Promoter for specific and high-efficiency expression in persimmon and its application

Abstract

The present invention discloses a promoter for specific and efficient expression in persimmon and its application, belonging to the field of genetic engineering technology. It provides a promoter for specific and efficient expression in persimmon, including a promoter 2×35S that drives Cas9 expression and / or a promoter DKU6-5 that drives sgRNA expression. The nucleotide sequence of the promoter 2×35S is shown in SEQ ID NO.1, and the nucleotide sequence of the promoter DKU6-5 is shown in SEQ ID NO.2. The present invention addresses the problem of low gene editing efficiency in persimmon trees in the prior art and provides a promoter that can efficiently drive persimmon gene editing to improve the efficiency of gene editing.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a promoter for specific and efficient expression in persimmon and its application. Background Art

[0002] Persimmon (Diospyros kaki Thunb.) is a representative species of cultivated fruit trees in the genus Diospyros. Existing persimmon varieties are divided into fully sweet persimmons (PCNA) and non-fully sweet persimmons (non-PCNA), depending on whether the fruit naturally loses its astringency on the tree during ripening and its genetic characteristics. Fully sweet persimmons are a natural mutation of non-fully sweet persimmons. Because their fruit can be harvested crisply after ripening without any processing, they have higher economic value than astringent persimmons and are currently the primary target of persimmon industry development and genetic improvement.

[0003] Persimmon is a perennial tree crop of the genus Diospyros in the family Ebenaceae. Its ploidy is complex, and conventional hybrid breeding is time-consuming and laborious. Although there are methods to shorten the breeding period, breakthroughs are still needed in the rapid and targeted creation of new germplasm.

[0004] CRISPR / Cas9 is a third-generation targeted genome editing technology. Its advantages include flexibility, efficiency, low production costs, and high specificity, greatly facilitating the study of gene function. Through continuous improvement and development, this technology has been widely applied in various fields.

[0005] A promoter is a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription. It contains conserved sequences required for specific RNA polymerase binding and transcription initiation. It is primarily categorized as constitutive, tissue-specific, and inducible. Constitutive promoters regulate the expression of structural genes to a certain degree, with little difference in expression levels in different parts or tissues (e.g., CMV, U6, etc.). Tissue-specific promoters cause genes to be expressed only in certain organs or tissues, and exhibit developmental regulation (e.g., CaMV 35S). Inducible promoters significantly increase gene transcription levels under the regulation of certain physical or chemical signals.

[0006] Gene editing in plants typically requires the simultaneous construction of both a Cas gene expression cassette and a sgRNA expression cassette. Cas gene expression is typically driven by an RNA polymerase II promoter, such as 35S or Ubi, terminated by a Nos terminator or other terminators. The sgRNA expression cassette is relatively small and is typically driven by an RNA polymerase III promoter, such as U3 or U6, terminated by six or more consecutive T bases. Alternatively, the strongly constitutive AtUBQ10 gene can be used to drive its expression.

[0007] Improving the expression levels of CRISPR gene-editing vector components in plant cells can improve gene editing efficiency. This can be achieved by replacing the promoters driving the Cas nuclease and sgRNA. Using the YAO promoter to replace the 35S promoter to drive SpCas9 in citrus increased mutation efficiency by nearly 25-fold, all other conditions remaining unchanged. Commonly used promoters driving sgRNAs in CRISPR gene-editing vectors in plants are AtU6-26, AtU6-29, and AtU3 from Arabidopsis thaliana, while using OsU6 and OsU3 from rice produces better editing results in grasses. Because sgRNA promoters are species-specific, identifying a species-specific promoter that effectively drives sgRNAs can improve gene editing efficiency. CRISPR / Cas gene editing technology has been applied to various horticultural crops, such as kumquat, apple, and grape, but no reports have been reported in Diospyros. Summary of the Invention

[0008] To solve the above technical problems, the present invention proposes a promoter that is specific and efficiently expressed in persimmon, wherein the promoter includes the promoter 2×35S that drives Cas9 expression and / or the promoter DKU6-5 that drives sgRNA expression. In view of the problem of low gene editing efficiency in persimmon trees in the prior art, a promoter that can efficiently drive gene editing in persimmon is provided to improve the efficiency of gene editing.

[0009] To achieve the above objectives, the present invention provides a promoter for specific and efficient expression in persimmon, wherein the promoter includes the promoter 2×35S that drives Cas9 expression and / or the promoter DKU6-5 that drives sgRNA expression, the nucleotide sequence of the promoter 2×35S is shown in SEQ ID NO.1, and the nucleotide sequence of the promoter DKU6-5 is shown in SEQ ID NO.2.

[0010] The invention also provides an expression vector containing the persimmon-specific and highly efficient expression promoter.

[0011] Preferably, the expression vector is pCambia1300 vector and / or pggk vector.

[0012] The present invention also provides a host bacterium containing the expression vector.

[0013] Preferably, the host bacteria is Escherichia coli DH5α or Agrobacterium tumefaciens EHA105 monoclonal cell line.

[0014] The present invention also provides application of the promoter in increasing the expression level of exogenous genes in persimmon.

[0015] The present invention also provides application of the expression vector in increasing the expression amount of exogenous genes in persimmon.

[0016] The present invention also provides application of the host bacteria in increasing the expression level of exogenous genes in persimmon.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects:

[0018] This study screened two promoters that are highly effective in driving gene editing in persimmons. The 2×35S promoter, used to drive SpCas9, is more efficient in driving SpCas9 expression in persimmons than other promoters commonly used in gene editing systems. The DKU6-5 promoter, used to drive sgRNA expression, is more efficient than the AtUBQ10 promoter in persimmons. This study optimizes the persimmon gene editing system to improve its efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 The figure shows the analysis of GUS activity driven by DlActin promoters of different lengths, where A is the pDIACT 500 bp promoter, B is the pDIACT 1000 bp promoter, and C is the pDIACT 1500 bp promoter;

[0021] Figure 2 Analysis of SpCas9 expression driven by different PolⅡ promoters in persimmon. In the figure, abc represents the significance of the difference.

[0022] Figure 3 The sequence alignment results of Arabidopsis thaliana U6 and U3 promoters and persimmon U6 promoter;

[0023] Figure 4 Analysis of sgRNA expression driven by different PolⅢ promoters in persimmon. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] Example 1

[0030] Screening of high-activity SpCas9 promoters driven by persimmon

[0031] 1. Construction of GUS vectors with different lengths of Junzi Actin gene promoter

[0032] Using Junqianzi DNA as a template, Actin promoters with lengths of 500 bp, 1500 bp, and 1000 bp were amplified starting from the transcription start site. The reaction system is shown in Table 1, and the PCR program is shown in Table 2.

[0033] Nucleotide sequence of DlActin500 (SEQ ID NO.3): GAACACGTTTTTAATTTTTAAAAAATAAATTATTAAAATAAAAAAATAA AAATAAATTTAAAAATCAAAACTAAAAATTAAAAATATAAAAAAAAACGCTACCTTAATAGTTTCCTCTGTTTTGAGTAATTCCCTCGTTACATAGATATCCGCCTATTCTTGACATTTCACCCCGTCTTGTATTCAATTTAGCCTAATTTCTCATCATCATTATCATTGTCAGTGTCCTATCATCGAAGCTGGAGCTTTCCTCACTCGACTCGGCTTAGCCGGCGCACCGTGGAGACACAAAGGACATTCTCCCAACCGAACCGAGCGCAAAATGTCGCGCTACACAGCCGCCCAATAAGCCCAGCAATACCCAAGATGCTCGTTAACGGTCCAGATTAAATCAAAAGCCTCGTCGTAAGATCGGATGGCTGAACATGCTGAAAGCCTGTCAAAGCCAGGTCACCATATCTTTTCTCGTGGTCCGAATGATTCCACTTCCCACTCTGTGTGTCAACATAAATTTAGCCTCATTTCAT。

[0034] Nucleotide sequence of D1Actin1000 (SEQ ID NO. 4): CAAAATAAATTTAAAATTCAAAATTCAAATCTAAAAACTGAAATATAA。

[0035]

[0036] The PCR products were subjected to agarose gel electrophoresis, and the correct target band was excised under ultraviolet light and recovered using an agarose gel purification and recovery kit (DR01, Beijing Adelaide Biotechnology Co., Ltd.). Since the vector was constructed using homologous recombination, primers were used to add homology arms on both sides of the recovered fragment to facilitate its construction into the GUS vector. The reaction system is shown in Table 1, and the PCR program is shown in Table 2. The PCR products were also subjected to agarose gel electrophoresis and gel excision.

[0037] The GUS vector DX2181 was double-digested with Hind III and BamH I enzymes to allow the promoter fragment to be recombined into the vector. The vector enzyme digestion system is shown in Table 3.

[0038] Table 1 Amplification reaction system

[0039] Reagents Volume (μL) PrimeSTARMax 25 Primer F (10 mM) 1.5 PrimerR (10mM) 1.5 DNA 1 <![CDATA[UltrapureH2O]]> to 50

[0040] Table 2 Gene cloning PCR amplification program

[0041]

[0042]

[0043] Table 3 Double enzyme digestion reaction system

[0044] Enzyme digestion system Volume (μL) HindⅢ 1 Bam HI 1 10×KBuffer 4 carrier 1-2 μg Total to 40

[0045] The reaction was carried out at 37℃ for 1.5h. After the vector was confirmed to be cut by enzyme by electrophoresis, the linearized GUS vector DX2181 was recovered by gel cutting. The recovered linearized GUS vector DX2181 fragment was connected with the promoter fragment with homology arms and recombinase was used. IIOne Step Cloning Kit (Nanjing Novozymes Biotechnology Co., Ltd.), the reaction system is shown in Table 4.

[0046] Table 4 In-fusion ligation reaction system

[0047] Reagents Volume (μL) Vector fragment (50ng / μL) 2.5 sgRNA fragment (50ng / μL) 2 Exnase 1 5×CEBuffer 2 <![CDATA[UltrapureH2O]]> to 10

[0048] 2. Agrobacterium Transformation

[0049] The recombinant vector was transformed into Agrobacterium tumefaciens GV3101 competent cells using the freeze-thaw method. The specific steps are as follows:

[0050] 1) Remove the Agrobacterium tumefaciens GV3101 competent cells and quickly thaw them on ice. After thawing, add 5 μL of the recombinant vector to the tube, flick gently to mix, and place on ice for 5 minutes.

[0051] 2) Quickly freeze in liquid nitrogen for 5 minutes, then immediately place in a 37°C water bath for 5 minutes;

[0052] 3) Place the tube on ice and let it stand for 5 minutes. Add 900 μL of LB liquid medium without any antibiotics to the tube.

[0053] 4) Incubate at 28°C, 250g with shaking for 5 h until turbidity occurs to allow the competent cells to recover;

[0054] 5) Centrifuge at 12000g for 1 min, and pipette the remaining 100 μL of cells to mix thoroughly.

[0055] 6) Use a sterile spreader to evenly spread the bacterial solution on the surface of LB solid medium supplemented with 50 mg / L Rif and 50 mg / L Kan antibiotics. Allow to dry, seal the medium, and invert it in a 28°C incubator for 3 days.

[0056] 7) After 2 days, single colonies can be selected and placed in a sterile centrifuge tube containing antibiotics (50 mg / L Rif and 50 mg / L Kan) and 1.0 mL of LB liquid medium. Shake for 12 hours until turbidity is achieved. PCR testing can then be performed (system and procedure are shown in Tables 1 and 2). The primers used are consistent with those used for the positive test for colon transformation.

[0057] For short-term use, the correctly detected bacterial solution can be placed in a 4°C refrigerator. For long-term use, add an equal volume of sterile glycerol, mix thoroughly by inversion, and then store in a -80°C refrigerator.

[0058] 3. Transient Transformation of Tobacco Leaves

[0059] Add pro-DlActin500::GUS Agrobacterium, pro-DlActin1000::GUS Agrobacterium, pro-DlActin1500::GUS Agrobacterium, DX2181 empty vector Agrobacterium (negative control), 35S::GUS Agrobacterium (positive control) into liquid LB culture medium containing vector-related antibiotics, shake overnight until golden yellow, and adjust OD 600 Adjust to 0.7, add 500 μL 1 mol / L MES buffer, 500 μL 1 mol / L MgCl2, and 75 μL 100 mol / L acetosyringone (AS) to each 50 mL injection solution, mix well, and then let it stand at room temperature and inject it into the leaf tissue of Nicotiana benthamiana. Repeat the injection of 4 to 6 leaves for the transgenic strain, GUS empty vector DX2181 strain (negative control), and 35S::GUS strain (positive control).

[0060] Nucleotide sequence of 35S promoter (SEQ ID NO. 6): TGAGACTTTTCAACAAAGGATAATTTCGGGAAACCTCCTCGGATTCCA TTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCATTCAAGATCTCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCA TCGTGGAAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGACATCTCCACTGACGTAAGGGATGACGCAACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTTCATTTCATTTGGAGAGGACA.

[0061] IV. GUS Histochemical Staining

[0062] After 3 days, the injected leaves were excised and immersed in GUS inoculum for vacuum infiltration until the leaves were visibly infiltrated. After infiltration, the leaves were replaced with fresh GUS inoculum at 37°C overnight. The chlorophyll was then removed with 75% ethanol. The decolorization solution was changed twice daily until complete decolorization was achieved. The formulation of the GUS inoculum is shown in Table 5.

[0063] Table 5 GUS infection solution formula

[0064] Staining solution composition Final concentration Mother liquor concentration 100mL dosage PBS 100mmol / L 200mmol / L 50mL <![CDATA[Na2EDTA]]> 100mmol / L 50mmol / L 20mL Ferric ferrocyanide 1mmol / L 40mmol / L 2.5mL Ferric ferrocyanide 1mmol / L 40mmol / L 2.5mL TritonX-10 0.5% (V / V) - 0.5mL Methanol 20% (V / V) - 20mL X-gluc 0.5 mg / mL - 50mg

[0065] PBS and Na2EDTA were sterilized by high temperature.

[0066] 5. Construction of SpCas9 vectors driven by different PolⅡ promoters

[0067] The ZmUbi, 2×35S, AtUBQ10, DlActin1500, and AtYAO promoters were amplified using primers with homology arms. The amplification system is shown in Table 1, and the PCR program is shown in Table 2. The PCR products were subjected to agarose gel electrophoresis and gel excision and recovery. The vector pCambia1300 was double-digested with enzymes SpeⅠ and BstbⅠ. The reaction system for double-digestion of the vector pCambia1300 is shown in Table 6 below.

[0068] Table 6. Double enzyme digestion reaction system of vector pCambia1300

[0069] Enzyme digestion system Volume (μL) SpeⅠ 1 BstbⅠ 1 10×rCutSmartBuffer 4 carrier 1-2 μg Total to 40

[0070] Nucleotide sequence of the 2×35S promoter (SEQ ID NO.1): TGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTC CATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGA。

[0071] Nucleotide sequence of the AtUBQ10 promoter (SEQ ID NO.7): CGACGAGTCAGTAATAAACGGCGTCAAAGTGGTTGCAGCCGGC ACACACGAGTCGTGTTTATCAACTCAAAGCACAAATACTTTTCCTCAACCTAAAAATAAGGCAATTAGCCAAAAACAACTTTGCGTGTAAACAACGCTCAATACACGTGTCATTTTATTATTAGCTATTGCTTCACCGCCTTAGCTTTCTCGTGACCTAGTCGTCCTCGTCTTTTCTTCTTCTTCTTCTATAAAACAATACCCAAAGAGCTCTTCTTCTTCACAATTCAGATTTCAATTTCTCAAAATCTTAAAAACTTTCTCTCAATTCTCTCTACCGTGATCAAGGTAAATTTCTGTGTTCCTTATTCTCTCAAAATCTTCGATTTTGTTTTCGTTCGATCCCAATTTCGTATATGTTCTTTGGTTTAGATTCTGTTAATCTTAGATCGAAGACGATTTTCTGGGTTTGATCGTTAGATATCATCTTAATTCTCGATTAGGGTTTCATAGATATCATCCGATTTGTTCAAATAATTTGAGTTTTGTCGAATAATTACTCTTCGATTTGTGATTTCTATCTAGATCTGGTGTTAGTTTCTAGTTTGTGCGATCGAATTTGTAGATTAATCTGAGTTTTTCTGATTAACAG。

[0072] The nucleotide sequence of the DlActin1500 promoter is shown in SEQ ID NO.5.

[0073]

[0074] Nucleotide sequence of AtYAO promoter (SEQ ID NO.9): CCCCCTCGACGATGGGAAATTCATTGAAAACCCTAAACCCAAATC。

[0075] The recovered linearized pCambia1300 vector fragment was ligated with the promoter fragment with homology arms and recombinase IIOne Step Cloning Kit (Nanjing Novozymes Biotechnology Co., Ltd.) was used. The reaction system was shown in Table 4. After the reaction, 5 μL was transferred into Escherichia coli and the single clone was positively identified. The constructed vector was transferred into Agrobacterium EHA105 and kept in an ultra-low temperature freezer at -80°C.

[0076] 6. Vacuum penetration

[0077] The specific steps are as follows:

[0078] 1) Take out proDlActin1500::SpCas9 Agrobacterium, pro2×35S::SpCas9 Agrobacterium, proZmUbi::SpCas9 Agrobacterium, proAtUBQ10::SpCas9 Agrobacterium, and proAtYao::SpCas9 Agrobacterium from a -80°C ultra-low temperature freezer, use an inoculation needle to pick up a small amount of bacterial liquid and streak it on solid LB medium containing 50 mg / L Rif and 50 mg / L of the corresponding carrier antibiotic, and culture in a constant temperature incubator at 28°C for 3 days;

[0079] 2) Pick a single colony of appropriate size and place it in 1 mL of LB liquid medium (containing 50 mg / L Rif and 50 mg / L of the corresponding carrier antibiotic) at 220 g, shaken at 28°C until the culture solution becomes turbid;

[0080] 3) After PCR-positive clones were identified, the entire bacterial suspension was inoculated into 50 mL of LB liquid medium (containing 50 mg / L Rif and 50 mg / L of the corresponding carrier antibiotic) and cultured with shaking at 220 g at 28°C until the suspension became turbid.

[0081] 4) Transfer the bacterial suspension into a sterile 50 mL centrifuge tube and centrifuge at 8000 g for 10 min. Retain the bacterial pellet and discard the supernatant.

[0082] 5) Prepare the infiltration solution: Add 10 mM MES, 10 mM MgCl2, and 150 μM acetosyringone (AS) to 100 mL of sterile water. Rinse the cells with sterile water and gently pour off the solution. Add the infiltration solution and pipette to mix thoroughly to resuspend the cells.

[0083] 6) Take a small amount of suspended bacterial solution to detect OD 600 value, OD 600 Adjust the value to 0.75;

[0084] 7) Place in a 28°C constant temperature incubator for 2 hours;

[0085] 8) Immerse the entire plantlet in the Agrobacterium infection solution and place it in a vacuum apparatus (0.8 MPa) for 25 minutes. Air-dry the solution on sterile filter paper and then inoculate it into subculture medium (DKW + 3% sucrose + 0.1 mg / L IAA + 1.0 mg / L ZT).

[0086] 9) After culturing for 3 days, the tissue culture seedlings were washed three times with 400 mg / L cephalosporin and three times with sterile water until the water was clear. The tissue culture seedlings were removed and placed on sterile filter paper to absorb the moisture on the leaf surface, and then inoculated into the subculture medium.

[0087] 10) Ten days after infection, the transiently transformed leaves were collected, immediately frozen with liquid nitrogen, and stored in a -80°C ultra-low temperature freezer.

[0088] VII. RNA Extraction

[0089] RNA was extracted from fruit pulp and leaves using a column-based plant total RNA extraction and purification kit (Shanghai Sangon Biotechnology Co., Ltd.) according to the manufacturer's instructions with minor modifications. The specific steps are as follows:

[0090] 1) Place 600 μL of Buffer Rlysis-PG (stored at 4°C) in a 1.5 mL RNase-free centrifuge tube and place on ice.

[0091] 2) Grind 0.1 g of frozen plant tissue into a powder using liquid nitrogen, add it to the 1.5 mL centrifuge tube, shake thoroughly, and let it stand at room temperature for 5 minutes (lay flat and mix thoroughly).

[0092] 3) Centrifuge at 12,000 g for 3 min at 4°C and transfer the supernatant to a new RNase-free centrifuge tube.

[0093] 4) Add 1 / 2 volume of anhydrous ethanol (stored at 4°C) to the supernatant and mix well;

[0094] 5) Place the adsorption column in the collection tube, add the solution to the adsorption column, let it stand for 1 minute, centrifuge at 12000g for 1 minute at room temperature, and discard the waste liquid in the collection tube;

[0095] 6) Place the adsorption column back into the collection tube, add 500 μL of GT Solution, let it stand for 1 minute, centrifuge at 10,000 g for 1 minute at room temperature, and discard the waste liquid in the collection tube;

[0096] 7) Place the adsorption column back into the collection tube, add 500 μL of NT Solution, let it stand for 1 minute, centrifuge at 10,000 g for 1 minute at room temperature, and discard the waste liquid in the collection tube;

[0097] 8) Centrifuge at 12,000 g for 10 min at 4°C, discard the supernatant, and add 1 mL of 75% ethanol to rinse the pellet;

[0098] 9) Centrifuge at 12,000 g for 10 min at 4°C, discard the supernatant, and air-dry at room temperature for 5 min;

[0099] 10) Place the adsorption column in a 1.5 mL RNase-free centrifuge tube, add 50 μL of diethylpyrocarbonate (DEPC) in water to the center of the adsorption membrane, let it stand for 5 minutes, and centrifuge at 12,000 g for 2 minutes;

[0100] (11) Detect RNA quality by 1% agarose gel electrophoresis or microspectrophotometer and store at -80°C until use.

[0101] 8. Reverse Transcription

[0102] Refer to the instructions of the PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, China) for cDNA synthesis. The specific steps are as follows:

[0103] 1) Removal of genomic DNA (gDNA)

[0104] Add the corresponding reagents according to the reaction system shown in Table 7, mix well, centrifuge briefly, incubate at 42°C for 2 minutes, and immediately place on ice.

[0105] Table 7 Reaction system for removing gRNA

[0106] Reagents Volume (μL) 5×gDNAEraserBuffer 2 gDNAEraser 1 Total RNA (1 μg) 2 <![CDATA[RNAfreeH2O]]> 5 Total 10

[0107] 2) Reverse transcription

[0108] Add the appropriate reagents according to the reaction system shown in Table 8, mix thoroughly, and briefly centrifuge. Reaction conditions are: incubate at 37°C for 15 minutes, 85°C for 5 seconds, and 4°C for 5 minutes to complete reverse transcription. Check the cDNA quality using a micro-spectrophotometer. Dilute the cDNA to 200-300 ng / μL with ultrapure water and store at -20°C until needed.

[0109] Table 8 Reverse transcription reaction system

[0110] Reagents Volume (μL) Step (1) reaction product 10 5×PrimeScriptBuffer2 4 PrimeScript RT Enzyme 1 RTPrimerMix 1 <![CDATA[RNAfreeH2O]]> 4 Total 20

[0111] IX. Real-time quantitative PCR

[0112] Real-time quantitative PCR (qRT-PCR) was used to detect the transcription level of SpCas9, and the designed quantitative primers are shown in Table 9. TB Pre mix Ex Taq II (TaKaRa, China) in ABIQuantStudio TM 7Flex real-time fluorescence quantitative PCR system (Applied Biosystems). Actin (accession no. AB473616) was used as the internal reference gene and the above cDNA was used as the template. qRT-PCR was repeated 4 times for each sample. T ( 2-△△CT ) method was used to calculate the expression level of the SpCas9 gene relative to the internal reference gene. The qRT-PCR reaction system is shown in Table 10.

[0113] PCR reaction program: 95°C for 30 s; 40 cycles of amplification including 95°C for 15 s, 58°C for 30 s, 72°C for 20 s, and finally 95°C for 15 s and 40°C for 30 s.

[0114] Table 9 Promoter amplification primers

[0115] Primers Primer sequence (5′-3′) p2×35S-F GCTTGCCAACATGGTGGAGC(SEQ ID NO.10) p2×35S-R TCAGCGTGTCCTCTCCAAATG(SEQ ID NO.11) pZmUbi-F TTTTCCTTTATTTCAATATATGCCGTGCAC(SEQ ID NO.12) pZmUbi-R GGCTATCGTTCGTAAATGGTGAAAATTT(SEQ ID NO.13) pAtUBQ10-F CGACGAGTCAGTAATAAACGGC(SEQ ID NO.14) pAtUBQ10-R CTGTTAATCAGAAAAACTCAGATTAATCTAC(SEQ ID NO.15) pDlActin-F TGTCATCCATACTTACCATTACCATCA(SEQ ID NO.16) pDlActin-R CTTTTATGACTGCAAAATAGT(SEQ ID NO.17) pYao-F CCCCCTCGACGATGGAAAT(SEQ ID NO.18) pYao-R TTCTTCTTCTCGTTGTTGTACTTCATTC(SEQ ID NO.19)

[0116] Table 10 qRT-PCR reaction system

[0117]

[0118] The results are as follows Figure 1 and Figure 2 As shown, Figure 1 Middle A, Figure 1 Middle B and Figure 1 As shown in C, the GUS staining results showed that the DlActin promoter with a length of 1500 bp had the strongest driving activity; Figure 2 As shown in Figure 3, the results of qRT-PCR quantitative experiments showed that the promoter with the strongest ability to drive SpCas9 expression in persimmon was the 2×35S promoter, followed by ZmUbi and AtUBQ10, and the weakest was AtYAO.

[0119] Screening of persimmon high-activity driven sgRNA promoters

[0120] 1. Driving sgRNA promoter amplification

[0121] Using Junqianzi and Diospyros kaki DNA as templates, primers were designed to amplify the PolⅢ promoter. The amplification system is shown in Table 1, and the PCR program is shown in Table 2. The reaction solution was subjected to agarose gel electrophoresis, and the correct target band was cut out, recovered from the gel, and ligated to the pTOPO-Blunt vector (CV17-Zero Background pTOPO-Blunt Simple Cloning Kit, Adelaide Biotechnology Co., Ltd.), and transformed into Escherichia coli competent DH5α. The next day, universal primers M13F / R (F: TGTAAAACGACGGCCAGT (SEQ ID NO. 20); R: CAGGAAACAGCTATGACC (SEQ ID NO. NO.21)) The single clones were positively identified and sent to Qingke for sequencing; the amplified PolⅢ promoters were named DlU6-1, DlU6-2, DlU6-3, DlU6-4, DlU6-5, DlU3, DkU6-1, DkU6-2, DkU6-3, DkU6-4, DkU6-5 and AtUBQ10.

[0122] Nucleotide sequence of DkU6-5 promoter (SEQ ID NO. 2): GGCATGCAAATCAAAGCAAGGACCAGCGATTGACTTGTTGTATCC ATGGCTAAATCAACCGCCAAATGTTCCTGCCAAGAATAGTCCTCTTCACAAACCACTGTCTCCTCTACCCACTTCGCACCTTCGCTTCTTGTGCTCCGCTGCCGCTGAAGCCTACAAAGCTCTCGCTCCTCGCCGACAAGTGCGCGTCGATGCACCAACTCAAACAAATCCACGCCCAAATGATCGTCTCAGCCAGGATTCACGACAACTACGCCGCCAGCCGATTTTTATCCTTCTGCG CTCTTTCGGAGTCGGGTGATCTCGCTTACGCTCTAAAGCTCTTTCAGCTTACTCAGGAACCCAATTCTTTCATGCGGAACAATCATTAGAGCTCTTGCCAGTAGCCCAAAACCCCATGAAGCTGTGTTTCTGTATGCTTCAACTACGTCGTTTTCTATCCTTGTTCTGTTAGCTATCCCACATCGCTGAGATAATAAGATTGCGCCTCGTTTATATATGTCGGGGCACGTAGAAGGTTTG.

[0123] Nucleotide sequence of the DlU6-1 promoter (SEQ ID NO.22): TTTATTGATTTGTTCTTAGCCTTGGTTTGAATGCTCTCAATTAAACT TTACTTAGATTATAATGCAGGATTAGATGATAATAACCTTAATCTGCTTGTTATTCTTTCTTTTTTTACTTATTTATTAAATTTTAAATATTAATTTAATTATCGAATATTATCCCCAGAAAACAAAAGCTCTGTGTTTATTTAAATTGAACAAATAATGCCTCAAATTTCAAGTGTTAACCAGATCAAACATTATTTCATTTAGGGCTATAGTCTTAACCGAATCAAATCAAGTAACTTAATCAAATTGCTTCCAATGAGCTGCTAAAGTTAATGCTTCAACGGCGTCGTTTTCTACCCTTCGGGGGAACGAAAATCAAGTAACTTGTTCTGTTTTCTATCCCACATCGTTGAGATAATAAGATTGCGCAAGGTTTATAGATGTCGGGGAACGAAGTAGGTTTG。

[0124] Nucleotide sequence of the DlU6-2 promoter (SEQ ID NO.23): TCCTGCCAAGAATACTCCTCTTCACAAACCACTGTCTCCTCCACC GCACCTTCGCTTCTTGTGCTCCCCCGCCGCTGAAGCCTACAAACCTCTCGCTCCTCGCCGACAAGTGCGCGTCGATGCACCAACTCAAACAAATCCATGCCCAAATGATCGTCTCAGCCAGGATTCACGACAACTACGCCGCCAGCCGATTATTATCCTTATGCGCTCTTTCGGAGTCGGGTGATCTCGCTTACGCTCTAAAGCTCTTTCAGTTTACTCAGGAACCCAATTCTTTCATGCGGAACAATCATTAGAGCTCTTGCCAGTAGCCCAGAACCCCATGAAGCTGTGTTTCTGTATGCTTCAACTACGTCGTTTTCTATCCTTGTTCTGTTAGCTATCCCACATCGCTGAGATAATAAGATTGCGCCTAGTTTATATATGTCGGGGCACGTAAAAGGGTTG。

[0125] Nucleotide sequence of the DlU6-3 promoter (SEQ ID NO.24): AAATATAATTATTTTTATATTTTAAGTATAATATCTTTGGTGATGGAC GACTGTAGCCCTTTGCTTAGGCGTGTCTGAAACTTTGCACAAGCAAAGAAATCTATCATATATTTTTAATTAAAAATATATATTTAATACATGAGATATTTTTTTTTATAAAAATTGAGAGAAGATCATATTTATAAGTAAATAAATTATTTTTTATTTTTATAAAATAAATTATTGTATTTATTTTTTACTGTTGCTAGTAAATAGTTATAAAATATTTTATTTAAATATCATTATAAAGTATATAAAAAATGGTGCCAATTTCTAAACCGTGAACGAGCCCAATTGCAGCTTACGCGGTGTCGTTTTAGTCTTCTTGTTCTTGGATGACTCCTCTTTCCCACATCGTGGAATTAGCAGATTTCTGATCGTTATATATATTTGCGACAGCAGATTAGGGTTG。

[0126] Nucleotide sequence of the DlU6-4 promoter (SEQ ID NO.25): GTATTAATCAAGAAATAAACATGATTATTTTTATATTTTAAGTTTAAT ATCTTTGGTGGTGAATGACTGGAGCCCTATCCTTAGGCATGTCTGAAATTTTGAACAAGGATATCATATATTTTTAATTAAAAAAATTTATCTAATACACGGGACACCTTTTTTTAGAAATTGAGAGAAGATCATGTTTATAAGTAAATAAATTATTTTTTATTTTTATAAAATCAATTTTTGTATTTATTTTTTACTGTTGCTAGTAAATAATTATAGAATATTTTATTTAAATATCATTATAAAATATATAAAAAATGGTGCCAATTGCACGGCAGCAGCTGCAATTGTAGTTTACACGGTGTCGTTTCAGTCTTCTTGTTCTTGGATGACTCCTCTTTCCCACATCGTGGAATTAGCAAATTCTGCTCGTTATATATGTTTGCGACAGCAGCTTAGGTTTG。

[0127] Nucleotide sequence of the DlU6-5 promoter (SEQ ID NO.26): TATTGATTTGTTCTTAGCCTTGGTTTGAATGCTCTCAATTAAACTTT ACTTAGATTATAATGCAGGATTAGATGATAATAACCTTAATCTGCTTGTTATTCTTTCTTTTTTTACTTATTTATTAAATTTTAAATATTAATTTAATTATCGAATATTATCCCCAGAAAACAAAAGCTCTGTGTTTATTTAAATTGAACAAATAATGCCTCAAATTTCAAGTGTTAACCAGATCAAACATTATTTCATTTAGGGCTATAGTCTTAACCGAATCAAATCAAGTAACTTAATCAAATTGCTTCCAATGAGCTGCTAAAGTTAATGCTTCAACGGCGTCGTTTTCTACCCTTCGGGGGAACGAAAATCAAGTAACTTGTTCTGTTTTCTATCCCACATCGTTGAGATAATAAGATTGCGCAAGGTTTATAGATGTCGGGGAACGAAGTAGGTTTG。

[0128] Nucleotide sequence of the DlU3 promoter (SEQ ID NO.27): GTTGGAAAATGAATTTTTAATAAGTATAATATAATCTTAAATAGGTAA AAATGAGCACAATTAGCATGACCCATCAAGACTCACTTGGTGGCTTGCCCTGTAGTAGCTCCAGTCTCCTTAAAAGGTCCAAATCATAAGTTAAACCGATAAAGCCAATTTATTTTTATGTCCAGATCTAATCGAATGGTCATTGGAGGTTCGAAAATAAAGCATATTACTAAAGAACAAGTATTAGTCCCACATCGGAATAATAGTCATAGAAAGTATAGAATATATTACTGGAGCAGAGGAGAGACGGACA。

[0129] Nucleotide sequence of the DkU6-1 promoter (SEQ ID NO.28): CCACAAATATATCACAGTCTCAATACTTTGTATAAGAACAAAAAA TACCCTTTAACAGTAAAGCCCCATAATCACAGACAAAGATCAGAGGCAAATAACCCTAAAACTAAAGAGGCTGTCGTGGTCACAACTCGAGGTGCCAGTAGAAATAAATAGACAATTCAATGGTTGGGATAAACTCAAATGAAAGTAAAATGACAACTGGACGCCAGCAGCATAAAATGACAATAAGGGTTGTCATTTTGGTCCTCCAGCCCATGGGCAGTTGTCAGAAGGCCGTCCACTCCTTGGCCCAAACAGAAGCTTGATTGGAGCTTCATAAATAATGCCAACATATAGTCTTAACCGAATCAAATTATAGATCATCAAATTAAGTAACTCAAGTCAAATTGCTTCCAATGAGCTGCTAAAGTTAATGCTTCAACGGCGTCGTTTTCTACCCTTCGGGGAACGAAAATCAAGTAACTGGTTCTGTTTTCTATCCCACATCGCTGAATAGCATGATTGCGCCTGGTTTATAGATGTCGGGGAACGGAAAAGG。

[0130] Nucleotide sequence of the DkU6-2 promoter (SEQ ID NO.29): GACTTTCCATGTGAATTAAAAATATATATATATAAGACGATTTGAA AAAATAATGCTTGAGATATTTTAAATTTTATTACATAAGACTTAAATTTTTTAGAGATAAAAAGTAATAATAGCCTAAATTTGCTTGTTACTCTTTCTTTTTACATATTTATTAAATTTTAAATATTAATTTAATTATCGAATATTATCCCCAAAAAAACAAAAGCTCTATGTTTATTTAAATTTAACAAATAATGCCTCAAATTTCAAGTCTTAACCAGATCAAAATTTATTTCATTTAGGGCTATAGTCTTAACCGAATCAAATTATAGATGATCAAATCAAGTAACTTAAATCAAATTGCTTCCAATGAGCTGCTAAAGTTAATGCTTCAACGGCGTCGTTTTCTACCCTTCGGGGAACGAAATCCAAGAAACTGGTTCTGTTTTCTATCCCACATCGCTGAATAGCATGACTGCGCCTGGTTTATACATCTCGGGGAACGGAGAAGGTTTG。

[0131] Nucleotide sequence of the DkU6-3 promoter (SEQ ID NO.30): TACAACCAAAATATCGAGAAAGAACAAAATACAATCTGATCAGG CGGAAGTTGCCGTGGTCACAATCTCGAGGTATCAGCAGAAATAAATAGGCAATTCAACAGATAGGATAAACTCGGATGAAGGCAAAAGGACAATTGGGCGCCAGTAGCATAAAACAACAAAGAGGGTTGTCGTTTTGACCCTCCAACCCATGGGCAGTTGCCAAAAGGCTGTCCATTCCTTGCCCAGACAAGAACTTGATTGGAGCTTTGGGTGTGGGGGCGTCTGAGATGGATGATTCATCTTTTTAGACAAGAACTTGATTGGAGCTTCATAAATAATGCCAACATATAGTCTTAACTGAATCAAATTATAGATCTCAAATCAAGTAACTTAAATCAAATTGCTTCCAATGAGCTGCTAAAGTTAATGCTTCAACGGCGTCGTTTTCTACCCTTCGGGGAACGAAATCCAAGAAACTGGTTCTGTTTTCTATCCCACATCGCTGAATAGCATGACTGCGCCTGGTTTATACATGTCGGGGAACGGAGAAGGTTTG。

[0132] Nucleotide sequence of the DkU6-4 promoter (SEQ ID NO.31): AGCTAAGCATTGTTCAAAGCCAAATTTATAGATTGTATATAGAAAA TAATTTAACAAACTAACGCTTGAGATATTTTAAATTTTATTATATAGGACTTAAATTTTTTAGAGATAAAAGATAATAATAACTTTAATTTATTTGCTATTCCTTTTTTTTATTTATTAAATTTTAAATATTAATTTAATTGTCAAATATTATCCCCCAAAAAACAAAAGCTTTATGTTTATTTAAATTTAACAAATAATGCCTCAAATTTCAAGTCTTAACCAGATCAAAATTTATTTCATTTAGGGCTATAGTCTTAACCGAATCAAATTATAGATTATCAAATCAAGTAACTTAAATCAAATTGCTTCGAATGAGCTGCTAAAGTTAATGCTTCAACGGCGTCGTTTTCTACCCTTCGGGGGAACGAAAATCAAGTAACTGGTTCTGTTTTCTATCCCACATCGCTGAATAGCATGATTGCGCCTGGTTTATAGATGTCGGGGAACGGAGGACGTTCG。

[0133] The nucleotide sequence of the DkU6-5 promoter is shown in SEQ ID NO.2.

[0134] II. Vector construction

[0135] The GoldenGate cloning method was used to construct vectors. DlU6-1, DlU6-2, DlU6-3, DlU6-4, DlU6-5, DlU3, DkU6-1, DkU6-2, DkU6-3, DkU6-4, DkU6-5 and AtUBQ10 were simultaneously constructed with the target sites T3 (TAAGGCCGGATATCAAGAGA (SEQ ID NO. 32)) and T5 (AACCCAACCTTCGAACAACA (SEQ ID NO. 33)) of DkMYB4 (BAI49721.1) into the expression vector pggk (Vector ID: 9_81; Decaesteck er & Buono et al. Plant Cell 2019). The PTG strategy (transfection of a vector containing a polycistronic sgRNA) was used to drive sgRNA expression. The reaction system is shown in Table 11, and the PCR program is shown in Table 12. 5 μL of the reaction solution was transferred into competent E. coli DH5α. The next day, the single clone was identified as positive using universal primers pgF / pgR (F: TGTGCCACTC CAAAGACATCAG (SEQ ID NO. 34); R: AAGCATCAGATGGGCAAACAAAGCACCAGTGGTCTAG (SEQ ID NO. 35)) and sent to Qingke for sequencing.

[0136] 3. Agrobacterium Transformation

[0137] The method is the same as the Agrobacterium transformation step in the screening of high-activity SpCas9 promoter driven by persimmon.

[0138] 4. Vacuum penetration

[0139] The method is the same as the vacuum infiltration step in the screening of high-activity SpCas9 promoters driven by persimmon, but the material is changed to tissue culture seedlings of 'Gongcheng Shuishi'.

[0140] 5. RNA Extraction

[0141] The method is the same as the RNA extraction step in the screening of high-activity driven SpCas9 promoters.

[0142] VI. cDNA Synthesis

[0143] cDNA synthesis was performed with reference to the instructions of the miRNA First-Strand cDNA Synthesis (Tailoring Method) Reverse Transcription Kit (Sangon Biotech Co., Ltd.). According to the reaction system shown in Table 11, the amplification program shown in Table 12, and the tailing method cDNA synthesis system shown in Table 13, the reaction mixture was added to a centrifuge tube in an ice bath to perform cDNA synthesis.

[0144] Table 11 Goldengate reaction system

[0145] Reagent Volume (μL) T4 DNA Ligase 1 T4 DNA Ligase Buffer 1.5 BsaⅠ 1 rCutSmart Buffer 1.5 PolⅢ promoter 1 T3 T5 1 <![CDATA[UltrapureH2O]]> up to 15

[0146] Table 12 PCR amplification program

[0147]

[0148] Table 13 Tailing method cDNA synthesis system

[0149]

[0150] VII. Real-time quantitative PCR

[0151] Using PowerUp TM SYBR TM Green premix (Thermo Scientific, USA) kit, the quantitative primers were sgRNA-R as shown in Table 15 and U6-R, persimmon U6 (derived from miRNA fluorescence quantitative PCR kit purchased from Sangon Biotech (Shanghai) Co., Ltd.) was used as the internal reference gene, and the quantitative primers were universal primers U6-F and U6-R. The method was the same as the real-time quantitative PCR in the screening of persimmon high-activity driven SpCas9 promoter.

[0152] Table 14 Promoter amplification primers

[0153] Primer Primer Sequence (5′-3′) pDkU6-1F GGTTCACCCGTTAATGAGAGCTAC (SEQ ID NO.36) pDkU6-1R CCTTTTCCGTTCCCCGAC (SEQ ID NO.37) pDkU6-2F CAATCGTTCACGGAGATTAGTCG (SEQ ID NO.38) pDkU6-2R CAAACCTTCTCCGTTCCCCG (SEQ ID NO.39) pDkU6-3F CAATCTGATCAGGCGGAAGTTG (SEQ ID NO.40) pDkU6-3R CAAACCTTCTCCGTTCCCCG (SEQ ID NO.41) pDkU6-4F GAGAAACGTGAGCTAAGCATTGTTC (SEQ ID NO.42) pDkU6-4R CGAACGTCCTCCGTTCCC (SEQ ID NO.43) pDkU6-5F GGCATGCAAATCAAAGCAAGGAC (SEQ ID NO.44) pDkU6-5R CAAACCTTCTACGTGCCCC (SEQ ID NO.45) p2×35S-F GCTTGCCAACATGGTGGAGC (SEQ ID NO.10) p2×35S-R TCAGCGTGTCCTCTCCAAATG (SEQ ID NO.11) pZmUbi-F TTTTCCTTTATTTCAATATATGCCGTGCAC (SEQ ID NO.12) pZmUbi-R GGCTATCGTTCGTAAATGGTGAAAATTT (SEQ ID NO.13) pAtUBQ10-F CGACGAGTCAGTAATAAACGGC (SEQ ID NO.14) pAtUBQ_{10}-R CTGTTAATCAGAAAAACTCAGATTAATCTAC (SEQ ID NO.15) pDlActin-F TGTCATCCATACTTACCATTACCATCA (SEQ ID NO.16) pDlActin-R CTTTTATGACTGCAAAATAGT (SEQ ID NO.17) pYao-F CCCCCTCGACGATGGGAAAT (SEQ ID NO.18) pYao-R TTCTTCTTCTCGTTGTTGTACTTCATTC (SEQ ID NO.19)

[0154] Table 15 qRT-PCR primers

[0155]

[0156] The results are as follows Figure 3 and Figure 4 As shown, the results of qRT-PCR quantitative experiments showed that the promoter with the strongest ability to drive sgRNA expression in persimmon was DKU6-5, which was nearly twice as high as the positive control AtUBQ10, followed by DkU6-3, and the weakest was DkU6-1.

[0157] The present invention screened out promoters p2×35S, pAtUBQ10 and pZmUbi with strong ability to drive SpCas9 expression in persimmon, providing a basis for the subsequent construction of CRISPR gene editing vectors. The present invention screened out a promoter DkU6-5 with strong ability to drive sgRNA expression in persimmon.

[0158] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A promoter for specific and efficient expression in persimmon, characterized in that: The promoter includes the promoter DKU6-5 that drives the expression of sgRNA, and the nucleotide sequence of the promoter DKU6-5 is shown in SEQ ID NO.

2.

2. An expression vector, characterized in that: Contains the persimmon-specific and highly efficient expression promoter according to claim 1.

3. The expression vector according to claim 2, characterized in that The expression vector is pCambia1300 vector and / or pggk vector.

4. A host bacterium, characterized in that Contains the expression vector according to claim 2.

5. The host bacteria according to claim 4, characterized in that The host bacteria is Escherichia coli DH5α.

6. Use of the promoter according to claim 1 in increasing the expression level of exogenous genes in persimmon.

7. Use of the expression vector according to claim 2 or 3 in increasing the expression level of exogenous genes in persimmon.

8. Use of the host bacteria as claimed in claim 4 or 5 in increasing the expression level of exogenous genes in persimmon.

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