Application and methods of the CaRHA2-Cap gene or its encoded protein in regulating capsanthin content in chili pepper varieties

Abstract

This application relates to the field of chili pepper variety breeding technology, specifically to a... CaRHA2‑Cap Applications and methods of genes or their encoded proteins in regulating capsanthin content in chili pepper varieties. CaRHA2‑Cap The application of genes or their encoded proteins in regulating capsanthin content in chili pepper varieties, the aforementioned CaRHA2‑Cap The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO: 2. The regulation described is positive regulation. CaRHA2‑Cap The application and methods of using genes or their encoded proteins to regulate capsanthin content in chili varieties can achieve precise and effective regulation of capsanthin content in chili fruits.

Description

Technical Field

[0001] This application relates to the field of chili pepper variety breeding technology, specifically to a... CaRHA2-Cap Applications and methods of genes or their encoded proteins in regulating capsanthin content in chili pepper varieties. Background Technology

[0002] Capsaicin is an important natural red pigment found in mature chili peppers, widely used in the food, cosmetics, and pharmaceutical industries. It is one of the main carotenoids accumulated in mature chili peppers. Breeding chili pepper varieties with high capsaicin content has significant economic value. Meanwhile, light-colored or novel-colored chili pepper varieties also have a broad market in ornamental and specialty agriculture, and varieties with diverse fruit colors are favored by home gardening and specialty agriculture. Therefore, precisely controlling the capsaicin content in chili pepper fruits is one of the core objectives of chili pepper molecular breeding.

[0003] Currently, traditional methods for improving the color of chili peppers mainly rely on hybridization breeding and artificial selection. However, this process is time-consuming, inefficient, and makes it difficult to achieve precise and targeted regulation of capsanthin content. With the development of molecular biology techniques, directly regulating key genes in the capsanthin synthesis pathway through genetic engineering has become an effective strategy for breeding crop varieties with specific colors. Molecular breeding and genetic engineering technologies have provided new avenues for improving crop color, but the acquisition and verification of key genes regulating capsanthin synthesis remain insufficient. Summary of the Invention

[0004] This invention aims to solve the problems in the prior art and provide a method that can accurately and effectively regulate the capsanthin content in chili pepper fruits. CaRHA2-Cap The application and methods of gene regulation of capsanthin content in chili pepper varieties.

[0005] To achieve the above objectives, the first aspect of this application provides: CaRHA2-Cap The application of a gene or its encoded protein in regulating capsanthin content in chili pepper varieties, characterized in that... CaRHA2-Cap The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO: 2. The regulation is positive regulation.

[0006] In one embodiment, by overexpressing the CaRHA2-Cap The expression of genes in chili varieties is used to cultivate chili varieties with high capsanthin content.

[0007] In one embodiment, by lowering the CaRHA2-Cap Gene expression or reduction in chili varieties CaRHA2- Cap The activity of genes in chili varieties can be used to breed chili varieties with low capsanthin content.

[0008] In one embodiment, the downregulation is achieved through virus-induced gene silencing technology or gene editing technology based on the CRISPR / Cas9 system. CaRHA2-Cap Gene expression or reduction in chili varieties CaRHA2-Cap The activity of genes in chili pepper varieties.

[0009] In one embodiment, when gene silencing technology is used, the target amplification primer sequences used to construct the silencing vector are shown in SEQ ID NO:11 and SEQ ID NO:12; when gene editing technology of the CRISPR / Cas9 system is used, the gene knockout target sequence used is shown in SEQ ID NO:15 or SEQ ID NO:16.

[0010] In one embodiment, the gene knockout target sequence used when constructing the overexpression vector is as shown in SEQ ID NO:13 or SEQ ID NO:14.

[0011] A second aspect of this application provides a method for amplifying as described above. CaRHA2-Cap The primer pairs for the gene, the nucleotide sequences of which are shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0012] A third aspect of this application provides a method for cultivating chili pepper varieties with high capsanthin content, comprising:

[0013] (1) As shown in SEQ ID NO: 1 CaRHA2-Cap The nucleotide sequence of the gene was constructed into a plant overexpression vector to obtain a recombinant vector; (2) The recombinant vector was transformed into pepper explants; (3) Tissue culture was performed on the transformed explants, and transgenic pepper plants were screened and obtained; (4) Select strains with increased capsanthin content and stable traits from the transgenic pepper plants.

[0014] The third aspect of this application provides a method for cultivating chili pepper varieties with low capsanthin content, wherein virus-induced gene silencing technology is used in chili pepper plants to downregulate the gene as shown in SEQ ID NO: 1. CaRHA2-Cap Gene expression specifically includes the following steps: (1) Construct a system containing primers amplified by primers shown in SEQ ID NO:11 and SEQ ID NO:12. CaRHA2-Cap VIGS silencing vector for gene-specific fragments; (2) The silencing vector was introduced into Agrobacterium and used to infect pepper plants; (3) Cultivate infected plants and achieve [the desired effect] in their fruits. CaRHA2-Cap Silencing of gene expression; (4) Select plants with significantly lighter pepper peel color.

[0015] Compared with the prior art, this application has the following beneficial effects: (1) This invention is the first to identify in chili peppers CaRHA2-Cap Genes influence capsanthin content. Capsanthin is the core component that determines the color of chili peppers. The higher the content, the brighter and more intense the red color of a ripe chili pepper. CaRHA2-Cap Genes provide key targets for the molecular regulation of pepper fruit color. This has been experimentally confirmed for the first time. CaRHA2-Cap Genes are key enzymes that regulate capsanthin synthesis in chili pepper fruits from an epigenetic perspective. CaRHA2-Cap This invention establishes a technical system for bidirectionally regulating capsanthin content through single-gene manipulation, utilizing gene regulation. By overexpressing this gene, dark-colored pepper varieties with significantly increased capsanthin content can be selectively bred; by downregulating... CaRHA2-Cap Gene expression or reduction in chili varieties CaRHA2-Cap The activity of genes in chili pepper varieties can be used to selectively breed lighter-colored chili pepper varieties with reduced capsanthin content. This method overcomes the drawbacks of traditional hybridization breeding, such as long cycles, low selection efficiency, and difficulty in precisely controlling the content of specific components, and has significant industrial application value. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is one embodiment of the present invention. CaRHA2-Cap Cloning electrophoresis diagram of a gene; Figure 2 This is one embodiment of the present invention. CaRHA2-Cap The cDNA sequence of a gene and the amino acid sequence it encodes; Figure 3 This is one embodiment of the present invention. CaRHA2-Cap Results of conserved domain analysis of the protein; Figure 4 This is one embodiment of the present invention. CaRHA2-Cap Phylogenetic analysis diagram of proteins; Figure 5 This is one embodiment of the present invention. CaRHA2-Cap Prediction of protein secondary structure; Figure 6 This is one embodiment of the present invention. CaRHA2-Cap Prediction of protein tertiary structure; Figure 7 This is one embodiment of the present invention. CaRHA2-Cap Subcellular localization map of genes; Figure 8 This is one embodiment of the present invention. CaRHA2-Cap Expression levels of genes at different developmental stages in pepper fruits with different color-changing characteristics; Figure 9 This is one embodiment of the present invention. CaRHA2-Cap VIGS Functional Verification Analysis: A. CaRHA2-Cap and pTRV2 unloaded silent fruit phenotype; B, CaRHA2-Cap Electrophoresis images of pTRV2 unloaded and silent fruits. Detailed Implementation

[0018] To facilitate understanding of this application, the following description will be more comprehensive and detailed in conjunction with the accompanying drawings and preferred embodiments, but the scope of protection of this application is not limited to the following specific embodiments.

[0019] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of this application.

[0020] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0021] Example 1: CaRHA2-Cap Gene cloning, bioinformatics analysis and subcellular localization 1. Materials and Methods Gene cloning was conducted using chili pepper inbred line 8214 as the experimental material. This material is an excellent inbred line bred by the chili pepper team of Hunan Agricultural University through continuous self-pollination and selection over many years. It is currently preserved in the germplasm resource bank of Hunan Agricultural University and is publicly accessible. Chili pepper inbred line 8214 produces red mature fruits and exhibits excellent characteristics such as high capsanthin content and disease resistance. Subcellular localization experiments were conducted using Nicotiana benthamiana as the experimental material.

[0022] 1.1 CaRHA2-Cap Gene cloning Based on the Ca_59 genome (GCA_021292125.1) CaRHA2-Cap Using the sequence as a reference, specific primers were designed. PCR amplification was performed using cDNA from pepper inbred line 8214 as a template, employing Takara's Prime STAR Max high-fidelity enzyme. The gene cloning primers were: F: 5'-ATGGGATTACAAAACCAGCTAAATGATATTTCTAC-3' (SEQ ID NO: 3); R: 5'-TCAGCTCAACGAAAACCAACCAAC-3' (SEQ ID NO: 4).

[0023] PCR reaction procedure: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 55℃ annealing for 10s, 72℃ extension for 15s, for a total of 35 cycles; final extension at 72℃ for 5min. After amplification, 5μL of product was taken for agarose gel electrophoresis to identify the target product.

[0024] 1.2 Bioinformatics Analysis The cloned gene sequences were sequenced and their encoded proteins were analyzed. BLAST alignment was performed using the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), and samples from other species were selected. CaRHA2-Cap Homologous amino acid sequences of the protein were obtained. A homology tree was then constructed using neighbor-joining in MEGA12 software, and multiple comparisons were performed using Snap Gene software. Amino acid sequence analysis was performed using BioXM2.6 software, and the constructed phylogenetic tree was evaluated using the Bootstrap method (repeated 1000 times). Physicochemical properties were analyzed using the ExPASy online tool, Prot Param tool (https: / / web.expasy.org / protparam / ).

[0025] The secondary structure of the protein was predicted using the SOPMA method provided by ExPASy (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page=npsa_sopma.html). The secondary structure was then analyzed using SWISS-MODEL (http: / / swissmodel.expasy.org / ). CaRHA2-Cap The tertiary structure of the protein was predicted using the WoLFPSORT online tool (https: / / wolfpsort.hgc.jp / ). CaRHA2-Cap Subcellular localization of proteins.

[0026] 1.3 Subcellular localization of genes 1.3.1 Carrier Construction against CaRHA2-Cap The stop codon of the gene was removed, a transient overexpression fragment was designed, and recombinant primers were designed: PSuper-1300-GFP- CaRHA2-Cap-F: 5'-tgcaggggcccggggtcgaTTCTATCGGGTCGGGTCTAACCAG-3' (as shown in SEQ ID NO:5); PSuper-1300-GFP- CaRHA2-Cap -R: 5'-ttgctcaccatggtaccGCTCAACGAAAACCAACCAACC-3' (as shown in SEQ ID NO:6) (lowercase letters are homologous arms of the PSuper-1300-GFP vector).

[0027] PCR was performed using cDNA from pepper inbred line 8214 as a template. The system is shown in Table 1 below: Table 1: PCR reaction system

[0028] PCR reaction procedure: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 55℃ annealing for 10 s, 72℃ extension for 10 s, for a total of 35 cycles; final extension at 72℃ for 5 min. After amplification, 5 μL of product was taken for agarose gel electrophoresis to identify the target product.

[0029] The amplified products were purified using the Fast Pure Gel DNA Extraction Mini Kit (Novizan, Nanjing, China). The purified fragment was then constructed into a linearized PSuper-1300-GFP vector. The purified target fragment and the linearized vector were ligated using the Clon Express II One Step Cloning Kit (Novizan, Nanjing, China). The recombinant plasmid was transformed into *E. coli* competent cells DH5α, plated on LB agar containing kanamycin, and single colonies were picked after 16 hours for PCR identification. Bacterial cultures confirming the presence of the target band were sequenced, and after confirmation with the vector, plasmid extraction was performed to obtain the desired fragment containing... CaRHA2-Cap The vector (PSuper-1300-GFP-) CaRHA2-Cap ).

[0030] 1.3.2 Transformation of Agrobacterium 100 μL of Agrobacterium tumefaciens competent cells (LGV3101) were removed from a -80°C freezer and placed on ice. Once the competent cells formed an ice-water mixture, 200 ng of the extracted PSuper-1300-GFP was added. CaRHA2-CapAfter mixing the plasmid, the cells were sequentially incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. Then, 700 μL of LYEB liquid medium was added, and the cells were incubated at 28°C with shaking for 2 h. After centrifugation at 4000 rpm for 5 min, 700 μL of supernatant was removed, and 100 μL of liquid was used to resuspend the cells. The bacterial culture was plated on YEB agar plates containing kanamycin (Km), rifampin (Rif), and gentamicin (Gent) for culture. After identifying correctly identified single clones, cells containing PSuper-1300-GFP- were obtained. CaRHA2-Cap Agrobacterium bacterial suspension on the carrier.

[0031] 1.3.3 Plant Infection and Cultivation Tobacco Benedict seedlings were cultivated in an artificial climate chamber and injected when they reached the 4-6 leaf stage. The specific steps are as follows: The product contains PSuper-1300-GFP, PSuper-1300-GFP-CaRHA2-Cap, and PSuper-1300-GFP. - The bacterial culture containing the AtH2b plasmid (PS-1300-GFP was an empty vector, used as an empty vector control after injection; PS-1300-GFP-AtH2b was a nuclear marker) was cultured on YEB medium containing Km, Rif, and Gent antibiotics (final concentration: Km 50 μg / mL). -1 Rif 50 μg·mL -1 Gent 50 μg·mL -1 Incubate at 28℃ with shaking at 200 rpm until OD600 = 1.0; Take the culture product into a centrifuge tube, centrifuge at 5000g for 8 min, discard the supernatant, adjust the pH of the infection solution (containing 200μM acetylsalicylic acid AS, 10mM MES and 10mM MgCl2·6H2O) to 5.6, suspend Agrobacterium and adjust OD600 to 0.8. The PS-1300-GFP-AtH2b strain was mixed 1:1 with other strains (PS-1300-GFP, PS-1300-GFP-CaRHA2-Cap), and after standing in the dark at 28°C for 3 hours, it was injected into tobacco. Using Nicotiana benthamiana as the target of infection, leaves were inoculated using the needleless infiltration method (dehydrated 2 days before inoculation). After inoculation, the leaves were cultured in the dark at 22°C for 24 hours in an artificial climate chamber. After exposure to light for 48 hours, the expression location of fluorescence in the lower epidermal cells of the tobacco was observed using a laser confocal microscope.

[0032] 1.4 Gene Expression Analysis Total RNA was isolated from the samples using the Eastep® Super Total RNA Extraction Kit (Promega, Beijing, China). RNA was reverse transcribed using the Hi Script IIQRT Super Mix for qPCR (+gDNA wiper) (Novizan, Nanjing, China). qRT-PCR analysis was performed using the Cham QU universal SYBR qPCR Master Mix (Novizan, Nanjing, China) on a Light Cycler® 96 Instrument Detection System (Roche, Basel, Switzerland). The pepper actin gene (NCBI ReferenceSequence: XM_016683691.2) was used as an internal reference. Each value represents the mean of three biological replicates.

[0033] Design quantitative PCR primers (5′→3′) online using NCBI (Primer designing tool (nih.gov)): Ca CaRHA2-Cap -qF: GGTGCTGGTTCGGGTACAT (as shown in SEQ IDN O:7); Ca CaRHA2-Cap -qR: AACCAAATCACTAGCCACGC (as shown in SEQ ID NO:8); CaActin-F: CCACCTCTTCACTCTCTGCTCT (as shown in SEQ ID NO:9); CaActin-R: ACTAGGAAAAACAGCCCTTGGT (as shown in SEQ ID NO: 10); The qPCR reaction system is shown in Table 2 below: Table 2: qPCR reaction system

[0034] The amplification program was: 95℃ pre-denaturation for 30s, 95℃ denaturation for 10s, 60℃ extension for 30s, for a total of 40 cycles.

[0035] 2. Results and Analysis 2.1 Gene Cloning and Sequence Analysis PCR amplification yielded a band of the expected size. Figure 1 Sequencing results showed that the full-length coding sequence of the gene is 480 bp (nucleotide sequence as shown in SEQ ID NO: 1), and the deduced protein sequence has 159 amino acids (amino acid sequence as shown in SEQ ID NO: 2). Figure 2 ).

[0036] Nucleotide sequence SEQ ID NO: 1: ATGGGATTACAAAACCAGCTAAATGATATTTCTACTGAATCAATCCCAATATTACTAATCACCCTTTTTGCTAATTCCATTAATTACCTCCGTTCCTTAATTCTCACTTCCTTCACTTCTCTCTCTCCTTTACCCCTCATCAAATCAACAATTCCTTTCCGATTCTATCGGGTCGGGTCTAACCAGCGTTATTTTCCTAACCGAACAGCTCAATTTAAACCGGTTATTATCTTAT CATCTAACCGGTGCTGGTTCGGGTACATGTGTGGTTTGTTGAACCGGTTGAGTGAAGGTGAACCGGTGCGTAAGTTAGCATGTCGGCATGTGTTTCATAAGGAGTGTTTGGATGGGTGGTTTAATACGTTGAATTTTAATTGTCCAATTTGTCGGAAGAATTTGGTCGCCGGTGAGCGCGTGGGAAGAGTGCGACGGCGCGTGGCTAGTGATTTGGTTGGTTGGTTTTCGTTGAGCTGA Amino acid sequence SEQ ID NO: 2: MGLQNQLNDISTESIPILLITLFANSINYLRSLILTFLHFSSSSHFTPHQINNSFSDSIGSGLTSVIFLTEQLNLNRLLSYHLTGAGSGTCVVCLNRLSEGEPVRKLACRHVFHKECLDGWFNTLNFNCPICRKNLVAGERVGRVRRRVASDLVGWFSLS

[0037] Use the ExPASy online analysis tool, Prot Param tool, to analyze... CaRHA2-Cap Physicochemical property analysis of the protein showed that its total average hydropathicity (GRAVY) was 0.134, i.e. CaRHA2-Cap It has extremely strong hydrophilicity; its instability index is 44.42, meaning... CaRHA2-Cap It is an unstable protein.

[0038] Conservative domain analysis results, such as Figure 3 As shown, the results indicate that CaRHA2-Cap The protein belongs to the RING-H2 type zinc finger protein family.

[0039] Using the online analysis software SOPMA to analyze... CaRHA2-Cap Predicting the secondary structure of proteins, such as Figure 5 As shown, the results are... CaRHA2-Cap The α-helices accounted for 37.7%, and random coils accounted for 55.97%. The predicted tertiary structure of the protein was obtained using the online software SWISS-MODEL. Figure 6 As shown. CaRHA2-Cap Protein polypeptide chains further coil or fold based on various secondary structures to form a regular three-dimensional spatial structure. The protein structure was established using a structure with PDB number A0A1J6HS36.1.A as a template, with a sequence identity of 72.96% and a GMQE value (global model quality estimate) of 0.66.

[0040] Subcellular localization results showed CaRHA2-Cap It is located on the cell membrane and nuclear membrane.

[0041] 2.2 Spatiotemporal expression analysis of CaMADS-RIN gene The qRT-PCR technique was used to analyze CaRHA2-Cap The gene expression patterns at different developmental stages of pepper pericarps in three different color-changing patterns: from yellow to orange to red, from green to brown to red, and from purple to red. Figure 8 As shown. The results indicate that, CaRHA2- Cap The gene is almost not expressed or expressed at a low level in the early stages of fruit peel development (before color change). After the fruit begins to color change (43 days)... [[ID= Gene expression levels were significantly increased, capsanthin began to accumulate, and the gene showed a strong correlation with capsanthin synthesis.

[0042] Example 2: Based on VIGS technology ​ Gene function verification (for breeding light-colored varieties) 1. Materials and Methods 1.1 Construction of VIGS silencing vector against ​ Genetic design of VIGS silencing fragments and design of recombinant primers: pTRV2- ​ -F: 5'-cggtgaggagaagagcccTTCTATCGGGTCGGGTCTAACCAG-3' (as shown in SEQ ID NO:11); pTRV2-​ -R: 5'-gctcgacgacaagacccCTCACCGGCGACCAAATTCTTCC-3' (as shown in SEQ ID NO:12) (lowercase letters are homologous arms of the pTRV2 vector).

[0043] PCR was performed using cDNA from pepper inbred line 8214 as a template, following the same system as described in Table 1 of Example 1. The vector construction method was the same as in Example 1, yielding a vector containing... ​ Silencing vector for the target (pTRV2-) ​ ).

[0044] 1.2 Transformation of Agrobacterium Same as Example 1.

[0045] 1.3 Plant Infection and Cultivation Chili seedlings were raised in an artificial climate chamber and inoculated after two weeks of age. The specific steps are as follows: Plants containing pTRV1, pTRV2, and pTRV2-PDS (PDS silencing causes plant leaves to turn white, serving as a positive control) were used. pTRV2-... ​ The plasmid was cultured using YEB medium containing three antibiotics: Km, Rif, and Gent (final concentration: Km 50 μg / mL). -1 Rif 50 μg·mL -1 Gent 50 μg·mL -1 Incubate at 28℃ with shaking at 200 rpm until OD600 = 1.0; Take the culture product into a centrifuge tube, centrifuge at 5000g for 8 min, discard the supernatant, adjust the pH of the infection solution (containing 200μM acetylsalicylic acid AS, 10mM MES and 10mM MgCl2·6H2O) to 5.6, suspend Agrobacterium and adjust OD600 to 0.2. pTRV1, pTRV2, and pTRV2-PDS bacterial cultures were prepared using the same method. pTRV1 strain was compared with other strains (pTRV2, pTRV2-PDS, pTRV2-) ​ Mix 1:1, let stand in the dark at 28℃ for 4 hours, and then inoculate. Pepper inbred line 8214 was used as the target for infection. Pepper cotyledons were inoculated using the needleless infiltration method (the leaves were dehydrated 2 days before inoculation). After inoculation, the leaves were cultured in a climate chamber at 18°C ​​in the dark for 3 days, then the day / night temperature was adjusted to 20°C / 18°C, and the light intensity was 375 μmol·m⁻¹. -2 ·s -1 (16h / 8h) under 60% relative humidity conditions.

[0046] 1.4 Phenotypic Observation and Detection Observe the fruit color change approximately 5 weeks after inoculation. Take photos on the day the fruit begins to change color, and collect samples (peel) on the 7th day.

[0047] 2. Results and Analysis Compared with the control plants, pTRV2- ​ The pepper pericarps of silent plants show a significantly lighter color (see...) ​ (The three types of pepper pericarp colors in A) indicate silence. ​ Genes can reduce the content of capsanthin, thereby controlling the lightening of the color of chili pepper peel.

[0048] The above results indicate that ​ Genes can regulate capsanthin biosynthesis by affecting the expression of key genes involved in capsanthin synthesis, and this can be achieved by silencing these genes in chili peppers. ​ Genes are used to cultivate lighter-colored peppers.

[0049] Example 3: Overexpression ​ Genetically bred chili varieties with high capsanthin content This embodiment provides an overexpression ​ Methods to obtain chili pepper plants with high capsanthin content through gene therapy.

[0050] 1. Construction of overexpression vectors Design overexpression primers containing restriction enzyme sites: ​ -OE-F:5'-atctctctcgagctttcgcgagctATGGGATTACAAAACCAGCTAAATGATATTTCTAC-3' (as shown in SEQ ID NO:13); ​ -OE-R:5'-tagaggatccccgggtaccgagctcTCAGCTCAACGAAAACCAACCAAC-3' (as shown in SEQ ID NO:14).

[0051] Using chili cDNA as a template, the above primers were used for amplification. ​ Gene coding region. The PCR product is cloned into an overexpression vector (such as pCAMBIA1300) to construct a recombinant overexpression vector.

[0052] 2. Genetic transformation and plant regeneration (1) Explant preparation Approximately 200 seeds were soaked in ultrapure water for 3-4 hours, followed by surface sterilization: immersion in 75% ethanol for 30 seconds, then in 1%-2% sodium hypochlorite solution for 10-15 minutes. The seeds were then rinsed 3-4 times with sterile water. The sterilized seeds were sown on 1 / 2 strength Murashige & Skoog (MS) medium (pH 5.8) with 15 g / L sucrose and 7.4 g / L agar. The seeds were incubated at 28°C in the dark for 4-5 days until germination, then transferred to a growth chamber under 16 h light / 8 h dark conditions at 25°C. Seedlings approximately 12 days old with fully expanded cotyledons were selected for explant preparation.

[0053] (2) Preparation of Agrobacterium The recombinant overexpression vector was transformed into Agrobacterium K599, and Agrobacterium K599 was cultured in standard TY medium supplemented with antibiotics (100 mg / L streptomycin, 50 mg / L kanamycin). The bacterial culture was prepared 4 days before transformation. One day before inoculation, the bacterial culture was transferred to fresh TY medium containing antibiotics. The culture was then incubated in the dark at 28°C with continuous shaking at 220 rpm for 8–10 h.

[0054] (3) Explant preparation and inoculation: Place the seedlings on moistened filter paper in a large petri dish. Remove the roots and carefully cut off the petioles and cotyledons. Make two incisions at both ends of each cotyledon to facilitate infection. Transfer the prepared explants to AKCMS solid medium (MS medium, 100 mg / L inositol, 1.3 mg / L vitamin B1, 0.2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 200 mg / L potassium dihydrogen phosphate, 0.1 mg / L KT, 200 μM acetylsuccinyl copper AS; 30 g / L sucrose; 7.4 g / L agar), pH 5.8, and separate them using sterile filter paper.

[0055] (4) Agrobacterium tumefaciens bacterial suspension was centrifuged at 5000 rpm for 5 minutes at room temperature to collect the bacterial cells. The Agrobacterium tumefaciens was resuspended in infection medium (MS medium containing 20 g / L sucrose, 100 mg / L inositol, 0.4 mg / L thiamine hydrochloride (vitamin B1), and 200 μM acetylsyringone) and adjusted to an OD600 of approximately 0.6. The bacterial suspension was transferred to a sterile container, and freshly prepared explants were immersed in the suspension. During the 15-minute inoculation period, vacuum infiltration (-0.6 MPa) was applied for 5 minutes to enhance bacterial penetration. After inoculation, the explants were carefully aspirated onto dry filter paper to remove excess bacterial solution. Finally, the explants were placed on AKCMS solid medium and incubated in the dark at 25°C for 2 days.

[0056] (5) Selective culture and subculture After co-culturing, the explants were transferred to callus induction medium (CIM) containing 4.4 g / L MS medium, 2 mg / L zeatin (ZR), 0.1 mg / L indole-3-acetic acid (IAA), 360 mg / L termethin, 75 mg / L kanamycin sulfate (Kan), 4 mg / L AgNO3, 30 g / L sucrose, and 7.4 g / L agar. Approximately 25-30 explants were placed in each culture dish. After about 20 days of culture on CIM medium, callus formation on the cotyledons was obvious, accompanied by the appearance of green shoot primordia.

[0057] (6) After culturing on CIM medium for 30 days, a large number of shoot primordia can usually be observed. At this stage, the explants that have differentiated into shoots are transferred to induction medium (SIM) consisting of 4.4 g / L MS medium, 0.5 mg / L ZR, 0.17 mg / L gibberellin (GA3), 360 mg / L termethin, 75 mg / L Kan, 30 g / L sucrose, 7.4 g / L agar and 100 mg / L activated charcoal. Explants that do not show shoot primordia but maintain good physiological condition can be subcultured on fresh CIM medium, while explants that grow poorly or die are discarded.

[0058] (7) Rooting culture When the regenerated shoots in SIM medium reach approximately 1 cm in length, they are cut and transferred to rooting medium (RIM) containing 4.4 g / L MS medium, 2 mg / L 3-indolebutyric acid (IBA), 360 mg / L termethin, 30 g / L sucrose, and 7.4 g / L agar. New roots can be observed after about 2 weeks. Within 3-4 weeks, plants with well-developed root systems can adapt to the environment and are then transplanted into soil.

[0059] Example 4: Knockout using CRISPR / Cas9 technology ​ Genetically bred light-colored pepper varieties This embodiment provides a knockout ​ Methods to obtain light-colored pepper plants using genetics.

[0060] 1. Construction of gene knockout vector Design for ​ The CRISPR / Cas9 knockout target sequences of the genes are shown in SEQ ID NO:11-12 respectively. Knockout target 1: 5'-GGAATTGTTGATTTGATGAG-3' (as shown in SEQ ID NO:15); Knockout target 2: 5'-GTGCTGGTTCGGGTACATGT-3' (as shown in SEQ ID NO:16).

[0061] The above target sequences were constructed into a CRISPR / Cas9 vector to form a gene knockout vector.

[0062] 2. Genetic transformation and plant regeneration Using Agrobacterium-mediated genetic transformation, the knockout vector was transformed into sterile explants of red peppers. Through tissue culture, the plants were screened, differentiated, rooted, and domesticated to obtain potential gene knockout pepper plants.

[0063] The above are merely preferred embodiments of this application. It should be noted that this application is not limited to the above embodiments. For those skilled in the art, several improvements and modifications can be made without departing from the principles of this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should also be considered within the scope of protection of this application.

Claims

1. CaRHA2-Cap The application of genes or their encoded proteins in regulating capsanthin content in chili pepper varieties is characterized by, The CaRHA2-Cap The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:

2. The regulation is positive regulation.

2. The application according to claim 1, characterized in that, By overexpressing the above CaRHA2-Cap The expression of genes in chili varieties is used to cultivate chili varieties with high capsanthin content.

3. The application according to claim 1, characterized in that, By lowering the aforementioned CaRHA2-Cap Gene expression or reduction in chili varieties CaRHA2-Cap The activity of genes in chili varieties can be used to breed chili varieties with low capsanthin content.

4. The application according to claim 3, characterized in that, The downregulation can be achieved through virus-induced gene silencing technology or gene editing technology based on the CRISPR / Cas9 system. CaRHA2-Cap Gene expression or reduction in chili varieties CaRHA2-Cap The activity of genes in chili pepper varieties.

5. The application according to claim 4, characterized in that, When gene silencing technology is used, the target amplification primer sequences used to construct the silencing vector are shown in SEQ ID NO:11 and SEQ ID NO:12; when gene editing technology using the CRISPR / Cas9 system is used, the gene knockout target sequence is shown in SEQ ID NO:15 or SEQ ID NO:

16.

6. The application according to claim 2, characterized in that, When constructing the overexpression vector, the gene knockout target sequence used is shown in SEQ ID NO:13 or SEQ ID NO:

14.

7. A method for amplifying the method described in claim 1 CaRHA2-Cap A primer pair for a gene, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID NO: 3 and SEQ ID NO:

4.

8. A method for cultivating chili pepper varieties with high capsanthin content, characterized in that, include: (1) As shown in SEQ ID NO: 1 CaRHA2-Cap The nucleotide sequence of the gene was constructed into a plant overexpression vector to obtain a recombinant vector; (2) The recombinant vector was transformed into pepper explants; (3) Tissue culture was performed on the transformed explants, and transgenic pepper plants were screened and obtained; (4) Select strains with increased capsanthin content and stable traits from the transgenic pepper plants.

9. A method for cultivating chili pepper varieties with low capsanthin content, characterized in that, In pepper plants, virus-induced gene silencing technology was used to downregulate the gene shown in SEQ ID NO:

1. CaRHA2-Cap Gene expression specifically includes the following steps: (1) Construct a system containing primers amplified by primers shown in SEQ ID NO:11 and SEQ ID NO:

12. CaRHA2-Cap VIGS silencing vector for gene-specific fragments; (2) The silencing vector was introduced into Agrobacterium and used to infect pepper plants; (3) Cultivate infected plants and achieve [the desired effect] in their fruits. CaRHA2-Cap Silencing of gene expression; (4) Select plants with significantly lighter pepper peel color.

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