. Experimental data evaluation: First, on the SD / -Ura-Leu medium after spotting, screen the AbA concentration at which pGADT7 cannot grow normally but pGADT7:CaBBX2 can grow;

[0072] like Figure 5-Figure 8 As shown, it is a bacterial flora diagram of the culture medium loaded with different antibiotic concentration gradients of the recombinant plasmids PAbai:AT3, PAbai:PAL3, PAbai:ACS, PAbai:COMT and their empty vector groups constructed by the method of step S6;

[0073] in, Figure 5 The two pictures 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 fine. The two pictures on the right show that PGADT7 (negative control) did not grow, while PGADT7:CaBBX2 could grow normally, which indicates that CaBBX2 interacts with AT3.

[0074] Figure 6 The two pictures 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 fine. The two pictures on the right show that PGADT7 (negative control) did not grow, while PGADT7:CaBBX2 could grow normally, which indicates that CaBBX2 interacts with PAL3.

[0075] Figure 7 The two pictures 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 fine. The two pictures on the right show that PGADT7 (negative control) did not grow, while PGADT7:CaBBX2 could grow normally, which indicates that CaBBX2 interacts with COMT.

[0076] Figure 8 The two pictures on the left prove that PGADT7 and PGADT7:CaBBX2 were successfully transferred into Y1Hgold bacteria containing PAbai:COMT, and the activity of the bacteria was fine. The two pictures on the right show that PGADT7 (negative control) did not grow, while PGADT7:CaBBX2 could grow normally, which indicates that CaBBX2 interacts with ACS.

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

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

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

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

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

[0082] The TRV:CaBBX2 silencing vector, TRV:00 empty vector, and TRV1 auxiliary expression vector were plated on LB solid medium containing 100 mg / mL kanamycin and rifampicin, respectively, and inverted at 28°C for 2-3 days before colony PCR detection; single clones with positive test results were picked and inoculated into liquid LB medium containing 100 mg / mL kanamycin and rifampicin for overnight amplification culture, and the bacteria were shaken at 28°C and 200 r / min. The OD value of the bacterial solution was measured by UV spectrophotometer until When OD600 is between 0.8 and 1.0, centrifuge and collect the bacteria, wash them twice with pre-cooled ddH2O, resuspend the bacteria with infection solution A containing 150 μM acetosyringone to obtain infection solution B containing TRV:CaBBX2 silencing vector and TRV:00 empty infection solution B1, and TRV1 auxiliary expression vector infection solution B2, stand at room temperature in the dark for 3 hours, then mix infection solution B and infection solution B1 with infection solution B2 in a 1:1 ratio to obtain infection solution raw material A and control group infection solution raw material B, wherein infection solution A is specifically 10 mM MES, 10 mM MgCl2, 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 h and then exposed to light at 50 μmol·m -2 ·s -1 The mice were adapted to low light conditions for 24 h, and then to a 12-h photoperiod, a day / night temperature of 22 / 20°C, and a light intensity of 200 μmol·m -2 ·s -1 Cultivate and grow the peppers in a nursery room under the conditions of; when the peppers begin to grow flower buds, silence the four new leaves around the pepper node 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 taken 16, 25, 33, and 38 days after pollination, with 25-30 fruits at each time point. The fruit stalks and pepper seeds were removed, and the peel and placenta were cut into pieces and quickly 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: 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°C, 10 min; 65°C, 15 min; the culture conditions of SD / -Ura medium and SD / -Ura-Leu medium are: 30°C constant temperature box, 2-3d; the reaction conditions of the bacterial solution obtained by shaking the bacteria are 30°C, 180rpm, 16h.

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

[0089] The qRT-PCR reaction system using 10 μL of the internal reference gene: SYBR 5 μL, ddH2O 3.4 μL, 10 μM PrimerF0.3 μL, 10 μM PrimerR 0.3 μL, cDNA 1 μL; the qRT-PCR reaction conditions using 10 μL of the internal reference gene were: 95°C pre-denaturation for 3 min, 95°C denaturation for 10 s, 40 cycles, 58°C annealing for 45 s, and 72°C extension for 30 s. The relative expression of the gene was calculated using the relative quantitative analysis 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 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; extract the filter residue and filter paper with 25mL of 95% ethanol solution through an ultrasonic extractor for 30min, filter, collect the filtrate, and repeat once; combine the filtrates collected by three filtrations, concentrate to 25mL using a rotary evaporator, transfer to a new 50mL centrifuge tube, make up to 30mL with 95% ethanol solution, filter through a 0.45μm organic phase filter membrane, and perform chromatographic analysis; determine the prepared test solution according to the chromatographic conditions, quantify by the peak area integral value, and calculate the final content of capsaicin, dihydrocapsaicin, and total capsaicin, where the chromatographic reference conditions are: Chromatographic column: Ultimate ODS-3 (4.6mm×250mm, 5μm); mobile phase: acetonitrile: water = 7:3, pass through 0.45μm filter membrane and deaerate before use; flow rate: 1mL / min; UV detector: wavelength 280nm; injection volume: 10μL; the preparation process of standard working solution is as follows: weigh natural capsaicin and dihydrocapsaicin, respectively, and prepare them into 1mg / mL mother solution with methanol, and dilute them into 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] Wherein, in step S1, the CaBBX2-TRV2 upstream primer sequence, the CaBBX2-TRV2 downstream primer sequence, the TRV2 upstream primer sequence, and the TRV2 downstream primer sequence are respectively: TGAGTAAGGTTACCGAATTCTCTAGAAAGGCAGAGAATTGAGTTTCCAG;

[0093] TTTAATGTCTTCGGGACATGCCCGGGTCATGATTGTTGCCACTACGCA;GATAATGGTTTGGTGGTC;GTTTAATGTCTTCGGGAC.

[0094] The CDS sequence of 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 primer sequence for AT3-PAbai-F was AGCTTGAATTCGAGCTCGGTACCGAAAAAAGACAAGTAAATTGAGAAATCAA;

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

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

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

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

[0104] Primer sequence for ACS-PAbai-R: ACATACAGAGCACATGCCTCGAGACCCTAAGATGTAATATAATTACATAACT;

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

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

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

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

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

[0110] CTCTCTGCCGTTCCTGTGA; GGCAGCAAGACCAACTCTTA.

[0111] This example proves that CaBBX2 can interact with genes related to capsaicin synthesis through comparative experiments and experiments on the interaction between CaBBX2 and capsaicin synthesis genes. The CaBBX2 gene is silenced in living plants to obtain experimental data on the capsaicin content and the expression of genes related to capsaicin synthesis in Zunla No. 1 pepper fruits 16, 25, 33, and 38 days after pollination when CaBBX2 is silenced. The results show that after silencing the CaBBX2 gene, the capsaicin content is significantly downregulated relative to TRV:00. With the silencing of CaBBX2, the expression of genes related to capsaicin synthesis also decreases to varying degrees, confirming that CaBBX2 can affect the synthesis of capsaicin by regulating the expression of genes related to capsaicin synthesis.

[0112] Embodiment 2

[0113] In vitro transient overexpression experimental method of pepper

[0114] The following steps are involved:

[0115] S1.35S: Construction of CaBBX2-flag overexpression vector, as follows:

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

[0117] The restriction endonuclease EcoR I was used to perform single restriction digestion on the 35S:flag vector. 20 μL of plasmid DNA, 5 μL of 10×NEB buffer, 3 μL of EcoR I, and 22 μL of ddH2O were added, and the mixture was mixed by pipetting and centrifuged instantaneously. The mixture was reacted at 37°C for 3 h. After the vector was confirmed to be cut by running on gel, the 35S:flag vector was purified and recovered.

[0118] Using homologous recombination ligase, CaBBX2-35-flag was connected to the cleaved 35S:flag vector to obtain the recombinant vector 35S:CaBBX2-flag;

[0119] The recombinant vector was transformed into Escherichia coli DH5α by heat shock method, and the positive single colonies were identified by 35S:flag universal primers, sequenced and analyzed. Finally, the successfully sequenced colonies were expanded and the plasmids were extracted by plasmid kit to obtain the recombinant plasmid overexpression vector 35S:CaBBX2-flag.

[0120] S2. Preparation of the impregnation solution A and B of the overexpression vector 35S:CaBBX2-flag group and the 35S:flag empty control group: The successfully constructed overexpression vector 35S:CaBBX2-flag and 35S:flag empty vector were respectively transferred into the competent cells of GV3101 Agrobacterium, plated on LB solid medium containing 100 mg / mL spectinomycin, inverted and cultured at 28°C for 2-3 days, and then colony PCR detection was performed;

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

[0122] Resuspend the bacteria in infection solution A (10 mM MES, 10 mM MgCl2, pH = 5.5) containing 150 μM acetosyringone to obtain infection solution A containing the overexpression vector 35S:CaBBX2-flag and 35S:flag empty control infection solution B, and let stand at 28°C in the dark for 3 hours.

[0123] S3. Treatment of disease-free Ganjiao No. 19 pepper fruits: Take disease-free Ganjiao No. 19 pepper fruits of uniform size and color, and lay them flat on a clean table in a disinfected laboratory to dissipate heat overnight at a room temperature of 23°C; wipe the fruit body and stalk with a paper towel dipped in sterilized alcohol for disinfection; use a 1mL syringe to draw up the original solutions A and B of the infection solution, and inject them into the pepper stalk, and inject the original solutions A and B of the infection solution into the pepper placenta respectively. The sampling time is 12h, 1d, 3d, 5d, and 7d after the injection. Take 25 pepper fruits at each time point, and the sampling site is the peel and placenta of the upper half of the Ganjiao fruit. Quick-freeze with liquid nitrogen, mix evenly and grind the samples for later use. For each treatment, weigh 4 0.5g samples in 2mL centrifuge tubes as molecular samples; use blue ink as a negative control test to confirm that the infection solution can be injected into the placenta by injecting from the fruit stalk.

[0124] S4. Detection of gene expression: Use Trizol method to extract RNA from the molecular sample obtained in step S3, use agarose gel electrophoresis to check RNA quality, and use UV spectrophotometer to detect RNA concentration and purity; use reverse transcription reagent to reverse transcribe the obtained RNA into cDNA and store it at 4°C; Real-time quantitative qRT-PCR of gene expression: Use the first chain of synthesized cDNA diluted 3 times as template, and use the primer pair consisting of qCaBBX2 upstream primer and qCaBBX2 downstream primer to detect CaBBX2 gene; Use pepper actin as the internal reference gene, design the primer of the internal reference gene as qActin, and use 10μL of the internal reference gene qRT-PCR reaction system: SYBR 5μL, ddH2O 3.4μL, 10μM PrimerF0.3μL, 10μM PrimerR 0.3μL, cDNA 1 μL; the qRT-PCR reaction conditions using 10 μL of the internal reference gene were: 95°C pre-denaturation for 3 min, 95°C denaturation for 10 s, 40 cycles, 58°C annealing for 45 s, 72°C extension for 30 s, and the relative expression of the gene was analyzed by relative quantitative analysis method 2 -ΔΔCt The method is used to calculate, and step S7 is repeated three times to obtain the final test data.

[0125] S5. Capsaicin determination method: Capsaicin was determined by high performance liquid chromatography, which specifically includes the following steps:

[0126] Accurately weigh 10g of the sample, put it in 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 together with the filter paper with 25mL of 95% ethanol solution through an ultrasonic extractor for 30min, filter, collect the filtrate, and repeat once;

[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, fixed to 30 mL with 95% ethanol solution, filtered through a 0.45 μm organic phase filter membrane, and then subjected to chromatographic analysis;

[0128] The prepared test solution was measured according to the chromatographic conditions, and the peak area integral value was used for quantification to calculate the final contents of capsaicin, dihydrocapsaicin and total capsaicin.

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

[0130] According to the capsaicin content data of the overexpression vector 35S:CaBBX2-flag group and the 35S:flag empty control group, after transient overexpression of the CaBBX2 gene in the detached pepper fruit, except for the 12th hour, at the other four time points after transient overexpression of CaBBX2, the contents of capsaicin, dihydrocapsaicin, and total capsaicin were significantly increased compared with the control group 35S:flag. It can be evaluated that transient overexpression of CaBBX2 can increase the spiciness of pepper fruit.

[0131] Since the synthesis of capsaicin involves two pathways, phenylpropanoid metabolism and fatty acid metabolism, the data of gene expression detection of pepper fruits with transient overexpression for 3d and 7d showed that the genes with significant differences between 3d and 7d were different because these genes existed in different positions of a pathway, and the response of gene expression should be faster than that of secondary metabolites;

[0132] According to the capsaicin expression data of the overexpression vector 35S:CaBBX2-flag group and the 35S:flag empty control group, after transient overexpression of the CaBBX2 gene in in vitro pepper fruits, the CaBBX2 expression level can be increased to 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 of step S5 are: chromatographic column: Ultimate ODS-3 (4.6 mm×250 mm, 5 μm);

[0134] Mobile phase: acetonitrile: water = 7:3, pass through 0.45μm filter membrane and deaerate before use; flow rate: 1mL / min; UV detector: wavelength 280nm; injection volume: 10uL; the preparation process of standard working solution is as follows: weigh natural capsaicin and dihydrocapsaicin, respectively, and use methanol to prepare 1mg / mL mother solution, and dilute to 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] Wherein, 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 respectively:

[0136] GATGACGATGACAAGGAATTCATGAGAACTCTCTGTGATGTTTTGT,

[0137] GTCCTTGTAATCCATGAATTCCTTCTCTGGTTCTCTTTTGAAGGG, ACGCACAATCCCACTATCCTTC, CATAAAAATACGATAGTAACGGGTG.

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

[0139] Example 3

[0140] In vitro transient silencing experimental method of pepper

[0141] In this embodiment, steps S1, S2, S4, and S5 are the same as those in Embodiment 2.

[0142] The difference between this embodiment and embodiment 2 is that the treatment of the disease-free pepper fruit of S3, Ganjiao No. 19, is specifically the following steps: take disease-free pepper fruit of Ganjiao No. 19 with uniform individual size and color, lay it flat on a clean table in a disinfected laboratory for one night to dissipate heat, and the room temperature is 23 degrees Celsius; wipe the fruit body and stalk with a paper towel dipped in sterilized alcohol for disinfection. Use a 1mL syringe to draw infection liquid B and B1 respectively, inject the pepper stalk, and inject the infection liquid into the pepper placenta. The sampling time is 12h, 1d, 3d, 5d, and 7d after the injection. Take 25 pepper fruits at each time point, and the sampling site is the peel and placenta of the upper half of the Ganjiao fruit, quick-freeze with liquid nitrogen, evenly mix and grind the sample for standby use, and weigh 4 0.5g samples in a 2mL centrifuge tube for each treatment as molecular samples.

[0143] The difference between this embodiment and embodiment 2 is that the S6 experimental data evaluation, 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 TRV:00 empty control group, after the isolated pepper was transiently silenced, the contents of capsaicin, dihydrocapsaicin, and total capsaicin 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 fruit;

[0145] According to the capsaicin-related gene detection data of the silencing vector TRV:CaBBX2 group and the TRV:00 empty control group, the difference between the experimental group and the control group was the largest 3 days after transient silencing, so the samples 3 days after transient silencing were selected to extract RNA for capsaicin synthesis-related gene detection. It can be seen that after transient silencing of CaBBX2, the expression levels of CaBBX2 and capsaicin synthesis-related genes decreased significantly. It can be evaluated that transient silencing of CaBBX2 will reduce the expression levels of capsaicin synthesis-related genes and thus reduce the capsaicin content.

[0146] According to the above embodiments, through the experimental method of silencing and overexpressing the CaBBX2 gene in isolated peppers, combined with the experimental method of silencing the CaBBX2 gene in living peppers, a comprehensive evaluation shows that the CaBBX2 gene affects capsaicin synthesis, and 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 promoting the increase of capsaicin synthesis.

Claims

1. A method for inducing capsaicin synthesis by light signal transcription factor CaBBX2, specifically comprising an experimental method for the effect of silencing CaBBX2 gene on capsaicin synthesis in living plants, characterized in that: The experimental steps are as follows: S1. Construction of CaBBX2-TRV2 silencing vector: The specific fragment of CaBBX2 was obtained from the pepper CaBBX2 gene sequence, and pepper cDNA was used as a template. Flash Master Mix high-fidelity enzyme was used to amplify the specific fragment of CaBBX2 by PCR, and then purified and recovered to obtain CaBBX2-TRV2; restriction endonucleases Xba I and Sma I were used to double-digest the TRV2 vector: reaction reagents were added separately, pipetted and mixed, and centrifuged instantaneously for reaction. After running gel to confirm that the vector was cut, it was purified and recovered to obtain the enzyme-digested TRV2 vector; CaBBX2-TRV2 was connected to the enzyme-digested TRV2 vector using homologous recombination ligase, and the recombinant vector was transformed into Escherichia coli DH5α by heat shock method, and positive single colonies were identified with TRV2 universal primers, sequenced and analyzed, and the colonies with successful sequencing were expanded and plasmids were extracted with a plasmid kit to obtain TRV:CaBBX2 silencing vector; S2. Preparation of impregnation liquid raw materials A and B for TRV:CaBBX2 silencing vector group and TRV:00 empty control group: The TRV:CaBBX2 silencing vector, TRV:00 empty vector and TRV1 auxiliary expression vector were respectively plated on LB solid culture medium containing kanamycin and rifampicin, inverted for culture, and single colony PCR detection was performed. The single clone with positive test results was picked and inoculated into liquid LB culture medium containing kanamycin and rifampicin for overnight amplification culture, and then the bacterial liquid was drawn from it to the liquid LB culture medium containing the corresponding antibiotics. The cells were propagated in liquid LB medium, the OD value of the bacterial solution was determined by ultraviolet spectrophotometer, the cells were centrifuged and collected, the cells were washed with pre-cooled ddH2O, and the cells were resuspended with infection solution A containing acetosyringone to obtain infection solution B containing TRV:CaBBX2 silencing vector and TRV:00 empty infection solution B1, and TRV1 auxiliary expression vector infection solution B2, and the cells were allowed to stand at room temperature in the dark, and then the infection solution B and the infection solution B1 were mixed with the infection solution B2 to obtain the infection solution raw material A and the control group infection solution raw material B; S3. Treatment of spicy pepper No. 1: When spicy pepper No. 1 grows cotyledons, the infection liquid is drawn with a syringe without a needle into the raw material A and the raw material B of the pepper cotyledons; S4. Treatment after infection: Improve the silencing efficiency by applying darkness, weak light and then normal light to peppers; S5. Pollination and sampling: artificial pollination is performed when the four-mother bucket blooms, and the pollination time is recorded on a sign. After pollination, pepper fruits at a corresponding time after pollination are taken, the fruit stalks and pepper seeds are removed, the peel and placenta are cut into pieces and quickly frozen in liquid nitrogen to obtain samples for measuring capsaicin content and gene expression; S6. The experimental method for the interaction between CaBBX2 and capsaicin biosynthesis gene is as follows: construct the vector: connect the promoter fragment of the gene to the pAbai vector, add the recognition sequence of KpnI - GGTACC and the linker in front of the front primer of the promoter fragment of the gene, add the recognition sequence of XhoI - TCTAGA in front of the back primer, use the genomic DNA of pepper CM334 as a template, amplify the promoter fragment of the gene, heat-shock transform Escherichia coli to connect it to the pAbai vector as bait, cut the pAbai recombinant plasmid with BstBI and BbsI, and at the same time Phosphorylase AP was added to perform linearization and dephosphorylation reaction in one step, and the reaction product was transferred into Y1HGold yeast cells, and spread on SD / -Ura medium to obtain a single clone; a single clone was selected and propagated in YPDA liquid medium for shaking to obtain a bacterial liquid, and the bacterial liquid was centrifuged to collect the bacteria; pGADT7 empty plasmid and pGADT7:CaBBX2 recombinant vector were transferred into the monoclonal yeast strain containing the promoter-PAbai vector, and spread on SD / -Ura-Leu medium to obtain a positive clone; spotting was performed on SD / -Ura-Leu medium in the presence of Aureobasidin A (AbA) antibiotic concentration gradient (0-200 ng / mL) to screen the AbA concentration at which pGADT7 could not grow normally but pGADT7:CaBBX2 could grow; S7. Gene expression detection: extract RNA from the sample molecule sample of step S5, check the RNA quality by agarose gel electrophoresis, and detect RNA concentration and purity by UV spectrophotometer; use reverse transcription reagent to reverse transcribe the obtained RNA into cDNA, and store it at 4°C; gene expression real-time quantitative qRT-PCR experiment: use the first chain of synthesized cDNA diluted 3 times as a template, and use the primer pair consisting of qCaBBX2 upstream primer and qCaBBX2 downstream primer to detect CaBBX2 gene; use pepper actin as the internal reference gene, and the primer designed for the internal reference gene is qActin; S8. Determination of capsaicin: Capsaicin was determined by high performance liquid chromatography; S9. Experimental data evaluation: First, by spotting on SD / -Ura-Leu medium, we screened the AbA concentration at which pGADT7 could not grow normally but pGADT7:CaBBX2 could grow, proving that CaBBX2 could interact with the gene; Secondly, the capsaicin content data of the TRV:CaBBX2 silencing vector group and the TRV:00 empty control group at different times after pollination showed that the accumulation of capsaicin reached its peak 33 days after pollination of live peppers. After silencing the CaBBX2 gene, the content of capsaicin was significantly downregulated compared with TRV2, which can be evaluated as a relationship between CaBBX2 and the biosynthesis of capsaicin. Finally, according to the data of capsaicin synthesis-related gene expression at different times after pollination in the TRV:CaBBX2 silencing vector group and the TRV:00 empty control group, CaBBX2 was effectively silenced with a silencing efficiency of 40%-60%, and with the silencing of CaBBX2, the expression of capsaicin synthesis-related genes also decreased to varying degrees. In the 33d data, the expression levels of CaCOMT and CaAT3 even decreased by 70% compared with the control group. It can be evaluated that CaBBX2 can affect the synthesis of capsaicin by regulating the expression of capsaicin synthesis-related genes.

2. The method for inducing capsaicin synthesis by light signal transcription factor CaBBX2 according to claim 1, characterized in that: The PCR reaction program in step S1 is: 98°C for 10s, 55°C for annealing for 5s, 72°C for 5s·1kb, and 34 cycles; the PCR reaction system is: Mix 25μL, F 2μL, R 2μL, template 2μL, and then adding ddH2O to 50μL; the reaction reagents are plasmid DNA 20μL, 10×NEBbuffer 5μL, Xba I 1.5μL, Sma I 1.5μL, and ddH2O 22μL; the reaction method after instantaneous centrifugation is 37°C for 3h.

3. The method for inducing capsaicin synthesis by light signal transcription factor CaBBX2 according to claim 1, characterized in that: The specific steps of step S2 are as follows: The TRV:CaBBX2 silencing vector, TRV:00 empty vector and TRV1 auxiliary expression vector were plated on LB solid culture medium containing 100 mg / mL kanamycin and rifampicin, respectively, and inverted at 28°C for 2-3 days before colony PCR detection; the single clone with a positive test result was picked and inoculated into liquid LB culture medium containing 100 mg / mL kanamycin and rifampicin for overnight amplification culture, and the bacteria were shaken at 28°C and 200 r / min. The OD value of the bacterial solution was measured by ultraviolet spectrophotometer until OD600 was between 0.8-1.0, centrifuged, and the bacteria were collected. The bacteria were washed twice with pre-cooled ddH2O, and the bacteria were resuspended in infection solution A containing 150 μM acetosyringone to obtain TR V: CaBBX The infection solution B of the silencing vector, the TRV:00 empty infection solution B1, and the TRV1 auxiliary expression vector infection solution B2 were allowed to stand in the dark at room temperature for 3 hours, and then the infection solution B and the infection solution B1 were mixed with the infection solution B2 in a 1:1 ratio to obtain the infection solution raw material A and the control group infection solution raw material B, wherein the infection solution A specifically contained 10mM MES, 10mM MgCl2, and pH=5.

5.

4. The method of inducing capsaicin synthesis by light signal transcription factor CaBBX2 according to claim 1, characterized in that: The specific steps of step S4 are as follows: The peppers were first cultured in the dark at 18°C ​​for 56 h and then exposed to light at 50 μmol·m -2 ·s -1 The mice were adapted to low light conditions for 24 h, and then to a 12-h photoperiod, a day / night temperature of 22 / 20°C, and a light intensity of 200 μmol·m -2 ·s -1 Cultivate and grow the peppers in a nursery room under the conditions of; when the peppers begin to grow flower buds, silence the four new leaves around the pepper node according to step S3 to ensure the silencing effect.

5. The method of inducing capsaicin synthesis by light signal transcription factor CaBBX2 according to claim 1, characterized in that: The specific steps of step S5 are as follows: Pepper fruits were taken 16, 25, 33, and 38 days after pollination, with 25-30 fruits at each time point. The fruit stalks and pepper seeds were removed, and the peel and placenta were cut into pieces and quickly frozen in liquid nitrogen to obtain samples for later use.

6. The method of inducing capsaicin synthesis by light signal transcription factor CaBBX2 according to claim 1, characterized in that: The one-step linearization and dephosphorylation reaction system in step S6 is: 10×Fast Digst Buffer, 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°C, 10 min; 65°C, 15min; the culture conditions of the SD / -Ura medium and the SD / -Ura-Leu medium are: 30°C constant temperature box, 2-3d; the reaction conditions of the bacterial solution obtained by shaking bacteria propagation are 30°C, 180rpm, 16h.

7. The method of inducing capsaicin synthesis by light signal transcription factor CaBBX2 according to claim 1, characterized in that: The specific reaction parameters of S7 are as follows: The qRT-PCR reaction system using 10 μL of internal reference gene was as follows: SYBR 5 μL, ddH2O 3.4 μL, 10 μM PrimerF 0.3 μL, 10 μM PrimerR 0.3 μL, and cDNA 1 μL. The qRT-PCR reaction conditions using 10 μL of internal reference gene were as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 10 s, 40 cycles, 58°C annealing for 45 s, and 72°C extension for 30 s. The relative expression of genes was analyzed by relative quantitative analysis method 2 -ΔΔCt The method is used to calculate, and step S7 is repeated three times to obtain the final test data.

8. The method of inducing capsaicin synthesis by light signal transcription factor CaBBX2 according to claim 1, characterized in that: The specific steps of step S8 are as follows: 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; extract the residue and filter paper with 25mL of 95% ethanol solution again with an ultrasonic extractor for 30min, filter, collect the filtrate, and repeat once; combine the filtrates collected by three filtrations, concentrate to 25mL using a rotary evaporator, transfer to a new 50mL centrifuge tube, and use 95% The ethanol solution was diluted to 30 mL, filtered through a 0.45 μm organic phase filter membrane, and then chromatographically analyzed; the prepared test solution was measured according to the chromatographic conditions, and the peak area integral value was used for quantification, and the final contents of capsaicin, dihydrocapsaicin, and total capsaicin were calculated, wherein the chromatographic reference conditions were: Chromatographic column: Ultimate ODS-3 (4.6mm×250mm, 5μm); mobile phase: acetonitrile: water = 7:3, pass through 0.45μm filter membrane and deaerate before use; flow rate: 1mL / min; UV detector: wavelength 280nm; injection volume: 10μL; the preparation process of standard working solution is as follows: weigh natural capsaicin and dihydrocapsaicin, respectively, and prepare them into 1mg / mL mother solution with methanol, and dilute them into 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.

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