CaNAC02 gene for regulating carotenoid synthesis in pepper fruits and application thereof

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a CaNAC02 gene that regulates carotenoid synthesis in chili pepper fruits and its applications. The amino acid sequence of the protein encoded by the CaNAC02 gene is shown in SEQ ID NO.1. The CaNAC02 gene of this invention promotes the synthesis of carotenoid compounds in chili pepper fruits, influencing fruit quality formation. The CaNAC02 gene of this invention is an NAC family transcription factor, which can provide a target gene for the breeding of new chili pepper varieties, and simultaneously provide genetic material and theoretical basis for the genetic improvement of chili pepper varieties.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a CaNAC02 gene that regulates the synthesis of carotenoids in pepper fruits and its applications. Background Technology

[0002] Chili peppers are annual or short-lived perennial herbaceous plants belonging to the Solanaceae family and the Capsicum genus. They comprise several species, with five currently cultivated varieties: annual chili pepper (Capsicum annuum L.), Chinese chili pepper (C. chinense Jacquin), drooping chili pepper (C. baccatum L.), hairy chili pepper (C. pubescens Keep), and shrub chili pepper (C. frutescens L.). In my country, annual chili peppers are the main crop, with smaller quantities of Chinese chili peppers and shrub chili peppers. As one of my country's important vegetable crops, chili peppers can be eaten fresh or processed into condiments such as chili oil, chili sauce, and pickled chili peppers. They have effects such as promoting metabolism, treating arthritis, and removing dampness, and can prevent the occurrence of various diseases, thus being widely used in the medical field. In addition, chili peppers also contain various nutrients such as vitamins, minerals, and soluble sugars, as well as natural pigments such as carotenoids and capsaicin.

[0003] Carotenoids are a class of natural pigments widely found in nature. First isolated in the early 19th century, nearly 800 natural carotenoids have been discovered to date, including more than 40 found in chili peppers. Carotenoids are isopentenyl polymers containing 40 or 30 carbon atoms, commonly found in the leaves, flowers, and fruits of plants. In some bacteria, fungi, and algae, they also exist at C45 and C50. In nature, carotenoids are mainly divided into two categories: one contains only carbon and hydrogen elements, without oxygen, such as α-carotene, β-carotene, γ-carotene, capsanthin, β-zeaxanthin, and lycopene. The other category consists of its oxidized derivatives, which contain at least one oxygen functional group, such as hydroxyl, ketone, epoxy, or methoxy groups, including lutein, zeaxanthin, astaxanthin, zeaxanthin, and fucoxanthin. These are all secondary metabolites produced by plants during photosynthesis. As a class of fat-soluble terpenoids synthesized via the isoprene pathway, carotenoids contain multiple conjugated double bonds. Due to the different positions and numbers of these conjugated double bonds, the absorbance of different types of carotenoids varies. Different parts of the plant exhibit colors ranging from colorless to yellow, orange, and red. The more conjugated double bonds a carotenoid has, the more red it tends to be.

[0004] The synthesis of carotenoids in plants is a complex process regulated by multiple factors. Key genes or enzymes involved in carotenoid synthesis have been identified in various plants, including tomatoes, potatoes, peppers, and barley. Studies have found that these genes directly or indirectly regulate carotenoid synthesis and are closely related to plant color traits. Among them, NAC (NAM, ATAF1 / 2, and CUC1 / 2) transcription factors, as one of the largest transcription factor families in plants, play an important role in plant growth and development, involving the regulation of multiple metabolic processes. They are involved not only in biological processes such as seed germination, lateral root development, shoot apical meristem formation, floral organ formation, as well as cell expansion, fiber development, secondary cell wall synthesis, leaf senescence, fruit ripening, and hormone synthesis, but also in mediating plant responses to abiotic and biotic stresses. To date, many NAC family transcription factors have been discovered in other horticultural plants, but research on their involvement in regulating carotenoid synthesis in pepper fruits has not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to provide a CaNAC02 gene that regulates the synthesis of carotenoids in chili pepper fruits and its application. The CaNAC02 gene promotes the synthesis of carotenoid compounds in chili pepper fruits, which is of great significance for breeding new chili pepper varieties with high carotenoid content, artificially regulating carotenoid biosynthesis, and improving the quality of chili pepper fruits.

[0006] This invention provides a CaNAC02 gene that regulates the synthesis of carotenoids in chili pepper fruits, and the amino acid sequence of the protein encoded by the CaNAC02 gene is shown in SEQ ID NO.1.

[0007] As a preferred embodiment, the nucleotide sequence of the CaNAC02 gene is shown in SEQ ID NO.2.

[0008] This invention provides a primer pair for amplifying the CaNAC02 gene, comprising an upstream primer with nucleotide sequences as shown in SEQ ID NO. 3 and a downstream primer with SEQ ID NO. 4.

[0009] This invention provides a recombinant plasmid for regulating carotenoid synthesis in pepper fruits, wherein the recombinant plasmid contains the aforementioned CaNAC02 gene.

[0010] As a preferred embodiment, the basic backbone vector of the recombinant plasmid includes pCambia1301.

[0011] This invention provides a recombinant engineered bacterium that regulates the synthesis of carotenoids in pepper fruits, wherein the recombinant engineered bacterium contains the CaNAC02 gene or a recombinant plasmid thereof.

[0012] This invention provides the application of the CaNAC02 gene, the recombinant plasmid, or the recombinant engineered bacteria in regulating carotenoid synthesis.

[0013] As a preferred embodiment, the carotenoids include β-carotene and / or zeaxanthin.

[0014] This invention provides the application of the CaNAC02 gene, the recombinant plasmid, or the recombinant engineered bacteria described above in improving the quality of pepper fruits.

[0015] This invention provides the application of the CaNAC02 gene, the recombinant plasmid, or the recombinant engineered bacteria described above in the creation of new chili germplasm.

[0016] Beneficial Effects: This invention provides a CaNAC02 gene that regulates carotenoid synthesis in chili pepper fruits. The amino acid sequence of the protein encoded by the CaNAC02 gene is shown in SEQ ID NO.1. The CaNAC02 gene of this invention promotes the synthesis of carotenoid compounds in chili pepper fruits and influences the formation of fruit quality. The CaNAC02 gene of this invention is an NAC family transcription factor, which can provide a target gene for the breeding of new chili pepper varieties and provide genetic material and theoretical basis for the genetic improvement of chili pepper varieties.

[0017] This invention provides a recombinant plasmid for regulating carotenoid synthesis in pepper fruits, wherein the recombinant plasmid contains the aforementioned CaNAC02 gene. This invention also provides a recombinant engineered bacterium for regulating carotenoid synthesis in pepper fruits, wherein the recombinant engineered bacterium contains the aforementioned CaNAC02 gene or a recombinant plasmid thereof. This invention, by constructing a recombinant plasmid containing the CaNAC02 gene and a transgenic engineered bacterium, provides construction materials for creating transgenic plants that regulate carotenoid synthesis in pepper fruits.

[0018] This invention provides the application of the CaNAC02 gene, the recombinant plasmid, or the recombinant engineered bacteria in regulating carotenoid synthesis. In the embodiments of this invention, it was verified that transgenic plants with suppressed CaNAC02 gene expression showed reduced carotenoid synthesis and significantly downregulated expression of related genes; while transgenic plants with overexpression of the CaNAC02 gene showed increased carotenoid synthesis and significantly upregulated expression of related genes.

[0019] This invention provides the application of the aforementioned CaNAC02 gene, recombinant plasmid, or recombinant engineered bacteria in improving the quality of chili pepper fruits. This invention also provides the application of the aforementioned CaNAC02 gene, recombinant plasmid, or recombinant engineered bacteria in creating new chili pepper germplasm. Using the CaNAC02 gene described in this invention, the regulatory mechanism of carotenoid accumulation in chili pepper fruits can be explored, enriching the understanding of secondary metabolism in chili pepper fruits. This is of great significance for future breeding of new chili pepper varieties with high carotenoid content, artificial regulation of carotenoid biosynthesis, and improvement of chili pepper fruit quality. Attached Figure Description

[0020] Figure 1 The graph shows the differences in CaNAC02 gene expression in different tissues at different developmental stages of pepper fruit, where a represents different developmental stages of pepper fruit and b represents the relative expression level.

[0021] Figure 2 The image shows the subcellular localization results of NAC02-GFP, where GFP is green fluorescent protein, RFP is red fluorescent protein, Chlorophyll fluorescence is chloroplast fluorescence, BrightField is bright field, and Merged is superimposed field.

[0022] Figure 3 Figure 1 shows the identification of fruits with the CaNAC02 gene silenced and the changes in β-carotene and zeaxanthin content. Figure 2 shows the phenotype of pepper fruits after silencing the gene, from left to right: recipient material, Agrobacterium tumefaciens injected with the empty vector pTRV2::00, and Agrobacterium tumefaciens injected with the pTRV2::CaNAC02 plasmid; Figure 3 shows the expression level of CaNAC02, with the left figure showing the expression level of CaNAC02 with the empty vector and the right figure showing the expression level of CaNAC02 with the silenced gene material; Figure 4 shows the β-carotene content, with the left figure showing the β-carotene content of the injected empty vector plasmid and the right figure showing the β-carotene content of the injected silenced plasmid pTRV2::CaNAC02; Figure 5 shows the zeaxanthin content, with the left figure showing the zeaxanthin content of the injected empty vector plasmid and the right figure showing the zeaxanthin content of the injected silenced plasmid pTRV2::CaNAC02.

[0023] Figure 4 A graph showing the differential expression of carotenoid synthesis-related genes in fruits where the CaNAC02 gene is silenced;

[0024] Figure 5This image shows the identification of fruits with transient overexpression of the CaNAC02 gene and the changes in β-carotene and zeaxanthin content. Image a shows GUS staining, from left to right: recipient material (unstained), injected with empty plasmid pCambia1301::00 (stained), and injected with Agrobacterium plasmid pCambia1301::CaNAC02 (stained). Image b shows the expression level of the CaNAC02 gene; the left image shows the CaNAC02 expression level with pCambia1301::00 (empty vector), and the right image shows the CaNAC02 gene expression level. The expression level of the overexpression material CaNAC02; c represents the content of β-carotene, the left figure shows the content of β-carotene after injection of pCambia1301::00 (empty vector) plasmid, and the right figure shows the content of β-carotene after injection of the overexpression plasmid pCambia1301::CaNAC02; d represents the content of zeaxanthin, the left figure shows the content of zeaxanthin after injection of pCambia1301::00 (empty vector) plasmid, and the right figure shows the content of zeaxanthin after injection of the overexpression plasmid pCambia1301::CaNAC02.

[0025] Figure 6 This is a graph showing the differential expression of carotenoid synthesis-related genes in fruits with transient overexpression of the CaNAC02 gene. Detailed Implementation

[0026] This invention provides a CaNAC02 gene that regulates carotenoid synthesis in chili pepper fruits. The amino acid sequence of the protein encoded by the CaNAC02 gene is shown in SEQ ID NO.1:

[0027] MGAVELRLPAGFRFHPTDEELVTHYLCRKCASQPIAVPIIAEIDLYKYNPWDLPDLALYGEKEWYFFSPRDRKYPNGSRPNRAAGNGYWKATGADKAIGRPKPMGIKKALVFYAGKAPKGEKTNWIMHEYRLAHVDRSARNKNNSL RLDDWVLCRIYNKKSTIEKSQLNSRKMNVAMSPVDIKPKIMAFPVSTRPSVTSQQVNNDFIYFDSSDSLPKLHTDSSCSEHVLSPEFTCEREVQSEPKVSEWEKNALDFPFNYSDDATTTMGELESCYEMSPLQDIFMYLQKPF*.

[0028] In a preferred embodiment, the nucleotide sequence of the CaNAC02 gene is shown in SEQ ID NO.2:

[0029] 5’-ATGGGAGCAGTGGAATTGCGATTGCCCGCTGGATTTCGATTTCACC CGACTGATGAAGAGCTTGTGACGCACTATTTATGTCGAAAATGCGCGTCACAGCCTATTGCTGTTCCTATTATAGCTGAAATTGACTTGTACAAGTACAATCCATGGGATCTTCCTGATTTGGCGTTGTATGGGGAGAAAGAGTGGTATTTCTTTTCACCGCGAGATCGGAAGTATCCGAATGGTTCACGGCCGAATCGAGCTGCCGGAAATGGGTACTGGAAGGCGACGGGTGCCGATAAGGCAATTGGTCGTCCAAAGCCAATGGGAATTAAGAAAGCTTTGGTATTTTACGCAGGGAAAGCTCCCAAAGGAGAAAAAACCAATTGGATTATGCACGAGTATCGGCTTGCTCACGTTGATCGATCTGCTCGTAACAAGAACAATAGCTTAAGGCTGGATGATTGGGTATTATGCCGAATATACAATAAGAAGAGTACAATTGAGAAGAGCCAATTGAATAGTCGAAAAATGAATGTTGCAATGTCACCCGTGGATATTAAACCAAAAATTATGGCATTTCCGGTATCGACAAGGCCATCGGTGACGTCTCAGCAAGTTAACAATGACTTCATCTACTTCGATTCATCGGATTCTCTCCCAAAACTTCATACTGATTCCAGCTGCTCCGAGCACGTGCTTTCACCGGAGTTCACGTGCGAGAGGGAAGTTCAGAGTGAGCCGAAAGTGAGTGAGTGGGAGAAAAACGCCCTTGATTTTCCTTTTAATTACAGTGATGATGCCACGACGACGATGGGGGAGTTGGAGAGCTGTTATGAGATGTCGCCGCTTCAAGATATATTCATGTATCTACAAAAGCCATTTTGA-3’。

[0030] In a preferred embodiment, the CaNAC02 gene described in this invention is a key transcription factor regulating carotenoid synthesis in chili pepper fruits. In one embodiment, the CaNAC02 gene is amplified from Yunnan chili pepper variety Dianjiao 13 [GPD chili pepper (2022) 530217] (Capsicum annuum L.).

[0031] This invention also provides a primer pair for amplifying the CaNAC02 gene, comprising an upstream primer with the nucleotide sequence shown in SEQ ID NO. 3 and a downstream primer shown in SEQ ID NO. 4. The upstream primer (as shown in SEQ ID NO. 3) is 5'-GCGCCTCCAATACACTTCAC-3', and the downstream primer (as shown in SEQ ID NO. 4) is 5'-GACCAACCCACTCTTTCTCATC-3'.

[0032] This invention provides a recombinant plasmid for regulating carotenoid synthesis in pepper fruits, wherein the recombinant plasmid contains the aforementioned CaNAC02 gene. In a preferred embodiment, the basic backbone vector of the recombinant plasmid includes pCambia1301. In one embodiment, the present invention can ligate the insert fragment of the CaNAC02 gene to the NcoI site of the pCambia1301 vector to construct an overexpression vector, and further construct the recombinant plasmid.

[0033] This invention provides a recombinant engineered bacterium that regulates the synthesis of carotenoids in pepper fruits, wherein the recombinant engineered bacterium contains the CaNAC02 gene or a recombinant plasmid thereof.

[0034] This invention provides the application of the CaNAC02 gene, the recombinant plasmid, or the recombinant engineered bacteria in regulating carotenoid synthesis. In a preferred embodiment, the carotenoids may include β-carotene and / or zeaxanthin. In this embodiment, silencing the CaNAC02 gene in pepper fruits using VIGS significantly reduced the content of zeaxanthin and β-carotene in the silenced samples, and significantly downregulated the expression of some carotenoid biosynthesis structural genes. Overexpression of the CaNAC02 gene in pepper fruits significantly increased the content of zeaxanthin and β-carotene in the overexpression samples, and significantly upregulated the expression of some carotenoid biosynthesis structural genes. This demonstrates that the CaNAC02 gene and its encoded protein are related to the regulation of carotenoid synthesis metabolism in pepper fruits, and can significantly improve the synthesis of carotenoid compounds in pepper fruits. The CaNAC02 gene can be applied to regulate carotenoid synthesis and the formation of pepper fruit quality.

[0035] This invention provides the application of the aforementioned CaNAC02 gene, recombinant plasmid, or recombinant engineered bacteria in improving the quality of chili pepper fruits. The expression pattern of the CaNAC02 gene described in this invention is highly correlated with carotenoid synthesis in chili pepper fruits. Transient overexpression of this gene in chili pepper fruits can promote the accumulation of carotenoids in plants, and the expression of carotenoid synthesis-related genes also increases significantly. Inhibiting the expression of the CaNAC02 gene using VIGS significantly reduces the content of carotenoid-related compounds in chili pepper fruits, and also significantly reduces the expression of carotenoid synthesis-related genes. Cloning the CaNAC02 gene described in this invention will not only help elucidate the regulatory mechanism of carotenoids in chili pepper fruits, but also contribute to the breeding of chili pepper varieties with higher carotenoid content, thereby improving the quality of chili pepper fruits and possessing significant application value.

[0036] This invention provides the application of the CaNAC02 gene, the recombinant plasmid, or the recombinant engineered bacteria described above in the creation of new chili pepper germplasm. The CaNAC02 gene described in this invention can provide a target gene for the breeding of new chili pepper varieties, and simultaneously provide genetic materials and theoretical basis for the genetic improvement of chili pepper varieties.

[0037] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of the CaNAC02 gene for regulating carotenoid synthesis in pepper fruits and its applications, should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] 1. Cloning and sequence structure analysis of the CaNAC02 gene

[0040] Yunnan chili pepper variety Dianjiao 13 [GPD chili pepper (2022) 530217] was cultivated in the planting greenhouse of Yunnan Agricultural University Diantai Center, Panlong District, Kunming City, Yunnan Province. Mature fruits were used for RNA extraction. 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 using a micro spectrophotometer.

[0041] use First-strand cDNA was synthesized using the 1st Strand cDNA Synthesis SuperMix reverse transcription kit. After optimization, an appropriate amount of the reverse transcription product was used for subsequent PCR. The CaNAC02 gene was amplified using the first-strand cDNA as an RT-PCR template via standard methods. The upstream and downstream primers were SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0042] The 25 μL PCR reaction system consisted of: 12.5 μL BlasTaq 2X PCR MasterMix, 1 μL each of forward and reverse primers, 1 μL template, and 9.5 μL ddH2O. The reaction program was: 95℃ for 3 min; 95℃ for 15 sec, 58.1℃ for 15 sec, 72℃ for 30 sec, for 35 cycles; 72℃ for 1 min.

[0043] After purification and recovery, the PCR product CaNAC02 gene was ligated into the pClone007 Versatile SimpleVector vector (Beijing Qingke Biotechnology Co., Ltd., China) to obtain the recombinant plasmid pMDTM19-T-CaNAC02. The plasmid was transformed into E. coli competent cells DH5α and sent to Sangon Biotech for sequencing. The nucleotide sequence of the CaNAC02 gene is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.1.

[0044] 2. Expression analysis of the CaNAC02 gene in different tissues of chili pepper fruit at different developmental stages and parts.

[0045] Yunnan pepper variety Dianjiao 13 was cultivated in a greenhouse at the Yunnan Agricultural University Taiwan Center in Panlong District, Kunming City, Yunnan Province. Artificial pollination was performed after the inflorescences unfolded, and the peppers were tagged and registered every ten days after pollination. Fruits of Dianjiao 13 at relatively concentric nodes were collected at seven post-pollination stages (10, 20, 30, 40, 50, 60, and 70 days). Samples of 0.5g, 0.1g, and 0.1g were taken from the pulp, placenta, and seeds, respectively. Ten fruits were randomly selected from each sample, with three replicates, for total RNA extraction. Primers for the CaNAC02 gene qRT-PCR were designed using Primer-blast online software: qRT-CaNAC02-F (as shown in SEQ ID NO. 5): 5'-GTATCGACAAGGCCATCGGT-3'; qRT-CaNAC02-R (as shown in SEQ ID NO. 6): 5'-CCACTCACTCACTTTCGGCT-3'. Using seeds harvested 0 days post-pollination as a control, the relative expression levels of the CaN AC02 gene in different parts (seeds, placenta, and pulp) at different developmental stages were determined by qRT-PCR. A relative quantification method was employed, with the ACTIN gene used as an internal control during amplification. ACTIN-F (as shown in SEQ ID NO.7): 5'-GCCTAGAAATTTGAGCCTCATT-3'; ACTIN-R (as shown in SEQ ID NO.8): 5'-AAGATGGTATTGTTGC TGATGG-3'. The relative expression levels were calculated using the 2-ΔΔCt method. Results are shown in [Figure number missing]. Figure 1 b.

[0046] The results of differential expression of the CaNACO2 gene in different tissues at different developmental stages of pepper fruit are as follows: Figure 1 As shown, the CaNAC02 gene is expressed in the seeds, placentas, and pericarps of pepper fruits, but the expression levels differ significantly. In the seeds, placentas, and pericarps of Dianjiao 13, the expression level of the CaNAC02 gene shows an increasing trend with developmental stage. Its expression level is significantly higher in the pericarps at 50 DPA (50 days after pollination, the color-changing stage), then remains relatively stable, reaching its highest level in the placentas at 70 DPA (70 days after pollination). Overall, the CaNAC02 gene has the highest expression level in the placentas of Dianjiao 13, followed by the pericarps, and the lowest expression level in the seeds.

[0047] 3. Subcellular localization of the CaNACO2 gene

[0048] The vector pC1300S-GFP (a commercially available plant transient fluorescent expression vector) was linearized using XbaI and BamHI restriction endonucleases. CaNAC02 ORF cloning primers with homologous arms of the pC1300S-GFP vector were designed to amplify the CDS coding region of the CaNAC02 gene as an insert. Specifically, pCaNAC02-1300S-GFP-F (as shown in SEQ ID NO. 9): 5'-GCTTTCGCGAGCTCGG TACCATGGGAGCAGTGGAATTGC-3'; pCaNAC02-1300S-GFP-R (as shown in SEQ ID NO. 10): 5'-CCCTTGCTCACCATGGATCCAAATGGCTTTTGTAGATACAT G-3'. The insert and linearized vector were recombined into the pCaNAC02-1300S recombinant vector using the In-Fusion™ HD Cloning kit according to the instructions.

[0049] The recombinant vector pCaNAC02-1300S-GFP and the empty vector pC1300S-GFP (commercially available product) were transformed into *E. coli*. After plating with antibiotic-resistant plates, positive bacteria were selected and cultured overnight at 37°C, and plasmids were extracted. The obtained recombinant plasmid pCaNAC02-1300S-GFP and the empty plasmid pC1300S-GFP were co-transformed into *Arabidopsis thaliana* protoplasts with nuclear markers, respectively. After culturing under low light for 10 h, the subcellular localization results of the CaNAC02 gene were observed under a laser confocal microscope. Figure 2 As shown.

[0050] from Figure 2It can be seen that the CaNAC02 gene has obvious transcription factor nuclear localization signals in Arabidopsis protoplasts, where GFP: green fluorescent protein fluorescence; RFP: red fluorescent protein fluorescence; Chlorophyll fluorescence: chloroplast fluorescence; BrightField: bright field; Merged: superposition field.

[0051] 4. Functional verification of the CaNAC02 gene in pepper fruit

[0052] (1) VIGS silencing the CaNAC02 gene in pepper fruit

[0053] Using the SGN VIGS Tool online gene silencing tool (https: / / vigs.solgenomics.net / ), the *C. annuum* zula v2 genome database was selected to screen for silencing fragments. The purified and amplified product of the *C. annuum* CaNAC02 gene was used as a template, and primers CaNAC02-VIGS were employed. The upstream primer (as shown in SEQ ID NO.11) was 5'-TAAGGTTACCGAATTACTTCCCTCTCGCACGTG-3', and the downstream primer (as shown in SEQ ID NO.12) was 5'-GCTCGGTACCGGATCTCGTAACAAGAACAATAGC TTAAGG-3'. The inserted fragment was cloned. The 25 μL PCR reaction system consisted of: 12.5 μL MegaFi™ Fidelity2XPCR MasterMix, 1 μL each of the upstream and downstream primers, 1 μL of the purified and amplified *C. annuum* CaNAC02 gene product, and 9.5 μL ddH2O. The PCR reaction program was as follows: 98℃ for 30 seconds; 98℃ for 10 seconds, 70.9℃ for 30 seconds, 72℃ for 10 seconds, 35 cycles; 72℃ for 2 minutes. The purified CaNAC02 gene silencing fragment was recombinated with the pTRV2 plasmid (commercially available product) using the In-Fusion™ HD Cloning kit to construct the silencing plasmid pTRV2::CaNAC02. The recombinant plasmid was then transformed into DH5α Escherichia coli, and after screening for positive bacteria, the plasmid was extracted and transformed into GV3101 Agrobacterium.

[0054] A solution containing 10 mM MES, 10 mM MgCl2, and 150 μM MAS was prepared, and the pH was adjusted to 5.6. After sterilization, this solution was used as the infection medium. Agrobacterium was collected by centrifugation at 4000 rpm for 10 min. The supernatant was discarded, and the bacteria were resuspended in the prepared infection medium. Using the infection medium as a control, the OD values ​​of the suspensions of each strain were finally adjusted. 600The concentration was 0.8A. The resuspension was incubated at 28℃ in the dark on a shaker for 3 hours. GV3101pTRV1 and pTRV2::CaNACO2, pTRV2::00 (empty) inoculum was prepared at a 1:1 v / v ratio. 0.5 mL of the inoculum was injected into the pulp of the pepper fruit using a 1 mL sterile needle-free syringe. The inoculated pepper fruits were cultured at 18℃ for 48 hours in the dark at 60% relative humidity, and then placed in an incubator at 18℃ with a 16-hour photoperiod and an 8-hour darkperiod. After 10 days of culture, samples were taken from the silenced fruits, and the phenotype of the silenced pepper fruits was photographed. The expression level of CaNACO2 was detected using quantitative real-time PCR. The contents of β-carotene and zeaxanthin were detected using liquid chromatography. The results are shown in [Figure number missing]. Figure 3 The primers in Table 1 were used to perform qRT-PCR to verify the successful silencing of fruit samples. The results are shown in [Table 1]. Figure 4 .

[0055] Table 1. qRT-PCR Primer List

[0056]

[0057]

[0058] from Figure 3 As can be seen, the results show ( Figure 3 (a) Ten days after inoculation, compared with the control, pepper fruits injected with Agrobacterium containing the pTRV2::CaNAC02 plasmid showed a distinct green color near the injection well. Fruits with CaNAC02 gene silencing were screened using qRT-PCR; downregulated CaNAC02 gene ( Figure 3 (b) Determination of carotenoid content in silent positive fruit tissues ( Figure 3 The results (d and e) showed that the contents of zeaxanthin and β-carotene were significantly reduced in the silent samples compared to pTRV2::00.

[0059] from Figure 4 It can be seen that when the CaNAC02 gene was silenced, compared with pTRV2::00, some carotenoid biosynthesis structural genes in pepper fruits were significantly downregulated (P<0.05), indicating that the carotenoid content decreased significantly.

[0060] (2) Overexpression of the CaNACO2 gene in pepper fruit

[0061] Seamless cloning primer pairs with homologous arms of the commercially available pCambia1301 plasmid (15bp) were designed at both ends of the CaNAC02 gene ORF using the In-Fusion Cloning Primer Design Tool online gene primer design software. The upstream primer (as shown in SEQ ID NO.41) was 5'-GGACTCTTGA CCATGATGGGAGCAGTGGAATTGCG-3', and the downstream primer (as shown in SEQ ID NO.42) was 5'-CTCAGATCTACCATGTCAAAATGGCTTTTGTAGATACATG-3'. The CaNAC02 gene sequence was used as a template for cloning and purification to obtain the insert fragment.

[0062] The CaNAC02 gene insert was ligated to the NcoI site of the pCambia1301 vector using a homologous recombination kit to construct an overexpression vector. The purified CaNAC02 gene overexpression fragment was then recombinated with the pCambia1301 plasmid using the In-Fusion™ HD Cloning kit to construct the overexpression recombinant plasmid pCambia1301::CaNAC02. This recombinant plasmid was transformed into DH5α *E. coli*, and after screening for positive colonies, the plasmid was extracted and transformed into *Agrobacterium GV3101*. Infection solution was prepared using the same silencing method, and the inoculum was injected into pepper fruits. Ten days later, samples were taken from the fruits, and tissue near the injection wells was stained with GUS. The expression level of CaNAC02 was detected using quantitative real-time PCR; the content of β-carotene and zeaxanthin was detected using liquid chromatography. The results are shown in the figure below. Figure 5 ; and the positive samples with successful overexpression were verified by qRT-PCR using the primers in Table 1. The results are shown in [Table 1]. Figure 6 .

[0063] from Figure 5 As can be seen, the results show ( Figure 5 (a) Compared to the control fruit, pepper fruit tissues injected with Agrobacterium containing pCambia1301::00 (empty vector) and pCambia1301::CaNAC02 plasmids were all stained, showing a distinct blue color. Screening of CaNAC02-overexpressing fruit tissues using qRT-PCR revealed that the CaNAC02 gene expression level in the overexpressing fruit tissues was significantly higher than that in the pCambia1301::00 fruit tissues, indicating successful CaNAC02 gene overexpression. Figure 5 (b) In the transient overexpression samples, the contents of zeaxanthin and β-carotene in pepper fruits were significantly increased (P>0.05). Figure 5 (c and d).

[0064] from Figure 6 It can be seen that, compared with the control, the expression levels of some carotenoid biosynthesis structural genes were upregulated to varying degrees, and the differences were significant (P<0.05).

[0065] In summary, the CaNAC02 gene is associated with the regulation of carotenoid synthesis and metabolism in pepper fruits, and can significantly increase the content of zeaxanthin and β-carotene in pepper fruits. The CaNAC02 gene can be applied to regulate carotenoid synthesis and the formation of pepper fruit quality.

[0066] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of the CaNAC02 gene, or a recombinant plasmid containing the CaNAC02 gene, or a recombinant engineered bacterium containing the CaNAC02 gene, in enhancing carotenoid synthesis in capsaicin, characterized in that... The amino acid sequence of the protein encoded by the CaNAC02 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The carotenoids are: β-carotene and / or zeaxanthin.

3. The application according to claim 1, characterized in that, The nucleotide sequence of the CaNAC02 gene is shown in SEQ ID NO.

2.

4. The application according to claim 1 or 3, characterized in that, The primer pair for amplifying the CaNAC02 gene includes 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.

5. The application according to claim 1, characterized in that, The basic scaffold vector for the recombinant plasmid includes pCambia1301.

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