CN112877330B - Method for efficiently expressing target gene in protoplast cell - Google Patents

Abstract

The invention discloses a method for efficiently expressing a target gene in a protoplast cell. The method comprises the following steps: using a DNA molecule as a promoter to start the expression of a target gene, wherein the DNA molecule is 1) or 2) or 3): 1) a DNA molecule shown as SEQ ID No. 3; 2) a DNA molecule having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the nucleotide sequence defined in 1) and having a promoter function; 3) a DNA molecule which is hybridized with the nucleotide sequence defined in 1) or 2) under strict conditions and has the function of a promoter. Experiments prove that: the DNA molecule can efficiently and stably drive a target gene to express in the apple protoplast cell, and breaks through the technical limitation that part of the gene is not expressed and part of the gene is weakly expressed when a CaMV 35S promoter is applied to the apple protoplast cell.

Description

Method for efficiently expressing target gene in protoplast cell

Technical Field

The invention relates to the field of plant biotechnology and biochemistry, in particular to a method for efficiently expressing a target gene in a protoplast cell.

Background

The apple is a plant of Malus of Maloideae of Rosaceae, has high nutritive value, is rich in minerals and vitamins, has rich calcium content, and is helpful for metabolizing excessive salt in vivo, and malic acid can metabolize heat and prevent obesity of lower body. The apple has high solubility of nutrient components and is easy to be absorbed by human body, so it is called "running water". It is beneficial to dissolving sulfur element and making skin smooth and tender. China is the biggest apple producing and consuming country in the world, and the area and the yield are the first place in the world.

The research on important functional genes in the apples is helpful for disclosing the molecular mechanism of cell signal transduction and the response mechanism to stress. The embryogenic callus is used as a good test material, is widely applied to researches such as genetic transformation, regeneration plants and the like of plants, and is a good test material of free protoplast; the metabolic process, signal transduction path, reaction to environmental factors and external stimulation of the protoplast without cell wall are basically the same as those of the whole plant cell or tissue, and by introducing a target gene or DNA fused with the target gene and a reporter gene into the protoplast cell, the transient expression of the reporter gene in the plant protoplast can be utilized to quickly and accurately research related gene functions, protein interaction, cell metabolic process and the like, thereby providing a simple and effective experimental system for researching physiological change, gene expression, signal transmission and the like of plants on the cellular level.

Disclosure of Invention

The invention provides a DNA molecule.

The provided DNA molecule is 1) or 2) or 3) as follows:

1) a DNA molecule shown as SEQ ID No. 3;

2) a DNA molecule having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the nucleotide sequence defined in 1) and having a promoter function;

3) a DNA molecule which is hybridized with the nucleotide sequence limited by 1) or 2) under strict conditions and has the function of a promoter.

The invention also provides a biological material containing the DNA molecule.

The biological material containing the DNA molecule provided by the invention is any one of the following B1) to B11):

B1) an expression cassette comprising the DNA molecule of claim 1;

B2) a recombinant vector comprising the DNA molecule of claim 1;

B3) a recombinant vector comprising the expression cassette of B1);

B4) a recombinant microorganism comprising the DNA molecule of claim 1;

B5) a recombinant microorganism comprising the expression cassette of B1);

B6) a recombinant microorganism containing the recombinant vector of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a transgenic cell line comprising the DNA molecule of claim 1;

B9) a transgenic cell line comprising the expression cassette of B1);

B10) a transgenic cell line comprising the recombinant vector of B2);

B11) a transgenic cell line comprising the recombinant vector of B3).

In the above biological material, the expression cassette is composed of the DNA molecule having a promoter function, a target gene whose expression is promoted by the DNA molecule, and a transcription termination sequence.

The recombinant vector may be a recombinant expression vector or a recombinant cloning vector.

Primer pairs for amplifying the above DNA molecules or any fragment thereof are also within the scope of the present invention.

The invention also provides a new application of the DNA molecule or the biological material.

The invention provides the application of the DNA molecule as a promoter.

The invention also provides the application of the DNA molecule or the biological material containing the DNA molecule in promoting the expression of target genes.

In the above application, the target gene expression is initiated in a plant cell.

The plant may be a dicot or a monocot; specifically, the dicotyledonous plant can be an Malus plant; more specifically, the Malus plant is Malus pumila.

The plant cell may be a protoplast cell; specifically, the protoplast cell is an apple protoplast cell.

The invention finally provides a method for expressing a target gene.

The method for expressing the target gene comprises the following steps: the expression of the target gene is promoted by using the DNA molecule as a promoter.

In the above method, the expression target gene is expressed in apple protoplast cells; the apple protoplast cell is prepared by separating an apple callus cell which grows for 10 days on an MS culture medium added with phytohormone as a test material. The apple protoplast cell can be Orin apple callus cell or purple red 2-4 apple callus cell. When the apple protoplast cells are Orin apple callus cells, the phytohormones are 2,4-D (1.5mg/L) and 6-BA (0.4 mg/L); when the apple protoplast cells are 2-4 purple red apple callus cells, the phytohormones are NAA (0.6mg/L) and 6-BA (0.5 mg/L). And the callus cells of day 10 are used for preparing the apple protoplast cells optimally.

In any of the above applications or methods, the target gene may be any one or several or all of the following genes: MdERF98, MdERF1, MdERF2, MdERF3, and MdERF 6.

The invention clones a nucleotide fragment with promoter function from apple. Experiments prove that: the nucleotide fragment can efficiently and stably drive and regulate the expression of a representative target gene in apple fruit maturation and immunoreaction in an apple protoplast cell, breaks through the technical limitation that part of genes are not expressed and part of genes are weaker when a CaMV 35S promoter is applied in the apple protoplast cell, and lays a foundation for applying an apple protoplast cell protein expression technology to apple signal transduction research.

Drawings

FIG. 1 shows apple callus cells used for isolation of cells for making apple protoplasts. a: orin apple callus cells grown on MS medium; b: 2-4 calli cells of prunus persica grown on MS medium; c: 4 initial cell lines are induced by hybrid progeny leaves of Fuji and Xinjiang wild apple. a. And b, the apple callus cells are used for separating the apple protoplast cells.

FIG. 2 shows apple protoplast cells isolated from apple callus cells. a: apple protoplast cells isolated from Orin apple callus cells grown on MS medium for different days; b: apple protoplast cells isolated from rhodopsin 2-4 apple callus cells grown on MS medium for different days. Days in the figure are days of apple callus cell growth on MS medium used to isolate apple protoplast cells. Scale bar 50 μm.

FIG. 3 shows the construction of the maternal expression vector using the Pro-BIUTNT promoter. a: an expression vector construction scheme; b: Pro-BIUTNT is cloned into the expression vector for illustration display.

FIG. 4 shows the construction of expression vectors of target genes to be tested using two types of CaMV 35S, Pro-BIUTNT promoters. a: constructing an expression vector; b: testing the full-length amplification result of the open reading frame of the target gene; c: enzyme digestion identification of the expression vector of the reference target gene; d: the dotted line is above the expression vector structure diagram of the target gene using CaMV 35S, Pro-BIUTNT promoter, the dotted line is below the expression vector structure diagram of the target gene using Pro-BIUTNT promoter, (left) is the mother carrier used in the carrier construction (plasmid 1, plasmid 3, plasmid 4 and plasmid 5), (middle) is the expression vector structure diagram of the reference target gene, (right) is the reference target gene set in the expression vector, CCCCT (shown in the legend) is the sequence formed by SmaI and StuI enzyme digestion end connection in the expression vector construction, and the sequence exists in the expression vectors of MdBAK1, MdFLS2, MdWRKY29, MdWRKY33-1 and AtFLS2 which are cloned by SmaI enzyme digestion.

FIG. 5 shows the construction of an expression vector for driving a target gene MdERF98 by using a nucleotide sequence in apple as a promoter. a: an expression vector construction scheme; b: cloning the target gene MdERF98 into a maternal vector 'plasmid 3' for legend display; c: cloning nucleotide sequences in 6 apples as promoters into an expression vector legend of a target gene MdERF98 for display; d: the structure diagram of MdERF98 expression vector (plasmid 7-plasmid 12) using apple nucleotide sequence as promoter and intermediate vector in the vector construction.

FIG. 6 is a comparison of the expression effects of Pro-BIUTNT and CaMV 35S promoter driving target genes MdERF1, MdERF2, MdERF3, MdERF6, MdERF98 and MdEIL 2. a: Pro-BIUTNT promoter is effective in driving MdERF1, MdERF3, MdERF6, MdERF98, MdEIL2(

Shown), high efficiency driving MdERF2 (shown by); b: the CaMV 35S promoter and the Pro-BIUTNT promoter drive the expression effects of target genes MdERF1, MdERF2, MdERF3 and MdERF6 in apple protoplast cells. Expression vectors driven by CaMV 35S promoter each select 3 clones, and 1#, 2#, and 3# are clone numbers. The end is marked by an arrow and the signal of the target protein is shown by a straight line.

FIG. 7 shows that the Pro-BIUTNT promoter can effectively drive the expression of MdWRKY33-1 and MdWRKY29, and can efficiently drive the expression of MdBAK 1.

FIG. 8 shows that the Pro-BIUTNT promoter is effective in driving the expression of MdFLS 2. a: comparing CaMV 35S and Pro-BIUTNT promoters in protoplast cells for driving target gene expression for different times; b: comparison of CaMV 35S and Pro-BIUTNT promoter to drive target gene expression in apple protoplast cells prepared from 6-day old callus cells cultured in MS medium.

FIG. 9 shows Pro-MdBIUTNT, Pro-MdBIUTNT-2 promoters with the ability to drive the expression of the target gene MdERF98 in apple protoplast cells, and Δ indicates the target protein signal.

FIG. 10 shows the construction of expression vectors for reference target genes using Pro-MdBIUTTNT-2 promoter. a: construction scheme of expression vector (pHBT: Pro-MdBIUTNT-2:: MdERF1/MdERF2/MdERF3/MdERF 6-HA); b: structure diagram of Pro-MdBIUTTNT promoter expression vector of 4 reference target genes (MdERF1, MdERF2, MdERF3 and MdERF 6); c: and (3) carrying out enzyme digestion identification mapping on the plasmids 17-20.

FIG. 11 is a comparison of the Pro-MdBIUTNT TNT-2 promoter and CaMV 35S promoter for the expression of target genes in apple protoplast cells.

Detailed Description

The following examples are presented to facilitate a better understanding of the invention, but are not intended to limit the invention thereto. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the following examples, all the names of the maternal expression vectors are unique, and the names of the parts are the same, i.e., the names refer to the complete identity. In the following examples, the promoter is named by adding Pro before the gene name to which the nucleotide fragment belongs, and spacing with a spacer "-" between the two, and adding a suffix "-2" or "-1", "-2" after the gene name indicates different nucleotide fragments from the same gene promoter region. Pro is English promoterProThe moter is abbreviated. The vector structure diagrams in the following examples include only the structural elements of the expression vectors referred to in this patent application, including the promoter, the driven gene of interest and the C-terminal tag of the gene of interest.

"plasmid 1" and "AvrPto-GFP" in the following examples are described in the literature "He P, Shan L, Lin NC, Martin GB, Kemmerling B, N ü rnberger T, Screen J.2006.specific bacterial supports of MAMP signaling upstream of MAPKKKK in Arabidopsis inmunity, 125: 563. Cell 575.", "plasmid 1" is the "AvrRpm 1" expression vector in Figure 2 of the literature, and AvrPto-GFP is the "Avro-GFP" expression vector in Figure 6A of the literature, publicly available from the Applicant (university of Shandong agriculture) for the sole purpose of repeating experiments relevant to the present invention, and are not used for other purposes.

The binary expression vector pCB302 in the following examples is described in the document "Cui F, Wu S, Sun W, Coaker G, Kunkel B, He P, Shan L.2013.the Pseudomonas syringae type III effect AvrRpt2 proteins methods relating to the use of microorganisms in the culture medium of adhering the amplification/induced acid protein turnover, Plant Physiology,162:1018 1029." the binary expression vector pCB302 "is described in the second part of the document 11. materials and methods, and is available from the present applicant (university of agriculture, Shandong), which is used only for repeating experiments relating to the present invention and is not applicable for other uses.

Example 1 separation method of apple protoplast cells

The apple callus cells are used as test materials, and three enzymes of cellulase, pectinase and eductase are used for mixed hydrolysis to separate and prepare the apple protoplast cells. The method comprises the following specific steps:

culture and preservation of apple callus cells

The apple callus cells are 2 types: 1 is Orin apple callus cells (fig. 1 a); 2 is 2-4 calli cells of apple (FIG. 1 b).

1. Origin, culture and preservation of Orin apple callus cells

1) Origin of Orin apple callus cells

Orin apple callus cells are derived from the references "AN JP, Yao JF, Xu RR, You CX, Wang XF, Hao YJ. apple bZIP transcription factor MdbZIP44 regulated abscisic acid-expressed anti-nocyanin acid-plant, Cell & Environment,2018, DOI: 10.1111/pce.13393".

2) Cultivation and preservation of Orin apple callus cells

Culture and preservation of Orin apple callus cells using MS (Murashige-Skooge) medium (M5519, Sigma) supplemented with 1.5 mg/L2, 4-D and 0.4 mg/L6-BA, subcultured every 2 weeks under the culture conditions: culturing in dark at 24 deg.C.

2. Source, culture and preservation of 2-4 purple apple callus cells

1) Sources of 2-4 Callicarpa Malaria callus cells

The calli cells of the purple red 2-4 are apple calli obtained by inducing superior leaves of a population separated from F1 filial generation of Fuji and Xinjiang wild apples, 2-4 is selected from 4 initial cell lines 2-3, 2-4, 2-2 obtained by induction and yellow cells (figure 1c), and the red calli cells which are loose, uniform and constitutively synthesized with anthocyanin are obtained by multi-generation subculture on MS culture medium under light (figure 1 b). The induction and biological properties of the cells of the calli refer to the references "Ji XH, Wang YT, Zhang R, Wu SJ, An MM, Li M, Wang CZ, Chen XL, Zhang YM, Chen XS. effects of auxin, cytokine and nitrogen on anticancer Cell biosynthesis in wells cultures of red-fleshed applets (Malus sievesius f. niedzetzyana)," Plant Cell Tissue Organ, Culture,2015,120: 325- "337".

2) Culture and preservation of calli 2-4 apple callus cells

Subculture, culture and maintenance are carried out on the calli cells of the purple red 2-4 apples by using an MS culture medium added with 0.6mg/L NAA and 0.5 mg/L6-BA, and subculture is carried out once every 2 weeks. The culture conditions were: the culture was incubated at 24 ℃ in 8 hours of light and 16 hours of darkness per day.

Second, separation of apple protoplast cells

And separating and preparing apple protoplast cells by taking the two apple callus cells growing on the MS culture medium added with the phytohormone for different days as test materials. The isolation of apple protoplast cells is described in the references "Gao X, Wheeler T, Li Z, Kenerley CM, He P, Shann L.2011.Silencing GhNDR1 and GhMKK2 microorganisms cotton resistance to Verticillium wilt.plant J,66:293 ″ (the reference species is cotton, the reference species is cotton leaves and hypocotyl, whereas the reference species is apple, the reference species is apple callus cells). The method comprises the following specific steps:

1. preparation of the desired solution

Enzymolysis liquid: 1.5% cellulase (Yakult Pharmaceutical, R-10), 0.4% Segrelase (Yakult Pharmaceutical, R-10), 0.05% pectinase (Kyowa Chemical, Y-23), 20mM KCl (Sigma, P9333), 10mM CaCl₂(Sigma, C1016), 2% sucrose (Tianjin Kanton), 20mM MES (Sigma, M3671, pH5.7), 0.4M mannitol (Sigma, M4125).

W5 solution: 0.154M NaCl (Chinese medicine, 10019318), 0.125M CaCl₂、0.005M KCl、0.002M MES(pH5.7)(Sigma，M3671)。

MMG solution: 0.4M mannitol, 0.015M magnesium chloride (national drug, 10012817), 0.004M MES (pH5.7) (Sigma, M3671).

2. Isolation of apple protoplast cells

1) Weighing 2g of apple callus cells in a sterile ultraclean workbench, adding the apple callus cells into 10mL of enzymolysis liquid, gently dispersing the callus cells uniformly by using a sterile medicine spoon, then putting the mixture into a vacuum drier, vacuumizing for 30 minutes at the vacuum degree of 0.06MPa, taking out, and placing the mixture at room temperature in a dark place for enzymolysis for 12 hours.

2) After enzymolysis, gently shaking the apple callus cells in the enzymolysis solution to release apple protoplast cells, adding an isovolumetric W5 solution, and uniformly mixing to obtain a mixed solution; the mixture was filtered through nylon cloth with a pore size of 100 μm into a 50mL round-bottom centrifuge tube, and the nylon cloth was washed sequentially 3 times with 5-10mL of W5 solution each time and collected in the above 50mL round-bottom centrifuge tube. Centrifuging the apple protoplast cells suspended in the enzymolysis solution and the W5 solution for 2 minutes at 900rpm on a Beckman centrifuge (J2-MC), and discarding the supernatant; apple protoplast cells were collected and resuspended in 15mL W5 solution, then placed on ice for 30 minutes, the supernatant was discarded, 2mL MMG solution was slowly injected into the bottom of the tube, the apple protoplast cells layered on the bottom of the tube were resuspended, 10. mu.L of the apple protoplast cell suspension was aspirated and dropped into the well of a hemocytometer, a cover glass was placed thereon, and observed, counted and photographed under an Olympus, BX53 optical microscope.

FIG. 2a shows apple protoplast cells isolated from Orin apple callus cells grown on MS medium supplemented with phytohormones for 6, 8, 10, 12, and 14 days, respectively, as test samples. FIG. 2b shows apple protoplast cells isolated from calli of Calf.purpurea 2-4 grown on a plant hormone-supplemented MS medium for 5 days, 10 days, 15 days, 20 days, and 25 days, respectively, wherein the protoplast cells isolated from calli of Calf.purpurea 20 days and 25 days grown on a plant hormone-supplemented MS medium are observed under a microscope, and no uniformly dispersed protoplast cells are observed, and the isolated protoplast cells aggregate into clumps and have fragmented pieces (FIG. 2b, 20 days, and 25 days), so the number of protoplast cells is not counted.

Through counting and calculating the number of the separated apple protoplast cells, the optimal separation conditions of the apple protoplast cells are found to be as follows: apple protoplast cells were isolated and prepared using apple callus cells grown on MS medium supplemented with phytohormones for 10 days as test samples, and the maximum amount of apple protoplast cells could be obtained (Table 1).

TABLE 1 statistics of apple protoplast cell numbers isolated from callus cells of Orin, purplish red 2-4 apples grown on phytohormone supplemented MS medium for different days

Example 2 amplification of Pro-BIUTNT promoter and construction of target Gene expression vector Using the same as promoter

Cloning of Pro-BIUTNT promoter

Extracting arabidopsis genome DNA, using arabidopsis genome DNA as a template, and carrying out PCR amplification on 1307bp nucleotide sequence of polyubiquitin coding gene (At4g05320.2) from A (excluding A) in translation initiation site ATG (excluding A) from arabidopsis genome DNA by using an amplification primer of Pro-BIUTNT promoter shown in Table 2, wherein the nucleotide fragment is named as Pro-BIUTNT in the patent application.

TABLE 2, name of 7 promoters, genes, source species, amplification primers and promoter fragment length

The amplification system is as follows: the total volume is 100. mu.L, including 4. mu.L of DNA template, 8. mu.L of dNTP, 5. mu.L of each of forward (F) and reverse (R) primers, 20. mu.L of 5 XHF Buffer, ddH₂O57. mu.L, 1. mu.L of Phusion Hi-Fi enzyme (Thermo SCIENTIFIC, # F-530L).

The amplification procedure was: firstly, the temperature is 98 ℃ for 3 min; ② 30s at 98 ℃; ③ 58 ℃ for 30 s; fourthly, 1min at 72 ℃ for 30 s; fifthly, go to for 35 cycles; sixthly, the temperature is 72 ℃ for 10 min; keeping at4 deg.C.

The result of the Arabidopsis thaliana genomic DNA extraction is shown by an arrow in lane 1 in FIG. 3b (I), and the result of the promoter amplification is shown by an arrow in lane 2 in FIG. 3b (I).

Secondly, constructing a maternal expression vector by using two types of promoters CaMV 35S, Pro-BIUTNT

The construction scheme of the maternal expression vector using both CaMV 35S, Pro-BIUTNT type promoters is shown in FIG. 3 a.

1. Plasmid 1

"plasmid 1" is the initial expression vector of all expression vectors in this patent application, is the expression vector of driving target gene AvrRpm1 by using CaMV 35S promoter, and HAs HA (HA: Hemagglutenin) at C terminal. The expression vector structure is shown on the left of the font of "plasmid 1" (FIG. 3 a).

2. Plasmid 2

Using a Thermo SCIENTIFIC (GeneArt, A13282) kit and primers listed in Table 3, the three sites of the structure of the plasmid 1 were mutated from XhoI to PstI, from BamHI to SmaI and from PstI to BamHI, respectively, using the plasmid 1 as a template, to obtain an expression vector with mutated cleavage sites, named "plasmid 2" (FIG. 3a, step 1).

TABLE 3 mutant primers for restriction sites in expression vectors

3. Plasmid 3

And (3) carrying out enzyme digestion cloning by using PstI/SmaI by using the plasmid 2 as a template, replacing a CaMV 35S promoter in the plasmid 2 with the Pro-BIUTNT promoter fragment obtained by amplification in the step one, obtaining an expression vector for driving a target gene AvrRpm1 by using the Pro-BIUTNT promoter, and naming the expression vector as a plasmid 3 (figure 3a, step 2). The preparation method comprises the following steps:

1) "plasmid 2" was digested with restriction enzymes PstI/SmaI in a volume of 15. mu.L, by adding 1.5. mu.L of 10 XNEB Cutsmart buffer, 7. mu.L of 100 ng/. mu.L "plasmid 2", 0.5. mu.L PstI (NEB, # R3104) and 5.5. mu.L of sterile water, then digesting in a 37 ℃ metal bath for 2 hours, adding 0.5. mu.L of SmaI (NEB, # R0141S), and finally digesting in a 25 ℃ metal bath for 2 hours.

2) After completion of the digestion, the digested product was electrophoresed on a 1.5% agarose gel. The electrophoresis results are shown in FIG. 3b (II). The lower arrow indicates the 564bp CaMV 35S promoter released from "plasmid 2"; the upper arrow indicates the remainder of the expression vector after release of the CaMV 35S promoter. The expression vector fragment shown by the upper arrow is cut into gel, the promoter is recovered and removed from the gel block by using a Kangji quick agarose gel DNA recovery kit (CW2302M), the rest part of the expression vector is connected with the Pro-BIUTNT amplified fragment which is recovered by PstI/SmaI double enzyme digestion, gel electrophoresis and gel cutting in the same way under the action of T4 DNA ligase (NEB, M0202) at 16 ℃ overnight, and the Escherichia coli is transformed.

3) And (3) selecting Escherichia coli for monoclonal culture, extracting plasmids, identifying Pro-BIUTNT promoters, and cloning into a target clone of an expression vector. The identification method comprises the following steps: and (3) digesting the target clone by using restriction enzyme PstI/SmaI, finishing digestion, and carrying out electrophoresis on a digestion product in 1.5% agarose gel. The electrophoresis results are shown in FIG. 3b (III). The upper arrow in the figure shows the rest of the expression vector after removal of the promoter, and the lower arrow shows the release of a fragment between 1000bp and 2000bp from the identified expression vector by using the enzyme PstI/SmaI. Sequencing the fragment cloned into the expression vector revealed that the Pro-BIUTNT fragment has been cloned into the expression vector, and the obtained expression vector was named "plasmid 3". The nucleotide sequence of the Pro-BIUTNT promoter is shown as a sequence 1 in a sequence table.

4. Plasmid 4

Using the Thermo SCIENTIFIC (GeneArt, A13282) kit and the primers listed in Table 3, 2 and 3 restriction sites on the plasmid 3 were mutated from SmaI to BamHI and BamHI to SmaI, respectively, to obtain an expression vector using the Pro-BIUTNT promoter, which was capable of cloning a target gene into the expression vector using the other restriction sites, and designated as "plasmid 4" (FIG. 3a, step 3).

5. Plasmid 5

The C-terminal tag of the target gene AvrRpm1 in the plasmid 3 is mutated into FLAG from HA by taking the plasmid 3 as a template, and the target gene AvrRpm1 is replaced by AtFLS2 to obtain an expression vector which is labeled FLAG and uses the Pro-BIUTNT promoter, and the expression vector is named as a plasmid 5. The preparation method comprises the following steps:

1) the C-terminal tag of the target gene AvrRpm1 in the plasmid 3 is mutated from HA to FLAG by using a Thermo SCIENTIFIC (GeneArt, A13282) kit and the primers listed in Table 3 and taking the plasmid 3 as a template, so as to obtain the expression vector with the C-terminal tag being FLAG, wherein in the forward primer sequence, the FLAG tag sequence is indicated by italics (Table 3).

2) The full-length sequence of the AtFLS2 open reading frame is obtained by amplifying the Arabidopsis thaliana cDNA as a template by a PCR amplification method by adopting the AtFLS2 amplification primers listed in Table 5, a PCR amplification reaction system and an amplification program are the same as those in the first embodiment 2, wherein the extension time at 72 ℃ in the amplification program is calculated according to 1-2kb/min, the amplification product of the AtFLS2 is shown in a figure 4b (II), and the length is 3519 bp.

3) Using SmaI single-enzyme restriction cloning, the full-length sequence of the AtFLS2 open reading frame obtained in 2) (GeneID: NM — 001344672.1) was cloned into an expression vector tagged with FLAG at the C-terminus, designated "plasmid 5" (fig. 4a, II). The method comprises the following specific steps: after the amplification product of the AtFLS2 is precipitated by alcohol and dissolved by sterile water, SmaI is used for single enzyme digestion for 2 hours, meanwhile, expression vector with the C terminal tag being FLAG is digested by SmaI/StuI, the target gene AvrRpm1 is released, the rest part of the vector is recovered from a gel block by using a Kangji quick agarose gel DNA recovery kit (CW2302M), and is connected with the AtFLS2 fragment digested by SmaI overnight under the action of T4 ligase, and the connection product is transformed into escherichia coli.

4) The BamHI/StuI enzyme digestion was used to identify the target clone of the AtFLS2 ORF full-length sequence cloned into the expression vector. The results of enzymatic identification of AtFLS2 are shown in FIG. 4c (I), demonstrating that AtFLS2 has been cloned into the expression vector.

"plasmid 3", "plasmid 4" and "plasmid 5" are the maternal expression vectors employing the Pro-BIUTNT promoter in this patent application; "plasmid 1" is the maternal expression vector employing the CaMV 35S promoter.

Thirdly, constructing expression vector of test target gene by using CaMV 35S, Pro-BIUTNT two types of promoters

The construction scheme of the expression vector of the test target gene using two types of CaMV 35S, Pro-BIUTNT promoters is shown in FIG. 4 a; the structure of the constructed expression vectors for the 13 reference target genes is shown in FIG. 4 d. The target vectors and target genes shown in FIG. 4a (I) correspond to the target vectors and target genes above the dotted line in FIG. 4d, and the target vectors and target genes shown in FIG. 4a (II) correspond to the target vectors and target genes below the dotted line in FIG. 4 d. The bold arrows between the expression vectors indicate the expression vector construction process. The target gene is arranged below the structure diagram of the target vector. Shown in the box are genes respectively constructed into expression vectors using a CaMV 35S promoter and a Pro-BIUTNT promoter; the bold boxes indicate the expression vectors constructed to the Pro-BIUTNT promoter; the thin line box represents the expression vector constructed to apply the CaMV 35S promoter; the genes with the same font color are the genes with the same enzyme cutting sites used in the construction of the expression vector.

1. Amplification of the full-length sequences of the open reading frames of 13 reference target genes

The cDNA was used as a template to amplify the full-length sequences of the open reading frames of the 13 reference target genes, respectively, using the primers shown in Table 4. AtFLS2 and AtBAK1 were amplified using Arabidopsis cDNA as template and apple cDNA as template for the remaining genes.

TABLE 4, 13 reference target Gene names, Gene codes and amplification primer sequences

The amplification results of the full-length sequence of the open reading frames of the 13 reference target genes are shown in FIG. 4 b. (I) Shown is the amplification result of 6 transcription factor genes, including 5 ERF family transcription factors (MdERF1, MdERF2, MdERF3, MdERF6 and MdERF98) and a transcription factor MdEIL2 which is an important regulatory gene in an ethylene signal transduction pathway. (II) shows the amplification results of 4 immunoreceptor genes (AtBAK1, AtFLS2, MdBAK1 and MdFLS2) and MdMAPK6, MdWRKY29 and MdWRKY33-1 genes in 3 immune signal pathways; the right one is an MdWRKY33-1 amplified fragment with SmaI enzyme cutting cloning sites at the 5 'and 3' ends, and is cloned into an expression vector plasmid 3 to obtain an expression vector driven by a Pro-BIUTNT promoter; the right two are MdWRKY33-1 amplified fragments with BamHI and SmaI enzyme cutting sites at the two ends of 5 'and 3' respectively, which are cloned into an expression vector 'plasmid 1' to obtain an expression vector driven by a CaMV 35S promoter.

2. Construction of expression vector of reference target gene

Respectively cloning the open reading frame full-length sequences of the 13 tested target genes amplified in the step 1 into an expression vector. The promoter type (CaMV 35S promoter, Pro-BIUTNT promoter), the parent vector (plasmid 1, plasmid 3, plasmid 4 or plasmid 5) and the restriction enzyme cloning site used for the expression vector into which each of the genes to be tested was cloned are shown in Table 5.

TABLE 5 promoter types of expression vectors into which each of the genes tested was cloned, the parental vectors and the restriction cloning sites used

Note: gene 1 is MdERF1 gene; gene 2 is the MdERF2 gene; gene 3 is the MdERF3 gene; gene 4 is the MdEIL2 gene; gene 5 is MdERF 6; gene 6 is the MdERF98 gene; gene 7 is mdpak 1 gene; gene 8 is the MdFLS2 gene; gene 9 is MdWRKY29 gene; the gene 10 is MdWRKY33-1 gene; gene 11 is AtFLS 2; gene 12 is the AtBAK1 gene; gene 13 is the MdMAPK6 gene. Filling the promoter type of the reference genes (directly facing the first row above the reference genes) cloned into the expression vector, the female parent vector (directly facing the left first row) and the used restriction enzyme cloning site (directly facing the left second row); black fill constructs to application C respectively_a MV 35S promoter and P_rGenes in the expression vector of the o-BIUTNT promoter (Gene 1-Gene 10); construction of purple fill to application P_rGenes (Gene 11 and Gene 12) in the expression vector for the o-BIUTNT promoter; orange fill construction to application C_aA gene in an expression vector of MV 35S promoter (gene 13); the numbers in the filled squares indicate the number of clones selected by the expression vector.

Because the open reading frame sequence of MdERF98 contains BamHI enzyme cutting site GGATCC, a Thermo SCIENTIFIC (GeneArt, A13282) kit and a mutation primer F are used for facilitating the subsequent construction of an expression vector: 5'-TATTTCCGGGCGTAGATCCTGGAAGGAG-3', respectively; r: 5 'AAGATGAGGAAGAAGCAGAAGATGAAGAC-3' the BamHI cleavage site of the gene was synonymously mutated in the expression vector to AGATCC. The nucleotide sequences of MdERF98 in the expression vector of MdERF98 in the patent application are all nucleotide sequences after synonymous mutation.

3. Enzyme digestion identification of expression vector of participating target gene

The enzyme digestion identification of the expression vector of the reference target gene uses BamHI/StuI, and the identification result is shown in FIG. 4 c. (I) Shown are restriction enzyme cutting identification (shown in the figure) of expression vectors driven by CaMV 35S promoter and Pro-BIUTNT promoter, and restriction enzyme cutting identification (shown in the figure) of expression vectors driven by MdMAPK6 and AtBAK1 and AtFLS2 expression vectors driven by the CaMV 35S promoter and the Pro-BIUTNT promoter respectively for 6 target genes; the gene names are shown in the figure. The number "1" indicates an expression vector using CaMV 35S promoter; the number "2" indicates an expression vector using a Pro-BIUTNT promoter; the number "3" indicates an expression vector using the Pro-BIUTNT promoter, C-terminally FLAG-tagged AtFLS 2. (II) shows that the enzyme digestion identification of expression vectors which use CaMV 35S promoters and Pro-BIUTNT promoters of MdERF1, MdERF2, MdERF3 and MdERF6 is carried out.

3 clones are selected from each of expression vectors driven by CaMV 35S promoters of MdERF1, MdERF2, MdERF3 and MdERF 6; 1 clone was selected for each expression vector using Pro-BIUTNT promoter. 1#, 2#, and 3# are different clone numbers of the expression vector. The names of the genes and promoters are shown in the figure.

FIG. 4c, (I) and (II) shows that all the genes to be tested are cloned into the expression vector.

Example 3 amplification of Pro-MdBIUTNT and Pro-MdBIUTNT-2 promoters in apple and construction of expression vectors Using the same as promoters

Amplification of 6 nucleotide fragments in apple

Using apple genome DNA as template, respectively adopting the primers corresponding to Pro-MdBIUTNT, Pro-MdBIUTNT-2, Pro-MdRP-1, Pro-MdRP-2, Pro-MdRC-1 and Pro-MdRC-2 fragments in Table 2 to amplify so as to respectively obtain Pro-MdBIUTNT, Pro-MdIUTNT-2, Pro-MdRP-1, Pro-MdRP-2, Pro-MdRC-1 and Pro-MdRC-2 nucleotide fragments.

The 6 nucleotide fragments were from the following 3 genes, respectively: MdBIUTNT (Pro-MdBIUTNT, Pro-MdBIUTNT-2), MdRP (Pro-MdRP-1, Pro-MdRP-2), MdRC (Pro-MdRC-1, Pro-MdRC-2). MdBi UTNT is a homologous gene of an arabidopsis polyubiquitin gene in apple; MdRP is 1, 5-ribulose two phosphoric acid carboxylase small subunit gene; MdRC is apple 1, 5-ribulose bisphosphate carboxylase activator gene; the numbering of the genes in the apple genome is shown in Table 2.

Wherein Pro-MdBi UTNT and Pro-MdBi UTNT-2 are two nucleotide fragments which are 1539bp and 2501bp upwards from A (excluding A) in the translation initiation site ATG of MdBi UTNT gene. The nucleotide sequence of the Pro-MdBi UTNT promoter is shown as a sequence 2 in a sequence table, and the nucleotide sequence of the Pro-MdBi UTNT-2 promoter is shown as a sequence 3 in the sequence table.

Pro-MdRP-1 and Pro-MdRP-2 are two nucleotide fragments which are 1972bp and 3050bp respectively from A (excluding A) in the translation initiation site ATG of the MdRP gene. The nucleotide sequence of the Pro-MdRP-1 promoter is shown as a sequence 4 in a sequence table, and the nucleotide sequence of the Pro-MdRP-2 promoter is shown as a sequence 5 in the sequence table.

Pro-MdRC-1 and Pro-MdRC-2 are two nucleotide fragments of 2025bp and 2542bp respectively from A (excluding A) in the translation initiation site ATG of MdRC gene. The nucleotide sequence of the Pro-MdRP-1 promoter is shown as a sequence 6 in a sequence table, and the nucleotide sequence of the Pro-MdRP-2 promoter is shown as a sequence 7 in the sequence table.

Pro-MdBIUTNT, Pro-MdBIUTNT-2, Pro-MdRP-1, Pro-MdRP-2 amplification products are shown in figure 5c (I); Pro-MdRC-1 and Pro-MdRC-2 amplification products are shown in FIG. 5c (I) by arrows.

Secondly, constructing an expression vector for driving a target gene MdERF98 by using a nucleotide sequence in the apple as a promoter

The construction scheme of the expression vector for driving the target gene MdERF98 by using the nucleotide sequence in the apple amplified in the step one as a promoter is shown in FIG. 5 a. The method comprises the following specific steps:

1. plasmid 6

Plasmid 6 was obtained by replacing the target gene AvrRpm1 in "plasmid 3" with the target gene MdERF 98. The preparation method comprises the following steps:

1) Pro-BIUTNT constructed as in example 2 (III): : the MdERF98 expression vector (see figure 4d for expression vector structure diagram) is used as a template, and the MdERF98 with the mark in the table 4 is used for amplification to obtain the target gene MdERF 98. FIG. 5b (I) shows the result of amplification of MdERF 98.

2) SmaI/StuI enzyme digestion cloning is applied, and a target gene MdERF98 is cloned into a parent vector 'plasmid 3' to obtain an expression vector which is named as 'plasmid 6' (figure 5a, step 1). FIG. 5b (II) shows the results of restriction enzyme identification of the expression vector MdERF98 using restriction enzyme SmaI/StuI. The cleavage showed that MdERF98 had been cloned into the expression vector.

2. Plasmid 7 "-" plasmid 12 "

And (2) taking the plasmid 6 as a template, carrying out enzyme digestion cloning by using PstI/SmaI, cloning the nucleotide fragments from the apples obtained in the step one into an expression vector as a promoter respectively, and obtaining an MdERF98 expression vector Pro-MdBIUTNT: : MdERF98 (plasmid 7), Pro-MdBIUTNT TNT-2: : MdERF98 (plasmid 8), Pro-MdRP-1: : MdERF98 (plasmid 9), Pro-MdRP-2: : MdERF98 (plasmid 10), Pro-MdRC-1: : MdERF98 (plasmid 11), Pro-MdRC-2: : MdERF98 (plasmid 12) (fig. 5a, step 2). FIG. 5c (II) shows the results of restriction enzyme identification of each MdERF98 expression vector using restriction enzymes PstI/SmaI. The expression vector and the restriction enzyme used in each lane are indicated on the right hand side of the figure. The enzyme cutting result shows that the 6 nucleotide fragments are all cloned into an expression vector to obtain the expression vector which uses the nucleotide sequence in the 6 apples as a promoter to drive a target gene MdERF 98. And the 6 nucleotide sequences cloned into the expression vectors are respectively sequenced and verified.

The 'plasmid 6' is an intermediate vector in vector construction, and the 'plasmid 7' -the 'plasmid 12' is an expression vector using a nucleotide sequence in apples as a promoter. The structure of each expression vector is shown in FIG. 5 d.

Example 4 apple protoplast cell transfection, target protein expression and detection

Apple protoplast cell transfection, target protein expression and detection

1. Purification of expression vectors

Cesium chloride gradient centrifugation was used to prepare and purify the expression vectors of the reference target genes constructed in examples 2 and 3 of this patent application, including 31 expression vectors of 13 target genes constructed in example 2 (table 5) and 7 expression vectors "plasmid 6" - "plasmid 12" constructed in example 3.The method for preparing and purifying expression vectors by cesium chloride gradient centrifugation is described in detail in molecular cloning, a laboratory Manual (third edition, scientific Press, Semikruke et al, Huangpetang), pages 53-56.

2. Apple protoplast cell transfection

In order to compare the effect of different promoters on the expression and translation of target genes in apple protoplast cells, the purified target vector from step one was transfected into apple protoplast cells prepared from Orin apple callus cells cultured in MS medium for 6 days in example 1 by PEG-mediated transient transfection, and the transfected cells were tested for expression and accumulation of target proteins after culturing for different periods of time. Wherein, the apple protoplast cells transfected with the expression vector of the target gene AtFLS2 were cultured for 2h, 4h, 6h, 8h and 10h, and then the expression and accumulation of the target protein were detected (FIG. 8 a); the apple protoplast cells transfected with the expression vectors of the remaining target genes were cultured for 6h (FIG. 6, FIG. 7, FIG. 8b) or 8h (FIG. 9) before the detection of the expression and accumulation of the target protein. Specific procedures for transfection of protoplast cells reference is made to the methods in "He P, Shan L, Sheen J.2006.the use of proplasts to study administration of microorganism responses. plant-Pathologen Interactions Volume 354: pps.1-10".

3. Target protein expression and detection

After the expression is finished, the apple protoplast cells are collected, protein denaturation is carried out by applying SDS (100mmol/L Tris-Cl, 4% SDS, 20% glycerol, 0.2% bromophenol blue and 200mmol/L dithiothreitol) sample buffer solution, and the translation level of the target gene in the apple protoplast cells is detected by applying western hybridization.

Comparison of the effects of Pro-BIUTNT, Pro-MdBIUTNT-2 promoter and CaMV 35S promoter on the expression of target gene in apple protoplast cells

1. Comparison of the Effect of Pro-BIUTNT and CaMV 35S promoters on Driving target Gene expression in apple protoplast cells

The Pro-BIUTNT promoter can stably drive 12 test-related target genes (MdERF1, MdERF2, MdERF3, MdERF6, MdEIL2, MdERF98, AtBAK1, AtFLS2, MdBAK1, MdFLS2, MdWRKY29 and MdWRKY33-1) to be efficiently expressed in apple protoplast cells. The results are detailed below:

(1) the CaMV 35S promoter cannot drive MdERF1, MdERF3 and MdERF6 to express in apple protoplast cells, and the Pro-BIUTNT promoter is used to obtain clear and specific target protein expression signals. Wherein the expression of MdERF2 is weaker by using CaMV 35S promoter, but a clear and specific target protein expression signal is obtained by using Pro-BIUTNT promoter (FIG. 6 a). Even among 3 clones selected per each of the expression vectors using CaMV 35S promoter, no vector clone driving efficient expression of MdERF1, MdERF3, MdERF6 and no vector clone of MdERF2 having the same signal strength as that using Pro-BIUTNT promoter were found (FIG. 6 b).

(2) MdERF98 is another transcription factor of ERFs (ethylene stress factors) family, the gene is not expressed (figure 6a, figure 7) or is weakly expressed (figure 9) by using CaMV 35S promoter, and clear and specific target protein expression signals are obtained by using Pro-BIUTNT promoter (figure 6a, figure 7, figure 9).

MdEIL2 is more than the above 5 ERFsThe transcription factor family gene has a longer nucleotide sequence of the amino acid coding region, CaMV 35S is used as a promoter, expression is not obtained, but Pro-BIUTNT promoter is used, a clear and specific target protein expression signal is obtained at the position of the target protein (figure 6a,

shown).

(3) MdWRKY33-1 and MdWRKY29 are important transcription factors of WRKY family. The expression of MdWRKY33-1 cannot be driven by CaMV 35S promoter, and a very weak expression signal is positioned at the position of MdWRKY29 band (CaMV 35S:: delta in MdWRKY29 in FIG. 7), while clear and specific target protein expression signals are obtained by MdWRKY33-1 and MdWRKY29 by using Pro-BIUTNT promoter (FIG. 7).

(4) MdFLS2 and MdBak1 are important immune genes in apples. By using CaMV 35S promoter, MdFLS2 is not expressed, and by using Pro-BIUTNT promoter, the signal accumulation of the target protein MdFLS2 is found after 2 hours of expression in apple protoplast cells (FIG. 8 a); furthermore, clear and specific MdFLS2 target protein expression signals were obtained from apple protoplast cells prepared from apple callus cells grown on MS medium for 6 days using Pro-BIUTNT promoter, while no protein expression signals were obtained using CaMV 35S promoter (fig. 8 b).

Although the CaMV 35S promoter was able to drive MdBak1 to obtain expression in apple protoplast cells, the signal intensity of the target protein was significantly lower than that of MdBak1 when Pro-BIUTNT promoter was used (FIG. 7

Showing the expression of mdpak 1), and using the Pro-bitnt promoter, one can clearly see the post-translational cleavage of mdpak 1 protein, while using the CaMV 35S promoter, one cannot see 3 specific bands modified by cleavage (fig. 7, showing the intracellular cleavage of mdpak 1).

The selected reference target genes in the application are all representative genes for regulating apple fruit ripening and immunoreaction, and comprise 5 ERF family transcription factors, 1 important transcription regulation factor gene in ethylene signal transduction, 4 immune receptor related genes and 3 important genes in MAPK signal pathways. Except that MdMAPK6 was not involved in the promoter strength comparison test, the Pro-BIUTNT promoter driven the expression of all 12 target genes tested, whereas with the CaMV 35S promoter, some of the genes were not expressed (MdERF1, MdERF3, MdERF6, MdEIL2, MdWRKY33-1, MdFLS2) and some were weakly expressed (MdERF2, mdky wr 29, MdERF98) (table 6, table 7).

TABLE 6 comparison of expression and translation effects of two promoters driving target genes in apple protoplast cells

MdERF1

MdERF3

MdERF6

MdEIL2

MdWRKY33-1

MdFLS2

MdERF2

CaMV

35S

Do not express

Do not express

Do not express

Do not express

Do not express

Do not express

Weak expression of

Pro-BIUTNT

Expression, Strong expression

Strong expression

Strong expression

Expression of

Strong expression

Expression of

Strong expression, expression

Source of results

FIGS. 6a, 6b, 7

FIGS. 6a and 6b

FIGS. 6a and 6b

FIG. 6a

FIG. 7

FIG. 8

FIGS. 6a, 6b, 7

Note: "/" shows that the construction of the corresponding expression vector and the comparative experiments were not performed.

TABLE 7 comparison of expression and translation effects of two promoters driving target genes in apple protoplast cells

Note: "/" shows that the corresponding expression vector construction and comparative experiments were not performed.

2. Comparison of the effects of Pro-MdBi, Pro-MdBi TNT-2 and CaMV 35S promoters on the expression of target genes in apple protoplast cells

The Pro-MdBIUTNT, Pro-MdBIUTNT-2 promoters have the ability to drive the expression of the target gene MdERF98 in apple protoplast cells (FIG. 9,. DELTA.; expression signal for the target protein MdERF 98). The cultivation in the dark for 8 hours and the cultivation in the light for 8 hours respectively means that after the transient transformation mediated by PEG4000, the protoplast cells are cultivated at 25 ℃ and under the light environment (figure 9, right) and at 25 ℃ and under the dark environment (figure 9, left) respectively to express the target protein for 8 hours.

The combination of the above results shows that: the Pro-BIUTNT promoter from the arabidopsis thaliana polyubiquitin coding gene and two nucleotide sequences Pro-MdBi UTNT and Pro-MdBi UTNT-2 from the promoter region of the apple polyubiquitin coding gene are promoters which can efficiently and stably drive the target gene to express in the apple protoplast cell and have the capacity of driving the target gene to express in the apple protoplast cell.

Example 5 application of Pro-MdBIIUTNT-2 promoter

Construction of expression vector of reference target gene by using Pro-MdBIUTTNT-2 promoter

The construction scheme of the expression vector of the reference target gene using Pro-MdBIUTIT-2 promoter (pHBT: Pro-MdBIUTIT-2:: MdERF1/MdERF2/MdERF3/MdERF6-HA) is shown in FIG. 10 a; respectively taking expression vectors of MdERF1, MdERF2, MdERF3 and MdERF6 genes driven by Pro-BIUTNT promoters as templates (see figure 4a (I)), amplifying Pro-MdBIUTTNT-2 by taking PstI/BamHI as enzyme cutting sites, replacing Pro-BIUTNT promoters to drive promoters Pro-BIUTNT in the expression vectors of MdERF1, MdERF2 and MdERF3 genes, amplifying Pro-MdBIUTTNT-2 by taking PstI/NcoI as enzyme cutting sites, and replacing Pro-BIUTNT promoters to drive promoters Pro-BIUTNT in the expression vectors of MdERF6 genes. The bold arrows between the expression vectors indicate the expression vector construction process; the genes constructed in the expression vectors using the Pro-MdBIUTNT promoter, respectively, are shown in boxes; the genes with the same font color are the genes with the same enzyme cutting sites used in the construction of the expression vector; the structure diagram of Pro-MdBITINTT-2 promoter expression vectors of the constructed 4 reference target genes (MdERF1, MdERF2, MdERF3 and MdERF6) is shown in FIG. 10b (plasmid 17-20); the restriction enzyme identification map of plasmid 17-20 is shown in FIG. 10c, and the restriction sites are PstI/BamHI (plasmid 17-19) and PstI/NcoI (plasmid 20), respectively.

Comparison of effects of Pro-MdBIUTNT-2 promoter and CaMV 35S promoter on driving target gene to express in apple protoplast cells

The steps of apple protoplast cell transfection, target protein expression and detection are the same as described in example 4.

The CaMV 35S promoter can not effectively drive MdERF1, MdERF3 and MdERF6 to express in apple protoplast cells, and a Pro-MdBIUTNT-2 promoter is used to obtain clear and specific target protein expression signals. Of these, MdERF2 expressed two clones (1# and 2#) slightly stronger than CaMV 35S promoter using Pro-MdBIUTNT-2 promoter, but no signal of the target protein was detected in clone CaMV 35S promoter 3# (FIG. 11 b). Even among the 3 clones selected for each expression vector using the CaMV 35S promoter, no vector clone was found that could drive efficient expression of MdERF1, MdERF3, MdERF6 (FIGS. 11a, c, d).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Shandong university of agriculture

<120> a method for expressing a target gene in protoplast cells with high efficiency

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tcacacatct ttttatttct cacatattcc ttgttaattt ttatccgtcg atcttcttca 1980

atttatcaaa cccgatacat gaaaattaaa aaggtgtgtg aaaggcaaaa aagtgtgtga 2040

atatcagatc tctttagtaa tccattagca ggtcatatac gatattgacg cgtgtaatcc 2100

acataacctg tcaacgtagc attctgcaag tccaaacaaa ttaggaatcc aattaccttc 2160

acaagcaagg aaaggggcaa caaaaatcca agatagagca ggtcaccttc tccctcatca 2220

aataaagaaa actaaaaatc agaggtcaac aattgttggc acttgcccac ctagaaacat 2280

ctgggccgtt cgtttcactg acttttctgg tccacgcgtt cctcgtccac tccggaatcc 2340

aacctaaaaa tgggatttga gtcggctcat ccttccccta tataactcct caccccctcc 2400

actcttcctc acaattctct caacaattct cgcccatcgt ccccaaatct ctctcaaatc 2460

cctaatttct ttcgaattac aaatccccaa tttctgaaac c 2501

<210>4

<211>1972

<212>DNA

<213>Artificial Sequence

<400>4

cccacaacat ctccatggct aagttgagaa aaccgaattg atttgtgccg ccgccgccgc 60

cgccgccttc agagacgaat atctctcgtc aacagaagct cagattcccc aattccttcg 120

gcatcaatca ggtatgggtt ttgaagaaat ttcgttttac tcaaaagtag acatacacaa 180

taggactttt tttatattta tttttgttga atgggttttt gagtaatttt actgagtatt 240

cagacccgaa agaaaagtcg taatattgat gaaaacattt gattccgatc gggttacaga 300

tatctggtta tttgttaggt ttaattgctt actatttgtt caatggggtt tacaagaact 360

ggctgaaaag aaggaactga ttatggcttt cataggactt tctcattcac ttgaaaccca 420

agtaagtata tgttctaata cttttcgttc agtttgttta aatgcttctg ggtttagggg 480

cgagtattgt tattggcaca ccaataacat aatcatgcgc tacttcatgt ataaattctc 540

ccctataaaa aagtacaagg ggtggggaat gacatttttg aagcgccaac aaggttctaa 600

aagacactag gcgctagttg ggcggcgggc tagggcctaa tgcttaggca gctaggcgga 660

tttaagtaaa tatattatat ttcgtataaa taagtgtctg cttatactta aagtacatat 720

aatttcatca taaactacaa aataaaatta catatatatt atgaagtatt taaacataat 780

gaaaacatag ggaacgatga tataatgtgt gttcatttaa gcatgcatca agtctcttac 840

aatcttttga aaaagataaa atgcaaaatg aaagttatct attttctatc taagagagtt 900

gcaacctagg taggtgtcta ggctggtttg ggcgggctag gcgggtgcct agatggtcta 960

ggcgggcacc tcaataggtc taggtaccct ttcttaattt tcaaatgcct aggcattaat 1020

cggggcagtg gctagccacc taccgcctac cccatgggcg gggattttta gaatagtaag 1080

caccaataac agctccatat aggatttaac atagcaagaa aaataatgtt agtgatcggg 1140

gccaaaagaa tgggaaggaa acatagtgtt ggtgaatggt gaggtatgag tatcagaaga 1200

tggtatgatg tttatgatgt ttagcgttta gagaaaaaat aagattaaga aggaaaaata 1260

gagttggcaa ggatcaaaag attttatggt cacttgtcag acgtcagatt aatttttgca 1320

atggaaatat aatattcaat acttttttgt ggtaaatggc aggccgaagc caaacaaaaa 1380

caacaaaaac taacacactg catcccaaac atgagctttt cccctcctgt catcatccaa 1440

aatacaagat ggatcaacac tgagcttcct cttaaacttt gaacattaat attcaatatt 1500

tttaagtgtt gtataacaga tgaaacatac ttattagtag tcagataatt atttataaaa 1560

aaggtcgtac ccagtgcaca aggctcccgc tttacgcagg gtatgggaga ggtgaatgtc 1620

ggctagcttt acccccattt attagtagtc agttaattat ttataacaac acataatact 1680

ttggccacaa caaggggtca aaattctttc caacacctgg ctttattttt gacacattaa 1740

ttaccccatc cattcgctct cgcgctctct cttttttttt cctctctttt gagctcaaga 1800

tctatttatt actttgtggc atactcagta tttgttttgt tgacagtggc ggatcttgga 1860

tatgatcatg ttcgagtgtc acacaaataa gcactttccc acaaagtctg ataaataaaa 1920

caggggctta aaatgggagc tgctgctttg tcagcttcaa agtgtgctcc ag 1972

<210>5

<211>3050

<212>DNA

<213>Artificial Sequence

<400>5

cttgtgtagg ttgatgaatc tcttatgctt aaactagaat tttaacctaa attaagattt 60

tttgggagtt gcttgtgtac ttttttggga tctttatagt agtgaaagtt catttccgta 120

gttcgacatc tgacgttctt cgggcaactg tgcatgttgt gtgacaagca tataggcatg 180

ttatttatta tggacattgc attcatatta ttattacttt ggttacatag ttatttcata 240

ttttgttttc cgcaaaaatt tacatggcaa gtaaatgagg aggtggcatc atcttattaa 300

atgatttaat ggacaagtga tcaaccccaa tttttcttgt ctcttagacc accaaaataa 360

cttacaaata aggacaacat gatggtcgta ttctaaacta atcatacaat gctatacaaa 420

aaaaaagaag ttaaaatata cttgtgaaat tgtaatagat ttgagatgac attaccacaa 480

cgaatgtgtc ttttgcaagt ctctcacact tttcaaatac attaatagat tttctacaag 540

aaaaatagaa atatataaat ttcaaactcg cctttgacaa aaactaatag caacaattaa 600

aaaaacggat ttcaattatg ctgtgcgaaa attacctgtg tgaaaccttt tttttaggaa 660

catgatcgta tccaaatcct ccttgtgata atctcacgga tcagttcatc aatcattctg 720

tggtcagaaa tcattttaaa ttttttattt aaaattgaat acaaacatca catagcaaaa 780

actaaccgcg caatatacaa tgaacgatta agatgaattg aggaccagga aaggatcctt 840

atccaatttt tttacacctg ttccttgtct tgacagtgtc agttttatat gaagttttta 900

acaaaaatac agaaaaaccc atcagccacc acgcctttgc acccacccaa cccagtgagc 960

cgcccaccac ccagtggccg gcaaaccctc ccttctcgat tccggcaaaa gctcatcctt 1020

tgaaccttct gcctcttcct ctcacctttt cttatctacc cagcgaaagc aaacccaaac 1080

cacaaaccca caacatctcc atggctaagt tgagaaaacc gaattgattt gtgccgccgc 1140

cgccgccgcc gccttcagag acgaatatct ctcgtcaaca gaagctcaga ttccccaatt 1200

ccttcggcat caatcaggta tgggttttga agaaatttcg ttttactcaa aagtagacat 1260

acacaatagg acttttttta tattgatttt tgttgagtgg gtttttgagt aattttactg 1320

agtattcaga cccgaaagaa aagtcgtaat attgatgaaa acatttgatt ccgatcgggt 1380

tacagatatc tggttatttg ttaggtttaa ttgcttacta tttgttcaat ggggtttaca 1440

agaactgact gaaaagaagg aacagattat ggctttcata ggactttgtc attcacttga 1500

aaccccagta agtatatgtt ctaatacttt tcgttcagat tgtttaaatg cttctgggtt 1560

taggggcgag tattgttatt ggcacacaaa taacataatc atgcgcaact ccatgtataa 1620

attctcccct ataaaaaagt acaaggaatg gggaatgaca tttttgaagc accaacaaga 1680

ttctaaaata cactaggcgc tagttggcta gggcctatcg cctagatggc taagccgatt 1740

taggtaaata tattatattt cgtataaata agtgtctgct tatacttaaa gtacatataa 1800

tttcatcata aattacaaaa taaaattaca tatatattat gaagtattta aacataatga 1860

aaacataggg aacgatgata taatgtgtgt tcatttaagc atgcatcaag tctcttacaa 1920

tcttttgaaa aagataaaat gcaaaatgaa agttatctat tttctatcta agagagttgc 1980

aacctaggta ggtgtctagg ctggtttggg cgggctaggc gggtgcctag atggtctagg 2040

cgggcacctc aataggtcta ggtacccttt cttaattttc aaatgcctag gcattaattg 2100

gggcagtggc cagccaccta ccgcctaccc catgggcggg gatttttaga atagtaagca 2160

ccaataacag ctccatatag gatttaacat agcaagaaaa ataatgttag tgatcggggc 2220

caaaagaatg ggaaggaaac atagtgttgg tgaatggtga ggtatgagta tcagaagatg 2280

gtatgatgtt tatgatgttt agcgtttaga gaaaaaataa gattaagaag gaaaaataga 2340

gttggcaagg atcaaaagat tttatggtca cttgtcagac gtcagattaa tttttgcaat 2400

ggaaatataa tattcaatac ttttttgtgg taaatggcag gccgaagcca aacaaaaaca 2460

acaaaaacta acacactgca tcccaaacat gagcttttcc cctcctgtca tcatccaaaa 2520

tacaagatgg atcaacactg agcttcctct taaactttga acattaatat tcaatatttt 2580

taagtgttgt ataacagatg aaacatactt attagtagtc agataattac ttataaaaaa 2640

ggtcgtaccc agtgcacaag gctcccgctt tacgcagggt atgggagagg tgaatgtcgg 2700

ctagctttac ccccatttat tagtagtcag ttaattattt ataacaacac ataatacttt 2760

ggccacaaca aggggtcaaa attctttcca acacctggct ttatttttga cacattaatt 2820

accccatcca ttcgctctcg cgctctctct ttttttttcc tctcttttga gctcaagatc 2880

tatttattac tttgtggcat actcagtatt tgttttgttg acagtggcgg atcttggata 2940

tgatcatgtt cgagtgtcac acaaataagc actttcccac aaagtctgat aaataaaaca 3000

ggggcttaaa atgggagctg ctgctttgtc agcttcaaag tgtgctccag 3050

<210>6

<211>2025

<212>DNA

<213>Artificial Sequence

<400>6

agccttagta ttattatgac tgacaaacaa aggttgtctc ttttatattc ccaattaatt 60

tacatgatta gaatggaaca tagggtttct catgttgtgt ttatgggtga aatagtctat 120

atatgtctcc atatgattga agagttttac ttgtgaattt tagtggtgtg tcgtgttata 180

aagagttaaa ggttgcatat aaaagcatgc ttaagcgtta actaattaac aacttaatac 240

ggtgctaact gttcgattag gtctccccta caaattgaac ttacgatccg ttcatttttt 300

agtgctcttt catattattc ttactgaaat tggaaatcat ttaatctttt aattattatt 360

gtcttttatg gaaacaaatg caaattcaga tatcaaacag atgtgtttgt ctaagagaga 420

gagagggggt ttggatgata gaaaggggac ctagtcatta attatcattg taaattgtat 480

tgtttattta tttatcaagt ccagttaaat aactcatctt caattttaat gaaattttgc 540

aagatgatct ttcattgata acctaaaact tgttagttca aattgtaaaa atattgtgaa 600

atgagtttaa acgagtgttt agcaccatat taagttacta actggtttga aaagtactct 660

gtaattttgt tttgttacaa atgctattct acactaatat acattaagaa gaggagaagc 720

tttgaactga agaagtaacg aataaagagg aagcttctct aacttccagt accagagcaa 780

tccattactt gtactagttt gataagtttt actttcacag tcaaattgtg ttatgtcgtg 840

ttaacgagtg aaattgttga taggcttgag atatgggcca ggtcaggact ctaagggggc 900

gtttgtttac cctcattaag tgggactaga ctggattgga ctagctgtta gtccaatact 960

gtgtttgttc catgctggga ctaacattaa tgagactaaa ggggactagc atggacaaaa 1020

cccttcacta agtggtctta gcgagacccc ccaataacca tgggactagc taagactatc 1080

ctctctcatc ctctcatgct caacaaacac tctcgataga ctcctcgtca tctccagtca 1140

cctagatcat aataaaatat taataattta tatattaaat aatataatat tagaatttgt 1200

tattatccag cttcttagtc caacactgca ccaaacgttt cactaagtta gtccagctta 1260

gtctagtcta agccagtcca acttagtcct tgaagctagt ccagtccgag atagtccagc 1320

gcaacaaacg ccccctaaga ggttacaatc agtgtatatt tctggggcca agactgtggc 1380

tcaaaggaac aaaataacca gcccaatgta atcttgtctt gcccttgctc taaacatttt 1440

gattgactga actttgtgcc ctagcataat atatacattt tttttttaaa ttcttccgca 1500

aatccaattt atttttaagg aaatgaaaat tgctgctggt cacaaacatt ttattcatgg 1560

ttcaccttat ctcttcatgc atcttcattc aagaaaacaa caaaataaaa ccccacaata 1620

atagacatgg gtttttccag tttcttccat tctcaattct gtggccaata aattgtctga 1680

attttatttt ttgtcacaaa gaaattgcct aatttttggt ggagaagaag agagcttaaa 1740

cactaacctc aggtcctcaa gcaccaagaa acgagtcaaa tgactgagtt tccaagcact 1800

ctttgtttga cacgtggcat caatgcaatc ttcattctat ggtctcaaac cattcaacaa 1860

gcaaatccaa acgatatcaa attctgtaca ctaacactat aaaggcaaac cagctctcat 1920

ttcttttccg ctcaaaacac aatagcatac tgtgcttgcg ggcaaccagg agtgcccccc 1980

tctgtcactc acactagaac acaaccgtcc gatccgcgag agatc 2025

<210>7

<211>2542

<212>DNA

<213>Artificial Sequence

<400>7

taacttgaga ccaagatagc cactttcgag ccgttttccg gccaaaccac tgtgagttag 60

ccactgaaaa aggtaccatt cttttcttct agtccagaag tatgattttc gtttttgaat 120

cacttaattt tgttgaatat tgaagaagtt atgaatgttt aaagtttatc cagttttcga 180

cgggttttcc aattttccct cagaccagtc aactttgagg ctattttcag accatctccg 240

acaaccattg ggcttccaaa taggtataat cttgttctaa actttcatat cttcattttg 300

atatattatg gctcgacttt gattaagaaa cgattgagtt acaaagcctt gaaaattgcc 360

caaacttccg gccaagaggt tcgggacact tgacagagtt tttaacggaa tgaccaaaat 420

gatggatgat ggacatctca gggaccactt ctattgattt gaatctcagg gaccaaagtg 480

aggagttatg cgaatttcat agaccacttt ggctaaaaag ccttagtatt attatgactg 540

acaaacaaag gttgtctctt ttatattccc aattaattta catgattaga atggaacata 600

gggtttctca tgttgtgttt atgggtgaaa tagtctatat atgtctccat atgattgaag 660

agttttactt gtgaatttta gtggtgtgtc gtgttataaa gagttaaagg ttgcatataa 720

aagcatgctt aagcgttaac taattaacaa cttaatacgg tgctaactgt tcgattaggt 780

ctcccctaca aattgaactt acgatccgtt cattttttag tgctctttca tattattctt 840

actgaaattg gaaatcattt aatcttttaa ttattattgt cttttatgga aacaaatgca 900

aattcagata tcaaacagat gtgtttgtct aagagagaga gagggggttt ggatgataga 960

aaggggacct agtcattaat tatcattgta aattgtattg tttatttatt tatcaagtcc 1020

agttaaataa ctcatcttca attttaatga aattttgcaa gatgatcttt cattgataac 1080

ctaaaacttg ttagttcaaa ttgtaaaaat attgtgaaat gagtttaaac gagtgtttag 1140

caccatatta agttactaac tggtttgaaa agtactctgt aattttgttt tgttacaaat 1200

gctattctac actaatatac attaagaaga ggagaagctt tgaactgaag aagtaacgaa 1260

taaagaggaa gcttctctaa cttccagtac cagagcaatc cattacttgt actagtttga 1320

taagttttac tttcacagtc aaattgtgtt atgtcgtgtt aacgagtgaa attgttgata 1380

ggcttgagat atgggccagg tcaggactct aagggggcgt ttgtttaccc tcattaagtg 1440

ggactagact ggattggact agctgttagt ccaatactgt gtttgttcca tgctgggact 1500

aacattaatg agactaaagg ggactagcat ggacaaaacc cttcactaag tggtcttagc 1560

gagacccccc aataaccatg ggactagcta agactatcct ctctcatcct ctcatgctca 1620

acaaacactc tcgatagact cctcgtcatc tccagtcacc tagatcataa taaaatatta 1680

ataatttata tattaaataa tataatatta gaatttgtta ttatccagct tcttagtcca 1740

acactgcacc aaacgtttca ctaagttagt ccagcttagt ctagtctaag ccagtccaac 1800

ttagtccttg aagctagtcc agtccgagat agtccagcgc aacaaacgcc ccctaagagg 1860

ttacaatcag tgtatatttc tggggccaag actgtggctc aaaggaacaa aataaccagc 1920

ccaatgtaat cttgtcttgc ccttgctcta aacattttga ttgactgaac tttgtgccct 1980

agcataatat atacattttt tttttaaatt cttccgcaaa tccaatttat ttttaaggaa 2040

atgaaaattg ctgctggtca caaacatttt attcatggtt caccttatct cttcatgcat 2100

cttcattcaa gaaaacaaca aaataaaacc ccacaataat agacatgggt ttttccagtt 2160

tcttccattc tcaattctgt ggccaataaa ttgtctgaat tttatttttg tcacaaagaa 2220

attgcctaat ttttggtgga gaagaagaga gcttaaacac taacctcagg tcctcaagca 2280

ccaagaaacg agtcaaatga ctgagtttcc aagcactctt tgtttgacac gtggcatcaa 2340

tgcaatcttc attctatggt ctcaaaccat tcaacaagca aatccaaacg atatcaaatt 2400

ctgtacacta acactataaa ggcaaaccag ctctcatttc ttttccgctc aaaacacaat 2460

agcatactgt gcttgcgggc aaccaggagt gcccccctct gtcactcaca ctagaacaca 2520

accgtccgat ccgcgagaga tc 2542

Claims (7)

1. A DNA molecule shown as SEQ ID No. 3.
2. 2. A biomaterial containing the DNA molecule of claim 1, which is any one of the following B1) to B8):

   B1) an expression cassette comprising the DNA molecule of claim 1;

   B2) a recombinant vector comprising the DNA molecule of claim 1;

   B3) a recombinant vector comprising the expression cassette of B1);

   B4) a recombinant microorganism comprising the DNA molecule of claim 1;

   B5) a recombinant microorganism comprising the expression cassette of B1);

   B6) a recombinant microorganism containing the recombinant vector of B2);

   B7) a recombinant microorganism comprising the recombinant vector of B3).
3. 3. A primer pair for amplifying the DNA molecule of claim 1.
4. 4. Use of the DNA molecule of claim 1 or the biomaterial of claim 2 for promoting expression of a target gene in a plant cell; the plant is apple.
5. 5. Use according to claim 4, characterized in that: the plant cell is a protoplast cell.
6. 6. A method of expressing a gene of interest comprising the steps of: the DNA molecule of claim 1 as a promoter to promote the expression of a target gene in apple.
7. 7. The method of claim 6, wherein: the expression target gene is expressed in the apple protoplast cell; the apple protoplast cell is prepared by separating an apple callus cell which grows for 10 days on an MS culture medium added with phytohormone as a test material.

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