CN102732536B - Cloning and transient expression method of persimmon deastringency related genes - Google Patents

Abstract

The invention provides a cloning and transient expression method of persimmon deastringency related genes. The method comprises: first acquiring gene 3'-terminal sequences, then employing 3' RACE (rapid amplication of cDNA ends) technology to acquire 3'-terminal sequences of ADH and PDC gene family members in persimmon fruits; adopting Q-PCR (quantitative polymerase chain reaction) technology to analyze the genetic expression of ADH and PDC gene family members in a persimmon fruit deastringency treatment; and conducting separation to obtain full-length sequences of ADH and PDC gene family members, then establishing a transient expression system of persimmon leaves. The method of the invention clones the persimmon deastringency related gene family members, analyze the expression patterns of the family members in the persimmon deastringency treatment, and finally verifies functions of the target genes through transient expression of the persimmon leaves. The invention clarifies the regulation mechanism for postharvest persimmon deastringency from the molecular mechanism aspective, and can be used for further functional verification (transgenosis, gene interaction and the like) study of the postharvest persimmon deastringency mechanism.

Description

A method for cloning and transient expression of genes related to persimmon astringency

technical field

The invention belongs to the technical field of bioengineering, and relates to the cloning and expression of genes related to persimmon deastringency and the establishment of a system for transient expression of persimmon leaf genes.

Background technique

Persimmon is native to China. It is delicious and good for human health. It is one of the most popular fruits in East Asia (China, Japan, Korea, etc.). The unique feature of persimmon is that its fruit can accumulate tannins (PAs) during development. ), tannin is a polymer substance, and soluble tannin is the main substance that presents the astringency of persimmon fruit.

Persimmons can be divided into four categories according to their deastringency type and whether the seeds affect the astringency and color of the fruit: complete sweet persimmon (PCNA); complete astringent persimmon (PCA); partial sweet persimmon (PVNA); partial astringent persimmon (PVA). ). Sweet persimmon fruit loses the ability to synthesize tannin in the early stage of development, so it can be naturally deastringent on the tree. When ripe and harvested, the fruit does not have astringent taste and can be eaten directly;

The research on deastringency of persimmon fruit has achieved great success, such as wrapping the fruit with ethanol vapor on the tree, treating with high concentration of carbon dioxide or nitrogen, soaking in warm water or alternately freezing and thawing, etc. can complete deastringency. Moreover, studies have shown that high-concentration nitrogen dioxide treatment can make persimmon fruit crisp while removing astringency.

The mechanism of persimmon deastringency has been studied a long time ago. It has been reported that a large amount of acetaldehyde is accumulated during the deastringency treatment. Matsuo and Ito (1977) proposed that two relatively independent processes are involved in CO2 deastringency treatment. First, fruits accumulate a large amount of acetaldehyde through anaerobic respiration under carbon dioxide treatment conditions. Secondly, acetaldehyde combines with soluble tannins to form a non-astringent insoluble substance, which promotes the removal of astringency from the fruit. Matsuo et al. (1991) also proved that acetaldehyde produced in anaerobic respiration directly participated in the astringency of persimmons. Therefore, the condensation reaction of soluble tannins mediated by acetaldehyde is the core of deastringency of persimmon fruit. Yamada et al. (2002) believed that ADH and PDC are the key enzymes of acetaldehyde synthesis in persimmon fruit, but so far, the regulatory mechanism of ADH and PDC in the study of persimmon fruit astringency has not been reported.

Contents of the invention

The object of the present invention is to provide a method for cloning and transient expression of genes related to persimmon deastringency, which is achieved by the following steps:

1. Obtaining the 3' end sequences of ADH and PDC gene family members in persimmon fruit:

Firstly, extract the RNA of persimmon tissue (pulp, stem, leaf, and flower), reverse transcribe the 3'CDNA and 5'CDNA to obtain the cDNA template, and search the known ADH and PDC gene family member sequences of other plants on the NCBI database, Then use ClustalX (v 1.81) software to find the conserved interval and design a degenerate primer (SEQ ID NO: 1; SEQ ID NO: 2) between them, and clone two ADH gene partial coding fragments (SEQ ID NO: 39; SEQ ID N0:40);

According to the previously merged and cloned ADH coding region fragments and the published ADH and PDC gene family EST sequences on NCBI, design primers (SEQ ID NO: 3~16), and use the 3'RACE (rapid amplification of cDNA ends) cloning technology system , PCR amplification, fragment recovery, ligation, transformation, plating, and testing, and finally obtained the 3' end sequences of three ADH and four PDC gene family members (SEQ ID NO: 41~47).

2. Gene expression analysis of ADH and PDC gene family members in persimmon fruit during deastringency treatment of persimmon fruit: Firstly, complete persimmon carbon dioxide and ethylene deastringency treatment, pulp RNA extraction and cDNA synthesis, and use the UTR of the 3' end sequence obtained in step 2 Specific primers (SEQ ID NO: 17~30) were designed for the region, primer specificity detection, melting point curve analysis, gel electrophoresis analysis and quantitative PCR product re-sequencing confirmed the correctness of the sequence. Real-time quantitative PCR analysis of the expression patterns of members of the ADH and PDC gene families in the treatment and control.

3. Acquisition of full-length sequences of ADH and PDC gene family members in persimmon fruit and construction of transient expression system in persimmon leaves: design 5'RACE primers (SEQ ID NO: 31~34) according to the obtained 3' end sequence, and use step 1 The obtained 5'RACE cDNA was used as a template, and the 5'RACE (rapid amplification of cDNA ends) cloning technology system was used to finally obtain an ADH and a PDC 5' end sequence respectively. Design upstream primers and downstream primers (SEQ ID NO: 35-38) across the initiator and terminator respectively, and use the cDNA obtained in step 2 as a template to clone and obtain the full-length genes of ADH and PDC (SEQ ID NO: 48; SEQ ID NO: 49), select the plasmid with the full-length genes of ADH and PDC returned by the company for the following double enzyme digestion, construct the expression vector, conduction transformation, Agrobacterium preparation, glycerol bacteria storage at minus 80°C, Inoculate the preserved glycerol bacteria onto LB (kan50) plates, culture them at 28 ^o C for 2 days, and then transfer them to new LB (kan50) plates

The plate was cultured for 1 day (bring to Beijing), and the day before the experiment, it was transferred to a new LB plate for culture, and brought to the experimental site (the first village of Mopanshi, Fangshan, Beijing) to transfer the Agrobacterium on the LB (kan50) plate to the Put it in a 50ml centrifuge tube for injection (preparation of injection solution: 10 mM MgCl ₂ and 0.5 mM acetosyringone), shake well. The leaves of "Mopan persimmon" with the same maturity were selected, and the back of the leaves took the veins as the axis of symmetry, and the two sides were injected with SK and the target gene (DkADH1 (SEQ ID NO: 48) or DkPDC2 (SEQ ID NO: 49) gene) respectively. Bacillus culture medium. Three days later, the leaves were picked, brought back to the laboratory, cut along the edge of the injection, and quick-frozen with liquid nitrogen to sample separately. The samples were stored at minus 80°C for the next experiment. Each gene was injected with 10 persimmon leaves. The samples were tested for soluble tannin content to analyze the effect of transient expression in leaves.

The present invention utilizes the base sequences of alcohol dehydrogenase and pyruvate dehydrogenase genes known in other plants such as Arabidopsis thaliana and tomato, and designs amalgamative primers in relatively conserved regions to clone ADH and PDC partial coding regions. The EST sequences of persimmon ADH and PDC genes were cloned using 3'RACE technology to clone the 3' end sequences of ADH and PDC genes, and specific primers were designed in the UTR region of the 3' end sequences to analyze the expression of members of the ADH and PDC gene families , and then using 5'RACE technology to clone the full-length sequences of ADH and PDC genes, construct expression vectors, transform Agrobacterium, and then perform transient expression in persimmon leaves to verify gene functions. The method of the invention is to clone gene family members related to persimmon astringency removal, analyze the expression patterns of these family members in persimmon astringency removal treatment, and finally verify the function of the target gene through transient expression of persimmon leaves. This has clarified the regulation mechanism of postharvest astringency removal of persimmon from the molecular mechanism, which can be used to further carry out functional verification (transgenic, gene interaction, etc.) research on the mechanism of postharvest astringency removal of persimmon.

Description of drawings

Figure 1 is the gene expression pattern of ADH gene family members in persimmon fruit in the treatment of persimmon fruit carbon dioxide deastringency.

Figure 2 is the gene expression pattern of the members of the PDC gene family in persimmon fruit in the treatment of persimmon fruit carbon dioxide deastringency.

Figure 3 is the gene expression pattern of ADH gene family members in persimmon fruit under ethylene deastringent treatment.

Figure 4 is the gene expression pattern of the members of the PDC gene family in persimmon fruit under ethylene deastringent treatment.

Figure 5 shows the results of transient expression in persimmon leaves: analysis of soluble tannin content in persimmon leaves.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments and accompanying drawings, but the embodiments do not limit the protection scope of the present invention.

The conventional gene manipulation methods in the following examples refer to the "Molecular Cloning Experiment Guide" (third edition).

Example 1: Real-time quantitative fluorescent PCR analysis of the expression patterns of ADH and PDC family members in persimmons in different treatments;

(1) Experimental method

1. Extraction of total RNA from persimmon fruit

At the designed sampling points, control, ethylene and carbon dioxide treated fruit pulp were frozen in liquid nitrogen and stored at -80°C. When extracting RNA, weigh 2g of the frozen sample and add liquid nitrogen to fully grind it, then add it to two centrifuge tubes with 4ml β-mercaptoethanol/CTAB (80ul/4ml) extraction buffer, heat at 65°C, and vortex to mix the cells Completely rupture, heat again at 65°C for 1-2min; then add 4ml of chloroform:isoamyl alcohol (24:1) extract to the centrifuge tube, vortex quickly to mix evenly; centrifuge at 10,000rpm at 15°C for 10min, absorb the supernatant to a new Use chloroform: isoamyl alcohol (24:1) to re-extract the centrifuge tube once, absorb about 3ml of the supernatant, combine the two tubes into one tube, add 1/4 volume of 10mol/l LiCl ₂ , and refrigerate overnight at 4°C ; Centrifuge at 10000rpm at 4 °C for 30min, pour off the supernatant, centrifuge the centrifuge tube again briefly, suck off the excess solution at the bottom, add 400μl 65°C SSTE, flick the tube wall with your fingers to fully dissolve the precipitate, and transfer the liquid to the In a 1.5ml centrifuge tube, add 400μl chloroform: isoamyl alcohol (24:1) extract, vortex to mix; centrifuge at 10000rpm at 20°C for 10min, draw the supernatant into a new centrifuge tube, add 2 times the volume of - Pre-cooled absolute ethanol at 20°C, mix up and down, place at -80°C for 30min-1hour; centrifuge at 10000rpm at 4°C for 20min, pour off the supernatant, suck out the residual liquid after short centrifugation, and dry the precipitate in a fume hood (approx. 10min); add 15-30μl DEPC water to each tube to dissolve the precipitate, and obtain a crude RNA sample; use 1% agarose gel electrophoresis to test the quality of the obtained sample, and the good RNA result shows that the sample has two bands, the brightness is the upper bar: the lower bar ≈2:1. Use an ultraviolet spectrophotometer to test the purity of the obtained samples. Take 1 μl of RNA sample and add 69 μl of DEPC water to mix evenly. Measure OD ₂₆₀ and OD ₂₈₀ . The analysis shows that OD ₂₆₀ /OD ₂₈₀ =1.8-2.0, indicating that the RNA purity meets the standard. According to the formula OD ₂₆₀ × 40 (RNA concentration coefficient) × 70 (sample dilution factor), the concentration of sample RNA (ng/μl) can be calculated for the next experiment.

2. Analysis of the expression patterns of ADH and PDC family members in persimmon

Genomic DNA contamination was first removed using TURBO DNase (Ambion, Applied Biosystems), and iScript cDNA Synthesis Kit (Bio-Rad) reverse-transcribed cDNA; then the reverse-transcribed cDNA was drawn into 5 μl and diluted 10 times, and the upstream and downstream amplification primers DkACtin-FP and DkACtin-RP of the internal reference gene were diluted to 2.5 μM respectively; SYBR Green/ROX qPCR Master Mix (10 μl/reaction), DkACtin-FP (1 μl/reaction) and DkACtin-RP (1 μl/reaction), water (6 μl/reaction) to prepare mixed working solution, mix well , dispensed into each reaction tube; then add the corresponding 10-fold diluted cDNA template (2μl) into each reaction tube; briefly centrifuge to collect the reaction solution at the bottom of the tube, and perform Q-PCR; the reaction program is 95°C pre-denaturation for 3 minutes ; Denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec, extension at 95°C for 10 sec, 45 thermal cycles; melting curve analysis from 65°C to 95°C. According to the Ct value (Cycle threshold) corresponding to each template, adjust the template stock solution again so that the Ct value of all templates is between 23-24.

Dilute the real-time quantitative PCR primers of each family member of persimmon ADH and PDC to 2.5 μM respectively, prepare the working solution according to the method of DkACtin gene above, the CDNA template is the working solution with adjusted CT value, and each template must be set with ACtin control gene and negative control. The Q-PCR procedure is the same as before; after the reaction, according to the Ct value obtained for each template, the relative expression level of the gene can be obtained by using the formula 2 ^-(Ct2-Ct1) .

(2) Experimental results

2. The results of real-time quantitative fluorescent PCR showed that DkADH1 (SEQ ID NO: 41), DkPDC1 (SEQ ID NO: 44), and DkPDC2 (SEQ ID NO: 45) were positively related to the removal of astringent taste of fruits in carbon dioxide treatment and ethylene treatment. Correlation, that is, with the deastringency treatment of persimmon fruit, the soluble tannins gradually decreased, and the relative expression levels of DKADH1 (SEQ ID NO: 41) and DkPDC1 (SEQ ID NO: 44), DkPDC2 (SEQ ID NO: 45) Gradually increased, after the treatment was removed, there were different degrees of down-regulation (Fig. 1, Fig. 2, Fig. 3, Fig. 4).

Example 2: Construction of transient expression system in persimmon leaves.

(1) Experimental method Obtaining the full-length sequences of ADH and PDC gene family members in persimmon fruit:

Firstly, 5'RACE primers were designed according to the obtained 3' end sequences, and the 5' RACE (rapid amplification of cDNA ends) cloning technology system was used to use the 5'RACE cDNA as a template, and finally the 5' end sequences of ADH and PDC gene family members were obtained . Then use the 5' end sequence as a template to design upstream primers and downstream primers spanning the initiator and terminator respectively, using tissue cDNA as a template, PCR reaction system: template cDNA: 1ul, DNTP mix (2.5mM): 1.6ul, Upstream primer (10mM): 0.8ul, downstream primer (10mM): 0.8ul, Pyrobest DNA Polymerase: 0.1ul, 10×pyrobest Buffer: 2.0ul, DEPC water: 13.7ul. Amplify by PCR: pre-denaturation at 94°C for 5 minutes, 35 cycles (95°C for 30 s, 55°C for 30 s, 72°C for 180 s), and finally 72°C for 10 m, recover the PCR target product, add A to the recovered product, the steps are as follows: 10 ×A-Tail Buffer: 2.5ul, DNTP mixture (2.5mM): 2ul, A-Tailing Enzyme: 0.25ul. Then mix 2.5ul of the DNA fragments recovered above, DEPC water: 17.25ul, mix evenly, 20min at 72°C, and 2min on ice. The product is connected to the pGEM-Teasy vector (Promega, A1360), the connection system: 2×Rapid Ligation Buffer: 5ul, PGEM-T EASY Vetor: 1ul, the above product: 3ul, T4 DNA ligase 1ul. Ligate overnight at 4°C, transform the product into DH5α competent cells, carry out resistance screening, bacterial liquid detection, shake the single colony with the target fragment to the company for sequencing (Invitrogen), sequence analysis and confirm the full-length genes of ADH and PDC .

Construction of transient expression system for persimmon leaves: Construction of transient expression vectors for persimmon leaves Select the correct plasmid returned by the company for the following double enzyme digestion, double enzyme digestion system: Not1: 1ul; Spe1: 1ul; 10×H Buffer: 2ul; 0.1% BSA: 2ul; plasmid: 3ul; DEPC water: 11ul. The digested products were detected by electrophoresis and recovered. Ligation system: digested pGreenII 0029 62-SK: 1ul, digested plasmid: 4ul, Solution: 5ul. Connect overnight at 4°C, transform the product into Agrobacterium, spread the plate, screen for resistance, detect single colony liquid, select the successfully transformed Agrobacterium and add 1:1 glycerol, store at minus 80°C, and use it for the next experiment.

Inoculate the above-mentioned preserved glycerol bacteria onto LB (kan50) plate, culture at 28 ^o C for 2 days, then transfer to a new LB (kan50) plate for 1 day (bring to Beijing), and transfer to a new plate the day before the experiment Cultivate and bring to the experimental site (Mopan Shi No. 1 Village, Fangshan, Beijing), transfer the Agrobacterium on the LB (kan50) plate to a centrifuge tube filled with injection solution (10 mM MgCl ₂ ; 0.5 mM acetosyringone 50ml) and shake well . Mopan persimmon leaves with the same maturity were selected, and the back of the leaves took the veins as the axis of symmetry, and the two sides were respectively injected with the Agrobacterium culture solution containing SK and the target gene ( DkADH1 (SEQ ID NO: 48) or DkPDC2 SEQ ID NO: 49). 10 persimmon leaves were injected with each gene, and the leaves were picked three days later, brought back to the laboratory, cut along the injected edge of the leaves, and quick-frozen with liquid nitrogen to sample respectively. The samples were stored at minus 80°C, and the soluble tannin content of the samples was determined to analyze the effect of transient expression on leaves (see Figure 5).

For those skilled in the art, improvements or transformations can be made according to the above description, and all these improvements and transformations should belong to the protection scope of the appended claims of the present invention.

<110> Zhejiang University

<120> A method for cloning and transient expression of a persimmon off-astringency related gene

<160> 49

<210> 1

<211> 19

<212> base sequence

<213> Artificial sequence

<400> 1

TGGGADGCYAAGGGMCARA

<210> 2

<211> 20

<212> base sequence

<213> Synthetic

<400> 2

CCCCACCATCRTGRACACA

<210> 3

<211> 25

<212> base sequence

<213> Synthetic

<400> 3

TTGGAGAGGGAGTGACGGATCTTGC

<210> 4

<211> 28

<212> base sequence

<213> Synthetic

<400> 4

AGGGGTCATGCTCCATGATGCCAAATCA

<210> 5

<211> 27

<212> base sequence

<213> Synthetic

<400> 5

GATCATGTCATACCTTGTTATCAGGCA

<210> 6

<211> 27

<212> base sequence

<213> Synthetic

<400> 6

TCAGGCAGAATGCAGGGAGTGTAAATT

<210> 7

<211> 27

<212> base sequence

<213> Synthetic

<400> 7

CCTTGAGATGTGCAGACTTCGTTGTTA

<210> 8

<211> 26

<212> base sequence

<213> Synthetic

<400> 8

TAGTGCCATGGTTCTCTTGCCAATCA

<210> 9

<211> 27

<212> base sequence

<213> Synthetic

<400> 9

GAGTCCCATTCTCTCTTGCACCATTTG

<210> 10

<211> 25

<212> base sequence

<213> Synthetic

<400> 10

GATCAAGAAAGAGAAAGCAGTGATC

<210> 11

<211> 27

<212> base sequence

<213> Synthetic

<400> 11

TCCGAAGTTGACTAATGAGATGGGTTT

<210> 12

<211> 25

<212> base sequence

<213> Synthetic

<400> 12

TAGAGTTAGCCGACGCCAGTGGTTT

<210> 13

<211> 27

<212> base sequence

<213> Synthetic

<400> 13

CTTTCCAGTCCGTAAGAATGGCGGATG

<210> 14

<211> 25

<212> base sequence

<213> Synthetic

<400> 14

AAGCGCTTAGCAAGCCGGACAATTC

<210> 15

<211> 27

<212> base sequence

<213> Synthetic

<400> 15

TAAGATCGGAGCCGTGGAAGTGAGTCC

<210> 16

<211> 27

<212> base sequence

<213> Synthetic

<400> 16

TGCGTTTCCACCATCCAGAACTCTTGC

<210> 17

<211> 20

<212> base sequence

<213> Synthetic

<400> 17

GAGAAGTTCATCACGCACGA

<210> 18

<211> 20

<212> base sequence

<213> Synthetic

<400> 18

ACCAACTTGAGCCACCACTC

<210> 19

<211> 20

<212> base sequence

<213> Synthetic

<400> 19

TGGCTCTTGCACAGATCAAC

<210> 20

<211> 20

<212> base sequence

<213> Synthetic

<400> 20

GTGACGAGGAGGTTCACCAT

<210> 21

<211> 20

<212> base sequence

<213> Synthetic

<400> 21

CAGAACATCTGGGCACTGAA

<210> 22

<211> 20

<212> base sequence

<213> Synthetic

<400> 22

GCCAGCCAAAGAAAAAGAAA

<210> 23

<211> 20

<212> base sequence

<213> Synthetic

<400> 23

CCCATAACAGTTCCGAGAAA

<210> 24

<211> 20

<212> base sequence

<213> Synthetic

<400> 24

CTGCACCATCAACTGCAAAT

<210> 25

<211> 20

<212> base sequence

<213> Synthetic

<400> 25

GCCCACCAAATCCTCAGTAA

<210> 26

<211> 20

<212> base sequence

<213> Synthetic

<400> 26

CCAGAAAAGATGGGCACAAA

<210> 27

<211> 20

<212> base sequence

<213> Synthetic

<400> 27

CTTAGCAAGCCGGACAATTC

<210> 28

<211> 20

<212> base sequence

<213> Synthetic

<400> 28

GGACTGCTCGTTATGGAAGG

<210> 29

<211> 20

<212> base sequence

<213> Synthetic

<400> 29

CCTCCGTCACACAAATTTCA

<210> 30

<211> 20

<212> base sequence

<213> Synthetic

<400> 30

CCACGGCTCCGATCTTAGTA

<210> 31

<211> 19

<212> base sequence

<213> Synthetic

<400> 31

ACGCAAACCAACTGCGTAT

<210> 32

<211> 20

<212> base sequence

<213> Synthetic

<400> 32

ACCAACTTGAGCCACCACTC

<210> 33

<211> 20

<212> base sequence

<213> Synthetic

<400> 33

GGGGATCATAGCCCAACAAT

<210> 34

<211> 20

<212> base sequence

<213> Synthetic

<400> 34

CCAGAAAAGATGGGCACAAA

<210> 35

<211> 19

<212> base sequence

<213> Synthetic

<400> 35

ACGCAAACCAACTGCGTAT

<210> 36

<211> 20

<212> base sequence

<213> Synthetic

<400> 36

ACCAACTTGAGCCACCACTC

<210> 37

<211> 20

<212> base sequence

<213> Synthetic

<400> 37

GGGGATCATAGCCCAACAAT

<210> 38

<211> 20

<212> base sequence

<213> Synthetic

<400> 38

CCAGAAAAGATGGGCACAAA

<210> 39

<211> 711

<212> DNA sequence

<213> Persimmon (Diospyros kaki)

<400> 39

TGGGATGCTAAGGGACAGAATCCTGTGTTTCCTCGAATCCTCGGCCACGAGGCAGGAGGGATTGTGGAGAGTGTTGGAGAGGGAGTGACGGATCTTGCACCGGGAGACCATGTGCTTCCGGTGTTCACAGGAGAATGCAAAGAATGCGCCCACTGCAAGTCAGAGGAAAGCAACATGTGTGATCTGCTCAGGATCAACACCGACAGAGGGGTCATGCTCCATGATGCCAAATCAAGGTTTTCCATCAACGGCAAGCCCATTTACCACTTTCTTGGAACCTCCACTTTCAGTGAATACACCGTTATTCATGTCGGCTGCCTTGCCAAGATCAACCCCCTTGCTCCTCTTGACAAAGTGTGCATTATGAGCTGTGGATTCTGCACAGGATTTGGGGCCACCGTGAATGTTGCCAAGCCAAAAAGGGGCTCCTCTGTGGCTATTTTCGGATTGGGAGCTGTGGGCCTTGCGGCTGCGGAAGGAGCTAGAGTTTCTGGGGCGTCAAGGATCATCGGTGTCGACATCAATCCCAGCAGATTTGAACAAGCAAAGAAGTTCGGCGTGACAGAGTTCGTGAACCCCAAAGACCATAAGAAACCTGTTCAAGAGGTGATTGCTGATATTACTGATGGAGGAGTCGACAGAAGCGTCGAATGCACAGGAAATATCGATGCCATGATCTCCGCATTCGAATGTGTCCACGATGGATGGGGA

<210> 40

<211> 505

<212> DNA sequence

<213> Persimmon (Diospyros kaki)

<400> 40

GCACGAGGAAGAAGAGAACATGGCGGCTTGGCTTCTCGGCATCAACACCCTCAAGATTCAGCCCTTCAAGCTGCCCACCCTTGGTCCCGGTGATGTTAGAGTTAGGATGAAAGCTGTTGGTATTTGTGGCAGTGATGTTCACTACCTGAAGACCTTGAGATGTGCAGACTTCGTTGTTAAGGAGCCTATGGTTATTGGGCATGAGTGTGCCGGGATTATAGAAGAAGTTGGGAGCGAGGTGAAATCACTGGTGCCGGGAGACCGAGTTGCCTTGGAGCCCGGCATCAGTTGTTGGCGCTGCTACCACTGCAAAGAGGGCCGCTACAATCTATGCCCCGATATGAAGTTCTTCGCGACTCCTCCTGTCCATGGTTCTCTTGCCAATCAGGTAGTGCACCCAGCAGACCTGTGCTTTAAGCTGCCTGAGAAATGTGAGCTTGGAGGAAGGGGCAATGTGCGAGCCCCTTAGTGTTGGCGTTCACGCTTGTCGGCGCGCCAACATTGG

<210> 41

<211> 1052

<212> DNA sequence

<213> Persimmon (Diospyros kaki)

<400> 41

GCAGGAGGGATTGTGGAGAGTGTTGGAGAGGGAGTGACGGATCTTGCACCGGGAGACCATGTGCTTCCGGTGTTCACAGGAGAATGCAAAGAATGCGCCCACTGCAAGTCAGAGGAAAGCAACATGTGTGATCTGCTCAGGATCAACACCGACAGAGGGGTCATGCTCCATGATGCCAAATCAAGGTTTTCCATCAGCGGCAACCCCATTTACCACTTTCTTGGAACCTCCACTTTCAGTGAATACACCGTTATTCATGTCGGTTGCGTTGCCAAGATCAACCCCCTTGCTCCTCTTGACAAAGTCTGCATTATGAGCTGTGGATTCTGCACAGGCTTTGGGGCCACCGTGAATGTTGCCAAGCCAAAAAGGGGCTCCTCTGTGGCTATTTTCGGACTGGGAGCTGTGGGCCTTGCGGCCGCGGAAGGAGCTAGAGTTTCTGGGGCTTCAAGGATCATCGGTGTTGACATCAATCCCAGCAGATTTGAACAAGCAAAGAAGTTCGGCGTGACAGAGTTCGTGAACCCCAAAGACCATAAGAAACCTGTTCAAGAGGTGATTGCTGAGATTACCGATGGAGGAGTCGACAGAAGCGTCGAATGCACCGGAAACGTCGATGCCATGATCTCAGCATTCGAATGCGTCCATGATGGCTGGGGCGTTGCTGTTCTTGTGGGTGTACCCCACAAAGAGGCTGTTTTCAAGACACACCCAGTGAACTTCCTCAACGAGAGGACCCTAAAGGGGACCTTCTTCGGCAACTTCAAGCCTCTCTCGGACCTTCCTGGTGTTGTGGAAAAATACATGAACAAGGAGCTGGAATTGGAGAAGTTCATCACGCACGAGGTGCCATTCTCGGAGATCAACAAGGCCTTCGATTACATGCTTAAAGGGGAAAGCCTTCGATGCATCATCCGAATGGAATAACAGAGTGGTGGCTCAAGTTGGTCATATCCGAGGATGTAGTGGAGTTGCATCGTCTGTAAACTGATGAATAAAC TAATGAATAAGAGTTGTTGCCCAGTAAAAAAAAAAAAAAAAAAAAAAAAAAA

<210> 42

<211> 1444

<212> DNA sequence

<213> Persimmon (Diospyros kaki)

<400> 42

GCACGAGGCACATTTCTGTCTCCCCCTTCTCTCTCTCTCTCTCTCTCTCTCGGTCTCTCTCTTCCGTCCTGTATCCATGGCTACTCAAGGTCAGGTCATAACCTGCAAAGCTGCGGTGGCCTGGGAACCCAACAAGCCTTTGGCGATCGAAGACGTCCAGGTGGCTCCGCCGCAGGCCGGCGAAGTTCGAATCCAGATCCTTTTCACCGCACTTTGCCACACTGATGCTTATACTTGGAGCGGCAAGGATCCTGAGGGTCTTTTTCCATGTATTCTTGGTCATGAAGCTGCTGGGATTGTAGAGAGTGTTGGTGAAGGTGTAACTGAGGTTCATCCTGGTGATCATGTCATACCTTGTTATCAGGCAGAATGCAGGGAGTGTAAATTCTGCAAATCAGGAAAAACAAACCTCTGTGGTAAAGTTAGGGCTGCCACAGGTGTTGGAGTCATGATGAATGATCGCAAGAGTCGTTTCTCCATCAATGGAACTCCAATTTATCATTTCATGGGAACTTCAACATTTAGTCAATACACTGTTGTCCATGATGTTAGTGTTGCAAAGATCGATCCAATAGCCCCCTTGGAGAAGGTGTGCCTTCTAGGTTGTGGTGTTCCTACCGGTCTAGGAGCGGTTTGGAACACAGCAAAAGTAGAGCCAGGTTCCATTGTTGCTATTTTTGGCCTAGGGACTGTTGGCCTTGCAGTGGCCGAGGGTGCAAAATCAGCTGGGGCTTCACGAATTATTGGTATTGATATTGATAGCAAAAAGTTTGATACAGCGAAGAATTTTGGAGTCACTGAATTTGTGAATCCAAAAGACCATGACAAACCAATACAGCAGGTTATCGTTGATCTCACAGATGGTGGTGTGGACTACAGTTTTGAGTGCATTGGAAATGTCTCTGTAATGAGATCTGCTTTGGAGTGCTGTCACAAAGGATGGGGTACCTCTGTTATTGTGGGCGTCGCTGCTTCTGGTCAAGAGATATCGACCCGCCCT TTCCAGTTGGTGACTGGCCGAGTCTGGAAAGGAACCGCATTTGGCGGGTTCAAGAGCCGCTCACATGTGCCTTGGCTTGTTGACAAGTACCTGAAGAAGGAAATCAAGGTGGACGAATACATCACCCACAATCTGGCTCTTGCACAGATCAACGAAGCTTTTCATTTGATGCATGAAGGGGGCTGTTTCCGCTGTGTTCTGAAGATGGCTTGAGCCTTGAGATGATTATCACACCATCCCGTCCTATCCCTTGTTTGGATATTATATGCTCATCTTTGATGATTGCCCTCTACCTACATGGTGAACCTCCTCGTCACTCTGACTGCTTACTTCTCAATAAGCAACACGTAAGGACGATGGTCTGTGTGCTTTGTTTTTGTGCTATTATTTATGTGTTTCTCTACTATTTTATGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

<210> 43

<211> 1348

<212> DNA sequence

<213> Persimmon (Diospyros kaki)

<400> 43

GCACGAGGAAGAAGAGAACATGGCGGCTTGGCTTCTCGGCATCAACACCCTCAAGATTCAGCCCTTCAAGCTGCCCACCCTTGGTCCCGGTGATGTTAGAGTTAGGATGAAAGCTGTTGGTATTTGTGGCAGTGATGTTCACTACCTGAAGACCTTGAGATGTGCAGACTTCGTTGTTAAGGAGCCTATGGTTATTGGGCATGAGTGTGCCGGGATTATAGAAGAAGTTGGGAGCGAGGTGAAATCACTGGTGCCGGGAGACCGAGTTGCCTTGGAGCCCGGCATCAGTTGTTGGCGCTGCTACCACTGCAAAGAGGGCCGCTACAATCTATGCCCCGATATGAAGTTCTTCGCGACTCCTCCTGTCCATGGTTCTCTTGCCAATCAGGTAGTGCACCCAGCAGACCTGTGCTTTAAGCTGCCTGAGAATGTGAGCTTGGAGGAAGGGGCAATGTGCGAGCCCCTTAGTGTTGGCGTTCACGCTTGTCGGCGCGCCAACATTGGTCACGAAACCAATGTTTTGATCATGGGAGCCGGTCCCATAGGTCTGGTAACGCTGCTTGCAGCTCGTGCCTTTGGAGCTCCCAGAATAGTCATCGTGGATGTAGATGACAACCGTCTCTCAGTTGCCAAGGAAGTCGGTGCAGATGAAACTATTAAAGTTTCAACAAGCATCCAGGATGTGAGCAAGGATGTAGAACAGATTCTTAAAACTATGGGGGGTGCAAGAGTGGATGTGACCTTTGATTGTGCCGGCTTTAACAAAACCATTTCGACAGCTCTAAGCTCCACTCGTTCAGGTGGAAGAGTTTgCATTGTGGGAATGGGTCACCACGAAGTAACTGTTCCCCTCACCCCAGCTGCTGCAAGGGAGGTTGATTTGATTGGAGTGTTCCGATACAAGAACACATGGCCGCTTTGCCTTGAATTCCTCAGCAGTGGAAAGATTGATGTGAAGCCCTTAATAACCCACAGATTCGGGTTCTCGCAGCAGGAGGTT GAAGAAGCTTTTGAGACCAGTGCTCGCGGTGGCAGCGCCATTAAGGTCATGTTCAATCTATAGACTCTGCAGAACATCTGGGCACTGAAGTTTGCCTCTTCTATGAAAAATTTGAAGTGAAAGGAATATGGTAGAGAATGAAGTTACCCTAAACTCTAAACCCTTAACATGGTGGAGAATGAAGTAATTGTATGAATAATGATCTCAGTAACCTTGAAAGCCTTTTATCTGTCTGGTTTCTTTTTCTTTGGCTGGCTAATAAaTTGGAGTTGTAAGCTTGCCATCTGTTCTTAGTTGGATGAGATTTATGAAATATACAATATTTCACTCCCAAAAAAAAAAAAAAAA

<210> 44

<211> 1552

<212> DNA sequence

<213> Persimmon (Diospyros kaki)

<400> 44

TGAAGGAAAGCAAGCCTGTATATATCAGCATAAGTTGTAATCTGCCTGGAATTCCTCATCCAACATTTGCCAGGGAGCCAGTCCCATTCTCTCTTGCACCATTTGTGAGCAATGAATGTGGATTAGAAGCTGCAGTGGAAGCAACAGCTGAGTTTTTGAACAAGGCTGTCAAGCCTGTCCTTGTGGGAGGTCCCAAGCTAAGGGTAGCAAAGGCACAGCAGGCGTTTATGGAGCTTGCTGATGCATGTGGATACCCAATTGCAGTAATGCCTTCAGGAAAAGGACTGGTTCAGGAGCACCACCCACACTTCATCGGGACATACTGGGGTGCTGTTAGCACCAGCTGCTGTGGGGAGATCGTGGAATCTGCTGATGCTTATGTTTTTGTCGGTCCAATCTTTAATGATTACAGTTCAGTTGGATATTCTTTGCTGATCAAGAAAGAGAAAGCAGTGATCGTACGGCCTAACCGTGTGACTATTGGTAATGGGCCGTCCCTGGGTTGGGTTTTTATGGCAGACTTCTTAAGGGCACTGGCCAAGAAGCTAAAGAAGAACAGTACAGCTCTGGAGAATTACCGCCGTATATATGTCCCTCCGGGCATTGCTCTGAAACGTGATAATAATGAACCTCTAAGAGTCAACATACTCTTCAAGCACATTCAGGACTTGCTAGGTGGAGACACTGCAGTGATTGCAGAGACAGGGGACTCATGGTTTAATTGTCAGAAGCTCAAGCTTCCAGAAAATTGCGGGTATGAATTCCAGATGCAGTATGGATCTATTGGGTGGTCTGTTGGTGCTACACTTGGTTATGCTCAGGCTGCCAAGGACAAGCGTGTGATTGCTTGCATTGGTGATGGAAGTTTCCAGGTGACAGCTCAGGATATTTCTACAATGATTCGCTGCGGGCAAAGGAGCATCATATTCCTCATCAACAATGGAGGCTATACGATCGAAGTTGAGATTCATGACGGACCATACAACGTGATCAAGAACTG GGACTACACTGGTCTCGTTGATGCTATTCACAACGGTGAAGGCAAATGCTGGACTGCCAAGGTATGCACAGAGGAACAGCTGGTTGAAGCAATTGCGACAGCAACGGGAGCTCACAAAGACTCGTTATGTTTCATTGAAGTTTTGGTTCACAAGGACGATACGAGCAAAGAGCTGCTGGAATGGGGATCCCGAGTCGCTTCTGCCAACAGCCGCCCTCCAAATCCTCAATAAAACCGGAGTTCTCCCAGACCAGGTTTTCCCAGGATCGATCGAGACTTGTGCCTTGACATTCAGGTAATCAGCTTTGTACCCATAACAGTTCCGAGAAATAAAAGAAAGCAGATAGCTACTTGATTACTGGCTGTACTTTGCAATATTCAGGAGAAGATAATGATCAGATACTGTAATAAAGAACCATTGTCTCTGTTTAGGACTAGTATGTTCCATATTAGTCGCACTTGTTTGTCTTTGAGCTGAATGTAATTTGCAGTTGATGGTGCAGTTAGAAGAATTCTTCATTATAGCCAAAAAAAAAAAAAAAAAAAAAAAAAA

<210> 45

<211> 1551

<212> DNA sequence

<213> Persimmon (Diospyros kaki)

<400> 45

GCACGAGGATCGACACAGCGATATCAACGGCATTGAAGGAGAGCAAGCCTGTTTATATCAGCGTCGGCTGTAACCTGCCGGCGATTCCACACCCTACTTTCAGCCACGACCCAGTTCCATTTGCTCTGTCTCCGAAGTTGACTAATGAGATGGGTTTGGAGGCTGCAGTGGAAGCAGCTGCAGCGTTCTTGAACAAGGCCGTGAAGCCAGTAATGGTGGGCGGGCCAAAGCTGCGAGTGGCGAAGGCTTCCGAGGCGTTCGTAGAGTTAGCCGACGCCAGTGGTTTCGCGCTGGCCGTGATGCCGTCGGCAAAAGGACTGGTGCCGGAGCACCATCCCCATTTCATCGGTACTTACTGGGGTGCGGTGAGCACGGCCTTCTGCGCAGAAATTGTGGAGTCTGCCGATGCCTACCTGTTTGCAGGACCCATTTACAACGACTACAGCTCCGTTGGCTACTCCCTGCTCCTGAAGAAGGAGAAGATGATCCTAGTCCAGCCCGATCGCGTGTTGATCGGAAATGGCCCTGCATTTGGGTGTATTCTGATGAAGGATTTCTTGAAAGCATTAGCCAAGCGGCTTAAGCACAACAAGACTGCCTTCGAGAACTATCATCGGATCTATGTTTCTGAGGGACATCCTCTCAAGTGTGAGCCTAGGGAGCCCTTGAGAGTCAATGTTCTGTTCCACCACATTCAGAATATGCTGTCGAGCGACACCGCCGTGATTGCCGAGACAGGGGATTCTTGGTTCAATTGCCAGAAGCTTAAATTACCAGAGGGATGTGGATATGAGTTCCAGATGCAGTATGGATCCATTGGCTGGTCAGTTGGTGCAACTCTGGGATATGCTCAGGCTGGGAAGAATAAGCGAGTGATTGCTTGCATTGGTGATGGTAGCTTCCAGGTGACTGCACAAGATATTTCAACAATGCTGAGATGTGGACAAAACACCATCATCTTCCTCATTAACAATGGAGGATACACTATAGAAGTCGAAAT CCATGATGGGCCTTACAATGTGATCAAGAACTGGAACTACACCGGATTGGTAGATGCCATCCACAACGGCCAAGGCAAATGCTGGACAACTAAGGTGCATTGTGAGGAAGACCTAATTGAAGCAATTGACACAGCAACAGGGGCCAAGAAAGATTGCCTCTGCTTCATAGAAGTGATCGTTCACAAGGATGACACAAGCAAAGAGCTGCTTGAATGGGGCTCCAGGGTCTCTGCTGCCAACAGCCGCCCACCAAATCCTCAGTAATTCTCTGGGCTTGCAGCTCTTGGGTGGATTAAGTCCTAATAAATATGTTTTGTAAATTTGTATTCAGCCACTGGCTTGCATCTCCTGTAACTATACGTAAACTGAGTTCCTTTTTTAGTTTTGTGCCCATCTTTTCTGGACCATTTTTAGTTATTTCCTGATCATTCTAGGAATTGGAGCCTAGAATATTAAGCGTTAAGACTCTTTAGAGTGAAGATTTATGTTTTTCAAAAAAAAAAAAAAAAAAAAAAAAAAA

<210> 46

<211> 1109

<212> DNA sequence

<213> Persimmon (Diospyros kaki)

<400> 46

ATTTGCTCTCTATCTATCGCTCTCGCGCTCTCTCTCTCTCTCTCACTGATTGAAAAGTTCCTTTCCAGTCCGTAAGAATGGCGGATGGCAGCCACCAAGCGCTTAGCAAGCCGGACAATTCGTCGCCGCTACTCGACGGCAACGCGATGGTGGCAAAGGCCCTGGCGCGCGCCGGGGTGGATCGGATGTTCGGTGTGGTGGGGATTCCGGTGACCTCCGTGGCCACCCGCGCCGTCGCCATGGGTGTCCGCTTCATCGCCTTCCATAACGAGCAGTCCGCCGGATACGCCGCCTCCGCCTACGGGTACCTCACTGGACGCCCCGGCGTGCTTCTCACCGTATCCGGCCCCGGCTGCGTCCACGGCTTAGCAGGCCTCTCGAACGCCGCCGTTAACGCCTGGCCGATGGTCCTGATCTCCGGCTCCTGCGATCAGAAAGATTTCGGCCGCGGCGACTTCCAGGAGCTGGATCAGATTGCCACCGTGAAGCCGTTCTCCAAATTCTCCGCCAAACCCACGAGTATTAAAGAAATCCCTAATTGCGTGTTCCAAGTTCTAGGTCACGCTGTCTCGGGTCGGCCCGGTGGGTGTTACTTGGACCTTCCGTCCGACGTACTTCACGAAACGATCACCGAATCTGAAGCCGAGAAGTTGCTTCTTGAAGCTGAGAGTGGTTGGCCACAACCGAATGCGATTGGAACTGTTGGCTGTTCCGAGATTGAGGCGGCGGTCTCGTTGCTCCGTCACGCTGAGCGGCCGTTGATTGTGTTCGGCAAAGGTGCTGCGTTTGCTCGGGCTGAGAATGGACTGAGGAAATTTGTAGAGAGTACTGGGATTCCGTTCTTGCCCACTCCAATGGGCAAGGGTTTGTTGCCGGACACCCACGAGCTCGCTGCCACGGCGGCAAGGTCGCTGGCAATCGGCAAATGCGACGTGGCGCTGGTAGTTGGTGCCCGGCTCAATTGGCTGTTACATTTTGGCGAGCCACCAAAGTGGTCCAA GGATGTGAAGTTTATCTTGGTTGATATAAGCAAGGAAGAGATTGAGATGCGAAATCCGTGTTTGGGATTGGTTGGTGATGCAGCAAAAAAAAAAAAAAAAAAAAAAAAA

<210> 47

<211> 1002

<212> DNA sequence

<213> Persimmon (Diospyros kaki)

<400> 47

AAGACGCCGCCCCTGGTTTCTTCGTCGTCACCCTTATAAATACTCTCTCCTCCGTCACACAAATTTCATCATAGCCCAACAATTCTTCATCATCCCAACCTTCTCTGTAATCCATTTCTGTAGATTTTCTAGCTTCTGGTCAAGTAATTAAGATGGATACTAAGATCGGAGCCGTGGAAGTGAGTCCAGGTCCCAGCAGCGACGCCATCGGCTGCCTGCCCGGTAACGGCTGCGTTTCCACCATCCAAAACTCTTGCCCCTCGACGGCGCTCGTCCCGTCCGAGGCGACGCTGGGGCGCCATCTAGCGCGGCGGCTGGTCCAGATTGGGGCAACGGACGTCTTCTCCGTCCCTGGGGACTTCAACCTGACGTTGCTGGACCACCTGATCGCCGAGCCGGGGCTCAACCTCATCGGCTGCTGCAACGAGCTCAACGCCGGGTATGCGGCCGATGGGTTCGCGCGGTGCCGCGGCGTCGGCGCCTGTGTTGTGACGTTCACCGTCGGCGGCCTGAGCGTGCTCAACGCCATCGCCGGGGCCTACAGCGAAAACCTTCCGGTGATCTGCATCGTTGGCGGCCCCAACTCCAACGATTACGGGACGAACAGAATCCTCCACCACACCATCGGACTTCCCGATTTCAGCCAGGAGCTTCGCTGCTTTCAAACTGTTACTTGCTACCAGGTCAGTTCACCAATATATGCGGCCATAGCCATATCCGCATCGTTAGGATAATTCTGATACACACTGAATTGGGTCTCTTAATCAGATCTGAATTTGAGAAGCTATCTAAGATACATTAATCTAGTTTTGTTAGGAGTGTTCAAAAAAATTAGTACACTGCCAAAAATTGACGAAATCAAACCGATCCGATCGATTTTTTTTTTGGCGGTCCAAAATGGTTTGGATTCTGTCCAAACCGTGCGGTTTGCGGTTTGGACAGTTGGCGGTTCGGTTTTAAACCGAACCGCCCAAGAGAAAAAAAAAAAAAAAAAAAAAAA AAA

<210> 48

<211> 1345

<212> DNA sequence

<213> Persimmon (Diospyros kaki)

<400> 48

ACATGGGGACCCCACGCAAACCAACTGCGTATATATTACATAAAGAAAGTTCTGAGAAAAAGCAAATTAAGCCCACAGAAATGGCAGCCACCGCTGGTCAAGTGATTGTTTGTAAAGCTGCCGTGGCATGGGAAGCGGGGAAACCCCTGGTGCTGGAAGATGTGGAGGTGGCGCCGCCACAGAAGATGGAGGTCCGCATCAAGATCCTCTATACCTCTCTCTGCCACACCGACGTCTACTTCTGGGAAGCCAAGGGTCAGAATCCTGTGTTTCCTCGAATCCTCGGCCACGAGGCAGGAGGGATTGTGGAGAGTGTTGGAGAGGGAGTGACGGATCTTGCACCGGGAGACCATGTGCTTCCGGTGTTCACAGGAGAATGCAAAGAATGCGCCCACTGCAAGTCAGAGGAAAGCAACATGTGTGATCTGCTCAGGATCAACACCGACAGAGGGGTCATGCTCCATGATGCCAAATCAAGGTTTTCCATCAGCGGCAACCCCATTTACCACTTTCTTGGAACCTCCACTTTCAGTGAATACACCGTTATTCATGTCGGTTGCGTTGCCAAGATCAACCCCCTTGCTCCTCTTGACAAAGTCTGCATTATGAGCTGTGGATTCTGCACAGGCTTTGGGGCCACCGTGAATGTTGCCAAGCCAAAAAGGGGCTCCTCTGTGGCTATTTTCGGACTGGGAGCTGTGGGCCTTGCGGCCGCGGAAGGAGCTAGAGTTTCTGGGGCTTCAAGGATCATCGGTGTTGACATCAATCCCAGCAGATTTGAACAAGCAAAGAAGTTCGGCGTGACAGAGTTCGTGAACCCCAAAGACCATAAGAAACCTGTTCAAGAGGTGATTGCTGAGATTACCGATGGAGGAGTCGACAGAAGCGTCGAATGCACCGGAAACGTCGATGCCATGATCTCAGCATTCGAATGCGTCCATGATGGCTGGGGCGTTGCTGTTCTTGTGGGTGTACCCCACAAAGAGGCTGTTTTCAAGAC ACACCCAGTGAACTTCCTCAACGAGAGGACCCTAAAGGGGACCTTCTTCGGCAACTTCAAGCCTCTCTCGGACCTTCCTGGTGTTGTGGAAAAATACATGAACAAGGAGCTGGAATTGGAGAAGTTCATCACGCACGAGGTGCCATTCTCGGAGATCAACAAGGCCTTCGATTACATGCTTAAAGGGGAAAGCCTTCGATGCATCATCCGAATGGAATAACAGAGTGGTGGCTCAAGTTGGTCATATCCGAGGATGTAGTGGAGTTGCATCGTCTGTAAACTGATGAATAAACTAATGAATAAGAGTTGTTGCCCAGTAAAAAAAAAAAAAAAAAAAAAAAAAAA

<210> 49

<211> 2173

<212> DNA sequence

<213> Persimmon (Diospyros kaki)

<400> 49

ACATGGGGATCATAGCCCAACAATTCTTCATCATCCCAGCTTTCTCTGTAATCCATTTCTGTAGATTTTCTAGCTTCTGGTCAAGTAATTAAGATGGATACTAAGATCGGAGCAGTGGAAGTGAGTCCAGGTCCCAGCAGCGACGCCATCGGCTGCCTGCCCGGTAACGGCTGCGTTTCCACCATCCAAAACTCTTGCCCCTCGACGGCGCTCGTCCCGTCCGAGGCGACGCTGGGGCGCCATCTAGCGCGGCGGCTGGTCCAGATTGGGGCAACGGACGTCTTCTCCGTCCCTGGGGACTTCAACCTGACGTTGCTGGACCACCTGATCGCCGAGCCGGGGCTCAACCTCATCGGCTGCTGCAACGAGCTCAACGCCGGGTATGCGGCCGATGGGTTCGCGCGGTGCCGCGGCGTCGGCGCCTGTGTTGTGACGTTCACCGTCGGCGGCCTGAGCGTGCTCAACGCCATCGCCGGGGCCTACAGCGAGAACCTTCCGGTGATCTGCATCGTTGGCGGCCCCAACTCCAACGATTACGGGACGAACAGAATCCTCCACCACACCATCGGACTTCCCGATTTCAGCCAGGAGCTTCGCTGCTTTCAAACTGTTACTTGCTACCAGGCTGTTGTAAATCACTTGGAGGATGCGCATGAACTGATCGACACAGCGATATCAACGGCATTGAAAGAAAGCAAGCCTGTTTATATCAGCGTCGGCTGTAACCTGCCGGCGATTCCACACCCTACTTTCAGCCACGACCCAGTTCCATTTGCTCTGTCTCCGAAGTTGACTAATGAGATGGGTTTGGAGGCTGCAGTGGAAGCAGCTGCAGCGTTCTTGAACAAGGCCGTGAAGCCAGTAATGGTGGGCGGGCCAAAGCTGCGAGTGGCGAAGGCTTCCGAGGCGTTCGTAGAGTTAGCCGACGCCAGTGGTTTCGCGCTGGCCGTGATGCCGTCGGCAAAAGGACTGGTGCCGGAGCACCATCCCCATTTCATCG GTACTTACTGGGGTGCGGTGAGCACGGCCTTCTGCGCAGAAATTGTGGAGTCTGCCGATGCCTACCTGTTTGCAGGACCCATTTACAACGACTACAGCTCCGTTGGCTACTCCCTGCTCCTGAAGAAGGAGAAGATGATCCTAGTCCAGCCCGATCGCGTGTTGATCGGAAATGGCCCTGCATTTGGGTGTATTCTGATGAAGGATTTCTTGAAAGCATTAGCCAAGCGGCTTAAGCACAACAAGACTGCCTTCGAGAACTATCATCGGATCTATGTTTCTGAGGGACATCCTCTCAAGTGTGAGCCTAGGGAGCCCTTGAGAGTCAATGTTCTGTTCCACCACATTCAGAATATGCTGTCGAGCGACACCGCCGTGATTGCCGAGACAGGGGATTCTTGGTTCAATTGCCAGAAGCTTAAATTACCAGAGGGATGTGGATATGAGTTCCAGATGCAGTATGGATCCATTGGCTGGTCAGTTGGTGCAACTCTGGGATATGCTCAGGCTGGGAAGAATAAGCGAGTGATTGCTTGCATTGGTGATGGTAGCTTCCAGGTGACTGCACAAGATATTTCAACAATGCTGAGATGTGGACAAAACACCATCATCTTCCTCATTAACAATGGAGGATACACTATAGAAGTCGAAATCCATGATGGGCCTTACAATGTGATCAAGAACTGGAACTACACCGGATTGGTAGATGCCATCCACAACGGCCAAGGCAAATGCTGGACAACTAAGGTGCATTGTGAGGAAGACCTAATTGAAGCAATTGACACAGCAACAGGGGCCAAGAAAGATTGCCTCTGCTTCATAGAAGTGATCGTTCACAAGGATGACACAAGCAAAGAGCTGCTTGAATGGGGCTCCAGGGTcTCTGCTGCCAACAGCCGCCCACCAAATCCTCAGTAATTCTCTGGGCTTGCAGCTCTTGGGTGGATTAAGTCCTAATAAATATGTTTTGTAAATTTGTATTCAGCCACTGGCTTGCATCT CCTGTAACTATACGTAAACTGAGTTCCTTTTTTAGTTTTGTGCCCATCTTTTCTGGACCATTTTTTAGTTATTTTCCTGATCATTCTAGGAATTGGAGCCTAGAATATTAAGCGTTAAGACTCTTTAGAGTGAAGATTTATGTTTTTCAAAAAAAAAAAAAAAAAAAAAAAAAA

Claims (2)

1. a cDNA who participates in the gene PDC that persimmon takes away the puckery taste, its nucleotide sequence is numbered: SEQ ID N0:49.

2. the application that cDNA according to claim 1 is preparing persimmon and takes away the puckery taste in enzyme.