CN116891859B - CaRAP2-12 gene and application thereof in regulating carotenoid synthesis in capsicum fruits - Google Patents

Abstract

The invention discloses CaRAP-12 genes and application thereof in regulating and controlling carotenoid synthesis in pepper fruits, belonging to the technical field of genetic engineering. The invention clones to key transcription factor CaRAP-12 for regulating and controlling the synthesis of carotenoid in capsicum fruits, and the nucleotide sequence is shown as SEQ ID No. 1. The invention constructs a transgenic plant for regulating and controlling the synthesis of carotenoid in pepper fruits by constructing recombinant plasmids containing CaRAP-12 genes and transgenic engineering bacteria. The CaRAP gene of the invention can enrich the cognition of the secondary metabolism of the pepper fruits, and can deeply understand the genetic mechanism of the pepper fruit quality formation, especially the color formation, thus providing theoretical and practical reference bases for realizing the selective agronomic character breeding of the pepper fruits.

Description

CaRAP2-12 gene and its application in regulating carotenoid synthesis in pepper fruit

Technical Field

The invention belongs to the technical field of genetic engineering, and in particular relates to a CaRAP2-12 gene and an application thereof in regulating the synthesis of carotenoids in pepper fruits.

Background Art

Pepper (Capsicum spp.) is a common spicy condiment and vegetable belonging to the Solanaceae family and an important agricultural and horticultural crop in my country. Fruit color, as one of the important characteristics of pepper, has attracted the attention of breeders and consumers. The pigments in pepper fruit mainly include chlorophyll, carotenoids and anthocyanins. The presence and relative content of these pigments determine the color of pepper fruit. Peppers of different varieties and maturity will show a variety of shades, from green, yellow, orange to red and purple. This rich and colorful color also provides different options and applications for peppers in cooking and decoration.

Carotenoids are widely found in the leaves, flowers and fruits of higher plants. They are natural yellow, orange and red lipophilic molecules belonging to the terpenoid pigments. The carotenoid content in pepper fruit is between 0.03 and 3 mg per 100 g fresh weight. It is mainly composed of β-carotene, capsanthin, lutein, zeaxanthin, etc., and plays an important role in the physiology, development, ecology and evolution of plants. Carotenoids in pepper are synthesized into digeranyl diphosphate (GGPP) through the methylerythritol phosphate pathway (MEP). In the first step, phytoene synthase (PSY) condenses two GGPP molecules head-on to form phytoene; in the second step, dehydrogenase and isomerase form lycopene; in the third step, lycopene is cyclized to form α-carotene or β-carotene; in the fourth step, α-carotene and β-carotene are hydroxylated to form lutein and zeaxanthin, respectively; finally, under the catalysis of capsanthin/capsanthin synthase (CCS), 5,6-epoxyzeaxanthin and violaxanthin are converted into capsanthin and capsanthin, respectively. This step is a unique synthesis pathway in peppers.

The AP2/ERF family can participate in regulating the synthesis of plant carotenoids. However, there are few studies on the regulation of carotenoid synthesis in pepper fruit by AP2/ERF family transcription factors.

Summary of the invention

The purpose of the present invention is to provide a CaRAP2-12 gene and its application in regulating carotenoid synthesis in pepper fruit. The CaRAP2-12 gene is an AP2/ERF family transcription factor, which can provide a target gene for the cultivation of new pepper varieties and provide genetic materials and theoretical basis for the genetic improvement of pepper varieties.

The present invention provides a CaRAP2-12 gene for regulating the synthesis of carotenoids in pepper fruits. The nucleotide sequence of the CaRAP2-12 gene is shown in SEQ ID No.1.

The present invention also provides a primer pair for amplifying the CaRAP2-12 gene, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID No.3 and a downstream primer as shown in SEQ ID No.4.

The present invention also provides a protein encoded by the CaRAP2-12 gene, and the amino acid sequence of the protein encoded is shown in SEQ ID No.2.

The present invention also provides the use of the CaRAP2-12 gene or the encoded protein in regulating the synthesis of carotenoids.

Preferably, the carotenoids include β-carotene, zeaxanthin and capsanthin.

The present invention also provides the use of the CaRAP2-12 gene or the encoded protein in improving the quality of pepper fruit.

The present invention also provides the use of the CaRAP2-12 gene or the encoded protein in creating new pepper germplasm.

The present invention also provides a method for regulating the carotenoid content in pepper fruits, comprising regulating the expression level of the CaRAP2-12 gene in the pepper fruits.

Preferably, the regulation includes any one of inhibition, knockdown and overexpression.

The invention provides a recombinant plasmid containing the CaRAP2-12 gene.

The present invention provides a transgenic engineering bacterium which is used for regulating the expression amount of the CaRAP2-12 gene and contains the recombinant plasmid.

Beneficial effects: The present invention cloned for the first time the key transcription factor CaRAP2-12 that regulates the synthesis of carotenoids in pepper fruits, and the nucleotide sequence of the CaRAP2-12 gene is shown in SEQ ID No. 1. The CaRAP2-12 gene of the present invention has the effects of promoting the synthesis of carotenoid compounds in pepper fruits and affecting the formation of pepper fruit quality. The present invention creates transgenic plants that regulate the synthesis of carotenoids in pepper fruits by constructing recombinant plasmids and transgenic engineering bacteria containing the CaRAP2-12 gene. In the examples of the present invention, it was verified that the transgenic plants in which the expression of the CaRAP2-12 gene was inhibited had reduced carotenoid synthesis, and the expression of related genes was significantly downregulated; the transgenic plants in which the CaRAP2-12 gene was overexpressed had increased carotenoid synthesis, and the expression of related genes was significantly upregulated.

The use of the CaRAP2-12 gene described in the present invention can enrich the understanding of the secondary metabolism of pepper fruits, deeply understand the genetic mechanism of pepper fruit quality formation, especially color formation, and provide a theoretical and practical reference basis for achieving selective agronomic trait breeding of pepper fruits.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the differences in CaRAP2-12 expression in different tissues of pepper fruit at different developmental stages;

FIG2 is a diagram showing the subcellular localization results of RAP2-12-GFP;

FIG3 is a graph showing changes in total carotenoid content and carotenoid synthesis-related genes in fruits with CaRAP2-12 gene silencing;

Figure 4 shows the changes in total carotenoid content and carotenoid synthesis-related genes in fruits overexpressing the CaRAP2-12 gene.

DETAILED DESCRIPTION

The present invention provides a CaRAP2-12 gene for regulating the synthesis of carotenoids in pepper fruit. The nucleotide sequence of the CaRAP2-12 gene is shown in SEQ ID No.1: ATGTGTGGTGGTTCAATAATCTCCGACTACATAGCTCCTAGCCGGACTTCTCGCCGACTTACCGCTGAGTTGCTATGGGGCCGCGCCGATCTGAGTAAAAAGCTCAAAAATCTTAGCAATTATCACTCTAAGCCCTTGCGATCTGAAGTAGTTGATGACTTCGAGGCTGATTTTCAGGACTTCAAAGAATTCTCTTATGATGAAGATGACGTTGAAGTCGATGTCAAGTCATTTACTTTCTCTGCTCCCAAAAAATCTACTGGCTCTAAATCTGTGAAAGCTGTTG ATTCAGACAACGATGCTGCTGATAGATCCTCTAAGAGAAAGAGGAAGAATCAGTATAGGGGGATCAGACAGAGACCTTGGGGTAAGTGGGCAGCTGAAATACGTGACCCAAGGAAAGGGGTTCGGGTCTGGCTGGGAACCTTCAATACTGCAGAAGAAGCTGCCAGAGCTTATGATGTTGAGGCTAGGAGGATCAGAGGCAATAAAGCTAAGGTAAACTTCCCTGATGAAGCTCCAGCGCCTGCATCCAGACACTCT GTTAAGCTGAATCCTCAGAATGGCCTTCCTAAGGAG AGCCTGGACTCGGTTCCGTCTGACTCAACTATCATGAACAGTTTGGGGGATGGTTATTATGATTCTTTGGGATTCCCTGAAGAGAAACCCCATGACAAAGCAGTATGCATATGGAAATGGGAGCAGTGCTTCTGCAGATACAGGTTTTGGCTCATTCACCCCTTCAGTTGGTGCTTCAGCTGGTGCTGATATCTACTTCAACTCTGATGTAGGAAGCAACTCTTTTGAGTGCTCTGATTTTGGTTGGGGGAGAGCCAT GCTCCAGGACTCCGGAGATATCATCAGTTCTGTCTGC TGCTGTTGAAAGTAATGAAGCTCAATTTGTTGTAGACGCCAATCCCCAGAAAAAAACTGAAATCATGCACCAACAATCCTGTAGCTGATGACGGAAACACCGTGAACAGGCTATCCGAAGAGCTCTCAGCTTATGAACCCCAGATGAATTTCTTTAATCTCCCATATATGGAGGGAAATTGGGATGCATCAGTTGATGCCTTCCTCAACACAAGTGCAACTCAGAATGGTGAAAATGCTATGGACCTATGGTCGTTTGA TGATGTTCCTTCTTTAATGGGGGGTGTCTTTTAA.

The CaRAP2-12 gene described in the present invention is a key transcription factor that regulates the synthesis of carotenoids in pepper fruits. The CaRAP2-12 gene described in the embodiment is preferably amplified from the Yunnan pepper variety Dianjiao No. 13 (registration number GPD Pepper (2022) 530217).

The present invention also provides a primer pair for amplifying the CaRAP2-12 gene, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID No.3 and a downstream primer as shown in SEQ ID No.4.

The upstream primer of the present invention is: 5'-AGCAAGCCAAATTGCTTACAA-3', and the downstream primer is: 5'-TCTTCCACCACCACACACAC-3'.

The present invention also provides a protein encoded by the CaRAP2-12 gene, wherein the amino acid sequence of the protein encoded is as shown in SEQ ID Shown in No.2: MCGGSIISDYIAPSRTSRRLTAELLWGRADLSKKLKNLSNYHSKPLRSEVVDDFEADFQDFKEFSYDEDDVEVDVKSFTFSAPKKSTGSKSVKAVDSDNDAADRSSKRKRKNQYRGIRQRPWGKWAAEIRDPRKGVRVWLGTFNTAEEAARAYDVEARRIRGNKAKVNFPDEAPAPASRHSVKLNPQNGLPKESLDSVPSD STIMNSLGDGYYDSLGFPEEKPMTKQYAYGNGSSADTGFGSFTPSVGASAGADIYFNSDVGSNSFECSDFGWGEPCSRTPEISSVLSAAVESNEAQFVVDANPQKKLKSCTNNPVADDGNTVNRLSEELSAYEPQMNFFNLPYMEGNWDASVDAFLNTSATQNGENAMDLWSFDDVPSLMGGVF.

The present invention also provides the use of the CaRAP2-12 gene or the encoded protein in regulating the synthesis of carotenoids.

In the embodiment of the present invention, the CaRAP2-12 gene in pepper fruit is silenced by VIGS, and the total carotene content in the silenced sample is significantly reduced, among which the expression level of the CaCRTZ1 gene in pepper fruit is significantly upregulated, and other carotenoid biosynthesis structural genes are significantly downregulated; the CaRAP2-12 gene in pepper fruit is overexpressed, and the expression levels of the carotenoid biosynthesis structural genes CaPSY2, CaPSY3, CaPDS, CaZDS, CaCrtISO1, CaCrtISO2, CaLYCB1, CaLYCB2, CaCRTZ1 and CaCRTZ2 are all upregulated to varying degrees, and the differences are significant, while the expression levels of CaVDE and CaZEP are significantly decreased, while there is no significant difference in CaPSY1, which proves that the CaRAP2-12 protein and its encoding gene are related to the regulation of the synthesis and metabolism of carotenoid compounds in pepper fruit, can significantly improve the quality formation of pepper fruit, and the CaRAP2-12 gene can be used to regulate the synthesis of carotenoids in pepper fruit and the formation of pepper fruit quality. The carotenoids of the present invention preferably include β-carotene, zeaxanthin and capsanthin.

The present invention also provides the use of the CaRAP2-12 gene or the encoded protein in improving the quality of pepper fruit.

The expression pattern of the CaRAP2-12 gene of the present invention is highly correlated with the synthesis of carotenoids in pepper fruits. Transient overexpression of the gene in pepper fruits can promote the accumulation of carotenoid synthesis in plants, and the expression of carotenoid synthesis-related genes is also significantly increased; inhibiting the expression of CaRAP2-12 by VIGS significantly reduces the content of carotenoid-related compounds in pepper fruits, and the expression of carotenoid synthesis-related genes is also significantly reduced. The cloning of the gene of the present invention will not only help to analyze the regulatory mechanism of pepper fruit carotenoids, but also help to cultivate pepper varieties with higher carotenoid content and improve the quality of pepper fruits, and has great application value.

The present invention also provides the use of the CaRAP2-12 gene or the encoded protein in creating new pepper germplasm.

The present invention also provides a method for regulating the carotenoid content in pepper fruits, comprising regulating the expression level of the CaRAP2-12 gene in the pepper fruits.

The regulation of the present invention preferably includes any one of inhibition, knockdown and overexpression. The present invention does not specifically limit the method of the regulation, and it can be carried out using conventional genetic engineering methods in the art.

The invention provides a recombinant plasmid containing the CaRAP2-12 gene.

The recombinant plasmid of the present invention preferably comprises connecting the insert fragment of the CaRAP2-12 gene to the NcoI site of the pCambia1301 vector to construct an overexpression vector.

The present invention provides a transgenic engineering bacterium used for regulating the expression amount of the CaRAP2-12 gene and comprising the recombinant plasmid.

To further illustrate the present invention, the CaRAP2-12 gene provided by the present invention and its application in regulating carotenoid synthesis in pepper fruit are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

Example 1

1. Cloning and sequence analysis of CaRAP2-12 gene

Yunnan pepper variety Dianjiao No. 13 was planted in the Yunnan-Taiwan Center of Yunnan Agricultural University in Panlong District, Kunming, Yunnan Province, and the mature fruits were used for RNA extraction. The total RNA was extracted using a rapid universal plant RNA extraction kit (Beijing Huayueyang) according to the instructions, and the RNA content and quality were detected by a spectrophotometer.

use Ⅲ1stStrandcDNASynthesisSuperMix reverse transcription kit was used to synthesize the first strand cDNA. After optimization, an appropriate amount of reverse transcription product was used for subsequent PCR. The first strand of cDNA was used as a template for RT-PCR and PCR was performed by conventional methods to amplify the CaRAP2-12 gene. The upstream primer and the downstream primer were SEQ ID No.3 and SEQ ID No.4, respectively.

The 25 μL PCR reaction system was: MegaFi ^TM Fidelity 2X PCR Master Mix 12.5 μL, upstream and downstream primers 1 μL each, template 1 μL, ddH ₂ O 9.5 μL. Reaction program: 98°C 2 min; 98°C 10 sec, 57°C 30 sec, 72°C 57 sec, 35 cycles.

The PCR product CaRAP2-12 gene was purified and recovered, and then connected to the pClone007 Versatile SimpleVector vector (Qingke Biotechnology, China) to obtain a recombinant plasmid, which was transformed into Escherichia coli competent cells DH5a and sent to Shanghai Biotechnology Company for sequencing. The nucleotide sequence of the obtained CaRAP2-12 gene is shown in SEQ ID No.1, and the amino acid sequence of the encoded protein is shown in SEQ ID No.2.

2. Expression analysis of CaRAP2-12 gene in different tissues of pepper fruit at different developmental stages in different parts of pepper fruit

The improved variety of Yunnan pepper Dianjiao No. 13 was planted in the planting greenhouse of Yunnan Agricultural University Diantai Center in Panlong District, Kunming City, Yunnan Province. Three different tissues at six different stages of pepper fruit development were used for total RNA extraction. Flowers were used as controls, and qRT-PCR was used to determine the relative expression levels of the CaRAP2-12 gene in different parts (seeds, placenta and pericarp) at different developmental stages.

Primers used for qRT-PCR:

CaRAP2-12-F (SEQ ID No. 37): AGCAAGCCAAATTGCTTACAA;

CaRAP2-12-R (SEQ ID No. 38): TCTTCCACCACCACACACAC;

Ubi-3-F (SEQ ID No. 39): TTGGCAAGCAACAATCAT;

Ubi-3-F (SEQ ID No. 40): GCAGATGCAGCAGGAC.

The results of the differential expression of the CaRAP2-12 gene in different tissues at different developmental stages of pepper fruit are shown in Figure 1. CaRAP2-12 is expressed in the seeds, placenta and pericarp of pepper fruit, but the expression levels are significantly different. The expression level of CaRAP2-12 in different developmental stages of seeds first increases and then decreases, with the lowest level at 40DPA and the highest level at 10DPA, but there is an abnormal increase at 50DPA; the expression level of CaRAP2-12 in different developmental stages of the placenta first increases, then decreases, and then increases, with the highest level at 20DPA and the lowest level at 50DPA; the expression level of CaRAP2-12 in different developmental stages of the pericarp first decreases and then increases, with the highest level at 60DPA and the lowest level at 20DPA. The expression level of CaRAP2-12 in the S01 placenta from 10DPA to 20DPA is higher, and that in the pericarp is lower; the expression level of CaRAP2-12 in the pericarp is higher from 30DPA to 60DPA.

3. Subcellular localization of CaRAP2-12 gene

The 1300-35S-GFP plant transient fluorescence expression vector was linearized using XbaI and BamHI restriction endonucleases, and the CaRAP2-12 ORF cloning primers Forward primer and Reverse primer with 1300-35S-GFP vector homology arms were designed to amplify the CaRAP2-12 gene CDS region as an insert fragment. The insert fragment and the linearized vector were recombined into the pCaRAP2-12-GFP recombinant vector using the In-FusionTM HD Cloning kit recombination kit according to the instructions.

Forwardprimer (SEQ ID No. 41): CTTGCTCACCATGTCGACTCTAGATAAATATCCTCCTCCTAGAGCTGG;

Reverseprimer (SEQ ID No. 42): GGAGAGAACACGGGGGACGAGCTCATGGTGGGCCAATTACTGTACGT.

The pCaRAP2-12-GFP recombinant vector and the empty vector 1300-35S-GFP were transformed into Escherichia coli, and after coating the resistant plate, the positive bacteria were selected and cultured at 37°C overnight and the plasmids were extracted. The pCaRAP2-12-GFP and the empty vector 1300-35S-GFP were co-transformed with the nuclear marker into Arabidopsis protoplasts, cultured under weak light for 8-10 hours, and the subcellular localization of CaRAP2-12 was observed under a laser confocal microscope.

The subcellular localization results of the CaRAP2-12 gene in Arabidopsis protoplasts are shown in Figure 2. CaRAP2-12 has obvious transcription factor nuclear localization signals in Arabidopsis protoplasts, which indicates that CaRAP2-12 may regulate the transcriptional expression of related genes in the cell nucleus.

4. Functional verification of CaRAP2-12 gene in pepper fruit

(1) VIGS silencing of CaRAP2-12 genes in pepper fruit

Based on the SGNVIGSTool online gene silencing tool (https://vigs.solgenomics.net/), the pepper genome database was selected to screen the silenced fragments, and the purified amplified product of pepper CaRAP2-12 was used as a template. The primers CaRAP2-12-VIGS (upstream primer, SEQ ID No.5: 5'-TAAGGTTACCGAATTCCCTGAAGAGAAACCCATGAC-3', downstream primer, SEQ ID No.6: 5'-GCTCGGTACCGGATCACAGGATTGTTGGTGCATG-3') were used to clone the insert fragment. The procedure and system were the same as before (annealing temperature was 65°C, and extension time was 10 sec). The purified CaRAP2-12 gene silencing fragment was recombined into the pTRV2 plasmid using the In-Fusion ^TM HDCloningkit recombination kit, and the recombinant plasmid was transformed into DH5α Escherichia coli. Finally, the positive colonies were screened and the plasmid was extracted and transferred into GV3101 Agrobacterium.

Prepare a solution containing 10mM MES, 10mM MgCl ₂ and 150μM AS, adjust the pH to 5.6, and use it as an infection solution after sterilization. Collect Agrobacterium by centrifugation at 4000rpm for 10min, pour off the supernatant, resuspend with the prepared infection solution, use the infection solution as a control, and finally adjust the OD ₆₀₀ of each strain suspension to 0.8A, and let the resuspension stand in a 28℃ shaker in the dark for 2-3h. Prepare GV3101pTRV1 and pTRV2::CaRAP2-12, pTRV2::00 inoculation solution in a 1:1 ratio, and inject 0.5ml of the inoculation solution into the flesh of pepper fruit with a 1ml sterile needleless syringe. The inoculated pepper fruit is cultured at 18℃ for 48h in a dark environment with a relative humidity of 60%, and then placed in an incubator at 18℃ with a light cycle of 16h/dark cycle of 8h. After pepper fruits were cultured for 15 days, samples of silenced fruits were taken, and qRT-PCR was used to verify the fruit samples with successful silencing.

Table 1 qRT-PCR primer list

The results showed (Figure 3) that after inoculation, pepper fruits injected with Agrobacterium containing the pTRV2::CaRAP2-12 plasmid showed obvious green color near the injection hole compared with the control. CaRAP2-12 silenced fruits were screened using qRT-PCR, and the CaRAP2-12 gene was downregulated (Figure 3b). The carotenoid content in the silenced positive fruit tissue was determined (Figure 3d), and the results showed that the total carotene content in the silenced sample was significantly reduced compared with pTRV2::00. When the CaRAP2-12 gene was silenced, the expression of the CaCRTZ1 gene in pepper fruits was significantly upregulated (P<0.05) compared with pTRV2::00, and other carotenoid biosynthesis structural genes were significantly downregulated (P<0.05) (Figure 3c).

(2) Overexpression of the CaRAP2-12 gene in pepper fruit

Based on the In-Fusion Cloning Primer Design Tool online gene primer design tool, seamless cloning primers with 15bppCambia1301 plasmid homology arms were designed for both ends of the CaRAP2-12 gene ORF (upstream primer SEQ ID No. 35: 5'-GGACTCTTGACCATGCCCCTTTGCTGAAGAAAAAACTTT-3', downstream primer SEQ ID No. 36: 5'-CTCAGATCTACCATGAGGAAGGCATGCCGACTTAAAAGA-3'), and the CaRAP2-12 gene sequence was used as a template for cloning and purification to obtain the inserted fragment.

The CaRAP2-12 gene insert was connected to the NcoI site of the pCambia1301 vector using a homologous recombination kit to construct an overexpression vector. The purified CaRAP2-12 gene overexpression fragment was recombined into the pCambia1301 plasmid using the In-Fusion ^TM HDCloningkit recombination kit, and the recombinant plasmid was transformed into DH5α Escherichia coli. Finally, the positive colonies were screened and the plasmid was extracted and transferred into GV3101 Agrobacterium. The infection solution was prepared in the same way as the silencing method, and the inoculum was injected into pepper fruits after obtaining it. After 10 days, the fruits were sampled, and the positive samples with successful overexpression were verified by qRT-PCR.

The CaRAP2-12 gene was overexpressed in the detached pepper fruit, and the tissue near the injection hole was taken for GUS staining 10 days after inoculation. The results showed (Figure 4a) that compared with the control fruit, the pepper fruit tissue injected with Agrobacterium containing pCambia1301::00 and pCambia1301::CaRAP2-12 plasmids showed obvious blue after staining, and the pCambia1301::CaRAP2-12 fruit tissue was slightly lighter than pCambia1301::00. qRT-PCR was used to screen the CaRAP2-12 overexpression fruit tissue, and it was found that the expression level of the CaRAP2-12 gene in the overexpression fruit tissue was significantly higher than that in the pCambia1301::00 fruit tissue, indicating that the CaRAP2-12 gene was successfully overexpressed (Figure 4b).

In the transient overexpression samples, the total carotenoid content of pepper fruit increased, but there was no significant difference (P>0.05) (Figure 4d). Compared with the control, the expression levels of carotenoid biosynthesis structural genes CaPSY2, CaPSY3, CaPDS, CaZDS, CaCrtISO1, CaCrtISO2, CaLYCB1, CaLYCB2, CaCRTZ1 and CaCRTZ2 were upregulated to varying degrees, and the differences were significant (P<0.05), while the expression levels of CaVDE and CaZEP were significantly decreased (P<0.05), while there was no significant difference in CaPSY1.

In summary, the CaRAP2-12 protein and its encoding gene are related to the regulation of the synthesis and metabolism of carotenoid compounds in pepper fruit, and can significantly improve the quality formation of pepper fruit. The CaRAP2-12 gene can be used to regulate the synthesis of carotenoids in pepper fruit and the formation of pepper fruit quality.

Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims (3)

1. The application of over-expression CaRAP-12 genes or encoded proteins in improving the synthesis of pepper carotenoids is characterized in that the nucleotide sequence of CaRAP-12 genes is shown as SEQ ID No. 1; the amino acid sequence of the coding protein of CaRAP-12 genes is shown as SEQ ID No. 2.

2. The use according to claim 1, wherein the carotenoids comprise β -carotene, zeaxanthin and capsanthin.

3. A method for improving carotenoid content in pepper fruits is characterized by comprising the step of over-expressing CaRAP-12 genes in pepper fruits, wherein the nucleotide sequence of the CaRAP-12 genes is shown as SEQ ID No. 1.