Cucumber CsCCD1 gene and application thereof in regulating and controlling cucumber pulp color

Abstract

The invention discloses a cucumber CsCCD1 gene and application of the cucumber CsCCD1 gene in regulating and controlling the color of cucumber pulp. According to the invention, a CCD family gene CsCCD1 is cloned from cucumber, and the cucumber CsCCD1 gene has the function of regulating and controlling the pulp color for the first time. A coding region of the CsCCD1 gene is edited by utilizing a CRISPR-Cas9 technology, and after the CsCCD1 is mutated, the color of the cucumber pulp is obviously yellowed, and the carotenoid content of the fruit is obviously improved. The invention not only lays an important foundation for revealing genetic and biological mechanisms of cucumber pulp color, but also provides a new gene resource for directional improvement of cucumber varieties and cultivation of high-quality new varieties, lays a theoretical foundation for later molecular breeding work, and has important application value.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering and molecular breeding, and in particular to a cucumber CsCCD1 gene and application thereof in regulating cucumber pulp color. Background Art

[0002] Carotenoids are essential for human health and can only be obtained through dietary sources. Among them, β-carotene is the main carotenoid and is a precursor of vitamin A. Lack of vitamin A intake can lead to dry eyes, increased infant morbidity and mortality, and reduced immune response. Improving the carotene 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. It can also be processed and used in various cooking methods. Flesh color is an important commercial feature of cucumber fruit. Although white is the main color of cucumber flesh, fruits with green, yellow and orange endocarp and mesocarp are also 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, breeding new yellow-fleshed varieties may improve the nutritional quality of cucumbers and increase the dietary source of vitamin A, which is of great significance to the global diet.

[0003] Regarding the genetic law of yellow flesh of cucumber, Kooistra et al. (1971) conducted the first genetic analysis of cucumber flesh color (orange, yellow, grayish white, bright white) genes and believed that flesh color is controlled by two pairs of genes. Qi et al. (1983) first described Xishuangbanna cucumber (Cucumis sativus L. var. xishuangbannanesis Qi et Yuan), whose ripe fruit flesh color is orange. It conforms to the genetic law of quantitative traits with major effect genes, and light color is dominant compared to dark color (Shen Di, 2009). Cuevas et al. (2010) conducted a genetic analysis of Xishuangbanna cucumber and believed that orange mesocarp is controlled by two recessive genes and orange endocarp is controlled by one recessive gene. In addition, PI200815 was described as yellow flesh (Kooistra, 1971). Lu et al. (2015) found that yellow flesh is controlled by a recessive gene (yf) and located it on chromosome 7.

[0004] At present, there have been some reports on the molecular level of research on yellow cucumber flesh. Song Hui et al. (2009) conducted QTL positioning for population flesh color, main carotenoid content in flesh, and lutein traits, and detected a total of 3 QTLs controlling the β-carotene content in the endocarp and mesocarp. In the same year, Shen Di also used the β-carotene content in Xishuangbanna cucumber as a morphological indicator for QTL positioning, and detected a total of 8 QTLs. Bo et al. (2012) found that the orange color of Xishuangbanna cucumber flesh was due to high levels of β-carotene accumulation, and located the orange gene on chromosome 3, named ore. Qi et al. (2013) found through GWAS analysis that in Xishuangbanna cucumber, CsaBCH1 can control the accumulation of β-carotene in mature fruits, resulting in orange flesh. Kishor et al. (2021) found through genetic positioning and whole genome sequencing that CsOr can also lead to an increase in the content of β-carotene in orange flesh. Wang et al. (2023) discovered yellow flesh mutants in European greenhouse cucumbers and found that abscisic acid 8'-hydroxylase Csyf2 regulates yellow flesh by regulating carotene synthesis.

[0005] The accumulation of carotenoids is determined by a biochemical pathway that has been largely elucidated. Carotenoid cleavage dioxygenases (CCDs) are a class of metabolic enzymes that affect carotenoid accumulation and control multiple carotenoid downstream processes. There are four carotenoid cleavage dioxygenase genes (CCD1, CCD4, CCD7, and CCD8) in Arabidopsis, of which CCD7 and CCD8 are associated with lateral branch growth, and CCD1 leads to the accumulation of carotenoids in seeds. Similarly, in chrysanthemum, CmCCD4 affects the color of chrysanthemum petals by controlling carotenoid content. In citrus, CCD4b is a key gene affecting the evolution of peel color. In strawberry, some scholars have found that CCD4 (4B) regulates the yellow flesh and carotenoid content of strawberry fruit. In tomatoes, CCD1 has been reported to be associated with the formation of flavor volatiles such as β-ionone, pseudoionone, and geranyl acetone. In addition, some scholars have found that SiCCD1 can catalyze the degradation of lutein in millet and affect the accumulation and color development of carotenoids in grains. Studies have shown that rice CCD1 (OsCCD1) affects the pigmentation of Golden Rice No. 2 (GR2), and found that carotenoids are substrates of OsCCD1. In cucumber, some scholars believe that CsCCD7 is related to the branching of cucumber, and have conducted bioinformatics analysis of the gene. However, there have been no reports on the cucumber CsCCD1 gene. Summary of the invention

[0006] The object of the present invention is to provide a cucumber CsCCD1 gene and its application in regulating cucumber pulp color in view of the above problems.

[0007] In order to achieve its purpose, the present invention adopts 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 present invention provides a CCD1 gene encoding the above-mentioned CCD1 protein.

[0010] The CCD1 gene is a DNA molecule of (a1) or (a2):

[0011] (a1) a DNA molecule whose coding region is shown in SEQ ID NO.4;

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

[0013] The 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-mentioned CCD1 protein or the above-mentioned CCD1 gene or the above-mentioned mutated CCD1 gene in any of the following:

[0015] (b1) regulating cucumber pulp color;

[0016] (b2) preparing yellow flesh cucumbers;

[0017] (b3) regulating the carotenoid content of cucumber fruits;

[0018] (b4) preparing cucumbers with high carotenoid content in fruits.

[0019] The application regulates the expression of the CCD1 gene in cucumbers, inhibits the expression and / or activity of the CCD1 gene in cucumbers, and causes the cucumber flesh to turn yellow; methods for inhibiting the CCD1 gene include gene editing and RNA interference.

[0020] In the above application, the CCD1 gene in the target cucumber genome is modified using gene editing technology to obtain a gene-edited plant, in which the function of the CCD1 gene is lost or weakened.

[0021] The following mutations occurred in the CCD1 gene in the genome of the gene-edited plant:

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

[0023] The CDS sequence of the CCD1 gene shown in SEQ ID NO.4 is missing from bases 19 to 23 at the 5' end;

[0024] Preferably, CRISPR / Csa9 gene editing technology is used to perform site-directed mutagenesis 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 yellow-fleshed cucumber breeding method, comprising the following steps: reducing the CCD1 protein content in cucumbers (white-fleshed cucumbers), or inhibiting the expression of the CCD1 gene by CRISPR / Csa9 gene editing of the CCD1 gene in the cucumber genome, thereby obtaining yellow-fleshed cucumbers.

[0026] The yellow-fleshed cucumber breeding method uses CRISPR / Csa9 gene editing technology to perform site-directed mutation on the CCD1 gene so that the CCD1 gene undergoes the following mutations:

[0027] The CDS sequence of the CCD1 gene shown in SEQ ID NO.4 is deleted from bases 22 to 23 at the 5' end, or the CDS sequence of the CCD1 gene shown in SEQ ID NO.4 is deleted from bases 19 to 23 at the 5' end;

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

[0029] The beneficial effects of the present invention are:

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

[0031] The present invention not only lays an important foundation for revealing the genetics and biological mechanisms of cucumber pulp color, but also provides new gene resources for the targeted improvement of cucumber varieties and the cultivation of high-quality new varieties, laying a theoretical foundation for subsequent molecular breeding work and having important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a structural map of the vector pKSE402.

[0033] Figure 2 This is a phylogenetic tree of CCD family genes in different species.

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

[0035] Figure 4 Comparison of fruit flesh color of transgenic plants after CsCCD1 gene mutation: (A) fruit flesh color; (B) carotenoid content in flesh; ** indicates P < 0.01. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0037] Unless otherwise specified, all technical and scientific terms used in the examples have the same meanings as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified; the biochemical reagents and raw materials used are commercially available conventional products.

[0039] Experimental Materials:

[0040] Cucumber material '9930': belongs to the North China type, with long and narrow fruits, dark green peel, and white flesh when ripe. As a reference genome material for cucumber, the whole genome has been sequenced. It is a known variety, and is recorded 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 grows vigorously, has strong branches, and the fruit is long and narrow. The peel and flesh of the mature fruit are both white. It is used as a material for cucumber transformation. It is an existing known variety and is recorded in the research paper "Targeted creation of new mutants with compact plant architecture using CRISPR / Cas9 genome editing by an optimized genetic transformation procedure in cucurbit plants" published by Tongxu Xin in the 9th volume of Horticulture Research in 2022.

[0042] The above cucumber materials are preserved in this laboratory and guaranteed to be released to the public for verification experiments within twenty years from the date of application.

[0043] Example 1: Identification of cucumber CsCCD1 gene

[0044] The amino acid sequence of CCD1 gene (Accession No. AT3G63520) in the Arabidopsis genome website (https: / / www.arabidopsis.org / ) was used as an information probe. The Csa7G428120 gene with a consistency of 81.54% with the information probe was obtained through the BlastP program in the cucumber reference genome (http: / / cucurbitgenomics.org / organism / 2, Chinese Long V2). The gene is located on cucumber chromosome 7. The Csa7G428120 gene sequence was used to design a specific primer pair for gene amplification: forward primer (CsCCD1-F, SEQ ID NO.1) and reverse primer CsCCD1-R, SEQ ID NO.2). Fresh leaves of cucumber material '9930' were used as materials, and genomic DNA was extracted using the modified CTAB method (Saghai-Maroof et al., 1984). The above-mentioned specific primers were used to amplify the genomic DNA by PCR. The amplified product was sent to a sequencing company for sequencing. The DNA sequence of the amplified product obtained is shown in SEQ ID NO.3, with a total length of 6778 bp, including 14 exons and 13 introns.

[0045] The sequences of the forward and reverse primers used for PCR amplification are:

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

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

[0048] Using the reported four Arabidopsis CCD subfamily proteins, we obtained four homologous proteins (CCD1, CCD4, CCD7 and CCD8) from the cucumber genome using the BlastP program (e value < 0.01). Then, we performed a phylogenetic analysis of the protein sequence encoded by SEQ ID NO.3 amplified from cucumber and the CCD subfamily proteins in Arabidopsis, corn, rice, sorghum, watermelon and melon. The results were ( Figure 2) showed 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 plant expression vector of cucumber CsCCD1 gene mutant

[0050] Using the software CRISPR-GE ( http: / / skl.scau.edu.cn / ) Design sgRNA for CsCCD1 gene, CRISPR / Csa9 target sequence is located in the first exon of the gene, sgRNA sequence anneals single-stranded Oligo DNA to form double-stranded DNA by PCR: 50μL PCR reaction system includes: Forword oligo (10uM): 15μL, Reverse oligo (10uM): 15μL, 10×NEB buffer 3.1: 5μL, dd H2O: 15μL. PCR instrument reaction program: 95℃ reaction for 4 minutes, then cool to 20℃ at a rate of 0.1℃ per second. After the reaction, dilute 10 times for use.

[0051] Target sequence of CRISPR / Csa9: 5′-GATCAGAAGCAGAAGCTCAACGG-3′ (SEQ ID NO. 6).

[0052] The pKSE402 vector was used as the backbone vector, and the double-stranded DNA formed by annealing was connected to the backbone vector by enzyme digestion to construct the knockout vector pCas9-CsCCD1. The vector pKSE402 used for gene editing comes from Huang Sanwen's research group at the Chinese Academy of Agricultural Sciences. Its structure map is shown in Figure 1 As shown, it contains Cas9 protein and sgRNA expression frame, which is modified on the basis of pKSE401 and inserted with eGFP. When the vector is successfully transferred 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, and then the luminescence of eGFP can be seen in living bodies without harming the plants, and the cell lines or plants with successful transformation can be screened.

[0053] The 15 μL ligation reaction system includes: 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 annealed double-stranded primer: 3 μL, dd H2O: supplemented to 15 μL. The PCR instrument reaction program of the ligation system: 37°C: 3 min, 16°C: 4 min, 40 cycles; 80°C: 5 min, 4°C stop reaction.

[0054] At this point, the target sgRNA was connected to the vector through PCR amplification, enzyme digestion and ligation. The ligation product was transformed into Escherichia coli competent cells, and positive clones were screened to construct the cucumber CsCCD1 editing vector pCas9-CsCCD1. The correctly detected recombinant plasmid was transformed into the EHA105 Agrobacterium strain and genetically transformed using the cucumber material 'CU2'.

[0055] Example 3. Obtaining transgenic cucumber plants

[0056] The pCas9-CsCCD1 constructed in Example 2 was transformed into Agrobacterium EHA105, and then cucumber transformation was performed.

[0057] 1. Preparation and transformation of competent Agrobacterium

[0058] Take 100 μL of Agrobacterium competent cells and freeze and thaw on ice, add 1 μg of plasmid DNA and mix gently, place on ice for 5 minutes, freeze in liquid nitrogen for 5 minutes, place in a 37°C water bath for 5 minutes, place on ice for 5 minutes, add 700 μL LB liquid culture medium, 28°C, 220rpm for 2-3 hours, and evenly spread the bacterial liquid on the solid culture medium containing the corresponding antibiotics. Invert and culture at 28°C for 2-3 days, pick a single colony for PCR identification of positive clones.

[0059] 2. Cucumber genetic transformation

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

[0061] (2) Seed germination: Sow the sterilized cucumber seeds on the germination medium and culture them in the dark at 28°C for about 28 hours.

[0062] (3) Agrobacterium infection:

[0063] The Agrobacterium transformed with the knockout vector was activated and cultured to about OD600=0.8, the cells were collected and resuspended in sterile MS culture medium, and diluted to about OD600=0.2 for later use.

[0064] In the clean bench, take the germinated cucumber seeds, remove the hypocotyls, cut off 1 / 3 of the cotyledons, and divide the cotyledons into two. Place the prepared explants in the reserved Agrobacterium solution, sonicate with an ultrasonic cleaner, evacuate, and infect for 30 minutes.

[0065] (4) Co-cultivation: After infection, the explants were evenly placed on IM solid culture medium covered 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 differentiation medium containing kanamycin for culture and screening. The cells were cultured at 25°C for about 25 days, and fluorescent buds were observed under a fluorescence microscope.

[0067] (6) Rooting culture: When the differentiated adventitious buds extend to about 2 cm, they are cut from the base and inoculated into rooting medium. When the roots are fully grown, the bottle caps can be opened for hardening, and then transplanted to the greenhouse for growth.

[0068] Example 4: Identification of transgenic positive strains

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

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

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

[0072] After sequencing and identification, a total of 2 T0 gene-edited plants were obtained, and the T0 gene-edited plants were self-pollinated to obtain T1 gene-edited plants. Fresh plant leaves of the T1 gene-edited plants were then taken, DNA was extracted using the CTAB method, PCR amplification was performed using the above-mentioned CsCCD1-JC-F and CsCCD1-JC-R primer pairs, and the PCR products were sequenced to select homozygous T1 gene-edited plants. The sequencing results of the T0 and T1 gene-edited plants are shown in Tables 1-2 and Figure 3 .CsCCD1 CR1 The CDS sequence shown in SEQ ID NO.4 lacks bases 22-23, CsCCD1 CR2 The CDS sequence shown in SEQ ID NO.4 lacks bases 19 to 23. CsCCD1 CR1 and CsCCD1 CR2 All of them caused the premature termination of amino acid translation. The specific sequencing results are shown in Figure 3 .

[0073] Table 1

[0074]

[0075] Table 2

[0076]

[0077] Example 5: Phenotypic Identification of Transgenic Cucumber

[0078] The cucumber material CU2 (i.e., wild type) and the one identified as CsCCD1 by sequencing in Example 4 were CR1 or CsCCD1 CR2 Homozygous mutant T1 plants were planted in a solar greenhouse, and the phenotype of the fruits at the same node 30 days after flowering was investigated. Fifteen plants of each experimental material were divided into three replicates, with five cucumbers in each replicate.

[0079] Visual inspection of pulp color: Cut the fruit in half lengthwise 30 days after flowering and observe the pulp color.

[0080] Determination of total pigment content: The carotenoid content was determined by light absorption method. 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-visible 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). 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 contents of the tested materials are shown in Table 3. Figure 4 As shown, after CsCCD1 mutation, in transgenic plants (CsCCD1 CR1 and CsCCD1 CR2 )'s flesh color turns significantly yellow and the carotenoid content accumulates significantly.

[0085] Table 3

[0086]

Claims

1. A CCD1 protein, the amino acid sequence of which is shown in SEQ ID NO.

5.

2. A CCD1 gene encoding the CCD1 protein according to claim 1.

3. The CCD1 gene according to claim 2, characterized in that: The CCD1 gene is a DNA molecule of (a1) or (a2): (a1) a DNA molecule whose coding region is shown in SEQ ID NO.4; (a2) A DNA molecule as shown in SEQ ID NO.

3.

4. A mutant CCD1 gene, characterized in that: 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.

5. Use of the CCD1 protein according to claim 1, or the CCD1 gene according to claim 2 or 3, or the mutated CCD1 gene according to claim 4 in any of the following: (b1) regulating cucumber pulp color; (b2) preparing yellow flesh cucumbers; (b3) regulating the carotenoid content of cucumber fruits; (b4) preparing cucumbers with high carotenoid content in fruits.

6. The use according to claim 5, characterized in that: Regulating 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 to inhibit the CCD1 gene include gene editing and RNA interference.

7. The use according to claim 6, characterized in that: The CCD1 gene in the target cucumber genome is modified using gene editing technology to obtain gene-edited plants, in which the function of the CCD1 gene is lost or weakened.

8. The use according to claim 7, characterized in that: The following mutations occurred in the CCD1 gene in the genome of the gene-edited plant: The CDS sequence of the CCD1 gene shown in SEQ ID NO.4 is missing from bases 22 to 23 at the 5' end, or, The CDS sequence of the CCD1 gene shown in SEQ ID NO.4 is missing from bases 19 to 23 at the 5' end; Preferably, CRISPR / Csa9 gene editing technology is used to perform site-directed mutagenesis on the CCD1 gene, 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 method comprises the following steps: reducing the CCD1 protein content described in claim 1 in cucumbers, or inhibiting the expression of the CCD1 gene by performing CRISPR / Csa9 gene editing on the CCD1 gene described in claim 2 or 3 in the cucumber genome, thereby obtaining yellow flesh cucumbers.

10. The method for breeding yellow-fleshed cucumbers according to claim 9, characterized in that: CRISPR / Csa9 gene editing technology was used to perform site-directed mutagenesis on the CCD1 gene, resulting in the following mutations in the CCD1 gene: The CDS sequence of the CCD1 gene shown in SEQ ID NO.4 is missing from bases 22 to 23 at the 5' end, or, The CDS sequence of the CCD1 gene shown in SEQ ID NO.4 is missing from bases 19 to 23 at the 5' end; The target sequence of CRISPR / Cas9 is shown in SEQ ID NO.6.

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