Cucumber csccd1 gene and its application in regulating cucumber fruit flesh color

Abstract

The application discloses a cucumber CsCCD1 gene and application thereof in regulating fruit flesh color of cucumber. A CsCCD1 gene is cloned from cucumber, and it is first disclosed that the cucumber CsCCD1 gene has the function of regulating fruit flesh color. The coding region of the CsCCD1 gene is edited by using a CRISPR-Cas9 technology, and it is found that after the CsCCD1 gene is mutated, the fruit flesh color of the cucumber is significantly yellow, and the carotenoid content of the fruit is significantly improved. The application not only lays an important foundation for revealing the genetic and biological mechanism of the fruit flesh color of the cucumber, but also provides a new gene resource for directional improvement and cultivation of new varieties with high quality of the cucumber, lays a theoretical foundation for the later molecular breeding work, and has important application value.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and molecular breeding technology, specifically to a cucumber CsCCD1 gene and its application in regulating cucumber flesh color. Background Technology

[0002] Carotenoids are essential for human health and can only be obtained through dietary sources. Beta-carotene is the main carotenoid and a precursor to vitamin A. Vitamin A deficiency can lead to dry eye, increased infant morbidity and mortality, and weakened immune responses. Increasing the carotenoid content in crop varieties through breeding is an important way to improve human health. Cucumber (Cucumis sativus L.) is an important vegetable crop, widely used as a fresh food, and can also be processed and used in various cooking methods. Flesh color is an important commercial characteristic of cucumbers. While white is the dominant flesh color, fruits containing green, yellow, and orange endocarps and mesocarps have also been found. The accumulation of carotenoids is the main reason for the formation of yellow and orange flesh. The concentration of carotenoids in white flesh is negligible. Therefore, developing new yellow-fleshed varieties may improve the nutritional quality of cucumbers, increase dietary sources of vitamin A, and have significant implications for global diets.

[0003] Regarding the genetic patterns of yellow flesh in cucumbers, Kooistra et al. (1971) first conducted a genetic analysis of the genes responsible for cucumber flesh color (orange, yellow, grayish-white, bright white), concluding that flesh color is controlled by two pairs of genes. Qi et al. (1983) first described the Xishuangbanna cucumber (Cucumis sativus L. var. xishuangbannanesis Qi et Yuan), whose mature fruit has orange flesh. This conforms to the quantitative trait inheritance pattern of major genes, with lighter colors being dominant over darker colors (Shen Di, 2009). Cuevas et al. (2010) conducted a genetic analysis of the Xishuangbanna cucumber, concluding that the orange inner pericarp is controlled by two recessive genes, while the orange inner pericarp is controlled by one recessive gene. Furthermore, PI200815 was described as having yellow flesh (Kooistra, 1971). Lu et al. (2015) found that yellow flesh is controlled by a single recessive gene (yf) and located it on chromosome 7.

[0004] Currently, there are some reports on molecular-level research on yellow cucumber flesh. Song Hui et al. (2009) performed QTL mapping on the flesh color, main carotenoid content, and lutein traits of a population, detecting three QTLs controlling β-carotene content in the endocarp and mesocarp. In the same year, Shen Di also performed QTL mapping using β-carotene content in Xishuangbanna cucumber as a morphological indicator, detecting eight QTLs. Bo et al. (2012) found that the orange color of Xishuangbanna cucumber flesh is due to high levels of β-carotene accumulation and located the orange gene on chromosome 3, naming it ore. Qi et al. (2013) found through GWAS analysis that CsaBCH1 can control the accumulation of β-carotene in mature fruit in Xishuangbanna cucumber, thus leading to orange flesh. Kishor et al. (2021) found through genetic mapping and whole-genome sequencing that CsOr can also lead to an increase in β-carotene content in orange flesh. Wang et al. (2023) discovered a yellow-fleshed mutant in European greenhouse cucumbers and found that abscisic acid 8'-hydroxylase Csyf2 regulates yellow flesh by modulating carotene synthesis.

[0005] Carotenoid accumulation is determined by a biochemical pathway, which has been largely elucidated. Carotenoid cleavage dioxygenases (CCDs) are metabolic enzymes that influence carotenoid accumulation and control multiple downstream carotenoid processes. Four carotenoid cleavage dioxygenase genes (CCD1, CCD4, CCD7, and CCD8) exist in Arabidopsis thaliana, with CCD7 and CCD8 related to lateral branch growth, and CCD1 leading to carotenoid accumulation in seeds. Similarly, in chrysanthemums, CmCCD4 affects petal color by controlling carotenoid content. In citrus, CCD4b is a key gene influencing the evolution of peel color. In strawberries, CCD4(4B) has been found to regulate the yellow flesh and carotenoid content of strawberry fruits. In tomatoes, CCD1 has been reported to be involved in the formation of flavor volatiles such as β-ketones, pseudoketones, and geraniol. Furthermore, some scholars have discovered that SiCCD1 can catalyze the degradation of lutein in millet and affect the accumulation of carotenoids and color development in cereals. Studies have shown that rice CCD1 (OsCCD1) affects pigment deposition in Jin Dao 2 (GR2) rice, finding that carotenoids are substrates for OsCCD1. In cucumbers, some scholars believe that CsCCD7 is related to branching and have conducted bioinformatics analysis on the gene. However, there are no reports on the cucumber CsCCD1 gene. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned problems by providing a cucumber CsCCD1 gene and its application in regulating cucumber flesh color.

[0007] To achieve its objective, the present invention employs the following technical solution:

[0008] The first aspect of the present invention provides a CCD1 protein, which is a protein with an amino acid sequence as shown in SEQ ID NO.5.

[0009] A second aspect of the invention provides a CCD1 gene encoding the aforementioned CCD1 protein.

[0010] The CCD1 gene is a DNA molecule that is either (a1) or (a2) as follows:

[0011] (a1) A DNA molecule with a coding region as shown in SEQ ID NO.4;

[0012] (a2) DNA molecules as shown in SEQ ID NO.3.

[0013] A third aspect of the present invention provides a mutated CCD1 gene, wherein the mutation refers to the deletion of bases 22-23 from the 5' end of the CDS sequence of the CCD1 gene shown in SEQ ID NO.4, or the deletion of bases 19-23 from the 5' end of the CDS sequence of the CCD1 gene shown in SEQ ID NO.4.

[0014] The fourth aspect of the present invention provides the use of the above-described CCD1 protein, the above-described CCD1 gene, or the above-described mutated CCD1 gene in any of the following:

[0015] (b1) Regulating the color of cucumber flesh;

[0016] (b2) Preparation of yellow-fleshed cucumbers;

[0017] (b3) Regulating the carotenoid content in cucumber fruits;

[0018] (b4) Prepare cucumbers with high carotenoid content in the fruit.

[0019] The application described above regulates the expression of the CCD1 gene in cucumbers. Inhibiting the expression and / or activity of the CCD1 gene in cucumbers will cause the cucumber flesh to turn yellow. Methods for inhibiting the CCD1 gene include gene editing and RNA interference.

[0020] The above application uses gene editing technology to modify the CCD1 gene in the genome of the target cucumber, resulting in gene-edited plants. The function of the CCD1 gene in the gene-edited plants is lost or weakened.

[0021] The CCD1 gene in the genome of the gene-edited plant underwent the following mutation:

[0022] The CDS sequence of the CCD1 gene shown in SEQ ID NO.4 is missing bases at positions 22-23 from the 5' end, or...

[0023] The CDS sequence of the CCD1 gene shown in SEQ ID NO.4 has bases deleted from position 19-23 at the 5' end;

[0024] The CRISPR / Cas9 gene editing technology is preferably used to perform site-directed mutations on the CCD1 gene, and the target sequence of the CRISPR / Cas9 is shown in SEQ ID NO.6.

[0025] 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 CCD1 protein in cucumbers (white-fleshed cucumbers), or inhibiting the expression of the aforementioned CCD1 gene by CRISPR / Csa9 gene editing in the cucumber genome, thereby obtaining yellow-fleshed cucumbers.

[0026] The above-mentioned breeding method for yellow-fleshed cucumbers uses CRISPR / Csa9 gene editing technology to perform site-directed mutagenesis on the CCD1 gene, resulting in the following mutations in the CCD1 gene:

[0027] The CDS sequence of the CCD1 gene shown in SEQ ID NO.4 has a deletion at positions 22-23 from the 5' end, or the CDS sequence of the CCD1 gene shown in SEQ ID NO.4 has a deletion at positions 19-23 from the 5' end;

[0028] The target sequence for CRISPR / Cas9 is shown in SEQ ID NO.6.

[0029] The beneficial effects of this invention are:

[0030] This invention cloned a CCD family gene, CsCCD1, from cucumber, revealing for the first time that the cucumber CsCCD1 gene has the function of regulating flesh color. Using CRISPR-Cas9 technology to edit the coding region of the CsCCD1 gene, it was found that after mutation of CsCCD1, the cucumber flesh color significantly turned yellow, and the carotenoid content of the fruit significantly increased.

[0031] 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

[0032] Figure 1 This is the structural diagram of the carrier pKSE402.

[0033] Figure 2 It is a phylogenetic tree diagram of the CCD family genes of different species.

[0034] Figure 3 This is a comparison of partial sequencing results of cucumber CsCCD1 mutant plants.

[0035] Figure 4 The comparison of fruit pulp color after CsCCD1 gene mutation in transgenic plants is as follows: (A) Fruit pulp color; (B) Carotenoid content in pulp; ** indicates P<0.01. Detailed Implementation

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Experimental materials:

[0040] 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 the cucumber, Cucumis sativus L." published by Sanwen Huang in *Nature Genetics*, Vol. 41, pp. 1275-1281, 2009.

[0041] 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*.

[0042] 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.

[0043] Example 1: Identification of the cucumber CsCCD1 gene

[0044] Using the amino acid sequence (Accession No. AT3G63520) of the CCD1 gene from the Arabidopsis genome website (https: / / www.arabidopsis.org / ) as an information probe, the Csa7G428120 gene, located on cucumber chromosome 7, was obtained using the BlastP program in the cucumber reference genome (http: / / cucurbitgenomics.org / organism / 2, Chinese Long V2). The Csa7G428120 gene sequence showed 81.54% identity with the information probe. Specific primer pairs for gene amplification were designed using the Csa7G428120 gene sequence: a forward primer (CsCCD1-F, SEQ ID NO.1) and a reverse primer (CsCCD1-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 then amplified by PCR using the aforementioned specific primers. The amplified product was sent to a sequencing company for sequencing. The DNA sequence of the amplified product is shown in SEQ ID NO.3. It is 6778 bp in length and contains 14 exons and 13 introns.

[0045] The forward and reverse primer sequences used for PCR amplification are as follows:

[0046] CsCCD1-F (SEQ ID NO.1): 5'-ATGGCTGATCAGAAGCAGAAGC-3';

[0047] CsCCD1-R (SEQ ID NO. 2): 5'-TTAAAGCCTTCCTTGTTCTTG-3'.

[0048] 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, a phylogenetic analysis was performed on the protein sequence encoded by SEQ ID NO.3 amplified from cucumber and compared with CCD subfamily proteins in Arabidopsis, maize, rice, sorghum, watermelon, and melon. The results (…) Figure 2The results show that it belongs to the CCD1 subfamily, so we named it CsCCD1. The CDS sequence of the CsCCD1 gene is shown in SEQ ID NO.4, and the amino acid sequence of the protein encoded by the CsCCD1 gene is shown in SEQ ID NO.5.

[0049] Example 2: Construction of a plant expression vector for cucumber CsCCD1 gene mutant

[0050] Using the software CRISPR-GE ( http: / / skl.scau.edu.cn / The sgRNA of the CsCCD1 gene was designed. The CRISPR / Csa9 target sequence is located on the first exon of the gene. The sgRNA sequence was annealed from single-stranded Oligo DNA to double-stranded DNA via PCR. The PCR reaction system consisted of: Forward oligo (10 μM): 15 μL, Reverse oligo (10 μM): 15 μL, 10×NEB buffer 3.1: 5 μL, and 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.

[0051] The target sequence of CRISPR / Csa9 is: 5'-GATCAGAAGCAGAAGCTCAACGG-3' (SEQ ID NO.6).

[0052] Using pKSE402 as the backbone vector, the annealed double-stranded DNA was ligated into the backbone vector via enzyme digestion to construct the knockout vector pCas9-CsCCD1. The gene editing vector pKSE402, derived from the research group of Huang Sanwen at the Chinese Academy of Agricultural Sciences, has 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.

[0053] The 15 μL ligation reaction system included: T4 ligase (high concentration 2,000,000 U): 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 dd H2O: 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, stop reaction at 4℃.

[0054] 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, positive clones were screened, and the cucumber CsCCD1 editing vector pCas9-CsCCD1 was constructed. The correctly identified recombinant plasmid was transformed into Agrobacterium tumefaciens strain EHA105, and genetic transformation was performed using cucumber material 'CU2'.

[0055] Example 3: Obtaining transgenic cucumber plants

[0056] After transforming Agrobacterium EHA105 with the pCas9-CsCCD1 constructed in Example 2, cucumber was transformed.

[0057] 1. Preparation and transformation of Agrobacterium competent cells

[0058] 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°C water bath for 5 min, place on ice for 5 min, add 700 μL of LB liquid medium, and thaw at 28°C and 220 rpm for 2-3 h. Spread the bacterial culture evenly on solid medium containing the appropriate antibiotic. Incubate upside down at 28°C for 2-3 days, and select single colonies for PCR identification of positive clones.

[0059] 2. Cucumber genetic transformation

[0060] (1) Seed disinfection: Select plump CU2 cucumber seeds, soak them in 55℃ warm water for 30 minutes, and peel off the seed coat. In a clean bench, first disinfect with 75% alcohol for 30 seconds, pour off the alcohol, then soak the seeds in 0.3% sodium hypochlorite solution for 15 minutes, pour off the liquid, and finally wash the seeds 5 times with sterile water.

[0061] (2) Seed germination: The sterilized cucumber seeds were sown on the germination medium and cultured at 28°C in the dark for about 28 hours.

[0062] (3) Agrobacterium infection:

[0063] 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.

[0064] 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.

[0065] (4) Co-culture: After infection, the explants were evenly placed on IM solid culture medium lined with sterile filter paper and cultured at 25°C in the dark for 3 days.

[0066] (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.

[0067] (6) Rooting culture: When the differentiated adventitious buds elongate to about 2cm, 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.

[0068] Example 4: Identification of transgenic positive lines

[0069] After the transgenic cucumbers in Example 3 had grown in a greenhouse for one week, fresh plant leaves were taken, and DNA was extracted using the CTAB method. PCR amplification was then performed on the location of the CsCCD1 sgRNA, and the PCR product was sequenced. The forward and reverse primer sequences used for PCR amplification are as follows:

[0070] CsCCD1-JC-F (SEQ ID NO.7): 5'-TTCGGAAGGAGAAAACACA-3';

[0071] CsCCD1-JC-R (SEQ ID NO. 8): 5'-CAGTATCCTCAGAAAGAAAGCC-3'.

[0072] Sequencing identification revealed two T0 generation gene-edited plants. T1 generation gene-edited plants were obtained through self-pollination of the T0 generation plants. Fresh leaves from the T1 generation plants were then collected, and DNA was extracted using the CTAB method. PCR amplification was performed using the aforementioned CsCCD1-JC-F and CsCCD1-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 Tables 1-2 and 1-2. Figure 3 CsCCD1 CR1 The CDS sequence shown in SEQ ID NO.4 is missing bases 22-23, CsCCD1. CR2 The CDS sequence shown in SEQ ID NO.4 is missing bases 19-23. CsCCD1 CR1 and CsCCD1 CR2 All of these resulted in premature termination of amino acid translation; see the specific sequencing results below. Figure 3 .

[0073] Table 1

[0074]

[0075] Table 2

[0076]

[0077] Example 5: Phenotypic Identification of Transgenic Cucumber

[0078] Cucumber material CU2 (i.e., wild type) and the one identified by sequencing in Example 4 as CsCCD1 were compared. CR1 Or CsCCD1 CR2 Homozygous 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.

[0079] Visually inspect the flesh color: Cut the fruit lengthwise in half 30 days after flowering and observe the flesh color.

[0080] 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.

[0081] Ca(mg / g FW)=(12.21×A663–2.81×A646)×V / (1000×W).

[0082] Cb(mg / g FW)=(20.13×A646–5.03×A663)×V / (1000×W).

[0083] 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.

[0084] The carotenoid content of the tested materials is shown in Table 3. Figure 4 As shown, after the CsCCD1 mutation, in transgenic plants (CsCCD1...) CR1 and CsCCD1 CR2 The flesh of the fruit turns significantly yellow, and the content of carotenoids accumulates significantly.

[0085] Table 3

[0086]

Claims

1. A mutated CCD1 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; The mutation refers to the one shown in SEQ ID NO.

4. CCD1 The CDS sequence of the gene is deleted from the 5' end at positions 22-23, or as shown in SEQ ID NO.

4. CCD1 The CDS sequence of the gene is deleted from the 5' end at positions 19-23.

2. The application according to claim 1, characterized in that: Regulating cucumber CCD1 Gene expression is suppressed in cucumbers. CCD1 Gene expression and / or activity can cause cucumber flesh to turn yellow; inhibition CCD1 Gene-related methods include gene editing and RNA interference.

3. The application according to claim 2, characterized in that: Gene editing technology was used to target the cucumber genome. CCD1 Genes are modified to obtain gene-edited plants. CCD1 The function of genes is lost or weakened.

4. A method for breeding yellow-fleshed cucumbers, characterized in that, The steps include: through analysis of the cucumber genome CCD1 CRISPR / Cas9 gene editing inhibits the genes described CCD1 Gene expression was used to produce yellow-fleshed cucumbers; CRISPR / Cas9 gene editing technology was employed to... CCD1 Site-directed mutation of genes enables CCD1 The gene undergoes the following mutation: As shown in SEQ ID NO.4 CCD1 The CDS sequence of the gene is deleted at positions 22-23 from the 5' end, or... As shown in SEQ ID NO.4 CCD1 The CDS sequence of the gene is deleted from the 5' end at positions 19-23.

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