Application of CaLSH10 gene or CaLSH10 protein in regulating capsanthin content in pepper

Abstract

The application provides a method for regulating the content of capsanthin in pepper CaLSH10 The application provides an application of a gene or CaLSH10 protein in regulating the content of capsanthin in pepper. CaLSH10 The nucleotide sequence of the gene is shown as SEQ ID NO:1, and the amino acid sequence of the CaLSH10 protein is shown as SEQ ID NO:2. The application can solve the problem that the regulating factor for regulating the synthesis of capsanthin in the prior art is not completely clear. By regulating the expression of the gene or protein in the pepper, the content of capsanthin in the pepper can be regulated, new gene resources and theoretical basis are provided for pepper germplasm innovation and molecular breeding, and the application is suitable for the field of genetic engineering breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering breeding, and more specifically, to a... CaLSH10 Application of gene or CalSH10 protein in regulating capsanthin content in chili peppers. Background Technology

[0002] Chili peppers are rich in various natural products, such as vitamin C, capsaicin, and capsanthin, which have significant production and application value. Capsanthin is a carotenoid that accumulates in high quantities in mature chili peppers, giving the fruit its red color and determining its appearance and nutritional quality. Capsanthin is also a fat-soluble natural pigment, classified as a Category A pigment by the Food and Agriculture Organization of the United Nations and the World Health Organization, meaning its use is not subject to limits. It is highly edible, has excellent coloring power, and possesses certain antioxidant, anti-radiation, and anti-inflammatory properties, thus it is widely used in food processing, animal feed, cosmetics, and the pharmaceutical industry.

[0003] In mature red pepper fruits, capsaicin accounts for over 60% of the total pigment content, with capsanthin and capsaicin being the main components. Capsanthin belongs to the carotenoid family, and its synthesis pathway is relatively well understood. Among these pathways, the capsanthin / capsaicin synthase gene... CCS (capsanthin-capsorubin synthase) is a key gene controlling the formation of red color in pepper fruits. It can catalyze the conversion of 5,6-epoxyzeaxanthin and purpuraxanthin into capsanthin / capsorubin, and its expression level is closely related to the content of capsanthin and capsorubin in pepper fruits.

[0004] In addition to genetic factors, the capsanthin content in peppers is regulated by environmental factors such as light, temperature, endogenous hormones, and cultivation conditions. Differences in environmental factors (e.g., light, temperature, humidity, soil) across different regions lead to significant variations in capsanthin content within the same variety, further limiting the promotion of varieties and industrial development. Furthermore, because pepper genetic transformation / gene editing systems are not yet mature, the regulatory factors controlling capsanthin synthesis are not fully understood.

[0005] Therefore, providing a regulatory factor that can effectively control capsanthin synthesis is crucial for the production and application of capsanthin. Summary of the Invention

[0006] The main objective of this invention is to provide a CaLSH10 The application of the gene or CalSH10 protein in regulating capsanthin content in chili peppers aims to address the issue that the regulatory factors controlling capsanthin synthesis are not fully understood in existing technologies.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a [missing information] is provided. CaLSH10 Application of gene or CalSH10 protein in regulating capsanthin content in chili peppers.

[0008] Furthermore, the above CaLSH10 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0009] Furthermore, the amino acid sequence of the CaLSH10 protein is shown in SEQ ID NO: 2.

[0010] To achieve the above objective, according to a second aspect of the present invention, a method for increasing the capsanthin content in chili pepper fruits is provided, the method comprising: increasing the expression level of CaLSH10 protein in chili pepper fruits.

[0011] Furthermore, the amino acid sequence of the CaLSH10 protein is shown in SEQ ID NO: 2.

[0012] Furthermore, the above-mentioned method increases the expression level of CaLSH10 protein in pepper fruits.

[0013] To achieve the above objective, according to a third aspect of the present invention, a method for reducing the capsanthin content in chili pepper fruits is provided, the method comprising: reducing the expression of CaLSH10 protein in chili pepper fruits or inactivating the CaLSH10 protein.

[0014] Furthermore, the amino acid sequence of the CaLSH10 protein is shown in SEQ ID NO: 2.

[0015] Furthermore, inactivating the CaLSH10 protein includes inhibiting the activity of the CaLSH10 protein in the pepper fruit.

[0016] Furthermore, inhibiting the activity of the CaLSH10 protein in the above-mentioned pepper fruit includes fusing the SUPERMAN repressor domain X to the C-terminus of the CaLSH10 protein.

[0017] By applying the technical solution of the present invention, a method is provided. CaLSH10 The application of genes or CalSH10 proteins in regulating capsanthin content in chili peppers effectively enriches the regulatory network of capsanthin synthesis, providing new gene resources and theoretical basis for the innovation of industrial pigment chili pepper germplasm and molecular breeding, thereby accelerating the technological upgrading of the traditional breeding industry. Attached Figure Description

[0018] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0019] Figure 1 The present invention is shown in Embodiment 1. 35S::FGFP-Sp Expression vector spectrum results.

[0020] Figure 2 The present invention, according to embodiment 3, is shown. Flag-CaLSH10 Overexpression transgenic pepper materials and their CaLSH10 The results of RNA and protein expression level analysis are shown in the figure. Figure 2 Figure A shows the results of overexpressing transgenic pepper materials. Figure 2 B represents WT and overexpression transgenic peppers. CaLSH10 Figure showing the results of RNA expression level analysis. Figure 2 C represents WT and overexpression transgenic peppers. CaLSH10 Figure showing the results of protein expression level analysis.

[0021] Figure 3 The present invention, according to embodiment 3, is shown. Flag-CaLSH10 Figure showing the results of T-DNA insertion site analysis in transgenic pepper materials.

[0022] Figure 4 The present invention, according to embodiment 3, is shown. CaLSH10-SRDX Overexpression transgenic pepper materials and their CaLSH10-SRDX The expression level analysis results are shown in the figure. Figure 4 A in the middle is CaLSH10-SRDX Results of overexpression transgenic and wild-type pepper materials. Figure 4 B is CaLSH10-SRDX Overexpression of genetically modified and wild-type peppers CaLSH10-SRDX Graph showing the results of expression level analysis.

[0023] Figure 5 The present invention, according to embodiment 3, is shown. CaLSH10-SRDX Figure showing the results of T-DNA insertion site analysis in transgenic pepper materials.

[0024] Figure 6 The present invention, according to embodiment 4, is shown. Flag-CaLSH10 The fruit phenotype and pericarp of genetically modified pepper materials CCS The results of gene expression level and capsanthin content analysis are shown in the figure. Figure 6 Image A shows phenotypic photos of fruits from different materials. Figure 6 B represents different developmental stages, in the pericarp CCS Graph of expression level analysis results Figure 6C represents the analysis of capsanthin content in the pericarp at different developmental stages.

[0025] Figure 7 The present invention, according to embodiment 4, is shown. CaLSH10-SRDX The fruit phenotype and pericarp of genetically modified pepper materials CCS The graph shows the results of gene expression and capsanthin content analysis. Figure 7 Photograph A shows the fruit phenotypes of different chili pepper strains; Figure 7 B represents different developmental stages, in the pericarp CCS Expression level analysis; Figure 7 C represents the analysis of capsanthin content in the pericarp at different developmental stages.

[0026] Figure 8 The present invention, according to embodiment 5, is shown. CCS Genome map results of ChIP-seq and RNA-seq signal distribution around genes. Detailed Implementation

[0027] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0028] As mentioned in the background section, there is a problem in the prior art that the regulatory factors controlling capsanthin synthesis are not yet fully understood. Therefore, in this application, the inventors attempt to provide a... CaLSH10 The application of the gene or CalSH10 protein in regulating capsanthin content in chili peppers effectively enriches the regulatory network of capsanthin synthesis, providing new gene resources and theoretical basis for germplasm innovation and molecular breeding of industrial pigment chili peppers, thereby accelerating the technological upgrading of the traditional breeding industry. Therefore, this application proposes a series of protection schemes.

[0029] In a first typical embodiment of this application, a method is provided. CaLSH10 Application of gene or CalSH10 protein in regulating capsanthin content in chili peppers.

[0030] In a preferred embodiment, the above CaLSH10 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0031] In a preferred embodiment, the amino acid sequence of the CaLSH10 protein described above is shown in SEQ ID NO: 2.

[0032] In a preferred embodiment, the above regulation includes increasing or decreasing.

[0033] In this application, the inventors discovered that increasing the yield of chili peppers in chili pepper plants... CaLSH10The expression level of genes can effectively increase the capsanthin content in chili peppers, and the use of techniques including, but not limited to, chimeric repressor silencing technology can significantly improve the expression of capsanthin in chili pepper plants. CaLSH10 Silencing gene expression effectively reduces the capsanthin content in pepper plants. The above experimental results fully demonstrate that... CaLSH10 This application highlights the important role of capsanthin in the regulatory network of capsanthin synthesis, providing new genetic resources and theoretical foundations for the innovation of industrial pigment pepper germplasm and molecular breeding.

[0034] In a second typical embodiment of this application, a method for increasing the capsanthin content in chili pepper fruits is provided, the method comprising: increasing the expression level of CaLSH10 protein in chili pepper fruits.

[0035] In a preferred embodiment, the amino acid sequence of the CaLSH10 protein described above is shown in SEQ ID NO: 2.

[0036] In a preferred embodiment, the above-mentioned method increases the expression level of CaLSH10 protein in pepper plants.

[0037] In this application, the inventors discovered that increasing the yield of chili peppers in chili pepper plants... CaLSH10 The expression level of the gene can effectively increase the capsanthin content in chili peppers. The above experimental results fully demonstrate that... CaLSH10 This application highlights the important role of capsanthin in the regulatory network of capsanthin synthesis, providing new genetic resources and theoretical foundations for the innovation of industrial pigment pepper germplasm and molecular breeding.

[0038] In a third typical embodiment of this application, a method for reducing the capsanthin content in chili pepper fruits is provided, the method comprising: reducing the expression of CaLSH10 protein in chili pepper fruits or inactivating the CaLSH10 protein.

[0039] In a preferred embodiment, the amino acid sequence of the CaLSH10 protein described above is shown in SEQ ID NO: 2.

[0040] In a preferred embodiment, the CaLSH10 protein includes an inhibitory effect on the activity of the CaLSH10 protein in the pepper fruit.

[0041] In a preferred embodiment, inactivation of the CaLSH10 protein includes, but is not limited to, competitive inhibition of the activity of the natural CaLSH10 protein in pepper fruit.

[0042] In a preferred embodiment, inhibiting the activity of the CaLSH10 protein in the chili pepper fruit includes fusing the SUPERMAN inhibitory domain X to the C-terminus of the CaLSH10 protein.

[0043] In a preferred embodiment, the SUPERMAN repressor domain X is fused to the C-terminus of the CalSH10 protein as shown in SEQ ID NO: 3.

[0044] In a preferred embodiment, the SUPERMAN repressor domain X is fused to the C-terminus of the CalSH10 protein as shown in SEQ ID NO: 4.

[0045] In this application, the inventors discovered that employing, but not limited to, chimeric repressor silencing techniques, including but not limited to fusing the SUPERMAN repressor domain X to the C-terminus of the CaLSH10 protein, can effectively reduce the capsanthin content in chili pepper plants. In chili pepper plants... CaLSH10 It is a positive regulatory factor. CaLSH10- SRDX It can competitively bind to the promoter of downstream target genes, thereby inhibiting the expression of downstream target genes, which include... CCS Gene. CaLSH10-SRDX The resulting phenotype is similar to silencing or suppression. CaLSH10 The phenotype of the gene. The above experimental results fully demonstrate the above. CaLSH10 This application highlights the important role of capsanthin in the regulatory network of capsanthin synthesis, providing new genetic resources and theoretical foundations for the innovation of industrial pigment pepper germplasm and molecular breeding.

[0046] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0047] Example 1

[0048] CaLSH10 Gene cloning and overexpression vectors 35S::Flag-CaLSH10-Sp Construction:

[0049] The genome of the CA59 pepper inbred line CaLSH10 Using the CDS sequence as a reference, cloning primers were designed, and the primer sequences are shown below:

[0050] CaLSH10 The nucleotide sequence of -XmaI-F is shown in SEQ ID NO: 5.

[0051] CaLSH10 The nucleotide sequence of -BamHI-R is shown in SEQ ID NO: 6.

[0052] In this application, the genome of the CA59 pepper inbred line is described in (reference: Liao Y, Wang J, Zhu Z, Liu Y, Chen J, Zhou Y, Liu F, Lei J, Gaut BS, Cao B, Emerson JJ, Chen C. The 3D architecture of the pepper genome and its relationship to function and evolution. Nat Commun. 2022 Jun 16;13(1):3479), which is available to the public from South China Agricultural University.

[0053] Using cDNA from the pericarp of the chili inbred line “CA59” at the color-changing stage as a template, PCR amplification was performed using high-fidelity KOD FX enzyme. The PCR program was as follows: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 30 sec, 56℃ annealing for 30 sec, 68℃ extension for 30 sec, for 35 cycles, followed by a final extension at 68℃ for 10 min. The PCR products were detected by agarose gel electrophoresis, and the target fragment was recovered.

[0054] Simultaneously, digestion with restriction endonucleases XmaI and BamHI was performed. 35S-FGFP-Sp carrier 35S-FGFP-Sp The sequence of the vector is shown in SEQ ID NO: 11. 35S-FGFP-Sp The structure of the carrier spectrum is as follows Figure 1 As shown, the digested vector fragment was then recovered, and the recovered target fragment was recombined with the vector fragment. The recombinant fragment was then transformed into *E. coli* to screen for positive clones. The positive clones were then sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO: 1. CaLSH10 The gene, whose encoded amino acid sequence is shown in SEQ ID NO: 2, is the chili pepper transcription factor CaLSH10, and the obtained expression vector is named 35S::Flag-CaLSH10-Sp .

[0055] Example 2

[0056] Fusion genes CaLSH10-SRDX Construction and genetic transformation of its overexpression vectors:

[0057] 1) SRDX and CaLSH10 Amplification of recombinant fragments:

[0058] According to the SUPERMAN suppression domain X ( SRDX ) nucleotide sequence, wherein the nucleotide sequence of SRDX is shown in SEQ ID NO: 7, 3 SRDXThe nucleotide sequence was sent to Qingke Biotechnology Co., Ltd. for primer design and synthesis, among which, 3 SRDX The nucleotide sequence is shown in SEQ ID NO: 8. Primers were designed, and their sequences are shown below:

[0059] 3 SRDX The nucleotide sequence of -BamHI-F is shown in SEQ ID NO: 9.

[0060] 3 SRDX The nucleotide sequence of -BamHI-R is shown in SEQ ID NO: 10.

[0061] With the synthesis of 3 SRDX Using the fragment as a template, PCR amplification was performed using high-fidelity KOD FX enzyme. The PCR program was as follows: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 30 sec, 56℃ annealing for 10 sec, 68℃ extension for 30 sec, for 35 cycles, followed by a final extension at 68℃ for 10 min. The PCR products were detected by agarose gel electrophoresis, and the target fragment was recovered.

[0062] according to CaLSH10 Based on the gene sequence, primers were designed, and the primer sequences are shown below:

[0063] CaLSH10 The nucleotide sequence of -XmaI-F is shown in SEQ ID NO: 5.

[0064] CaLSH10-SRDX The nucleotide sequence of -BamHI-R is shown in SEQ ID NO: 12.

[0065] by 35S::Flag-CaLSH10-Sp Using the vector as a template, PCR amplification was performed using high-fidelity KOD FX enzyme. The PCR program was as follows: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 30 sec, 56℃ annealing for 30 sec, 68℃ extension for 30 sec, for 35 cycles, followed by a final extension at 68℃ for 10 min. The PCR products were detected by agarose gel electrophoresis, and the target fragment was recovered.

[0066] 2) Fusion genes CaLSH10-SRDX Overexpression vector 35S::Flag-CaLSH10-SRDX-Sp Construction:

[0067] Digested with restriction endonucleases XmaI and BamHI 35S-FGFP-Sp The vector was then recovered, and the digested vector fragments were further processed with the recovered... CaLSH10 , SRDXThe fragment was recombined, then transformed into E. coli to screen for positive clones, and the positive clones were sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO: 3, which is... CaLSH10-SRDX The nucleotide sequence, whose encoded amino acid sequence is shown in SEQ ID NO: 4, is... CaLSH10-SRDX The obtained expression carrier is 35S::Flag-CaLSH10-SRDX-Sp .

[0068] Example 3

[0069] 1) Genetic transformation of chili peppers and the acquisition of transgenic chili pepper materials:

[0070] The obtained expression vector 35S::Flag-CaLSH10-Sp and 35S::Flag-CaLSH10-SRDX-Sp They were transferred into Agrobacterium strain GV3101 Biomed (BC304) respectively.

[0071] Pepper seedlings of the cultivar "CA59" at approximately 10 days old were selected, and cotyledon segments were prepared as explants for transformation. The explants were then placed in a -0.1 MPa negative pressure environment and subjected to OD... 600 Agrobacterium suspension with a pH of 0.6–0.8 was used for direct infection, followed by co-culture in the dark for 2 days. After co-culture, the explants were sequentially transferred to callus induction medium and adventitious shoot induction medium for regeneration culture. Both media were supplemented with 50 mg / L spectinomycin (Spe) as a selection marker for transgenic positive plants. When the adventitious shoots grew to a suitable length, they were individually cut and transferred to rooting medium, and cultured for another 3–4 weeks until the plants developed robust root systems.

[0072] In this application, CA59 is also referred to as the CA59 pepper inbred line.

[0073] 2) Genotyping of transgenic pepper materials:

[0074] The obtained regenerated plants were analyzed by PCR, qRT-PCR, WB, etc., and positive plants were selected for phenotypic analysis.

[0075] 1. PCR:

[0076] According to the expression carrier ( 35S-FGFP-Sp Based on the nucleotide sequence of the vector T-DNA fragment, primers were designed, and the primer sequences are shown below:

[0077] The nucleotide sequence of Sp-JF is shown in SEQ ID NO: 13.

[0078] The nucleotide sequence of Sp-JR is shown in SEQ ID NO: 14.

[0079] Genomic DNA was extracted from regenerated plants using the CTAB (Hexadecyltrimethylammonium bromide) method. PCR amplification was performed using a 2 × Taq Master Mix (Dye Plus) (P112, Vazyme) with the following program: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 sec, 56℃ annealing for 30 sec, 72℃ extension for 50 sec, 35 cycles, followed by a final extension at 72℃ for 10 min. PCR products were detected by agarose gel electrophoresis, and positive plants were selected for further validation.

[0080] 2. qRT-PCR:

[0081] RNA was extracted from leaves of WT and overexpressing transgenic materials, and reverse transcribed using EasyScript® One-Step gDNARemoval and cDNA Synthesis SuperMix (AE311, TransGene). The resulting cDNA was used as a template for qRT-PCR detection.

[0082] according to CaLSH10 and CaActin Based on the nucleotide sequence of the gene, qRT-PCR primers were designed, and the primer sequences are shown below:

[0083] CaLSH10 The nucleotide sequence of -RT-F is shown in SEQ ID NO: 15.

[0084] CaLSH10 The nucleotide sequence of -RT-R is shown in SEQ ID NO: 16.

[0085] CaActin The nucleotide sequence of -RT-F is shown in SEQ ID NO: 17.

[0086] CaActin The nucleotide sequence of -RT-R is shown in SEQ ID NO: 18.

[0087] qRT-PCR analysis was performed using SupRealQ Ultra Hunter SYBR qPCR Master Mix (U+) (Q713, Vazyme) on a CFXOpus 96 Real-Time PCR System (#120113119, Roche). CaActin As an internal reference gene, each value represents one biological replicate.

[0088] 3. Western Blot:

[0089] Total protein was extracted from the leaves of WT and transgenic plants using non-grinding buffer (120 mM Tris-HCl pH 6.8, 100 mM EDTA, 4% SDS, 10% β-mercaptoethanol, 5% glycerol, 0.05% bromophenol blue). After separation by 4-20% FastPAGE™ protein precast gel (TSP024, Tsingke), the protein was transferred to an Amersham Protran 0.2 NC nitrocellulose membrane (10600001, Cytiva) using a transfer apparatus. After successful transfer, the membrane was blocked with 5% skim milk powder for 2 h, then incubated with anti-flag antibody (F1804, Sigma-Aldrich) at room temperature for 2 h. After incubation, the membrane was washed three times (10 min each time) with PBST buffer. Goat Anti-Mouse IgG was then added, HRP Conjugated (CW0102S, CWBIO), and incubated at room temperature for 2 hours. After incubation, the membrane was washed again to remove unbound secondary antibody. Finally, the prepared SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Cat. 34580, ThermoFisher) was added, and the membrane was exposed using an Amersham ImageQuant 800 Fluor (Cytiva). Anti-HSP82 antibody was used as an internal control antibody. Positive identification was performed using an anti-flag antibody, with Anti-HSP82 (AbM51099-31-PU, Beijing Protein Innovation) serving as the internal control antibody.

[0090] 35S::Flag-CaLSH10 ( Flag-CaLSH10 Transgenic pepper materials overexpressing lines #23 and #25 and WT pepper (CA59) such as Figure 2 As shown in A, CaLSH10 RNA expression levels, such as Figure 2 As shown in B, CaLSH10 Protein expression levels, such as Figure 2 As shown in Figure C. The results for the WT, #23, and #25 pepper lines are shown in the figure below. Figure 2 As shown in A, where WT represents wild type, and #23 and #25 are... 35S::Flag-CaLSH10 ( Flag-CaLSH10 Two overexpression lines. Figure 2 The experimental results in B and C show that: 35S::Flag-CaLSH10 ( Flag-CaLSH10 Overexpression in strains #23 and #25 CaLSH10 Both RNA and protein expression levels were significantly higher than those of WT, further... 35S::Flag-CaLSH10 ( Flag-CaLSH10 Whole-genome resequencing data of overexpression lines #23 and #25 are as follows: Figure 3 As shown, Figure 3 The experimental results showed that the T-DNA in both strains was stably integrated into the pepper genome. In strain #23, the T-DNA was inserted into chromosome 5, and in strain #25, the T-DNA was inserted into chromosome 12. Neither strain was inserted into the coding gene region.

[0091] CaLSH10-SRDX Overexpression transgenic pepper materials and their CaLSH10-SRDX The results of expression level analysis are as follows Figure 4 As shown, where Figure 4 A in the middle is CaLSH10-SRDX Overexpression of transgenic and wild-type pepper (CA59) materials, Figure 4 B is CaLSH10-SRDX Overexpression of genetically modified and wild-type peppers CaLSH10-SRDX Expression level analysis results Figure 4 WT indicates wild-type chili peppers, #59 and #190 indicate wild-type chili peppers. 35S::Flag-CaLSH10-SRDX ( CaLSH10-SRDX Two strains. Figure 4 The experimental results showed that in transgenic lines #59 and #190 CaLSH10 RNA expression levels were upregulated by 9-fold and 160-fold compared to WT, respectively. CaLSH10 -Analysis of T-DNA insertion sites in SRDX transgenic pepper materials #59 and #190 as follows: Figure 5 It is said that, Figure 5 The experimental results showed that T-DNA was integrated into the pepper genome in both strains. In strain #59, T-DNA was inserted into chromosome 3, and in strain #190, T-DNA was inserted into chromosome 7. However, in strain #59, T-DNA was inserted into an unknown NF-X1-type zinc finger protein gene sequence.

[0092] Finally obtained 35S::Flag-CaLSH10 ( Flag-CaLSH10 Overexpression lines #23 and #25, 35S:: Flag-CaLSH10-SRDX ( CaLSH10-SRDX Transgenic lines #59 and #190.

[0093] Example 4

[0094] Phenotypic analysis of transgenic lines:

[0095] 1) CCS Gene expression level analysis:

[0096] RNA was extracted from the pericarps of WT (a chili pepper variety CA59) and overexpressing transgenic materials at the red-ripe stage. Reverse transcription was performed using EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (AE311, TransGene), and the obtained cDNA was used as a template for qRT-PCR detection.

[0097] according to CCS Based on the nucleotide sequence of the gene, qRT-PCR primers were designed, and the primer sequences are shown below:

[0098] CCS The nucleotide sequence of -RT-F is shown in SEQ ID NO: 19.

[0099] CCS The nucleotide sequence of -RT-R is shown in SEQ ID NO: 20.

[0100] qRT-PCR analysis was performed using SupRealQ Ultra Hunter SYBR qPCR Master Mix (U+) (Q713, Vazyme) on a CFXOpus 96 Real-Time PCR System (#120113119, Roche). CaActin was used as an internal control gene, and each value represents one biological replicate.

[0101] 2) Determination of capsanthin content in fruit peel:

[0102] Pepper pericarps from different developmental stages of WT and overexpression transgenic materials were selected, rapidly frozen in liquid nitrogen, and thoroughly ground into powder. The powder was then dried using a freeze dryer (Cat. CTFD-10S-U, CREATRUST). Approximately 100 mg of sample was weighed and added to an extraction solvent (acetone:n-hexane:anhydrous ethanol (V / V / V=1:1:1, containing 0.1% BHT) at a mass-to-volume ratio of 50 mg / ml. After vortexing, the mixture was sonicated on ice for 30 min, centrifuged at 7830 rcf and 4℃ for 10 min, and 1.5 ml of the supernatant was collected into a centrifuge tube. The sample was lyophilized and reconstituted with 200 μL of a reconstitution solvent (acetonitrile:isopropanol (v / v=1:1, containing 0.1% BHT) by vortexing. After centrifugation at 12700 rpm and 4℃ for 5 min, the mixture was filtered through a 0.22 μm organic filter membrane and transferred to a sample vial. The sample was then sent to the mass spectrometry platform of Peking University Modern Agricultural Research Institute for analysis.

[0103] Flag-CaLSH10 The fruit phenotype and pericarp of genetically modified pepper materials CCS Gene expression levels and capsanthin content analysis, such as Figure 6 As shown, where Figure 6 Image A shows phenotypic photos of fruits from different materials. Figure 6 B represents different developmental stages, in the pericarp CCS Expression level analysis, Figure 6 C represents the analysis of capsanthin content in pepper pericarps at different developmental stages. Among them, #23 and #25 are... Flag-CaLSH10 Two overexpression lines. G, B, and R represent the green-ripe stage, the color-changing stage (half-ripe), and the red-ripe stage, respectively. The method for distinguishing the color of pepper fruits at different stages in this application is as follows: immature fruits are green, green-ripe fruits are dark green, and from the color-changing stage to the red-ripe stage (maturity stage), the color of pepper fruits changes completely from brownish-red to red.

[0104] Figure 6 The experimental results show that Flag-CaLSH10 The peppers overexpressing the transgenic material had a deeper color during the color-changing and ripening stages compared to the WT peppers. CCS Gene expression was significantly increased, and the capsanthin content in the peel increased significantly during the color change and ripening stages.

[0105] CaLSH10-SRDX The fruit phenotype and pericarp of genetically modified pepper materials CCS The results of gene expression and capsanthin content analysis are as follows: Figure 7 As shown, Figure 7 Photograph A shows the fruit phenotypes of different chili pepper strains; Figure 7 B represents different developmental stages, in the pericarp CCS Expression level analysis; Figure 7 C represents the analysis of capsanthin content in the pericarp at different developmental stages. Figure 7 In the middle, #59 and #190 are CaLSH10-SRDX Two overexpression lines. B and R represent the color-changing stage and the red-ripe stage, respectively.

[0106] Figure 7 The experimental results of A show that, CaLSH10-SRDX The peppers overexpressing the transgenic material had lighter fruit color during the color change and ripening stages compared to the WT peppers. Figure 7 The experimental results of B show that CaLSH10-SRDX The peppers overexpressing the transgenic material showed significantly different fruit colors during the color-changing and ripening stages compared to the WT material. CCS Gene expression levels decreased significantly. Furthermore, the capsanthin content in the pericarp at the red-ripe stage was measured. Figure 7 The experimental results of C show that CaLSH10-SRDX The capsanthin content in the pericarp of the overexpression strain was significantly lower than that in the WT group.

[0107] Example 5

[0108] transcription factors CaLSH10 Mechanism of action verification:

[0109] Select WT (chili variety CA59) and Flag-CaLSH10 ChIP-seq experiments were performed on the pericarp of (strain #23) during the color-changing stage. First, the sample material was cross-linked with 1% formaldehyde, thoroughly ground in liquid nitrogen, and then the cell nuclei were extracted. Genomic DNA was fragmented to 200-500 bp using an ultrasonic homogenizer. 5% of the sonicated sample was used as input. The remaining sample was enriched with anti-flag antibodies and Protein A / G magnetic beads to collect the DNA complex bound to the CaLSH10 target protein, followed by multiple washes to remove non-specifically bound components. The immunoprecipitated complex was eluted with elution buffer, and then decross-linked, protein and RNA were removed, and the DNA fragments were purified and recovered. DNA libraries were constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V4 (Vazyme, Cat. ND610) and the VAHTS Multiplex Oligos Set 4 for Illumina (Vazyme, Cat. N321) kits, and then sent to Beijing Berry Genomics Co., Ltd. for high-throughput sequencing.

[0110] CCS Genomic maps showing the distribution of ChIP-seq and RNA-seq signals around genes, as shown below. Figure 8 As shown, Figure 8 The experimental results show that during the color-changing stage of pepper fruit, the transcription factor CalSH10 can directly bind to... CCS The promoter region of a gene.

[0111] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: The present invention creates... CaLSH10 Phenotypic analysis of transgenic pepper materials with overexpressed gene showed that overexpression... CaLSH10 The capsanthin content in the fruit peel of the gene-expressing organism was significantly higher than that in fruits with normal expression. Furthermore, using chimeric repressor silencing technology, a new gene was created... CaLSH10-SRDX Phenotypic analysis of overexpression-modified chili pepper transgenic materials showed that: overexpression CaLSH10-SRDX The capsanthin content in the pericarp of fruits with the gene expression was significantly lower than that in fruits with normal expression.

[0112] In summary, this invention enriches the regulatory network of capsicum red pigment synthesis, providing new gene resources and theoretical basis for the innovation of industrial pigment pepper germplasm and molecular breeding, thereby accelerating the technological upgrading of the traditional breeding industry.

[0113] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. CaLSH10 The application of the gene or CalSH10 protein in regulating capsanthin content in chili peppers, characterized by: In the application The CaLSH10 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; The amino acid sequence of the CalSH10 protein is shown in SEQ ID NO:

2.

2. A method for increasing the capsanthin content in chili pepper fruits, characterized in that, The method includes: increasing the expression level of CaLSH10 protein in pepper fruits; The amino acid sequence of the CalSH10 protein is shown in SEQ ID NO:

2.

3. A method for reducing the capsanthin content in chili pepper fruits, characterized in that, The method includes: reducing the expression of CaLSH10 protein in pepper fruit or inactivating the CaLSH10 protein; The amino acid sequence of the CalSH10 protein is shown in SEQ ID NO:

2.

4. The method for reducing capsanthin content in chili pepper fruits according to claim 3, characterized in that, Inactivating the CaLSH10 protein includes inhibiting the activity of the CaLSH10 protein in the pepper fruit.

5. The method for reducing capsanthin content in chili pepper fruits according to claim 4, characterized in that, Inhibiting the activity of the CalSH10 protein in the pepper fruit involves fusing the SUPERMAN repressor domain X to the C-terminus of the CalSH10 protein.

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