Application and method of transcription factor CaBTF3 for regulating synthesis of capsorubin

Abstract

The invention belongs to the technical field of genetic engineering breeding, and particularly relates to application and a method of a transcription factor CaBTF3 for regulating capsorubin synthesis. According to the capsicum transcription factor CaBTF3 and the coding gene thereof provided by the invention, the biosynthesis of capsorubin can be influenced by adjusting the expression level of a key enzyme gene in a capsorubin synthesis route and regulating and controlling the development and quantity of fruit chloroplast, the expression of the CaBTF3 gene in capsicum fruits is inhibited, the accumulation amount of capsorubin in the capsicum fruits can be reduced, and the yield of capsorubin is increased. The color of the pepper fruit is further influenced. By enhancing the expression level of the CaBTF3 gene, the content of capsorubin in capsicum fruits can be increased, the gene is applied to plant genetic engineering breeding, a new effective way is provided for creating capsicum varieties with high capsorubin content, and the gene plays an important role in improving the economic benefits of the capsicum industry and popularizing and applying the capsorubin.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering breeding technology, and in particular to the application and method of CaBTF3, a transcription factor that regulates capsanthin synthesis. Background Technology

[0002] Chili peppers (Capsicum annuum L.) are an important vegetable crop and a popular condiment, ranking first among vegetable crops in terms of planting area and economic benefits in my country. Capsaicin, the main coloring substance in chili peppers, belongs to the carotenoid family and is a unique metabolite found in mature chili pepper fruits. It is mainly synthesized in the plastids of the epidermal cells of chili peppers and is a key indicator for evaluating chili pepper quality. Furthermore, in the food industry, capsaicin is widely used for coloring various foods such as aquatic products, meats, pastries, salads, canned goods, and beverages due to its bright color, high color value, strong coloring power, good color retention, and high safety. In the pharmaceutical field, capsaicin also possesses a wide range of biological activities, including antioxidant, anti-inflammatory, and metabolic regulation effects, and can help protect the cardiovascular system, enhance immunity, delay aging, and inhibit tumors. Capsaicin is also attracting attention in military, agricultural, and cosmetic fields. Currently, the demand for capsaicin is continuously increasing. Capsaicin is mainly extracted from mature red chili peppers, therefore, increasing the capsaicin content in chili pepper fruits is particularly important.

[0003] Currently, although the synthetic pathway of capsanthin is largely understood, its related regulatory mechanisms are still poorly reported. Transcription factors and their encoding genes are important regulators of plant growth and development. Enriching and exploring key functional genes controlling capsanthin content and studying their impact on capsanthin accumulation will provide scientific guidance for applied research such as breeding and improving chili fruit quality, and will have significant practical implications for cultivating new chili varieties with high capsanthin content. Summary of the Invention

[0004] To address the aforementioned technical problems in the prior art, this invention provides a transcription factor CaBTF3 and its encoding gene, which can influence capsanthin biosynthesis by regulating the expression activity of key enzyme genes in the synthetic pathway and controlling chloroplast development and quantity, thus exhibiting a positive regulatory effect on capsanthin synthesis. Therefore, this invention provides the application of the transcription factor CaBTF3 and its encoding gene in regulating capsanthin synthesis or color regulation in chili pepper fruits, and provides a method for increasing the capsanthin content in chili pepper fruits. Specifically, this invention is implemented through the following scheme:

[0005] The first aspect of this invention provides the application of transcription factor CaBTF3 in regulating capsanthin synthesis in chili pepper fruit, and the nucleotide sequence of the CaBTF3 gene is shown in SEQ ID NO.1.

[0006] Furthermore, the regulation includes positive regulation or negative regulation; the positive regulation includes: increasing the expression level of the CaBTF3 gene in wild-type pepper plants to obtain pepper plants with increased CaBTF3 gene expression level; the negative regulation includes: decreasing the expression level of the CaBTF3 gene in wild-type pepper plants to obtain pepper plants with decreased CaBTF3 gene expression level.

[0007] Furthermore, the reagent used to increase the expression level of the CaBTF3 gene is selected from one of the following (A)-(C):

[0008] (A) A nucleic acid molecule containing the full-length coding region of the CaBTF3 gene, wherein the nucleotide sequence of the CaBTF3 gene is shown in SEQ ID NO.1;

[0009] (B) An expression vector containing the nucleic acid molecules described in (A);

[0010] (C) Recombinant microorganisms containing the expression vector described in (B).

[0011] Furthermore, the reagent used to reduce the expression level of the CaBTF3 gene is selected from one of the following (D)-(F):

[0012] (D) A nucleic acid molecule for silencing the CaBTF3 gene, the nucleotide sequence of which is shown in SEQ ID NO. 18;

[0013] (E) A silencing vector containing the nucleic acid molecules described in (D);

[0014] (F) Recombinant microorganisms containing the silencing vector described in (E).

[0015] Furthermore, the method for increasing the expression level of the CaBTF3 gene includes: constructing an overexpression vector of the CaBTF3 gene, transforming it into wild-type pepper, and culturing pepper plants with increased CaBTF3 gene expression levels.

[0016] Furthermore, the construction of the overexpression vector includes the following steps: using pepper genomic cDNA as a template, PCR amplification is performed using primer pairs OE-BTF3-F and OE-BTF3-R to obtain the full-length coding region sequence of the CaBTF3 gene; the full-length coding region sequence is ligated to the pHELLSGATE8 expression vector digested with XmaI and XbaI using homologous recombination technology to construct the overexpression vector; wherein, the nucleotide sequences of OE-BTF3-F and OE-BTF3-R are shown in SEQ ID NO. 31-32, respectively.

[0017] Furthermore, the method for reducing the expression level of the CaBTF3 gene includes: constructing a CaBTF3 gene silencing vector, transforming it into wild-type pepper, and culturing pepper plants with reduced CaBTF3 gene expression levels.

[0018] Furthermore, the construction of the CaBTF3 gene silencing vector includes the following steps: using pepper genomic cDNA as a template, PCR amplification is performed using the TRV2-BTF3-F and TRV2-BTF3-R primer pairs to obtain the target sequence targeting the CaBTF3 gene; the target sequence is then ligated into the pTRV2 expression vector digested by SmaI enzyme using homologous recombination technology to construct the CaBTF3 gene silencing vector; wherein, the nucleotide sequences of TRV2-BTF3-F and TRV2-BTF3-R are shown in SEQ ID NO. 19-20, respectively.

[0019] Furthermore, the transformation method employed was Agrobacterium-mediated transformation.

[0020] A second aspect of the present invention provides a method for increasing the capsanthin content in chili pepper fruits, comprising the following steps: increasing the expression level of the CaBTF3 gene in chili pepper plants, wherein the nucleotide sequence of the CaBTF3 gene is shown in SEQ ID NO.1.

[0021] A third aspect of the present invention provides the application of transcription factor CaBTF3 in regulating the color of pepper fruit, wherein the nucleotide sequence of the CaBTF3 gene is shown in SEQ ID NO.1.

[0022] The advantages and positive effects of this invention are as follows:

[0023] The chili pepper transcription factor CaBTF3 and its encoding gene provided by this invention can influence capsanthin biosynthesis by regulating the expression levels of key enzyme genes in the capsanthin synthesis pathway and controlling the development and number of chloroplasts in fruits. Inhibiting the expression of the CaBTF3 gene in chili pepper fruits reduces the accumulation of capsanthin and further affects fruit color. Conversely, enhancing the expression level of the CaBTF3 gene increases the accumulation of capsanthin in chili pepper fruits. Applying this gene to plant genetic engineering breeding provides a new and effective approach for creating chili pepper varieties with high capsanthin content, playing a significant role in improving the economic benefits of the chili pepper industry and promoting the widespread application of capsanthin. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments are briefly introduced below.

[0025] Figure 1This is an agarose gel electrophoresis image of positive clones from the CaCCS promoter yeast one-hybrid screening library in this embodiment of the invention, obtained by PCR detection.

[0026] Figure 2 Figure 1 shows the functional analysis results of transcription factor CaBTF3 in an embodiment of the present invention; wherein, Figure (A) is the subcellular localization result of transcription factor CaBTF3, Figure (B) is the result of transcriptional activation activity detection of transcription factor CaBTF3, and Figure (C) is the tissue expression pattern of CaBTF3 in pepper plants, flowers and fruits.

[0027] Figure 3 This is a diagram showing the interaction results of transcription factor CaBTF3 with the promoters of CaPSY1, CaBCH1 and CaCCS genes in an embodiment of the present invention;

[0028] Figures (A)-(C) show the transient luciferase detection results after CaBTF3 binds to the promoters of CaPSY1, CaBCH1 and CaCCS, respectively. The fluorescence intensity is expressed as CPS. Figures (D)-(F) show the statistical values ​​of fluorescence intensity in Figures (A)-(C).

[0029] Figure 4 Phenotypic diagrams of CaBTF3-silenced plants and control plants in embodiments of the present invention;

[0030] Figure 5 The images show chlorophyll fluorescence phenotypic diagrams and chlorophyll fluorescence statistical diagrams of leaves from CaBTF3-silenced plants and control plants in this embodiment of the invention.

[0031] Figure 6 Figure 1 shows the relative expression levels of the transcription factor CaBTF3 silencing plant and the capsanthin content in the control plant, as well as the results of the measurement. Figure 2 shows the relative expression level of the CaBTF3 gene, Figure 3 shows the capsanthin content, and Figure 4 shows the relative expression levels of the CaCCS, CaPSY1, and CaBCH1 genes in the fruit of the silencing plant.

[0032] Figure 7 The figures show the number and development of chloroplasts in the fruits and leaves of CaBTF3-silenced plants and control plants in this embodiment of the invention; wherein, Figures (A)-(B) show chloroplasts in the pericarp cells of the control plants, Figures (C)-(D) show chloroplasts in the leaf cells of the control plants, Figures (E)-(F) show chloroplasts in the pericarp cells of CaBTF3-silenced plants, and Figures (G)-(H) show chloroplasts in the leaf cells of CaBTF3-silenced plants.

[0033] Figure 8 The PCR detection results of CaBTF3 gene overexpressing plants transformed according to an embodiment of the present invention are shown.

[0034] Figure 9 Phenotypic diagrams of plants and fruits transformed with CaBTF3 gene overexpression and control plants, as well as fruits, in embodiments of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments described herein are for illustrative purposes only and are not intended to limit the invention. All figures and other numerical values ​​used in this invention to indicate amounts, percentages, etc., should be understood in all cases to be modified by the word "approximately". Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and by conventional rounding methods. The meanings of words such as "comprising," "including," "containing," "having," etc., are non-limiting, allowing for the addition of other steps and other components that do not affect the result. "And / or" should be considered as a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" would be considered to include (i) A, (ii) B, and (iii) A and B.

[0036] Capsaicin is a unique metabolite found in mature chili peppers and is a key indicator for evaluating chili pepper quality. Currently, the biosynthetic pathway of capsaicin is largely understood. Its biosynthesis begins with geraniol-geraniol pyrophosphate (GGPP), which, under the catalysis of phytoene synthase (PSY), converts two molecules of GGPP into phytoene. This phytoene then undergoes a series of dehydrogenation and isomerization steps, eventually being oxidized to lycopene. Lycopene, through the combined action of a series of enzymes including LCYE, LCYB, BCH, ZEP, and CCS, and with the aid of oxygen, is ultimately converted into capsaicin and capsanthin. However, the regulatory mechanisms of key enzyme genes in the capsaicin biosynthetic pathway remain unclear. Current technologies lack methods to cultivate varieties with high capsaicin content by intervening in the expression regulation of these genes.

[0037] This invention utilizes a yeast library specific to chili pepper fruits to screen and obtain the transcription factor CaBTF3, which binds to the promoters of key enzyme genes involved in capsanthin synthesis. Silencing the CaBTF3 gene in chili peppers resulted in reduced chloroplast numbers, decreased chlorophyll content, and decreased expression of genes involved in the capsanthin synthesis pathway in both leaves and immature fruits. During the ripening stage, the chili pepper pericarp changed from red to orange-yellow. Further analysis of the biological function of the transcription factor CaBTF3 revealed that it binds to the promoters of the PSY1, BCH1, and CCS genes involved in the capsanthin synthesis pathway and positively regulates their expression. This invention preliminarily elucidates the mechanism by which the transcription factor CaBTF3 indirectly regulates capsanthin biosynthesis, not only by directly regulating the expression activity of key enzyme genes in the synthesis pathway but also by influencing chloroplast number and development.

[0038] Transforming the cotyledons of pepper PC69 with the CaBTF3 gene yielded transformed plants overexpressing the CaBTF3 gene. Phenotypic analysis of these transformed plants revealed that enhanced CaBTF3 gene expression did not affect normal plant growth and development. At the fruit ripening stage, the capsanthin content in the pericarp was measured. The results showed that compared to the control material (wild-type pepper), the capsanthin content in the CaBTF3-overexpressing line at ripening stage was significantly increased. This further confirms that the transcription factor CaBTF3, as a regulator of a key enzyme gene in capsanthin synthesis, can positively regulate capsanthin synthesis. The findings of this invention provide a theoretical basis and scientific guidance for the application of the transcription factor CaBTF3 in the study of capsanthin synthesis regulation.

[0039] Based on this, one embodiment of the present invention provides the application of transcription factor CaBTF3 in regulating capsanthin synthesis in chili pepper fruit, wherein the nucleotide sequence of the CaBTF3 gene is shown in SEQ ID NO.1.

[0040] The CaBTF3 gene in chili peppers plays a positive regulatory role in capsanthin synthesis. Applying this gene to plant genetic engineering breeding, by enhancing the expression level of the CaBTF3 gene, can increase the accumulation of capsanthin in chili pepper fruits. This provides a new and effective approach for creating chili pepper varieties with high capsanthin content and offers new breeding ideas for cultivating high-quality chili pepper varieties, playing an important role in improving the economic benefits of the chili pepper industry and promoting the application of capsanthin. Conversely, inhibiting the expression of the CaBTF3 gene can reduce the accumulation of capsanthin in chili pepper fruits and further affect the fruit color, providing a feasible approach for breeding chili pepper germplasm with different fruit colors.

[0041] The aforementioned regulation includes both positive and negative regulation. Positive regulation of capsanthin synthesis can be achieved by increasing the expression level of the CaBTF3 gene in wild-type pepper plants; negative regulation of capsanthin synthesis can be achieved by decreasing the expression level of the CaBTF3 gene in wild-type pepper plants. It should be noted that the expression level of the CaBTF3 gene includes both gene-level (mRNA) expression and / or protein-level expression. Gene-level expression can be achieved through RNA interference (RNAi) technology, virus-mediated gene silencing (VIGS) technology, CRISPR-Cas9 technology, or overexpression vectors, while protein-level expression can be achieved through protein activators or inhibitors. These are conventional techniques in this field and will not be described in detail here.

[0042] Optionally, the application of transcription factor CaBTF3 in positively regulating capsanthin synthesis in pepper fruit includes: transforming wild-type pepper plants with a reagent that increases the expression level of the CaBTF3 gene to obtain pepper plants with increased CaBTF3 gene expression levels. The reagent used to increase the expression level of the CaBTF3 gene is selected from one of the following (A)-(C):

[0043] (A) A nucleic acid molecule containing the full-length coding region of the CaBTF3 gene, wherein the nucleotide sequence of the CaBTF3 gene is shown in SEQ ID NO.1;

[0044] (B) An expression vector containing the nucleic acid molecules described in (A);

[0045] (C) Recombinant microorganisms containing the expression vector described in (B).

[0046] The expression vector mentioned above can be the commonly used plant expression vector pHELLSGATE8, and the starting strain of the recombinant microorganism is Agrobacterium.

[0047] Specifically, the method for increasing the expression level of the CaBTF3 gene includes: constructing an overexpression vector of the CaBTF3 gene, transforming it into wild-type pepper, and culturing pepper plants with increased CaBTF3 gene expression levels. More specifically, the construction of the CaBTF3 gene overexpression vector includes the following steps: using pepper genomic cDNA as a template, performing PCR amplification using primer pairs OE-BTF3-F and OE-BTF3-R to obtain the full-length coding region sequence of the CaBTF3 gene, and ligating the full-length coding region sequence into the pHELLSGATE8 expression vector digested with XmaI and XbaI using homologous recombination technology to construct the CaBTF3 gene overexpression vector; wherein, the nucleotide sequences of OE-BTF3-F and OE-BTF3-R are shown in SEQ ID NO.31-32, respectively.

[0048] Optionally, the application of the transcription factor CaBTF3 gene in negatively regulating capsanthin synthesis in pepper fruit includes transforming wild-type pepper plants with a reagent that reduces the expression level of the CaBTF3 gene to obtain pepper plants with reduced CaBTF3 gene expression levels. The reagent used to reduce the expression level of the CaBTF3 gene is selected from one of the following (D)-(F):

[0049] (D) A nucleic acid molecule for silencing the CaBTF3 gene, the nucleotide sequence of which is shown in SEQ ID NO. 18;

[0050] (E) A silencing vector containing the nucleic acid molecules described in (D);

[0051] (F) Recombinant microorganisms containing the silencing vector described in (E).

[0052] In a preferred embodiment of the present invention, the method for silencing the gene is virus-mediated gene silencing (VIGS). Therefore, the silencing vector is preferably pTRV2, and the recombinant microorganism is Agrobacterium.

[0053] Specifically, the method for reducing the expression level of the CaBTF3 gene includes: constructing a CaBTF3 gene silencing vector, transforming it into wild-type pepper, and culturing pepper plants with reduced CaBTF3 gene expression levels. More specifically, the construction of the CaBTF3 gene silencing vector includes the following steps: using pepper genomic cDNA as a template, performing PCR amplification using TRV2-BTF3-F and TRV2-BTF3-R primer pairs to obtain the target sequence targeting the CaBTF3 gene, and ligating the target sequence into a pTRV2 expression vector digested with SmaI enzyme using homologous recombination technology to construct the CaBTF3 gene silencing vector; wherein, the nucleotide sequences of TRV2-BTF3-F and TRV2-BTF3-R are shown in SEQ ID NO.19-20, respectively.

[0054] The transformation of wild-type pepper plant cells or tissues by the CaBTF3 gene overexpression vector and silencing vector described above can be performed using conventional techniques well known to those skilled in the art, such as calcium phosphate coprecipitation, Ti plasmid method, Ri plasmid method, viral vector method, gene gun method, microinjection method, electroporation method, or Agrobacterium-mediated transformation.

[0055] Another embodiment of the present invention provides a method for increasing the capsanthin content in chili pepper fruits, comprising the following steps: increasing the expression level of the CaBTF3 gene in chili pepper plants, wherein the nucleotide sequence of the CaBTF3 gene is shown in SEQ ID NO.1.

[0056] The methods for increasing the expression level of the CaBTF3 gene have been described previously and will not be repeated in this embodiment.

[0057] This invention also provides the application of transcription factor CaBTF3 in regulating pepper fruit color, and the nucleotide sequence of the CaBTF3 gene is shown in SEQ ID NO.1.

[0058] The method for regulating the expression level of the CaBTF3 gene is the same as in the above embodiments, and will not be repeated in this embodiment.

[0059] Breeding chili pepper varieties with different fruit colors to meet the needs of diverse consumer groups is one of the important breeding goals for chili peppers. Capsaicin is one of the main carotenoids in chili peppers, and the color of mature chili pepper fruits mainly depends on the accumulation of capsaicin and capsanthin. The transcription factor CaBTF3 of this invention plays a positive regulatory role in capsaicin synthesis. By changing its expression level, it can affect capsaicin accumulation, thereby altering fruit color. Specifically, inhibiting or silencing the expression of this gene results in a lighter green peel during the color-changing stage and an orange-yellow color during the red-ripe stage.

[0060] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or conditions recommended by the manufacturer.

[0061] Plant: The chili pepper material PC69 used for genetic transformation is disclosed in the literature “Tang,Y.,Shen,X.,Deng,X.,Song,Y.,Zhou,Y.,Lu,Y.,Li,F.and Ouyang,B.(2025),Establishment ofan efficientAgrobacterium-mediated transformation system for chili pepper and its application in genome editing.PlantBiotechnol.J.(access link: https: / / doi.org / 10.1111 / pbi.70216)”. The aforementioned germplasm resources are available to the public from the applicant for use in replicating the experiments of this invention, but may not be used for other purposes.

[0062] Genes and primers: The sequences of the pepper CaBTF3 gene and the primers used in this invention are shown in Table 1, with the numbers corresponding to the sequence numbers in the sequence listing (SEQ ID NO.). Primer synthesis and PCR fragment sequencing were both performed by Youkang Biotechnology Co., Ltd.

[0063] Table 1. DNA sequences of the CaBTF3 gene and related primers in the embodiments of the present invention.

[0064]

[0065]

[0066] Data processing: Each group was set up with 3 biological replicates (n=3). The error was expressed as standard error, **p<0.01, ***p<0.001, ****p<0.0001.

[0067] 1. Screening of the transcription factor CaBTF3 interacting with the pepper CaCCS gene using yeast one-hybrid assay and its functional analysis

[0068] A chili pepper fruit-specific yeast library (constructed by OY Biotech) was used to screen transcription factors expressed in the yeast library using the promoter of CaCCS, a key gene in the capsanthin synthesis pathway. After two rounds of screening on selection medium, positive yeast colonies were subjected to PCR detection using universal T7 promoter primers (nucleotide sequence TAATACGACTCACTATAG). The length of genes binding to the promoter was observed by agarose gel electrophoresis. The results are shown below. Figure 1 Lane M represents the molecular marker, and lanes 1-15 show the PCR results of single clones selected from the yeast library. Gel analysis results were similar to those of the yeast library quality control; most genes were between 1kb and 2kb in length and appeared as single bright bands, with nonspecific amplification observed only in a few lanes. The transcription factor CaBTF3, which may be involved in the regulation of CaCCS, was obtained from the yeast one-hybrid library, and its regulatory mechanism was preliminarily investigated.

[0069] 1.1 Subcellular localization analysis of transcription factor CaBTF3

[0070] Using chili pepper “PC69” cDNA as a template, primer pairs CaBTF3-N-F1 and CaBTF3-C-R1, CaBTF3-N-F1 and CaBTF3-N-R1, and CaBTF3-C-F1 and CaBTF3-C-R1 were used to amplify the full-length CaBTF3 gene sequence without a stop codon, the coding sequence at the 5' end (1-278 bp, encoding the N-terminus of the CaBTF3 protein), and the coding sequence at the 3' end (279-498 bp, encoding the C-terminus of the CaBTF3 protein) using the Planta kit (Novozymes, Nanjing, China). The amplified target fragments were then bound with NcoI and SpeI (Thermopolymer). The linearized plant expression vector PRI101, digested with Fisher's enzymes, was ligated to obtain expression vectors for different fragments of the CaBTF3 gene. A green fluorescent protein (GFP, as a reporter protein) encoding gene was then fused downstream of the CaBTF3 gene in these vectors. The expression vectors carrying the GFP and CaBTF3 fusion proteins were named CaBTF3-GFP, CaBTF3-N-GFP, and CaBTF3-C-GFP, respectively. The expression vectors, along with either a nuclear labeling (NLS-mCherry) vector or a cell membrane labeling (PM-mCherry) vector, were co-microinjected into *Nicotiana benthamiana* cells. After a period of culture, the fluorescence signals of GFP and mCherry were observed under a fluorescence microscope.

[0071] The results are as follows Figure 2 As shown in Figure A, the red fluorescent proteins PM-mCherry and NLS-mCherry are positive controls; the former is located in the cell membrane, and the latter in the cell nucleus. The green fluorescence represents the fusion protein of different fragments of CaBTF3 with GFP. The yellow fluorescence (Merged) is produced by the superposition of the CaBTF3-GFP fusion protein and mCherry protein. Bright represents a bright-field tobacco protoplast microscopic image. Scale bar: 10 μm. It can be seen that the green fluorescence of CaBTF3-N-GFP mainly appears in the cell membrane and cell nucleus. The green fluorescence of CaBTF3-C-GFP co-localizes with the red membrane protein maker on the cell membrane. The full-length CaBTF3-GFP fusion protein, in addition to co-localizing with the red mCherry nuclear localization signal protein, is also expressed on the cell membrane.

[0072] 1.2 Analysis of the transcriptional activation activity of CaBTF3 protein

[0073] The full-length and truncated sequences of the CaBTF3 coding region were ligated into the yeast two-hybrid system. Using chili cDNA as a template, the full-length, N-terminal, and C-terminal coding sequences of CaBTF3 were amplified using the Planta kit with primer pairs CaBTF3-N-F2 and CaBTF3-C-R2, CaBTF3-N-F2 and CaBTF3-N-R2, and CaBTF3-C-F2 and CaBTF3-C-R2, respectively. The amplified target fragments were then ligated into the EcoRI-digested linearized vector pGBKT7 (BD vector, purchased from Eurobio) using the Exnase II kit (Novozymes, Nanjing, China) via homologous recombination technology to obtain the recombinant vectors pGBKT7-CaBTF3, pGBKT7-CaBTF3-N, and pGBKT7-CaBTF3-C. pGBKT7-CaBTF3, pGBKT7-CaBTF3-N, and pGBKT7-CaBTF3-C were co-transfected with AD empty vector pGADT7 (purchased from Ouyi Biotechnology) and yeast strain AH109 (a yeast strain carrying the α-galactosidase (MEL1) gene, which appears blue on medium containing X-α-Gal). The cultures were plated on SD / -Trp plates and incubated at 30℃ for 2-3 days. After colony growth, single colonies were selected and spotted onto SD / -Trp-His (SD / -TH, indicating tryptophan- and histidine-deficient medium) plates. After incubation at 30℃ for 2-3 days, the presence of transcriptional activation activity was determined based on colony growth. The control vectors BD-P53 and BD-Lam carry P53 and laminin, respectively, while AD-T carries a T antigen with a large SV40. P53 and T antigen interact with each other. The pGBKT7-P53+pGADT7-T combination was used as a positive control, while the non-interacting pGBKT7-Lam+pGADT7-T was set as a negative control.

[0074] The activation activity was verified by adding X-α-Gal to SD / -HT selection medium. The results are shown below. Figure 2 Figure B in the middle shows that, compared with the negative control, CaBTF3 expressing the full length and having an N-terminus turned into a blue spot on the yeast selection medium containing X-α-Gal, indicating that CaBTF3 has self-transcriptional activation activity in yeast, and that the transcriptional activation domain is located at the N-terminus of the CaBTF3 protein.

[0075] 1.3 Analysis of CaBTF3 tissue expression patterns

[0076] To clarify the expression pattern of CaBTF3 in different tissues of pepper, the tissue expression profile of CaBTF3 was analyzed using the PepperHub database (access link: http: / / lifenglab.hzau.edu.cn / PepperHub / index.php). The results are shown below. Figure 2 As shown in Figure C, CaBTF3 is expressed throughout the entire plant and at all developmental stages of chili peppers, but the FPKM value is not high. The gene is most highly expressed in young flower buds and the placenta during the early stages of fruit development, suggesting that this gene may have a specific function in the development of chili pepper flowers and fruits.

[0077] 1.4 Interaction analysis of transcription factor CaBTF3 with the promoter of key gene for capsaicin synthase

[0078] The binding activity of CaBTF3 to the promoters of CaPSY1, CaBCH1 and CaCCS genes was verified by transient expression assays using luciferase. Using chili genomic cDNA as a template, the CaCCS promoter (ProCaCCS) sequence was amplified using primer pairs CaCCS-F and CaCCS-R, the CaPSY1 promoter (ProCaPSY1) sequence was amplified using primer pairs CaPSY1-F and CaPSY1-R, and the CaBCH1 promoter (ProCaBCH1) sequence was amplified using primer pairs CaBCH1-F and CaBCH1-R. The ProCaCCS, ProCaPSY1, and ProCaBCH1 promoter sequences were then cloned into the vector pK7LIC containing the LUC reporter gene (vector information and construction process can be found in the literature "Tang Y, Gan Y, Zhang G, et al. Identification of carotenoids and candidate genes shaping high pigment chili pepper variety[J]. Scientia Horticulturae, 2024, 327(000):11.DOI:10.1016 / j.scienta.2023.112799."). Simultaneously, the CaBTF3 gene was amplified using primer pairs CaBTF3-F and CaBTF3-R, and then ligated downstream of the 35S promoter of the expression vector pHELLSGATE8 to obtain the recombinant vector pHELLSGATE8-CaBTF3. The recombinant vector pHELLSGATE8-CaBTF3 expressing the CaBTF3 gene and the reporter vector containing the promoter were transiently transformed into native tobacco leaves via microinjection, and fluorescence signals were observed after 72 hours.

[0079] See interaction results Figure 3Figures (A)-(C) show the transient luciferase detection results after CaBTF3 binds to the promoters of CaPSY1Pro, CaBCH1Pro, and CaCCS, respectively. Fluorescence intensity is expressed in CPS. The vector injection combinations are shown in the right-hand captions. The vector before the plus sign (+) is the LUC reporter vector, and the vector after the plus sign (+) is the empty pHELLSGATE8 vector or the pHELLSGATE8 recombinant vector carrying the CaBTF3 gene. Figures (D)-(F) show the statistical values ​​of fluorescence intensity in Figures (A)-(C), with the horizontal axis representing the injected vector combination and the vertical axis representing fluorescence intensity. It is evident that CaBTF3 can successfully activate the promoters of CaCCS, CaPSY1, and CaBCH1 in tobacco leaves, leading to a significant enhancement of the LUC signal, indicating that the transcription factor CaBTF3 interacts with the promoters of CaCCS, CaPSY1, and CaBCH1.

[0080] 2. Phenotypic analysis of pepper plants silenced by VIGS technology using CaBTF3

[0081] Virus-mediated gene silencing (VIGS) technology was used to silence the expression of the CaBTF3 gene. In the VIGS system, pTRV1 and pTRV2 express fragments of the viral gene and the target gene, respectively. When these RNAs enter plant cells, they trigger the RNAi mechanism, leading to the silencing of the expression of the target gene and its endogenous genes.

[0082] Construction of the silencing vector: Using pepper genomic DNA as a template, a 300bp target fragment of the CaBTF3 gene was amplified using primer pairs TRV2-BTF3-F and TRV2-BTF3-R. This 300bp target fragment was then ligated into the linearized pTRV2(EV) vector digested with SmaI using the Exnase II kit via homologous recombination technology to obtain the recombinant vector pTRV2-CaBTF3. The pTRV2-EV (empty vector control), pTRV2-CaBTF3, and pTRV1 plasmids (helper plasmids) were transformed into Agrobacterium GV3101 via electroporation. The OD values ​​of the Agrobacterium suspensions were adjusted. 600 The value was set at 0.5, and then an equal volume of Agrobacterium suspension containing pTRV1 and pTRV2-EV (TRV1+TRV2-EV combination) or its derivative vector (TRV1+TRV2-CaBTF3 combination) was mixed. The cotyledons were microinjected into the PC69 pepper variety, which is high in capsanthin and has high VIGS efficiency, at approximately 12 days of seedling age. After injection, the plants were incubated in the dark for 3 days before being returned to normal light conditions.

[0083] Three to four weeks after injection, significant phenotypic differences were observed between CaBTF3-silenced plants and control plants. Compared to the control plants, CaBTF3-silenced plants exhibited slower growth, smaller leaves, and paler color. As the pepper fruits developed, a lighter green phenotype was observed in the silenced plants 7-9 days after color change. Figure 4 (Left image, middle section) In ripe red fruit, the peel changes from red to orange-yellow. Figure 4 (Right image in the middle) Figure 4 Each small image in the middle, from left to right, shows an empty (EV) plant injected with TRV1+TRV2 and an RV1+TRV2-CaBTF3 plant, respectively. Scale bar: 1cm.

[0084] Using a chlorophyll fluorometer, the fluorescence signal of leaves in CaBTF3-silenced plants changed from blue to yellow, and the fluorescence value also decreased significantly, indicating that the photosynthetic capacity of chloroplasts was affected. Figure 5 The left and right images show leaves of plants injected with TRV1+TRV2 (empty, EV) and RV1+TRV2-CaBTF3, respectively. Statistical analysis of the maximum photochemical quantum yield (FV / FM) of PSII showed that the FV / FM value of CaBTF3-silenced plants was significantly lower than that of the control, indicating that plant photosynthesis was affected. Figure 5 (Right image in the middle)

[0085] The expression of CaBTF3 in fruits was detected by quantitative real-time PCR (qRT-PCR). Fruit samples were collected 7-9 days after fruit color change. qRT-PCR analysis confirmed that the relative expression of CaBTF3 was significantly decreased in phenotypically shaped fruits, with a reduction of approximately 70%. Figure 6 Figure A shows that VIGS successfully suppressed the expression of the CaBTF3 gene. Further analysis of the expression levels of three key genes in the capsaicin synthesis pathway—CaCCS, CaPSY1, and CaBCH1—revealed a significant decrease in the expression levels of all three genes in CaBTF3-silenced plants, with CaCCS and CaPSY1 showing the most significant reductions. Figure 6 (See Figure C). It is evident that CaBTF3 silencing affected the expression of these genes, confirming that CaBTF3 may directly participate in regulating key genes in the capsaicin synthesis pathway, thereby affecting capsaicin synthesis. The capsaicin content in the pericarp of silenced fruits was determined using a capsaicin enzyme-linked immunosorbent assay (ELISA) kit (Yuanju, Shanghai, China). The results showed that the capsaicin content in the pericarp of CaBTF3-silenced plants was significantly lower than that of the control. Figure 6 (Figure B in the middle)

[0086] VIGS silencing of CaBTF3 resulted in a decrease in chlorophyll content in the leaves and pericarps of plants, suggesting that it might affect chloroplast development. Therefore, transmission electron microscopy (TEM) was used to further observe the chloroplast morphology in the leaves and pericarps of CaBTF3-silenced plants. Results are shown below. Figure 7 Figures (A)-(B) show chloroplasts in the pericarp cells of the control plant (injected with TRV2-EV), Figures (C)-(D) show chloroplasts in the leaf cells of the control plant, Figures (E)-(F) show chloroplasts in the pericarp cells of the CaBTF3-silenced plant (injected with TRV2-CaBTF3), and Figures (G)-(H) show chloroplasts in the leaf cells of the CaBTF3-silenced plant. Scale bar: 10 μm. As can be seen from the figures, compared with the control plant, the chloroplasts in the fruit and leaves of the CaBTF3-silenced plant are smaller, and the number of chloroplasts per cell is reduced. The internal structure of the chloroplasts, such as the arrangement of thylakoids and the number of chloroplast grana, shows significant abnormalities. These results demonstrate that CaBTF3 plays an important role in the morphological development of chloroplasts in the leaves and fruits of peppers.

[0087] The results reveal the overall impact of transcription factor CaBTF3 on chloroplast function and the synthesis of plant metabolites in pepper fruits and leaves. Chloroplasts are crucial sites for carotenoid synthesis, where various compounds, including capsanthin, are synthesized. Chloroplast integrity is essential for maintaining plant metabolic pathways and producing secondary metabolites. These data indicate that CaBTF3 can indirectly influence capsanthin biosynthesis by affecting chloroplast quantity and structure. Furthermore, in fruits where CaBTF3 was silenced, the expression levels of key genes in the three capsanthin biosynthetic pathways—CaPSY1, CaBCH1, and CaCCS—were significantly reduced. Therefore, the role of CaBTF3 in capsanthin synthesis extends beyond directly regulating key genes in the biosynthetic pathway; it also includes a comprehensive influence on chloroplast quantity and development.

[0088] 3. Phenotypic analysis of pepper plants overexpressing CaBTF3

[0089] The chili pepper material PC69 was selected as the background material for genetic transformation, and the CaBTF3 gene was overexpressed to verify the effect of enhanced expression of this transcription factor on capsanthin synthesis.

[0090] Overexpression vector construction: Using cDNA from pepper PC69 as a template, primers OE-BTF3-F and OE-BTF3-R were constructed using the overexpression vector to amplify the full-length coding region sequence of the CaBTF3 target gene. The linearized vector pHellstage8, obtained by double digestion with XmaI and XbaI (Thermo Fisher), was then ligated to the linearized vector pHellSGATE8 via homologous recombination using an Exnase II kit.

[0091] Using cotyledons of PC69 as explants, without pre-culture, Agrobacterium-mediated transformation of expression vectors carrying the CaBTF3 gene was performed under a vacuum pressure of 0.06 MPa. Regenerated plants were obtained 100 days after Agrobacterium transformation. Genomic DNA was extracted from peppers transformed with the CaBTF3 gene, and the transformation results were detected by PCR. Primer pairs BTF3-JC-F and BTF3-JC-R were used to detect the transformed plants. All 10 transformed plants showed positive bands in the detection of the transformed 35S promoter and the target gene CaBTF3. The results are as follows: Figure 8 As shown, lane M is the molecular marker, lanes 1-10 are positive transformed plants, and lane 11 is the negative control.

[0092] Phenotypic observation of the T0 generation plants transformed with the CaBTF3 gene showed that overexpression of CaBTF3 in peppers did not affect plant growth and development throughout the entire growth cycle (see...). Figure 9 (Left image, scale bar: 5cm) Furthermore, the peppers from plants overexpressing the CaBTF3 gene exhibit a deeper biological fruit color during color change and ripening compared to wild-type PC69 peppers (see image). Figure 9 (Right image in the middle, scale bar: 1cm)

[0093] During the ripening stage of chili peppers, the capsanthin content in the pericarp was measured. The capsanthin content of the control material's ripe fruit was 950.40 μg / g, while after overexpression of the CaBTF3 gene, the capsanthin content increased to 1108.06 μg / g, an increase of 16.58% compared to the control. This indicates that enhancing the expression level of the CaBTF3 gene in chili peppers is beneficial to promoting capsanthin synthesis and increasing capsanthin accumulation.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of transcription factor CaBTF3 in regulating capsaicin synthesis in chili pepper fruit, characterized in that, The nucleotide sequence of the CaBTF3 gene is shown in SEQ ID NO.

1.

2. The application of transcription factor CaBTF3 according to claim 1 in regulating capsaicin synthesis in chili pepper fruit, characterized in that, The regulation includes positive regulation or negative regulation; The positive regulation includes: increasing the expression level of the CaBTF3 gene in wild-type pepper plants to obtain pepper plants with increased CaBTF3 gene expression levels. The negative regulation includes: reducing the expression level of the CaBTF3 gene in wild-type pepper plants to obtain pepper plants with reduced CaBTF3 gene expression levels.

3. The application of the transcription factor CaBTF3 according to claim 2 in regulating capsaicin synthesis in chili pepper fruit, characterized in that, The reagent used to increase the expression level of the CaBTF3 gene is selected from one of the following (A)-(C): (A) A nucleic acid molecule containing the full-length coding region of the CaBTF3 gene, wherein the nucleotide sequence of the CaBTF3 gene is shown in SEQ ID NO.1; (B) An expression vector containing the nucleic acid molecules described in (A); (C) Recombinant microorganisms containing the expression vector described in (B); The reagent used to reduce the expression level of the CaBTF3 gene is selected from one of the following (D)-(F): (D) A nucleic acid molecule for silencing the CaBTF3 gene, the nucleotide sequence of which is shown in SEQ ID NO. 18; (E) A silencing vector containing the nucleic acid molecules described in (D); (F) Recombinant microorganisms containing the silencing vector described in (E).

4. The application of transcription factor CaBTF3 according to claim 2 in regulating capsaicin synthesis in chili pepper fruit, characterized in that, Methods for increasing the expression level of the CaBTF3 gene include: An overexpression vector for the CaBTF3 gene was constructed, and wild-type peppers were transformed to obtain pepper plants with increased CaBTF3 gene expression levels.

5. The application of the transcription factor CaBTF3 according to claim 4 in regulating capsaicin synthesis in chili pepper fruit, characterized in that, The construction of the overexpression vector includes the following steps: Using chili genomic cDNA as a template, PCR amplification was performed using primer pairs OE-BTF3-F and OE-BTF3-R to obtain the full-length coding region sequence of the CaBTF3 gene. The full-length coding region sequence was then ligated into the pHELLSGATE8 expression vector, which was digested with XmaI and XbaI, to construct the overexpression vector. The nucleotide sequences of OE-BTF3-F and OE-BTF3-R are shown in SEQ ID NO. 31-32, respectively.

6. The application of the transcription factor CaBTF3 according to claim 2 in regulating capsaicin synthesis in chili pepper fruit, characterized in that, The method for reducing the expression level of the CaBTF3 gene includes: constructing a CaBTF3 gene silencing vector, transforming it into wild-type pepper, and culturing pepper plants with reduced CaBTF3 gene expression levels.

7. The application of the transcription factor CaBTF3 according to claim 6 in regulating capsaicin synthesis in chili pepper fruit, characterized in that, The construction of the CaBTF3 gene silencing vector includes the following steps: Using chili genomic cDNA as a template, PCR amplification was performed using primer pairs TRV2-BTF3-F and TRV2-BTF3-R to obtain the target sequence targeting the CaBTF3 gene. The target sequence was then ligated into the pTRV2 expression vector digested by SmaI via homologous recombination technology to construct the CaBTF3 gene silencing vector. The nucleotide sequences of TRV2-BTF3-F and TRV2-BTF3-R are shown in SEQ ID NO.19-20, respectively.

8. The application of the transcription factor CaBTF3 according to any one of claims 4-7 in regulating capsanthin synthesis in chili pepper fruit, characterized in that, The transformation method employed was Agrobacterium-mediated transformation.

9. A method for increasing the capsanthin content in chili pepper fruits, characterized in that, Includes the following steps: To increase the expression level of the CaBTF3 gene in pepper plants, the nucleotide sequence of the CaBTF3 gene is shown in SEQ ID NO.

1.

10. The application of transcription factor CaBTF3 in regulating pepper fruit color, characterized in that, The nucleotide sequence of the CaBTF3 gene is shown in SEQ ID NO.1.

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