Specific high-efficiency expression promoter in persimmon and application thereof

Abstract

The invention discloses a specific high-efficiency expression promoter in persimmon and application thereof, belongs to the technical field of gene engineering, and provides the specific high-efficiency expression promoter in persimmon, the specific high-efficiency expression promoter comprises a promoter 2 * 35S for driving Cas9 expression and / or a promoter DKU6-5 for driving sgRNA expression, the nucleotide sequence of the promoter 2 * 35S is as shown in SEQ ID NO.1, and the nucleotide sequence of the promoter DKU6-5 is as shown in SEQ ID NO.2. Aiming at the problem of low persimmon tree gene editing efficiency in the prior art, the invention provides the promoter capable of efficiently driving persimmon gene editing so as to improve the gene editing efficiency.

Description

Technical Field

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

[0002] Persimmon (Diospyros kaki Thunb.) is a representative species of Diospyros kaki cultivated as a fruit tree. According to whether the fruit naturally loses its astringency on the tree when ripe and its genetic characteristics, the existing persimmon varieties can be divided into fully sweet persimmon (PCNA) and non-fully sweet persimmon (non-PCNA). Fully sweet persimmon is a natural mutation of non-fully sweet persimmon. Since its fruit can be picked and eaten crisp without any treatment after ripening, it has higher economic value than astringent persimmon and is the main target of the current persimmon industry development and genetic improvement.

[0003] Persimmon is a perennial tree crop of the genus Diospyros in the family Ebenaceae. It has a complex ploidy 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 / Cas technology is a third-generation new targeted genome editing technology that has the advantages of flexibility, high efficiency, low production cost and high specificity, which has greatly promoted the research on gene function. After continuous improvement and development, this technology has been widely used in various fields.

[0005] A promoter is a DNA sequence that RNA polymerase recognizes, binds to, and starts transcription. It contains conserved sequences required for RNA polymerase specific binding and transcription initiation. It is mainly divided into constitutive promoters, tissue-specific promoters, and inducible promoters. Constitutive promoters are mainly able to regulate the expression of structural genes to a certain extent, and the expression levels in different parts or tissues are not significantly different (such as CMV, U6, etc.); tissue-specific promoters are mainly to make genes often only expressed in certain specific organs or tissue parts, and show the characteristics of developmental regulation (such as CaMV 35S); inducible promoters are that after certain specific physical or chemical signal stimulation, the gene transcription level is greatly increased under the regulation of this type of promoter.

[0006] Gene editing in plants usually requires the construction of both Cas gene expression cassettes and sgRNA expression cassettes. The expression of Cas genes is usually driven by RNA polymerase II promoters such as 35S or Ubi, and terminated by Nos terminator or other terminators; the sgRNA expression cassette is relatively small, generally driven by RNA polymerase III promoters such as U3 or U6, and terminated by more than 6 consecutive T bases. The strong constitutive AtUBQ10 can also be used to drive its expression.

[0007] Improving the expression level of the components of CRISPR gene editing vectors in plant cells can improve the efficiency of gene editing, which can be achieved by replacing the promoters that drive the Cas nuclease and s gRNA. In citrus, the YAO promoter was used to replace the 35S promoter to drive SpCas9, which increased the mutation efficiency by nearly 25 times under the condition that other conditions remained unchanged. The promoters that drive sgRNA in the CRISPR gene editing vectors commonly used in plants are AtU6-26, AtU6-29, and At U3 from Arabidopsis, while OsU6 and OsU3 from rice can produce better editing effects in Gramineae plants; because the promoters that drive sgRNA are species-specific, identifying the promoters that drive sgRNA in this species can effectively improve the efficiency of gene editing. CRISPR / Cas gene editing technology has been applied in many horticultural crops such as kumquat, apple, and grape, but no relevant reports have been seen in Diospyros plants. Summary of the invention

[0008] In order to solve the above technical problems, the present invention proposes a promoter for specific and efficient expression in persimmon, wherein the promoter includes the promoter 2×35S for driving Cas9 expression and / or the promoter DKU6-5 for driving sgRNA expression. In view of the problem of low efficiency of gene editing 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 promoter 2×35S for driving Cas9 expression and / or promoter DKU6-5 for driving 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 promoter for specific and efficient expression in persimmon.

[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 invention also provides application of the promoter in increasing the expression amount of foreign genes in persimmon.

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

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

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

[0018] The present invention screened out two promoters that efficiently drive persimmon gene editing. 2×35S is used as a promoter to drive SpCas9. Compared with promoters commonly used in other gene editing systems, it can efficiently drive the expression of SpCas9 in persimmon; DKU6-5 is used as a promoter to drive sgRNA. Compared with the AtUBQ10 promoter, it can efficiently drive the expression of sgRNA in persimmon. The present invention optimizes the persimmon gene editing system to improve the efficiency of persimmon gene editing. 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

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

[0021] Figure 2 Analysis of the expression of SpCas9 driven by different PolⅡ promoters in persimmon, abc in the figure represents the significant 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are 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 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): GAACACGTTTTTAATTTTTAAAAAATAAATTATTAAAATAAAAAAATAAAAAATAAATTTAAAAATCAAAACTAAAAATTAAAAATATAAAAAAAAACGCTACCTTAATAGTTTCCTCTGTTTTGAGTAATTCCCTCGTTACATAGATATCCGCCTATTCTTGACATTTCACCCCGTCTTGTATTCAATTTAGCCTAATTTCTCATCATCATTATCATTGTCAGTGTCCTATCATCGAAGCTGGAGCTTTCCTCACTCGACTCGGCTTAGCCGGCGCACCGTGGAGACACAAAGGACATTCTCCCAACCGAACCGAGCGCAAAATGTCGCGCTACACAGCCGCCCAATAAGCCCAGCAATACCCAAGATGCTCGTTAACGGTCCAGATTAAATCAAAAGCCTCGTCGTAAGATCGGATGGCTGAACATGCTGAAAGCCTGTCAAAGCCAGGTCACCATATCTTTTCTCGTGGTCCGAATGATTCCACTTCCCACTCTGTGTGTCAACATAAATTTAGCCTCATTTCAT。

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

[0035]

[0036] The PCR product was subjected to agarose gel electrophoresis, and the correct target band was cut out under ultraviolet light and recovered using an agarose gel purification and recovery kit (DR01, Beijing Aidelai 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 construct it into the GUS vector. The reaction system is shown in Table 1, and the PCR program is shown in Table 2; the PCR product was also subjected to agarose gel electrophoresis and gel excision recovery.

[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]]> Up 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 Up to 40

[0045] The reaction was carried out at 37°C for 1.5 hours. After the vector was confirmed to be cut by enzyme by electrophoresis, the linearized GUS vector DX2181 was recovered and separated by gel cutting. The recovered linearized GUS vector DX2181 fragment was connected with the promoter fragment with homology arms and recombinase IIOne Step Cloning Kit (Nanjing Novozyme 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]]> Up to 10

[0048] 2. Agrobacterium Transformation

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

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

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

[0052] 3) Place the tube in ice and let it stand for 5 min, then add 900 μL of LB liquid culture medium without any antibiotics into the tube;

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

[0054] 5) Centrifuge at 12000g for 1 min, and mix the remaining 100 μL of cells by pipetting;

[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, let it stand and blow dry, then seal the film and invert it in a 28°C incubator for 3 days;

[0056] 7) After 2 days, single clones 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 LB liquid culture medium. After shaking for 12 hours until turbidity, PCR detection can be performed (the system and procedure are shown in Tables 1 and 2). The primers used are consistent with those for the positive detection of large E. coli transformation.

[0057] The correctly tested bacterial solution can be placed in a 4°C refrigerator for short-term use. For long-term use, add an equal volume of sterile glycerol, invert and mix, 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 increase OD 600 Adjust to 0.7, add 500μL 1mol / L MES buffer, 500μL 1mol / L MgCl2, and 75μL 100mol / L acetosyringone (AS) to every 50mL injection solution, mix well, and then let stand at room temperature and inject into the leaf tissue of Nicotiana benthamiana. Repeat the injection of 4 to 6 leaves of 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 cut and immersed in GUS infection solution for vacuum infiltration until the leaves were obviously infiltrated. After infiltration, they were replaced with new GUS infection solution at 37°C overnight, and then chlorophyll was removed with 75% ethanol. The decolorization solution was changed twice a day until the decolorization was complete. The formula of GUS infection solution is shown in Table 5 below.

[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 0.5mg / mL - 50mg

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

[0066] 5. Construction of SpCas9 vector 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, the PCR program is shown in Table 2, and the PCR products were subjected to agarose gel electrophoresis and gel recovery; the vector pCambia1300 was double-digested with enzymes SpeⅠ and BstbⅠ, and the reaction system for double-digestion of the vector p Cambia1300 is shown in Table 6 below.

[0068] Table 6 Vector pCambia1300 double restriction enzyme digestion reaction system

[0069] Enzyme digestion system Volume (μL) SpeⅠ 1 BstbⅠ 1 10×rCutSmartBuffer 4 Carrier 1-2 μg Total Up 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 to the promoter fragment with homology arms and recombinase IIOne Step Cloning Kit (Nanjing Novozyme Biotechnology Co., Ltd.), the reaction system is shown in Table 4, after the reaction, 5 μL was transferred into Escherichia coli and the single clone was positively identified, and the constructed vector was transferred into Agrobacterium EHA105 and kept in an ultra-low temperature refrigerator 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 the -80°C ultra-low temperature freezer, use an inoculation needle to take a small amount of bacterial liquid and streak it on a solid LB medium containing 50 mg / L Rif and 50 mg / L corresponding carrier antibiotics, and culture it 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 LB liquid medium (containing 50 mg / L Rif and 50 mg / L corresponding carrier antibiotic), shake and culture the bacterial solution at 220 g and 28°C until it becomes turbid;

[0080] 3) After PCR positive clones were identified, all bacterial suspension was inoculated into 50 mL LB liquid medium (containing 50 mg / L Rif and 50 mg / L corresponding carrier antibiotics), and the bacterial suspension was shaken and cultured at 220 g and 28°C until it became turbid;

[0081] 4) Transfer the bacterial solution into a sterile 50 mL centrifuge tube and centrifuge at 8000 g for 10 min. Keep 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 pour out gently, add the infiltration solution and mix by pipetting to resuspend the cells;

[0083] 6) Take a small amount of the suspended bacterial solution and test the OD 600 value, OD 600 The value is adjusted to 0.75;

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

[0085] 8) The whole plant of Junqianzi tissue culture seedling was infiltrated into the Agrobacterium infection solution, and placed in a vacuum device (0.8MPa) for 25 minutes, the bacterial solution was dried on sterile filter paper, and then inoculated into the subculture medium (DKW+3% sucrose+0.1mg / L IAA+1.0mg / 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 no longer turbid; the tissue culture seedlings were taken out and placed on sterile filter paper to absorb the moisture on the surface of the leaves, and then inoculated into the subculture medium;

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

[0088] VII. RNA Extraction

[0089] The RNA from the pulp and leaves was extracted using the column-type plant total RNA extraction and purification kit (Shanghai Shenggong Biotechnology Co., Ltd.) according to the instructions with slight modifications. The specific steps are as follows:

[0090] 1) Take 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) Take 0.1 g of frozen plant tissue and grind it into powder with liquid nitrogen, add it to the above 1.5 mL centrifuge tube, shake it thoroughly and place it at room temperature for 5 min (lay it flat and mix it thoroughly);

[0092] 3) Centrifuge at 12000g for 3 min at 4°C and transfer the supernatant to a new RNAase-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 min, centrifuge at 12000g for 1 min 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 GT Solution, let stand for 1 min, centrifuge at 10,000 g for 1 min 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 NT Solution, let stand for 1 min, centrifuge at 10,000 g for 1 min at room temperature, and discard the waste liquid in the collection tube;

[0097] 8) Centrifuge at 12000 g for 10 min at 4°C, discard the supernatant, and add 1 mL of 75% ethanol to rinse the precipitate;

[0098] 9) Centrifuge at 12000 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 diethyl pyrocarbonate (DEPC) water to dissolve in the center of the adsorption membrane, let stand for 5 minutes, and centrifuge at 12000 g for 2 minutes;

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

[0101] 8. Reverse Transcription

[0102] Refer to the instructions of 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 and centrifuge briefly, incubate at 42°C for 2 min, 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 corresponding reagents according to the reaction system shown in Table 8, mix well and centrifuge briefly. The reaction conditions are incubation at 37℃ for 15min; 85℃ for 5s; 4℃ for 5min, and reverse transcription is completed. Use a micro-spectrophotometer to detect the quality of cDNA, dilute the cDNA with ultrapure water to 200-300ng / μL, and store it in a -20℃ refrigerator for later use.

[0109] Table 8 Reverse transcription reaction system

[0110] Reagents Volume (μL) Step (1) Reaction product 10 5×PrimeScriptBuffer2 4 PrimeScriptRTEnzyme 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. qRT-PCR was performed using 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 to calculate the expression of 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 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 Medium 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, the results of qRT-PCR quantitative experiments showed that the promoter with the strongest ability to drive SpCas9 expression in persimmon is 2×35S promoter, followed by ZmUbi and AtUBQ10, and the weakest is AtYAO.

[0119] Screening of sgRNA promoter driven by high activity in persimmon

[0120] 1. Driving sgRNA promoter amplification

[0121] Using Junqianzi and Diospyros kaki DNA as templates, primers were designed to amplify 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 connected 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 the vector, and the target sites T3 (TAAGGCCGGATATCAAGAGA (SEQ ID NO.32)) and T5 (AACCCAACCTTCGAACAACA (SEQ ID NO.33)) of DlU6-1, DlU6-2, DlU6-3, DlU6-4, DlU6-5, DlU3, DkU6-1, DkU6-2, Dk U6-3, DkU6-4, DkU6-5 and AtUBQ10 and DkMYB4 (BAI49721.1) were simultaneously constructed into the expression vector pggk (Vector ID: 9_81; Decaesteck er & Buono et al. Plant Cell 2019). The PTG strategy (transfection of vectors containing 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 positively identified 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 the high-activity driven SpCas9 promoter of persimmon.

[0138] 4. Vacuum penetration

[0139] The method was the same as the vacuum infiltration step in the screening of high-activity SpCas9 promoter driven by persimmon, but the material was 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 (Tail Method) Reverse Transcription Kit (Sangon Biotechnology Co., Ltd.), and the reaction mixture was added to an ice-bathed centrifuge tube 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 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) pAtUBQ10-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 only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A promoter for specific and efficient expression in persimmon, characterized in that: The promoter includes promoter 2×35S that drives Cas9 expression and / or 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.

2. An expression vector, characterized in that: The invention comprises the persimmon-specific and highly efficient expression promoter as claimed in 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 or Agrobacterium tumefaciens EHA105 monoclonal cell line.

6. Use of the promoter as claimed in claim 1 in increasing the expression level of exogenous genes in persimmon.

7. Use of the expression vector as claimed in claim 2 or 3 in increasing the expression level of foreign 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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