Cucumber ccd4 gene and its application in regulating cucumber fruit flesh color

Abstract

This invention discloses the cucumber CCD4 gene and its application in regulating cucumber flesh color. This invention clones a CCD family gene from cucumber. CsCCD4 For the first time, cucumbers were revealed CsCCD4 Genes have the function of regulating fruit flesh color. Using CRISPR-Cas9 technology... CsCCD4 Editing the coding region of a gene revealed... CsCCD4 After the mutation, the cucumber flesh turned significantly yellow, and the content of carotenoids in the fruit increased significantly, all of which was purpuric acid. This invention not only provides key theoretical support for elucidating the genetic mechanism and biological basis of cucumber flesh color, but also opens up a gene resource reserve for the targeted improvement of this trait and the breeding of high-quality new varieties, thus laying a solid foundation for subsequent molecular breeding practices and possessing significant scientific research and application value.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and molecular breeding technology, specifically to a cucumber CCD4 gene and its application in regulating cucumber flesh color. Background Technology

[0002] Carotenoids are a vital class of dietary phytonutrients that play an indispensable role in maintaining human health, including supporting immune function and slowing the progression of age-related degenerative diseases (Fiedor and Burda, 2014). Among them, β-carotene is the most abundant provitamin A carotenoid and a major precursor for dietary vitamin A synthesis (Nisar et al., 2015). Vitamin A deficiency remains a significant global public health challenge, closely associated with dry eye, impaired immune responses, and increased childhood morbidity and mortality (Underwood, 2004). Therefore, biofortification of crops through selective breeding to increase carotenoid content has become a sustainable, food-based strategy for improving nutritional security.

[0003] Cucumber is a globally important vegetable crop, with a total production exceeding 94.7 million tons in 2022 (FAOSTAT, 2022). As a major fresh-eating commodity, its flesh color is determined by the relative accumulation of chlorophyll and carotenoids, a key quality trait influencing market acceptance and consumer preference (Ranganath et al., 2022). Typically, cucumber flesh color ranges from white to green due to variations in chlorophyll content (Bo et al., 2019); while the accumulation of carotenoids constitutes its distinctive yellow or orange flesh. This unique coloration phenomenon has only been documented in limited genetic resources, such as the semi-wild Xishuangbanna type (XIS), germplasm PI200815 and PI163217, and the mutant yf-343 (Kooistra, 1971; Qi et al., 1983; Kishor et al., 2021; Wang et al., 2023). Recent genetic studies have begun to reveal the molecular basis of cucumber flesh color variation. For example, the orange endocarp of XIS cucumber is controlled by the recessive locus ore (Cuevas et al., 2010; Bo et al., 2012), and Qi et al. (2020) identified Csa3G183920 as a candidate gene through genome-wide association analysis (GWAS). Similarly, a single recessive gene, CsaV3_6G040750, was identified as the cause of the orange endocarp in PI163217 (Kishor et al., 2021). For yellow flesh, Csyf2 regulates the flesh color of the European greenhouse cucumber mutant yf-343 by modulating carotenoid biosynthesis (Wang et al., 2023), while CsCCD1 regulates the yellow endocarp of PI200815 by degrading β-cryptoxanthin (Dai et al., 2025).

[0004] The plant carotenoid metabolic pathway comprises three key stages: precursor synthesis via the methyl erythritol phosphate (MEP) pathway, carotenoid biosynthesis, and carotenoid degradation. Carotenoid accumulation is determined by a biochemical pathway, which has been largely elucidated. Carotenoid cleavage dioxygenases (CCDs) are a class of metabolic enzymes that influence carotenoid accumulation and control multiple downstream carotenoid processes. Carotenoid homeostasis in plants is regulated by a dynamic balance between its biosynthesis, degradation, and stable storage in plastids (Sun et al., 2022). The degradation pathway is primarily initiated by CCDs, which catalyze the oxidative cleavage of the carotenoid skeleton, producing abscisic carotenoids—a class of compounds that influence multiple processes, including pigment formation, aroma, and stress responses (Walter et al., 2010). The CCD family mainly includes four members—CCD1, CCD4, CCD7, and CCD8—and the nonacis-epoxycarotenoid dioxygenase (NCED) subfamily. CCD1 primarily utilizes substrates such as α / β-carotene, lutein, and zeaxanthin to produce volatile abscissorptive carotenoids such as α / β-ionone and pseudoionone (Floss et al., 2009; Simkin et al., 2021). CCD7 and CCD8 mediate the biosynthesis of strigolactones, influencing root architecture, tillering, and flower development (Auldridge et al., 2006; Guan et al., 2012; Kulkarni et al., 2014). CCD4 is a core regulator of fruit coloration in multiple species. In Silent White Chrysanthemum, CmCCD4a induces carotenoid accumulation and yellow petal coloration (Ohmiya et al., 2006). StCCD4 cleaves β-carotene in potato to produce β-ionone (Bruno et al., 2015), and inhibiting its expression can increase iodine expression by 2-5 times, resulting in yellow tubers (Campbell et al., 2010). CCD4b imparts a red color to citrus peel by degrading carotenoids to produce β-limonene and β-limonene (Zheng et al., 2019). Knockdown of FaCCD4(4B) in strawberries enhances carotenoid accumulation, resulting in yellow flesh (Amaya et al., 2025). Although CCD4 functions are conserved across multiple species, the function of CsCCD4 in cucumber remains unclear. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned problems by providing a cucumber CCD4 Genes and their application in regulating cucumber flesh color.

[0006] To achieve its objective, the present invention employs the following technical solution: A first aspect of the present invention provides a CCD4 protein, which is a protein with an amino acid sequence as shown in SEQ ID NO.5.

[0007] A second aspect of the present invention provides a protein encoding the aforementioned CCD4 protein. CCD4 Gene.

[0008] The CCD4 A gene is a DNA molecule that is (a1) or (a2) as follows: (a1) A DNA molecule with a coding region as shown in SEQ ID NO.4; (a2) DNA molecules as shown in SEQ ID NO.3.

[0009] The third aspect of the present invention provides a mutation CCD4 The mutation refers to the gene shown in SEQ ID NO.4. CCD4 The CDS sequence of the gene is deleted from the 5' end at positions 53-74.

[0010] The fourth aspect of the present invention provides the above-described CCD4 protein or the above-described... CCD4 Genes or the above-mentioned mutations CCD4 The application of genes in any of the following: (b1) Regulating the color of cucumber flesh; (b2) Preparation of yellow-fleshed cucumbers; (b3) Regulating the carotenoid content in cucumber fruits; (b4) Prepare cucumbers with high carotenoid content; Preferably, the carotenoid is azadirachtin.

[0011] The aforementioned application regulates the properties of cucumbers. CCD4 Gene expression is suppressed in cucumbers. CCD4 Gene expression and / or activity can cause cucumber flesh to turn yellow; inhibition CCD4 Gene-related methods include gene editing and RNA interference.

[0012] The above applications utilize gene editing technology to target specific cells in the cucumber genome. CCD4 Genes are modified to obtain gene-edited plants. CCD4 The function of genes is lost or weakened.

[0013] In the genome of the gene-edited plant CCD4 The gene undergoes the following mutation: As shown in SEQ ID NO.4 CCD4 The CDS sequence of the gene is deleted from the 5' end at positions 53-74; CRISPR / Csa9 gene editing technology is preferred for use. CCD4 The gene is subjected to site-directed mutation, and the target sequence of the CRISPR / Cas9 is shown in SEQ ID NO.6.

[0014] The fifth aspect of the present invention provides a method for breeding yellow-fleshed cucumbers, comprising the following steps: reducing the content of the aforementioned CCD4 protein in cucumbers (white-fleshed cucumbers), or by adjusting the content of the aforementioned CCD4 protein in the cucumber genome. CCD4 CRISPR / Csa9 gene editing inhibits the genes described CCD4 Gene expression results in yellow-fleshed cucumbers.

[0015] The above-mentioned breeding method for yellow-fleshed cucumbers uses CRISPR / Csa9 gene editing technology. CCD4 Site-directed mutation of genes enables CCD4 The gene undergoes the following mutation: As shown in SEQ ID NO.4 CCD4 The CDS sequence of the gene is deleted from the 5' end at positions 53-74; The target sequence for CRISPR / Cas9 is shown in SEQ ID NO.6.

[0016] The beneficial effects of this invention are: This invention clones a CCD family gene from cucumber. CsCCD4 For the first time, cucumbers were revealed CsCCD4 Genes have the function of regulating fruit flesh color. Using CRISPR-Cas9 technology... CsCCD4 Editing the coding region of a gene revealed... CsCCD4 After the mutation, the cucumber flesh turned significantly yellow, and the content of carotenoids in the fruit increased significantly, all of which was purpuric acid.

[0017] This invention not only lays an important foundation for revealing the genetic and biological mechanisms of cucumber flesh color, but also provides new gene resources for the targeted improvement of cucumber varieties and the breeding of high-quality new varieties, laying a theoretical foundation for subsequent molecular breeding work and having important application value. Attached Figure Description

[0018] Figure 1 This is the structural diagram of the carrier pKSE402.

[0019] Figure 2 It is a phylogenetic tree diagram of the CCD family genes of different species.

[0020] Figure 3 It's a cucumber. CsCCD4 Comparison of sequencing results of mutant plants.

[0021] Figure 4 yes CsCCD4 Comparison of fruit pulp color after gene mutation in transgenic plants: (A) Fruit pulp color; (B) Carotenoid content in the pulp; ** indicates P <0.01, (C) content of azadirachtin in the pulp; ** indicates P <0.01. Detailed Implementation

[0022] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention. Unless otherwise specified, all technical and scientific terms used in the embodiments have the same meaning as commonly understood by one of ordinary skill in the art described in this invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the biochemical reagents and raw materials used are all commercially available products.

[0024] Experimental materials: Cucumber material '9930': Belongs to the North China type, with elongated fruit, dark green peel, and white flesh on mature fruit. Its whole genome has been sequenced as a reference genome for cucumber. It is a known variety, described in the research paper "The genome of thecucumber, Cucumis sativus L." published by Sanwen Huang in *Nature Genetics*, Vol. 41, pp. 1275-1281, 2009.

[0025] CU2: The plant exhibits vigorous growth and strong branching. The fruit is elongated, with both the peel and flesh of mature fruits being white. It is used as material for cucumber transformation. It is a known variety, described in the 2022 research paper "Targeted creation of new mutants with compact plant architecture using CRISPR / Cas9 genome editing by an optimized genetic transformation procedure in cucumber plants" published by Tongxu Xin in Volume 9, uhab086 of *Horticulture Research*.

[0026] The above cucumber materials are stored in our laboratory and will be distributed to the public for verification experiments within twenty years from the date of application.

[0027] Example 1: Cucumber CsCCD4 Gene identification Using the amino acid sequence (Accession No. AT4G19170) of the CCD4 gene from the Arabidopsis genome website (https: / / www.arabidopsis.org / ) as an information probe, the Search Gene program in the cucumber reference genome (http: / / 60.30.67.245:8070 / # / home, Chinese Long V4) was used to obtain a sequence with 69.81% consistency with the information probe. CsaV4_4G000731 The gene is located on chromosome 4 of the cucumber. Utilizing... CsaV4_4G000731 Gene sequence was used to design specific primer pairs for gene amplification: a forward primer (CsCCD4-F, SEQ ID NO.1) and a reverse primer (CsCCD4-R, SEQ ID NO.2). Genomic DNA was extracted from fresh leaves of cucumber material '9930' using a modified CTAB method (Saghai-Maroof et al., 1984). The genomic DNA was amplified by PCR using the aforementioned specific primers. The amplified products were sent to a sequencing company for sequencing. The obtained DNA sequence of the amplified product is shown in SEQ ID NO.3, with a full length of 2,103 bp, containing 1 exon and 0 introns.

[0028] The forward and reverse primer sequences used for PCR amplification are as follows: CsCCD4-F (SEQ ID NO.1): 5'-TCAGTTCAAGTTTGGTATGT-3'; CsCCD4-R (SEQ ID NO. 2): 5'-AAAGTTTCACGGATGTATCT-3'.

[0029] Using four reported Arabidopsis CCD subfamily proteins, four homologous proteins (CCD1, CCD4, CCD7, and CCD8) (e-value < 0.01) were obtained from the cucumber genome using the BlastP program. Then, we performed a phylogenetic analysis on the protein sequence encoded by SEQ ID NO. 3 amplified from cucumber and compared it with CCD subfamily proteins from Arabidopsis, maize, rice, sorghum, watermelon, and melon. The results showed that it belongs to the CCD4 subfamily, therefore we named it... CsCCD4 ( Figure 2 ), CsCCD4 The CDS sequence of the gene is shown in SEQ ID NO.4. CsCCD4 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.5.

[0030] Example 2, Cucumber CsCCD4 Construction of plant expression vectors for gene mutants Using the software CRISPR-GE ( http: / / skl.scau.edu.cn / )design CsCCD4 The sgRNA of the gene, the CRISPR / Csa9 target sequence, is located on the exon of the gene. The sgRNA sequence was used to anneal single-stranded Oligo DNA to form double-stranded DNA via PCR: 50 μL. The PCR reaction system included: Forward oligo (10 μM): 15 μL, Reverse oligo (10 μM): 15 μL, 10×NEB buffer 3.1: 5 μL, dd H2O: 15 μL. The PCR reaction program was: 95℃ for 4 minutes, then cooled to 20℃ at a rate of 0.1℃ per second. After the reaction, the solution was diluted 10-fold for later use.

[0031] The target sequence of CRISPR / Csa9 is: 5'-CAATTTCCAGTTCCCTCCCA-3' (SEQ ID NO.6).

[0032] Using pKSE402 as the backbone vector, the double-stranded DNA formed by annealing was ligated into the backbone vector via enzyme digestion, and the pCas9 knockout vector was then executed. Construction of CsCCD4. The gene-editing vector pKSE402, from the Functional Gene Research Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, is shown in the following structural diagram. Figure 1 As shown, the expression cassette containing Cas9 protein and sgRNA is modified based on pKSE401 and inserts eGFP. When the vector is successfully transformed into plant cells, the eGFP sequence will be expressed and translated to produce green fluorescent protein. The fluorescence emitted by eGFP can be seen under a fluorescence microscope. Thus, the luminescence of eGFP can be observed in vivo and without harming the plant, and successfully transformed cell lines or plants can be screened.

[0033] The 15 μL ligation reaction system consisted of: T4 ligase (high concentration 2000000U): 1 μL, Bsal enzyme (NEB): 1 μL, 10×NEB T4 buffer: 1.5 μL, 10×BSA: 1.5 μL-2 μL (0.2 mg), empty vector: 1 μL, diluted and annealed double-stranded primers: 3 μL, and ddH2O: to a final volume of 15 μL. The PCR reaction program for the ligation system was: 37℃: 3 min, 16℃: 4 min, 40 cycles; 80℃: 5 min, reaction stopped at 4℃.

[0034] Thus, the target sgRNA was ligated into the vector via PCR amplification, enzyme digestion, and ligation. The ligation product was transformed into competent E. coli cells, and positive clones were screened to construct the cucumber vector. CsCCD4 Editing carrier pCas9 CsCCD4. The correctly identified recombinant plasmid was transformed into Agrobacterium strain EHA105 and genetically transformed using cucumber material 'CU2'.

[0035] Example 3: Obtaining transgenic cucumber plants The pCas9 constructed in Example 2 After CsCCD4 was transformed into Agrobacterium EHA105, cucumber was transformed.

[0036] 1. Preparation and transformation of Agrobacterium competent cells Take 100 μL of Agrobacterium competent cells, freeze and thaw them on ice, add 1 μg of plasmid DNA, mix gently, place on ice for 5 min, flash freeze in liquid nitrogen for 5 min, incubate in a 37 ℃ water bath for 5 min, place on ice for 5 min, add 700 μL of LB liquid medium, and thaw at 28 ℃ and 220 rpm for 2-3 h. Spread the bacterial culture evenly on solid medium containing the appropriate antibiotic. Incubate upside down at 28 ℃ for 2-3 days, and select single colonies for PCR identification of positive clones.

[0037] 2. Cucumber genetic transformation (1) Seed disinfection: Select plump CU2 cucumber seeds, soak them in 55 °C warm water for 30 min, and peel off the seed coat. In a clean bench, first disinfect with 75% alcohol for 30 s, pour off the alcohol, then soak the seeds in 0.3% sodium hypochlorite solution for 15 min, pour off the liquid, and finally wash the seeds 5 times with sterile water.

[0038] (2) Seed germination: The sterilized cucumber seeds were sown on the germination medium and cultured at 28 °C in the dark for about 28 h.

[0039] (3) Agrobacterium infection: Agrobacterium that has been transformed with the knockout vector was activated and cultured to an OD600 of approximately 0.8. The bacterial cells were collected, resuspended in sterile MS medium, and diluted to an OD600 of approximately 0.2 for later use.

[0040] In a clean bench, germinated cucumber seeds were taken, the hypocotyl was removed, and one-third of the cotyledons were cut off, dividing the cotyledons in half. The prepared explants were placed in the prepared Agrobacterium tumefaciens solution, ultrasonically cleaned, and then vacuum-sealed for 30 minutes.

[0041] (4) Co-culture: After infection, the explants were evenly placed on IM solid culture medium lined with sterile filter paper and cultured at 25 ℃ in the dark for 3 days.

[0042] (5) Differentiation culture: After 3 days of dark culture, the explants were transferred to a differentiation medium containing kanamycin for culture and screening. After culturing at 25 ℃ for about 25 days, fluorescent buds were observed using a fluorescence microscope.

[0043] (6) Rooting culture: When the differentiated adventitious buds elongate to about 2 cm, cut them off from the base and inoculate them into the rooting culture medium. When the roots have fully grown, the bottle cap can be opened to harden the seedlings, and then they can be transplanted to the greenhouse for growth.

[0044] Example 4: Identification of transgenic positive lines After the transgenic cucumbers in Example 3 had grown in the greenhouse for one week, fresh plant leaves were taken, and DNA was extracted using the CTAB method. CsCCD4 PCR amplification was performed at the location of the sgRNA, and the PCR product was sequenced. The sequences of the forward and reverse primers used for PCR amplification are as follows: CsCCD4-JC-F (SEQ ID NO.7): 5'-GAAGAATCTCGCCGGTAG-3'; CsCCD4-JC-R (SEQ ID NO. 8): 5'-GAAGAAAGAGAGGAAGATGTGA-3'.

[0045] Sequencing identification yielded one T0 generation gene-edited plant. Self-pollination of the T0 generation plant yielded a T1 generation gene-edited plant. Fresh leaves from the T1 generation plant were then collected, and DNA was extracted using the CTAB method. PCR amplification was performed using the aforementioned CsCCD4-JC-F and CsCCD4-JC-R primer pairs. The PCR products were sequenced, and homozygous T1 generation gene-edited plants were selected. Sequencing results for the T0 and T1 generation gene-edited plants are shown in Table 1 and [Table data missing]. Figure 3 . CsCCD4 CR The CDS sequence shown in SEQ ID NO.4 is missing bases 53-74. CsCCD4 CR This caused premature termination of amino acid translation; see the specific sequencing results below. Figure 3 .

[0046] Table 1

[0047] Example 5: Phenotypic Identification of Transgenic Cucumber Cucumber material CU2 (i.e., wild type) and the material identified by sequencing in Example 4 were compared. CsCCD4 CRHomozygous mutant T1 generation plants were transplanted into a greenhouse, and the phenotypic characteristics of fruits 30 days after flowering at the same node were investigated. Fifteen plants of each experimental material were divided into three replicates, with five cucumber plants in each replicate.

[0048] Visually inspect the flesh color: Cut the fruit lengthwise in half 30 days after flowering and observe the flesh color.

[0049] Determination of total pigment content: The content of carotenoids was determined by light absorption. Briefly, 2 g of pulp sample was soaked in 40 mL of acetone:deionized water (4:1, v / v) mixture for 6 h, and the absorbance of the reaction mixture was measured at 663 nm (A663), 646 nm (A646), and 470 nm (A470) using a UV-Vis spectrophotometer.

[0050] Ca(mg / g FW)=(12.21×A 663–2.81×A 646)×V / (1000×W). Cb(mg / g FW)=(20.13×A 646–5.03×A 663)×V / (1000×W). Carotenoid content (mg / g FW) = [1000×A470-3.27×Ca–104×Cb) / 229]×V / (1000×W), where Ca, Cb, FW, V, and W represent chlorophyll a content, chlorophyll b content, fresh weight, sample volume (ml), and sample mass (g), respectively.

[0051] Carotenoid component analysis: This was performed by Wuhan Maiwei Biotechnology Co., Ltd. using an AB Sciex QTRAP 6500LC-MS / MS platform. Fruit pulp tissue was collected 30 days after flowering, flash-frozen in liquid nitrogen, and stored at -80℃. Before analysis, the frozen sample was ground into a fine powder in liquid nitrogen, freeze-dried, and 50 mg of the dried powder was weighed and added to 0.5 mL of a hexane-acetone-ethanol mixture (volume ratio 1:1:1) containing an internal standard. The mixture was vortexed (12,000 × g, 10 min, 4℃), and the supernatant was collected. The extraction was repeated twice, and the combined supernatants were dried under a gentle nitrogen stream. The residue was redissolved in dichloromethane, filtered through a 0.22 μm PTFE membrane, and injected. Chromatographic separation was performed using an ExionLC™ AD ultra-high performance liquid chromatography system with an APCI heated nebulizer ion source. Mobile phase A was an acetonitrile-water solution (3:1, v / v) containing 0.01% BHT and 0.1% formic acid, and mobile phase B was methyl tert-butyl ether containing 0.01% BHT. The flow rate was 0.8 mL / min. -¹, Column temperature 28℃. Elution gradient: 0–3 min hold at 0% B, 3–5 min increase to 70% B, 5–9 min increase to 95% B, 9–11 min return to 0% B. Mass spectrometry was performed using an AB Sciex 6500 QTRAP mass spectrometer in APCI positive ion mode, with data acquisition and processing controlled by Analyst® 1.6.3 software.

[0052] Carotenoids are a large family, including two main categories: carotenes (such as β-carotene) and lutein (such as violaxanthin and zeaxanthin). Violaxanthin belongs to the lutein class of compounds and is an oxygen-containing derivative, typically appearing yellow or orange. Violaxanthin is not only a type of carotenoid but also a key intermediate in the entire carotenoid metabolic network, connecting photoprotection mechanisms and hormone synthesis pathways, playing an irreplaceable role in plant physiology. Simultaneously, violaxanthin exhibits significant antioxidant, anti-inflammatory, anti-tumor, and anti-skin photoaging activities, making it valuable for applications in nutrition, health, and functional foods (Pasquet et al., 2011; Soontornchaiboon et al., 2012; Kim et al., 2019). Therefore, elucidating the metabolic regulatory mechanisms of violaxanthin is crucial for its further development and utilization in food science and human nutrition.

[0053] The carotenoid and xanthophyll contents of the tested materials are shown in Tables 2 and 3. Figure 4 As shown, CsCCD4 After mutation, in transgenic plants ( CsCCD4 CR The pulp of the 10 ...

[0054] Table 2 Results of carotenoid content

[0055] Table 3 Results of xanthophyll content

[0056] Table 4 Differentially detected metabolites

[0057] It is worth noting that another member of the CCD family has been reported in cucumbers. CsCCD1Its function has been confirmed to regulate the formation of yellow pulp by degrading β-cryptoxanthin (Dai et al., 2025, An intronic SNP in the Carotenoid Cleavage Dioxygenase 1 ( CsCCD1 ) controls yellow fleshformation in cucumber fruit ( Cucumis sativus L. (doi: 10.1111 / pbi.70034). The CsCCD4 involved in this invention has a fundamentally different mechanism of action from CsCCD1: CsCCD4 specifically targets and cleaves another carotenoid—vioolaxanthin. Although β-cryptoxanthin and violaxanthin both belong to the lutein family of carotenoids, they differ significantly in chemical structure, biological activity, metabolic pathways, and main sources.

[0058] As shown in the data of the embodiments of the present invention, CsCCD4 After functional loss, the cucumber pulp specifically accumulates large amounts of purpuricin and its esterified derivatives, accounting for 100% of the total differentially expressed carotenoids, rather than β-cryptoxanthin. This clearly reveals... CsCCD4 and CsCCD1 They are completely different in substrate specificity, regulated metabolites, and final pigment composition. Therefore, CsCCD4 This represents a completely new genetic target, and its technical approach, metabolite profile, and nutritional functional characteristics of the bred varieties are substantially different from existing technologies based on CsCCD1.

[0059] The nucleotide sequence of the CDS of the CsCCD4 gene is as follows (SEQ ID NO.4): CsCCD4 The amino acid sequence of the protein encoded by the gene is as follows (SEQ ID NO.5): MDSISSPFLSGRNLILSPPISSSLPPISTPIYSVLTEQNVKKNTPPPDADSPSPPLPRPSPPSPPMPRVSSTRRVQPSLPARFFNAFDDLINNFINPPVSPSVDPRYILADNFAPVDELPPTECEVIYGSLPSSLNGAYIRNGPNPQYLPRGPYHLFDGDGMLHSLRISDGRAVLCSRYVKTYKYTLERDAGHPVFPNVFSGFNGLTASAARGAVAVGRILTGQYNPANGIGLANTSLAFFGDRLYALGESDLPYPIRLTPNGDIETLARHDFDGKLTLSMTAHPKVDSDTGEAFAFRYGPLPPFLTYFRFDKNGAKHSDVPILSMNRPSFLHDFAITKKYAVFTDIQIGINPTQMIIEGGSPVGSDPSKISRVGLIPRYANDESKMKWFDVPGLNLIHAINAWDEDDAVVIVAPNILSVEHALERMDLVHALVEKIRIDLKTGIVTRTPLSTRNLDFGVIHPSYVGKKHRFVYAGVGDPMPKISGVVKLEISQEERRDCIVACRIFGPGCYGGEPFFVPRERESSDETEAEEDDGYVVSYVHDENSGESRFIVMDAKSPELEIIAAVKLPRRVPYGFHGLFVKESDLNKL。

Claims

1. A CCD4 protein, having the amino acid sequence shown in SEQ ID NO.

5.

2. Encoding the CCD4 protein of claim 1 CCD4 Gene.

3. As described in claim 2 CCD4 Genes are characterized by: The CCD4 A gene is a DNA molecule that is (a1) or (a2) as follows: (a1) A DNA molecule with a coding region as shown in SEQ ID NO.4; (a2) DNA molecules as shown in SEQ ID NO.

3.

4. A mutated type CCD4 Genes are characterized by: The mutation refers to the one shown in SEQ ID NO.

4. CCD4 The CDS sequence of the gene is deleted from the 5' end at positions 53-74.

5. The CCD4 protein according to claim 1, or the protein according to claim 2 or 3. CCD4 Gene or the mutation as described in claim 4 CCD4 The application of genes in any of the following: (b1) Regulating the color of cucumber flesh; (b2) Preparation of yellow-fleshed cucumbers; (b3) Regulating the carotenoid content in cucumber fruits; (b4) Prepare cucumbers with high carotenoid content; Preferably, the carotenoid is azadirachtin.

6. The application according to claim 5, characterized in that: Regulating cucumber CCD4 Gene expression is suppressed in cucumbers. CCD4 Gene expression and / or activity can cause cucumber flesh to turn yellow; inhibition CCD4 Gene-related methods include gene editing and RNA interference.

7. The application according to claim 6, characterized in that: Gene editing technology was used to target the cucumber genome. CCD4 Genes are modified to obtain gene-edited plants. CCD4 The function of genes is lost or weakened.

8. The application according to claim 7, characterized in that: In the genome of the gene-edited plant CCD4 The gene undergoes the following mutation: As shown in SEQ ID NO.4 CCD4 The CDS sequence of the gene is deleted from the 5' end at positions 53-74; CRISPR / Csa9 gene editing technology is preferred for use. CCD4 The gene is subjected to site-directed mutation, and the target sequence of the CRISPR / Cas9 is shown in SEQ ID NO.

6.

9. A method for breeding yellow-fleshed cucumbers, characterized in that, The steps include: reducing the content of the CCD4 protein as described in claim 1 in cucumber, or by adjusting the content of the protein as described in claim 2 or 3 in the cucumber genome. CCD4 CRISPR / Csa9 gene editing inhibits the genes described CCD4 Gene expression results in yellow-fleshed cucumbers.

10. The method for breeding yellow-fleshed cucumbers according to claim 9, characterized in that: Using CRISPR / Csa9 gene editing technology to CCD4 Site-directed mutation of genes enables CCD4 The gene undergoes the following mutation: As shown in SEQ ID NO.4 CCD4 The CDS sequence of the gene is deleted from the 5' end at positions 53-74; The target sequence for CRISPR / Cas9 is shown in SEQ ID NO.6.

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