Application of CaNAC100 gene in regulating synthesis of plant carotenoid

Abstract

The invention discloses application of a CaNAC100 gene in regulation of synthesis of carotenoids in plants, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the CaNAC100 gene is as shown in SEQ ID NO. 1; a protein sequence coded by the gene is as shown in SEQ ID NO. 2. The expression mode of CaNAC100 is highly related to synthesis of carotenoid in pepper fruits, and the gene can significantly promote synthesis of plant carotenoid. Cloning of the gene not only facilitates analysis of a regulation mechanism of carotenoids in pepper fruits, but also facilitates cultivation of pepper varieties with high carotenoid content and improvement of pepper fruit quality, and the gene has great application value.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to the application of CaNAC100 gene in regulating the synthesis of plant carotenoids. Background Art

[0002] 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.

[0003] 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 molecules of GGPP head-on to form phytoene; in the second step, lycopene is formed through dehydrogenase and isomerase; 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.

[0004] NAC 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 NAC family transcription factors. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the CaNAC100 gene in regulating the synthesis of plant carotenoids to solve the problems existing in the above-mentioned prior art. Genetic verification provides a target gene for the cultivation of new pepper varieties, and at the same time provides genetic materials and theoretical basis for the genetic improvement of pepper varieties.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is the use of the CaNAC100 gene in regulating the synthesis of plant carotenoids. The nucleotide sequence of the CaNAC100 gene is shown in SEQ ID NO.1.

[0008] The second technical solution of the present invention is the use of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the CaNAC100 gene in regulating the synthesis of carotenoids in pepper plants.

[0009] The third technical solution of the present invention is the use of the protein encoded by the CaNAC100 gene in regulating the synthesis of plant carotenoids, and its amino acid sequence is shown in SEQ ID NO.2.

[0010] A fourth technical solution of the present invention is a method for improving the quality of pepper fruit, which promotes the synthesis of carotenoids in pepper fruit by regulating the expression level of the CaNAC100 gene in pepper plants.

[0011] A fifth technical solution of the present invention is the use of the CaNAC100 gene in cultivating transgenic pepper plants with high carotenoid content.

[0012] The sixth technical solution of the present invention is a method for cultivating transgenic pepper plants with high carotenoid content, comprising the following steps: introducing the CaNAC100 gene into the target plant.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] The present invention cloned and verified for the first time the key transcription factor CaCaNAC100 that regulates the synthesis of carotenoids in pepper fruit. The transcription factor promotes the synthesis of carotenoid compounds in pepper fruit and affects the formation of pepper fruit quality. The present invention also provides a recombinant plasmid, a transgenic engineering bacterium and a transgenic plant containing the CaCaNAC100 gene. The present invention enriches the understanding of the secondary metabolism of pepper fruit and the genetic mechanism of pepper fruit quality formation, especially color formation. The present invention provides a theoretical and practical reference basis for realizing selective breeding of pepper fruit agronomic traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1This is a graph showing the expression of CaNAC100 in different tissues of pepper fruit at different developmental stages and the expression differences of CaNAC100 and carotenoid synthesis genes.

[0017] Figure 2 This is a diagram of the subcellular localization analysis of the CaNAC100 gene in tobacco leaves.

[0018] Figure 3 This is a comparison of the synthesis of carotenoid compounds in pepper fruits promoted by the CaNAC100 gene. Among them, a is the silenced fruit of different silencing systems, CK is the pepper fruit without inoculation, pTRV2::00 is the fruit inoculated with the pTRV2 empty vector strain, and pTRV2::CaNAC100 is the CaNAC100 silenced fruit; b is the expression of CaNAC100 in the CaNAC100 silenced fruit, in the figure pTRV2::00 is the fruit inoculated with the pTRV2 empty vector strain, and pTRV2::CaNAC100 is the CaNAC100 silenced fruit; c is the expression of carotenoid biosynthesis-related genes in the CaNAC100 gene silenced fruit tissue; d is the total carotenoid content, in the figure pTRV2::00 is the fruit inoculated with the pTRV2 empty vector strain, and pTRV2::CaNAC100 is the CaNAC100 silenced fruit.

[0019] Figure 4 The figure shows the accumulation of carotenoid compounds in pepper fruit regulated by the CaNAC100 gene. Among them, a is the GUS staining image of different silenced fruits in the overexpression system, CK is the pepper fruit tissue without inoculation, 1301::00 is the pCambia1301::00 empty vector strain inoculated, and 1301::CaNAC100 is the CaNAC100 overexpression fruit tissue; b is the CaNAC100 expression level in CaNAC100 overexpression fruit, in the figure 1301::00 is the pCambia1301::00 empty vector strain inoculated, 1301::CaNAC100 is the CaNAC100 overexpression fruit tissue; c is the expression of carotenoid biosynthesis-related genes in the CaNAC100 gene overexpression fruit tissue; d is the total carotenoid content, in the figure 1301::00 is the pCambia1301::00 empty vector strain inoculated, and 1301::CaNAC100 is the CaNAC100 overexpression fruit tissue. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0026] The embodiment of the present invention provides the use of the CaNAC100 gene in regulating the synthesis of plant carotenoids. The nucleotide sequence of the CaNAC100 gene is shown in SEQ ID NO.1.

[0027] In some specific embodiments, overexpression of the CaNAC100 gene promotes the synthesis of carotenoids, and silencing or knocking out the CaNAC100 gene inhibits the synthesis of carotenoids.

[0028] In some specific embodiments, the plant comprises Capsicum annuum.

[0029] The embodiments of the present invention also provide the use of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the CaNAC100 gene in regulating the synthesis of carotenoids in pepper plants.

[0030] The embodiment of the present invention also provides the use of the protein encoded by the CaNAC100 gene in regulating the synthesis of plant carotenoids, and the amino acid sequence thereof is shown in SEQ ID NO.2.

[0031] In some specific embodiments, the plant comprises Capsicum annuum.

[0032] The embodiment of the present invention also provides a method for improving the quality of pepper fruit, which promotes the synthesis of carotenoids in pepper fruit by regulating the expression level of the CaNAC100 gene in the pepper plant.

[0033] The embodiment of the present invention also provides the use of the CaNAC100 gene in cultivating transgenic pepper plants with high carotenoid content.

[0034] The embodiment of the present invention also provides a method for cultivating transgenic pepper plants with high carotenoid content, comprising the following steps: introducing the CaNAC100 gene into the target plant to obtain a transgenic pepper plant.

[0035] In some specific embodiments, the CaNAC100 gene is introduced into the target plant via the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria.

[0036] Preferably, the carotenoids include β-carotene, zeaxanthin and capsanthin.

[0037] Example 1

[0038] 1Cloning and sequence analysis of CaNAC100 gene

[0039] CaNAC100 gene is a NAC-type transcription factor gene of pepper, and its cloning and sequence structure analysis were carried out.

[0040] Yunnan pepper variety Dianjiao No. 13 (registration number GPD(2022)530217) was planted in the greenhouse of the Sericulture and Bee Research Institute in Caoba Town, Mengzi City, Honghe Prefecture, Yunnan Province, and the 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 its RNA content and quality were detected by a spectrophotometer.

[0041] use Ⅲ1st Strand cDNA Synthesis SuperMix 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 using conventional methods to amplify the CaNAC100 gene.

[0042] The upstream primer: (5'-TCCTTAGAGCAATGGAAGAA-3');

[0043] Downstream primer: (5'-CCGAGTCAATCATTACAAGT-3').

[0044] 25μL PCR reaction system: MegaFi TM Fidelity 2X PCR Master Mix 12.5μL, upstream and downstream primers 1μL each, template 1μL, ddH2O 9.5μL.

[0045] The reaction program was as follows: 98°C for 2 min, 1 cycle, 98°C for 10 sec, 57°C for 30 sec, 72°C for 57 sec, 35 cycles.

[0046] The PCR product CaNAC100 gene was purified and recovered, and then connected to the pClone007 Versatile SimpleVector vector (Qingke Biotechnology, China) to obtain the pMDTM19-T-CaNAC100 plasmid, which was transformed into Escherichia coli competent cells DH5a and sent to Shanghai Biotechnology Company for sequencing. The nucleotide sequence of the CaNAC100 gene obtained is shown in the sequence table SEQ ID NO.1:

[0047] SEQ ID NO.1:ATGGAAGAAAATCTCCCTCCAGGGTTTAGGTTCCATCCAAGT。

[0048] The amino acid sequence encoded by the CaNAC100 gene is shown in the sequence listing SEQ ID NO.2:

[0049] SEQ ID NO.2:MEENLPPGRFHPSDEELITYYLSNKVSDFNFTCRAIADVDLNK SEPWDLPAKASMGEKEWYFFSQKDRKYPTGLRTNRATEAGYWKTTGKDKEVYRGGVHLVGMKKTLVFYRGRAPKGEKTNWVMHEYRLETNLGFKPSKEEWVVCRVFHKNSTAKKPQPTSSSQQSDHEDQSTCDTYHH TLPNEFGDMDLTNNFNLATPIGNGSLTNISLHNYSNIQENMNIMNLAALPSVNSLLFRALQLRGNFNNQPRDDIIPATTITSTSNYPFNIQPQGTISAFGVDFDPVLEPPSSSSHVVFNPLQPQGEQSYKLDSNIWE.

[0050] 2 Expression analysis of CaNAC100 gene in different tissues of pepper fruit at different parts and developmental stages

[0051] The improved Yunnan pepper variety Dianjiao No. 13 (registration number GPD(2022)530217) was planted in the greenhouse of the Sericulture and Bee Research Institute in Caoba Town, Mengzi City, Honghe Prefecture, Yunnan Province. Mature fruits were used for RNA extraction, and 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 CaNAC100 gene in different parts (seeds, placenta, and pericarp) at different developmental stages.

[0052] Figure 1 The following is a graph showing the expression differences of CaNAC100 in different tissues of pepper fruit at different developmental stages. Figure 1As shown in the results, CaNAC100 was expressed in the seeds, placenta and pericarp of pepper fruit, but the expression levels were significantly different. After 10 days of flowering, the expression level of CaNAC100 gene in the placenta was significantly higher than that in the pericarp and seeds (P<0.05); after 20 days and 50 days of flowering, the expression levels of this gene in the placenta and seeds were significantly higher than those in the pericarp (P<0.05); after 30 days and 60 days of flowering, the expression levels of this gene in the seeds were significantly higher than those in the placenta and pericarp (P<0.05); but in the samples with 40 days of flowering, the expression level of this gene in the pericarp was significantly higher than that in the seeds and placenta (P<0.05). Overall, the highest developmental period of the expression level of pepper CaNAC100 gene in the placenta and seeds was 20 days after flowering, while the highest developmental period of the expression level in the pericarp was 30 days after flowering.

[0053] 3Subcellular localization of CaNAC100 gene

[0054] The PC1300S-EGFP plant transient fluorescence expression vector was linearized by double restriction digestion with KpnI and BamHI, and CaNAC100 ORF cloning primers F and R (see Table 1) with homology arms of the PC1300S-EGFP vector were designed to amplify the CaNAC100 gene CDS region as an insert. TM HD Cloning kit was used to reconstitute the insert fragment and linearized vector into pCaNAC100-GFP recombinant vector according to the instructions.

[0055] Table 1

[0056]

[0057]

[0058] The pCaNAC100-GFP recombinant vector and the empty vector PC1300S-EGFP 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. pCaNAC100-GFP and the empty vector PC1300S-EGFP were co-transformed with the nuclear marker and injected into tobacco leaves, and then cultured under weak light for 8-10 hours, and the subcellular localization of CaNAC100 was observed under a laser confocal microscope.

[0059] Figure 2 The figure shows the subcellular localization analysis of CaNAC100 gene in tobacco leaves injected with 50% ethanol. Figure 2 As shown, CaNAC100 has an obvious transcription factor nuclear localization signal in Arabidopsis protoplasts, which indicates that CaNAC100 may regulate the transcriptional expression of related genes in the cell nucleus.

[0060] Functional verification of CaNAC100 gene in pepper fruit

[0061] (1) VIGS silencing of the CaNAC100 gene in pepper fruit

[0062] Based on the SGN VIGS Tool online gene silencing tool (https: / / vigs.solgenomics.net / ), the pepper genome database was selected to screen the silenced fragments, and the purified amplification product of pepper CaRAP2-12 was used as a template. The amplification primers are as follows:

[0063] Upstream primer: 5′-TAAGGTTACCGAATTCTTACTCCCAAATATTAGAGTC-3′;

[0064] Downstream primer: 5'-GCTCGGTACCGGATCCCAAGAGAACATGAATATCAT-3').

[0065] The insert fragment was cloned using primers CaNAC100-VIGS, and the procedure and system were the same as before (annealing temperature was 65°C, extension time was 10 sec). TM The HD Cloning kit recombined the purified CaNAC100 gene silencing fragment into the pTRV2 plasmid, and transformed the recombinant plasmid into DH5α Escherichia coli. Finally, after screening the positive colonies, the plasmid was extracted and transferred into GV3101 Agrobacterium.

[0066] Prepare a solution containing 10mM MES, 10mM MgCl2 and 150μM AS, adjust the pH to 5.6, and use it as the infection solution after sterilization. Collect Agrobacterium by centrifugation at 4000rpm / min for 10min, discard the supernatant, and resuspend it with the prepared infection solution. Use the infection solution as a control, and finally adjust the OD of each strain suspension 600nm The inoculation solution was 0.8A, and the resuspension was placed in a 28°C shaker in the dark for 2-3 hours. GV3101 pTRV1 and pTRV2::CaNAC100, pTRV2::00 inoculation solution were prepared in a 1:1 ratio, and 0.5 ml of the inoculation solution was injected into the flesh of the pepper fruit with a 1 ml sterile needleless syringe. The inoculated pepper fruit was cultured at 18°C ​​for 48 hours in a dark environment with a relative humidity of 60%, and then placed in an incubator at 18°C ​​with a light cycle of 16h / dark cycle of 8h. After the pepper fruit was cultured for 15 days, the silent fruit was sampled, and the fruit samples with successful silencing were verified by qRT-PCR.

[0067] Table 1 qRT-PCR primer list

[0068]

[0069]

[0070] The results show that Figure 3 )After inoculation, pepper fruits injected with Agrobacterium containing pTRV2::CaNAC100 plasmid showed obvious green color near the injection hole compared with the control. CaNAC100 silenced fruits were screened using qRT-PCR, and the CaNAC100 gene was downregulated ( Figure 3 b). Determination of carotenoid content in silent positive fruit tissues ( Figure 3 d). The results showed that the total carotene content in the silenced samples was significantly reduced compared with pTRV2::00. When the CaNAC100 gene was silenced, the expression levels of CaZISO, CaZDS, CaCRTZISO02, CaLCYB, CaCHYB ​​and CaCRTY genes were significantly upregulated in pepper fruits compared with pTRV2::00 (P<0.05), while the other carotenoid biosynthesis structural genes were significantly downregulated (P<0.05) ( Figure 3 (c).

[0071] (2) Overexpression of the CaNAC100 gene in pepper fruit

[0072] Based on the In-Fusion Cloning Primer Design Tool online gene primer design tool, seamless cloning primers with 15 bp pCambia1301 plasmid homology arms were designed for both ends of the CaNAC100 gene ORF:

[0073] Upstream primer: 5′-GGACTCTTGACCATGGCCCTTCCGAGTCAATCATTACA-3′;

[0074] Downstream primer: 5'-CTCAGATCTACCATGGCCCTTAAATAATCTCCTTAGAGCAA-3'.

[0075] The CaNAC100 gene sequence was used as a template for cloning and purification to obtain the insert fragment. The CaNAC100 gene insert fragment was connected to the NcoI site of the pCambia1301 vector using a homologous recombination kit to construct an overexpression vector. TMThe HD Cloning kit recombined the purified CaNAC100 gene overexpression fragment into the pCambia1301 plasmid, and transformed the recombinant plasmid 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.

[0076] The CaNAC100 gene was overexpressed in pepper detached fruits. GUS staining was performed on tissues near the injection hole 10 days after inoculation. The results showed that ( Figure 4 (a) Compared with the control fruit, pepper fruit tissues injected with Agrobacterium containing pCambia1301::00 and pCambia1301::CaNAC100 plasmids showed obvious blue color after staining. qRT-PCR was used to screen the CaNAC100 overexpression fruit tissues, and it was found that the expression level of CaNAC100 gene in the overexpression fruit tissues was significantly higher than that in the pCambia1301::00 fruit tissues, indicating that the CaNAC100 gene was successfully overexpressed ( Figure 4 In the transient overexpression samples, the total carotenoid content of pepper fruit increased significantly (P<0.05) ( Figure 4 (d) Compared with the control, except for the CaLCYE gene, the expression levels of other carotenoid biosynthesis structural genes were up-regulated to varying degrees, and the differences were significant (P<0.05).

[0077] In summary, CaNAC100 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 CaNAC100 gene can be used to regulate the synthesis of carotenoids in pepper fruit and the formation of pepper fruit quality.

[0078] The expression pattern of CaNAC100 is highly correlated with carotenoid synthesis in pepper fruit. Transient overexpression of this gene in pepper fruit 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 CaNAC100 by VIGS can significantly reduce the content of carotenoid-related compounds in pepper fruit, and the expression of carotenoid synthesis-related genes is also significantly reduced. The cloning of this gene will not only help to analyze the regulatory mechanism of carotenoids in pepper fruit, but also help to cultivate pepper varieties with higher carotenoid content and improve the quality of pepper fruit, which has great application value.

[0079] In the present invention, the key transcription factor CaNAC100 regulating the synthesis of carotenoids in pepper fruit was cloned and verified for the first time. The transcription factor promotes the synthesis of carotenoid compounds in pepper fruit and affects the formation of pepper fruit quality. The present invention also provides a recombinant plasmid containing the CaNAC100 gene, a transgenic engineering bacterium and a transgenic fruit. The present invention enriches the cognition of the secondary metabolism of pepper fruit and the genetic mechanism of pepper quality formation. The present invention provides a theoretical and practical reference basis for realizing selective agronomic trait breeding of pepper.

[0080] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Application of CaNAC100 gene in regulating plant carotenoid synthesis, characterized in that: The nucleotide sequence of the CaNAC100 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Overexpression of the CaNAC100 gene promotes the synthesis of carotenoids, while silencing or knocking out the CaNAC100 gene inhibits the synthesis of carotenoids.

3. The use according to claim 1, characterized in that: The plants include peppers.

4. Application of recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the CaNAC100 gene in regulating carotenoid synthesis in pepper plants.

5. Application of the protein encoded by the CaNAC100 gene in regulating the synthesis of carotenoids in plants, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

6. The use according to claim 5, characterized in that: The plants include peppers.

7. A method for improving the quality of pepper fruit, characterized in that: By regulating the expression level of the CaNAC100 gene in pepper plants, the synthesis of carotenoids in pepper fruits is promoted.

8. Application of CaNAC100 gene in cultivating transgenic pepper plants with high carotenoid content.

9. A method for cultivating transgenic pepper plants with high carotenoid content, characterized in that: The method comprises the following steps: introducing the CaNAC100 gene described in claim 1 into a target plant.

10. The method according to claim 9, characterized in that The CaNAC100 gene described in claim 1 is introduced into the target plant via the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria described in claim 4.

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